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Les strigolactones (SL) constituent la neuvième classe d'hormones végétales. Chez les Angiospermes, ces hormones jouent divers rôles dans le développement végétatif et sont des régulateurs majeurs de l'architecture aérienne et racinaire. Leur effet le plus notable est la répression de l'activité des bourgeons axillaires qui se traduit par une moindre ramification des tiges.

Pour autant, les SL n'ont été caractérisées en tant que phytohormones qu'en 2008. En effet, cette famille de molécules avait été auparavant mise en évidence en sa qualité de signal interspécifique, libéré dans la rhizosphère. Les SL jouent ce rôle dans le cadre de deux types d'interactions : le parasitisme inter-végétal et la symbiose mycorhizienne à arbuscules (AMS). L'implication des SL lors du parasitisme fut le premier mécanisme découvert dans les années 1960, lorsque C.E. Cook et ses collègues isolèrent des molécules jusque-là inconnues à partir d'exsudats racinaires de coton. Ces molécules qui faisaient germer les graines de l'Angiosperme parasite Striga lutea furent nommées strigol et strigyl acétate, en l'« honneur du parasite ». Une trentaine d'années plus tard, suite à l'isolement de plusieurs composés proches chez de nombreux hôtes des Striga, une équipe japonaise mit en évidence l'action similaire d'une molécule structurellement apparentée, l'orobanchol, exerçant son effet sur la germination des graines de la plante parasite Orobanche minor (autre famille que Striga parmi les Orobanchaceae). Du fait de leur structure proche de celle du strigol, contenant deux groupements lactones, ces molécules furent collectivement baptisées strigolactones.

Ces découvertes soulevèrent une question qui tarauda de nombreux scientifiques jusqu'au milieu des années 2000 et qui peut être résumée ainsi : « Pourquoi diable la sélection naturelle n'a-t-elle pas mis un terme à la production de ces molécules chez les plantes hôtes ? ». C'est donc en 2005, que l'équipe de Kohki Akiyama mit en évidence qu'une autre strigolactone exsudée dans la rhizosphère du lotier, le 5-désoxystrigol, induisait la ramification des hyphes du champignon Gigaspora margarita, symbiote de nombreuses plantes dans le cadre de l'AMS. Ce résultat fut ensuite répliqué avec d'autres strigolactones et l'effet positif des SL sur la croissance fongique partiellement élucidé. Ces découvertes indiquent que l'effet positif des SL sur la symbiose l'emporte sur leur effet négatif dans le cadre du parasitisme par les Orobanchaceae et expliquent la conservation des SL au cours de l'évolution des plantes terrestres.

Etant donné le rôle majeur des SL dans l'induction de l'AMS, une symbiose à l'origine ancienne et quasiomniprésente parmi les plantes terrestres, il n'est pas étonnant de retrouver la capacité de biosynthèse de SL au sein de tous les taxons. En effet, les gènes codant les premières enzymes de biosynthèse des SL (D27, CCD7 et CCD8) sont retrouvés chez les Bryophytes (mousses, hépatiques et anthocérotes), les Lycophytes (sélaginelles, lycopodiales et les isoëtes), les Monilophytes (fougères et prêles) et les Spermatophytes (Gymnospermes et Angiospermes), et même chez certaines algues Spermatophytes (algues les plus proches des plantes terrestres). Il est donc tentant de penser que la fonction ancestrale des SL était de promouvoir la symbiose chez les premières plantes terrestres. De plus, cette hypothèse semble être confirmée par l'absence d'homologues canoniques des gènes codant le récepteur des SL (D14) et les acteurs régulant ensuite la transduction du signal (SMXL7) en dehors des Spermatophytes (voire des Angiospermes). Seule la protéine à boîte F MAX2, agissant comme corécepteur des SL, est retrouvée chez toutes les plantes terrestres. L'absence du récepteur D14 et du répresseur SMXL7 indiquerait que les SL n'ont pas de fonction phytohormonale chez les plantes terrestres souvent présentées comme « basales » (non-Spermatophytes, qui ne produisent pas de graines).

Au contraire, toutes les plantes terrestres possèdent, en plus de MAX2, un homologue plus lointain de D14 appelé KAI2. Chez les Angiospermes, KAI2 et MAX2 agissent ensemble avec SMAX1 (un autre homologue SMXL) dans la voie de réponse à un autre signal, le KAI2-ligand (KL). Le KL est une phytohormone encore non-identifiée qui peut être mimée par les karrikines (KAR), des molécules produites à partir de la combustion de matériel végétal lors des feux de forêt. Les KAR induisent la germination des graines chez certaines espèces, notamment chez la plante modèle Arabidopsis. Hormis ce rôle de promotion de la germination, KAR et KL régulent le développement précoce des plantules et il a récemment été montré que la voie KAI2-MAX2-SMAX1 est nécessaire à l'instauration de l'AMS chez les Angiospermes. Les études phylogénétiques récentes semblent indiquer que cette voie KL est ancestrale. Cependant, le rôle de cette voie en dehors des plantes à graines reste à déterminer, ainsi que celui qu'elle jouait chez les plantes terrestres ancestrales. Ces éléments d'introduction sont développés dans le chapitre III. A ce jour, seule une espèce de Bryophytes a fait preuve d'une réponse développementale à un traitement avec des SL : la mousse Physcomitrium patens (P. patens). Le développement de P. patens et son utilisation en tant que plante modèle en biologie sont décrits dans le chapitre I. Les rôles et voies cellulaires associées aux phytohormones chez P. patens sont détaillés dans le chapitre II. En outre, il est important de souligner que les mousses ne recourent pas à la symbiose avec des champignons endomycorhiziens. Donc, la réponse développementale observée chez cette espèce n'est pas le fait d'un défaut d'association symbiotique. P. patens réagit notamment à la présence de faibles quantités de SL par une diminution de la ramification et de la croissance de son protonema (phase développementale filamentaire caractéristique des mousses). Lorsque le gène de biosynthèse des SL CCD8 est muté chez P. patens, la plante développe un phénotype de déficience en SL, révélé notamment par une croissance et une ramification exacerbée du protonema. Comme toutes les Bryophytes, P. patens ne possède pas d'homologue proche de D14 et de SMXL7, mais possède un homologue de MAX2 (PpMAX2), ainsi que de nombreux homologues de KAI2 (PpKAI2Like ou PpKAI2L-A à -M) et quatre homologues SMAX1/SMXL7 (PpSMXLA à D). L'expansion des familles KAI2L et SMXL est une spécificité des mousses parmi les plantes non-Spermatophytes. Etant donné que P. patens est capable de répondre aux SL, une hypothèse possible est que PpMAX2, certaines copies PpKAI2L et certaines copies PpSMXL seraient impliquées dans la voie de signalisation des SL à la manière de ce qui est connu chez les Angiospermes. Cependant, le mutant perte-de-fonction Ppmax2 ne présente pas un phénotype développemental similaire à Ppccd8, ce qui est pourtant attendu pour un mutant de réponse aux SL. Au contraire, Ppmax2 a un protonema peu développé et passe rapidement à la phase développementale suivante, c'est-à-dire la formation de tiges feuillées (gamétophores) qui vont porter les organes reproducteurs. En outre, Ppmax2 peut toujours répondre à un ajout de SL exogènes, ce qui démontre clairement que la protéine PpMAX2 n'est pas nécessaire pour cette réponse. Une caractérisation fine de ce mutant suggère que PpMAX2 est impliquée dans la réponse à la lumière et probablement dans la voie de signalisation du KL, ce qui est également appuyé par les travaux de thèse ici présentés (chapitres IV, VI et VII). Une étude préliminaire portant sur les protéines PpKAI2L suggère pourtant que si certaines sont largement similaires à KAI2 et donc potentiellement impliquées dans la perception du KL, d'autres peuvent être des récepteurs de SL car leur structure les rapproche de D14. Les prédictions de cette étude sont ici largement confirmées dans le chapitre IV. P. patens possède quatre clades de PpKAI2L : le clade (A-E) est impliqué dans la voie PpMAX2dépendante, le clade (G, J, M) est nécessaire à la perception des SL, alors que les clades (F, K) et (H, I, L) exercent des fonctions restant à élucider. Ces conclusions reposent sur l'étude de mutants multiples Ppkai2-L et de leur capacité à répondre à une SL artificielle ou à un mime de KL artificiel, ainsi que sur la caractérisation biochimique de certaines protéines PpKAI2L. Dans le chapitre VI, le rôle des protéines PpSMXL dans la voie de réponse aux SL et dans la voie PpMAX2dépendante est exploré, par la caractérisation du développement de mutants Ppsmxl et de lignées sur-exprimant les gènes PpSMXL, ainsi que par des approches de recherche d'interactions protéine/protéine. Les protéines PpSMXL sont séparées en deux clades (A/B et C/D) mais semblent toutefois avoir la même fonction d'inhibiteurs de la voie PpMAX2dépendante. Cependant, il apparaît que la voie de signalisation des SL agit également via PpSMXLC/D car les doubles mutants Ppsmxlcd ne répondent plus à un traitement avec des SL artificielles. Dans le chapitre VII, le rôle des protéines PpSMXL en situations de stress (blessure et exposition au froid) est investigué et achève de mettre en évidence un rôle primordial et particulier de la protéine PpSMXLC dans l'équilibre entre croissance et tolérance au stress. Les rôles hypothétiquement opposés des SL et du KL sur la division cellulaire sont également explorés.

En conclusion, il apparaît que la mousse P. patens n'utilise pas la même voie de signalisation des SL que les Angiospermes. L'expansion de la famille des KAI2L au fil des duplications de génomes dans la lignée évolutive des mousses a permis l'émergence d'une nouvelle fonction : la perception des SL. Il est intéressant de noter que l'émergence de la germination SL-dépendante chez les plantes Angiospermes parasites est expliquée par un mécanisme analogue.

Les copies additionnelles de SMXL chez les mousses, ainsi que l'unique copie de MAX2, n'ont pas été recrutées pour transduire le signal SL au cours de l'évolution des mousses, à l'inverse de ce qui est probablement advenu au cours de l'évolution des Angiospermes. Par ailleurs, l'existence d'une voie similaire à la voie KAI2-MAX2-SMAX1 des Angiospermes chez la mousse P. patens appuie l'hypothèse selon laquelle cette voie KL est ancestrale chez les plantes terrestres. En outre, la voie KL semble jouer un rôle majeur dans le développement de P. patens, ce qui pourrait indiquer qu'elle jouait également un rôle très important chez les premières plantes terrestres, probablement plus semblables aux Bryophytes qu'aux Angiospermes dans leur développement.

Firstly, I would like to thank Sandrine and Mauricio for giving me the opportunity to get invested in their interesting project, and all CORAM team members for the warm and laid-back atmosphere I could enjoy at the lab for 4 years now. I give my respectful regards to Philippe, the Master of Molecular Biology, for his technical support and our funny (often coming out of nowhere) conversations. My thanks also go to Catherine for her numerous advices and for sneezing on Sandrine instead of me! I warmly thank all people at the IJPB who helped me prepare for the Doctoral School exam, as I probably would not be writing a ~200 pages manuscript now without their help ^^ Thanks to Alex for teaching me the bases of biochemistry, even though I (strangely) seem incompatible with proteins … But hey that is only because SMXL are so difficult to work with! ^^ More seriously, I thank the members of my thesis committee, Annie, Yoan and Patrick, for their helpful comments and constructive criticism. I also add Fabien to this list of helpful people, for always taking the time to check my guide RNAs and helping me to prepare presentations, and Gladys for training me on almost every microscope at the IJPB. I thank Marion, Florence and Anouchka for their nice help on several occasions. I dedicate a special thanks to the two interns I had the chance to supervise during this thesis, especially Malo without whom I would have abandoned regeneration experiments. Beyond their helpful participation to experiments and the mentoring skills I gained; it was always a pleasure to talk about science (among other things) with them. I also thank Carine and Anouchka for sometimes almost forcing me to participate in presenting the IJPB to middle/high school students. It often proved to be quite funny in the end and it enabled me to think of my research in a more "out-ofthe box" fashion.

Finally, I thank all my family and friends, from my Master years or from (far!) before, for supporting me during these three years (+2 months) of quite intense work, furthermore during lockdown even though we could only see each other on screen … I telepathically send my most embarrassing good vibes to my comrades Hayat, Mathilde, Pierre, Gwilherm and especially Houda for their thesis finish line. Bryophytes are non-vascular descendants of the earliest diverging land plants. Land plants emergence is dated around 500 Mya, while the vascular plants (tracheophytes) lineage diverged approximately 450 Mya [START_REF] Morris | The timescale of early land plant evolution[END_REF]. Phylogenetic relationships between the three classes of bryophytes, namely mosses (Bryopsida), liverworts (Marchantiopsida) and hornworts (Anthocerotopsida), have long been a subject of debate. Nevertheless, bryophytes now tend to be considered as a monophyletic clade, named Bryophyta [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF][START_REF] De Sousa | Nuclear protein phylogenies support the monophyly of the three bryophyte groups (Bryophyta Schimp.)[END_REF][START_REF] De Sousa | The Chloroplast Land Plant Phylogeny: Analyses Employing Better-Fitting Tree-and Site-Heterogeneous Composition Models[END_REF], relative to vascular plants (see figure I-1). Together with the liverwort Marchantia polymorpha, the moss Physcomitrium (or Physcomitrella) patens has since long outreached from the field of bryology into diverse plant biology studies as a major model species [START_REF] Rensing | The moss Physcomitrium (Physcomitrella) patens: A model organism for non-seed plants[END_REF]. This phenomenon is explained by the relatively simple life cycle and development of this moss, while still showing numerous similarities to other land plants, particularly at the cellular level [START_REF] Schaefer | The moss Physcomitrella patens, now and then[END_REF][START_REF] Wood | Bryophytes as Model Systems[END_REF]. In addition, P. patens has a particularly high regeneration ability, making in vitro propagation quick and convenient [START_REF] Cove | The Moss Physcomitrella patens[END_REF]. Thanks to the sequencing of its genome [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF], as well as to its high frequency of homologous recombination [START_REF] Schaefer | Efficient gene targeting in the moss Physcomitrella patens[END_REF][START_REF] Schaefer | Gene targeting in Physcomitrella patens[END_REF], P. patens is particularly amenable to genome modifications and thus to reverse genetics. While P. patens has been shown to be transformable by diverse methods including Agrobacterium mediated transfection (Cove et al., 2009b;[START_REF] Li | Genetic transformation of Physcomitrella patens mediated by Agrobacterium tumefaciens[END_REF] and biolistic based methods [START_REF] Smidkova | Efficient biolistic transformation of the moss Physcomitrella patens[END_REF], its robust protoplasts [START_REF] Cove | The Moss Physcomitrella patens[END_REF] are most often transformed through poly-ethylene-glycol (PEG)-mediated membrane permeabilization (Hohe and Reski, 2002). Moreover, modification of several loci at once has been achieved very efficiently via the use of CRISPR-Cas based systems in P. patens, that is seemingly becoming more prevalent over homologous recombination (Lopez-Obando et al., 2016b;[START_REF] Collonnier | CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51independent gene targeting in the moss Physcomitrella patens[END_REF][START_REF] Mallett | Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens[END_REF][START_REF] Yi | Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens[END_REF][START_REF] Pu | A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens[END_REF][START_REF] Veillet | Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops[END_REF]. Forward genetics methods have also been proven to work well in this species, as collections of randomly mutagenized P. patens mutants exist, obtained using transposons [START_REF] Egener | High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library[END_REF][START_REF] Vives | Highly efficient gene tagging in the bryophyte Physcomitrella patens using the tobacco (Nicotiana tabacum) Tnt1 retrotransposon[END_REF][START_REF] Mohanasundaram | Agrobacteriummediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte[END_REF] or T-DNA (Cove et al., 2009b). Several reports also showed that P. patens is amenable to chemical (alkylating agents such as EMS), genotoxins [START_REF] Holá | Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens[END_REF], and radiation (X-rays and UV) triggered mutagenesis [START_REF] Engel | THE INDUCTION OF BIOCHEMICAL AND MORPHOLOGICAL MUTANTS IN THE MOSS PHYSCOMITRELLA PATENS[END_REF]Cove et al., 2009a). Most importantly, the genome of P. patens is in a haploid state for the longest part of the life cycle. In other words, the gametophytic stage is dominant over the sporophytic stage in bryophytes, contrarily to vascular plants. That makes mutant generation even more convenient since possible mutant phenotypes are visible immediately (at least when the mutant is regenerated from a single mutagenized cell, e.g. a protoplast) without there being a need for a round of self-fertilization to generate homozygotes. This is especially valuable since the fertility of the laboratory multiplied Gransden ecotype has significantly decreased along years [START_REF] Rensing | The moss Physcomitrium (Physcomitrella) patens: A model organism for non-seed plants[END_REF]. However, this advantage can become a hindrance when vital genes are targeted. Fortunately, knock-down alternatives are a part of the well-tried toolkit in P. patens [START_REF] Khraiwesh | Specific Gene Silencing by Artificial MicroRNAs in Physcomitrella patens : An Alternative to Targeted Gene Knockouts[END_REF][START_REF] Nakaoka | An Inducible RNA Interference System in Physcomitrella patens Reveals a Dominant Role of Augmin in Phragmoplast Microtubule Generation[END_REF] and can circumvent this issue, as well as the generation of conditional mutants using inducible promoters [START_REF] Kubo | System for Stable β-Estradiol-Inducible Gene Expression in the Moss Physcomitrella patens[END_REF]. Additionally, somatic diploids can be induced to resolve this issue [START_REF] Rensing | The moss Physcomitrium (Physcomitrella) patens: A model organism for non-seed plants[END_REF].

Figure I-2 -Physcomitrium patens cycle of life I-C) Physcomitrium patens cycle of life

P. patens gametophytic life stage starts from the germination of a spore under appropriate environmental conditions, the imbibition level and amount of red light being crucial [START_REF] Cove | GENETIC AND PHYSIOLOGICAL STUDIES OF THE EFFECT OF LIGHT ON THE DEVELOPMENT OF THE MOSS[END_REF]. The first tissue that is generated from the germinating spore is primary chloronema, composed of cells with a high chloroplast content and disposed in a single cell file (filaments) (figure I-2). This filament elongates via division and longitudinal elongation of the apical cell (tip-growth) and branches via division of subapical cells. Apical cells of these first produced filaments eventually switch to producing another type of tip-growing filament, which is faster growing and has a decreased chloroplast content: the caulonema. Caulonema branches similarly to chloronema and gives rise mainly to secondary chloronema. Aside from the aforementioned differences between the two types of filaments, another divergence is the orientation of cross walls: chloronema's are perpendicular to the elongation axis, while caulonema's are oblique [START_REF] Crandall-Stotler | Morphology of Mosses (Phylum Bryophyta)[END_REF]. Taken together, chloronema and caulonema make up the protonema, which can be seen as P. patens first stage of gametophytic life, enabling superficial colonization of the medium [START_REF] Cove | The Moss Physcomitrella patens[END_REF]. Environmental cues that are not clearly understood, together with better known endogenous signals (notably cytokinins [START_REF] Reski | Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine[END_REF], see the following literature review on phytohormones in mosses in chapter II), can trigger the differentiation of branch cells initials emerging from caulonema into bud progenitor cells. These cells acquire the ability to divide asymmetrically and eventually lead to the formation of leafy shoots called gametophores, switching P. patens development pattern to three-dimensional growth. Gametophores develop initial cells of phyllids (proto leaves) by successive divisions of their apical meristematic cell [START_REF] Harrison | Local Cues and Asymmetric Cell Divisions Underpin Body Plan Transitions in the Moss Physcomitrella patens[END_REF]. Phyllids develop following a spiral phyllotaxis on the gametophore stem and are made of two main parts: the lamina, which is a monolayer of highly photosynthetic cells, and the central multilayered midrib which contains water-conducting tissues (hydroids) [START_REF] Crandall-Stotler | Morphology of Mosses (Phylum Bryophyta)[END_REF]. The gametophore stem epidermis, at the axil of phyllids in the continuity of the midrib [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF], generates another type of filaments: the anchoring rhizoids. In P. patens, rhizoids are unbranched, reddish-brown pigmented filaments, and structurally very similar to caulonema, also having oblique cross walls [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF]. Also at the axil of phyllids, but on the adaxial side, axillary hairs develop from the epidermis [START_REF] Eklund | Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens[END_REF]. However, the function of these structure is still under investigation. The stem epidermis can moreover occasionally give rise to adventitious gametophores (or branches) [START_REF] Eklund | Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens[END_REF][START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF]. Ultimately, each gametophore differentiates several male (antheridia) around female (archegonia) reproductive organs at its apex, when environmental conditions become suitable (low temperature and short daylength (Hohe et al., 2002;[START_REF] Landberg | The moss physcomitrella patens reproductive organ development is highly organized, affected by the two SHI/STY genes and by the level of active auxin in the SHI/STY expression domain[END_REF]). After fertilization of the oosphere in the archegonia (usually self-fertilization) a diploid zygote is formed, most often from a single archegonia per apex (Hohe et al., 2002). The success rate of fertilization is highly reliant on a high hygrometry level so that the sperm cells can access the egg cell in the archegonia [START_REF] Kofuji | Antheridial development in the moss physcomitrella patens: Implications for understanding stem cells in mosses[END_REF][START_REF] Hiss | Sexual reproduction, sporophyte development and molecular variation in the model moss Physcomitrella patens: introducing the ecotype Reute[END_REF]. The zygote undergoes several rounds of mitosis, giving rise to the diploid sporophytic stage of P. patens life. However, the sporophyte never lives independently from its maternal gametophyte and keeps getting nutrients from it through their interconnecting seta. This stage is very brief as specific cells of the sporangium soon undergo meiosis and produce haploid spores. When the sporophyte becomes mature and dry, it ruptures, freeing the spores in the environment and starting a new gametophytic cycle. The whole cycle usually takes around 4 months to be completed in the so far most used Gransden ecotype [START_REF] Engel | THE INDUCTION OF BIOCHEMICAL AND MORPHOLOGICAL MUTANTS IN THE MOSS PHYSCOMITRELLA PATENS[END_REF].

I-D) Physcomitrium patens usefulness in evo-devo studies

Owing to its early divergence from other land plants as a bryophyte, P. patens is widely employed as a model plant species to explore research topics relative to plant evolution. As a matter of facts, despite its ancient divergence with vascular plants, P. patens shares many characteristics with them, making it particularly amenable to evo-devo (Evolutionary Developmental Biology) approaches. Studying P. patens can hence give precious insight into both the ancestral state and the possible evolutionary fates of diverse conserved processes and structures. Indeed, while organs are notably different between this moss and vascular plants, many parallels can be traced between them. For instance, rhizoids can be associated to vascular plants' root hair, phyllids to leaves photosynthetic parenchyma, and the basic mechanisms underlying meristem maintenance and function can be compared. Moreover, P. patens is widely comparable to vascular plants model species at the cellular level, being equally sensitive and displaying similar responses to most growth-regulating compounds (e.g. phytohormones, see the following review in chapter II) and environmental stimuli (light, gravitropism, nutrient deficiency, dehydration, etc.). It is also important to note that even points of divergence between bryophytes and vascular plants can be addressed by using P. patens as a model, notably the switch between gametophytic and sporophytic dominance [START_REF] Pires | Morphological evolution in land plants: new designs with old genes[END_REF][START_REF] Bowman | Evolution in the Cycles of Life[END_REF].

There is however a cautionary statement to remember: P. patens is not more representative of the ancestral land plants than any other extant land plant [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF]. Each plant species is the result of a mix of conserved primitive features and derived characters distinctive of its lineage. Only a joint effort, ideally with comparative studies including plants from diverse lineages (from bryophytes and tracheophytes), can give a reliable view of ancestral processes.

INTRODUCTION

Hormones make up a class of signaling molecules, specific to multicellular organisms. They enable communication between somewhat distant cells of the same organism and thus coordination of distant cells/tissues to trigger given physiological and/or developmental processes. Classically, hormones have been defined in animals as signaling molecules produced by particular tissues or even organs (glands) that act on other cells at a distance, by moving through the blood flow, are perceived at low concentration by specific receptors and induce diverse physiological responses in the target cells (historical definition of Sterling in 1905, also establishing the term hormone). These effects are attained via the activation of an intracellular transduction pathway, and notably act via modulation of gene transcription. The main divergence between animal hormones and plant hormones (also called phytohormones, which concept even predates that of animal hormones as it can be traced back to the 18 th century) is that all plant cells are potentially able to synthesize and excrete phytohormones, while only highly specialized cells are dedicated to this function in animals, for each given hormone. Phytohormones can be transported through the vasculature (xylem or phloem) in Tracheophytes, and even by cell-to-cell transport (see [START_REF] Park | Plant hormone transporters: What we know and what we would like to know[END_REF] for a review on this subject).

Perception usually occurs at the plasma membrane or in the cell, mostly depending on the hydrophilic/hydrophobic nature of the signal. Generally speaking, hydrophobic signals act directly in the target cell nucleus and thus do not require the generation of a secondary messenger. Chemical identity of molecules acting as hormones can be very variable, in both animals and plants.

This chapter is presented as a review paper that was submitted, in a less detailed form, to Plant Molecular Biology (https://www.springer.com/journal/11103/updates/17618428). It can be read independently from chapter I and all following chapters without preventing understanding.

Authors contributions:

Both A.G and S.B contributed to compiling and reading appropriate literature and to the writing of the present review paper.

Phytohormones have first been discovered and studied in Angiosperms, notably auxin which presence was evidenced in the 1920s [START_REF] Went | On growth-accelerating substances in the coleoptile of Avena sativa[END_REF]. Soon, they were found to also be produced in early-diverging, non-vascular plants, which raised new interrogations about their involvement in plant evolution. Pioneer work of Bopp et al. starting for the 1950s on Funaria hygrometrica (F. hygrometrica), and complementary work by Ashton et al. and Wang et al. and then by Reski et al. on Physcomitrium (Physcomitrella) patens (P. patens), starting from the end of the 1970s, paved the way to understanding phytohormones' biosynthesis and action in mosses. The specific ease of P. patens' use in genetic studies gradually made this species the favorite model for mosses and even one main model in plant biology [START_REF] Engel | THE INDUCTION OF BIOCHEMICAL AND MORPHOLOGICAL MUTANTS IN THE MOSS PHYSCOMITRELLA PATENS[END_REF][START_REF] Ashton | The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens[END_REF]. The sequencing of P. patens genome [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF] added to the potential of this species by broadening its use to reverse genetics studies. In addition to this, most of the decades-old knowledge gained from early studies focused on Funaria hygrometrica and Ceratodon purpureus has now been transferred to P. patens.

Hence, we chose to focus on this model species and findings emerging from other mosses will not be reported here unless the information is especially meaningful and/or missing in P. patens.

Phytohormones classically refer to nine groups of compounds primarily identified in Angiosperms: auxins (AUX), cytokinins (CK), gibberellins (GA), abscisic acid (ABA), ethylene (ETH), brassinosteroids (BR), salicylic acid (SA), jasmonic acid (JA) and strigolactones (SL). A comprehensive study of phytohormone content on a broad spectrum of 30 Bryophytes species (among which 24 mosses, [START_REF] Drábková | Phytohormone profiling across the bryophytes[END_REF] highlighted the presence of AUX, CK, ABA, SA and JA. GA and BR were identified in low quantities and their occurrence in mosses was deemed dubious. SL and ETH were not addressed in this study. Since most of these groups are present in mosses, all nine classical phytohormones groups will be discussed in the present review, as well as CLE signaling peptides, and a possible tenth class of phytohormones that is so far referred to as KAI2-ligand (KL). Other compounds that are sometimes referred to as plant hormones, such as polyamines, strictly intracellular lipidic signals, signaling peptides aside from CLE, some signaling messenger RNAs, cyclic monophosphate nucleotides and small molecules such as Ca 2+ , nitric oxide (NO) and hydrogen peroxide will not be explored here. The reason for ignoring these is either that these molecules are most often considered as second messengers instead of hormones proper, or that evidence of their occurrence and/or function is fragmentary in mosses. Likewise, the signaling function of sugar and nitrate will not be explored here, as they are above all vital compounds.

AUXINS

The predominant and most potent natural auxin is indole-3-acetic acid, shortened as IAA . Auxin has been identified in evolutionarily diverse species, from filamentous brown algae to flowering plants, and is a wellknown regulator of cell growth and division [START_REF] Bail | Auxin metabolism and function in the multicellular brown alga ectocarpus siliculosus[END_REF]. While five different pathways for auxin biosynthesis have been proposed in flowering plants, only two appear to exist in P. patens. In 2010, Eklund and colleagues showed that genes involved in auxin biosynthesis from tryptophan through the indole-3-pyruvic acid pathway (TRYPTOPHAN AMINOTRANSFERASE 1 (TAA1)-likes) and from the tryptamine pathway (YUCCAs) have homologs in P. patens genome. It has not yet been formally proven that these genes are involved in auxin production, but some indirect evidence points to the tryptamine pathway being active. Indeed, PpSHI/STY proteins, which homologs in Angiosperms are inducers of YUCCA expression, do have a positive role on auxin biosynthesis in P. patens [START_REF] Eklund | Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens[END_REF]. The major auxin produced in many mosses is IAA [START_REF] Drábková | Phytohormone profiling across the bryophytes[END_REF], but it has not been confirmed in P. patens itself.

Auxin biosynthesis likely takes place all along protonema and its production in gametophores can be ensured by axillary hairs [START_REF] Eklund | Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens[END_REF]. A balance between active and inactive auxin can be achieved through conjugation to amino acids via GRETCHEN HAGEN3 (GH3) enzymes, which number four in P. patens [START_REF] Ludwig-Müller | Moss (Physcomitrella patens) GH3 proteins act in auxin homeostasis[END_REF]. The two PpGH3 genes studied are both widely expressed starting early in development but are not induced by auxin application. Nevertheless, PpGH3 genes seem to play a role in auxin homeostasis, as Ppgh3 loss of function mutants are hypersensitive to auxin and accumulate free auxin (Ludwig- [START_REF] Ludwig-Müller | Moss (Physcomitrella patens) GH3 proteins act in auxin homeostasis[END_REF].

The first demonstrated effect of auxin on moss development was induction of the chloronema to caulonema transition [START_REF] Johri | Auxin regulation of caulonema formation in moss protonema[END_REF][START_REF] Ashton | Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants[END_REF] . In 2006, Decker et al. further demonstrated that caulonema promotion by auxin is associated to an arrest of the cell cycle in the G1 phase, a specificity of caulonema.

Moreover, Thelander and colleagues recently showed that auxin inhibits protonema branching [START_REF] Thelander | Auxin-mediated developmental control in the moss Physcomitrella patens[END_REF].

Aside from this, auxin has many other developmental roles in mosses, such as the promotion of rhizoid formation [START_REF] Ashton | Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants[END_REF][START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF]. Exogenously supplied 1-Naphthaleneacetic acid (NAA, a synthetic artificial auxin analog) not only increases rhizoid number, but also induces apical rhizoids [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF].

Besides, expression of the HD-ZIP class I transcription factor gene PpHB7 is rapidly induced by NAA. HD-ZIP transcription factors are largely known as potent regulators of cell differentiation in Angiosperms. PpHB7 is partly responsible for the effect of auxin on rhizoid differentiation, notably by down-regulating total chloroplast mass per cell [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF]. Rhizoid determination, on the other hand, is likely PpHB7-independent. Yet another role of auxin is the regulation of gametophore morphogenesis: at low concentrations, NAA promotes elongation of gametophores stems [START_REF] Fujita | Convergent evolution of shoots in land plants: Lack of auxin polar transport in moss shoots[END_REF][START_REF] Bennett | Plasma membrane-targeted PIN proteins drive shoot development in a moss[END_REF] and cell elongation in phyllids [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF], but both tissues display a different level of sensitivity [START_REF] Bennett | Plasma membrane-targeted PIN proteins drive shoot development in a moss[END_REF].

Polar auxin transport (PAT) in Angiosperms relies on PIN (PIN-FORMED) efflux transporters, and four PIN genes (PpPINA to PpPIND) have been identified in P. patens [START_REF] Bennett | Plasma membrane-targeted PIN proteins drive shoot development in a moss[END_REF]. PpPIN genes are expressed in protonema with a maximum level in apical cells [START_REF] Viaene | Directional auxin transport mechanisms in early diverging land plants[END_REF], and the PpPIN proteins themselves are located both at the basal and at the apical membrane in these cells, suggesting auxin is exported acropetally but also exported out of the tip in the medium. In protonema, PpPINA-C limit intracellular auxin content, thereby delaying the onset of caulonema differentiation [START_REF] Viaene | Directional auxin transport mechanisms in early diverging land plants[END_REF]. Bennett et al. have demonstrated that PpPINA and PpPINB function is also needed for proper gametophore development. Interestingly, the gametophores of the Pppina/b double mutant are agravitropic and aphototropic (to blue light) but not its filaments, suggesting PpPIN function is most important in gametophores [START_REF] Bennett | Plasma membrane-targeted PIN proteins drive shoot development in a moss[END_REF]. In gametophores, auxin produced by the main stem exerts an inhibitory effect on branches emergence [START_REF] Von Maltzahn | Interaction between Kinetin and Indoleacetic Acid in the Control of Bud Reactivation in Splachnum ampullaceum (L.) Hedw[END_REF][START_REF] Nyman | Auxin-cytokinin interaction in the inhibition, release, and morphology of gametophore buds of Plagiomnium cuspidatum from apical dominance[END_REF][START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF]. However, auxin production in the apex and PpPIN-mediated exportation cannot explain alone the branching pattern of gametophores.

Indeed, the Pppina/b mutant is only minorly affected in gametophore branching and PIN inhibitors have no impact on this phenotype [START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF]. Computer modelling proved that only a bidirectional transport of auxin in the stem can generate a realistic branching pattern. Further investigation by [START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF] identified regulation of plasmodesmal conductivity through callose deposition as a mean for bidirectional auxin transport. As for the auxininduced emergence of rhizoids, Thelander and colleagues suggest that a combination of PpPIN-mediated auxin export from the leaves [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF], and bidirectional plasmodesmal transport in the stem, might allow sufficient auxin accumulation at the base of phyllids to promote rhizoid initiation [START_REF] Thelander | Auxin-mediated developmental control in the moss Physcomitrella patens[END_REF]. Auxin production and export have also been shown to be important for apical opening of both archegonia and antheridia, and beforehand during the development of the egg cell [START_REF] Landberg | The moss physcomitrella patens reproductive organ development is highly organized, affected by the two SHI/STY genes and by the level of active auxin in the SHI/STY expression domain[END_REF]. Auxin response seems even more tightly spatially and temporally regulated across embryo development which indicates a complex role of auxin during zygote maturation.

Finally, PpPINA and PpPINB are required for proper sporophyte development and notably prevent sporophyte branching [START_REF] Bennett | Plasma membrane-targeted PIN proteins drive shoot development in a moss[END_REF].

Many genes homologous to auxin signaling components in flowering plants are found in P. patens genome [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF] . In vascular plants, auxin is perceived by receptors of the TIR1/AFB family. Then the auxin signal is transduced via the degradation of Aux/IAA repressors, which frees transcription factors of the ARF family, regulating the expression of auxin-responsive genes. P. patens possesses four TIR1/AFB genes, two more distant homologs (PpXFB1 and PpXBF2), three Aux/IAA genes (PpIAA1A, PpIAA1B and PpIAA2, [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF]) and a dozen of ARF genes [START_REF] Paponov | The evolution of nuclear auxin signalling[END_REF][START_REF] Lavy | Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins[END_REF][START_REF] Kato | Evolution of nuclear auxin signaling: lessons from genetic studies with basal land plants[END_REF]. From the study of transient knock-down RNAi lines, it appears that if PpXFBs are not required for response to auxin, each PpAFB is necessary for response to NAA and therefore a good auxin receptor candidate [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF]. Despite being larger proteins than flowering plants Aux/IAAs, PpIAA contain all known functional domains, including a consensus EAR motif required for transcriptional regulation and a degron motif necessary for proteasomal degradation [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF]. Prigge and colleagues examined 17 NAR mutants (NAA-resistant mutants (Ashton et al., 1979a)) and found that the more severe ones were mutated in the DII degron motif of PpIAAs (VGWPPV). In Angiosperms, similar mutations prevent association with the SCF TIR1/AFB ubiquitylation complex, therefore making the Aux/IAA more stable and preventing auxin signal transduction. Thus, PpIAAs do act as negative regulators of auxin response. Moreover, NAA treatment induces expression of all three PpIAA genes within one hour [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF], revealing that auxin signaling is subjected to negative feedback regulation via PpIAAs, which was later evidenced as PpARF-dependent [START_REF] Tao | Mutational studies of the Aux/IAA proteins in Physcomitrella reveal novel insights into their function[END_REF]. Furthermore, the degron motif is necessary for auxin triggered PpAFB/PpIAA interaction [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF].

Degron deletion in PpIAA1A results in resistance to exogenous NAA but interestingly does not cause a mutant phenotype in the absence of treatment, e.g. in response to basal levels of endogenous IAA [START_REF] Tao | Mutational studies of the Aux/IAA proteins in Physcomitrella reveal novel insights into their function[END_REF].

Transduction of the auxin signal also requires TPL/TPRs, one of the two major classes of plant transcriptional corepressors, in P. patens: PpTPL1 and PpTPL2 proteins both interact with all 3 PpIAAs, in an EAR motif-dependent fashion [START_REF] Causier | TOPLESS co-repressor interactions and their evolutionary conservation in plants[END_REF]. However, P. patens lines expressing an EAR-deleted version of PpIAA1A are not as impacted as the triple ppiaa mutant, suggesting that EAR is not necessary for all PpIAAs functions, including some aspects of PpIAA-mediated transcriptional repression [START_REF] Tao | Mutational studies of the Aux/IAA proteins in Physcomitrella reveal novel insights into their function[END_REF]. PB1 domains predicted to enable proteinprotein interactions are found both in PpARFs and PpIAAs. Constitution of PpIAA oligomers and PpIAA-PpARF interactions contribute to repressive function of PpIAA1A but PpIAA1A monomers can also have a repressive activity on transcription [START_REF] Tao | Mutational studies of the Aux/IAA proteins in Physcomitrella reveal novel insights into their function[END_REF]. According to a recent study by Lavy et al., P. patens possesses 16 ARF genes, divided in 4 subgroups, with one being non-seed plant specific [START_REF] Lavy | Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins[END_REF]. As in Angiosperms, there are PpARFs that have a repressive effect on auxin-induced transcriptional response (negative ARFs) and PpARFs that have a positive role (positive ARFs). Both functional types target the same promoter elements and therefore the same genes.

Additionally, they both act in coordination with PpIAAs. Moreover, the effect of PpARFs on transcription also relies on TPLs: PpTPL1 and/or PpTPL2 interact in an EAR-dependent manner with negative PpARFs (PpARFe and PpARFf).

Another negative PpARF (PpARFb4) does not need the EAR motif to interact with PpTPLs, suggesting that it acts by competitive DNA binding instead of direct repression [START_REF] Lavy | Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins[END_REF]. The repressive effect of negative PpARFs is less stable than that of PpIAAs, which partly explains how PpARFs enable fine-tuning of the auxin response. On the other hand, PpIAAs ensure a long-term repression of transcriptional response to auxin [START_REF] Lavy | Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins[END_REF]. Indeed, Lavy and colleagues showed that IAA treatment has a huge effect on P. patens transcriptome, as more than 700 genes are induced and 700 more are repressed. This is also confirmed by the observation that in the triple Ppiaa mutant, which displays a constitutive auxin response and is completely insensitive to auxin, as much as one third of all P. patens genes are differentially expressed relative to WT. PpIAA1A is likely the main responsible for this huge effect, since the Ppiaa1b Ppiaa2 double mutant is phenotypically indistinguishable from WT [START_REF] Lavy | Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins[END_REF]. Two mechanisms permitting PpIAA-mediated auxin-induced transcriptional repression probably coexist: the first via association with PpARF that could interact with chromatin remodelers, the second via EAR-mediated interaction with PpTPLs that could sustain repression by recruiting histone deacetylases [START_REF] Tao | Mutational studies of the Aux/IAA proteins in Physcomitrella reveal novel insights into their function[END_REF]. Paponov et al. also reported the existence of a PpARF lacking the C-terminal dimerization domain, thus unable to interact with PpIAA [START_REF] Paponov | The evolution of nuclear auxin signalling[END_REF]. This specific PpARF could therefore regulate the basal, auxin-independent expression of auxin responsive genes. The caulonemal differentiation response to auxin is specifically dependent on the function of several bHLH transcription factors: PpRSL1 and 2 [START_REF] Jang | Auxin promotes the transition from chloronema to caulonema in moss protonema by positively regulating PpRSL1and PpRSL2 in Physcomitrella patens[END_REF], and PpLRL1 and 2 [START_REF] Tam | Conserved regulatory mechanism controls the development of cells with rooting functions in land plants[END_REF], all four being transcriptionally induced by auxin. Auxin signaling also leads to AP2-type transcription factors gene expression (PpAPB), required for apical stem cell genesis prior gametophore bud formation [START_REF] Aoyama | AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens[END_REF]. Auxin-induced PpAPBs act synergistically with cytokinins to enable bud establishment, while PpAPB4 is suggested to repress auxin biosynthesis [START_REF] Aoyama | AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens[END_REF]. Hence, PpAPBs constitute a major crosstalk node between cytokinins and auxin ensuring finetuned regulation of the control of bud induction. Thanks to the interplay between PpIAAs and PpARFs, auxin can have different effects on different cell types and/or at different doses, partly explained by the induction of different transcriptional responses [START_REF] Lavy | Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins[END_REF]. Cell susceptibility to auxin in P. patens is moreover regulated through an expression gradient of negative PpARFs, owing to PpARF transcripts processing consequently to TAS3 tasiRNAs action, generated notably from the action of miR390, similarly to what is known in Angiosperms [START_REF] Axtell | Common functions for diverse small RNAs of land plants[END_REF][START_REF] Plavskin | Ancient trans-Acting siRNAs Confer Robustness and Sensitivity onto the Auxin Response[END_REF]. This mechanism enables regulation of auxin signaling independently from auxin levels and could potentially be more sensitive to the internal state of the cell.

Auxin signaling evolution has recently been reviewed [START_REF] Paponov | The evolution of nuclear auxin signalling[END_REF][START_REF] Kato | Evolution of nuclear auxin signaling: lessons from genetic studies with basal land plants[END_REF]. All constituents of the canonical nuclear signaling pathway are conserved in bryophytes (P. patens and M. polymorpha), but are not found in algae genomes, even though algae do display responses to auxin. Therefore, this pathway originated in land plants ancestry. The ancestral effect of auxin is perhaps the induction of cell elongation, which is observed in all land plants. The dependence of moss rhizoid development on auxin signaling probably represents an ancient role given the involvement of auxin in rhizoid development in many earlier-diverging Streptophyte plants, including Chara and liverworts [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF]. On the contrary, the role of this hormone in caulonemal differentiation in mosses could be specific to mosses, and even to the most widely represented bryopsids mosses, as other Bryophytes and early diverging mosses like Takakia do not display comparable protonema development [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF].

CYTOKININS

Natural cytokinins are adenine derivatives with a substitution on N 6 , best known in vascular plants for their promoting effect on cell division, also demonstrated in mosses [START_REF] Szweykowska | The Cell Division Response to Cytokinins in Isolated Cell Cultures of the Protonema of Funaria hygrometrica and its Comparison with the Bud Induction Response[END_REF]Szweykowska and Korcz, 1972;Szweykowska et al., 1972) (Figure II-1). The structure of the substituent on N 6 is distinctive of the cytokinin's type: isoprenoid (iP type) or aromatic (kinetin type). There is an ongoing controversy about which cytokinins are majorly produced by P. patens. Still, it has been shown that most known types of cytokinins are produced in P. patens, among which N 6 -(∆ 2 -isopentenyl)-adenine (iP) [START_REF] Wang | Isopentenyladenine from mutants of the moss, Physcomitrella patens[END_REF][START_REF] Von Schwartzenberg | Cytokinins in the bryophyte Physcomitrella patens: Analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins[END_REF], zeatins, aromatic cytokinins (notably benzyl-adenine (BA)), as well as derived ribosides and O-glucosides [START_REF] Von Schwartzenberg | Cytokinins in the bryophyte Physcomitrella patens: Analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins[END_REF]. Cytokinins produced by P. patens are secreted into the medium [START_REF] Schumaker | HORMONE-INDUCED SIGNALING DURING MOSS DEVELOPMENT[END_REF][START_REF] Von Schwartzenberg | The moss Physcomitrella patens releases a tetracyclic diterpene[END_REF][START_REF] Von Schwartzenberg | Cytokinins in the bryophyte Physcomitrella patens: Analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins[END_REF], but selectively: aromatic cytokinins are not secreted while iP and derivatives are the major cytokinins found in the medium. In flowering plants, two pathways enable iP biosynthesis, relying on different IPT (isopentenyl transferases) enzymes: adenylate-IPT and tRNA-IPT, which catalyse the first and limiting step of the pathway (see the review by [START_REF] Spíchal | Cytokinins -Recent news and views of evolutionally old molecules[END_REF]. Cytokinins biosynthesis in P. patens relies solely on the tRNAisopentenylation pathway, where isopentenylation and following tRNA degradation liberates cytokinin nucleotides during RNA translation [START_REF] Yevdakova | Characterisation of a prokaryote-type tRNAisopentenyltransferase gene from the moss Physcomitrella patens[END_REF][START_REF] Frébort | Evolution of cytokinin biosynthesis and degradation[END_REF]. Cytokinin inactivation might be achieved in P. patens via phosphorylation of isopentenyl-adenosine (iPR) into isopentenyl-adenosine monophosphate (iPRMP). Indeed, P. patens possesses a single adenosine kinase (ADK), expressed in chloronema, which can produce iPRMP from iPR in vitro [START_REF] Von Schwartzenberg | Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism[END_REF]. On another note, cytokinin degradation is enabled by the activity of CKX enzymes (cytokinin oxidases), which predictably have a preferential activity on cis-zeatin [START_REF] Von Schwartzenberg | Cytokinins in the bryophyte Physcomitrella patens: Analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins[END_REF]. [START_REF] Frébort | Evolution of cytokinin biosynthesis and degradation[END_REF] reported 6 PpCKX genes, all encoding enzymes with conserved catalytic and ligand binding domains [START_REF] Hyoung | Cytokinin oxidase PpCKX1 plays regulatory roles in development and enhances dehydration and salt tolerance in Physcomitrella patens[END_REF]. Interestingly, these proteins are predicted to localize in diverse subcellular compartments, and even the extracellular space, perhaps hinting at slightly different function, although these predictions are not consensual [START_REF] Gu | Comparative Expression and Phylogenetic Analysis of Maize Cytokinin Dehydrogenase/Oxidase (CKX) Gene Family[END_REF][START_REF] Hyoung | Cytokinin oxidase PpCKX1 plays regulatory roles in development and enhances dehydration and salt tolerance in Physcomitrella patens[END_REF].

Early studies in mosses revealed a positive role of cytokinins on bud induction and subsequent maintaining of bud identity [START_REF] Gorton | Development of the Gametophyte in the Moss Tortella caespitosa[END_REF][START_REF] Brandes | Studies on Cytokinin-Controlled Bud Formation in Moss Protonemata[END_REF][START_REF] Christianson | The Quantitative Response to Cytokinin in the Moss Funaria hygrometrica Does Not Reflect Differential Sensitivity of Initial Target Cells[END_REF]. In P. patens also, cytokinins induce bud formation at low concentrations [START_REF] Bopp | Control of differentiation in fern-allies and bryophytes[END_REF]Ashton et al., 1979a;[START_REF] Wang | Cytokinins from the Moss Physcomitrella patens[END_REF]

(Figure II-2).
Interestingly, von Schwartzenberg et al. (2007) discovered that extracellular iP and iPR were responsible for cytokinininduced bud formation, rather than intracellular cytokinins as was previously suggested by [START_REF] Wang | Cytokinins from the Moss Physcomitrella patens[END_REF]. At first, cytokinins rapidly induce an increase in intracellular calcium ion levels, which mediates induction of asymmetric cell division [START_REF] Saunders | Calcium Mediation of Cytokinin-Induced Cell Division[END_REF]. Earlier, Szweykowska and colleagues [START_REF] Szweykowska | The Cell Division Response to Cytokinins in Isolated Cell Cultures of the Protonema of Funaria hygrometrica and its Comparison with the Bud Induction Response[END_REF]Szweykowska and Korcz, 1972;Szweykowska et al., 1972) had suggested that an increase of cell divisions is not the main specifier of bud induction. Thus, these two effects of cytokinins can be dissociated. Then, Doonan et al. showed that microtubules are diffusely organized in bud initials compared to side branches initials [START_REF] Doonan | Pre-prophase band of microtubules, absent from tip-growing moss filaments, arises in leafy shoots during transition to intercalary growth[END_REF]. They hence raised the interesting hypothesis that cytokinins rather promote bud formation by modifying the direction of cell divisions, instead of increasing cell division rates. This hypothesis conveniently explains another effect of cytokinins that is the induction of chloronema branching [START_REF] Thelander | Effect of the energy supply on filamentous growth and development in Physcomitrella patens[END_REF]. Still, the cell division induction by cytokinins is more directly linked to other effects of cytokinins, notably the promotion of protoplasts' regenerative ability [START_REF] Von Schwartzenberg | Cytokinins in the bryophyte Physcomitrella patens: Analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins[END_REF] and the induction of meristematic cell formation and proliferation along the gametophore axis, leading to increase gametophore branching [START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF]. Moreover, exogenous cytokinins treatment was shown to elicit the formation of ectopic apical meristems on gametophores and to induce cell divisions in phyllids [START_REF] Cammarata | Cytokinin and CLE signaling are highly intertwined developmental regulators across tissues and species[END_REF].

Another evidence of this is the induction of callus-like buds as a presumably toxic effect of cytokinins [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF]. Additionally, cytokinins inhibit the formation of rhizoids (Ashton et al., 1979a;[START_REF] Hyoung | Cytokinin oxidase PpCKX1 plays regulatory roles in development and enhances dehydration and salt tolerance in Physcomitrella patens[END_REF] and growth of the stems (Ashton et al., 1979a). [START_REF] Reski | Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine[END_REF] suggested that chloronema and caulonema cells have a different susceptibility to cytokinins, P. patens caulonema being sensitive only to higher doses of iP for bud induction.

Buds typically develop from caulonema but the experiments from [START_REF] Reski | Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine[END_REF] showed that both caulonema cells and chloronema cells are competent for cytokinin-induced bud formation [START_REF] Reski | Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine[END_REF]. Overall, cytokinins favor the development of primary chloronema and buds, both chlorophyll-rich tissues. This is consistent with the findings that cytokinins can act alongside light to boost plastids metabolism, notably increasing expression of plastid polypeptides as well as rbcL transcript levels [START_REF] Reski | Cytokinin and Light Quality Interact at the Molecular Level in the Chloroplast-Mutant PC22 of the Moss Physcomitrella[END_REF]. Moreover, cytokinins were reported to induce chloroplast division in protonema [START_REF] Abel | A cytokinin-sensitive mutant of the moss,Physcomitrella patens, defective in chloroplast division[END_REF][START_REF] Reutter | Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division[END_REF]. Von Schwartzenberg also communicated on a role of cytokinins in repressing brachycytes and tmema development [START_REF] Von Schwartzenberg | Hormonal Regulation of Development by Auxin and Cytokinin in Moss[END_REF]. Brachycytes are small round, thick-walled cells, where vacuolation and cell expansion is suppressed (they are also called brood cells) and tmema cells are the accompanying empty, thin-walled cells, enabling dispersal of mature brachycytes after programmed cell death (Goode et al., 1993a(Goode et al., , 1993b)). Finally, more recently, cytokinins were shown to be required for reproductive organs development and spore production [START_REF] Von Schwartzenberg | CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens[END_REF][START_REF] Hyoung | Cytokinin oxidase PpCKX1 plays regulatory roles in development and enhances dehydration and salt tolerance in Physcomitrella patens[END_REF].

Cytokinins are active at very low concentrations relative to other phytohormones (such as ABA): 1µM BA was reported as being enough to trigger a toxic growth-inhibiting effect, possibly stemming from an increase in senescence and PCD [START_REF] Thelander | Effect of the energy supply on filamentous growth and development in Physcomitrella patens[END_REF][START_REF] Von Schwartzenberg | CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens[END_REF].

As underlined by Pils and Heyl, the cytokinin signaling pathway in plants is very alike a bacterial twocomponent system of signal transduction, using successive phosphorylations to finally trigger changes in target genes' expression [START_REF] Pils | Unraveling the evolution of cytokinin signaling[END_REF]. Sequencing of P. patens genome [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF] indicated that homologs of cytokinin signaling pathway components, as CHASE-domain receptors, as well as type A and type B ARRs are present . This was further investigated by Ishida and colleagues in 2010: P. patens has 3 PpHK genes among which one putative cytokinin receptor PpHK4b (most similar to AtCRE1, one of the cytokinin receptor from Arabidopsis thaliana) [START_REF] Ishida | Classification of the genes involved in the twocomponent system of the moss Physcomitrella patens[END_REF]. When expressed in Escherichia coli, the PpHK4b protein is indeed able to sense cytokinins. By looking for P. patens and F. hygrometrica genes encoding putative proteins where a histidine kinase domain is coupled with a CHASE domain and a RR domain, Gruhn et al. found 3 "canonical" PpCHK (1,2,3,CHK stands for CHASE-domain containing Histidine Kinase) that group with Angiosperms cytokinin receptors (likely corresponding to the 3 PpHK genes from [START_REF] Ishida | Classification of the genes involved in the twocomponent system of the moss Physcomitrella patens[END_REF]) and 8 "divergent" PpCHK (4 to 11) that belong to a Bryophytes specific CHK clade [START_REF] Gruhn | A subfamily of putative cytokinin receptors is revealed by an analysis of the evolution of the two-component signaling system of plants[END_REF]. They selected PpCHK4, which is expressed both in P. patens and in F. hygrometrica, for functional characterization. They demonstrated in vitro the ability of the PpCHK4 protein to bind tZ but also to transduce a signal via phospho-relay from tZ, cZ, iP and BA. Thus, at least two out of the 11 PpCHK are serious cytokinin receptors candidates. The 3 canonical CHK genes from P. patens were functionally characterized only recently [START_REF] Von Schwartzenberg | CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens[END_REF] and for the first time their function was actually directly assessed in planta thanks to characterization of all combinations of Ppchk1-3 loss-of-function mutants. Their conclusions were that:

(1) the three receptors mediate a cytokinin-dependent signal independently from each other; (2) PpCHK3 has a minor role in cytokinin-induced bud formation; (3) PpCHK1 and PpCHK2 play a major role in triggering this developmental process, PpCHK2 responding preferentially to iP, while CHK1 has a broader ligand range ; (4) divergent PpCHK alone cannot ensure cytokinin response, at least for bud induction in protonema, since loss of function of PpCHK1 to PpCHK3 was sufficient for complete cytokinin insensitivity. Moving downwards the signaling pathway, Gruhn and colleagues (2014) found only two putative PpHPt genes where the histidine kinase domain and the conserved phosphorylation target histidine residue are both present, much less than the 6 genes reported by [START_REF] Ishida | Classification of the genes involved in the twocomponent system of the moss Physcomitrella patens[END_REF]. On the other hand, they found more RR: 7 PpRRA and 5 PpRRB, which all retain the conserved aspartate residue necessary for phosphotransfer. Additionally, 3 PpRRC and 4 PpPRR genes are present in P. patens genome, but they are probably not involved in cytokinin signaling. However, these genes have not been functionally characterized yet. Recently, a possible negative feedback mechanism on cytokinin signaling was hinted at [START_REF] Von Schwartzenberg | CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens[END_REF]. As a matter of facts, the Ppchk1-3 mutant has elevated PpCHK2/3 transcript levels, suggesting that cytokinin perception inhibits expression of genes encoding cytokinin receptors. On the other hand, there is probably no such feedback on cytokinin biosynthesis, as the Ppchk123 cytokinin-insensitive mutant produces similar amounts and types of cytokinins as the WT P. patens [START_REF] Von Schwartzenberg | CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens[END_REF].

Analysis of genes associated with cytokinin biosynthesis and metabolism [START_REF] Frébort | Evolution of cytokinin biosynthesis and degradation[END_REF][START_REF] Spíchal | Cytokinins -Recent news and views of evolutionally old molecules[END_REF] and signaling [START_REF] Pils | Unraveling the evolution of cytokinin signaling[END_REF][START_REF] Gruhn | A subfamily of putative cytokinin receptors is revealed by an analysis of the evolution of the two-component signaling system of plants[END_REF] show that all protein domains and enzymatic activities needed were already present in bacteria and were seemingly acquired by the plant lineage by gene transfer. Then, across plant evolution, these functional domains were assembled in novel ways and eventually led to a functional signal transduction mechanism in response to the phytohormones cytokinins.

ABSCISSIC ACID

ABA is produced from carotenoids following abiotic stress in flowering plants . Aside from promoting tolerance to said abiotic stress, ABA is known for regulating several developmental processes, as seed germination and root growth. P. patens constitutively accumulates ABA in its protonema, and ABA levels increase approximately 3-fold under hyperosmotic stress [START_REF] Minami | Physiological and Morphological Alterations Associated with Development of Freezing Tolerance in the Moss Physcomitrella patens[END_REF][START_REF] Takezawa | Epoxycarotenoid-mediated synthesis of abscisic acid in Physcomitrella patens implicating conserved mechanisms for acclimation to hyperosmosis in embryophytes[END_REF]. A 10 μM ABA treatment corresponds to the endogenous levels of ABA produced by WT P. patens under abiotic stress and has been shown to induce a molecular response but little phenotypic changes [START_REF] Arif | ABA-induced vegetative diaspore formation in physcomitrella patens[END_REF]. To our knowledge, only Takezawa and colleagues (2015) characterized an ABA biosynthesis gene: PpABA1. This gene encodes a zeaxanthin epoxidase (ZEP), likely responsible for the first step of ABA biosynthesis in plastids. The Ppaba1 loss of function mutant does not synthesize ABA anymore, so this epoxides pathway is likely the only one leading to ABA production. Recently, [START_REF] Arif | ABA-induced vegetative diaspore formation in physcomitrella patens[END_REF] suggested the involvement of two PpNCEDs (9-cis epoxy-carotenoid dioxygenases, which homologs in Angiosperms cleave 9-cis xanthophylls to xanthoxin in plastids) in ABA biosynthesis.

Abscisic acid (ABA) is accumulated in slowly drying tissues of Funaria hygrometrica [START_REF] Lankester | Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw[END_REF], which can, after rehydration, then survive a rapid dehydration. ABA treatment of protonema enables tolerance to rapid drying, which is not possible in the absence of treatment. Thus, the induction of drought tolerance in this moss is mediated by ABA. ABA does not seem to act by preventing water loss but rather by stimulating the synthesis of protective proteins, since cycloheximide prevents ABA-mediated tolerance. These proteins were later shown to be dehydrins, some belonging to the LEA (Late Embryogenesis Abundant) superfamily [START_REF] Bopp | Abscisic Acid and Desiccation Tolerance in Mosses[END_REF][START_REF] Oliver | Desiccation tolerance in bryophytes: A reflection of the primitive strategy for plant survival in dehydrating habitats?[END_REF]. Specific mechanism of LEA action is unknown, but they likely act in stabilization and reconstitution (both during rehydration) of membranes [START_REF] Oliver | Desiccation tolerance in bryophytes: A reflection of the primitive strategy for plant survival in dehydrating habitats?[END_REF]. ABA treatment also grants freezing tolerance in P. patens [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF][START_REF] Komatsu | Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens[END_REF], accompanied with alterations of organelle morphology and accumulation of free soluble sugars [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF]. [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF] also suggested that ABA grants tolerance to high salinity. This implies a common ABA-mediated response to drought, freezing and osmotic stresses. Indeed, by comparing gene expression after NaCl and ABA treatment, Richardt et al. showed that both signals lead to overlapping expression profiles and that NaCl treatment induces the up regulation of ABA (putative at the time) biosynthesis genes [START_REF] Richardt | Microarray analysis of the moss Physcomitrella patens reveals evolutionarily conserved transcriptional regulation of salt stress and abscisic acid signalling[END_REF]. Thus, ABA likely also mediates salt responses in P. patens [START_REF] Qudeimat | A PIIB-type Ca2+-ATPase is essential for stress adaptation in Physcomitrella patens[END_REF][START_REF] Richardt | Microarray analysis of the moss Physcomitrella patens reveals evolutionarily conserved transcriptional regulation of salt stress and abscisic acid signalling[END_REF].

In P. patens, treatment with exogenous ABA causes a dramatic reduction of growth rate, leading to dwarf plants with a somehow twisted protonema. This tremendous change in morphology is caused by ABA-induced differentiation of chloronema cells into brachycytes and accompanying tmema cells (Goode et al., 1993a;[START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF][START_REF] Arif | ABA-induced vegetative diaspore formation in physcomitrella patens[END_REF]. Development of these specific cell types could stem from the earlier promoting effect of ABA on perpendicular cell-divisions in protonema filaments [START_REF] Sakata | Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens[END_REF], which suggests that ABA might regulate the cell cycle. ABA also apparently restricts the height of gametophores stems, which has been interpreted as an efficient mean of keeping a sufficient moisture level of the plant by keeping close to the substrate [START_REF] Sakata | Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens[END_REF][START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF]. The same study from Sakata and colleagues (2009) additionally suggests that ABA induces archegonia production and thus increases the number of sporophytes, sometimes leading to multiples sporophytes per apex. But ABA also has an effect earlier, inhibiting gametophore bud formation [START_REF] Christianson | ABA prevents the second cytokinin-mediated event during the induction of shoot buds in the moss Funaria hygrometrica[END_REF], see the following part on crosstalks) . It has been shown this year that ABA restricts macromolecular trafficking between protonemal cells in P. patens [START_REF] Tomoi | Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens[END_REF], in a way seemingly independent from callose deposition. This might be linked to the cell-wall thickening preconditioning brachycytes differentiation. Fitting with its classification as a stress hormone, ABA has a dosedependent inhibiting effect on P. patens spore germination, and inhibition of ABA biosynthesis with norflurazon has the same effect [START_REF] Moody | An ancient and conserved function for Armadillo-related proteins in the control of spore and seed germination by abscisic acid[END_REF][START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF].

While Saruhashi et al. have reported the identification of four PYR/PYL/RCAR (pyrabactin resistance 1like/regulatory component of ABA receptor) homologs in P. patens' genome [START_REF] Saruhashi | Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2[END_REF], ABA perception by PYL/PYR/RCAR receptors or other unknown proteins has not been yet experimentally investigated. On the other hand, most of the following steps of the ABA signaling pathway in P. patens have been extensively investigated (Figure . The first level of ABA signaling that was studied in P. patens is ABA-dependent transcriptional regulation via ABI3 (ABA Insensitive 3) transcription factors. P. patens genome encodes three ABI3 homologs: PpABI3A, B and C [START_REF] Marella | Characterization and functional analysis of ABSCISIC ACID INSENSITIVE3-like genes from Physcomitrella patens[END_REF]. PpABI3A, the most potent inducer of PpLEA1 expression out of the three PpABI3s [START_REF] Kamisugi | The Evolution of the Abscisic Acid-response in Land Plants: Comparative Analysis of Group 1 LEA Gene Expression in Moss and Cereals[END_REF], is indeed a nuclear protein, hinting again at a role in transcriptional regulation [START_REF] Marella | Characterization and functional analysis of ABSCISIC ACID INSENSITIVE3-like genes from Physcomitrella patens[END_REF]. Sakata et al. reported that induction of gene expression by PpABI3A relies on direct binding to RY-element in promoters [START_REF] Sakata | Regulation of the ABA-responsive Em promoter by ABI3 in the moss Physcomitrella patens: role of the ABA response element and the RY element[END_REF]. Dozens of genes encoding transcription-associated proteins are differentially expressed after ABA treatment, indicating that response to ABA has a dramatic effect on transcription [START_REF] Richardt | Microarray analysis of the moss Physcomitrella patens reveals evolutionarily conserved transcriptional regulation of salt stress and abscisic acid signalling[END_REF][START_REF] Arif | ABA-induced vegetative diaspore formation in physcomitrella patens[END_REF].

Dependence of a gene expression on ABA in P. patens correlates well with the presence of ABRE and CE1 elements in promoter regions notably for genes encoding ABI3, bZIP and AP2 transcription factors [START_REF] Qudeimat | A PIIB-type Ca2+-ATPase is essential for stress adaptation in Physcomitrella patens[END_REF][START_REF] Richardt | Microarray analysis of the moss Physcomitrella patens reveals evolutionarily conserved transcriptional regulation of salt stress and abscisic acid signalling[END_REF][START_REF] Timmerhaus | Prediction and validation of promoters involved in the abscisic acid response in physcomitrella patens[END_REF]. ABA-induced genes in P. patens are mostly involved in several stress response pathways, indicating overlapping pathways in the control of stress-responsive genes [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF]. For instance, PpLEA1 transcripts accumulate in response to both ABA and osmotic stress [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF]. This induction is permitted by the ACGT core motif of the ABRE (ABA-responsive element) contained in flowering plants LEA genes' promoters [START_REF] Kamisugi | The Evolution of the Abscisic Acid-response in Land Plants: Comparative Analysis of Group 1 LEA Gene Expression in Moss and Cereals[END_REF]. PpLEA proteins are hence accumulated in response to ABA [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF]. Concomitantly, two PP2C (Protein Phosphatase 2C) genes involved in ABA signaling in P. patens were characterized: PpABI1A and PpABI1B [START_REF] Komatsu | Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens[END_REF][START_REF] Sakata | Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens[END_REF][START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF]. Among these two ubiquitously expressed genes, PpABI1A is quickly and durably induced by ABA treatment, osmotic stress and cold [START_REF] Sakata | Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens[END_REF]. Simple Ppabi1 loss of function mutants are hypersensitive to ABA treatment but display a WT phenotype in control conditions, while the double Ppabi1a/b mutant has a slow growth and spontaneously differentiates brachycytes, thus displaying a constitutive response to ABA [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF]. Therefore, PpABI1A/B act as negative regulators of ABA signaling acting upstream of PpABI3A-induced PpLEA1 expression. Unexpectedly, bud formation was not impacted in the Ppabi1a/b double mutant. Therefore, PpABI1 genes are not involved in all responses to ABA, but they do limit constitutive tolerance of protonema to diverse water-associated stresses when not enough ABA is present. However, while PpABI1s play a major role in regulating the transcriptional response to ABA (~65%), they are not responsible for all aspects of it [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF]. Indeed, ABA treatment induces kinase activity of PpSnRK2 enzymes [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF][START_REF] Saruhashi | Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2[END_REF][START_REF] Bressendorff | An innate immunity pathway in the moss Physcomitrella patens[END_REF][START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF] in a PpABI1A/B-independent manner [START_REF] Amagai | Phosphoproteomic profiling reveals <scp>ABA</scp> -responsive phosphosignaling pathways in Physcomitrella patens[END_REF]. Since P. patens possesses 49 other PP2C genes, phylogenetically more distant from Angiosperms ABI1s [START_REF] Sakata | Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens[END_REF], a possible explanation is that PpABI1A/Bindependent PpSnRK2-dependent ABA signaling relies on these other PP2Cs. However, another player was identified more recently [START_REF] Saruhashi | Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2[END_REF][START_REF] Stevenson | Genetic Analysis of Physcomitrella patens Identifies ABSCISIC ACID NON-RESPONSIVE, a Regulator of ABA Responses Unique to Basal Land Plants and Required for Desiccation Tolerance[END_REF]: PpARK/ANR (ABA and abiotic stress responsive Raflike Kinase/ABA Non-Responsive). This group B3 Raf-like MAP kinase kinase kinase (B3-MAPKKK) is a major positive regulator of ABA-responsive gene expression in P. patens. Interestingly, this gene is actually identical to the PpCTR1L protein acting in ethylene signaling, characterized in 2015 by Yasumura and colleagues (see the following part on ethylene [START_REF] Yasumura | An ancestral role for CONSTITUTIVE TRIPLE RESPONSE1 proteins in both ethylene and abscisic acid signaling[END_REF]). In order to simplify our writing, this gene will be consistently noted as PpARK in this review. PpARK is necessary for PpSnRK2 ABA-induced activity, suggesting it acts upstream of these kinases in the ABA signaling pathway. PpARK colocalizes with the ABA-activated PpSnRK2B in the cytosol, where it phosphorylates and activates PpSnRK2B [START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF]. PpARK is itself activated by phosphorylation in response to ABA, but the enzyme responsible for this modification is unknown. Interestingly, characterization of the Ppark loss-of-function mutant (AR7) implies that PpARK is also involved in signaling of ABA-independent hyperosmotic stress signals. Four subclass III SnRK2 are present in P. patens genome [START_REF] Saruhashi | Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2[END_REF], which homologs in flowering plants are involved in ABA signaling and are targets of ABI1 PP2Cs repressive activity. Loss of function of these genes results in variable levels of ABA insensitivity, ranging from loss of sporophyte stomata ABA sensitivity in the simple Ppost1-1/Ppsnrk2a mutant [START_REF] Chater | Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution[END_REF] to complete ABA-insensitivity and hypersensitivity to diverse stresses (freezing, desiccation and hyperosmosis) in the quadruple Ppsnrk2a/b/c/d mutant [START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF]. Hence, PpSnRK2 activity is absolutely necessary for ABA-associated responses to stress in P. patens, mediating the induction of PpLEA genes' expression and accumulation of PpLEA proteins. Gene expression analyses showed that, while a majority of osmostress-responsive genes overlapped with ABA-responsive genes, there was a partial independence between these two transcriptional programs [START_REF] Stevenson | Genetic Analysis of Physcomitrella patens Identifies ABSCISIC ACID NON-RESPONSIVE, a Regulator of ABA Responses Unique to Basal Land Plants and Required for Desiccation Tolerance[END_REF][START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF].

The study of [START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF] moreover led to the identification of SAGs (SnRK2-regulated and ABAupregulated Genes), among which most ARK-regulated genes were retrieved. Hence, it underlines the major role of the ABA/ARK/SnRK2 module in the regulation of ABA-responsive gene expression. Recently, Amagai and colleagues used a phosphoproteomics approach to identify PpABI1A/B-dependent and PpARK-dependent phosphopeptides [START_REF] Amagai | Phosphoproteomic profiling reveals <scp>ABA</scp> -responsive phosphosignaling pathways in Physcomitrella patens[END_REF]. They found that, whereas most PpABI1A/B-dependent phosphopeptides are regulated in an ABAindependent manner, the majority of PpARK-dependent phosphopeptides are regulated in an ABA-dependent manner [START_REF] Komatsu | Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance[END_REF][START_REF] Saruhashi | Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2[END_REF]. Moreover, an alternative SnRK2-independent phosphorylation cascade in response to ABA appears to exist, as loss of function of the 4 PpSnRK2 genes (QKO mutant) prevented phosphorylation of only a portion of ABA targets [START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF]. Nonetheless, 24 proteins are common targets of PpARK and PpSnRK2s-dependent phosphorylation cascades, among which an ABA-responsive bZIP transcription factor (ABF)-related protein was found [START_REF] Amagai | Phosphoproteomic profiling reveals <scp>ABA</scp> -responsive phosphosignaling pathways in Physcomitrella patens[END_REF][START_REF] Shinozawa | SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment[END_REF]. This means PpARK and PpSnRK2 together regulate ABA signaling at least partially through phosphorylation of PpABFs. Ultimately, ABA signaling leads mainly to upregulation of gene expression (almost 600 genes), with most genes being either late responding or stably induced from earlier time-points [START_REF] Arif | ABA-induced vegetative diaspore formation in physcomitrella patens[END_REF]. Amongst them, cell wall related genes and programmed cell death associated genes are found, which is undoubtedly linked respectively to the cell wall thickening of brachycytes and to the formation of tmema cells. On the other hand, ABA has a relatively modest effect on the proteome of P. patens, with only 65 proteins being impacted by a long-term ABA treatment [START_REF] Wang | Conservation between higher plants and the moss Physcomitrella patens in response to the phytohormone abscisic acid: a proteomics analysis[END_REF]. Nonetheless, it has been reported that ABA treatment induces an overall reduction in secreted proteins, notably for proteins regulating cell wall composition, again pointing to a role in membrane remodeling [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF].

Interestingly, it was shown that the PpNCED1 gene expression is lower in Ppaba1 compared to WT, suggesting the existence of a positive feedback mechanism on ABA biosynthesis [START_REF] Takezawa | Epoxycarotenoid-mediated synthesis of abscisic acid in Physcomitrella patens implicating conserved mechanisms for acclimation to hyperosmosis in embryophytes[END_REF]. Accordingly, Arif and colleagues found that 2 PpNCEDs, as well as almost all genes known to be involved in ABA-dependent signaling are upregulated after an ABA treatment [START_REF] Arif | ABA-induced vegetative diaspore formation in physcomitrella patens[END_REF]. Therefore, it appears not only ABA biosynthesis but also ABA signaling is subjected to such a positive feedback.

Comparison of ABA signaling pathways between Angiosperms and Bryophytes implies that ABA has been ancestrally used as a stress hormone in land plants and that the ABA signal was transduced using the same proteins (Wang et al., 2015a). However, ABA signaling is not totally the same in P. patens and Angiosperms, notably at the level of ABA regulated gene expression, where the targets are quite different, as well as the interplay with ethylene using ARK (see following part on crosstalks). This could very well result from a divergent evolution specific to Bryophytes or even to mosses.

ETHYLENE

Ethylene acts as a major stress hormone in flowering plants, notably involved in resistance to flooding, but also regulates several aspects of development such as floral senescence, fruit ripening and abscission of leaves. In mosses, gaseous ethylene is most probably produced via 1-aminocyclopropane-1-carboxylic acid (ACC) and not via possible precursors such as 2-ketoglutarate or glutamate, but the exact biosynthetic pathway remains to be demonstrated [START_REF] Rohwer | Ethylene Synthesis in Moss Protonema[END_REF]. Existence of such an ACC-based biosynthesis pathway is also suggested by the finding of two putative genes encoding ACC-synthases in P. patens genome [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF].

Very few studies have focused on the effect of ethylene and its precursor ACC in mosses. The earliest report is one by [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF] focusing on rhizoid development. This study concluded that ACC does not increase the number of rhizoids, although both ACC and ethylene induce root hair formation in Angiosperms. However, they did find that ACC treatment caused immediate senescence [START_REF] Sakakibara | Involvement of auxin and homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens[END_REF]. Almost ten years later, it was shown that ethylene treatment replicates submergence response in P.patens [START_REF] Yasumura | Studies of Physcomitrella patens reveal that ethylenemediated submergence responses arose relatively early in land-plant evolution[END_REF], characterized by increased protonema extension via caulonema growth and a more distal development pattern of gametophores, with them developing mainly at the periphery of the protonema. Ethylene promotes gametophore elongation and permits more apical rhizoid development on gametophores, belying previous reports (Yasumura et al., 2015) (Figure II-2). Yasumura and colleagues concluded that the role of ethylene relies primarily on the regulation of water status, working partially in opposition to ABA [START_REF] Yasumura | Studies of Physcomitrella patens reveal that ethylenemediated submergence responses arose relatively early in land-plant evolution[END_REF](see the last part on crosstalks). Exogenous ACC was also shown to inhibit spore germination, although it is more likely ethylene produced from this ACC in planta that holds this effect, since cotreatment with ACC-oxidase inhibitor amino-isobutyric acid has no more impact on spore germination [START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF]. P. patens genome encodes proteins resembling angiosperm ethylene signaling components [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF]. Evidence for ethylene binding in P. patens has been reported almost fifteen years ago (Wang et al., 2006), and legitimate ethylene receptor candidates were identified in 2012 (Figure II-3). Among the seven ETR genes in P. patens, PpETR1, 3, 6 and 7 are phylogenetically closer to functionally characterized ethylene receptors from flowering plants and are therefore considered as best candidates [START_REF] Yasumura | Studies of Physcomitrella patens reveal that ethylenemediated submergence responses arose relatively early in land-plant evolution[END_REF]. Still, all 7 predicted PpETR proteins contain a conserved histidine kinase domain and C-terminal regulator domain. Yasumura and colleagues focused on PpETR7 for functional characterization: Overexpression of a mutated version in the putative N-terminal ethylene-binding domain (Ppetr7-1) leads to ethylene-insensitivity, while overexpression of the WT version results in WT, submergence-like response [START_REF] Yasumura | Studies of Physcomitrella patens reveal that ethylenemediated submergence responses arose relatively early in land-plant evolution[END_REF]. This shows that PpETR7 is likely a functional ethylene receptor and that it plays a major role in ethylene perception amongst PpETRs. It also proves that the mutated version has a dominant effect over the WT copy, suggesting the involvement of PpETR oligomers in perception. Moreover, the higher proportion of caulonema and shorter branches of the Ppetr7-1 plants is phenocopied by 1-MCP (1-methylcyclopropene, an inhibitor of ETR ethylene binding) application on WT P. patens. Ishida and colleagues also previously reported ethylene binding by the PpETR1c homolog in vitro (we could not retrace whether this protein is PpETR7), which inhibits the histidine kinase activity of the receptor [START_REF] Ishida | Classification of the genes involved in the twocomponent system of the moss Physcomitrella patens[END_REF]. This step occurs on the endoplasmic reticulum membrane in Angiosperms, but its localization has not been investigated yet in mosses. Possibly transducing the ethylene signal downstream of perception by ETRs are a lone CTR1 (Constitutive Triple Response 1) homolog (PpCTR1L/PpARK, cf. previous part on ABA), two EIN3/EIL1 (Ethylene Insensitive 3/EIN3-like 1) and dozens of ERF/EREBP (Ethylene Response Factors/Ethylene-Responsive Element (ERE)-Binding Proteins) transcription factors [START_REF] Yasumura | Studies of Physcomitrella patens reveal that ethylenemediated submergence responses arose relatively early in land-plant evolution[END_REF].

Further inspection of P. patens EIN3 homologs revealed that both PpEIN3a and b do contain the EIN3 DNA-binding domain and could thus be functional transcription factors [START_REF] Chang | Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis[END_REF]. Similarly, all PpERFs contain the AP2/ERF domain granting DNA binding ability to their Angiosperms' counterparts. In Angiosperms, PpERF expression is typically induced by EIN3 binding to their promoter region. In P. patens, PpERFa is repressed and PpERFb is slightly upregulated by ethylene [START_REF] Yasumura | Studies of Physcomitrella patens reveal that ethylenemediated submergence responses arose relatively early in land-plant evolution[END_REF], in a PpETR7-dependent manner. However, the involvement of PpEIN3a/b in this regulation has not been explored. Yasumura and colleagues further demonstrated that the PpCTR1L/PpARK protein does bind to PpETR7 in yeast two hybrid experiments, so it should logically be involved in ethylene signaling [START_REF] Yasumura | An ancestral role for CONSTITUTIVE TRIPLE RESPONSE1 proteins in both ethylene and abscisic acid signaling[END_REF]. The Ppctr1l/Ppark mutant is indeed insensitive to exogenous ethylene and displays a constitutive ethylene response phenotype, showing PpCTR1L/PpARK is very likely a negative regulator of ethylene signaling. Moreover, the Ppctr1l/Ppark mutation was determined to be epistatic to Ppetr7-1 overexpression, proving that PpCTR1L/PpARK acts downstream of PpETR7 in the ethylene signaling cascade. However, fine characterization of the Ppctr1l/Ppark Ppetr7-1 line suggested that PpETR7 might induce some aspects of the ethylene response independently from PpCTR1L/PpARK [START_REF] Yasumura | An ancestral role for CONSTITUTIVE TRIPLE RESPONSE1 proteins in both ethylene and abscisic acid signaling[END_REF]. These other pathways remain to be discovered.

In Angiosperms, CTR1 typically phosphorylate the EIN2 protein, the master regulator of ethylene signaling, that will transduce the signal to the nucleus. To date, no study has focused on EIN2 homologues of P. patens, although there are at least two of them (Wang et al., 2015a).

Although ethylene's use as a phytohormone appears quite ancestral and relies on a phospho-transfer signaling cascade reminiscent of the bacterial two component system (much like cytokinins), not much is known about ethylene signaling in mosses. The role of the bifunctional PpCTR1L/PpARK homolog is very puzzling and could either represent the ancestral state of the ethylene/ABA interplay or be the product of a moss specific evolutionary route.

JASMONIC ACID and OPDA

In plants, one major class of defense hormone is composed of plant-specific oxylipins collectively known as jasmonates . In Angiosperms, jasmonates are also critical for fertility and reproduction. Production of oxylipins is very conserved across evolution, between plants and animals (Ponce de [START_REF] Ponce De León | Oxylipins in moss development and defense[END_REF]. However, the presence of jasmonates per se (jasmonic acid itself or its derivates) in mosses is the subject of ongoing debate. In 2009 two research teams reported the synthesis of jasmonic acid in P. patens, furthermore in higher amounts than in flowering plants [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF][START_REF] Bandara | Cloning and functional analysis of an allene oxide synthase in Physcomitrella patens[END_REF]. However, recent profiles of produced oxylipins and a study of oxylipins biosynthesis genes in P. patens both point to oxylipins not being further metabolized into jasmonates in this species [START_REF] Senger | A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens[END_REF][START_REF] Stumpe | Biosynthesis of C9-aldehydes in the moss Physcomitrella patens[END_REF][START_REF] Chico | JAZ repressors set the rhythm in jasmonate signaling[END_REF]. Nevertheless, all studies agree on the production of the jasmonate precursor 12-oxo-phytodienoic acid (OPDA) in P. patens. Oxylipins and thus jasmonates biosynthesis starts with the release of precursor fatty acids from membrane glycerolipids. In most plants, this fatty acid is almost invariably α-linolenic acid, but oxylipins biosynthesis in mosses involves a broader range of precursors than in flowering plants, notably arachidonic acid [START_REF] Wichard | Unprecedented Lipoxygenase/Hydroperoxide Lyase Pathways in the Moss Physcomitrella patens[END_REF]Anterola et al., 2009a). These free fatty acids are then oxidized into hydroperoxides by LOX enzymes (lipoxygenases). Among the eight LOX genes found in P. patens, seven encode catalytically active PpLOX enzymes with different substrate specificity. PpLOX1/2 predominant action on arachidonic acid gives rise to 12S-hydroperoxy eicosatetraenoic acid (12-HPETE), hence starting the eicosanoid pathway, while PpLOX3-7 selective action on α-linolenic acid produces 13S-hydroperoxy octadecatrienoic acid , starting the octadecanoid pathway [START_REF] Wichard | Unprecedented Lipoxygenase/Hydroperoxide Lyase Pathways in the Moss Physcomitrella patens[END_REF]Anterola et al., 2009a). Following PpLOX action, two putative allene oxide synthases (AOS, OPDA biosynthesis enzymes) could follow up: PpAOS1 and PpAOS2 [START_REF] Bandara | Cloning and functional analysis of an allene oxide synthase in Physcomitrella patens[END_REF], PpAOS1 being more highly expressed. Recombinant PpAOS1 protein can indeed produce racemic OPDA from 13-HPOTE. PpAOS1 can also use hydroperoxy-fatty acid substrates from the eicosanoid pathway, such as 12-HPETE, as substrates, which is the main activity of PpAOS2 [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF]. The allene oxides produced via AOS activity are very unstable and readily non-enzymatically degraded into racemic OPDA [START_REF] Bandara | Cloning and functional analysis of an allene oxide synthase in Physcomitrella patens[END_REF]. However, only the cis-(+)-OPDA enantiomer can give rise to natural jasmonates. The proper enantiomeric structure of OPDA is set by AOC enzymes (allene oxide cyclases) which activity is certainly temporally and spatially coupled with AOS in order to protect allene oxides from spontaneous hydrolysis [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF]. There are three PpAOC genes in P. patens and all three have been shown to produce cis-(+)-OPDA from 13-HPOTE in vitro, given an AOS enzyme is provided as a helper [START_REF] Stumpe | The moss Physcomitrella patens contains cyclopentenones but no jasmonates: Mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology[END_REF][START_REF] Hashimoto | Cloning and characterization of an allene oxide cyclase, PpAOC3, in Physcomitrella patens[END_REF]. Interestingly, the seven active PpLOXs and the three PpAOCs collectively localize in plastids [START_REF] Stumpe | The moss Physcomitrella patens contains cyclopentenones but no jasmonates: Mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology[END_REF][START_REF] Hashimoto | Cloning and characterization of an allene oxide cyclase, PpAOC3, in Physcomitrella patens[END_REF] along with PpAOS2 [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF], whereas PpAOS1 is cytosolic [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF], suggesting the OPDA biosynthesis pathway in P. patens might involve this subcellular compartment, which is not the case in Angiosperms. In Angiosperms, OPDA is transported into peroxisomes, where it is reduced by OPDA reductases (OPR) enzymes into 3-oxo-2-(2′(Z)-pentenyl)-cyclopentane-1octanoic acid (OPC-8:0). After three cycles of β-oxidation that shorten the carboxyl side chain, jasmonic acid is obtained. P. patens possesses at least six OPR genes, and while they have not been experimentally characterized, predicted substrate affinity and phylogenetical evidence suggest that some are relevant candidates for jasmonates' biosynthesis [START_REF] Breithaupt | Structural Basis of Substrate Specificity of Plant 12-Oxophytodienoate Reductases[END_REF][START_REF] Li | Phylogenetic analysis, structural evolution and functional divergence of the 12-oxo-phytodienoate acid reductase gene family in plants[END_REF]. cis-(+)-OPDA is accumulated under infection with both oomycetes [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF] and fungi, where free linolenic acid levels are also increased [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF]. Some cis-(+)-OPDA biosynthesis genes (PpLOX6 and PpAOS1, PpAOC1) are indeed induced by fungal infection, along with a PpOPR gene, suggesting this homolog might be involved in the generation of defense-promoting compounds, perhaps a jasmonate [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF][START_REF] Toshima | Proteomic analysis of Physcomitrella patens treated with 12-oxo-phytodienoic acid, an important oxylipin in plants[END_REF].

cis-(+)-OPDA globally inhibits P. patens growth, by restricting protonema extension and rhizoid growth, but increasing rhizoid number . This effect could result from a block in cell cycle progression [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF]. According to the phenotypes of the Ppaoc1 and Ppaoc2 simple mutants, cis-(+)-OPDA (or its derivative(s)) is necessary for fertility [START_REF] Stumpe | The moss Physcomitrella patens contains cyclopentenones but no jasmonates: Mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology[END_REF]. However, this defect in fertility is not restored by a cis-(+)-OPDA treatment, neither during sporophyte induction, nor on already formed capsules, leading to the hypothesis that either cis-(+)-OPDA is not the active molecule or that its effect takes place much earlier in development. Hence, Stumpe and colleagues concluded that cis-(+)-OPDA must act at the level of gametes development [START_REF] Stumpe | The moss Physcomitrella patens contains cyclopentenones but no jasmonates: Mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology[END_REF].

Interestingly, when Scholz and colleagues characterized the Ppaos1 and Ppaos2 simple loss-of-function mutants, they did not observe these types of defects, suggesting cis-(+)-OPDA can still be synthesized in sufficient amounts if only one PpAOS enzyme is active [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF]. However, induction of cis-(+)-OPDA accumulation by wounding was impaired in Ppaos1, suggesting PpAOS1 has a prevalent effect in this case [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF]. cis-(+)-OPDA mediates defense responses against pathogenic microorganisms such as oomycetes [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF] and fungi, and even has a direct antimicrobial activity against the latter [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF]. cis-(+)-OPDA accumulation triggers PpPAL2 (phenylalanine ammonia-lyase) expression, possibly inducing the biosynthesis of phenylpropanoids and salicylic acid as in Angiosperms [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF]. Puzzlingly, even though evidence for jasmonates' production is lacking, P. patens is sensitive to methyl-jasmonate (MeJA), as this molecule induces PpPAL2 [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF] and has the same effects on growth as cis-(+)-OPDA, albeit higher concentrations are necessary [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF]. An effect of jasmonates as inducers of bifunctional phytoalexic and allelopathic compounds production (momilactone A and B) has also been described, albeit not in P. patens but in Hypnum plumaeforme [START_REF] Kato-Noguchi | Allelopathy of the moss Hypnum plumaeforme by the production of momilactone A and B[END_REF].

To our knowledge, there is no experimental evidence of a working signaling pathway for cis-(+)-OPDA in P.

patens. It has been reported that all components of the JA signaling pathway, namely COI1 (Coronatine Insensitive 1, an F-box protein that serves as the jasmonic acid receptor in Angiosperms), JAZ transcriptional corepressors (Jasmonate-ZIM-domain proteins) and NINJA transcriptional repressors (Novel Interactor of JAZ), are present in P.

patens [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF]Han, 2017)(Figure II-3). P. patens possesses several homologs of COI1 [START_REF] Chico | JAZ repressors set the rhythm in jasmonate signaling[END_REF][START_REF] Han | Evolution of jasmonate biosynthesis and signalling mechanisms[END_REF], but we could very well hypothesize that these PpCOI-like proteins recognize other oxylipins rather than jasmonic acid, notably cis-(+)-OPDA. As written before, P. patens can also respond to jasmonic acid and its methylated derivative MeJA [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF], even though it is supposedly unable to synthesize these molecules. This could imply that these compounds are transformed into a bioactive molecule which can be produced from cis-(+)-OPDA in P. patens, or simply mimic the endogenous ligand of P. patens, and eventually exert similar effects. The events following signal transduction have been further explored by Toshima and colleagues when they examined the effect of a cis-(+)-OPDA treatment on P. patens proteome [START_REF] Toshima | Proteomic analysis of Physcomitrella patens treated with 12-oxo-phytodienoic acid, an important oxylipin in plants[END_REF]. Almost a hundred soluble proteins are impacted by this treatment, the majority being less accumulated. Notably, carbon fixation, as well as translation and amino acid synthesis, are repressed by cis-(+)-OPDA, which is highly coherent with cis-(+)-OPDA role as a stress hormone and explains how it represses growth [START_REF] Toshima | Proteomic analysis of Physcomitrella patens treated with 12-oxo-phytodienoic acid, an important oxylipin in plants[END_REF]. This effect on the proteome likely prevents unnecessary energy consumption in response to adverse environmental conditions such as infection or wounding. Interestingly, the PpAOC1 enzyme itself was over-accumulated after cis-(+)-OPDA treatment, showing that a positive feedback regulation by OPDA likely takes place in P. patens. Such a mechanism is also supported by the accumulation of proteins likely involved in generation of ROS in plastids, which could thus oxidize polyunsaturated lipids in plastid membranes, and produce the free fatty acids necessary for cis-(+)-OPDA production. [START_REF] Toshima | Proteomic analysis of Physcomitrella patens treated with 12-oxo-phytodienoic acid, an important oxylipin in plants[END_REF] also suggest that cis-(+)-OPDA can regulate gene expression in plastids. Last year, Fesenko and colleagues showed that MeJA treatment of P. patens protonema induces the production of new small peptides especially from chloroplastic precursor proteins, possibly by action of the ubiquitin-proteasome system, with some being released extracellularly [START_REF] Fesenko | Phytohormone treatment induces generation of cryptic peptides with antimicrobial activity in the Moss Physcomitrella patens[END_REF].

Certain peptides from this secretome had a bacteriostatic effect on E. coli and B. subtilis and one could induce the expression of pathogenesis-related genes in P. patens, hinting at a possible signaling ability [START_REF] Fesenko | Phytohormone treatment induces generation of cryptic peptides with antimicrobial activity in the Moss Physcomitrella patens[END_REF].

cis-(+)-OPDA might not have been used as a hormone per se in ancestral plants, but rather as defense compounds against pathogens and wounding (this hypothesis is further discussed in the review by [START_REF] Ponce De León | Oxylipins in moss development and defense[END_REF].

However, the effects of this molecule in P. patens fertility, at micromolar doses, do suggest a hormonal role. Indeed, a derivate from this molecule (dinor OPDA) has a demonstrated hormonal role in another Bryophyte, the liverwort Marchantia polymorpha [START_REF] Monte | Ligand-receptor co-evolution shaped the jasmonate pathway in land plants[END_REF]. Elucidation of a cis-(+)-OPDA (or derivative(s))-specific signaling pathway would definitely help determining whether this (these) molecule(s) are bona fide hormones in mosses.

SALICYLIC ACID

Salicylic acid (SA) is a phenolic compound (Figure II-1) acting as a major stress hormone, protecting Angiosperms against pathogens, notably by enabling pre-emptive defense reaction in cells located at a distance from the plant/pathogen interaction point (namely Systemic Acquired Resistance or SAR). SA can be produced in plants through two different pathways, both starting from chorismate: the ICS (isochorismate synthase) plastidial pathway and the PAL (phenylalanine ammonia-lyase) cytosolic pathway (see the recent review by [START_REF] Lefevere | Salicylic Acid Biosynthesis in Plants[END_REF]. Neither pathways have been characterized in mosses, nor possible mechanisms regulating SA catabolism, transport or storage. However, P. patens can produce SA [START_REF] Richter | Polyphenol oxidases in Physcomitrella: functional PPO1 methylation and chromatin patterning knockout modulates cytokinin-dependent development in the moss Physcomitrella patens[END_REF]. P. patens moreover possesses several PpPAL genes, but it is worth to remember that PAL enzymes are also involved in phenylpropanoid biosynthesis and not specific to SA biosynthesis [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF][START_REF] Wolf | The molecular and physiological responses of Physcomitrella patens to Ultraviolet-B Radiation[END_REF].

To our knowledge, only one study by Christianson and Duffy investigated the impact of salicylates on P. patens development [START_REF] Christianson | Dose-Dependent Effect of Salicylates in a Moss, Funaria hygrometrica[END_REF]. They determined that both salicylic acid itself, as well as acetylsalicylic acid, can inhibit bud formation in a dose-dependent way . They further established that this effect likely does not result from an impairment of cytokinin-induced bud induction as it occurs much later in bud formation and is neither explained by an inhibition of young gametophores outgrowth. Several elements suggest that SA is involved in defense promotion in P. patens. Firstly, Salicylic acid (SA) levels increase rapidly after fungal infection, and treatment with SA enhances transcript accumulation of one of the phenylalanine ammonia-lyase (PAL) defense genes in P. patens [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF]. SA levels are increased 7-fold in a few hours following the onset of B. cinereal infection, suggesting that SA is synthesized and perceived during pathogen infection. In contrast, there was no induction of SA accumulation after oomycetes infection according to a study by [START_REF] Oliver | Pythium infection activates conserved plant defense responses in mosses[END_REF], so the SA pathway might not be involved in defense against all microbial pathogens.

Studies about SA signaling in mosses are scarce and recent. Peng et al. identified two putative NPR (Nonexpresser of PR genes) SA receptors in P. patens genome [START_REF] Peng | Perception of salicylic acid in physcomitrella patens[END_REF], confirming previous predictions (Wang et al., 2015a). However, one of them appeared to be a pseudogene and only PpNPR1 was characterized. It contains both conserved protein interaction domains: The N-terminal BTB/POZ domain and the central ankyrin-repeat domain, as well as a C-terminal domain with nuclear localization signals. Functional complementation of the Arabidopsis Atnpr1 mutant suggested that PpNPR1 is indeed a functional receptor of SA and is even able to properly interact with other players in the SA signaling pathway of Angiosperms. Thus, SA signaling is likely similar between Angiosperms and mosses. Nevertheless, in Angiosperms, paralogs of NPR1 called NPR3/4 act as negative regulators of response to SA. This function of NPRs does not exist in P. patens, as the Atnpr3/4 mutant is not complemented by PpNPR1 unlike Atnpr1. It would imply that mosses rely on other mechanisms to fine-tune the SA signaling level of activation. Other cellular events downstream of PpNPR1 are not known yet in P. patens or other mosses. Whether PpNPR1 can, similarly to Angiosperms NPR proteins, interact with WRKY and TGA transcription factors in the nucleus to transduce the SA signal remains undetermined. Nevertheless, the fact that SA does rapidly induce the expression of defense genes, namely PpPAL1 (Ponce De [START_REF] Ponce De León | Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection[END_REF] and PpPR gene(s) [START_REF] Peng | Perception of salicylic acid in physcomitrella patens[END_REF], implies that PpNPR1 is indeed interacting with (a) transcription factor(s) to induce SAR (Systemic Acquired Resistance). SA also triggers effects at the protein level, as Filippova et al. have shown that a SA treatment activates specific proteases to generate a pool of almost 150 new active peptides in protonema, some being secreted [START_REF] Filippova | Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss physcomitrella patens[END_REF]. SA treatment notably induced proteolysis of small stress-related proteins, and overall results suggest that SA caused a significant increase in proteasome activity. However, the potential roles of these new peptides, either as signaling elements in defense, or as molecules with a direct effect against possible causes of stress, is not known.

Despite the lack of a decisive number of studies on SA biosynthesis and signaling, present evidence suggests that the use of SA as a defense hormone is ancestral to land plants. Nonetheless, further investigation might reveal some minor divergences between Bryophytes and vascular plants.

GIBBERELLINS and KAURENE Derivatives

Gibberellins are tetracyclic diterpene acids (Figure II-1) synthesized in plants but also in fungi and bacteria. In flowering plants, they are major regulators of diverse aspects of development, notably inducing the elongation of stems by triggering cell elongation, which was their first discovered role, and inducing seed germination. The first steps of gibberellic acid (GA) biosynthesis in plastids lead to the formation of the key intermediate ent-kaurene, which was shown to be also true in P. patens (Anterola et al., 2009b;[START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. However, moss relies on a single, bifunctional enzyme copalyl-diphosphate synthase/ent-kaurene synthase (PpCPS/KS), to catalyze the two steps necessary for ent-kaurene synthesis [START_REF] Hayashi | Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens[END_REF]Anterola et al., 2009b). This makes ent-kaurene biosynthesis in moss more alike to what has been shown in fungi [START_REF] Hedden | Gibberellin Biosynthesis in Plants and Fungi: A Case of Convergent Evolution?[END_REF][START_REF] Davidson | Cytochromes P450 in gibberellin biosynthesis[END_REF][START_REF] Ross | Evolution of growth-promoting plant hormones[END_REF] rather than what happens in vascular plants where the two enzymatic activities are separate. The CPS enzymatic activity of the PpCPS/KS protein converts geranyl-geranyl-diphosphate (GGDP) into ent-copalyl-diphosphate, and then the KS activity transforms the ent-copalyl-diphosphate into ent-kaurene. Then, ent-kaurene is likely oxidized into ent-kaurenoic acid, this molecule being produced at high levels in protonema [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. Indeed, P. patens possesses a gene that could encode a CYP450 with ent-kaurene oxidase activity (PpKO/PpCYP701B1) and could thus be able to synthesize ent-kaurenoic [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. Interestingly, loss-of-function of PpCPS/KS results in complete absence of ent-kaurene and ent-kaurenoic acid, thus this enzyme is responsible for the biosynthesis of the whole pool of entkaurene in P. patens [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. ent-kaurenoic acid is then transformed into ent-3b-hydroxy-kaurenoic acid (3OH-KA) and ent-2a-hydroxy-kaurenoic acid (2OH-KA), both being produced along protonema development [START_REF] Miyazaki | An Ancestral Gibberellin in a Moss Physcomitrella patens[END_REF]. 2OH-KA biosynthesis is provided by the activity of an ent-kaurenoic acid oxidase (PpKA2ox), however the enzyme enabling 3OH-KA production was not identified. Interestingly, PpKA2ox was eventually demonstrated to be an ent-kaurenoic acid inactivation enzyme, in accordance with 2OH-KA being less bioactive than 3OH-KA. Surprisingly, PpKA2ox transcription is decreased by ent-kaurenoic acid, implying that 3OH-KA accumulation is subjected to positive feedback [START_REF] Miyazaki | An Ancestral Gibberellin in a Moss Physcomitrella patens[END_REF]. GA biosynthesis steps following 3OH-KA are far less clearly established. In 2010, Hayashi and colleagues underlined that, as several "canonical" gibberellins have similar effects on P. patens as ent-kaurenoic acid, P. patens is expected be able to synthesize such compounds. But they also acknowledged that the biosynthesis steps leading to these GA, most likely needing enzymes of the CYP450 superfamily, remain to be elucidated. Very recently, a whole genome comparison study by Cannell et al. suggested that genes potentially encoding canonical gibberellins' biosynthesis enzymes (production of GA12 and all derived GA) are missing in both P. patens and Sphagnum fallax genomes, whereas they are present in other Bryophytes [START_REF] Cannell | Multiple Metabolic Innovations and Losses Are Associated with Major Transitions in Land Plant Evolution[END_REF].

They hence concluded on a secondary loss of these genes along mosses evolution. However, an earlier study reported that P. patens possesses homologs of GA20 oxidase and GA3 oxidase encoding genes, suggesting respectively GA20 and GA3 (gibberellic acid) can be produced [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF]. Nevertheless, GA production or GA oxidase activity could not be detected in this moss [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF].

GA3 seems to have no effect on P. patens protonemal development, even at high concentrations [START_REF] Vandenbussche | Evolutionary conservation of plant gibberellin signalling pathway components[END_REF]. Moreover, the gibberellin biosynthesis inhibitor paclobutrazol (PAC) inhibits protonemal growth, but GA3 addition does not reverse this effect, further suggesting that P. patens is insensitive to this molecule [START_REF] Yasumura | Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution[END_REF]). Yet, GA3 can inhibit P. patens spores' germination , although not as efficiently as ent-kaurene (Anterola et al., 2009b). Unexpectedly, an ent-kaurene biosynthesis inhibitor (AMO-1618) severely decreases germination, but its effect is alleviated by addition of ent-kaurene only (and not by GA3). This further shows that the active gibberellin-like molecule is not GA3 but likely an ent-kaurene derivative or ent-kaurene itself (Anterola et al., 2009b). Both ent-kaurene and ent-kaurenoic acid are major inducers of the transition from chloronema to caulonema, but otherwise do not seem to affect cell size and growth rate in filaments [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]

(Figure II-2).
Interestingly, GA9-methyl ester could also promote caulonema differentiation, albeit being ~10 times less potent than ent-kaurenoic acid [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF], implying that P. patens could produce active compounds similar to "canonical" GA. On another note, inhibiting synthesis of ent-kaurenoic acid from ent-kaurene with uniconazole decreased caulonema number in WT, and this effect is lost when ent-kaurenoic acid was co-applied [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. Therefore, it suggests that ent-kaurene itself is not the active compound, but rather ent-kaurenoic acid or a derivative, which was confirmed later as Ppko and Pcps/ks mutants display the same phenotype (lack of blue light avoidance) [START_REF] Miyazaki | Hormonal diterpenoids derived from ent -kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens[END_REF]. On the other hand, neither ent-kaurene or ent-kaurenoic acid appear to have a significant effect on gametophores development, according to the lack of defects of the Ppks mutant. Puzzlingly, the Ppks mutant has WT fertility and spore germination rate [START_REF] Miyazaki | Hormonal diterpenoids derived from ent -kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens[END_REF], suggesting ent-kaurene and its derivatives are not necessary for proper reproduction, contrary to what was hypothesized before (Anterola et al., 2009b).

Hayashi and colleagues also showed that AMO-1618 neither impair ent-kaurene production in the protonema, nor affect chloronema to caulonema transition [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. Therefore, the previously described effect of AMO-1618 on spore germination is likely imputable to another pathway rather than to an ent-kaurene response defect. This was infirmed later, as Ppks mutants were revealed to have a reduced germination speed compared with WT, although they eventually attained 100% germination [START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF]. Moreover, germination speed was reduced after application of either GA9-methyl ester or ent-kaurene, but consistently not GA3 (contradicting the previous observation of Anterola et al.). Taken together, these studies demonstrate that P. patens can respond in a similar fashion to ent-kaurenoic acid and to some other, likely derived from the former, compounds more similar to "canonical" GA such as GA9-methyl ester. However, P. patens cannot respond to GA3, which is the most bioactive GA is flowering plants, thus GA perception likely has a different specificity in mosses.

The gibberellin signaling pathway was extensively investigated by Hirano and colleagues [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF], who identified two genes encoding putative receptors of gibberellins in P. patens: PpGID1L1 (GIBBERELLIN INSENSITIVE DWARF1 Like1) and PpGID1L2 (Figure . PpGID1Ls, like their Angiosperms' homologs are alphabeta-hydrolases/hormone sensitive lipases (HSL). However, contrary to Angiosperms, PpGID1Ls retain the Ser-Asp-His catalytic triad, which suggest they might still be catalytically active [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF], but this point is debated as another team found the His residue of the triad to be replaced by a tryptophan in PpGID1Ls [START_REF] Vandenbussche | Evolutionary conservation of plant gibberellin signalling pathway components[END_REF]. Moreover, two residues that are known to be necessary for gibberellins perception are lost in these two proteins, thus their function as gibberellins receptors cannot be assumed. These proteins are not able to bind neither GA4, GA9, GA12 nor 3-epi-GA4 in vitro [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF], which retrospectively appears coherent with the lack of physiological response of P. patens to these molecules. ent-kaurene and ent-kaurenoic acid binding to PpGID1Ls ought to be explored, as well as with the impact of PpDELLA-Likes on this binding, since DELLAs strengthen interaction between GID1 and gibberellins in Angiosperms [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF]. PpGID1L1 (PpGLP1 for [START_REF] Yasumura | Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution[END_REF] could not interact with PpDELLA-L1 in a yeast two hybrid assay with or without GA3, however it could interact with a Lycophyte DELLA even in the absence of GA3, suggesting the lack of interaction of moss proteins stems from PpDELLA-Ls. Therefore, affinity of GID1-like proteins for DELLAs could be ancestral and has been lost in Bryophytes, while DELLAs affinity for GID1-likes would have evolved after the split of Bryophytes and vascular plants. The necessity for gibberellins to trigger this interaction would have appeared even later, after Lycophytes diverged from other vascular plants.

Alternatively, this interaction might rely on other compounds, notably ent-kaurenoic acid, which was not tested in these studies. Three genes encoding putative F-box proteins (PpGID2L1 to 3) are found in P. patens genome [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF]. Analysis of their phylogeny and domain composition however suggests that, while they likely play a role in targeting some proteins for proteasomal degradation, it is not in the context of gibberellins signaling [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF][START_REF] Vandenbussche | Evolutionary conservation of plant gibberellin signalling pathway components[END_REF]. Another evidence that P. patens might not be able to sense gibberellins is that the PpDELLA-L proteins do not contain a conserved DELLA motif, responsible for the gibberellins-induced interaction with GID1 and subsequent proteasomal degradation. Moreover, PpDELLA-Ls lack half of the conserved tyrosine residues known as necessary for gibberellins-induced degradation of DELLAs from A. thaliana. Despite this discrepancy, PpDELLA-Ls do possess the C-terminal GRAS domain involved in protein-protein interactions and possibly in DNA binding [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF]. Also, they group with Angiosperms' DELLA proteins phylogenetically.

It can be argued that the absence of strict homology of domain and the absence of residues characterized as necessary in Angiosperms do not necessarily mean that these proteins are not involved in gibberellin signaling in mosses. However, some experimental evidences go along with this hypothesis. Notably, in a yeast two hybrid experiment, PpDELLA-Ls are unable to interact with either PpGID1L, even triggered by gibberellins addition [START_REF] Hirano | The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens[END_REF]. Since DELLA proteins are homologs of the other GRAS proteins SLR-like (SCARECROW-like), it can be hypothesized that PpDELLA-Ls are in fact more functionally related to SLR-likes and rather play a role in development regulation [START_REF] Vandenbussche | Evolutionary conservation of plant gibberellin signalling pathway components[END_REF]. The lack of developmental phenotype of the Ppdella-l1/2 loss of function mutant, along with its WT-like sensitivity to PAC, suggests that biosynthesis of gibberellin-like molecules in P. patens is irrelevant to the function of PpDELLA-Ls [START_REF] Yasumura | Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution[END_REF]. Hence, PpDELLA-Ls are not involved in the response to GA in P. patens, and they are not potent regulators of development either. When expressed in Arabidopsis roots, GFP-fused PpDELLA-L1 localizes to nuclei and its levels are not impacted by GA3 treatment [START_REF] Yasumura | Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution[END_REF]. However, its introduction apparently corrects the dwarf phenotype of the DELLA ga1-3 mutant of Arabidopsis, suggesting it can properly replace GA1. It is puzzling that PpDELLA-L1 can restrain growth in Arabidopsis but not in P. patens itself.

Yasumura and colleagues raise the interesting hypothesis that this phenomenon could be explained by growth controlling genes becoming DELLA responsive along evolution via changes in cis-regulatory regions. Thus, in P. patens, these genes controlling growth would not be under the transcriptional control of PpDELLA-Ls [START_REF] Yasumura | Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution[END_REF]. More recently, coexpression networks involving DELLA proteins were compared between P. patens and flowering plants (A. thaliana and Solanum lycopersicon), revealing that DELLAs likely act as hubs of transcriptional regulation in P. patens [START_REF] Briones-Moreno | Evolutionary analysis of DELLA-associated transcriptional networks[END_REF]. Functional annotation of genes involved in PpDELLA-Ls networks suggest a major implication in response to stress.

Taken together, results cited before suggest that P. patens is unable to respond to canonical gibberellins but that the gibberellin precursor ent-kaurenoic acid might be relevant during early development of the gametophyte. Since some gibberellins trigger similar responses to ent-kaurenoic acid, it is possible that the signaling cascade involved in P. patens works in a way that cannot be inferred by taking the Angiosperms pathway as a basis. For instance, even if the homologs of GID and DELLA genes are actually involved in a gibberellin-signaling cascade, the exact residues involved in these proteins have evolved independently so they might be quite different from residues necessary in Angiosperms. Mosses might have secondarily lost the ability to sense gibberellins [START_REF] Vandenbussche | Evolutionary conservation of plant gibberellin signalling pathway components[END_REF][START_REF] Yasumura | Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution[END_REF], as early studies reported that both a liverwort and a Charophyte can respond to gibberellins [START_REF] Asprey | Effect of Gibberellin and lndoleacetic Acid on Seta Elongation in Pellia epiphylla[END_REF][START_REF] Kwiatkowska | Effect of GA3 treatment on the number of spermatozoids and endopolyploidy levels of non-generative cells in antheridia of Chara vulgaris L[END_REF]. This loss of response ability is relevant if we consider the possible secondary loss of canonical gibberellins' biosynthesis in mosses [START_REF] Cannell | Multiple Metabolic Innovations and Losses Are Associated with Major Transitions in Land Plant Evolution[END_REF]. However, that could imply that the demonstrated effect of ent-kaurenoic acid and/or derived compounds is not perceived and transduced through the same pathway as canonical gibberellins.

BRASSINOSTEROIDS

Brassinosteroids (BRs) are a group of hydroxylated steroid-derived compounds (Figure II-1) undertaking very diverse roles in Angiosperms, from the promotion of senescence to ensuring pollen fertility, and from regulation of cell growth to protection of plants against abiotic stress. In contrast to auxins and GAs, there is no report yet of BRs in fungi or bacteria, thus BRs appear to be strictly plant-specific compounds (see the review by [START_REF] Ross | Evolution of growth-promoting plant hormones[END_REF]. Much like jasmonates, the presence of BRs in moss is still under debate. While sterol biosynthesis upstream of BRs is highly conserved across land plants, no clear homologs of BR biosynthesis genes are found in P. patens genome, except DET2 (involved in campestanol (CN) generation from campesterol (CR), [START_REF] Cannell | Multiple Metabolic Innovations and Losses Are Associated with Major Transitions in Land Plant Evolution[END_REF]. Two cytochrome P450 enzymes, CYP710A13, and CYP710A14, were identified by Morikawa et al. (2009) as sterol C22-desaturases in P. patens. These enzymes can use β-sitosterol in vitro to produce stigmasterol, but they cannot use campesterol and 24-epi-campesterol as substrates. This evidence again indicated the presence of an entire sterol biosynthetic pathway in this moss, although the sterols produced are different from flowering plants. These two enzymes are hence unlikely to be a part of a BR biosynthesis pathway. More recently, Yokota et al. reported that P. patens possesses both CYP85 and many CYP72 enzymes, respectively required for biosynthesis and inactivation of BR. Cannell and colleagues disagreed on this point as they noted that non-seed plants lack clear homologues of these inactivating CYP450 enzymes [START_REF] Yokota | Occurrence of brassinosteroids in non-flowering land plants, liverwort, moss, lycophyte and fern[END_REF][START_REF] Cannell | Multiple Metabolic Innovations and Losses Are Associated with Major Transitions in Land Plant Evolution[END_REF]. Despite these uncertainties, P. patens produces castasterone (CS), which is a bioactive BR in Angiosperms [START_REF] Yokota | Occurrence of brassinosteroids in non-flowering land plants, liverwort, moss, lycophyte and fern[END_REF]. Therefore, P. patens can likely synthesize and inactivate BR, but the enzymes involved might not be close homologs to those of Angiosperms and could therefore not be recovered by BLAST.

No homolog of the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is found in P. patens [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF]. In facts, the only partially similar sequences found lack the key extracellular domain involved with BR binding [START_REF] Ross | Evolution of growth-promoting plant hormones[END_REF]. In their review on phytohormones signaling evolution, [START_REF] Wang | Insights into the Origin and Evolution of the Plant Hormone Signaling Machinery[END_REF] reported that several other components of the BR signaling pathway are present in P. patens, notably the co-receptor BRI1-Associated receptor Kinase (BAK1). However, this species is missing the key regulator Brassinosteroid Insensitive1 Kinase Inhibitor (BKI1), which appears to be specific to Angiosperms. Other molecular players of the BR signaling pathway are more ancient, as they are similarly found in flowering plants and in P. patens. This is the case for the kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) and for the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1). But they have not been experimentally assigned to BR signaling in P. patens. Similarly, BIL4 (BZR-Insensitive Long hypocotyl 4), a seven transmembrane-domains protein similar to G protein-coupled receptors, has a homolog in P. patens. In Arabidopsis, this protein interacts with the BR receptor BRI1 in endosomes to prevent its relocation and degradation in vacuoles, but in P. patens, BIL4 function is likely independent of BR signaling [START_REF] Yamagami | Evolutionarily conserved BIL4 suppresses the degradation of brassinosteroid receptor BRI1 and regulates cell elongation[END_REF].

The evidence on biosynthesis, effects and signaling are thus too fragmentary to establish whether BRs are actually used as phytohormones in mosses.

Consistently, in their 2010 review, Ross and Reid emitted the hypothesis that BRs or BR-like compounds could also simply be present as specialized metabolites in mosses, rather than act as actual hormones. That could explain the lack of a specific signaling pathway in the Bryophytes species studied until now, despite their synthesizing BR-like compounds.

SIGNALING PEPTIDES

To date, sixteen families of plant signaling peptides have been identified in Angiosperms. These peptides belong to two major groups: Group I consists of cysteine-rich peptides for which proteolytic processing is not always necessary, while group II includes cysteine-poor, smaller, peptides, and must be proteolytically processed to maturity (see the review by [START_REF] Ghorbani | Signaling Peptides in Plants[END_REF]. Most signaling peptides belong to group II, notably the CLE peptides (Clavata3/Endosperm Surrounding Region (ESR)-Related), which have been the focus of extensive research effort. In Angiosperms, CLE peptides have been shown to have dramatic effects on stem cells maintenance. Two classes of CLEs have been described: CLV3-like and TDIF-like (Tracheary element Differentiation Inhibitory Factor-like). The CLV3 (Clavata3) peptide hormone has been especially well characterized. CLE peptides production is initiated by the translation of a short (~200 residues) pre-propeptide containing a N-terminal secretion signal. This pre-propeptide contains the CLE domain, which is only 12-13 amino acid long. The inactive pre-propeptide must be enzymatically cut by serine-or carboxy-peptidase for the CLE peptide to be freed, although some CLE peptides need to be further post-translationally modified to become biologically active and be ultimately secreted in the extracellular space [START_REF] Goad | Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function[END_REF].

Genes encoding CLE peptides are present in P. patens genome, even though CLE genes are difficult to identify since only the CLE domain is usually highly conserved, and since most are lowly expressed and thus not annotated in genomes [START_REF] Sawa | RPK2 functions in diverged CLE signaling[END_REF][START_REF] Goad | Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function[END_REF]. More precisely, only 9 PpCLE genes were identified, but more divergent ones might have been missed [START_REF] Goad | Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function[END_REF]. Further research by Whitewoods et al. led to the identification of only 7 non-divergent PpCLE, giving rise to four different peptides: PpCLE1/2/3, PpCLE4, PpCLE5/6 and PpCLE7 (named from their encoding gene(s)) [START_REF] Whitewoods | CLAVATA Was a Genetic Novelty for the Morphological Innovation of 3D Growth in Land Plants[END_REF]. All four peptides contain most of the residues necessary for interaction with CLV1 receptor [START_REF] Goad | Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function[END_REF]. PpCLE1, PpCLE2 and PpCLE7 are mainly expressed in gametophores, while PpCLE6 expression is protonema specific. On the other hand, PpCLE3, PpCLE4 and PpCLE5 transcripts could not be detected [START_REF] Whitewoods | CLAVATA Was a Genetic Novelty for the Morphological Innovation of 3D Growth in Land Plants[END_REF]. Both studies agree that all PpCLEs belong to the CLV3-like class. In P. patens as in Angiosperms, the precise enzymes involved in CLE peptides generation have not been formally identified.

CLV3 in Angiosperms can potentially be sensed via three different family of RECEPTOR-LIKE KINASES (RLK) receptors: CLV1 (Clavata1), CLV2 (Clavata2, along with the SOL2/CRN (Suppressor Of LLP1 2/Coryne) coreceptor) and RPK2 (Receptor-like Protein Kinase 2). P. patens has 2 homologs of CLV1 (PpCLV1a and PpCLV1b), as well as one of RPK2 (PpRPK2), but no CLV2 and SOL2/CRN [START_REF] Sawa | RPK2 functions in diverged CLE signaling[END_REF][START_REF] Whitewoods | CLAVATA Was a Genetic Novelty for the Morphological Innovation of 3D Growth in Land Plants[END_REF].

Whitewoods et al. report the expression of all three genes in gametophores stems and phyllids. Earlier, PpCLV1a transcripts are accumulated in protonema cells around buds, while PpCLV1b and PpRPK2 accumulate in buds themselves, especially at the apex. Further confirming that CLE signaling is involved in transition to gametophore 3D growth, Whitewoods and colleagues noted that Ppcle knock down lines and Ppclv1a/b knock out lines developed far less, dwarf, gametophores. Close inspection of buds revealed misdirection of division planes in the first divisions of the buds. However, since gametophores eventually develop, the disruption likely occurs after "bud" cell fate is specified, while still taking place at the single-celled stage. In gametophores, all lines had defective phyllids development [START_REF] Whitewoods | CLAVATA Was a Genetic Novelty for the Morphological Innovation of 3D Growth in Land Plants[END_REF]. Moreover, Pprpk2 and Ppclv1a/b lines developed calluses at the gametophore base, resulting from the activity of ectopic apical cells. Puzzlingly, exogenous application of PpCLEs also caused gametophore dwarfing and a decrease in phyllids size, but these were not the results of the same developmental arrest and meristematic over-proliferation as in Ppcle lines. Therefore, it could be concluded that a very narrow range in PpCLEs concentration must be achieved to permit WT development of gametophores. Finally, Whitewoods and colleagues demonstrated that Pprpk2 is indeed unable to respond to neither PpCLE1,2,3 nor PpCLE7, in terms of gametophore dwarfing [START_REF] Whitewoods | CLAVATA Was a Genetic Novelty for the Morphological Innovation of 3D Growth in Land Plants[END_REF]. Interestingly, P. patens does not respond to the TDIF peptide from Arabidopsis, while it does respond to the CLV3 peptide from this same species. This again points to mosses not using TDIF-like CLEs, which likely stems from a secondary loss in their evolutionary lineage (see the recent review by [START_REF] Whitewoods | Evolution of CLE peptide signalling[END_REF].

STRIGOLACTONES and KAI2-ligand(s)

Strigolactones (SL) are cyclic lactones originating from apocarotenoids . They are the most recently established class of phytohormones and play many roles in development in Angiosperms, notably repressing axillary branching, promoting secondary growth and regulating root architecture. In addition, SL are involved in the regulation of interspecific interactions between plants and micro-organisms, of both symbiotic and pathogenic nature, and help the plant cope with several abiotic stresses (see the review by [START_REF] Mishra | The Role of Strigolactones and Their Potential Cross-talk under Hostile Ecological Conditions in Plants[END_REF]. Sequencing of P. patens genome has shown that this moss possesses homologs genes to those encoding the first enzymes of the SL biosynthetic pathway, leading to carlactone (CL, Figure II-1) production: the isomerase D27 (DWARF 27), and the CAROTENOID CLEAVAGE DIOXYGENASES CCD7 and CCD8 [START_REF] Rensing | The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants[END_REF]. On the other hand, no clear CYP711A1 candidate for catalyzing the transformation of the CL precursor into SL has been identified. Recent quantifications of SL in P. patens revealed only the presence of CL, in accordance with the absence of CYP711A1 enzymes [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]Yoneyama et al., 2018b). However, it does not necessarily mean that no other compound with SL activity is synthesized by P. patens: it cannot be excluded that enzymes apart from CYP711A1 (maybe even other classes of CYP450) could use CL as a substrate. Furthermore, several compounds beside the precursor of CL can be obtained from PpCCD7 enzymatic activity in vitro [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF] and add a layer in the putative structural diversity of SL like compounds in P. patens. Decker et al. also confirmed PpCCD8 activity as a CL synthase in vitro. It is widely admitted that SL are unstable molecules and are furthermore produced at very low concentrations. These characteristics make their identification (and use) quite difficult, even in flowering plants, and is part of why there is no certainty about the exact SL molecules produced by P. patens. This, along with the fact that SL receptors break their substrates down into inactive molecules in vitro [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF], also makes enzymatic inactivation of SL dispensable, unlike for other phytohormones such as cytokinins.

In 2011, the first homolog of a SL biosynthesis gene in P. patens, PpCCD8, was functionally characterized by revealed that SL in P. patens repress spore germination (Proust et al., 2011;[START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF]. SL also repress branching of primary chloronema, while they do not appear to affect caulonema branching until 21 days after spore germination, when Ppccd8 caulonema filaments continue to develop secondary chloronema and buds at the periphery, unlike WT (Proust et al., 2011). This suggests that SL do repress caulonema branching at this later time point and/or at the apex of filaments . Proust and colleagues suggested this effect on branching could be explained by SL inhibiting the cell division rate, at least in chloronema. Effects of SL on cell division and cell elongation were clarified a few year later, when closer inspection of the Ppccd8 mutant revealed that SL likely repress cell division rate, and less potently cell elongation, in both types of protonemal filaments (Hoffmann et al., 2014). This was further confirmed by Decker and colleagues when they demonstrated that the Ppccd7 loss of function mutant displays the same increase in filaments length, resulting of an increase in cell number, as Ppccd8 [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. A study by Coudert et al. suggested that this inhibitory effect on cell division is also active in gametophores , as the Ppccd8 mutant displays strongly increased branching of gametophores at their base and this phenotype was suppressed by racGR24 [START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF]. This branching phenotype is reminiscent of SL insensitive mutants from Angiosperms (affecting MORE AXILLARY BRANCHES, MAX genes), suggesting that inhibition of branching, albeit involving different tissues and processes in mosses and Angiosperms, is an ancestral role of SL. Puzzlingly, SL increase rhizoid length in P. patens, which could mean that they have a positive effect on cell division and/or cell elongation in this tissue, contrary to their effect on protonema [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF]. On another note, it is interesting to underline that, similarly to cytokinins, SL are liberated in the medium (Proust et al., 2011). Proust et al. suggested that SL act as bacterial quorum sensing molecules, regulating growth of close neighboring plants, as their concentration in the medium reflects plant density. Decker et al. described a protective effect in P. patens against the phytopathogenic fungi Fusarium oxysporum and Sclerotinia sclerotiorum [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. This effect is most probably explained by an enhancement of P. patens resistance, but the mechanism has not been investigated yet. Moreover, they can have an effect in protecting whole P. patens populations from pathogens, as they increase resistance of the plants [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. Furthermore, Vesty et al. suggested that this quorum sensing mechanism could be particularly of use during spore germination: plants developing form the first germinating spores would produce SL and therefore prevent germination of close spores [START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF]. This could alleviate intraspecific competition for resources. Taken together, these results point to SL being a major mean of shaping and organizing P. patens whole populations. P. patens responds similarly to the synthetic analog of SL racGR24 and to WT exudates, suggesting racGR24 is sensed through the same signaling cascade as compounds derived from PpCCD8 activity (Proust et al., 2011). CL itself rescues Ppccd7 and Ppccd8 hyper-branched phenotype [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF], implying either that CL itself is the bioactive compound in P. patens, or that CL can be converted to a bioactive SL in vivo. P. patens can also respond to some natural canonical SL, notably solanacol and 5-deoxystrigol (Hoffmann et al., 2014), hence this (these) CL-derived compound(s) might have a similar structure.

In Angiosperms, two different subclasses of related KAI2L (KARRIKIN INSENSITIVE 2-like) α/β-hydrolases, euKAI2 and DDK subclasses, are involved in the perception of two types of phytohormones: DDKs handle SL perception, whereas euKAI2s tackle KL (KAI2-ligand) perception (Bythell-Douglas et al., 2017). KL is a conceptual term used to refer to unknown endogenous compounds that are perceived by euKAI2s and are widely considered as a new type of phytohormones. In 2016, a study by Lopez-Obando and colleagues identified thirteen KAI2L genes in P.

patens . Investigation of these genes' expression and modelling of the putative proteins denoted PpKAI2L-G and -J as the best candidates for SL perception, whereas PpKAI2L-B, -C, -D and -E are closer to euKAI2 and therefore are candidate for KL perception. Karrikins are natural compounds found in fire smoke and stimulating seed germination and seedling emergence after a fire, which are usually used as KL mimics [START_REF] Flematti | A compound from smoke that promotes seed germination[END_REF]. Biochemical characterization by [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF] however showed that PpKAI2L-C, -D and -E cannot bind the karrikin KAR1 (Figure II-1), while PpKAI2L-H, -L and -K can. Nevertheless, it must be noted that the biological relevance of these results is for the moment lacking, especially since P. patens has been shown to be non-responsive to KAR1 (Hoffmann et al., 2014). Characterized euKAI2/DDK proteins in Angiosperms both interact with the same F-box protein MAX2 (MORE AXILLARY BRANCHES 2) and facilitate the involvement of MAX2 in a SCF E3-ligase ubiquitination complex [START_REF] Yao | Perception of karrikins by plants: A continuing enigma[END_REF]. This complex then triggers the proteasomal degradation of proteins from the SMXL (SUPPRESSOR OF MAX2-1-like) family. In flowering plants, SMXL proteins of different groups are associated with either euKAI2-dependent signaling or DDK-dependent signaling [START_REF] Yao | Perception of karrikins by plants: A continuing enigma[END_REF]. However, in non-seed plants such as P. patens, this divide in function has not been proved and phylogeny cannot predict in which pathway they might act [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF]Walker et al., 2019) (Figure II-3). In addition to this, a recent study of the sole MAX2 homolog from P. patens [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF] demonstrated that PpMAX2 is most probably not involved in SL signaling. Indeed, Ppmax2 loss-of-function mutants have a striking developmental phenotype that is completely different from that of SL biosynthesis mutants (Ppccd7 and Ppccd8). Additionally, these Ppmax2 mutants can respond to exogenous SL, even more significantly than WT. PpMAX2 is instead involved in photomorphogenesis, which is hypothesized to be linked to KL signaling [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. If PpMAX2 is involved in KL signaling, then this response in P. patens likely takes place where PpMAX2 is expressed: chloronema and young caulonema, the basal portion of gametophores and sporophytes [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. Given the apparent roles of PpMAX2 in promoting chloronema to caulonema transition and gametophores development, KL likely has similar roles in P. patens, maybe through an induction of cell division, contrary to SL. Going back to the SL response pathway, although signaling events downstream of perception are unresolved at the moment, it is known that SL signaling leads to an inhibition of both PpCCD7 and PpCCD8 expression (Proust et al., 2011). This negative feedback on SL biosynthesis via corepression of PpCCD7 and PpCCD8 can partly be explained by their close colocalization on chromosome 6. SL production in P. patens is moreover induced by phosphate depletion, but it remains to be investigated whether this results from an induction of PpCCD7 and PpCCD8 expression at the transcriptional level [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF].

Like what is hypothesized for jasmonates, there is an ongoing debate that SL might not have been used as hormones per se in ancestral plants, but rather interspecific signals, notably permitting symbiosis with symbiotic fungi [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF][START_REF] Waters | Strigolactone Signaling and Evolution[END_REF]. On another note, common use of racGR24 in experiments often makes results unclear, as this enantiomeric mixture not only stimulates the SL signaling pathway but also the euKAI2-MAX2dependent pathway [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF], which might also be the case in P. patens.

CROSSTALKS Phytohormonal crosstalks involving auxin and/or cytokinins:

The phytohormonal crosstalk that is perhaps the most extensively documented is the interplay of auxin and cytokinins in the induction of gametophore buds. Cytokinin-insensitive bar mutants can produce initial cells but no buds in P. patens [START_REF] Schumaker | HORMONE-INDUCED SIGNALING DURING MOSS DEVELOPMENT[END_REF]. However, addition of auxin can trigger the development of gametophores in these mutants, showing that auxin is also needed for bud assembly, probably at higher concentration than for caulonema differentiation. Conversely, gametophores fail to form in nar auxin insensitive mutants in control conditions. However, when nar mutants producing auxin-resistant degron-less PpIAAs are cultured with exogenous cytokinins under low-fluence red light, gametophore formation is partly restored [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF]. This suggests that cytokinins acts downstream of auxin to promote gametophores formation. Accordingly, BAP treatment results in increased PpIAA gene expression, suggesting that this aspect of the auxin/cytokinins crosstalk, also found in Angiosperms, is ancient.

The moss PpRR10, a direct target of cytokinin signaling, is repressed both in the degron-less PpIAA nar mutants and in WT when treated with NAA. Hence, this suggests that in P. patens auxin regulates ARR expression in a PpIAAindependent way [START_REF] Prigge | Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway[END_REF]. On the other hand, Thelander et al. suggested that auxin might sensitize future bud-forming caulonema cells to cytokinins, thereby easing bud initiation [START_REF] Thelander | Auxin-mediated developmental control in the moss Physcomitrella patens[END_REF]. Therefore, auxin would act earlier than cytokinins. One notable evidence for that is that the PpAPB1-4 transcription factors that are required for bud initiation are transcriptionally induced by auxin and not by cytokinins [START_REF] Aoyama | AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens[END_REF].

Puzzlingly, auxin and cytokinins then have opposite effects in gametophores branching [START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF]. Auxin is also linked to ethylene, although this was only reported once and not further investigated. In facts, both ACC contents and ethylene formation are promoted by exogenous IAA [START_REF] Rohwer | Ethylene Synthesis in Moss Protonema[END_REF].

Both cytokinins and CLE peptides are potent regulators of cell division and cell differentiation. In 2019, Cammarata et al. described numerous contexts where CLE and cytokinin signaling converge on the same developmental processes in P. patens. CLE signaling appears to inhibit the effects of cytokinins at different levels: bud production, regulation of apical meristem identity in gametophores, and cell divisions in phyllids. However, the level(s) at which this inhibition takes place remain(s) to be identified. Still, it is interesting to note that such interplays between cytokinins and CLEs have also been reported in Angiosperms, suggesting these processes are ancestral [START_REF] Cammarata | Cytokinin and CLE signaling are highly intertwined developmental regulators across tissues and species[END_REF].

Phytohormonal crosstalks involving stress hormones:

In Funaria hygrometrica, cytokinin-mediated bud induction can be inhibited by ABA, in a concentrationdependent manner [START_REF] Christianson | ABA prevents the second cytokinin-mediated event during the induction of shoot buds in the moss Funaria hygrometrica[END_REF]. More precisely, ABA does not interfere with the initial perception of cytokinin but rather acts by blocking the second step of cytokinins action on bud differentiation, that is the stable commitment to "bud state". Another level of ABA/cytokinins interplay was evidenced later in P. patens. Hyoung and colleagues found that the cytokinin inactivation gene PpCKX1 holds a positive role in resistance to dehydration and salt stress, linked to a higher expression of ABA signaling genes such as PpABI1A and B [START_REF] Hyoung | Cytokinin oxidase PpCKX1 plays regulatory roles in development and enhances dehydration and salt tolerance in Physcomitrella patens[END_REF]. Thus, cytokinins interact with ABA signaling and likely have an opposite effect on resistance to abiotic stress. ABA signaling is also linked to ethylene signaling, primarily at the level of their shared kinase: PpCTR1L/PpARK. The Ppctr1l/Ppark loss of function mutant is completely insensitive to ABA and displays a constitutive response to ethylene, revealing that it plays antagonistic roles in the two pathways [START_REF] Yasumura | An ancestral role for CONSTITUTIVE TRIPLE RESPONSE1 proteins in both ethylene and abscisic acid signaling[END_REF]. That ancestral dual function for CTR1-like proteins in mediating ABA signaling has likely been lost in the Angiosperms lineage, as it has not been reported in these plants.

In seed plants, ABA and gibberellins have a key antagonistic effect in regulating seed germination. In P. patens, exogenously applied ent-kaurene similarly reverses the inhibitory effect of ABA on spore germination, however the effects of both hormones are less potent than in seed germination [START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF]. Indeed, all ABA biosynthesis genes are expressed in dry spores and then at lower levels during imbibition, whereas the ent-kaurene biosynthesis gene PpCPS/KS is expressed only after imbibition, during germination and growth. Likewise, putative ABA signaling genes are for most expressed at higher levels in dry spores than imbibed spores, which is puzzlingly also the case for putative GA/ent-kaurene receptors [START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF].

Crosstalks between light signaling and phytohormones signaling:

A chloronema cell has four possible developmental fates: 1) divide and give rise to new chloronema cells; 2) differentiate into a caulonema cell; 3) give rise to a bud; 4) differentiate into brachycytes and tmema cells [START_REF] Decker | Moss systems biology en route: Phytohormones in Physcomitrella development[END_REF]. Naturally, the energy status has a great impact on the development of the plant in P. patens. This has perhaps been exemplified by the study of the hexokinase mutant Pphkx1 [START_REF] Olsson | A Novel Type of Chloroplast Stromal Hexokinase Is the Major Glucose-phosphorylating Enzyme in the Moss Physcomitrella patens[END_REF]. Glucose produced through photosynthesis is a vital raw material for most of the metabolic pathways and biomass accumulation in plants. However, glucose needs to be phosphorylated via a hexokinase activity before it can enter primary carbon metabolism. PpHXK1 encodes the major, plastid-located, hexokinase in P. patens [START_REF] Olsson | A Novel Type of Chloroplast Stromal Hexokinase Is the Major Glucose-phosphorylating Enzyme in the Moss Physcomitrella patens[END_REF]. In WT, glucose treatment induces caulonema formation, showing there is indeed a link between developmental regulation and the energy supply.

Thelander and colleagues showed that, as expected, Pphkx1 has a reduced growth rate and displays a lack of caulonema differentiation, even when glucose is provided [START_REF] Thelander | Effect of the energy supply on filamentous growth and development in Physcomitrella patens[END_REF]. More surprisingly, cytokinin mediated induction of chloronema branching is completely lost in the Pphxk1 mutant, raising the possibility that cytokinin acts to relieve a PpHXK1-dependent inhibition of chloronema branching. This is further supported by the fact that Pphxk1 develop much more buds (even ectopically on primary chloronema) than the cytokinin-treated WT, and that cytokinins do not further increase bud formation in Pphxk1. Therefore, cytokinins and hexokinase affect bud formation through a common mechanism, with HXK1 acting downstream [START_REF] Thelander | Effect of the energy supply on filamentous growth and development in Physcomitrella patens[END_REF]. Interestingly, the inhibitory effect of auxin on bud development is apparently epistatic to the effect of the Pphxk1 mutation, which could mean that the auxin-controlled step in bud formation is downstream of the step affected by hexokinase and cytokinins [START_REF] Thelander | Effect of the energy supply on filamentous growth and development in Physcomitrella patens[END_REF]. Carbon status is also directly regulated by light and, as discussed therebefore, most phytohormones have been linked to light in a way or another. Partly because of this, response(s) to light is (are) a major crosstalk node involving several hormones in plants. For instance, intact auxin and ent-kaurenoic acid signaling are both necessary for red-light induced caulonema transition [START_REF] Hayashi | Endogenous diterpenes derived from entkaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens[END_REF]. Indeed, ent-kaurenoic acid deficient mutants have a deficient caulonema differentiation phenotype in red light, rescued by ent-kaurenoic acid. In white light also, the ent-kaurene deficient Ppks mutant is less responsive to NAA than WT and its response is restored when co-treated with ent-kaurenoic acid or ent-kaurene.

Moreover, response to blue light is also disturbed in ent-kaurenoic acid deficient mutants: while WT P. patens displays an avoidance phenotype to unilateral blue light, both the Ppks and the Ppko mutants do not, unless exogenous entkaurenoic acid is provided [START_REF] Miyazaki | Hormonal diterpenoids derived from ent -kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens[END_REF]. PpKS gene expression itself is induced by blue light, suggesting GAlike levels might be increased in blue light [START_REF] Miyazaki | Blue-light irradiation upregulates the ent-kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens[END_REF][START_REF] Miyazaki | Hormonal diterpenoids derived from ent -kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens[END_REF]. Thus, ent-kaurenoic acid (or active derived compound(s)) in P. patens are involved in the well-known crosstalk between light and auxin. Blue light sensed via the cryptochrome PpCRY1/b induces side branch formation in the protonema, induces leaf growth and represses stem elongation [START_REF] Imaizumi | Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens[END_REF]. Also, since Ppcry mutants develop gametophores earlier than the WT, blue light signalled through cryptochromes light likely inhibits the transition to bud, on the opposite of red light. On the other hand, red and blue light both induce caulonema formation in WT, and NAA further increases caulonema formation under white light, red light, and blue light [START_REF] Imaizumi | Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens[END_REF]. Ppcry mutants being more sensitive to NAA except in red light, thus it seems that cryptochrome-mediated blue light signals inhibit auxin responses, whereas red light either induces or does not affect auxin responses [START_REF] Imaizumi | Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens[END_REF]. This inhibition of auxin response is at least partly dependent on IAAs: the PpIAA1 gene is induced by exogenous auxin in WT and constitutively over-expressed in the double Ppcry mutant, which accumulates PpIAA1 transcripts faster than in the WT under NAA treatment [START_REF] Imaizumi | Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens[END_REF]. Hence, cryptochromes in P. patens inhibit auxin signaling by interfering with the induction of auxin responsive genes.

In P. patens, cytokinin-induced bud induction requires light, but it has not been determined whether it is initial cell formation or bud assembly (or both) that is light-dependent. Buds cannot develop if the light intensity is too low, even when cytokinins are applied, and this probably depends mainly on the red component of white light. However, rather than light itself, it might be a component only formed in light in some species that is necessary and that might be sucrose, as suggested by Chopra and Gupta for Funaria hygrometrica [START_REF] Chopra | Dark-Induction of Buds in Funaria hygrometrica Hedw[END_REF]. Indeed, it has been shown for a closely related species to P. patens, Physcomitrium turbinatum, that there is a relatively large cumulative light dose required for bud formation, suggesting that light energy may be used for the synthesis of a product that must accumulate before buds form. Thus, sugars are the most logical candidates. In P. patens, further support for the accumulation of such a product was obtained: some buds are formed when dark-grown cultures are exposed to light for several hours and then simultaneously treated with cytokinin and returned to darkness, suggesting that P. patens somehow "remembers" the light signal (and/or cytokinins perpetuate the response to light) [START_REF] Schumaker | HORMONE-INDUCED SIGNALING DURING MOSS DEVELOPMENT[END_REF]. Interestingly, it was found recently that ABA can be added to the mix, as sucrose inhibits spore germination in a dose-dependent manner and acts synergistically with ABA [START_REF] Vesty | The decision to germinate is regulated by divergent molecular networks in spores and seeds[END_REF].

CONCLUSION

Appearance of phytohormones groups coincide with (or shortly precede) major transitions in plants evolutionary history and are therefore thought to be particularly relevant for adaptation to new environments and/or new developmental programs. Evolution of biosynthesis and signaling pathways relies on the assembling of pre-existent functions, which is eventually kept by natural selection when it grants a survival asset. These "building blocks", following a long process of co-evolution and possible events of duplication, neofunctionalization and such, finally make up coherent pathways, linking a given signal, synthesized and "freed" in appropriate conditions, to the most appropriate responses.

To study these evolutionary processes, in addition to the fossil records that is often fragmentary or even missing, comparison of plants from diverse lineages is necessary. Physcomitrium patens has long been a widely used model species for studies on hormones, thanks to the ease of its in vitro propagation and genetic transformation. It has more recently been joined by the liverwort Marchantia polymorpha, which also seems to be a promising model for this other bryophyte lineage. We can also note the multiplying of studies on Selaginella (Lycophyte), which will undoubtedly help in giving us more insight from this seedless vascular plants' lineage.

Recent reviews gathered much of the current knowledge about the state of phytohormones signaling pathways in various lineages of plants to give us a clearer view of this step-by-step acquisition of signaling components [START_REF] Bowman | Something ancient and something neofunctionalized-evolution of land plant hormone signaling pathways[END_REF][START_REF] Blázquez | Evolution of Plant Hormone Response Pathways[END_REF].

Chapter III -A short history of strigolactones III-A) Discovery of the first strigolactones and of their multiple roles in flowering plants

Strigolactones (SL) are a group of apocarotenoids that make up the tenth class of phytohormones, with over 20 molecules identified to date [START_REF] Xie | Structural diversity of strigolactones and their distribution in the plant kingdom[END_REF][START_REF] Wang | Structural diversity in the strigolactones[END_REF]. However, SL were first identified and studied not as phytohormones but as rhizosphere signals. As a matter of facts, strigol was identified in the 1960s as a potent inducer of germination for seeds of the parasitic plant Striga lutea, active and produced at hormonal concentrations by its host plant cotton [START_REF] Cook | Germination of Witchweed (Striga lutea Lour.): Isolation and Properties of a Potent Stimulant[END_REF]. The chemical structure of this compound was soon resolved [START_REF] Cook | Germination stimulants. II. Structure of strigol, a potent seed germination stimulant for witchweed (Striga lutea)[END_REF] and revealed a tricyclic lactone moiety (ABC tricycle) connected to another lactone ring (the methylbutenolide D ring) by an enol-ether bridge. This structure exemplifies the organization of canonical SL, in opposition to non-canonical SL which can bear various groups in place of the ABC moiety (Yoneyama et al., 2018b). However, the enol-ether bridge and D ring are constant features found in all SL [START_REF] Alder | The Path from β-Carotene to Carlactone, a Strigolactone-Like Plant Hormone[END_REF] (figure III-1). Canonical SL can be further divided, depending on the orientation of the C ring, into strigol-type (β orientation) and orobanchol-type (α orientation) (Yoneyama et al., 2018b) and Figure III-1, orobanchol being a germination stimulant for parasitic plants of the Orobanche family instead of Striga [START_REF] Müller | Germination stimulants produced by Vigna unguiculata Walp cv Saunders Upright[END_REF]. It was then demonstrated that SL were derived from carotenoids [START_REF] Matusova | The Strigolactone Germination Stimulants of the Plant-Parasitic Striga and Orobanche spp. Are Derived from the Carotenoid Pathway[END_REF]. The discovery of SL role in parasitism, highly disadvantageous for the producing plant, raised the question as to why SL biosynthesis and exudation into the rhizosphere had not been eliminated by natural selection.

A compound very similar to strigol, 5-deoxystrigol (5DS), was then identified as the sought-after "branching factor" inducing arbuscular mycorrhizal fungi (AMF) hyphal branching [START_REF] Buee | The Pre-Symbiotic Growth of Arbuscular Mycorrhizal Fungi Is Induced by a Branching Factor Partially Purified from Plant Root Exudates[END_REF][START_REF] Akiyama | Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi[END_REF]. As such, SL are particularly important for this symbiosis, since hyphal branching is recognized as the host recognition step [START_REF] Yoneyama | Strigolactones, host recognition signals for root parasitic plants and arbuscular mycorrhizal fungi, from Fabaceae plants[END_REF]. This effect of SL is explained by a rapid increase in AMF mitochondrial division and ATP production, in phylogenetically distant AMF species (Gigaspora rosea, Glomus intraradices and Glomus claroideum) [START_REF] Besserer | Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria[END_REF]. Nevertheless, the mitochondria is not the only cell compartment that is affected by SL in the fungi and SL likely have an even broader effect on fungal gene expression and metabolism [START_REF] Lanfranco | Strigolactones cross the kingdoms: plants, fungi, and bacteria in the arbuscular mycorrhizal symbiosis[END_REF].

Moreover, SL also have a positive impact on AMF growth earlier, by promoting AMF spores germination, probably by inducing remobilization of carbon stored in lipids [START_REF] Besserer | GR24, a Synthetic Analog of Strigolactones, Stimulates the Mitosis and Growth of the Arbuscular Mycorrhizal Fungus Gigaspora rosea by Boosting Its Energy Metabolism[END_REF][START_REF] Lanfranco | Strigolactones cross the kingdoms: plants, fungi, and bacteria in the arbuscular mycorrhizal symbiosis[END_REF]. SL also initiate the molecular dialogue between the host plant and the fungal partner [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca 2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF][START_REF] Bonfante | Arbuscular mycorrhizal dialogues: do you speak 'plantish' or 'fungish'?[END_REF].

However, the question as to how SL are perceived by these fungi is still open. A role of SL in the regulation of symbiosis between legumes and Rhizobia nitrogen-fixing bacteria has also been investigated [START_REF] Foo | The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway[END_REF][START_REF] Van Zeijl | The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis[END_REF][START_REF] De Cuyper | From lateral root density to nodule number, the strigolactone analogue GR24 shapes the root architecture of Medicago truncatula[END_REF][START_REF] López-Ráez | Strigolactones in Plant Interactions with Beneficial and Detrimental Organisms: The Yin and Yang[END_REF][START_REF] Mcadam | Determining the Site of Action of Strigolactones during Nodulation[END_REF], with most studies concluding that SL have an early positive effect on nodulation, by inducing infection thread formation from the bacterial partner end [START_REF] Mcadam | Determining the Site of Action of Strigolactones during Nodulation[END_REF]. Also, SL biosynthesis in the host plant seems to be induced by its perception of the bacterial Nod factors [START_REF] Mcadam | Determining the Site of Action of Strigolactones during Nodulation[END_REF]. These roles in symbiosis are highly beneficial to plant growth, and probably outweigh the negative impact of SL-mediated promotion of parasitism in most cases. Hence, the conservation of SL production and exudation along land plants evolution would have been permitted by the evolutive advantage granted by enhanced symbiosis ability. SL. The stereochemistry of ent5DS and ent4DO enantiomers of the artificial SL GR24 is hence said to be non-natural (red inset with dotted outlines). GR24 enantiomers are usually named after the natural SL to which they are the most similar (first notation, similarity is illustrated by the green background). For convenience, GR24 enantiomers will consistently be referred to using the second notation (e.g. (+)-GR24 and so on) in the following chapters.

SL first uncovered hormonal role as repressors of axillary shoot branching was established later, simultaneously by two teams [START_REF] Gomez-Roldan | Strigolactone inhibition of shoot branching[END_REF][START_REF] Umehara | Inhibition of shoot branching by new terpenoid plant hormones[END_REF]. This finding was permitted by the identification and characterization of hyper-branched mutants in rice [START_REF] Umehara | Inhibition of shoot branching by new terpenoid plant hormones[END_REF], pea [START_REF] Gomez-Roldan | Strigolactone inhibition of shoot branching[END_REF] and Arabidopsis [START_REF] Gomez-Roldan | Strigolactone inhibition of shoot branching[END_REF][START_REF] Umehara | Inhibition of shoot branching by new terpenoid plant hormones[END_REF]. These mutants were subsequently shown to be affected either in SL biosynthesis or signaling. Interestingly, the existence of a plant hormone regulating shoot branching along auxin and cytokinins had been suspected for a long time and previous identification of mutants in genes encoding carotenoid cleavage dioxygenases had shown that this signal was derived from carotenoids (reviewed by Ongaro and Leyser [START_REF] Ongaro | Hormonal control of shoot branching[END_REF]). Over the years, more developmental roles have been attributed to SL, for some together with other phytohormones (reviewed in [START_REF] Brewer | Diverse roles of strigolactones in plant development[END_REF][START_REF] Al-Babili | Strigolactones, a Novel Carotenoid-Derived Plant Hormone[END_REF][START_REF] Lopez-Obando | Strigolactone biosynthesis and signaling in plant development[END_REF][START_REF] Yang | Comparing and Contrasting the Multiple Roles of Butenolide Plant Growth Regulators: Strigolactones and Karrikins in Plant Development and Adaptation to Abiotic Stresses[END_REF]. Notably, SL induce internode elongation and secondary growth of stems, regulate root architecture depending on the plant phosphate nutritional status, promote senescence in leaves and repress hypocotyl elongation in seedlings. Taken together, these findings suggest that SL generally fine-tune growth and morphogenesis of plants according to the availability of mineral nutrients in the soil, notably inorganic phosphate and nitrogen [START_REF] Marzec | Regulation of Root Development and Architecture by Strigolactones under Optimal and Nutrient Deficiency Conditions[END_REF]. Furthermore, several other studies point to SL as defense phytohormones against both biotic [START_REF] Torres-Vera | Do strigolactones contribute to plant defence?[END_REF][START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF][START_REF] Xu | Strigolactones positively regulate defense against root-knot nematodes in tomato[END_REF][START_REF] Nasir | Strigolactones positively regulate defense against Magnaporthe oryzae in rice (Oryza sativa)[END_REF]Li et al., 2020b) and abiotic stresses [START_REF] Pandey | Emerging Roles of Strigolactones in Plant Responses to Stress and Development[END_REF][START_REF] Shirani Bidabadi | Strigolactone and Methyl Jasmonate-Induced Antioxidant Defense and the Composition Alterations of Different Active Compounds in Dracocephalum kotschyi Boiss Under Drought Stress[END_REF][START_REF] Saeed | Strigolactones Biosynthesis and Their Role in Abiotic Stress Resilience in Plants: A Critical Review[END_REF].

III-B) Strigolactones biosynthesis and signaling in flowering plants

SL biosynthesis starts with transformation of all-trans-β-carotene into carlactone (CL, see its structure in figure III-1) in plastids, via the successive action of the DWARF 27 (D27) isomerase and of CAROTENOID CLEAVAGE DIOXYGENASE 7 and 8 (CCD7 and CCD8) [START_REF] Sorefan | MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea[END_REF][START_REF] Booker | MAX3/CCD7 Is a Carotenoid Cleavage Dioxygenase Required for the Synthesis of a Novel Plant Signaling Molecule[END_REF][START_REF] Alder | The Path from β-Carotene to Carlactone, a Strigolactone-Like Plant Hormone[END_REF]. CL, the precursor of all active natural SL identified so far [START_REF] Seto | Carlactone is an endogenous biosynthetic precursor for strigolactones[END_REF], then moves to the cytosol where it is transformed into SL by CYP450 enzymes from the CYP711A/MORE AXILLARY BRANCHING 1 (MAX1) family. This last step(s) are less well known and appear to vary between species, the different MAX1 enzymes likely having very specialized reaction and substrate specificities [START_REF] Challis | A Role for MORE AXILLARY GROWTH1 (MAX1) in Evolutionary Diversity in Strigolactone Signaling Upstream of MAX2[END_REF][START_REF] Abe | Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro[END_REF][START_REF] Zhang | Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis[END_REF][START_REF] Flematti | Stereospecificity in strigolactone biosynthesis and perception[END_REF]Yoneyama et al., 2018a;[START_REF] Iseki | Evidence for species-dependent biosynthetic pathways for converting carlactone to strigolactones in plants[END_REF][START_REF] Zhang | The tomato MAX1 homolog, SlMAX1 , is involved in the biosynthesis of tomato strigolactones from carlactone[END_REF]. Other types of enzymes have also been identified as SL biosynthesis enzymes but have only been found in specific plants. This is notably the case for the 2-oxoglutarate and Fe(II)-dependent dioxygenases LATERAL BRANCHING OXYDOREDUCTASE (LBO) in Arabidopsis [START_REF] Brewer | LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis[END_REF] and LOTUS-LACTONE DEFECTIVE (LLD) in Lotus japonicus [START_REF] Mori | Identification of two oxygenase genes involved in the respective biosynthetic pathways of canonical and non-canonical strigolactones in Lotus japonicus[END_REF], as well as for LOW GERMINATION STIMULANT 1 (LGS1), a sulfotransferase found in sorghum [START_REF] Gobena | Mutation in sorghum LOW GERMINATION STIMULANT 1 alters strigolactones and causes Striga resistance[END_REF]. More recently, another family of CYP450 enzymes, CYP722C, was found to be involved in the generation of canonical SL in cowpea [START_REF] Wakabayashi | Direct conversion of carlactonoic acid to orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis[END_REF] and cotton [START_REF] Wakabayashi | CYP722C from Gossypium arboreum catalyzes the conversion of carlactonoic acid to 5-deoxystrigol[END_REF]. SL signaling has been extensively investigated in flowering plants (Arabidopsis, but also pea, petunia and rice).

In these species, SL are perceived in the cytosol by DWARF 14 (D14), a peculiar receptor that has a catalytic activity as an α/β-hydrolase [START_REF] Arite | D14, a strigolactone-Insensitive mutant of rice, shows an accelerated outgrowth of tillers[END_REF][START_REF] Yao | DWARF14 is a non-canonical hormone receptor for strigolactone[END_REF][START_REF] De Saint Germain | An histidine covalent receptor and butenolide complex mediates strigolactone perception[END_REF]Yao et al., 2018b). Using this catalytic activity, D14 can cleave its SL ligand, generating a covalently linked D-ring to the histidine residue of the catalytic triad of D14, also called CLIM (for covalently linked intermediate molecule). Moreover, D14 itself adopts a destabilized conformation following its interaction with SL and is eventually degraded by the 26S proteasome [START_REF] Hu | DWARF14, A Receptor Covalently Linked with the Active Form of Strigolactones, Undergoes Strigolactone-Dependent Degradation in Rice[END_REF]. Before its degradation, the conformational change of D14 enables it to interact in the nucleus with the F-box protein MORE AXILLARY BRANCHING 2 (MAX2) and with proteins of the SUPPRESSOR OF MAX2 1 LIKE (SMAX1-like or SMXL) family [START_REF] Yao | DWARF14 is a non-canonical hormone receptor for strigolactone[END_REF]. This multipartite interaction triggers the formation of a SCF MAX2 E3 ubiquitin ligase complex that directs SMXL proteins towards proteasomal degradation . SMXL can thus be seen as repressors of SL responses, which must be removed for the SL signal to be transduced [START_REF] Jiang | DWARF 53 acts as a repressor of strigolactone signalling in rice[END_REF][START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF][START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Wang | Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation[END_REF]). An ongoing debate about SL signaling is about which state of the D14-SL interaction, either before or after cleavage of the SL and CLIM formation, is the one able to transduce the signal [START_REF] De Saint Germain | An histidine covalent receptor and butenolide complex mediates strigolactone perception[END_REF][START_REF] Yao | DWARF14 is a non-canonical hormone receptor for strigolactone[END_REF][START_REF] Carlsson | The elusive ligand complexes of the DWARF14 strigolactone receptor[END_REF][START_REF] Marzec | Binding or Hydrolysis? How Does the Strigolactone Receptor Work?[END_REF]. Even though both states seem able to bind MAX2 and SMXLs, the outcome of these interaction patterns are likely different [START_REF] Shabek | Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling[END_REF]. However, SMXL molecular function(s) are not well understood yet, even more so at the molecular level (more details are given in chapters V, VI and VII). Eventually, SMXL removal has major effects on gene expression (Wang et al., 2020b), notably lifting the transcriptional block on BRC1 [START_REF] Braun | The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching[END_REF], a TCP transcription factor acting as a major repressor of axillary branching. Nonetheless, some effects of SL signaling are transcription independent, such as the relocation of PINs auxin efflux transporters, which additionally does not require de novo protein synthesis [START_REF] Shinohara | Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane[END_REF][START_REF] Kumar | Arabidopsis response to low-phosphate conditions includes active changes in actin filaments and PIN2 polarization and is dependent on strigolactone signalling[END_REF]. This rapid effect also has a negative effect on branching, since it prevents exportation of auxin out of axillary buds, blocking them in a dormant state. Both transcription-dependent and transcription-independent processes play together in SL response, probably at variable levels depending on the mechanism. Notably, it seems that SL effect on branching relies mainly on the former, whereas SL regulation of stem secondary growth is more dependent on the latter [START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF].

III-C) The mystery of the doppelgänger KL pathway

Simultaneously to the progressive elucidation of the SL signaling pathway, evidence for another pathway having multiple levels of convergence with the former emerged. In this second pathway, the same MAX2 F-box protein and close homologs of D14 (KAI2, also an α/β-hydrolase) and SMXL proteins (SMAX1/SMXL2) are respectively acting as receptor and negative regulator [START_REF] Scaffidi | Carlactoneindependent seedling morphogenesis in Arabidopsis[END_REF][START_REF] Waters | Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis[END_REF]. On another note, SMAX1 has been identified in Arabidopsis in a suppressor screen in max2 [START_REF] Stanga | SUPPRESSOR OF MORE AXILLARY GROWTH2 1 Controls Seed Germination and Seedling Development in Arabidopsis[END_REF], the same year as the SL pathway SMXL D53 in rice [START_REF] Jiang | DWARF 53 acts as a repressor of strigolactone signalling in rice[END_REF][START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF]. However, the smax1 mutation only suppresses the max2 phenotypes that are not associated with SL signaling, that is germination and seedling photomorphogenesis [START_REF] Stanga | SUPPRESSOR OF MORE AXILLARY GROWTH2 1 Controls Seed Germination and Seedling Development in Arabidopsis[END_REF].

The KAI2-MAX2-SMAX1 pathway enables response to karrikins (KAR), exogenous butenolides found in smoke generated from burning vegetation (figure III-3). In fire-following species, these compounds trigger rapid seed germination, even when other positive environmental signals are lacking (especially light). In regards to that, it is interesting to note that the KAI2 (KARRIKIN INSENSITIVE 2) gene had first been characterized as HTL (HYPOSENSITIVE TO LIGHT) [START_REF] Sun | HYPOSENSITIVE TO LIGHT , an Alpha / Beta Fold Protein , Acts Downstream of ELONGATED HYPOCOTYL 5 to Regulate Seedling De-Etiolation[END_REF].

Figure III-3 -Structure of compounds perceived through the KAI2-MAX2 pathway

The f-box protein MAX2 itself had been independently identified as KAI1 (KARRIKIN INSENSITIVE 1) and its dual role in SL and KAR pathways has been shown in Arabidopsis already 10 years ago [START_REF] Nelson | F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana[END_REF]. Interestingly, MAX2 had been identified in earlier screens as a gene regulating senescence (as ORE9) [START_REF] Woo | ORE9, an F-Box Protein That Regulates Leaf Senescence in Arabidopsis[END_REF] and photomorphogenesis (as PPS) [START_REF] Shen | The F-Box Protein MAX2 Functions as a Positive Regulator of Photomorphogenesis in Arabidopsis[END_REF]. Recognition that kai2 and max2 loss-of-function mutants have major developmental defects, together with the observation that these genes exist and have similar developmental functions in plants that are not fire-following, or even insensitive to KAR, pointed to this pathway likely recognizing an endogenous molecule [START_REF] Flematti | Karrikin and cyanohydrin smoke signals provide clues to new endogenous plant signaling compounds[END_REF][START_REF] Conn | Evidence that KARRIKIN-INSENSITIVE2 (KAI2) Receptors may Perceive an Unknown Signal that is not Karrikin or Strigolactone[END_REF]. Still, this endogenous KAI2 ligand (KL) has not been identified yet, even though several studies sought to clarify its nature [START_REF] Scaffidi | Carlactoneindependent seedling morphogenesis in Arabidopsis[END_REF][START_REF] Conn | Evidence that KARRIKIN-INSENSITIVE2 (KAI2) Receptors may Perceive an Unknown Signal that is not Karrikin or Strigolactone[END_REF]. Some divergences between the two pathways exist: for instance, enzymatic activity of KAI2 does not seem to be necessary for KL signaling (Yao et al., 2018a;[START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF]. But they work similarly in the way that SMAX1 is degraded in a MAX2-dependent manner after its interaction with KAR-activated KAI2 [START_REF] Khosla | Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2[END_REF][START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF]. Extensive use of an unspecific racemic mixture of the synthetic SL analogue GR24 (racGR24), as well as studies relying only on the max2 loss-of-function mutants, have made attribution of diverse physiological functions to either SL or KL signaling challenging [START_REF] De Cuyper | Strigolactones, karrikins and beyond[END_REF]. Studies interested in the evolution of these twin pathways seem to converge on the KL pathway (KAI2-MAX2-SMAX1, figure III-4) being ancestral relative to the SL pathway Douglas et al., 2017;[START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. This particular point on evolution is further discussed hereafter in chapters V and VIII. Another ongoing investigation is how MAX2 can differentiate between SL and KL signals to target different SMXL and whether it relies only on which receptor (KAI2 or D14) is activated at a given time (given possible co-expression of the D14, KAI2, MAX2, SMAX1 and SMXL7 genes in some tissues, according to the Arabidopsis eFP Browser expression atlas). Also, if them both being activated at the same time is possible, does MAX2 « prioritize » one signal over the other?

(D14-MAX2-SMXL, Figure III-2) (Bythell-

III-D) Strigolactones biosynthesis and signaling outside of flowering plants

All extant land plants explored so far, including those that are hosts neither for parasitic plants nor for AMF, possess the basic genetic toolkit for SL biosynthesis until carlactone production (thus until CCD8 function) [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF][START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Moreover, the loss of MAX1 and LBO close homologs seems specific to P. patens lineage and is not even a common feature of mosses, which could imply that most bryophytes are able to synthesize SL downstream of carlactone using enzymes of the MAX1 and LBO families [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. This conservation of "CCD8-derived compounds" biosynthesis also infers that such non-host plant species either use these compounds in other types of interspecific communications, or that these compounds are "restrained" to their phytohormonal functions. This second hypothesis is especially supported by the current view that canonical SL act mainly in the rhizosphere as interspecific signals, whereas non-canonical SL are the actual phytohormones (Yoneyama et al., 2018b). Answering this question is further complicated by the extreme difficulty to identify SL molecules, as they are produced in very low amounts and furthermore highly unstable in aqueous solutions. Broadened to ancestral land plants, these interrogations are aiming to determine what was the first role of SL: an interspecific signal acting in the environment or an endogenous growth-regulating signal?

According to a recent study [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF], the moss P. patens can produce several molecules from PpCCD7 and PpCCD8 activity. When expressed in vitro and fed with all trans-β-carotene, PpCCD7 is indeed able to synthesize the 9-cis-beta-apo-10'-carotenal SL precursor from 9-cis-β-carotene. But, PpCCD7 is also able to cleave other types of carotenoids: it can convert both 9-cis-zeaxanthin and 9'-cis-cryptoxanthin into 9-cis-3-OH-β-apo-10'-carotenal, 9-cis-lutein into 9-cis-3-OH-α-apo-10'-carotenal, and 9-cis-cryptoxanthin into 9-cis-3-β-apo-10'-carotenal.

However, the biological relevance of these other apocarotenoids, also produced by CCD7 in several angiosperms, remains to be investigated. In vitro, PpCCD8 can produce carlactone from 9-cis-beta-apo-10'-carotenal, thus it is a functional carlactone synthase. PpCCD8 can also transform all-trans-β-apo-10'-carotenal into β-apo-13-carotenone (better known as d'orenone). Whether these molecules are all produced in planta is unknown, in addition to their function in most plant species, especially P. patens. Nevertheless, findings presented in chapter IV do suggest that P. patens synthesizes non-canonical SL from PpCCD7 and PpCCD8 conjoined action, putatively after the isomerization of all-trans-β-carotene by a PpD27-like (PpD27L) homolog. P. patens possesses five PpD27L genes (table III-1).

Following the consideration that genes involved in the same pathway are usually co-expressed, comparison of expression profiles suggests that PpD27L2 is most probably acting upstream of PpCCD7 and PpCCD8. Indeed, these three genes are collectively most highly expressed in rhizoids whereas their transcript levels are very low in sporophytes and spores (figure III-5). However, if we consider that the most promising candidate is the one subjected to negative feedback by PpCCD8-derived compounds, as D27 homologs from Angiosperms (Waters et al., 2012a) then PpD27L1 might be the best candidate (our unpublished data).

Genes homologous to aforementioned constituents of the SL signaling toolkit are found in all land plants examined so far [START_REF] Knack | Microbiomes of Streptophyte Algae and Bryophytes Suggest That a Functional Suite of Microbiota Fostered Plant Colonization of Land[END_REF]Wang et al., 2015a;[START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF] (table III-1). Still, some of these homologs are very distant, so them having a similar function to their characterized angiosperms' counterparts is far from being obvious. This is particularly the case for the putative SL receptors, and this issue is further complexified by the existence of a highly similar pathway in response to another putative class of phytohormones. Globally, the mechanisms underlying SL perception and signal transduction outside of flowering plants are still very unclear. The results presented herein in chapters IV and VI refine current knowledge about these processes in the model moss species P. patens, as well as signaling associated with the elusive KL. [START_REF] Waters | Strigolactone Signaling and Evolution[END_REF]. SLs exuded from plant roots can also act in the rhizosphere as a signal molecule inducing the germination of seeds of parasitic plants [START_REF] Cook | Germination of Witchweed (Striga lutea Lour.): Isolation and Properties of a Potent Stimulant[END_REF][START_REF] Delavault | Communication Between Host Plants and Parasitic Plants[END_REF]. SLs have been found in most land plants, including Bryophytes, Lycophytes, Gymnosperms, and Angiosperms (Yoneyama et al., 2018b). However, their synthesis and signaling pathways are mainly described in the latter, where a core pathway of enzymes among which two CAROTENOID CLEAVAGE DEOXYGENASE (CCD7 and CCD8) convert carotenoids into carlactone (CL), the reported precursor of all known SLs. CL is the substrate for further enzymes as the CYTOCHROME-P450 MORE AXILLARY GROWTH1 (MAX1) (for review: [START_REF] Alder | The Path from β-Carotene to Carlactone, a Strigolactone-Like Plant Hormone[END_REF][START_REF] Al-Babili | Strigolactones, a Novel Carotenoid-Derived Plant Hormone[END_REF]. Depending on plant species, CL is modified into canonical or non-canonical SLs, that differ in the structure attached to the conserved enol ether-D ring moiety, shared by all the SLs, and essential for biological activity (Yoneyama et al., 2018b;[START_REF] Yoneyama | Recent progress in the chemistry and biochemistry of strigolactones[END_REF]. In Angiosperms, SLs are perceived by an α/β hydrolase DWARF14 (D14)/DECREASED APICAL DOMINANCE2 (DAD2) that, in interaction with an F-box protein MORE AXILLARY GROWTH2 (MAX2), targets repressor proteins for proteasome degradation [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Wang | Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation[END_REF]. SLs perception originality is that the D14 protein is both a receptor and an enzyme that cleaves its substrate (and covalently binds part of the SL), in a mechanism that is still debated [START_REF] Yao | DWARF14 is a non-canonical hormone receptor for strigolactone[END_REF][START_REF] De Saint Germain | An histidine covalent receptor and butenolide complex mediates strigolactone perception[END_REF][START_REF] Shabek | Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling[END_REF][START_REF] Seto | Strigolactone perception and deactivation by a hydrolase receptor DWARF14[END_REF] for review: Bürger and Chory, 2020).

The evolutive origins of SLs, and in particular the identification of their primary role, as hormones or rhizospheric signals are still elusive. SLs identification and quantification are challenging in many species, due to very low amounts of the molecules present in plants and its exudates, and their high structure diversification [START_REF] Xie | Structural diversity of strigolactones and their distribution in the plant kingdom[END_REF]Yoneyama et al., 2018b). Therefore, occurrence of SLs in a species often relies on the presence of the core biosynthesis enzymes in its genome [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF][START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Besides, it has been recently proposed that SLs are only produced in land plants [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. As for perception, evidence of an ancestral pathway came from the finding during the screening of Arabidopsis mutants of an ancient paralog of D14, named KARRIKIN INSENSTIVE2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) [START_REF] Nelson | F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana[END_REF][START_REF] Waters | Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis[END_REF]. KAI2 is an α/β hydrolase as D14, that also interacts with the MAX2 F-box protein, in a pathway regulating Arabidopsis seed germination and seedling development. However, the endogenous signal perceived through this pathway remains unknown and is reported thus far as KAI2-Ligand (KL) [START_REF] Conn | Evidence that KARRIKIN-INSENSITIVE2 (KAI2) Receptors may Perceive an Unknown Signal that is not Karrikin or Strigolactone[END_REF]. KAI2 is also involved in stress tolerance, drought tolerance and AM symbiosis [START_REF] Gutjahr | Rice perception of symbiotic arbuscular mycorrhizal fungi requires the karrikin receptor complex[END_REF]Wang et al., 2018;[START_REF] Villaécija-Aguilar | SMAX1/SMXL2 regulate root and root hair development downstream of KAI2mediated signalling in Arabidopsis[END_REF][START_REF] Li | Strigolactone signaling regulates specialized metabolism in tobacco stems and interactions with stem-feeding herbivores[END_REF].

To gain insight into SLs signaling evolution, we focused our investigations on a model for earliest land plants,

namely Physcomitrium (Physcomitrella) patens (P. patens). As a moss, P. patens belongs to bryophytes, which also include two other clades, hornworts and liverworts [START_REF] Bowman | Something ancient and something neofunctionalized-evolution of land plant hormone signaling pathways[END_REF]. Bryophytes are currently assigned as a monophyletic group of embryophytes sharing an ancestor with vascular plants [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF][START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF].

Extant bryophytes are therefore considered as the descendants of the first plants which became able to survive out of water, and conquer land, 450 million years ago [START_REF] Bowman | Something ancient and something neofunctionalized-evolution of land plant hormone signaling pathways[END_REF][START_REF] Blázquez | Evolution of Plant Hormone Response Pathways[END_REF][START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF]. In P.

patens, both CAROTENOID CLEAVAGE DEOXYGENASE7 and 8 (PpCCD7 and PpCCD8) enzymes required for SL synthesis are found (Proust et al., 2011), and carlactone has been detected as the product of PpCCD8 [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. The extended phenotype of the Ppccd8 mutant plants indicates that PpCCD8-derived molecules are required for moss filament growth regulation, and more broadly for interaction with neighboring plants (Proust et al., 2011).

Application of the artificial SL (±)-GR24 does complement the Ppccd8 mutant phenotype (Proust et al., 2011). PpCCD8derived molecules likely play also a role in rhizoid elongation and gametophore shoot branching [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF][START_REF] Coudert | Three ancient hormonal cues co-ordinate shoot branching in a moss[END_REF]. The exact nature of PpCCD8-derived molecules is still elusive (Yoneyama et al., 2018b) and the absence of MAX1 homologs in P. patens suggests that the synthesis pathway in moss may differ from that in vascular plants. However, phylogenetic analysis of MAX1 homologs highlights the presence of this gene in other mosses and suggest a conservation of the biosynthesis pathway in land plants [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Contrary to its flowering plants homolog, we previously showed that PpMAX2 is not involved in the perception of PpCCD8-derived molecules, as the corresponding mutant does respond to (±)-GR24 [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. The PpMAX2 F-box protein is rather involved in a light dependent pathway required for moss early development and gametophore development regulation published the crystal structure of PpKAI2L-C, -E and -H and showed that in vitro purified PpKAI2L proteins -C, -D

and -E bind (-)-5-deoxystrigol, a canonical SL with non-natural stereochemistry, while PpKAI2L proteins -H, -K and -L could adapt the karrikin KAR1 [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF]. However, proteins from the (J,G,M) clade were not studied, and no evidence for a role of one (or several) PpKAI2L as receptor for CCD8-derived molecules was brought, nor in vivo (in moss) experiments validating the results.

Several questions remain to understand the evolution of the SL signaling pathway, and we address the following two in this paper: what is the nature of PpCCD8-derived molecules in moss? What are the receptors for these compounds? Answering these questions would be facilitated if one could mimic these molecules in assays. So far, the racemic (±)-GR24 has been used as an analog to SLs, but recent works indicate that the different enantiomers present in this synthetic mixture do not have the same effect [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF]. Indeed, (+)-GR24 (also called GR24 5DS ), with a configuration close to natural strigol is mostly perceived by D14 and mimics CCD8-derived SLs (e.g. carlactone), while (-)-GR24 (also called GR24 ent5DS ) has a configuration that so far has not been found in natural SLs. However, the (-)-GR24 analog is better perceived by KAI2 than by D14 proteins and has been described as a KL mimic [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF][START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF].

Here we tested the activity of moss as a stimulant for P. ramosa germination to shed light on the chemical nature of PpCCD8-derived compounds. We characterized the expression patterns of all 13 PpKAI2L genes in moss, at various developmental stages. Refining the findings of [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF], we assessed the biochemical activity of PpKAI2L

proteins in vitro by testing their cleavage activity and binding towards both GR24 enantiomers. We expressed some of these proteins in Arabidopsis d14-1 kai2-2 double mutant to question the conservation of the SL and/or KL perception function. Finally, we used CRISPR-Cas9 technology to isolate moss mutants amongst the four clades of PpKAI2L genes and analyzed their phenotype and their response to both GR24 enantiomers. We propose that clade (A-E) PpKAI2L

proteins could be moss KL receptors, while clade (J,G,M) PpKAI2L would function as PpCCD8-derived compounds receptors.

52

Results

PpCCD8-derived compounds induce the germination of a hemp-specific population of Phelipanche ramosa

Although [START_REF] Yoneyama | Which are the major players, canonical or non-canonical strigolactones?[END_REF] noted that the PpCCD8-derived molecules, previously reported in moss tissues by Proust et al. (2011), were likely contaminants, it is still a valuable hypothesis that P. patens does synthesize SL like products. Indeed, the synthetic SL analog (±)-GR24 or carlactone do complement the Ppccd8 phenotype (Proust et al., 2011;[START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. Unfortunately, quantification of SL and related compounds is still a challenge in many species [START_REF] Boutet-Mercey | Validated Method for Strigolactone Quantification by Ultra High-Performance Liquid Chromatography -Electrospray Ionisation Tandem Mass Spectrometry Using Novel Deuterium Labelled Standards[END_REF]Yoneyama et al., 2018b;[START_REF] Rial | A new UHPLC-MS/MS method for the direct determination of strigolactones in root exudates and extracts[END_REF][START_REF] Floková | An improved strategy to analyse strigolactones in complex sample matrices using UHPLC-MS/MS[END_REF]. An alternative to evaluate the SL-like activity of PpCCD8-derived molecules is to test the effect of moss exudates as stimulant of parasitic seeds germination. Parasitic plants as Phelipanche ramosa can parasitize various host plants, in response to specific exuded germination stimulants (GS). Indeed, different genetic groups of P. ramosa seeds can be the identified, depending on the crop grown in the field where seeds have been collected [START_REF] Huet | Populations of the Parasitic Plant Phelipanche ramosa In fl uence Their Seed Microbiota[END_REF]. Seeds from two populations of P. ramosa harvested in hemp (P. ramosa group 2a) and oilseed rape (P. ramosa group 1) fields [START_REF] Stojanova | Genetic differentiation and host preference reveal non-exclusive host races in the generalist parasitic weed Phelipanche ramosa[END_REF][START_REF] Huet | Populations of the Parasitic Plant Phelipanche ramosa In fl uence Their Seed Microbiota[END_REF] the addition of (±)-GR24 to the medium restores seed germination. Thus, PpCCD8-derived compounds induce the germination of a specific population of P. ramosa seeds, responding to GS exuded by hemp.

54

P. patens strongly responds to (+)-GR24 and carlactone application, but poorly to (-)-GR24 and KAR2 in the dark

So far, the length of caulonema filaments grown in the dark was used as a proxy to quantify the P. patens phenotypic response to SL using (±)-GR24 in Petri dishes assays (Hoffmann et al., 2014;[START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF].

With 24-well tissues culture plates, we reduced the amount of compound required for phenotypic assays (Guillory and Bonhomme, methods chapter given in annex 1) and the number of caulonemal filaments per plant ended up as a more robust proxy of the response to compounds than caulonema length ( Strikingly, no significant changes of caulonema filaments number is observed with (-)-GR24, except in WT for which 0.1 M and 10 M doses leads to a significant increase of this number (more pronounced at 0.1 M, Figure IV-2A).

Higher concentrations of (-)-GR24 are not significantly active in WT, nor in Ppccd8. However, in further assays (see below, Figure , a slight but significant decrease of Ppccd8 caulonema filament number is observed following (-)-GR24 application. To summarize, the phenotypic response of P. patens is significant and marked with the (+)-GR24 that induces a decrease of both filament number and length in WT and Ppccd8. The response to (-)-GR24 is less clear for both genotypes, with sometimes contrary effects (increase of number in WT, see above).

A previous study concluded on the absence of response to KAR1 in both WT and Ppccd8 mutant, when testing the caulonema length in the dark or using a transcriptional marker (Hoffmann et al., 2014). In the present work, we tested the KAR2 molecule described as more active than KAR1 in Arabidopsis [START_REF] Sun | Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds[END_REF]

(Figure IV-2A-B).
KAR2 has an unmethylated butenolide group, unlike KAR1. In WT, no significant effect on caulonema number is observed following application of increasing doses of KAR2 (Figure IV-2A). In Ppccd8, we observe an increase of filament number, as with (-)-GR24 in WT, and this increase is significant at 10 M (Figure IV-2B). Surprisingly, the length of WT filaments is significantly decreased by the 1 M dose only, while higher concentration (10 M) has no significant effect (Supplemental figure IV-1). Caulonema length is also diminished in the Ppccd8 mutant by KAR2 application but at low concentrations (0.1 M and 1 M, Supplemental Figure IV-1). To conclude, KAR2 phenotypic effects on P. patens are slight and not clearly dose responsive, similarly to those of (-)-GR24.

We also tested racemic Z-carlactone (CL), described as the natural product of PpCCD8 in P. patens [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. CL application has a negative effect on caulonema filament number of both WT and Ppccd8 mutant, however significant only at 10 M (Figure IV-2C).

56

With these phenotypic assays, we can conclude to separate effects of GR24 enantiomers in P. patens, as in Arabidopsis [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF]. Indeed, the (+)-GR24 analog mimics CL effects, though it is far more potent, and can thus be used to mimic the PpCCD8 compounds effects, while the (-)-GR24 analog has slight phenotypic effects, that resemble those of KAR2 molecule. We obtained the expression pattern of all PpKAI2L genes in P. patens, thanks to a cDNA library from various organs/tissues (Supplemental Figure 3 A), including spores, protonema of increasing age and different tissue composition (6-day-old: primarily chloronema; 11-day-old: mix of chloronema and caulonema; 15-day-old: mix of chloronema, caulonema and gametophores buds), and gametophores from 5-week-old plants. PpKAI2L genes transcripts are found in all tested tissues, at relatively low levels compared to the control genes. Notably, we also find that PpKAI2L-A is expressed, thus confirming it is not a pseudogene. This could not be assessed by qPCR for PpKAI2L-M, as its predicted transcript is almost identical to PpKAI2L-G, and reported transcript levels are attributed to both PpKAI2L-G and -M. Thus PpKAI2L-G/M probably arose recently as a local duplication in the genome of P. patens. In spores, PpKAI2L-F and -J have higher transcript levels than other PpKAI2L genes. In protonema and gametophores however, PpKAI2L-D from clade (A-E) shows the highest transcript levels compared to any other PpKAI2L genes.

PpKAI2L-I transcript levels are the lowest, in all tested tissues. When considering the clades separately (Supplemental (ratio F350nm/F330nm) with temperature, whereas the bottom panels show the first derivatives for the F350nm/F330nm curve against the temperature gradient from which the apparent melting temperatures (Tm) for each sample was determined.

The experiment was carried out twice. To summarize, all proteins from clade (A-E) have a similar biochemical behavior toward SL analogs, with a stereoselectivity toward (-)-GR24, similar to AtKAI2. As to the proteins from the other tested clades, (H,I,L) and (F,K), no specific feature can be highlighted, indicating that these proteins need to be considered independently regarding their interaction with the ligand. Furthermore, these results show that (+)-GR24 and (-)-GR24 stereoisomer can be bound by PpKAI2L proteins from different clades, and even lead to opposite effects, in terms of stability. µM.min -1 vs 0,01465 µM.min -1 ), suggesting that the L 28 residue affects the velocity of catalytic activity. Thus, the PpKAI2L-H protein lacks a Phenylalanine residue that partly explains its strong enzymatic activity. Continued.

Moss PpKAI2L proteins covalently link GR24 enantiomers, as vascular plants receptors

To further question the role of PpKAI2L proteins as receptors, we looked for covalent attachment of the GR24 proteins show strongest affinity for the (-)-GR24, reported in vascular plants as a mimic for KL unknown compound [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF][START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF].

Biochemical analysis of PpKAI2L proteins thus highlights some resemblances with the AtD14 and even more with the AtKAI2 protein, in accordance with a possible role as SL (or related compounds) and/or KL receptors. In planta studies were next led to further test this possible role.

None of the PpKAI2L gene complements the Arabidopsis d14-1 kai2-2 double mutant

We used cross species complementation assays to test if some of the PpKAI2L proteins could ensure similar function to that of AtD14 and/or AtKAI2 in Arabidopsis SL and/or KL signaling. PpKAI2L (C, D, F, G, J, H) genes were cloned downstream the AtD14 or AtKAI2 promoters, and resulting constructs were expressed in the Arabidopsis double mutant Atd14-1 kai2-2, that shows both hyperbranched phenotype and elongated hypocotyls (Supplemental Compared to WT, the double mutant shows longer hypocotyls in control conditions, as the single kai2-2 mutant, and neither (+)-GR24 nor (-)-GR24 addition has an effect on this phenotype. In contrast, 1 µM (+)-GR24 in the medium leads to shorter kai2-2 hypocotyls, likely due to perception and transduction by the AtD14 protein still active in the single mutant. Accordingly, 1 µM (-)-GR24 in the medium has no effect on kai2-2 hypocotyls. Expressing AtKAI2 under the control of AtKAI2 promoter surprisingly does not fully restore the hypocotyl length of the double mutant in our control conditions, but it does restore the response to (-)-GR24 as expected. similar function to AtD14 in a kai2-2 background, for hypocotyl development but not for rosette branching.

More surprising, PromAtKAI2:AtD14 expressing line shows longer hypocotyls than the double mutant in control conditions (DMSO), and similar phenotypes of longer hypocotyls in control conditions were found in lines for further analysis (Supplemental Table IV-2). The three other clades (H,I,L), (F,K), and (J,G,M) were targeted in separate experiments using combinations of specific guide-RNAs for each gene (PpKAI2L-F to -L). The same guide-RNA was used to target PpKAI2L-G and -M. Several mutants were obtained for clade (J,G,M) genes, including a single KO Ppkai2L-j mutant (j1*) (Supplemental Table IV-2 leading to ∆h-i-l and ∆h-f-k-j mutants (Supplemental Table IV-2). Eventually, a 7X mutant was obtained (∆h-i-f-k-j-gm) where all but mutations in PpKAI2L-J and -M genes were null mutations. (Supplemental Table IV-2 and see hereafter). Our rationale was that a mutant affected in the response to PpCCD8-derived compounds should show a phenotype similar to that of the Ppccd8 synthesis mutant. We first performed a phenotypic analysis of the mutants in light conditions. After 4 weeks of culture, the Ppccd8 mutant plant size is slightly larger than that of the WT (Proust et al., 2011, Figure IV-9), while the Ppmax2-1 mutant is smaller, with fewer but bigger gametophores (Lopez-Obando et

Clade (A-E) quintuple mutants are affected in photomorphogenesis, as Ppmax2

Mutants in clade (A-E) genes show the typical phenotype of the Ppmax2-1 mutant in white light. We previously showed that Ppmax2-1 mutant is affected in photomorphogenesis under red light [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. In conclusion, the phenotype of the Ppkai2L mutants in red light allows to separate clade (A-E) genes, likely involved in a PpMAX2 dependent pathway, related to photomorphogenesis, and genes from the three other clades, likely independent from this pathway.

Mutants in (J,G,M) clade do not respond to (+)-GR24 application

We report above the response of WT and Ppccd8 mutant to SLs analogs (Figure IV-2). To determine among the Ppkai2L mutants those carrying mutations in potential receptors for PpCCD8-compounds (SL-related) or other (KLrelated) compounds, we tested their phenotypic response in the dark to GR24 enantiomer application, using 0.1 M concentration in all assays, and counting the number of caulonema filaments per plant.

For the (A-E) clade, in control conditions, all Ppkai2L mutants show a number of filaments equivalent to that of WT, except for the quintuple mutant, which tends to have less filaments in control conditions, like Ppmax2-1 ( ), but has no significant effect on the Ppmax2-1 mutant, though the same tendency to a decrease is observed. At this higher dose, the quintuple mutant affected in all five PpKAI2L-A to E genes shows a significant decrease of caulonema filaments number, like Ppccd8, and opposite to WT. Thus, similarly to PpMAX2 loss of function, mutating clade (A-E) PpKAI2L genes does not abolish a response to the (-)-GR24 enantiomer. A significant negative effect of (+)-GR24 on the number of filaments is observed for the quintuple Ppkai2L the two mutants where j mutation is not null, but PpKAI2L-G gene is KO (7x and j6-g5*-m1 mutants), the response to (+)-GR24 is also abolished (Supplemental Figure IV-14 C,D). Thus, from phenotypic assays on mutants' caulonema, both PpKAI2L-J and -G (and presumably -M) genes are likely involved in the response to (+)-GR24, and therefore the best candidates for receptors to PpCCD8-derived molecules.

To confirm that clade (J,G,M) PpKAI2L are likely receptors for PpCCD8-derived molecules, we tested the transcript levels of SL responsive genes in the corresponding mutants . We have shown previously that in WT and Ppccd8 mutant, PpKUF1LA gene transcript abundance is increased 6 h after plant transfer on medium containing 3 µM (±)-GR24, and that this response is enhanced in dark conditions [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. We used this marker along with the Pp3c6_15020 gene, previously found upregulated by (±)-GR24 (our unpublished data). Using GR24 enantiomers, we confirm the increase of both genes' transcript levels, following 1 µM (+)-GR24 addition in WT and Ppccd8, but not in Ppmax2-1. Strikingly, an increase of transcript level following (-)-GR24 application is observed for both markers in the Ppccd8 mutant, and for PpKUF1LA only in WT (Figure IV-12). In the quintuple mutant of clade (A-E), PpKUF1LA and Pp3c6_15020 transcript levels are highly increased by the (+)-GR24 addition, but unchanged by (-)-GR24. In contrast, in the Ppkai2L-j1* mutant, both genes transcript levels are increased by (-)-GR24 addition, and slightly increased (PpKUF1LA) or unchanged (Pp3c6_15020) by (+)-GR24 application. In the Ppkai2L j3-g3*-m1 mutant, the response markers transcript levels are slightly increased (PpKUF1LA) or unchanged by (+)-GR24 addition, while unchanged by (-)-GR24. Thus, the transcriptional response of the tested mutants confirms that clade (A-E) genes are not involved in the response to (+)-GR24, while this response is impaired in clade (J,G,M) mutants. Only Ppccd8

and Ppkai2L-j1* mutants show a clear and significant transcriptional response, with both markers, to (-)-GR24 addition. and noted in corresponding colors (cyan for (+)-GR24 and red for (-)-GR24).

Discussion

Are PpCCD8-derived products non-canonical SLs?

The PpCCD8-derived products are germination stimulants of P. ramosa group 2a seeds, harvested in a hemp field, but do not induce the germination of P. ramosa group 1 seeds, collected from an oilseed rape field. [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF] also reported the induction of Orobanche ramosa (old denomination of Phelipanche ramosa) seed germination by P.

patens exudates, however without specifying the origin of the tested population. Differences in root parasitic weeds susceptibility can be attributed to the chemical nature of host plant exudates (Yoneyama et al., 2018b). Our results suggest that the PpCCD8 products share similarities with hemp secondary metabolites. So far, no known canonical SL has been isolated from hemp [START_REF] Huet | Populations of the Parasitic Plant Phelipanche ramosa In fl uence Their Seed Microbiota[END_REF]. Since P. patens likely produces carlactone [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF], but lacks a true MAX1 homolog (Proust et al., 2011), we can hypothesize that PpCCD8-derived compounds may correspond to non-canonical SLs, derived from carlactone or hydroxyl carlactones [START_REF] Yoneyama | Recent progress in the chemistry and biochemistry of strigolactones[END_REF]. Indeed, among analogs showing bioactivity on P. patens caulonema length, we previously showed that GR5, a non-canonical analog, was as active as (±)-GR24 (Hoffmann et al., 2014). As mimics of SLs for the presented work, we used the (+)-and (-)-GR24 artificial analogs, available at the time of our study. It is to note that both isomers are active on P. ramosa group 1 and group 2a seeds. However, as GS, the (+)-GR24 isomer, which has a canonical SL structure, is similar to (±)-GR24, while the (-)-GR24 isomer is far less active [START_REF] De Saint Germain | Contalactone, a contaminant formed during chemical synthesis of the strigolactone reference GR24 is also a strigolactone mimic[END_REF]. For future identification of PpCCD8-derived compounds, non-canonical SL analogs such as the recently described Methyl Phenlactonoates [START_REF] Jamil | A New Series of Carlactonoic Acid Based Strigolactone Analogs for Fundamental and Applied Research[END_REF] would certainly be more adapted.

Looking for the best mimic of SLs or KL

The (-)-GR24 analog has a non-natural configuration, meaning a configuration that has so far never been isolated from plant exudates, contrary to the (+)-GR24 enantiomer, that bears configurations as 5-deoxystrigol ((+)-5DS) and strigol-type canonical SLs [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF]. In our bioassays on moss phenotype, CL application decreases the number of caulonema of both WT and Ppccd8 mutant, in a dose-response manner. A similar (though much stronger) effect is observed with the (+)-GR24, that we thus consider as the best mimic of P. patens CCD8-derived compounds.

It is not surprising that (+)-GR24 is more potent than CL, as assays are realized in a wet medium and natural SLs are described as far less stable than synthetic analogs in aqueous medium [START_REF] Akiyama | Structural requirements of strigolactones for hyphal branching in AM fungi[END_REF][START_REF] Boyer | Structure-Activity Relationship Studies of Strigolactone-Related Molecules for Branching Inhibition in Garden Pea: Molecule Design for Shoot Branching[END_REF]. In contrast, the effects of (-)-GR24 are weak, not dose responsive, and sometimes opposite in WT versus Ppccd8 mutant.

Indeed, in several assays, we observed a significant increase of caulonema number in WT ( Interestingly, we also observed an increase of caulonema number when testing KAR2, though only significant at 10 µM on Ppccd8. So far, we had not observed any effect of karrikins (KAR1) on P. patens phenotype (Hoffmann et al., 2014), and this is thus the first hint of a possible effect of some karrikins on moss, that needs to be confirmed. It is puzzling however that the effect of KAR2 is better seen in Ppccd8 (thus in the absence of SLs), while the effect of (-)-GR24 is better seen in WT versus Ppccd8. Thus, the (-)-GR24 is not very robust as a mimic of yet-to-be-identified moss KL.

This could also suggest that the moss KL is somehow different from Angiosperm KL.

Biochemical characterization of the PpKAI2L proteins highlights those from the (A-E) subclade as KL receptors

In biochemistry experiments, among used analogs, we observed that the (-)-GR24 enantiomer is better bound by AtKAI2 and all tested PpKAI2L proteins, from (A-E) clade. This confirms a recent report [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF] of preferential binding of (-)-5DS by PpKAI2L-C, -D and -E. Surprisingly, the (+)-GR24 poorly interacts with these PpKAI2L proteins. As the (-)-GR24 enantiomer is a mimic of KL in Arabidopsis, this result suggests that clade (A-E)

PpKAI2L proteins may share a perception mechanism with AtKAI2, and furthermore may recognize KL-like compounds. It is to note that AtKAI2 is not degraded following KAR perception (Waters et al., 2015a), and this could be tested on clade (A-E) PpKAI2L proteins. As to the two other tested clades (F,K) and (H,I,L), none of the interaction assays allowed to highlight a preferential binding of GR24 enantiomers. Unfortunately, none of the PpKAI2L proteins from clade (J,G,M) could be purified for interaction assays, as also experienced by [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF]. In the future, overexpression in P. patens or in other heterologous systems (yeast, insect cells) may be a solution to produce these proteins and permit their biochemistry studies.

PpKAI2L-H is the most efficient hydrolase among PpKAI2L proteins

The PpKAI2L-H protein shows a high cleavage activity towards all four GR24 stereoisomers as well as towards the synthetic probe (±)-GC242, compared to any other PpKAI2L protein, but also compared to Arabidopsis AtKAI2 and AtD14 proteins. Mutating the Leu28 residue into a Phe is enough to reduce the efficiency of the enzymatic activity (strong reduction of the kcat and of the Vmax), but has no effect on the K1/2 towards (±)-GC242. The efficient cleavage activity of PpKAI2L-H is therefore likely not due to a stronger affinity of this protein for the substrate. This is in accordance with previous hypothesis that the Leu 28 residue (as the Phe 181 residue), that is unique to PpKAI2L-H, does not particularly enlarge the pocket size of PpKAI2L-H [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF]. Among other plant D14/KAI2 proteins, hydrolytic activity is observed for the pea PsD14/RMS3 towards three out of four GR24 enantiomers (this study, Figure 6A), and has also been reported for two KAI2 homologs, respectively from Selaginella moellendorffii (SmKAI2b, towards (+)-GR24) and Marchantia polymorpha (MpKAI2b, towards (-)-GR24 (Waters et al., 2015b)). The conserved strong enzymatic activity of PpKAI2L-H would thus have a specific (and ancient) role in plants, may be as a cleaning enzyme, to eliminate an excess of signaling molecules [START_REF] Seto | Strigolactone perception and deactivation by a hydrolase receptor DWARF14[END_REF].

When mutating the PpKAI2L-H gene alone (∆h mutant), no striking phenotype is observed (Supplemental

Arabidopsis Atd14-1 kai2-2 mutant complementation assay is more than anecdotal

Using the endogenous AtD14 promoter, we confirmed the incapacity of PpKAI2L-C and H proteins to fulfill Arabidopsis D14 function in rosette branching, previously reported using the 35S promoter [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF]. We can extend this observation to PpKAI2L-D, PpKAI2L-F and PpKAI2L-G, never tested before for rosette branching complementation. Interestingly, one line expressing PpKAI2L-J shows partial complementation of the branching that could be the hint of SL perception function for this protein. However, given the phenotype of moss mutants in clade (J,G,M), one would have expected PpKAI2L-G expressing lines also to complement the branching phenotype, unless the PpKAI2L-J is more active than its counterparts from the same clade. Using the endogenous AtKAI2 promoter, we also confirmed as observed by [START_REF] Bürger | Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens[END_REF], that PpKAI2L-C and -H proteins cannot complement the kai2-2 mutation effect, and extend this observation to PpKAI2L-D, and -J. However, expressing moss PpKAI2L-G reduces the size of Atd14-1 kai2-2 hypocotyls, suggesting that PpKAI2L-G may be able to perceive and transduce the endogenous KL signal, though it is not able to respond to (-)-GR24. Strikingly, when expressed under the control of AtKAI2 promoter, the AtD14 protein but also the moss PpKAI2L-C, -J or -H proteins in Arabidopsis Atd14-1 kai2-2 exacerbates the defect induced by the kai2-2 mutation by leading to even more elongated hypocotyls. This suggests a putative interaction of these proteins with the Arabidopsis KAI2/KL pathway, that needs further investigations. Even more strikingly, (+)-GR24-induced shorter hypocotyls are observed in PpKAI2L-H expressing lines, as in AtD14 expressing lines, which may indicate that the strong hydrolytic activity of PpKAI2L-H can fulfil AtD14 function in the seedling.

Still, it is clear that none of the PpKAI2L protein fully complements the AtD14, nor the KAI2 function.

Genetic analysis suggests that genes from the (A-E) clade are likely involved in the PpMAX2 dependent pathway

Mutants phenotype clearly distinguish clade (A-E) from the three other clades. Indeed, the 5x PpKAI2L-A to -E mutant shows a phenotype in white light quite similar to that of Ppmax2-1 mutant, as well as elongated gametophores under red light, and low number of caulonema filaments in the dark, suggesting that clade (A-E) PpKAI2L and PpMAX2

proteins could be part of a same pathway. As PpKAI2L proteins from the (A-E) clade preferentially bind the (-)-GR24 enantiomer, we expected the mutants in this clade to be blind to (-)-GR24 application. This is what we observe when testing the transcriptional response markers, which transcript levels are unchanged by (-)-GR24 application in both the Ppmax2-1 and the 5x (clade A-E) Ppkai2l mutant, while transcript levels of the response markers are increased in the Ppccd8 mutant (but not in WT, Figure 12). However, as mentioned above, it should be noted that the (-)-GR24 is apparently not a good mimic of the unknown moss KL, and therefore other transcriptional response markers need to be found, that would reflect the moss KL response, as DLK2 or STH7 for Arabidopsis KL [START_REF] Nelson | Karrikins enhance light responses during germination and seedling development in Arabidopsis thaliana[END_REF][START_REF] Waters | KAI2-and MAX2-mediated responses to karrikins and strigolactones are largely independent of HY5 in arabidopsis seedlings[END_REF]. As for the phenotypic response, application of 0.1 µM (-)-GR24 had no effect on clade (A-E) mutants caulonema number in the dark, nor on WT, but significantly decreased both Ppccd8 and Ppmax2-1 caulonema number (Figure IV-11A). Strikingly, a 1 µM concentration of (-)-GR24, that has opposite effects on WT (increases the number of caulonema) and Ppccd8 (decreases the number of caulonema), led to no response of the Ppmax2-1 mutant, while the 5x (clade A-E) mutant showed a significant decrease of caulonema number (Supplemental Figure . Thus, the 5x (clade A-E) mutant is still able to perceive (-)-GR24, as well as the Ppmax2-1 mutant. This does not rule out the hypothesis of clade (A-E) PpKAI2L and PpMAX2 proteins being in a same pathway, but further indicates that the (-)-GR24 enantiomer is a poor mimic of a moss KL. In addition, a dual effect of (-)-GR24, promoting both the KAI2 and the D14 pathways has been reported in Arabidopsis roots [START_REF] Villaecija-Aguilar | KAI2 regulates root and root hair development by modulating auxin distribution[END_REF], and could explain the residual response of the clade (A-E) Ppkai2l mutant, through other PpKAI2L proteins, in a PpMAX2 independent pathway. mutants, suggesting a response to the SL analog. Thus, while the KO mutation of either PpKAI2L-J or PpKAI2L-G is sufficient to abolish the phenotypic response in the dark, mutations in both genes are necessary to completely abolish the transcriptional response to (+)-GR24. The transcriptional markers first identified using (±)-GR24 (Lopez-Obando et al., 2016a, 2018) may not be fully specific to assay the response to enantiomers. It could also suggest that the transcriptional response, which is assessed far earlier than the phenotypic response (6 hours versus 15 days), is perhaps more sensitive to a very slight activation of the CCD8-compound pathway by PpKAI2L proteins.

PpKAI2L proteins are likely receptors in two separate pathways, respectively dependent and independent from the PpMAX2 F-box protein.

Our previous results on the PpMAX2 F-box protein indicated that, in contrast to its homolog in flowering plants, it is not involved in response to CCD8-derived compounds [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. Like MAX2 in flowering plants however, PpMAX2 plays a role in gametophore early development and photomorphogenesis. We suggested that PpMAX2 could play a role in the moss KL signalling pathway, but we lacked evidence for other actors in this pathway in P. patens. The present work indicates that PpKAI2L-A to -E are α/β hydrolases involved in the same pathway as PpMAX2, since mutating these genes lead to similar light-related phenotypes as those of Ppmax2 mutant. Specific mimics for the moss KL are however still missing for further evidence that PpKAI2L-A-E are receptors of the moss KL. Still, these results are consistent with the view that the KL pathway is ancestral relative to the SL pathway, and that the ancestral role of MAX2 in the land plants lineage is the transduction of the KL signal (Bythell-Douglas et al., 2017;[START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF].

As to SL-related pathway, PpKAI2L-J and PpKAI2L-G proteins are likely receptors to CCD8-compounds, which we suspect to be non-canonical SLs. Strikingly, these receptors are not particularly closer to D14 than other PpKAI2L proteins. As hypothesized earlier by Lopez-Obando ( 2016) and Bythell-Douglas ( 2017), the expansion of the PpKAI2L family might have enabled in moss (and not in other bryophytes as Marchantia polymorpha, that counts 2

MpKAI2 genes), as in parasitic Angiosperms [START_REF] Conn | Convergent evolution of strigolactone perception enabled host detection in parasitic plants[END_REF][START_REF] Toh | Structure-function analysis identifies highly sensitive strigolactone receptors in Striga[END_REF][START_REF] De Saint Germain | A Phelipanche ramosa KAI2 Protein Perceives enzymatically Strigolactones and Isothiocyanates[END_REF]), the emergence of SL sensitivity. Neofunctionalization of additional KAI2 copies in P. patens ancestry towards SL perception is therefore a possible explanation for what we observe in this moss and would reveal a convergent evolution process, relative to the emergence of D14 in seed plants. We can also imagine that these neo-functionalized PpKAI2L lost the ability to interact with MAX2 in P. patens and established a different protein network that potentially integrates new factors like an alternative F-box, since PpMAX2 is not necessary for SL sensitivity. The remaining question is therefore to determine how the SL signal is transduced downstream of perception by PpKAI2L-J -G -M.

Consequently, the search for interactants to these moss KL and CCD8-derived compounds receptors should be a priority in the next future. As SMXL proteins are key players of both SL and KL pathways in flowering plants [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Wang | Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation[END_REF][START_REF] Khosla | Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2[END_REF] specific involvement of PpSMXL homologs (4 genes)

was also examined (chapters VI and VII).

Five more PpKAI2L proteins are present and expressed in moss (Lopez-Obando et al, 2016), and mutant analyses indicate that those are neither KL nor CCD8-derived compounds receptors. Three of them however, among which the efficient hydrolase PpKAI2L-H, are likely involved in moss development, through pathways that remain to be discovered.

Methods

Plant Materials and Growth Conditions. The Physcomitrium (Physcomitrella) patens Gransden wild-type (WT) strain was used and grown as previously described (Hoffmann et al, 2014;[START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF], at 24°C in long day (16h) conditions, (22°C 8h night), except for assays in red light. Unless otherwise explicitly stated in legends, experiments were always carried out on PpNO3 medium (corresponds to the minimal medium described by Ashton et al., 1979), in the following control conditions: 25°C during daytime and 23°C at night, 50% humidity, long days conditions with 16 hours of day and 8 hours of night (quantum irradiance of ~80 µmol/m 2 /s). Multiplication of tissues from young protonema fragments prior to every experiment is done in the same conditions but using medium with higher nitrogen content (PpNH4 medium, PpNO3 medium supplemented with 2.7 mM NH4 tartrate). For red light experiments, plants were grown on PpNO3 medium in Magenta pots at 25°C, in continuous red-light (~45 µmol µmol/m 2 /s).

Germination assay on root parasitic plant seeds. P. patens WT plants were grown on PpNH4 plates with cellophane disks for 2 weeks then the plants (and cellophane) were transferred on PpNO3 medium without phosphate (Phosphate buffer was replaced by 1g/L of MES buffer and the pH adjusted to 5.8) for another 2 week. Moss exudates were collected by transferring the plants (still on cellophane disks) on plates with 10 mL distilled water, placed in growth chamber with gentle agitation. After 48h, exudates were pipeted and filtered (0.2 µm). Exudates were diluted twice prior testing their germination stimulant activity on preconditioned seeds of parasitic plants, as described previously [START_REF] Pouvreau | A high-throughput seed germination assay for root parasitic plants[END_REF]. Distilled water was used as control. For germination assays on plates (Figure 1B-C), WT and Ppccd8 mutant were cultivated as above, and P. ramosa seeds were added on the plates after 10 days of phosphate starvation. Seeds were counted out of 3 plates, with 7-10 microscope fields per plate.

CRISPR-Cas9 mediated mutagenesis and homologous recombination. P. patens mutants were obtained as described in Lopez-Obando et al (2016a), using CRISPR-Cas9 technology. Coding sequences of PpKAI2L were used to search for CRISPR RNA (crRNA) contiguous to a PAM motif recognized by Streptococcus pyogenes Cas9 (NGG), using the webtool CRISPOR V4 against P. patens genome Phytozome V9 (http://crispor.tefor.net/). crRNAs located in the first third of the coding sequence, with highest possible specificity score, and fewest possible predicted off-targets, were selected. Small constructs containing each crRNA fused to either the proU6 or the proU3 snRNA promoter in 5' U3 or U6 promoter [START_REF] Collonnier | CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51independent gene targeting in the moss Physcomitrella patens[END_REF], and to the tracrRNA in 3', encased between attB1/attB2 GateWay recombination sequences, were synthesized by Twist Biosciences. These inserts were then cloned into pDONR207 vectors.

Polyethylene glycol-mediated protoplast transformation was performed with multiple pDONR207-sgRNA according to the protocol described by (Lopez-Obando et al., 2016b). Mutations of the PpKAI2L genes were confirmed by PCR amplification of PpKAI2L loci around the recognition sequence of each guide RNA and sequencing of the PCR products.

The deletion mutant in PpKAI2L-H (∆h) was obtained through homologous recombination. The full coding sequence of PpKAI2L-H from ATG to stop was replaced by a resistance cassette. A 550 bp PpKAI2L-H 5' CDS flanking sequence was cloned into the pBNRF vector [START_REF] Thelander | The moss genes PpSKI1 and PpSKI2 encode nuclear SnRK1 interacting proteins with homologues in vascular plants[END_REF] cut with BstBI/XhoI. Then a 500 bp PpKAI2L-H 3' CDS flanking sequence was cloned into the BNRF-PpKAI2L-H 5' construct digested with BcuI, so that the kanamycin resistance cassette of the vector was surrounded by PpKAI2L-H 5' and 3' flanking sequences. Moss WT protoplasts were transformed with the resulting construct as described previously (Lopez-Obando et al., 2016b), and transformants selected on 50mg/L Geneticin/G418. Transient expression of the CRE recombinase [START_REF] Trouiller | MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens[END_REF] in a confirmed transformant allowed to remove of the resistance cassette and to obtain the Ppkai2l-∆h mutant, as described Phenotypic assays on moss. Analysis of caulonema growth in the dark was performed in 24-well plates, starting from very small pieces of protonema, with ~2 weeks of growth in control conditions before incubation (± treatment) in the dark and placed vertically for ~10 days (See also Guillory and Bonhomme, contribution to the book "Methods in strigolactone research" in annex).

Chemicals. Racemic and pure enantiomers of GR24, (±)-GC242 probe were produced by F-D Boyer (ICSN, France).

Racemic Z-CL was kindly provided by A. Scaffidi (University of Western Australia, Perth, Australia). Chemicals were diluted in DMSO or acetone as indicated in legends to figures. KAR2 was purchased from Chiralix.

RT-qPCR analysis. Freshly grinded WT (Gransden) tissues were inoculated in Petri dishes of PpNO3 medium, overlaid with a cellophane sheet. Protonema tissues were harvested after 6 days, 10 days or 15 days of growth in long days conditions (25°C during the day and 22°C during the night, 50% hygrometry, quantum irradiance of 75 µmol m -2 s -1 ).

To obtain older gametophores and spores, WT were regenerated from spores for approximately two weeks and then transferred to Magenta pots containing PpNO3 medium (9 plants per pot) and cultivated in the same conditions as written above. Gametophores were harvested after 35 days and then from different plants after 70 days. After 70 days, rhizoids were also harvested after being separated from gametophores' shoots by dissection. Both protonema and gametophores samples were immediately flash frozen in liquid nitrogen and kept at -80°C until RNA extraction. Remaining pots were transferred at 35 days to short days conditions (15°C, 100% hygrometry, quantum irradiance of 15 µmol m -2 s -1 ) for approximately two months until capsule maturity. Capsules were sterilized (90% chlore and 10% pure ethanol) then rinsed with sterile water. Each of the four biological replicates consisted of 10-20 capsules from which spores were freed by mechanical disruption and separated from capsules' debris by filtering through a 25µm nylon mesh. Spores were kept in sterile water and flash frozen in liquid nitrogen and kept at -80°C until RNA extraction. For all samples except for spores, tissues were grinded in liquid nitrogen using a mortar and pestle and RNA were extracted and subsequently treated with DNAseI using the Plant RNeasy Mini extraction kit (Qiagen) following the manufacturer's instructions. Spores were recovered in 1mL TRIzol reagent (Invitrogen) and crushed manually using fine pestles. RNA was separated from cell debris and protein using chloroform and then precipitated with isopropanol and washed with ethanol 70%. RNA pellets were dissolved in RLT buffer from the Qiagen Plant RNeasy Mini kit and treated with DNAse I on columns following the manufacturer's instructions. 500 ng of each RNA sample was used for retro-transcription using the RevertAid H Minus Reverse Transcriptase from Thermo Fisher. Quality of obtained cDNA extracts was checked by semi-quantitative RT-PCR using the reference gene PpAPT. Quantitative RT-PCR were carried out in 5µL using the SsoAdvanced Universal SYBR Green Supermix from BioRad and the following program on QuantStudio TM 5 (ThermoFisher): initial denaturation at 95°C for 3 minutes, then 45 cycles with 95°C 10 seconds and 60°C 30 seconds.

Using the CTi (for the genes of interest) and CTref (mean for the 2 reference genes) values obtained, relative expression is given by RE = 2 -CTi /2 -CTref . Note that in Supplemental Figure IV-3 the values given are Log (RE).

Constructs, generation of transgenic lines. The expression vectors for transgenic Arabidopsis were constructed by MultiSite Gateway Three-Fragment Vector Construction kit (Invitrogen). All the PpKAI2L constructs were tagged with 6xHA epitope tag at their C-terminus. Lines were resistant to hygromycin. AtD14 native promoter (0.8 kb) and AtKAI2

native promoter (0.7 kb) were amplified by PCR from Col-0 genomic DNA and cloned into pDONR-P4P1R, using Gateway recombination (Invitrogen) (see Supplementary Table IV-2 for primers). AtD14 CDS and AtKAI2 CDS were PCR amplified from Col-0 cDNA, PpKAI2cds were PCR amplified from Physcomitrella patens cDNA and recombined into pDONR221 (Invitrogen). 6xHA with a linker (gift from U. Pedmale) was cloned into pDONR-P2RP3 (Invitrogen).

The suitable combination of promoters, CDS and 6xHA was cloned into the pH7m34GW final destination vectors by using three fragments recombination system [START_REF] Karimi | Recombinational cloning with plant gateway vectors[END_REF], and named pD14::cds-6xHA or pKAI2::cds-6xHA.

Transformation of Arabidopsis Atd14-1kai2-2 double mutant, Landsberg background (gift from M. Waters) was performed according to the conventional dipping method [START_REF] Clough | Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF], with Agrobacterium strain GV3101.

For all constructs, more than 12 independent T1 lines were isolated and between 2 to 4 representative single-insertion lines were selected in T2. Only 2-3 lines per constructs were shown in these analyses. Phenotypic analysis shown in Expression and purification of AtD14, RMS3 and AtKAI2, with cleavable GST tag was performed as described in de Saint Germain et al, 2016. For PpKAI2L proteins expression, the full-length coding sequences from Physcomitrella patens were amplified by PCR using cDNA template and specific primers (see Supplementary Table IV-1) containing a protease cleavage site for tag removal, and subsequently cloned into the pGEXT-4T-3 expression vector. For the PpKAI2L-L, the N-terminal 47 amino acids have been removed. The expression and purification of PpKAI2 proteins followed the same method as for AtD14 and AtKAI2.

Site-directed mutagenesis. Site-directed mutagenesis experiments were performed using QuickChange II XL Site Directed Mutagenesis kit (Stratagene), performed on pGEX-4T-3-PpKAI2L-H (see Supplementary Table IV-1 for primers). Mutagenesis was verified by systematic DNA sequencing.

Enzymatic degradation of GR24 isomers by purified proteins. The ligand (10 µM) was incubated without and with purified RMS3/AtKAI2/PpKAI2L proteins (5 µM) for 150 min at 25 ºC in PBS (0.1 mL, pH = 6.8) in presence of (±)-1-indanol (100 µM) as internal standard. The solutions were acidified to pH = 1 by addition of trifluoroacetic acid ( 2µL) to quench the reaction and centrifugated (12 min, 12,000 tr/min). Thereafter the samples were subjected to RP-UPLC-MS analyses. The instrument used for all the analysis was an Ultra Performance Liquid Chromatography system equipped with a PDA and a Triple Quadrupole mass spectrometer Detector (Acquity UPLC-TQD, Waters, USA). RP-UPLC (HSS C18 column, 1.8 μm, 2.1 mm × 50 mm) with 0.1% formic acid in CH3CN and 0.1% formic acid in water (aq. FA, 0.1%, v/v, pH 2.8) as eluents [10% CH3CN, followed by linear gradient from 10 to 100% of CH3CN (4 min)] at a flow rate of 0.6 mL/min. The detection was performed by PDA and using the TQD mass spectrometer operated in Electrospray ionization positive mode at 3.2 kV capillary voltage. The cone voltage and collision energy were optimized to maximize the signal and was respectively 20 V for cone voltage and 12 eV for collision energy and the collision gas was argon at a pressure maintained near of 4.5.10 -3 mBar.

Enzymatic assay with pro-fluorescent probes and p-nitrophenyl acetate has been performed as described in (de

PCR-Based mutagenesis of PpKAI2L-H

PpKAI2L-H-L28F GTGGTGCTGGGGCATGGCTTTGGAACCGACCAATCAG qPCR primers Supplementary table IV-3 -List of gene sequences used in this study.

PpKAI2L-A_qF468 ATGGCAGTGCAGGAGTTTAG PpKAI2L-A_qR571 GTAACACGCTGCGCAAATC PpKAI2L-B_qF631 CTTGCCACATCTTGCAAAGC 110 PpKAI2L-B_qR735 ATGCAGCACCTCAACAATGC PpKAI2L-C_qF525 GGAGTTTGGTAGGACGCTATTC PpKAI2L-C_qR632 GCACAGTCACCTTTGGTAGAA PpKAI2L-D_qF623 CACCGTCCCTTGCCATATT PpKAI2L-D_qR736 TCTGCAACACCTCGACAATC PpKAI2L-E_qF667 CCTTGGTAGTCGCGGATTAT PpKAI2L-E_qR761 GAACTGAGCTGAGGCAAATG PpKAI2L-F_qF602 TGAAAGTGCCAGTGCATCTC PpKAI2L-F_qR744 TCACTCAAATGCGGCAAG PpKAI2L-K_qF589 TCTTCCAGAGCGATCTACGTTC PpKAI2L-K_qR738 GTTCAGAACCTCCATCATCGTC PpKAI2L-H_qF671 AATTGAAGTGGCGGAGTACC PpKAI2L-H_qR779 ACCACCAATTCTGGACAACTC PpKAI2L-I_qF509 TGACGACAAAGCAGTGCAAG PpKAI2L-I_qR643 TATGGCAAGGCACTGTAACCTC PpKAI2L-L_qF453 AGGTTGCCTTGCATCTCTTG PpKAI2L-L_qR569 AGGTCATGCTGCTCAAATCC PpKAI2L-J_qF393 CTCATTCTCATGGCAGCATCTC PpKAI2L-J_qR538 CCATCGCCTTAGGTACAAAACC PpKAI2L-G/M_qF638 GCGACCAGATATTGCCCTTA PpKAI2L-G/M_qR743 ACTCCACTTTGCACTAGATAGC PpKUF1LA_qF GGAGGTGCTCATTGGAACTAAA PpKUF1LA_qR GGTGCATCCGAAGCAATATCTA Pp3c6_15020_qF CAGAACGGCTTTGTGGATTTG Pp3c6_15020_qR GTCCGAGTTGGTAGAGGTAGTA PpAPT_qF ACTTGCCGTGGCGAGCTAC PpAPT_qR CATCCTTGGAGGCCGACATC PpACT3_qF AGCGAGTACGATGAATCTGG PpACT3_qR ACACAGCAAGAGCTCAATCC Supplementary table IV-2 -

Sequence ID Genebank GI number

Phytozome ID Splicing variants (underlined variant has been used in this study) CHAPTER V -What are SMXL proteins putative molecular functions and how can it be integrated in SL and KL signaling?

V-A) ClpATPases proteins in plants and beyond

SMXL proteins display high structural similarity to a broader family of proteins called Clp-ATPases, which are present in all three parts of the living realm: Bacteria, Archea and Eukaryotes. Clp-ATPases are specific members of the AAA+-ATPases superfamily [START_REF] Snider | The AAA+ superfamily of functionally diverse proteins[END_REF] which take their name from their first characterized role in casein degradation (caseinolytic protease, Clp) [START_REF] Schirmer | HSP100/Clp proteins: a common mechanism explains diverse functions[END_REF]. Unlike what their name implies, they do not carry a protease catalytic activity, but rather enable proteolytic activity. Indeed, these proteins were first discovered in bacteria, where some of these ATPases form a multimeric complex with the unrelated serine-protease ClpP component [START_REF] Katayama-Fujimura | A multiple-component, ATP-dependent protease from Escherichia coli[END_REF][START_REF] Mimiaga | Reactions and Receptivity to Framing HIV Prevention Message Concepts About Pre-Exposure Prophylaxis for Black and Latino Men Who Have Sex with Men in Three Urban US Cities[END_REF]. These complexes have similar organization and function as the proteasome and have hence been called "proto-proteasomes" by some authors [START_REF] Snider | The AAA+ superfamily of functionally diverse proteins[END_REF][START_REF] Budenholzer | Proteasome Structure and Assembly[END_REF][START_REF] Clarke | ATP-dependent Clp Proteases in Photosynthetic Organisms-A Cut Above the Rest![END_REF][START_REF] Ali | Protective Roles of Cytosolic and Plastidal Proteasomes on Abiotic Stress and Pathogen Invasion[END_REF]. In such complexes, Clp-ATPases act as chaperones to recognize and, using the energy taken from ATP hydrolysis, unfold proteins that will be degraded by the complex [START_REF] Schirmer | HSP100/Clp proteins: a common mechanism explains diverse functions[END_REF]. This similar molecular mechanism was shown to enable very diverse functions in bacteria, always related to the regulation of other proteins' stability and/or activity [START_REF] Wawrzynow | The Clp ATPases define a novel class of molecular chaperones[END_REF]. Close homologs of these ATPases are also found in plants, where ClpP partners are also present, therefore it was early suggested that such protease complexes could also exist in plants [START_REF] Liebeherr | Does Link Scheduling Matter on Long Paths?[END_REF]. However, as we shall see hereafter, not all Clp-ATPases are involved in such complexes. The function of Clp-ATPases is often brought down to the removal of protein aggregates (Singh et al., 2010).

V-B) Clp-ATPases/HSP100 classification

More precisely, SMXL fall into type I Clp-ATPases (HSP100 using yeast nomenclature), as they contain two nucleotide binding domains (NBD), contrary to type II which contain only one NBD (more similar to NBD2 from type I). The two NBD are very different in sequence and in structure, except for Walker A (or P-loop) and Walker B motifs (necessary for ATP binding and hydrolysis) (figure V-1), suggesting a functional split between the two NBD [START_REF] Gottesman | The ATP-dependent Clp Protease of Escherichia[END_REF]. Indeed, one NBD ensures ATP hydrolysis while the other permits oligomerization [START_REF] Schirmer | HSP100/Clp proteins: a common mechanism explains diverse functions[END_REF].

Oligomerization is a key feature of both type I and type II Clp-ATPases and is induced by either ADP or ATP binding.

As exemplified by Escherichia coli proteins, Clp-ATPases usually form hexameric rings, with target proteins being unraveled through the very tiny central pore of the ring [START_REF] Duran | Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions[END_REF]. Across all living organisms, Clp-ATPases can be further classified into four subtypes in both type I and type II.

According to the nomenclature of Schirmer and colleagues [START_REF] Schirmer | HSP100/Clp proteins: a common mechanism explains diverse functions[END_REF], type I contains subtypes ClpA, found in Gram negative bacteria, ClpB which is ubiquitous, ClpC that is found in land plants, algae, cyanobacteria and Gram positive bacteria, and ClpD which is specific to land plants (figure V-I). Type II contains subtypes ClpM, N, X and Y, where ClpX is ubiquitous, ClpM is specific to mammals and protozoa, and ClpY/HslU and ClpN are found in some bacteria [START_REF] Mishra | ClpB/Hsp100 proteins and heat stress tolerance in plants[END_REF][START_REF] Clarke | ATP-dependent Clp Proteases in Photosynthetic Organisms-A Cut Above the Rest![END_REF]. This classification in subfamilies relies on the differences in consensus sequence of Walker A and Walker B motifs, as well as specific signature sequences in the more variable Nterminal, C-terminal and middle domains [START_REF] Schirmer | HSP100/Clp proteins: a common mechanism explains diverse functions[END_REF].

V-C) Clp proteolytic complexes

The first Clp proteolytic complex that was discovered, and perhaps the most described since, is ClpAP. ClpP association with ClpA changes ClpP substrate specificity (see paragraph V-A), but also enhances its catalytic efficiency in E. coli (Thompson and Maurizi, 1994;Thompson et al., 1994). ATP binding, but not its hydrolysis, is necessary for ClpAP complex formation and activity on small peptides. However, to degrade larger protein substrates, the complex needs intact ClpA ATP hydrolysis activity (Thompson et al., 1994;Thompson and Maurizi, 1994;[START_REF] Wawrzynow | The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone[END_REF]. ClpP can be seen as a real Heat Shock Protein (HSP) in E. coli, as its expression is highly induced by heat stress, compared to its basal level in physiological conditions [START_REF] Squires | ClpB is the Escherichia coli heat shock protein F84.1[END_REF][START_REF] Squires | The Clp proteins: proteolysis regulators or molecular chaperones?[END_REF]. On the other hand, ClpA is produced at similar levels along development in bacteria, consistent with its role in housekeeping/physiological growth [START_REF] Squires | The Clp proteins: proteolysis regulators or molecular chaperones?[END_REF]. Still, ClpA has a protective role against other stresses besides heat and is effectively induced by anaeroby and high density cultivation [START_REF] Gottesman | The ATP-dependent Clp Protease of Escherichia[END_REF]. In bacteria, ClpP can also form proteolytic complexes with the other Clp ATPases ClpC [START_REF] Porankiewicz | New insights into the ATP-dependent Clp protease: Escherichia coli and beyond[END_REF] and ClpX [START_REF] Wickner | Posttranslational Quality Control: Folding, Refolding, and Degrading Proteins[END_REF][START_REF] Horwich | Chaperone rings in protein folding and degradation[END_REF]. The resulting ClpCP and ClpXP complexes are not functionally equivalent to ClpAP and are more relevant to heat stress. For some Clp complexes, adapter proteins might be needed to target specific substrates [START_REF] Schmidt | ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway[END_REF]. The stoichiometry of these three Clp complexes is most often one hexameric ring of Clp-ATPases (containing the same number of adaptors if needed [START_REF] Kirstein | Adapting the machine: Adaptor proteins for Hsp100/Clp and AAA+ proteases[END_REF]) at one extremity of two stacked heptameric rings of ClpP [START_REF] Duran | Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions[END_REF][START_REF] Baker | ClpXP, an ATP-powered unfolding and protein-degradation machine[END_REF].

V-D) Bi-functionality of Clp-ATPases

Several clues lead to the view that both type I and II Clp-ATPases act as chaperones in the absence of ClpP, and help to degrade abnormal proteins in complex with ClpP [START_REF] Wawrzynow | The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone[END_REF][START_REF] Wawrzynow | The Clp ATPases define a novel class of molecular chaperones[END_REF]. The switch between these two "roles" would be dependent on ClpP presence but also on the energy balance, since substrate degradation depends on ATP hydrolysis. It was also hypothesized that this functional switch depends on the motifs recognized on their protein substrates [START_REF] Wawrzynow | The Clp ATPases define a novel class of molecular chaperones[END_REF]: the hexamer of Clp-ATPase would recognize a specific motif or structure on various partially denatured proteins which would activate the ATPase activity and hence permit local unfolding of the protein. If the protein does not contain an additional motif/structure, it would be released, and the unfolded site would spontaneously correctly refold. On the other hand, if the engaged protein contains further "signatures of abnormality", a further change in the Clp-ATPase conformation would permit the recruitment of the protease ClpP partner and the now stable complex would proceed to complete degradation of the protein substrate.

V-E) Clp chaperone complexes

Among both types Clp-ATPases, ClpB subtypes have a different operating mode, as they do not associate with proteases [START_REF] Clarke | ATP-dependent Clp Proteases in Photosynthetic Organisms-A Cut Above the Rest![END_REF]. Indeed, ClpB subtypes are the only Clp-ATPases that do not contain the conserved IGF/L motif enabling interaction with ClpP [START_REF] Gottesman | ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli[END_REF][START_REF] Wojtkowiaks | Isolation and Characterization of ClpX , a New ATP-dependent Specificity of the Clp Protease of Escherichia coli[END_REF] (figure V-1). Interestingly, the association in oligomers is as important for ClpB function as for the others [START_REF] Duran | Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions[END_REF], so this feature is not only crucial for protease activity of Clp complexes, but also for the chaperone activity of Clp-ATPases per se. Moreover, ClpB (HSP104 in Saccharomyces cerevisiae) interact with other chaperones such as DnaK heat-shock proteins (Hsp70) and DnaJ (Hsp40) to disaggregate protein aggregates [START_REF] Duran | Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions[END_REF]. ClpB by itself has a limited disaggregation ability, which is dramatically increased by association with these co-chaperones.

In E. coli, ClpA and B are closely related, but unlike ClpA, ClpB is a real HSP whose gene transcription is induced by heat stress [START_REF] Kitagawa | Expression of ClpB, an analog of the ATP-dependent protease regulatory subunit in Escherichia coli, is controlled by a heat shock sigma factor (sigma 32)[END_REF][START_REF] Squires | ClpB is the Escherichia coli heat shock protein F84.1[END_REF]. Moreover, the clpB mutant is hypersensitive to high temperature, suggesting ClpB has a protective role against heat stress [START_REF] Squires | ClpB is the Escherichia coli heat shock protein F84.1[END_REF]. The ClpB homolog in the yeast S. cerevisiae is called HSP104 and is also necessary for both basal long-term thermotolerance (especially in the highly thermotolerant spores) and for induced thermotolerance [START_REF] Sanchez | Hsp104 is required for tolerance to many forms of stress[END_REF]. Furthermore, HSP104 accumulation grants increased tolerance to many other stresses and HSP104 expression is induced by low glucose, ethanol, arsenite and cadmium. Therefore, HSP104 has a protective, cross-tolerance, effect against many abiotic stresses [START_REF] Sanchez | Hsp104 is required for tolerance to many forms of stress[END_REF]. In mammals ClpB (HSP110) have been suggested to protect rRNA transcription from heat stress, which has also been suggested for E. coli ClpB [START_REF] Squires | The Clp proteins: proteolysis regulators or molecular chaperones?[END_REF].

The roles of Clp-ATPases as chaperones likely explains why the phenotypes of KO Clp mutants are often dim, as other chaperones families can ensure survival when a single Clp is absent [START_REF] Squires | The Clp proteins: proteolysis regulators or molecular chaperones?[END_REF].

V-F) ClpATPases in plants

In plants, subtypes ClpB, C and D (type I) and ClpX (type II) are present, as well as ClpP serine proteases (table V-1). Almost all genes that encode Clp components are found in the nucleus genome, as exemplified by Arabidopsis, where only ClpP1 (ATCG00670) is encoded by the plastidial genome [START_REF] Ali | Protective Roles of Cytosolic and Plastidal Proteasomes on Abiotic Stress and Pathogen Invasion[END_REF]. Therefore, it appears that Clp genes, like many protein-encoding genes originating from ancestral endosymbionts, have been transferred to the nuclear genome along plants evolution (table V-1).

One of the firsts that was characterized is the plastidial ClpD/ERD1 in Arabidopsis [START_REF] Kiyosue | Characterization of cDNA for a Dehydration-Inducible Gene That Encodes a CLP A, B-like Protein in Arabidopsis thaliana L[END_REF]. While it is a homolog of E. coli ClpB and S. cerevisiae HSP104, ERD1 expression is specifically and quickly induced only by dehydration, and not by other stresses like heat, cold, carbon deficiency or metals. Later, ERD1 expression was found to be also induced by salt stress and dark-induced senescence and etiolation, in a partially ABA-dependent way [START_REF] Nakashima | A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana[END_REF].

It was soon established that ERD1 has at least three homologues in Arabidopsis [START_REF] Kiyosue | Characterization of cDNA for a Dehydration-Inducible Gene That Encodes a CLP A, B-like Protein in Arabidopsis thaliana L[END_REF].

Subsequently, other proteins that could be bound by anti-HSP104 antibodies were found to be accumulated in various heat-stressed angiosperms. Accumulation of AtClpB/HSP101 transcripts occurs under heat shock conditions but is not triggered by ABA treatment or by cold or osmotic stress [START_REF] Nieto-Sotelo | Characterization of a maize heat-shock protein 101 gene, HSP101, encoding a ClpB/Hsp 100 protein homologue[END_REF][START_REF] Agarwal | Plant Hsp100 proteins: Structure, function and regulation[END_REF]. These proteins correspond to bona fide ClpB/HSP104 homologs and were named HSP101 proteins. Arabidopsis AtHSP101 was first characterized and was found to be able to complement the yeast hsp104 mutant [START_REF] Schirmer | An Arabidopsis heat shock protein complements a thermotolerance defect in yeast[END_REF]. The same restoration of yeast induced thermotolerance by plant HSP101 was observed with a soybean protein [START_REF] Lee | A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance[END_REF], and later with wheat, tobacco and rice HSP101 [START_REF] Agarwal | Plant Hsp100 proteins: Structure, function and regulation[END_REF]. AtHSP101 is cytosolic, unlike most of Arabidopsis ClpATPases [START_REF] Agarwal | Plant Hsp100 proteins: Structure, function and regulation[END_REF], however it was afterwards shown that most plants possess several ClpB homologs with diverse subcellular localizations (Singh and Grover, 2010).

On the other hand, ClpC is the functional equivalent of bacterial ClpA in flowering plants plastids [START_REF] Schirmer | An Arabidopsis heat shock protein complements a thermotolerance defect in yeast[END_REF][START_REF] Shanklin | The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease[END_REF]. In vitro, plant ClpC can interact with the bacterial ClpP and degrade the same substrates, suggesting this complex was conserved across evolution, between bacteria and plastids. Indeed, like ClpD/ERD1, ClpC proteins have a long N-terminal leader domain containing a chloroplast transit peptide (Figure V-1) [START_REF] Mishra | ClpB/Hsp100 proteins and heat stress tolerance in plants[END_REF]. The existence of ClpCP complexes was also supported by the observation that plants possess several ClpP homologs, most being directed to plastids. Much like ClpA and ClpP in E. coli, Arabidopsis ClpC and ClpP are expressed in all tissues in basal conditions. Unlike in E. coli however, neither ClpC nor ClpP are HSP. ClpCP complexes in plastids could permit to degrade neosynthesized proteins containing wrong amino acids or incorrectly folded, therefore preventing accumulation of potentially toxic plastidial proteins in excess. It was also suggested that ClpCP degrades functional plastidial proteins normally acting in complexes when their nucleus-encoded partners are not present. Since the Ubiquitin-Proteasome System (UPS) is not present in plastids, the ClpCP complex could play a similar role in housekeeping as bacterial ClpAP complexes. Indeed, approximately 30% of plastidial neosynthesized proteins are almost immediately degraded if their partners imported from the cytosol are not present [START_REF] Shanklin | The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease[END_REF]. ClpC can associate with the internal plastidial membrane, thereby permitting recognition of misfolded proteins directly from their translocation into plastids, possibly acting on some subunits of Rubisco and of the cytochrome b6f photosynthetic complex [START_REF] Clarke | ATP-dependent Clp Proteases in Photosynthetic Organisms-A Cut Above the Rest![END_REF]. Highlighting the vital role of ClpCP-regulated protein turnover in photosynthetic organisms, loss of function of ClpC is lethal both in plants and cyanobacteria [START_REF] Clarke | ATP-dependent Clp Proteases in Photosynthetic Organisms-A Cut Above the Rest![END_REF].

Some plant ClpP homologs are encoded by the nuclear genome (nClpP) and others are encoded by the plastidial genome (pClpP). The function of pClpP was hypothesized to be vital, since it is one of the few genes still present in a parasitic plant plastidial genome [START_REF] Squires | The Clp proteins: proteolysis regulators or molecular chaperones?[END_REF]. While pClpP proteins are retained to plastids, at least some nClpP are targeted to mitochondria [START_REF] Sokolenko | Clp protease complexes and their diversity in chloroplasts[END_REF][START_REF] Clarke | ATP-dependent Clp Proteases in Photosynthetic Organisms-A Cut Above the Rest![END_REF][START_REF] Porankiewicz | New insights into the ATP-dependent Clp protease: Escherichia coli and beyond[END_REF]. Plants ClpX are also directed to this compartment, where they form ClpXP proteolytic complexes with nClpP, with similar structure and function as bacterial ClpXP complexes [START_REF] Halperin | Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease[END_REF]. Such ClpXP proteolytic complexes are found in the mitochondria of animals as well, suggesting it is an ancestral eukaryotic character [START_REF] Halperin | Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease[END_REF]

119

Clp proteolytic complexes acting in organelles are major players in one of the two coexisting main plant systems of bacterial origin for degrading aberrant or damaged proteins, together with the proteasome complex that is active in the cytosol and nucleus [START_REF] Ali | Protective Roles of Cytosolic and Plastidal Proteasomes on Abiotic Stress and Pathogen Invasion[END_REF]. Among systems of bacterial origin, Clp complexes are particularly important as loss of function mutants are often lethal [START_REF] Ali | Protective Roles of Cytosolic and Plastidal Proteasomes on Abiotic Stress and Pathogen Invasion[END_REF]. On the other hand, much like in bacteria, loss of function of Clp-ATPases that are not in proteolytic complexes (ClpB and ClpD) is not lethal in physiological conditions. However, their function becomes vital when the plant is subjected to stress.

Interestingly, while ClpB and ClpD gene number is low in both Arabidopsis thaliana and Physcomitrium patens, there is in the latter a noticeable expansion of the ClpC and ClpP encoding genes, but not of ClpX genes (table V-1).

Moreover, this expansion is not found for other protease systems of bacterial origin such as FtsH genes and Deg/HtrA genes (respectively around 20 members and 15 members for both species). This could mean that the ClpCP system in P. patens chloroplasts is particularly relevant to protein monitoring and turnover in P. patens.

120 configured for highest accuracy (MUSCLE with default settings). After alignment, regions containing gaps (and/or poorly aligned) were removed with Gblocks (v0.91b). The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT). The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.012) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (gamma=1.951).

Reliability for internal branch was assessed using the aLRT test (SH-Like). Graphical representation and edition of the phylogenetic tree were performed with TreeDyn (v198.3) [START_REF] Dereeper | Phylogeny.fr: robust phylogenetic analysis for the nonspecialist[END_REF].

V-G) SMXL proteins as ClpATPases

The first described protein of the SMXL family, D53/OsSMXL7, was found to be structurally similar to type I ClpATPases, even though the sequence is divergent [START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF] (figure V-2). Indeed, considering the whole groups taken as wholes, SMXL proteins and type I Clp-ATPases are only around 20 to 30% identical in sequence [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF]. Nevertheless, key structural elements such as Walker A and B motifs are conserved (figure V-3), implying that SMXL proteins could have a similar molecular function as type I ClpATPases [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF].

Moreover, the turnover of most SMXL proteins is likely dependent on their ATPase activity, as the stabilizing mutation of the degron motif is actually a loss of the second NDB Walker A motif. Such degron-dependent turnover has been proven at least for Arabidopsis and rice SMAX1 [START_REF] Khosla | Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2[END_REF]Wang et al., 2020a;[START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF] and SMXL6/7 [START_REF] Wang | Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation[END_REF][START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF][START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF]. Still, the most notable characteristics differentiating D53 from other Clp-ATPases are the presence of an EAR transcriptional repression motif between the two NBDs (see paragraph V-H) and D53 strictly nuclear subcellular localization (figure V-3). However, as made apparent by the limited sequence homology between plant type I Clp-ATPases and SMXL, SMXL clearly group independently from Clp-ATPases and they are also restricted to land plants (figure V-2). It is also unlikely that SMXL proteins take part in proteolytic complexes with ClpP proteases, as they lack the IGF/L interaction motif [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF]. Therefore, a possible molecular role of SMXL proteins would be to ensure the proper folding, and thus activity, of target proteins. Under this hypothesis, SMXL would have a similar molecular function as ClpB and ClpD, their specific sets of targets leading to very different outcomes. Notably, the role of D53/SMXL7 on inhibiting BRC1 transcription could be understood as a stabilization of the TPL co-repressor under an active conformation [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF]. On the contrary, we could imagine that SMXL interaction with their targets could sequester the targets in nuclear speckles (see paragraph V-H), hence rendering them inactive.

V-H) The multiple roles of SMXL proteins

In angiosperms, SMXL proteins can be subdivided into four functional types [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF][START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]: SMAX1/SMXL2, SMXL3, SMXL45 and SMXL678 (here named according to their respective Arabidopsis homologs). As written in chapter III, SMAX1 homologs are involved in KL signaling as repressors, while SMXL678

homologs have a corresponding role in the SL signaling pathway (see figures II-2 and III-4 from chapter III). On the other hand, SMXL45 homologs (at least in Arabidopsis) have been shown to act independently of SL or KL to promote early differentiation of phloem cells [START_REF] Wallner | Strigolactone-and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation[END_REF][START_REF] Wallner | SUPPRESSOR OF MAX2 1-LIKE 5 promotes secondary phloem formation during radial stem growth[END_REF], whereas the role of SMXL3 homologs has not been resolved yet. SMAX1 and SMXL678 contain a conserved RGKT motif (or degron) that has been shown to be necessary for their degradation [START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF][START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF][START_REF] Khosla | Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2[END_REF][START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF]. These proteins also contain the EAR (Ethylene-responsive element binding factor-Associated amphiphilic Repression) hydrophobic motif (LxLxL or [F/L]DLN), which permits interaction of SMXL with the CTLH domain of TPL/TPR (Topless/Topless Related) transcriptional corepressors [START_REF] Martin-Arevalillo | Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression[END_REF]. SMXL/TPL interactions are documented in Arabidopsis, where AtSMXL6 interacts with AtTPR3 (Causier et al., 2012a), while both AtSMAX1 and AtSMXL7 interact with several AtTPLs [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF]. This interaction has also been demonstrated in rice, where OsSMXL78/D53 interacts with all three OsTPLs [START_REF] Jiang | DWARF 53 acts as a repressor of strigolactone signalling in rice[END_REF]. It is interesting to note that SMXL from different functional subclades can interact with the same TPL, therefore these interactions do not completely explain the different effects triggered by the SL and KL pathways, as already noted by Soundappan and colleagues [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF]. Alternatively, it can be argued that most of SMXL/TPL interactions were demonstrated through in vitro approaches and might therefore not faithfully reflect what happens in planta. At least in rice, OsSMXL78/D53 also interact with IPA1/SPL14 [START_REF] Song | IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice[END_REF] (IDEAL PLANT ARCHITECTURE 1/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14), a transcription factor from the SPL family. This interaction enables D53 to block IPA1-induced TB1 (TEOSINTHE BRANCHED 1, the rice ortholog of BRC1)

expression. As such, IPA1 is hence a positive actor in the rice SL signaling pathway, downstream of D53. Furthermore, IPA1 can induce the expression of D53 itself, thereby mediating SL-triggered D53 increase in expression [START_REF] Song | IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice[END_REF]. This mechanism seems a conserved feature of monocots, as it was also demonstrated in bread wheat [START_REF] Liu | miR156-Targeted SBP-Box Transcription Factors Interact with DWARF53 to Regulate TEOSINTE BRANCHED1 and BARREN STALK1 Expression in Bread Wheat[END_REF]. However, in dicots, SPL transcription factors and the SL pathway seem to operate in parallel in the control of axillary branching, at least in Arabidopsis (Wang et al., 2020b) (Figure V-3). Nevertheless, the EAR motif is probably not necessary for all roles of SMXL proteins, as was shown for AtSMXL7 [START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF].

V-I) SMXL proteins phylogeny

The SMXL family evolutionary history appears quite complicated. Two recent studies on this subject came to the conclusions that SMXL genes are exclusively found in land plants and that they extensively diversified in the angiosperm lineage [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF][START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Outside of seed plants, only one type of SMXL is found, which is most similar to angiosperms and gymnosperms SMAX1 (aSMAX1 and gSMAX1). One notable exception to that is the presence of a second SMXL type in bryopsids mosses [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF] (that is, most of mosses), probably as the result of a Whole Genome Duplication (WGD) event (Bythell-Douglas et al., 2017) (see figure V-6).

Gymnosperms only possess two clades: gSMAX1 and gSMXL4 [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. gSMAX1 and gSMXL4 do not broadly differ from non-seed plants sequences and from one another, therefore they did not diverge a lot from the ancestral SMXL. On the contrary, SMXL are much more diversified in angiosperms, in which they fall into 4 clades corresponding to their demonstrated functions in Arabidopsis [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF][START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF]

(figure V-5).
Interestingly, phylogenetical classification of angiosperms SMXLs is also reflected by their gene expression profiles in Arabidopsis [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF]. As a matter of facts, AtSMAX1 is broadly expressed at similar (high) level except in roots, AtSMXL3 is mostly expressed in roots, AtSMXL7 is mainly expressed in axillary branches, and AtSMXL4/5 are lowly expressed everywhere and associated with the vasculature [START_REF] Stanga | SUPPRESSOR OF MORE AXILLARY GROWTH2 1 Controls Seed Germination and Seedling Development in Arabidopsis[END_REF][START_REF] Wallner | Strigolactone-and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation[END_REF][START_REF] Yang | Nuclear-localized AtHSPR links abscisic acid-dependent salt tolerance and antioxidant defense in Arabidopsis[END_REF]. It is puzzling that all Arabidopsis SMXL genes seem to be expressed in vascular tissues [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Wallner | Strigolactone-and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation[END_REF].

The following evolutionary scenario is predicted [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]: SMAX1 and SMXL4 superclades present in the seed plants common ancestor were duplicated in the angiosperms lineage: SMAX1 gave rise to aSMAX1 and 125 aSMXL78, while aSMXL4 and aSMXL39 arose from SMXL4. Further duplications in dicots produced SMXL7 and SMXL8, and SMXL3 and SMXL9. Emergence of SMXL2 from aSMAX1 duplication and SMXL5 from aSMXL4, as well as the loss of SMXL9, are specificities of the Brassicaceae [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. These increases in SMXL family size correlate well with known WGD events, notably the ζ WGD predating the split between gymnosperms and angiosperms, as well as the ε WGD at the base of the angiosperms lineage (Clark and Donoghue, 2018) (see figure V-6).

Figure V-5 -Phylogeny of SMXL proteins. Adapted from [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Angiosperms SMXL clades are named following Walker et al., 2019 (red) and Moturu et al., 2018 (blue). SMXL origin at the base of land plants, together with their structural similarity to ClpATPases, suggest that these proteins are indeed derived from a ClpATPase ancestor. The question as to why such highly neofunctionalized proteins were evolutionary conserved and then recruited in diverse processes including response pathways to two phytohormones (chapter III) is very intriguing.

Loss of the degron is specific to aSMXL39 and aSMXL4 [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF][START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. aSMXL39 and aSMXL4 are probably neofunctional respective to the ancestral seed plants SMXL4, because loss of the degron implies both increased stability and loss of the interaction with KAI2/D14. On another note, the EAR motif is very conserved in aSMXL39 and aSMXL4, thus they probably retain a transcriptional role [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF]. Moreover, they likely play similar molecular functions as other SMXL clades, since expression of AtSMAX1 under the AtSMXL5 promoter restores Atsmxl5 mutant phenotypes [START_REF] Wallner | Strigolactone-and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation[END_REF].

The degron (RGKT) and EAR motifs are probably ancestral features, the degron being probably responsible for interaction with proteins from the KAI2/D14 family (figure V-4) [START_REF] Shabek | Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling[END_REF][START_REF] Struk | Quantitative Tandem Affinity Purification, an Effective Tool to Investigate Protein Complex Composition in Plant Hormone Signaling: Strigolactones in the Spotlight[END_REF]. The ancestral SMXL was probably closer to aSMAX1 and gSMAX1 and was likely involved in KL signaling and degraded in a MAX2 dependent fashion (see chapter III). This function is also currently assumed for the lone SMXL homolog of nonseed plants [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Alternatively, gSMAX1 and non-seed plants SMXL proteins might be common, bifunctional targets of KL and SL signaling.

In order to settle between these two main hypotheses on SMXL function in mosses, we used P. patens as a model (chapter VI).

Introduction

Strigolactones are recently characterized phytohormones with an early origin in land plants evolution. These molecules have first been identified as rhizospheric signals with both negative and positive outcomes for the producing plant: strigolactones stimulate parasitic plants seed germination [START_REF] Cook | Germination of Witchweed (Striga lutea Lour.): Isolation and Properties of a Potent Stimulant[END_REF] but also promote AM symbiosis (Akiyama et al.) by boosting AM fungi mitochondrial metabolism and thus hyphae growth. Strigolactones are also employed as phytohormones in Angiosperms, where they have been linked to diverse roles in regulating plant architecture, in response to nutrient availability (see [START_REF] Waters | Strigolactone Signaling and Evolution[END_REF] for a recent review). One of the most extensively studied roles of strigolactones is their ability to repress axillary branching, by inhibiting axillary bud activity, denoting them as the sought-after second player in the apical dominance theory. Strigolactones involvement in mycorrhization and probably later in plant architecture imply that these molecules played major roles in land plants evolution, for land colonization and then for the emergence of Angiosperms. However, strigolactones roles and associated cellular pathways in early-diverging plants are pretty much unknown, whilst this knowledge is necessary to understand how strigolactones could have contributed to land colonization. Still, it has been previously reported that strigolactones repress filaments branching and elongation (Proust et al., 2011;Hoffmann et al., 2014) and enhance resistance to phytopathogenic fungi [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF] in the moss Physcomitrium patens (P. patens), showing this moss possesses a functional strigolactones signaling pathway. Phylogenetic studies suggest that strigolactones' biosynthesis pathway could be ancient, genes encoding most SL biosynthesis hormones being found both in land plants and some charales [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF]. Notably, it has been demonstrated that the CCD8 enzyme has the same role in P. patens as in the Angiosperm pea (Proust et al., 2011;[START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. But the conservation is more contrasted when considering the signaling pathway genes. As a matter of facts, it has been shown that the P. patens homolog of MAX2, a F-box protein that plays a key role in strigolactones signaling, is not necessary for response to strigolactones but is rather involved in the response to red light and possibly in KAI2-Ligand (KL) signaling (Lopez-Obando et al., 2018, chapter IV). This might extend to role of the ancestral MAX2 protein. SMXL678/D53 proteins, which are type I Clp-ATPases described as repressors of strigolactones signaling in Angiosperms [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Wang | Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation[END_REF], also have homologs in P. patens. In the former, these repressors must be degraded by the 26S proteasome, via the action of an SCF complex containing MAX2, for responses to SL to occur. Their degradation relies on their having a specific degron motif (RGKT) and their activity as repressors partly relies on an EAR motif mediating transcriptional repression.

Angiosperms possess two other subclades of SMXL proteins: the SMAX1/SMXL2 subclade is involved in the transduction of the KL signal in a MAX2-dependent pathway, while the SMXL345 subclade is involved in cell differentiation [START_REF] Moturu | Molecular evolution and diversification of the SMXL gene family[END_REF][START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Here, we show that PpSMXL proteins of P. patens possess a similar structure as subclades SMXL678 and SMAX1/SMXL2 proteins, with a conserved EAR motif and similar domain organization. Despite only two of them carrying a conserved degron motif, all four homologs are involved in the same pathway as PpMAX2 where they play a negative role downstream of PpMAX2. Moreover, two out of the four PpSMXL proteins could act as positive actor of SL signaling and thus constitute a level of crosstalk between this 129 pathway and the PpMAX2-dependent pathway. The finding that none of these SMXL homologs are repressors in the SL signaling pathway supports the hypothesis that SL sensitivity has an independent origin in moss and seed plants, even though the same family of receptors was recruited in both lineages (Bythell-Douglas et al., 2017, chapter IV).

130 On the other hand, the age of the plant had little effect for PpSMXLA. We could not obtain proPpSMXLB:GUS lines, as the PpSMXLB promoter region could not be amplified by PCR.

Nevertheless, using these GUS lines, we confirmed the difference of expression levels given by the P. patens eFP-Browser (Supplemental Figure VI-3, original data from Ortiz-Ramírez et al., 2016). We then investigated these genes' expression along vegetative development, using RT qPCR on samples from different organs: protonema at 6 days (mostly chloronema in our culture conditions), at 10 days (mix of chloronema and caulonema) and at 14 days (mix of chloronema, caulonema and buds), and mature gametophores and rhizoids at 35 days. Using this method, we could determine PpSMXLB level of expression and found that it is higher than that of PpSMXLA and lower than that of PpSMXLC and PpSMXLD (Figure VI-2B). We could not distinguish any significant expression differences across protonema development, for either of the four genes. However, PpSMXL genes all tended to be more expressed later,

Loss-of-function of all four PpSMXL genes causes a dramatic loss of viability

Our first mutagenesis strategy also granted us some higher order Ppsmxl mutants, of which we characterized one triple mutant (Ppsmxla1b1d1) and one quadruple mutant (Ppsmxla2b2c1d2). It should be noted that the Ppsmxlc1 mutation in the quadruple mutant probably does not cause a loss of function of PpSMXLC, as it does not induce a frameshift . The triple and quadruple mutants displayed highly restrained growth and browning of the protonema, and only rare stunted gametophores were produced (Supplemental Figure . Moreover, these higher order mutants grew few agravitropic curled caulonema filaments in the dark . These mutants appeared sterile, as the very few capsules formed were empty. Thus, it appears that loss of function of both clades has a dramatic effect on viability, maybe linked to an increase of senescence.

Due to the severe growth defect of these mutants, we could hardly work on them anymore after several rounds of vegetative propagation: even when cultures were restarted exclusively from green tissues, growth was increasingly difficult.

Loss of either clade A/B or C/D PpSMXL function leads to (-)-GR24 insensitivity, whereas only the loss of C/D function results in (+)-GR24 insensitivity

We then explored whether Ppsmxl(∆) mutants have a modified phenotypic response to (±)-GR24 enantiomers.

To investigate this response, we relied on the test of caulonema growth in the dark (Hoffmann et al. 2014, see also

Chapter IV). When treated with 1µM (+)-GR24, WT plants develop significantly less caulonema filaments, while 1µM (-)-GR24 treatment tends to increase filaments' number . Consistent with its lack of endogenous SL, the Ppccd8 mutant has more filaments than WT in control conditions, and (+)-GR24 treatment has a stronger repressive effect on caulonema number compared to WT. Response to (-)-GR24 is however either completely abolished in Ppccd8, or sometimes even tends to be reversed in some experiments . As for Ppmax2-1, it responds as Ppccd8 with a similar tendency towards a decrease in filaments number after (-)-GR24 treatment . When investigating the response of Ppsmxl double mutants to (+)-GR24, it appears that ab mutants can still respond to this molecule, apparently as much as the WT, while the response of clade C/D Ppsmxl double mutants is virtually abolished . We can also note that these double mutants develop much more filaments than WT or even Ppccd8 in the absence of treatment, supporting the negative role of PpSMXLC/D in filaments growth. When we look at simple mutants however (Supplemental Figure , we find that response to (+)-GR24 is not disturbed, suggesting PpSMXLC and PpSMXLD play redundant roles in this response. Moreover, simple Ppsmxlc mutants develop slightly but significantly more filaments than WT in control conditions, which indicates that PpSMXLC likely plays a predominant role over PpSMXLD in the regulation of protonema growth.

145 As for response to (-)-GR24, we can see that both clade A/B and clade C/D double mutants tend to be less sensitive than , while none of the simple Ppsmxl mutants responds differently to (-)-GR24

(Supplemental Figure . This observation leads to the hypothesis that all four PpSMXL genes play a redundant role in response to (-)-GR24 in this experimental setup (in the dark). Aiming to clarify the involvement of PpSMXL genes in the response to endogenous compounds, and investigating this at an earlier time point, we looked at the expression of SL-responsive genes in Ppsmxl mutants in the dark. We found that PpCCD7 expression level, which is highly increased in the Ppccd8 background (Proust et al., 2011) Interactions with DEFICIENS and GLOBOSA proteins from Antirrhinum majus are given as negative controls.

PpSMXL proteins interact with players of the PpMAX2-dependent pathway

In bimolecular fluorescence complementation (BiFC) assays, all four PpSMXL proteins interact with PpMAX2

when respectively fused to complementary parts of the eYFP fluorescent protein and overexpressed in the same Nicotiana benthamiana cells . Although the eYFP signal is quite low for PpSMXLA and PpSMXLC, we can notice a nuclear interaction of both with PpMAX2 (the previously published GLOBOSA/DEFICIENS interaction was herein consistently used a positive control of eYFP reconstruction, see [START_REF] Bouchez | Arabidopsis TONNEAU1 Proteins Are Essential for Preprophase Band Formation and Interact with Centrin[END_REF]. Interestingly, we also found that PpSMXLB and PpSMXLD could potentially form homo-oligomers. On the other hand, formation of PpSMXL hetero oligomers between themselves remains to be investigated fully, nonetheless we have already found that PpSMXLA was not involved in such structures. In order to confirm these interactions, we tested them in vitro by yeast two hybrid (Y2H) assays, where interactions are semi-quantitatively revealed by blue coloration. Unfortunately, PpMAX2/PpSMXL interactions were not observed in yeast, using either protein as the bait or the prey (Supplemental Figure . Moreover, PpMAX2/PpSMXL interactions were not triggered in this system by addition of (±)-GR24

(even at the high concentration of 50µM). We could not replicate the homo-interactions of PpSMXLB and PpSMXLD either and, while there was a PpSMXLB/PpSMXLD interaction, it was interestingly weakened in the presence of (±)-GR24 (Supplemental Figure . This interaction is reliable only with PpSMXLD taken as the bait, as slight autoactivation was observed when using PpSMXLB as the bait.

Using the same two techniques, we investigated the interactions of PpSMXL proteins with PpKAI2L proteins.

Among the PpKAI2L proteins we tested (at least one from each clade: PpKAI2L-C, -G, -J, -F, -H, -L), only PpKAI2L-C could interact with PpSMXL proteins, moreover the four of them . Here again, we had some issue with the replication of these results in Y2H (Supplemental Figure . Indeed, we had a severe autoactivation problem with most of clade A-E PpKAI2L when taken as baits. Still, we did note that blue coloration was darker when PpSMXLB/C/D (B especially) were used as preys, thus suggesting an interaction between PpSMXLB/C/D and clade

A-E PpKAI2L as a whole. This seems consistent with our previous demonstration that PpSMXL proteins act in the PpMAX2-dependent pathway, since clade (A-E) PpKAI2L genes are also involved in this pathway (see chapter IV).

Additionally, we found PpKAI2L-F appeared to also interact with PpSMXLB/C/D (Supplemental Figure VI-24), which was not seen in biFC. Another new interaction was PpSMXLB/PpKAI2L-K (Supplemental Figure VI-24), which we did not explore in biFC. Globally, it is interesting to take note that all these interactions are not dependent on (±)-GR24 addition. This implies either that they do not need a PpKAI2L-perceived ligand to occur, or that this ligand (or molecule(s) with the same activity and close structure) is present in yeast. Both biFC and Y2H revealed only weak interactions involving PpSMXLC (despite both methods using overexpression of proteins of interest), while it seems to be the main PpSMXL player in P. patens, given mutants phenotypes.

PpSMXLC interactome reveals a major implication in regulation of photosynthesis and protein turnover

In order to clarify the interactions of PpSMXLC, we used a GFP-trap pull-down strategy to recover proteins interacting in planta with GFP tagged PpSMXLC, with or without a 6h 3µM (±)-GR24 treatment, and then identified signature peptides recovered with GFP-PpSMXLC by MS/MS . Using this without a priori method, we did not recover PpMAX2/PpSMXLC or PpKAI2L/PpSMXLC interactions, suggesting that these interactions are indeed transient and/or too weak to endure the experimental procedure of protein complex recovery here. However, we found 7). One could argue that the consensus RGKT sequence is also modified in some flowering plants SMXL where the motif is nonetheless functional (OsSMAX1 [START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF], however among sequences examined, PpSMXLA/B are the only one lacking the K residue, which is functionally relevant in Walker A motifs as it is necessary for phosphate binding [START_REF] Bianchi | A synthetic hexapeptide designed to resemble a proteinaceous p-loop nest is shown to bind inorganic phosphate[END_REF]. Inversely, the EAR motif is conserved in all four PpSMXL proteins, thus transcriptional regulation could be a uniting feature between the two clades.

Additionally, expression level of PpSMXLA/B is lower than that of PpSMXLC/D in protonema.

PpSMXL proteins function at least partly involves the nuclear compartment

The presence of predicted NLS sequences, as well as an EAR motif in the four PpSMXL proteins hint at a nuclear localization (Supplemental Figure . Indeed, all four PpSMXL are located in the nucleus, albeit not only (Figure 7,8 and 9). Still, this partial nuclear localization is enough to enable the interaction of PpSMXLs with proteins in this organelle (PpMAX2 and PpKAI2L-C, Figures . It would be interesting in the future to explore whether this EAR motif grants PpSMXL proteins a function in transcriptional regulation. Notably, we could determine if they are able to interact directly with DNA, like has recently been demonstrated for AtSMXL6 (Wang et al., 2020b), and which loci are targeted. Such effect could also 158 be reliant on PpSMXL interaction with TOPLESS transcriptional repressors (TPL) and we accordingly project to test the interaction between PpSMXL and the two TPL homologs from P. patens.

PpSMXL proteins are not highly sensitive to SL levels and their sensitivity to KL probably depends on light

We report herein that none of the four PpSMXL proteins are rapidly degraded in response to (+)-GR24, not even PpSMXLA/B which transcripts are increased by treatment with this enantiomer in the dark (supplemental Figures . The absence of (+)-GR24 triggered degradation is probably not the result of an insufficient amount of (+)-GR24 and/or treatment duration, as (1) AtSMXL6 is almost completely degraded after 20 minutes of (±)-GR24 2µM in p35S:AtSMXL6-GFP seedling [START_REF] Wang | Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation[END_REF] PpMAX2 is involved in the induction of PpSMXL expression, while PpSMXL proteins are likely targets of PpMAX2-dependent degradation. Therefore, PpSMXL could constitute a level of negative feedback regulation in the PpMAX2-dependent pathway. Since this induction by PpMAX2 is especially prevalent in the dark, negative feedback might be more active in the absence of light. On the other hand, the short-term induction of PpSMXLA/B/C by red light also relies on PpMAX2. Hence the PpMAX2-dependent regulation of PpSMXL expression needs to be further clarified.

PpSMXL could be a bridge linking SL signaling and PpMAX2-dependent signaling

It is interesting to note that "de-repression" of PpMAX2-dependent signaling (Ppsmxlcd mutants) and "silencing" of SL signaling (Ppccd8 mutant) have very similar effects on developmental pattern of the protonema (more extended) and gametophores (smaller) . This, together with the opposite effects of (±)-GR24 enantiomers on dark grown caulonema in WT, indicates that these two pathways likely regulate the same processes in opposite manners. One of these processes, as demonstrated for the Ppccd8 mutant (Hoffmann et al., Cloning of PpSMXL CDS and promoters. Coding sequence of each PpSMXL gene was amplified on WT P. patens Gransden cDNA, using Phusion DNA polymerase (ThermoFisher), following provided instructions and using primers with attB1 and attB2 extensions (respectively on the forward and reverse primer). A similar strategy was used to amplify promoter sequences (full 5'UTR and 1000 bp upstream, in V3.1 sequences available on Phytozome). Both CDS and promoters were then integrated into the pDONR207 plasmid using BP clonase II mix (Thermofisher). pDONR207 plasmids containing PpSMXL CDS were submitted to PCR-mediated mutagenesis to obtain ΔRGK/RT versions.

CRISPR-Cas9 mediated mutagenesis. Coding sequences of PpSMXL and PpMAX2 were used to search for CRISPR RNA (crRNA) contiguous to a PAM motif recognized by Streptococcus pyogenes Cas9 (NGG), using the webtool CRISPOR V4 against P. patens genome Phytozome V9 (http://crispor.tefor.net/). crRNAs located in the first third of the coding sequence, with highest possible specificity score, and fewest possible predicted off-targets, were selected.

Small constructs containing each crRNA fused to either the proU6 or the proU3 snRNA promoter in 5' U3 or U6

promoter [START_REF] Collonnier | CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51independent gene targeting in the moss Physcomitrella patens[END_REF], and to the tracrRNA in 3', encased between attB1/attB2 GateWay recombination sequences, were synthesized by Twist Biosciences. These inserts were then cloned into pDONR207 vectors.

Polyethylene glycol-mediated protoplast transformation was performed with multiple pDONR207-sgRNA according to the protocol described by Lopez-Obando et al., 2016. Mutations of the PpSMXL genes were confirmed by PCR amplification of PpSMXL loci around the recognition sequence of each guide RNA and sequencing of the PCR products.

Alternatively, for PpSMXL genes, a second strategy was employed where crRNAs were designed in the 5' and 3'UTR sequences, to completely remove the coding sequence of PpSMXL genes from the genome when used together. Mutants obtained from this second strategy were genotyped by monitoring the size and sequence of amplicons spanning from the 5'UTR to the 3'UTR.

Generation of proPpSMXL:GUS, proZmUbi:GFP-PpSMXL and control proZmUbi:flag-GFP lines.

proPpSMXL:GUS constructs were obtained by LR recombination of pDONR207 plasmids containing PpSMXL promoters with the pMP1301 destination vector previously described [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. This method could not be employed for the promoter of PpSMXLA, which had to be amplified with a NotI forward primer and a AscI reverse primer and was subcloned into the pTOPO-blunt II vector (ThermoFisher), and then directly inserted into a NotI-AscI digested pMP1301. proZmUbi:GFP-PpSMXL constructs were obtained by LR recombination of pDONR207 plasmids containing PpSMXL coding sequences with the pMP1335 destination vector (http://labs.biology.ucsd.edu/estelle/Moss_files/pK108N+Ubi-mGFP6-GW.gb). Similarly, the proZmUbi:flag-GFP construct was obtained using the pMP1382 destination vector. These plasmids were used independently to transform WT P. patens Gransden, together with pDONR207 containing sgRNA recognizing Pp108 homology sequences contained in the three pMP vectors and appropriate Cas9 and selection plasmids (Lopez-Obando et al., 2016b). Obtained G418 resistant lines were screened for insertion using PCR (with proPpSMXL forward and GUS reverse, GFP forward and PpSMXL reverse, or proZmUbi forward and GFP reverse primers, respectively).

GUS staining. Two to six independent G418 resistant lines with verified GUS insertion into the genome were obtained for each PpSMXL genes except PpSMXLB and used for histochemical analyses. GUS staining of two-week-old P. patens plants was carried out following the protocol detailed by Yuji Hiwatashi on the NIBB PHYSCObase website (http://moss.nibb.ac.jp/protocol.html). As we have seen in caulonema growth assays that the effects of (-)-GR24 are often quite discrete compared to those of (+)-GR24, we switched to more sensitive protoplasts regeneration assays to assess the effects of separate (±)-GR24 enantiomers (Lopez-Obando et al., 2018) (figure VII-2). This change also enabled us to keep using low concentrations of enantiomers (3, 30 and 300 nM). Moreover, by slightly modifying our previous protoplasts preparation protocol, we could obtain enough robust protoplasts for Ppmax2-1.

(+)-GR24 had a dose-dependent repressive effect on regeneration from protoplasts at 72 hours, both in WT and Ppccd8 (figure VII-2A). Strangely, unlike in caulonema growth tests, while Ppccd8 regenerated better than WT in the absence of treatment, it did not respond more strongly to (+)-GR24, similarly to what was noted in phyllids regeneration tests carried out in the light (figure VII-1A). On the contrary, a higher dose was necessary to induce a significant drop in regeneration. This could be explained by the different light conditions. Indeed, both in transcriptional response and caulonema growth assays, which are monitored in the dark, Ppccd8 has a clearer response to (+)-GR24 compared to WT. Hence, we can hypothesize that the well-documented positive impact of light on protoplast regeneration [START_REF] Jenkins | Light requirements for regeneration of protoplasts of the moss Physcomitrella patens[END_REF]) is somehow more intense in Ppccd8 than in WT and thus that PpCCD8-derived compounds interfere with this response to light in WT. (-)-GR24 also had a dose-dependent effect on protoplasts regeneration in WT (figure VII-2B and D). As we hypothesized, this effect was opposite to that of (+)-GR24 and its amplitude was lower, possibly because (-)-GR24 also stimulate the SL pathway (see chapters IV and VI). Response of Ppmax2-1 was impossible to assess after only 72 hours of regeneration (figure VII-1A and B). Therefore, we observed the protoplasts again after an additional 72 hours (6 days in total, figure . At this later time point, the opposite response of WT to both enantiomers was still evident, and we could note that Ppmax2-1 does respond to (+)-GR24 (figure VII-2C) but this response is decreased compared to WT. On the other hand, Ppmax2-1 does not respond to (-)-GR24 (figure VII-2D).

The dose-dependent response to (±)-GR24 enantiomers in protoplasts regeneration assays apparently validates our previous hypothesis that, (±)-GR24 has a non-dose-dependent effect in phyllids regeneration assays because of the influence of the (-)-GR24 enantiomer. However, this phenomenon could also stem from a bias specific to our phyllids regeneration assays, since we previously noted a dose-dependent effect of (±)-GR24 in protoplasts assays [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. Hence, we had to fine-tune the experimental setup for phyllids regeneration assays, notably carrying them out in the dark where response to (+)-GR24 become dose-dependent (see methods chapter in the annex).

Until now, phyllids assays could not reveal the effect of (-)-GR24, which suggests that these assays are better suited to study the effect of SL mimics rather than that of KL mimics.

Although regeneration from phyllids and protoplasts can be broken down into two main processes (dedifferentiation followed by cell division) and our experiments cannot really determine which out of the two is impacted by treatment with (±)-GR24 enantiomers, we can infer that at least the cell division process is affected. Indeed, cell division is specifically affected in opposite ways by the two enantiomers in dark grown caulonema (see chapters IV and VI, as deduced from the number of caulonema filaments). Moreover, oppositely modified cell division rates in the Ppccd8 and Ppmax2-1 mutants could also explain their respective protonema growth in control long-days conditions.

These experiments moreover underline the major impact of light on response to (±)-GR24 enantiomers. Indeed, contrary to what we thought until now based on tests using the (±)-GR24 mixture and from testing the enantiomers on caulonema growth in the dark, (-)-GR24 does have a dose-dependent effect on WT, which is opposite to the effect of (+)-GR24. The effect of the (-)-GR24 enantiomer could be clearer in light conditions, which might extend to the effect of endogenous KL and of karrikins. Thus, it would be interesting to check whether protonema extension in control longdays conditions is affected in response to (-)-GR24 and KAR2 addition, in a dose-dependent manner.

VII-B) Higher order Ppsmxl loss of function mutants have a decreased regeneration ability

As both SL and KL signaling appeared to play a role in regeneration, we investigated the ability of Ppsmxl mutants phyllids to regenerate in the light, in the absence of GR24 treatments. Ppsmxlab double mutants tended to regenerate more slowly than WT, whereas Ppsmxlcd double mutants appeared to regenerate more quickly (figure . This tendency for Ppsmxlcd was supported by our previous observations that, in the process of obtaining Ppsmxlc/d mutants, WT protoplasts transformed with guide RNAs targeting PpSMXLC and PpSMXLD regenerated better than untransformed controls. Higher order Ppsmxl mutants were much more affected than double mutants and their regenerative ability was decreased. This observation is quite difficult to explain, as higher order mutants' phenotype does not correspond to an additive effect of the mutation of clades A/B and C/D. However, since we have established before that these higher order mutants have a highly restricted protonema growth, certainly linked to a senescence phenotype, it is possible that this senescence interferes with the regeneration process here.

The observation that both clades have apparently opposite roles in regeneration is quite surprising considering our previous finding that both clades similarly act as negative actors in the PpMAX2-dependent pathway. However, since both clades are not preferentially expressed in the same tissues and life-stages, the effects of PpSMXL function loss we report in this experiment might not hold biological significance in physiological growth. Still, this finding does raise questions about potential different interactors between the two clades, that could explain this opposite outcome on regeneration. The regeneration ability of Ppsmxl simple mutants should also be investigated to clarify the role of each PpSMXL gene in the context of regeneration. Protoplasts regeneration of Ppsmxl mutants will also be assessed, moreover in response to (±)-GR24 enantiomers, as we found that this type of assay is more sensitive than phyllids regeneration.

201 In order to determine the physiological implications of these gene expression profiles, we investigated the growth of Ppsmxl mutants in cold stress conditions (at 10°C) and after a one-week recovery period (at 25°C) (our experimental setup is illustrated in panel A of figure VII-5). Our starting basis was that tolerance to the cold treatment would be reflected by a moderate decrease in growth during the cold period and a rapid resumption of growth after return to control conditions.

203

204

The Ppccd8 mutant had a higher initial loss of growth (after 1 week in cold conditions) but then an equivalent relative limitation of growth as WT during the second week. Moreover, its recovery was better than WT (as shown by the slope of the second part of the curve in figure . This behavior implies that PpCCD8-derived compounds (and by extension SL) initially repress growth limitation in reaction to cold and limit recovery but do not impact the acclimation ability. Therefore, PpCCD8-derived compounds tend to keep growth stable when temperature changes, which could be a form of tolerance to cold. On the other hand, growth of Ppmax2-1 was only slightly more limited than WT initially but decreased much more after the second week in cold conditions. However, recovery seemed equivalent to WT. Together, these observations suggest that PpMAX2 limits decreases in growth in the second part of our cold treatment. Thus, PpMAX2 could play a role in acclimation to cold.

As suggested by the four genes' expression profiles, simple mutants of the same clade did not display the same response to the cold-stress treatment (figure VII-5B and Supplemental Figure VII-3). Starting with clade A/B, the Ppsmxla5 simple mutant growth was less restricted by cold compared to WT during the first week, however it was more affected during the second week. Furthermore, its recovery slope was less steep than for WT. Hence, it seems that PpSMXLA rapidly limits growth in cold conditions and is necessary for acclimation to cold and then for quick recovery when stress is alleviated. The simple PpsmxlΔb7 mutant is only slightly more impacted than WT by cold. However, during its recovery, slope is comparable to that of Ppsmxla5 (see percentages given in table VII-1). Therefore, while

PpSMXLB could play a minor opposite role to PpSMXLA in cold-triggered limitation of growth, it most importantly plays an equivalent role in promotion of recovery. The double Ppsmxla8Δb6 mutant indeed was more alike Ppsmxla5 in its reaction to cold stress, underlining the predominant effect of PpSMXLA. Moreover, recovery of the double mutant was completely abolished, illustrating an additive action of PpSMXLA and PpSMXLB during this process. It is surprising to observe such a major effect of PpSMXLA loss of function by itself. Indeed, since this protein is virtually not expressed except in spores, we initially thought that this gene might be in the process of pseudogenization. However, PpSMXLA function might be very relevant in particular cases such as response to stresses. While we did not investigate yet the role of PpSMXL genes in tolerance to desiccation, it would be very interesting to do it soon, as all four PpSMXL are highly expressed in spores which are desiccation-resistant cells.

205 When we focused on the C/D clade, we found that the PpsmxlΔc4 simple mutant growth was initially more decreased by cold than for WT, but this mutant eventually resumed growth (see the 2 weeks timepoint). This suggests that PpSMXLC function is important to maintain growth during cold stress, but that role becomes reversed under longterm cold stress. This suggests that acclimation to cold is repressed by PpSMXLC in WT plants. The recovery of PpsmxlΔc4 was only slightly decreased compared to WT, so PpSMXLC might not play an important role during this step. As for the Ppsmxld1 mutant, its reaction to cold stress was almost identical to that of WT, but recovery was completely abolished. The capital function of PpSMXLD for recovery to cold-stress fits with the induction of this gene expression by cold. We could hypothesize that the PpSMXLD protein accumulates in cold-stressed plants and acts as a chaperone to either protect proteins necessary for recovery or maybe prevents the action/accumulation of defective proteins that could hamper recovery. The PpsmxlΔc7Δd4 double mutant was initially more affected by cold than WT, even more than PpsmxlΔc4. Under long-term cold stress however, it reacted similarly to WT, like the Ppsmxld1 mutant.

Finally, PpsmxlΔc7Δd4 recovered better than WT and both simple mutants of the C/D clade. Therefore, the simultaneous loss of PpSMXLC function alleviates the dramatic effect of PpSMXLD loss of function on recovery.

Strikingly, the phenotype of PpsmxlΔc7Δd4 is here again akin to the SL deficient Ppccd8 mutant, suggesting the molecular processes disturbed in the two mutants are the same. This observation is very puzzling because we have seen that loss of PpCCD8 function results in increased transcript levels of PpSMXLC and PpSMXLD (chapter VI).

Alternatively, similar reaction to cold of Ppccd8 and PpsmxlΔc7Δd4 could be explained by the prevalence of protonema over gametophores in these two mutants. It is also worthy of note that Ppsmxl mutants and Ppmax2-1 do not display opposite responses to cold, which could imply that in these conditions PpSMXL proteins exert PpMAX2-independent roles.

Relative loss week 1 Relative loss week 2 Relative gain recovery

WT -28% -17% 10% Ppccd8 -37% -17% 17% Ppmax2-1 -33% -20% 13% Ppsmxla8∆b6 -17% -21% 0% Ppsmxl∆c7 ∆d4 -39% -15% 17% Ppsmxla5 -15% -22% 6%
Ppsmxl∆b7 -33% -16% 5%

Ppsmxl∆c4 -35% -11% 8%

Ppsmxld1 -26% -18% -1%

Table VII-1 -Comparison of growth in response to cold. Percentages of relative growth loss/gain were calculated as the difference of growth between two successive time-points. These values are proportional to the slopes of curves given in figure VII-5. Differences with WT equal or superior to 5% are deemed significant: green = higher loss/gain than WT, red = lower loss/gain than WT.

VII-D) PpSMXL proteins play important roles in regeneration and stress tolerance

We found that loss of Ppsmxl function, and furthermore disturbance of either the SL pathway (in Ppccd8) or the KL pathway (in Ppmax2-1), has major implications on regeneration and growth regulation in response to cold stress.

However, while the effects of the two pathways are clearly opposite in the case of regeneration, their respective roles in response to cold is more ambiguous.

In order to clarify these findings, we must replicate the cold stress experiment (notably including the triple Ppsmxla1b1d1 mutant) and to devise similar experiments to assess the effect of Ppsmxl mutations in the context of heatstress and in the context of dehydration. As for regeneration assays, we ought to carry them out on simple Ppsmxl mutants in order to determine the role of each PpSMXL gene in this process. As the cold stress experiment showed, these genes might have different functions even within the same clade. Given the complex impact of combinations of Ppsmxl mutations, one possible explanation is that the PpSMXL proteins might be able to interact together, maybe forming oligomeric complexes as other Clp-ATPases (see chapter V). We can imagine that the different complexes have slightly different function, putatively by interacting with different sets of target proteins. Also, it is worthy of note that these functions of PpSMXL proteins are exerted when there is no KL signal transduction, and reinforced in the presence of SL in the case of PpSMXLC/D. Hence, SL can be read as stress hormones in P. patens (as was suggested already in Angiosperms), while KL would be a growth promoting signal limiting stress response.

Interestingly, we show here that PpSMXLC seems to have a divergent role compared to the other three PpSMXL Ppccd8 mutant, the PpKAI2L(JGM)-dependent pathway is off (in the absence of exogenous SL or mimics). Thus, PpSMXLC/D are not as stabilized/activated as in WT and PpSMXL levels/activity is globally decreased. This leads a de-repression on protonema growth and therefore Ppccd8 develops extended protonema. In Ppsmxlabd triple mutants, the effect of SL is lost and the putative specific function of PpSMXLC in senescence promotion is not downregulated by the other PpSMXL anymore. Senescence eventually even overruns the effects of de-repression on protonema growth.

In Ppsmxl simple mutants, no major developmental phenotype like in Ppccd8 is observed because PpSMXL proteins play a redundant role in preserving the protonema/gametophores balance. Nonetheless, the slightly more affected Ppsmxlc mutants reveal that PpSMXLC has a prominent effect on this developmental balance. In the double Ppsmxlcd mutants, the stabilizing/inductive effect by SL signaling is lost. Subsequent decrease of PpSMXLC/D activity leads to a de-repression on protonema growth, replicating the phenotype of Ppccd8. The slight effect of PpSMXLA/B cannot ensure sufficient repression of protonema growth in the absence of PpSMXLC/D. Consequently, in the double Ppsmxlab mutants, no major effect is noted on the protonema/gametophores balance, however other phenotypes might be disturbed (notably spore germination given PpSMXLA/B expression patterns). Treatment with the unspecific (-)-GR24 enantiomer in Ppmax2 further limits protonema growth because it can only be perceived through the SL signaling pathway, which leads to even higher PpSMXLC/D levels/activity. Hence, (-)-GR24 has an opposite effect on caulonema number on WT and Ppmax2-1. In Ppccd8, PpKAI2L(JGM) receptors are not occupied by endogenous SL, thus (-)-GR24 can easily "spill out" on the SL signaling pathway, here again leading to an opposite effect on caulonema number compared to WT.

VIII-B) Current views on SL signaling evolution

Specific receptors (KAI2/DDK) and SMXL proteins associated with either the KAR/KL pathway or the SL pathway seem to be an Angiosperms innovation (Bythell-Douglas et al., 2017;[START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Indeed, the canonical D14 SL receptor appeared after the emergence of seed plants, and the SMXL was only recently expanded in Angiosperms. This raises several questions: (1) Did ancestral land plants perceive and transduce the SL signal? (and if yes, how?); (2) How can SL perception and transduce occur in extant SL-sensitive non-Angiosperms?

It is important to note that, for the moment, SL sensitivity outside of Angiosperms is only documented in P.

patens, albeit SL biosynthesis is shared by all land plants and even some algae. Indeed, while other studies explored the effects of exogenous (±)-GR24 on development in other Bryophytes or even in algae, the use of the racemic mixture does not permit to known if it is actually an effect of SL that is observed or a stimulation of the KL pathway [START_REF] Delaux | Origin of strigolactones in the green lineage[END_REF].

In the moss P. patens, two main, non-exclusive, hypotheses can explain SL sensitivity: (1) Multi-functionality of ancestral DDK and SMXL proteins, which has been conserved in P. patens lineage; (2) Convergent evolution: DDK and SMXL homologs were independently recruited to act in SL signaling in P. patens lineage and Angiosperms.

Previous studies have already supported the convergent evolution hypothesis in the case of KAI2/DDK receptors (Lopez-Obando et al., 2016a;Bythell-Douglas et al., 2017). Indeed, in bryopsids mosses, the expansion of the KAI2L protein family following WGD would have been followed by a neofunctionalization event much like in parasitic mechanism enables accumulation of the molecule [START_REF] Waters | Quorum sensing: Cell-to-cell communication in bacteria[END_REF]. Therefore, we could argue that SL in the medium act more as a negative allelopathic signal.

Still, as we have seen in chapters IV and VI, as well as was already suggested by previous studies in P. patens (Proust et al., 2011;[START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF], SL (or rather PpCCD8 derived compounds) actually have an hormonal activity in this species. Indeed, while SL are liberated into the medium where they affect other individuals of the same species (Hoffmann et al., 2014), they also hold an endogenous effect. Indeed, the Ppccd8 SL deficient mutant displays its extension phenotype even when grown alone, which shouldn't be the case if SL were only relevant in the environment.

Along its evolutive history, P. patens lost the SL biosynthesis gene MAX1 [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF], most probably involved in the specific generation of canonical SL. Canonical SL have been shown to be the major actors in the positive role of SL towards AMF symbiosis, which might be linked to their increased stability relative to non-canonical SL, likely making them slightly more persistent in the soil (Yoneyama et al., 2018b). Thus, the loss of this canonical SL biosynthesis gene seems coherent with the lack of AMS ability in this moss. However, loss of MAX1 is not common to all mosses [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. Thus, considering MAX1 as a purely symbiosis associated gene might be far-fetched.

Indeed, when MAX1 activity is not followed up by another enzyme (see chapter III), non-canonical SL might be the end-product of SL biosynthesis, as shown in Arabidopsis and hemp [START_REF] Abe | Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro[END_REF][START_REF] Huet | Populations of the Parasitic Plant Phelipanche ramosa In fl uence Their Seed Microbiota[END_REF] (see chapter IV).

It was suggested that two main drivers can explain the loss of AMS and associated genes: a nutrient rich ecological niche (makes symbiosis too costly for a meager usefulness), or a high pathogen pressure (makes intracellular symbiosis risky as it can be diverted by pathogenic micro-organisms) [START_REF] Radhakrishnan | An ancestral signalling pathway is conserved in intracellular symbioses-forming plant lineages[END_REF]. We cannot know whether any of these happened in P. patens evolutive history. The loss of CCD7 and CCD8 in P. patens evolutive history would have been prevented by the emergence of a new pathway responding to SL, after neofunctionalization of KAI2L proteins. Furthermore, SL biosynthesis confers resistance against phytopathogenic fungi [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF], so it is tempting to assume that CCD7 and CCD8 were kept because of a selection pressure from pathogenic origin.

Given these considerations, we can hypothesize that the ability to excrete SL into the medium is ancestral, as it is needed for inducing symbiosis, and was conserved in P. patens lineage with another purpose: co-regulation of growth across a local population. That might permit this species to limit intraspecific competition and instead redirect growth (protonema extension) towards patches where this species is absent, thus increasing its ability for rapid colonization of the environment. Moreover, it is interesting to note that induction of CCD7 expression in low phosphate growth conditions is a conserved feature also reported in P. patens [START_REF] Decker | Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens[END_REF]. Thus, we could imagine that SL in this species inhibits growth of the protonema and hastens switching to the reproductive stage when the medium contains suboptimal amounts of inorganic phosphate, thereby adapting growth to the soil phosphate content.

For the moment, we can only hypothesize that the same reflection can be applied to other extant mosses (except Takakia), as experimental evidence is lacking.

VIII-D) About the ancestral role(s) of SL

To date, there is not clear element refuting an ancestral role of SL as interspecific rhizosphere signals, although no consensus has been reached. These molecules would have been recruited as phytohormones later in the ancestry of seed plants, possibly under a new selection pressure faced by these plants. Indeed, players of the canonical SL signaling pathway seem to have been acquired all together either in seed plants or later in the Angiosperms lineage (this cannot be resolved yet owing to the inexistent data on SL response in Gymnosperms). The nature of this new selection pressure might be linked to the increasing complexity of branching forms/meristems. Moreover, outside of bryopsids mosses, most bryophytes are able to accommodate fungal symbionts, including the divergent moss Takakia, suggesting this ability is indeed ancestral and was lost in the moss lineage after the divergence of Takakia [START_REF] Wang | Phylogenetic distribution and evolution of mycorrhizas in land plants[END_REF]. Given this unique role in symbiosis, there was no need for a SL signaling pathway in ancestral land plants.

Introduction

In this chapter, we present a scaled-up and economical twist for two widely used methods for characterizing hormones' effects on the moss Physcomitrella patens (P. patens). We also introduce a new experiment that can demonstrate even slight phenotypic response to hormones. The first method relies on vertical growth of the moss in the dark, which triggers specifically the elongation of caulonemal filaments upwards (negative gravitropism, as we previously described (1).

Also see (2) for a recent review). Growth in the dark also enables to get rid of the potential interference of light with the response to strigolatone (SL)-like molecules of interest (1). We previously showed (1,3) that both natural SLs as well as widely used SL synthetic analogues enantiomeric mixtures such as rac-GR24 repress caulonema growth in a dosedependent way in such experimental set-ups. The second method aims at testing the transcriptional response of P. patens to SL-like molecules of interest. It involves growth and subsequent incubation of P. patens tissues with the molecule(s)

This annex is a method chapter that is to be published in the Methods in Molecular Biology book series.

It details some of the experiments used to generate results presented in chapters IV, VI and VII.

1 µM (picture on the right). You can note that this molecule decreases both the number and length of caulonema filaments, in a dose-dependent manner. Indicated percentages reflect the effect of the molecule on the number of caulonema filaments.

Testing P. patens transcriptional response to SL-like compounds

1.

Prepare the 6-well plates: 3-4 plates per genotype and for three treatments (including the control one). You will need to pour 6 mL of PpNO3 medium in each well. Let the medium polymerize under the hood with the plate's lid off.

3.

Ensure the medium is completely polymerized by slightly shaking the last plate you poured. After checking that, you can place the cellophane disks in the wells: put sterile MilliQ water in the sterile container with your cellophane disks. Using sterile pliers take one cellophane disk at a time from the water and try to lay it flat on the medium in the well (without trapping air bubbles underneath). Store your closed multi-well plates under the hood and proceed to step 4 as soon as possible.

4.

Start cultures: In each well of the 6-well plates, deposit ~ 500 µL of freshly ground tissues. Whenever you finish a plate, put the lid back on, seal it with Micropore tape and carefully swirl the plate to evenly distribute the tissues in the wells.

5.

Incubate the plates in the culture chamber for ~2 weeks. Check from time to time that there are no contaminations under the binocular.

6.

Transfer the plates in the dark and let the tissues grow for at least one more week under the same temperature and hygrometry conditions (see Note 5). Panel B displays the ectopic regeneration of a phyllid that was wounded during handling. The black star underlines ectopic hollowing of cells that precedes regeneration of a filament. In both panels, regeneration loci are pointed by black arrows.

Notes

1.

The agar must have high water-retaining ability, especially for method 3.1 where mosses are grown vertically, also its salt composition should be adapted to plant culture (for instance, we use 10 g/L Phytoblend agar from Caisson labs).

2.

Acetone can also be used but tends to evaporate and thus solutions must be used immediately in this case.

3.

Light grinding with a mortar and pestle in a small volume of MilliQ sterile water can also be employed.

Fragmented tissues can then be transferred to a sterile tube or pot.

4.

If you re-start your cultures from spores (advised after numerous cycles of fragmentation, e.g. once a year) it will take much longer to obtain protonema tissues at the proper stage. Usually, spores take 1-2 weeks to germinate and need at least 1 additional week to give rise to a sufficient amount of protonema. Then, the protonema needs at least one cycle of grinding followed by a 1 week-long culture before you can use it in your experiment). Abstract: Strigolactones (SL) make up a novel class of phytohormones that are found across the whole land plant lineage. In vascular plants, the main hormonal role of SL is the repression of shoot axillary branching. However, SL are also a major symbiotic signal, granting the plant increased access to the nutrients and water contained in the rhizosphere. These two functions of SL led to the hypothesis that these molecules have been instrumental at the time of land colonization by plants, approximately 450 million years ago. Studying SL biosynthesis and signaling in the bryophyte Physcomitrium patens (P. patens, a non-vascular plant), and comparing these processes with the available knowledge in vascular plants, enables to investigate the evolution of SL cellular pathways in land plants. As a matter of facts, SL biosynthesis and signaling pathways are quite extensively described in vascular plants. Notably, SL signaling starts in the cytosol where the SL molecule binds to the D14 receptor. D14 cleaves the SL and stays covalently linked to a part of the SL. Under this conformation, D14 can then interact with two partners in the nucleus: the MAX2 F-box protein and SMXL proteins. SMXL proteins act as repressors of the SL response, as their interaction with D14 and MAX2 will trigger their ubiquitination and subsequent proteasomal degradation. Most SL biosynthesis and signaling genes have homologs in P. patens genome, sometimes in different numbers.

INTRODUCTION

Nevertheless, only reverse genetics approaches can clearly establish these homologs function. Previous characterization of P. patens SL deficient mutant Ppccd8 revealed that SL biosynthesis is broadly conserved between mosses and vascular plants. Furthermore, SL play a similar role in the regulation of plant architecture in P. patens as demonstrated in vascular plants.

On the other hand, results presented herein show that SL signaling is only partly conserved between P. patens and vascular plants, supporting the prior observation that the sole MAX2 homolog in P. patens is not needed for SL response. Indeed, only 2 out of the 13 P. patens D14 homologs (PpKAI2L genes) are involved in SL perception according to the characterization of Ppkai2l CRISPR knock-out mutants and to biochemistry analyses. Moreover, instead of D14, PpKAI2L proteins are closer to the KAI2 protein, which is not involved in SL perception in vascular plants. In addition, the phenotype of CRISPR knock-out mutants for the PpSMXL genes, together with genetic linkage analysis of PpSMXL with PpMAX2 and PpCCD8, show that none of the 4 PpSMXL proteins play a major role in SL response. However, the two PpSMXL homologs that are closer to the ancestral land plants SMXL seem to be important regulators of growth, which as per recent phylogenetic studies could be the ancestral role of the SMXL family. This effect on growth would actually be the main response to the ancestral KL (KAI2-ligand) signal, an endogenous signal different from SL. Transduction of the KL signal would hence be conserved across land plants and would be achieved in P. patens via some PpKAI2L proteins, together with the PpMAX2 and at least two PpSMXL proteins. To date, the identity of the KL molecule(s) remains under debate. As SL signaling is not conserved in P. patens, it appears that the known SL signaling pathway results from a vascular plants specific innovation. Likewise, SL response in P. patens would be the product of a convergent evolution. Therefore, the question as to how P. patens transduces the SL signal, downstream of perception by specific PpKAI2L proteins, remains open.

  Physcomitrium patens life cycle and usefulness in research I-A) Physcomitrium patens phylogenetic location

Figure

  Figure I-1 -Phylogenetic relationships in the green lineage (Viridiplantae)

Figure II- 1 -

 1 Figure II-1 -Representative molecules of each phytohormonal group. The structure of the molecule that is the most widely distributed in land plants is given, for each phytohormone family. Molecules (or analogs) from the same family are noted in the same colour: auxins in blue, cytokinins in red, jasmonates in yellow, strigolactones in purple. IAA= Indole-3-Acetic Acid; NAA= 1-Naphthaleneacetic acid ; BA= benzyl adenine; N 6 -iP= N 6 -(∆ 2 -isopentenyl)-adenine; SA= Salicylic Acid; ABA= Abscisic acid; BR= Brassinosteroid ;JA= Jasmonic Acid; OPDA= 12-oxo-phytodienoic acid; CL= Carlactone; KAR1= Karrikin 1 = Karrikinolide.

Figure II- 2 -

 2 Figure II-2 -Principal hormone effects on P. patens organs and tissues. (A): Sexual organs; (B):Spore; (C): Bud; (D): Protonemal filaments, chloronema (ch) and caulonema (ca); (E): Phyllids; (F) : Branched gametophore and sporophytes (s); (G):Gametophores (g) and rhizoïds (r). Arrows indicate positive effects, dashes negative effects. Dotted lines in (E) symbolizes an effect on cell division, while square bracket an effect on cell length. Square bracket in (G) symbolizes an effect on gametophore stem length. Thickness of the arrows reflects number of experimental evidences.

Proust

  et al. Study of the loss-of-function Ppccd8 mutant and use of the synthetic SL analog racGR24 (Figure II-1)

Figure II- 3 -

 3 Figure II-3 -Occurrence of plant hormone signaling components in P. patens. Schematic diagrams are shown for each hormone class, with the number of genes found in P. patens genome. Receptors are shown as sectors, signal transduction components as ovals and transcription factors as triangles. Components drawn with dotted outlines and question marks indicate still hypothetical involvement in signaling.

Figure III- 1 -

 1 Figure III-1 -General view of strigolactones structural diversity. Opposite stereochemistry of the C ring in orobanchol type SL (blue inset) and strigol type SL (violet inset) is highlighted by blue/violet ovals. Note that the orientation of the D ring (highlighted by red ovals on orobanchol and strigol structures) is always the same in natural

Figure III- 2 -

 2 Figure III-2 -Schematic overview of SL biosynthesis and signaling pathways. SL responses include repression of axillary branching and regulation of many other developmental processes detailed in part III-B.

Figure III- 4

 4 Figure III-4 Schematic overview of the putative KL signaling pathway

Figure III- 5 -

 5 Figure III-5 -Expression profile of putative SL biosynthesis genes in Physcomitrium patens Absolute levels of expression are given according to the data from Ortiz-Ramirez available on P. patens eFP Browser (Ortiz-Ramírez et al., 2016) (http://bar.utoronto.ca/efp_physcomitrella/cgi-bin/efpWeb.cgi). Genes ID are respectively Pp3c9_19810 (PpD27L1), Pp3c26_11090 (PpD27L2), Pp3c4_23850 (PpD27L3), Pp3c24_12440 (PpD27L4), Pp3c4_23900 (PpD27L5), Pp3c6_21550 (PpCCD7) and Pp3c6_21520 (PpCCD8).

(

  number and size)[START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. In a first attempt no true homolog for the D14 SL receptor was found in P. patens genome, whereas 11-13 PpKAI2-LIKE (PpKAI2L) candidate genes, named PpKAI2L-A to PpKAI2L-M, were described(Lopez-Obando et al., 2016a). These genes are split in 4 subclades (i), (i.i-i.ii), (ii) and (iii), hereafter renamed respectively asPpKAI2L-(A-E), (F,K), (H,I,L), and (J,G,M). A comprehensive phylogenetic assessment described the presence of moss clades (F,K), (H,I,L), and (J,G,M)) into a super clade called DDK (D14/DLK2/KAI2) containing spermatophyte (Angiosperm and Gymnosperm) D14 clades, while clade (A-E) belongs to the EuKAI2 clade, common to all land plants(Bythell-Douglas et al., 2017). Nevertheless, the characteristics of moss proteins from DDK clade were found to be as different from D14 as from KAI2(Bythell-Douglas et al., 2017). Structure prediction of the PpKAI2L proteins indicated various pocket sizes, as observed for D14 and KAI2 from vascular plants(Lopez-Obando et al, 2016). Consequently, those (or some of those) genes could encode receptors for various molecules, including the PpCCD8-derived compounds, or the elusive KL[START_REF] Conn | Evidence that KARRIKIN-INSENSITIVE2 (KAI2) Receptors may Perceive an Unknown Signal that is not Karrikin or Strigolactone[END_REF]. Accordingly, our work on PpMAX2 allowed us to hypothesize that this F-box protein might be involved in a moss KL pathway[START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. But the question as to the involvement of PpKAI2L proteins in the PpMAX2 pathway remains open. Last year,Bürger et al.

Figure IV- 1 -

 1 Figure IV-1 -PpCCD8-derived compounds are germination stimulant (GS) of a specific group of Phelipanche ramosa. (A) GS activities of exudates of P. patens on seeds of P. ramosa group 1 and 2a relative to (±)-GR24 0,1 µM. (B) Germination rate of seeds of P. ramosa group 2a on plates with P. patens WT, Ppccd8 mutant plants, or with culture medium only, with or without (±)-GR24 0.1 µM. (C) Seeds from P. ramosa group 1 (left) and group 2a (right) on plates with WT (top) or Ppccd8 mutant (bottom), with or without (±)-GR24 0,1 µM. Arrows point at germinating seeds. Scale bar = 0.5 mm.

Figure IV- 2 -

 2 Figure IV-2 -Phenotypic response to (+)-and (-)-GR24 enantiomers and natural compounds: number of caulonema filaments. Caulonema filaments were counted in WT (A) and Ppccd8 SL synthesis mutant (B) grown 10 days vertically in the dark, following application of increasing concentrations (0.1, 1 and 10 µM) of (+)-GR24 (cyan boxes), (-)-GR24 (red boxes) and KAR2, (blue boxes). Control is 0.01% DMSO. (C) Caulonema filament numbers of WT and Ppccd8 mutant grown 10 days vertically in the dark, following application of increasing concentrations (0.1, 1and 10 µM) of (±)-Z-CL (noted Z-CL, green boxes). Control is 0.01% DMSO. (+)-GR24 (cyan boxes) and (-)-GR24 (red boxes) were applied at 1 µM. Significant differences between control and treated plants within a genotype based

  Figure IV-2 and Supplemental Figure IV-1). Sinceit is now well established that the (-)-GR24 can activate nonspecific responses[START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF], we tested separately the (+) and (-)-GR24 enantiomers on both WT and Ppccd8 mutant. As in previous studies(Hoffmann et al., 2014;[START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF], we predict a clearer response in the synthesis mutant than in the WT, due to absence of endogenous PpCCD8-derived compounds potentially mimicked by (+)-GR24. Both the number and the length of caulonema filaments significantly decrease following application of (+)-GR24 in WT and Ppccd8 mutant, in a doseresponse manner. A dose of 0.1 M is enough to see a clear and significant response in terms of number in both genotypes. Application of (-)-GR24 leads to a significant decrease of Ppccd8 filaments length, as (+)-GR24, but has no effect on WT filament length (Supplemental FigureIV-1).

Figure IV- 3 -Figure 2 ,

 32 Figure IV-3 -Phylogeny and models of the PpKAI2L gene family. (A) Phylogenic tree of PpKAI2-LIKE proteins and paralogs from Arabidopsis thaliana (At) using the Maximum Likelihood method based on the Dayhoff matrixbased model. The tree with the highest log likelihood is shown. Numbers are percent bootstrap values for 1000 replicates. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA5. (B) Gene diagrams. Exons are displayed as grey boxes, introns and UTRs are depicted as thin black lines. Start and Stop codons are written in bold, while plain text indicates the start/end position for each feature, relative to the start codon. Only 5'-UTR regions are not represented true to scale. Transcript versions that were

Figure 59 Figure IV- 4 -

 594 Figure3B-E), PpKAI2L-D has higher transcript levels among clade (A-E) genes, while PpKAI2L-H transcript levels are highest compared to that of PpKAI2L-I and -L (clade (H,I,L)). PpKAI2L-F and -K (clade (F,K)) show comparable transcript levels, and PpKAI2L-J has slightly higher transcript levels than PpKAI2L-G/M (clade (J,G,M)). The data are consistent with those previously reported(Ortiz-Ramirez et al, 2016, shown in Supplemental Figure IV-4).

Figure IV- 4 -

 4 Figure IV-4 -PpKAI2L proteins response differential to the GR24 isomers. Continued.

Figure IV- 5 -

 5 Figure IV-5 -SL isomers bind PpKAI2L proteins based on intrinsic tryptophan fluorescence with different affinity. Plots of fluorescence intensity versus SL concentrations. The change in intrinsic fluorescence of AtD14 (A);

Figure IV- 6 -

 6 Figure IV-6 -PpKAI2L enzymatic activities confirm stereoselectivity and reveal PpKAI2L-H special feature. (A) PpKAI2L enzymatic activity towards GR24 isomers. (+)-GR24, (-)-GR24, (+)-2'-epi-GR24 and (-)-2'-epi-GR24 at 10 µM were incubated with RMS3, AtKAI2 and 7 PpKAI2 proteins at 5 µM for 150 min at 25 °C. UPLC-UV (260 nm) analysis was used to detect the remaining amount of GR24 isomers. Columns represent the mean value of the hydrolysis rate calculated from the remaining GR24 isomers, quantified in comparison with (±)-1-indanol as internal standard. Error bars represent the SD of three replicates (means ± SD, n = 3). The asterisks indicate statistical significance from the AtKAI2 values, for each isomer, as ***p ≤ 0.001; **p ≤ 0.01 and n.s., p > 0.05, as measured by Dunnett nonparametric relative contrast effects test, with AtKAI2 taken as the control group, for each compound. (B) Enzymatic kinetics for PpKAI2L, AtD14 and AtKAI2 proteins incubated with (±)-GC242 (structure shown Supplemental Figure IV-8A). Progress curves during probe hydrolysis, monitored (λem 460 nm) at 25 °C. Protein catalyzed hydrolysis with 330 nM of protein and 4 µM of probe. These traces represent one of the three replicates and the experiments were

Figure IV- 6 -

 6 Figure IV-6 -PpKAI2L enzymatic activities confirm stereoselectivity and reveal PpKAI2L-H special feature.

  isomers to the PpKAI2L proteins.. Mass spectrometry analyses highlight 96 Da increments (corresponding to the D ring mass), when incubating AtKAI2 and (-)-GR24, and all PpKAI2L-C, -D, -E, -F or -L with (-)-GR24. Strikingly, 96 Da increments are also observed when incubating PpKAI2L-E -F -L and -K with the other isomer (+)-GR24. However, for PpKAI2L-E, the peak intensity is much lower with (+)-GR24 than with (-)-GR24, confirming the better affinity for the latter (Figure IV-5). PpKAI2L-H does not covalently bind the D ring, following incubation with either of both enantiomers, further arguing for a Michaelian enzymatic activity and a specific enzymatic role for this protein. Poor interactions are observed with (+)-GR24, reported as mimicking SLs, and showing the more potent effect in our phenotypic assays (Figure IV-2 and Supplemental Figure IV-1). Strikingly, all clade (A-E) PpKAI2L

Figure

  Figure IV-10A). As controls, the double mutant was transformed with AtKAI2 or AtD14 CDS under the control of endogenous promoters. Only lines expressing AtD14 under the control of the AtD14 promoter fully restored the rosette branching to WT (Ler) values (Figure IV-7A). Under the control of AtD14 promoter, neither AtKAI2 nor any of the PpKAI2L can significantly restore the branching phenotype of the Atd14-1 kai2-2 mutant, to the exception of one line expressing PpKAI2L-J (#4.6), that shows a significant lower number of rosette branches (still higher to that of WT and AtD14 expressing line, Figure IV-7A). We conclude that none of the PpKAI2L genes can fully complement the AtD14 function in shoot branching. We tested possible complementation of AtKAI2 function in the Atd14-1 kai2-2 mutant, by monitoring hypocotyl length under low light conditions, with or without 1 µM (+)-GR24 or (-)-GR24 in the culture medium (Figure IV-7B).

expressingFigure IV- 7 -

 7 Figure, to the striking exception of lines expressing PpKAI2L-H, that showed a clear response to (+)-GR24 addition (shorter hypocotyls). To conclude with these assays, PpKAI2L-G is able to mediate the Arabidopsis KL signaling in hypocotyls, though it does not fully ensure AtKAI2 response function; PpKAI2L-H is able to ensure similar function to AtD14 in a kai2-2 background, for hypocotyl development but not for rosette branching.

  ). As biochemistry experiments suggested a pure enzymatic role for PpKAI2L-H, a deletion mutant was obtained through homologous recombination, where the full CDS was removed from the moss genome (∆h mutant, Figure IV-8 and Supplemental Figure IV-12A). This ∆h mutant was chosen for further transformation experiments with guide-RNAs from the same (H,I,L) clade and/or from clade (F,K) and (J,G,M),

Figure IV- 8 -

 8 Figure IV-8 -Mutations obtained in all 13 PpKAI2L genes. For all 13 PpKAI2-Like genes, WT nucleotide and protein sequences are shown, above altered sequences found in various CRISPR-Cas9 lines (in italics, numbered). The number

  al., 2018, Figure IV-9). The diameter of mutants in clade (A-E) genes is significantly smaller than that of Ppccd8 and WT, and a bit larger than that of Ppmax2-1 (Figure IV-9A-B and Supplemental Figure IV-12B). The phenotype of the clade (A-E) mutants, with early and big gametophores resembles that of Ppmax2-1 mutant despite not as strong (Figure IV-9A). To the naked eye, 3 week-old plants from mutants of all three clades (F,K) (J,G,M) and (H,I,L) genes are indistinguishable from WT (Figure IV-9A). These observations also stand for very young plants (10-day-old, Supplemental Figure IV-12B). After a month growth however, all mutants affecting genes from clade (F,K) and (J,G,M) show slightly larger diameter, intermediate between that of WT and Ppccd8, the triple Ppkai2L j3-g3*-m1 being even larger than Ppccd8 (Figure IV-9C and D). Mutants in clade (H,I,L) as Ppkai2L ∆h, Ppkai2L ∆h-i2* and Ppkai2L ∆h-i3*-l1 are not different from WT (Figure IV-9D and Supplemental Figure IV-12B).

Figure IV- 10 -

 10 Figure IV-10 -Ppkai2L mutant gametophores in red light. (A) Gametophore height of Ppkai2L mutants affecting clade (A-E) genes (a2*-b4*-c2*; c2*-d4*-e1; a1-b1-c1-d1-e2*) and clade (H,I,L) genes (∆h-i2* and ∆h-i3*-l1), compared to that of WT, Ppccd8 and Ppmax2-1 mutants, following 2 months growth under red light. Mutant genotypes carry mutations as indicated in Figure IV-8 and Supplemental Table IV-2, with asterisks for null mutations. Box plots of n = 32-36 gametophores, grown in 3 Magenta pots, harboring between 15 and 25 leaves. Statistical groups (all genotypes comparison) are indicated by letters and were determined with a Kruskal-Wallis test followed by a Dunn post hoc test (p<0.05). (B) Gametophore height of Ppkai2L mutants affecting clade (H,I,L) gene (∆h), both clades (F,K) and (J,G,M) (f2*-j5*-k2*) and all 3 clades (H,I,L) (F,K) and (J,G,M) (∆h-f1*-j4-k1* and ∆h-f1*-j4-k1*-g2*-m1). Mutant genotypes carry mutations as indicated in Figure IV-8 and Supplemental Table IV-2, with asterisks for null mutations. Box plots of n = 11-15 gametophores, grown in 3 Magenta pots, harboring between 15 and 25 leaves. Statistical groups (all genotypes comparison) are indicated by letters and were determined with a Kruskal-Wallis test followed by a Dunn post hoc test (p < 0.05). (C) Examples of gametophores following 2 months growth under red light, from WT, Ppccd8, Ppmax2-1, and Ppkai2L mutants as shown in (A) and (B). Scale bar = 5 mm.

Following 2 1 (

 21 months growth under red light, Ppmax2-1 gametophores are much more elongated than WT gametophores, whereas Ppccd8 mutant gametophores are shorter (Figure IV-10). Among mutants affecting clade (A-E) genes, both triple mutants Ppkai2L a2*-b4*-c2* and Ppkai2L c2*-d4*-e1 gametophores show similar height to WT. Interestingly, the quintuple mutant (Ppkai2L a1-b1-c1-d1-e2*) shows significantly elongated gametophores, similar to Ppmax2-Figure 10 A and C). The other tested quintuple mutants (Ppkai2L a3*-b1-c3*-d3*-e2* and a1-b1-c1-d1-e2*) also show elongated gametophores under red light, intermediate between WT and Ppmax2-1 (Supplemental Figure IV-13). The weak phenotype of both triple mutants suggests a functional redundancy among clade (A-E) genes, as KO mutations for PpKAI2L-A, B, C and/or D do not lead to as elongated gametophores as in Ppmax2-1 mutant. Gametophores from mutants where genes from clade (F,K) and/or (J,G,M) were mutated (Ppkai2L f2*-j5-k2*, Ppkai2L ∆h-f1*-j4-k1* and ∆h-f1*-j4-k1*-g2*-m1, Figure 10B) are similar in height to WT, suggesting that genes from clade (F,K) and clade (J,G,M) have no role in photomorphogenesis in red light. The Ppkai2L ∆h-i2*and Ppkai2L ∆h-i3*-l1 mutants, affected in clade (H,I,L) genes show shorter gametophores under red light, similar to Ppccd8 (Figure IV-10A, C), while the single Ppkai2L ∆h mutant shows gametophores intermediate in height between WT and Ppccd8 (Figure IV-10B). This may suggest a specific role for the (H,I,L) clade genes, opposite to that of PpMAX2, or unrelated to the PpMAX2 pathway.

Figure IV- 11 -

 11 Figure IV-11 -Phenotypic response of Ppkai2L mutants to (+)-GR24 and (-)-GR24 application in the dark. (A)(B) Caulonema numbers from mutants affecting clade (A-E) genes following application of 0.1 µM (-)-GR24 (in red, A) or 0.1µM (+)-GR24 (in cyan, B). DMSO was applied as control treatment (ctl, dark grey). WT and both Ppccd8 and Ppmax2-1 mutants were used as control genotypes. (C-E) Caulonema numbers from from mutants affecting clade

  Figure IV-11A-B and Supplemental Figure IV-14A). No significant effect of (-)-GR24 0.1 M addition is observed on clade (A-E) mutants, as for WT (Figure IV-11A). In this assay, both Ppccd8 and Ppmax2-1 mutants show a significant decrease of caulonema filaments number after (-)-GR24 application. In a separate experiment, a dose of 1 M of (-)-GR24 leads to opposite effects on caulonema filaments number of WT (increased) and Ppccd8 (decreased) (Supplemental Figure IV-14 A, see also Figure IV-2

  a3*-b1-c3*-d3*-e2* mutant, as for WT and the Ppccd8 and Ppmax2-1 mutants (Figure IV-11 B). Thus, mutating any of clade (A-E) PpKAI2L gene does not hamper the response to (+)-GR24, and therefore likely the response to CCD8derived compounds. We then tested the effect of GR24 enantiomers on Ppkai2L mutants from the 3 other clades (Figure IV-11 C-E and Supplemental Figure IV-14). Strikingly, in control conditions, all mutants have more filaments than WT, as Ppccd8, except for clade (H,I,L) mutants which tend to have less filaments (Figure IV-11C-E and Supplemental Figure IV-14). Both the single mutant Ppkai2L-j1* and the quintuple Ppkai2L j1*-g1-m6*-i3*-l2 show a significant response to (-)-GR24 (less caulonema), as Ppccd8 (Figure IV-11C and Supplemental Figure IV-14 A,B). Mutants with KO mutations in PpKAI2L-F, -K, -H, -G, -M, -I or -L show no clear response to (-)-GR24, as for WT. When testing the response to (+)-GR24 (Figure IV-11 D-E and Supplemental Figure IV-14 C,D), the number of caulonema is clearly reduced in WT and Ppccd8, and in mutants carrying the Ppkai2L Δh mutation, alone or in combination with f*, k* i* or l* null mutations. Thus clade (F,K) and clade (H,I,L) genes do not play a role in the response to (+)-GR24. However, no more response to the (+)-GR24 enantiomer is observed for all mutants where the PpKAI2L-J gene is KO (Figure IV-11 D: j1* and j1*-g2*-m2*, and Supplemental Figure IV-14 C,D). Interestingly, in

Figure IV- 12 -

 12 Figure IV-12 -Ppkai2L mutant transcriptional response to (+)-and (-)-GR24. Transcript abundance analysis of the SL-responsive gene PpKUF1LA and Pp3c6_15020 , in WT, Ppccd8, Ppmax2-1 and Ppkai2Like mutants a1-b1-c1-d1-e2* (clade (A-E)), j1* and j3-g3*-m1 (clade (J,G,M)), grown for 2 weeks in light, then transferred for 1 week in the dark, 6 hours after treatment with DMSO (control, grey plots), 1 µM (+)-GR24 (cyan plots) or 1 µM (-)-GR24 (red plots). Box plots of at least 4 biological repeats are shown, relative to mean (PpAPT-PpACT3) transcript abundance. 2fold differences in median values of transcript levels between control and treated plants are estimated as significant (DE)

  Figure IV-14A) while this number consistently decreases in Ppccd8 (Figure IV-11, Supplemental Figure IV-14 A, B).

Figure

  Figure IV-12B), and in particular, the phenotypic response to (+)-GR24, that mimics CCD8-derived compounds, is similar to that of WT plants, contrary to other mutants. In red light however, the gametophores of ∆h-i2* and ∆h-i3*-l1 mutants are less elongated than WT gametophores, as it is also observed in Ppccd8. However, if, for Ppccd8, this can be related to the higher number of filaments, leading to the initiation of more (but smaller) gametophores, the number of filaments in the dark is not higher in clade (H,I,L) mutants. Even more, clade (H,I,L) mutants tend to have less filaments than. Altogether, these phenotypes, although quite tenuous, could suggest that clade (H,I,L) genes may undertake a specific role in P. patens development. The relation of this role to PpKAI2L-H enzymatic activity remains to be discovered.

Finally, both phenotypic

  response in the dark and transcriptional response to the (+)-GR24 enantiomer are unaffected in clade (A-E) Ppkai2l mutant, indicating that clade (A-E) PpKAI2L proteins are likely not receptors to CCD8-derived compounds. PpKAI2L-J, -G, -M mediate PpCCD8 derived (SL-related) response In white light conditions (Figure IV-9), mutants affecting other clades than (A-E) show either similar to WT phenotype (mutants affecting clade (H,I,L) genes), or intermediate phenotype between WT and Ppccd8 (mutants in clade (F,K) and (J,G,M) clades). In the dark (Figure IV-11), the caulonema number in control conditions is also intermediate between WT and Ppccd8 mutant for mutants in clades (F,K) and (J,G,M), while it is similar to WT (or slightly smaller) in clade (H,I,L) mutants. Based on the hypothesis that synthesis and response mutants show similar phenotypes, genes from clades (F,K) and (J,G,M) are thus the best candidates for PpCCD8-derived compounds receptors. When testing the phenotypic response of these mutants to (+)-GR24 application, plants with KO mutations for PpKAI2L-J or PpKAI2L-G/M (Figure IV-11, j1*, and j1*-g2*-m2*, and Supplemental Figure IV-14, j7*-g1-m1, j8*-g1 -m5*, and j6-g5*-m1) no more respond to this compound. In contrast, both Δh-f1*-k1*-j4 and Δh-f3*-k3*-j6 mutants, show a significant response to (+)-GR24 application (Figure IV-11 and Supplemental Figure IV-14), indicating that KO mutations in both PpKAI2L-F and PpKAI2L-K, or deletion of PpKAI2L-H do not abolish the response to the CCD8-derived compound mimic, not even additively. The absence of response in higher order mutants where either PpKAI2L-J or PpKAI2L-G are KO confirms the prominent role of both genes in the response to (+)-GR24. However, if, as expected, transcript levels of the Pp3c6_15020 response marker gene are unchanged in both j1* and j3-g3*-m1 mutants following (+)-GR24 application (Figure IV-12), transcript levels of the PpKUF1LA gene are increased in both

Figure

  Figure

Figure IV- 7 and

 7 Figure IV-7 and Supplementary Figure IV-10 were performed on T3 homozygous segregating lines.

  concentration. Ligand was tested at the concentration of 200 µM. The intrinsic fluorescence signal was measured as a function of increasing temperature in Prometheus NT.48 fluorimeter (Nanotemper™), with 55% excitation light intensity and 1 °C/minute temperature ramp. Analyses were performed on capillaries filled with 10 µL of respective samples. Intrinsic fluorescence signal expressed by the 350 nm/330 nm emission ratio, which increases as the proteins unfold, is plotted as a function of temperature. The plots are one of the three independent data collections that were performed for each protein.Intrinsic tryptophan fluorescence assays and determination of the dissociation constant KD has been performed as described in[START_REF] De Saint Germain | An histidine covalent receptor and butenolide complex mediates strigolactone perception[END_REF], using Spark® Multimode Microplate Reader from Tecan.Direct ESI-MS in denaturant conditions. Mass spectrometry measurements were performed with an electrospray Q-TOF mass spectrometer(Waters) equipped with the Nanomate device (Advion, Inc.). The HD_A_384 chip (5 μm I.D. nozzle chip, flow rate range 100-500 nL/min) was calibrated before use. For ESI-MS measurements, the Q-TOF instrument was operated in RF quadrupole mode with the TOF data being collected between m/z 400-2990. Collision energy was set to 10 eV and argon was used as collision gas. Mass spectra acquisition was performed after denaturation of PrKAI2 ± ligand in 50% acetonitrile and 1% formic acid. The Mass Lynx 4.1 (Waters) and Peakview 2.2 (Sciex) softwares were used for acquisition and data processing, respectively. Deconvolution of multiply charged ions was performed by applying the MaxEnt algorithm (Sciex). The protein average masses are annotated in the spectra and the estimated mass accuracy is ± 2 Da. External calibration was performed with NaI clusters (2 μg/μL, isopropanol/H2O 50/50, Waters) in the acquisition m/z mass range.Localization of the fixation site of ligands on PpKAI2L. PpKAI2L-ligand mixtures were incubated for 10 min before to be submitted overnight to Glu-C proteolysis. Glu-C-generated peptides mixtures were analyzed by nanoLC-MS/MS with the Triple-TOF 4600 mass spectrometer (AB Sciex) coupled to the nanoRSLC ultra performance liquid chromatography (UPLC) system (Thermo Scientific) equipped with a trap column (Acclaim PepMap 100 C18, 75 μm i.d. × 2 cm, 3 μm) and an analytical column (Acclaim PepMap RSLC C18, 75 μm i.d.× 25 cm, 2 μm, 100 Å). Peptides caulonema number in the dark. Caulonema numbers from Ppkai2L mutants following application of (A) 1 µM (-)-GR24 (in red), (B) 0.1 µM (+)-GR24 (in turquoise) (C,D) 0.1 µM (-)-GR24 (in red). DMSO was applied as control treatment (ctl, dark grey) except in (A), where acetone was applied. WT and both Ppccd8 and Ppmax2-1 mutants were used as control genotypes. Mutant genotypes carry mutations as indicated in Figure

  g1-m1 J,G,M STOP in PpKAI2Like-J (27); PpKAI2Like-G: -*-k4* -j2* F,K ; J,G,M ; H,I,L PpKAI2L-H: Full CDS deletion; STOP in PpKAI2Like-F (41); STOP in PpKAI2L-K (239); STOP in PpKAI2Like-J (47) Δh-i1*-f1*-k1*-j4-g4*-m1 F,K ; J,G,M ; H,I,L PpKAI2L-H: Full CDS deletion; STOP in PpKAI2Like-I (75); STOP in PpKAI2Like-F (31); STOP in PpKAI2L-K (233); PpKAI2Like-J:

Figure V- 1 -

 1 Figure V-1 -Domain architecture of major Clp-ATPases subtypes. N = amino-terminal domain (also called double Clp-N domain), NBD1 = first ATPase domain, M = middle domain, NBD2 = second ATPase domain, CTP = chloroplast transit peptide. bClp stands for bacterial Clp, eClp for eukaryotic Clp and pClp for plant Clp. In the absence of such precision, all members of the subfamily have a similar architecture. Note that NBD1 usually contains two Walker B motifs. Domains are not represented true to scale.

Figure V- 2 -

 2 Figure V-2 -Phylogeny of SMXL proteins relative to other type I ClpATPases. Phylogeny analysis was performed on the Phylogeny.fr platform and comprised the following steps. Sequences were aligned with MUSCLE(v3.8.31)

Figure V- 3 -

 3 Figure V-3 -Scheme of the different feedback regulation mechanisms of SL-associated SMXL genes expression in Arabidopsis and rice. Homologs are shown as identical symbols: SMXL6 (Arabidopsis) ≈ D53 (rice, Oryza sativa), SPL9/15 ≈ IPA1, BRC1 ≈ TB1. Adapted from[START_REF] Song | IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice[END_REF] Wang et al., 2020b).

Figure V- 4 -

 4 Figure V-4 -Domain structure and motifs of a typical SMXL protein. Domain names written in blue are SMXLspecific notations. The N-terminal double ClpN domain and approximately the first half of the second NBD (NTPase 1 subdomain) are highly conserved between SMXL proteins, as well as with other type I Clp-ATPases. The first NBD is variable, and its Walker motifs are often difficult to spot (as in figure V-1, Walker B motifs are shown by red bars). The NBD2 is elongated in C-terminal relative to other Clp-ATPases (NTPase 2 subdomain) but is not highly conserved across SMXL proteins. The M domain is the most versatile, including amongst SMXL proteins, together with the spacer sequence between the two subdomains of NBD2, which nevertheless contains the conserved EAR motif. Highly variable regions are shown as pale boxes with dotted outlines, conserved regions are depicted as vivid colored boxes with continuous outlines.

Figure V- 6 -

 6 Figure V-6 -Global view of SMXL proteins evolutionary history. The number of functional SMXL subtypes are indicated at the bottom and color-coded. WGD = Whole Genome Duplication.

Figure VI- 1 -

 1 Figure VI-1 -Model of PpSMXL genes. Genomic sequences of the four PpSMXL genes were recovered from Phytozome (V3.6 of P. patens genome). Architecture of the primary (V3.1) transcript is shown for each. Only exons and inter-exonic introns are represented true to scale. Exons are shown as grey boxes, inter-exonic introns as black angles, UTRs as solid black lines and introns located in the 5'UTR are depicted as blue angles. Guide RNAs used for mutagenesis are depicted as black triangles (they are named according to their location relative to the ATG and according to their orientation), see supplementary table for their sequences. Degron (RGK/RT) and EAR motifs are shown by red and purple small boxes, respectively. Positions are indicated relative to the START codon.

  in mature gametophores and/or rhizoids. Taken together, these analyses show another divergence between the two PpSMXL clades, with A/B being lowly expressed and C/D being expressed at an average level (compared to the mean expression level of P. patens genes which is around 16 RPKM, PpSMXLC/D levels are approximately 18 RPKM and PpSMXLA/B levels are around 2 and 6, respectively[START_REF] Perroud | The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data[END_REF]).

Figure VI- 2 -

 2 Figure VI-2 -Expression of PpSMXL genes along P. patens vegetative development. (A) GUS staining of 2-week old proPpSMXL:GUS plants. Scale bars are 500µm. (B) Transcript levels of the four PpSMXL genes, relative to the two reference genes PpACT3 (Pp3c10_17080) and PpAPT (Pp3c8_16590). RT-qPCR data used for the analysis was extracted from 4 biological replicates, each with two technical repeats. Point represents the mean of two technical repeats. Some points were excluded from analysis following an outliers identification test carried out in GraphPad Prism (version 8.4.2). Statistical significance scores (p<0.05) of comparisons among tissues are indicated as bold letters (Kruskal-Wallis test followed by a Dunn post-hoc test). Note the differences in expression value on y axes.

Figure VI- 5 -

 5 Figure VI-5 -Subcellular localization of GFP-PpSMXL fusion proteins in protonema of transgenic proZmUbi:GFP-PpSMXL P. patens lines. Scale bars are 50µm. Arrow points to a nucleus.

Figure VI- 7 -

 7 Figure VI-7 -Plant extension of Ppsmxl mutants. (A) Phenotype of three-week-old plants on low nitrogen content medium (without underlying cellophane). Scale bars are 5mm. (B) and (C) Diameters were measured each week for the same 35-49 plants of each genotype. Statistical significance of differences between mutants and WT at the last time

Figure VI- 8 -

 8 Figure VI-8 -Phenotypic response of Ppsmxl double mutants to (+)-GR24 and (-)-GR24 in the dark. 24 plants of each genotype were cultivated for two weeks and then treated with 0,01% DMSO (control, grey), 1µM of (+)-GR24 (blue) or 1µM (-)-GR24 (red). Plants were incubated vertically in the dark for ten days. Negatively gravitropic caulonema filaments were enumerated for each plant. Statistical significance of comparisons of control groups relative to WT is shown as bold black symbols (Kruskal Wallis followed by a Dunn post-hoc test, * 0.01≤p<0.05, **

PpSMXLFigure VI- 9 -Figure VI- 10 - 1

 9101 Figure VI-9 -Genetic analysis of PpSMXL relationship with PpCCD8. The mutagenesis strategies used on WT were re-enacted in the Ppccd8 mutant background. (A) Phenotype of three-week-old plants on low nitrogen content medium (without underlying cellophane). Scale bars are 5 mm. (B) Extension phenotype of these mutants and mutants carrying equivalent Ppsmxl mutations in the WT background, on low nitrogen content medium (with underlying cellophane), along a 5-week kinetic. Statistical significance of comparisons relative to WT at the last time point is shown as bold black letters (Kruskal Wallis followed by a Dunn post-hoc test, p<0.05).

Figure VI- 11 -

 11 Figure VI-11 -Genetic analysis of PpSMXL relationship with PpMAX2. The mutagenesis strategies used on WT were re-enacted in the Ppmax2-1 mutant background (Lopez-Obando et al. 2018). A Ppmax2 CRISPR mutagenesis using 5 guide RNAs was employed in parallel in the Ppsmxl∆c7∆d4 mutant background, giving rise to the Ppmax2-16 mutation. (A) Representative individuals for those measured in Figure VI-11-B, at the end of the experiment (week 5).

Figure VI- 12 -

 12 Figure VI-12 -PpSMXL proteins interact with PpMAX2 and can form homo-oligomers. The first indicated protein is fused to the N-terminal part of eYFP, while the second protein is fused to the C-terminal part (both tags are fused at the N-terminal end of P. patens proteins). Both fusion proteins are transiently overexpressed in N. benthamiana leaf

Figure VI- 13 -Figure VI- 14 -

 1314 Figure VI-13 -PpSMXL proteins interact with some PpKAI2L proteins. The first indicated protein is fused to the N-terminal part of eYFP, while the second protein is fused to the C-terminal part (both tags are fused at the N-terminal end of P. patens proteins). Both fusion proteins are transiently overexpressed in N. benthamiana leaf epidermis. Colocalization of CFP-H2b and eYFP biFC signals are pointed at with white arrows. Scale bars are 50µm. Interactions with DEFICIENS and GLOBOSA proteins from Antirrhinum majus are given as negative controls.

  37 signature peptides that were significantly enriched in untreated GFP-PpSMXLC samples relative to untreated flag-GFP samples, which we considered as specific interactors of PpSMXLC. Most of them are involved in photosynthesis, energetic metabolism (glycolysis and mitochondrial respiration) and protein turnover (translation and protein degradation), together making up 72% of interacting proteins. The remaining proteins are involved in oxidative stress (14%), cytoskeleton (5%), specialized metabolism (4%, notably OPDA and possibly kaurene biosynthesis enzymes) and finally 5% belong to other categories or have no predictable function. (±)-GR24 had virtually no impact on the interactions of PpSMXLC, with only 3 interactions induced by (±)-GR24, and 3 others diminished by (±)-GR24 (Supplemental Figure. profiles a differential regulation of clade A/B and clade C/D PpSMXL It has already been shown that most of mosses have an additional SMXL clade compared to other non-seed plants[START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. We report evidence of this in P. patens, in which PpSMXLA/B belong to this divergent clade, which could potentially be neofunctional, as we have shown earlier for some PpKAI2-L homologs (see chapter IV). The split between PpSMXLA and PpSMXLB, and between PpSMXLC and PpSMXLD on the other hand, is probably quite recent given the high similarity of encoded proteins in each given clade (supplemental Figure VI-1). PpSMXLA/B moreover have shorter D1 and M domains compared to PpSMXLC/D (supplemental Figure VI-1) and lack the typical degron motif (supplemental Figure VI-

  Figure VI-4). Puzzlingly, only the C/D clade appears sensitive to PpCCD8-derived compounds (SL), in the light, when these compounds are theoretically mimicked by (+)-GR24 (supplemental Figure VI-4). This suggests that natural SL produced by P. patens normally repress PpSMXLC/D gene expression, which might represent a level of crosstalk between PpCCD8-derived compounds signaling and PpMAX2-dependent signaling. However, all PpSMXL genes are regulated in a PpMAX2-dependent manner, especially PpSMXLA and PpSMXLC which are also the main targets of regulation by light (both on the long-term (Figure VI-2) and on the short-term by red light (Figure VI-3)).

Figure

  Figure VI-7).

Figure VII- 2 -

 2 Figure VII-2 -Effect of (+)-GR24 and (-)-GR24 on protoplasts regeneration in the light. (A) Effect of (+)-GR24, at 3, 30 and 300 nM. Control is DMSO 0.3%. Points represent the percentage of regenerating protoplasts in a drop (n=27 drops across 3 plates), 72h after the start of cultivation. (B) Effect of (-)-GR24 at the same concentrations. Control is DMSO 0.3%. Points represent the percentage of regenerating protoplasts in a drop (n=24 drops across 4 plates), 72h after the start of cultivation. (C) and (D) Follow up of the experiments respectively presented in (A) and (B), 6 days after the start of cultivation. (A) (B) (C) and (D) Bars indicate median values. Bold red symbols indicate statistical significance of comparisons of GR24 treated samples to control samples within genotype (Kruskal-Wallis test followed by a Dunn post-hoc test, * 0.01≤p<0.05, ** 0.001≤p<0.01, *** p<0.001). Blue bold symbols give the statistical significance of comparisons of mutants control groups to WT (Mann-Whitney test, * 0.01≤p<0.05, *** p<0.001). Note the difference in overall regeneration levels between panels A and B, which reveals a difference in the quality of protoplasts preparation between the two experiments.

Figure VII- 3 -

 3 Figure VII-3 -Regeneration from phyllids in the light is affected in Ppsmxl mutants. Points represent the percentage of regenerating phyllids out of 16 from a given plant (n=3 individual plants). Bars are the median of the 3 replicates. Symbols in bold red indicate the statistical significance of the regeneration difference relative to WT at each time point (Kruskal-Wallis test followed by a Dunn post-hoc test, # 0.1<p<0.05, * 0.01≤p<0.05).

Figure VII- 4 -

 4 Figure VII-4 -Transcript levels of PpSMXL genes are affected by temperature. Transcript levels of the four PpSMXL genes are given relative to the two reference genes PpElig2 (Pp3c14_21480) and PpAPT (Pp3c8_16590). RT-qPCR data used for the analysis was extracted from 6 biological replicates, each with 2 technical repeats (in a given experiment). Each point represents the mean of these 2 technical repeats. Bars indicate median values. Statistical significance scores of differences relative to 25°C are indicated by bold symbols for each gene (Kruskal-Wallis test followed by a Dunn post-hoc test, # 0.1<p<0.05, * 0.01≤p<0.05, ** 0.001≤p<0.01, *** p<0.001).

Figure VII- 5 -

 5 Figure VII-5 -Ppsmxl mutations in the same clade have a different impact on reaction to cold stress and subsequent recovery. (A) Scheme of the experimental setup. All plants (n=6 plates x 9 plants = 54) were grown at 25°C in for 2 weeks. Then half of the plants (n=27 plants, 3 plates) were transferred at 10°C, in the same lighting and hygrometry conditions, while the other half remained at 25°C as a control. After 2 weeks at 10°C, cold-stressed plants were returned to 25°C for 1 week. Diameter of each plant was assessed just before transfer, then in the middle of the cold stress period, at the end of the cold stress period and finally at the end of the experiment after 1 week of recovery at 25°C. (B) Mean relative decrease of growth in the cold-stressed groups compared to the control group, for each

  genes, which is coherent with our previous concluding statements in chapter VI. While its loss has the most predominant effect on growth in control conditions (see chapter VI), it is the less transcriptionally regulated by temperature and the one with the less predominant effect on cold tolerance (see figure. Hence, the PpSMXLC protein might have a different scope of action compared to the other PpSMXL proteins. This specificity could be explained by PpSMXLC having specific interactors, replication of our GFP-trap experiment (see figure VI-14 and supplemental figures VI-25 and VI-26) for PpSMXLA, B and D could reveal such discrepancy.

  Please keep in mind that you will need at least 6 wells for each treatment, distributed across at least 3 different plates, to have proper biological replicates. So, in the instance where you would like to test one new SL-like molecule along with a negative control (diluted solvent) and a positive control ((+)-GR24), which makes up for three treatments, you will need a minimum of 3 plates per genotype (figure1-3).

Figure 1 - 3 -

 13 Figure 1-3 -Layout of 6-well plates 2.Prepare the cellophane disks for your 6-well plates. You need a cellophane sheet and a tool to cut it in the right format. We typically use a scrapbooking punch producing disks with a diameter of 30 mm. Place the cellophane disks in a heat-resistant closed container and spread them to prevent stacking of the cellophane disks. Autoclave.

Figure 1 - 6 -

 16 Figure 1-6 -Kinetics of phyllid regeneration. Regeneration of chloronema filaments from the cells bordering the cut is assessed every 24 hours. Localized regeneration is usually a slow process and the first filaments are most often seen only from the 48h/72h time points on. This figure shows two instances of WT phyllid regeneration in the same culture conditions (with no specific treatment): panel A displays the usual regeneration process when the phyllid is cleanly cut.

Figure I- 1 -Figure I- 2 -

 12 Figure I-1 -Phylogenetic relationships in the green lineage (Viridiplantae). (Page 2)

Figure II- 3 -Figure IV- 3 -Figure V- 4 -Figure V- 5 -Figure V- 6 -Figure VII- 5 -ANNEXFigure 1 - 1 -Figure 1 - 2 -Figure 1 - 3 -Figure 1 - 4 -Figure 1 - 5 -Figure 1 - 6 -Figure 2 - 1 -Figure 2 - 2 -Figure 2 - 3 -

 334565111213141516212223 Figure II-3 -Occurrence of plant hormone signaling components in P. patens. (Page 33)

Title:

  Strigolactones perception and signal transduction in the moss Physcomitrium patens Keywords: Strigolactones, phytohormone, bryophytes, moss, signaling, SMXL

Table III -1 -Homologs of SL related genes in flowering plants and the moss Physcomitrium patens. 47 RESULTS AND DISCUSSION Introduction Strigolactones

 III

(SLs) are butenolide compounds with dual roles in plants: exuded in soil, SLs signal the presence of a host to Arbuscular Mycorrhizal (AM) fungi

[START_REF] Akiyama | Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi[END_REF][START_REF] Besserer | Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria[END_REF]

, and thus favor the establishment of symbiosis; as endogenous compounds, SLs (or derived compounds) play a hormonal role in developmental program

[START_REF] Gomez-Roldan | Strigolactone inhibition of shoot branching[END_REF][START_REF] Umehara | Inhibition of shoot branching by new terpenoid plant hormones[END_REF] for review: Lopez-Obando et al., 2015;

Table 1 )

 1 allowed to isolate mutants affected in all of clade (A-E) genes, as well as other mutants affected in fewer genes of this clade (seeLopez-Obando et al., 2016b and Supplemental table IV-2).

	We chose two triple (Ppkai2L-a2*-b4*-

  Table IV-2, with asterisks for null mutations. For each genotype, caulonema were counted after 2 weeks in the dark, from 24 individuals, grown in 3 different 24-well plates. Statistical

	groups (comparing genotypes in control conditions) are indicated by letters and were determined by a one-way ANOVA
	with Welch test (95% Cl). Significant differences between control and treated plants within a genotype based on one-
	way ANOVA with Welch test: ***, p < 0.001; **, p < 0.01; *, p < 0.5; . , p < 0.1.

  SaintGermain et al., 2016), using a TriStar LB 941 Multimode Microplate Reader from Berthold Technologies. Rad Laboratories, Inc., Hercules, California, USA) as described in de Saint Germain et al 2016. nanoDSF.

	Temperature melts proteins.
	Differential Scanning Fluorimetry (DSF) experiments were performed on a CFX96 Touch TM Real-Time PCR Detection
	System (Bio-

Proteins were diluted in Phosphate buffer saline (PBS) (100 mM Phosphate, pH 6.8, 150 mM NaCl) to ∼10μM

Table IV -

 IV 2, with asterisks for null mutations. For each genotype, caulonema were counted after 2 weeks in the dark, from 24 individuals, grown in 3 different 24-well plates. Statistical groups (comparing genotypes in control conditions) are

	indicated by letters and were determined by a one-way ANOVA with Welch test (95% Cl). Significant differences
	between control and treated plants within a genotype based on one-way ANOVA with Welch test: ***, p < 0.001; **,
	p < 0.01; *, p < 0.5.
	Supplementary table IV-1 -Oligonucleotides used in this study.
	Primer name/purpose	Sequence (5ʹ-3ʹ)
	Cloning for protein expression (Gateway recombination sites are underlined, protease cleavage sites are in italic)
	PpKAI2L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGGATGAGCTACCATCACTC
	C_attb1_HRV3C
	PpKAI2L-C_attb2	ggggaccactttgtacaagaaagctgggtctcaTCAACAAGACTCCAGATTCC
	PpKAI2L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGGAGGAAGGACCAACACT
	D_attb1_HRV3C
	PpKAI2L-D_attb2	ggggaccactttgtacaagaaagctgggtctcaTTACAGACTTCCTGCGAGGT
	PpKAI2L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGGAGGAGCCATCCTTGTT
	E_attb1_HRV3C
	PpKAI2L-E_attb2	ggggaccactttgtacaagaaagctgggtctcaTTATAGACTTCCAGCGAGGT
	PpKAI2L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGCAGTCCCACAATGTGAT
	F_attb1_HRV3C
	PpKAI2L-F_attb2	ggggaccactttgtacaagaaagctgggtctcaTCATGATGCAAAACATCGCAA
	PpKAI2L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGCCGAGCCCGTTGCTCTC
	H_attb1_HRV3C
	PpKAI2L-H_attb2	ggggaccactttgtacaagaaagctgggtctcaTCATGAGTCGATGCAGTGGAG
	PpKAI2L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGATTCCGCAATCGAGCTC
	K_attb1_HRV3C
	PpKAI2L-K_attb2	ggggaccactttgtacaagaaagctgggtctcaTCACGGCGCAAGGCAGCGGAGA
	PpKAI2L-L-	ggggacaagtttgtacaaaaaagcaggctccctggaagtgctgtttcagggcccgATGGTGGTGTCCGAGTCCTTG
	∆47_attb1_HRV3C
	PpKAI2L-L_attb2	ggggaccactttgtacaagaaagctgggtctcaTTAATCTTCAATGCAGCGAA
	Cloning for complementation assay
	AtD14_promo_attB4	ggggacaactttgtatagaaaagttgccCCTCTTGTTGGATTCTTGGC
	AtD14_promo_attB1R	ggggactgcttttttgtacaaacttgcTTTTTTATGTGTTTGGGTTTGAGG
	AtD14_CDS_attB1	ggggacaagtttgtacaaaaaagcaggcttcATGAGTCAACACAACATCTT
	AtD14_CDS_attB2_ΔS	ggggaccactttgtacaagaaagctgggtcCCGAGGAAGAGCTCGCCGGA
	AtKAI2_promo_attB4	ggggacaactttgtatagaaaagttgccTTCACGACCAGTATGGTTTACTCA
	AtKAI2_promo_attB1R	ggggactgcttttttgtacaaacttgcCTCTCTAAAGAAGATTCTTCTCTGGTT
	AtKAI2_CDS_attB1	ggggacaagtttgtacaaaaaagcaggcttcATGGGTGTGGTAGAAGAAGC
	AtKAI2_CDS_attB2_ΔS	ggggaccactttgtacaagaaagctgggtcCATAGCAATGTCATTACGAAT

  .

		Protein type (localization)		Arabidopsis thaliana		Physcomitrium patens
		ClpB (cy)	1	HSP101/AT1G74310
	Chaperone	ClpB (cp) ClpB (mt)	2 1	AT5G15450 and AT4G14670 AT2G25140	2	Pp3c24_9060 and Pp3c8_12320
		ClpB (?)			2 Pp3c19_4790 and Pp3c12_10120
						Pp3c16_17640, Pp3c25_7180,
		ClpC (cp)	2	AT5G50920 and AT3G48870	5	Pp3c3_18360, Pp3c5_23010 and
						Pp3c6_7360
		ClpD (cp)	1	ERD1/AT5G51070
		ClpD (?)			2 Pp3c18_15840 and Pp3c21_5430
		ClpX (mt)	3	AT5G53350, AT5G49840 and AT1G33360	1	Pp3c1_30360
		ClpX (?)			2 Pp3c1_40090 and Pp3c17_15650
	Clp	ClpP (mt)	1	AT5G23140	1	Pp3c4_31230
	proteolytic					Pp3c23_19030,
	complex
				ATCG00670, AT1G66670,
		ClpP (cp)	5	AT1G02560, AT1G11750 and	15
				AT5G45390

Table V -1 -Comparison of Clp proteins content in Arabidopsis thaliana and in Physcomitrium patens

 V

Subcellular localization of P. patens proteins are only in silico predictions (annotation on Phytozome and prediction by DTU.dk TargetP-2.0 server). Localization is given between parentheses: cytosol (cy), plastids (cp), mitochondria (mt), undetermined (?).

  , is not impacted by loss of PpSMXL function. Thus, SL biosynthesis is likely not regulated by PpSMXL proteins. We also compared the levels of expression of Pp3c6-15020, a gene that is induced by both (+)-GR24 and endogenous PpCCD8-derived compounds. Contrary to PpCCD7, expression of this gene is disturbed in Ppsmxl mutants (Supplemental Figure VI-16):It is overexpressed in the simple Ppsmxld mutant and (non-significantly) in the simple Ppsmxlb mutant as well as in both double mutants. This would imply that PpSMXL genes redundantly repress SL response on the short-term, contrary to what is hypothesized in the long-term (caulonema growth assays).

  GFP degradation in rice roots[START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF]. Thus, PpSMXL proteins do not behave similarly to AtSMXL6/7 or D53 proteins and they seem insensitive to both the SL mimic (+)-GR24 and endogenous SL from both P. patens (GFP lines) and N. benthamiana (RFP transient expression lines). While their stability in N. benthamiana could be explained by an incompatibility with the Angiosperm MAX2, PpSMXLs are also stable in P. patens. This indicates that PpSMXL proteins are likely not degraded in the context of SL signaling. Likewise, deletion of the degron motif (or degron-like for PpSMXLA/B) has no effect since these proteins are stable (supplemental

	; (2) AtSMXL7 is significantly decreased after only 10 minutes
	of a 1µM (±)-GR24 treatment in p35S:AtSMXL7-YFP Arabidopsis roots (Soundappan et al., 2015); (3) as little as 12
	minutes are necessary for pOsAct:D53-

202 VII-C) PpSMXL are not bona fide heat-shock proteins but they play an important role in temperature- mediated growth regulation

  Since we found that PpSMXL proteins are not repressors in the SL signaling pathway, we wanted to clarify the molecular function of these proteins in development. Notably, we sought whether they have similar properties as more distant ClpATPases such as ClpB/HSP101 (see chapter V). First, we investigated the profile of PpSMXL genes expression when WT P. patens was subjected to 3 hour-long incubations at different temperatures. Two different experiments were carried out, to extend the range of temperatures examined. In a preliminary in silico search, we found that PpSMXL promoter sequences and 5'UTR regions contain several heat-shock elements and/or similar sequences (Supplemental Figure VII-1), which seem broadly conserved between P. patens and Angiosperms[START_REF] Schöffl | The DNA sequence analysis of soybean heat-shock genes and identification of possible regulatory promoter elements[END_REF][START_REF] Elzanati | Dynamics of the Transcriptome Response to Heat in the Moss, Physcomitrella patens[END_REF]. Hence, we thought that expression PpSMXL might be induced by heat. However, we observed that none of the four PpSMXL display the characteristic profile of HSPs e.g. a steep increase of expression at high temperature (see Supplemental Figure VII-2 for the profile of PpHSP19). Instead, we discovered that, within A/B and C/D clades, each gene seems to have a different reaction to temperature: PpSMXLA is induced by heat (42°C) and repressed by cold (8°C and 15°C), PpSMXLB is repressed by heat (42°C) and seemingly also repressed by cold (15°C) (figureVII-4). On the other hand, PpSMXLC expression is induced by cold (8°C) but does not seem sensitive to heat, and PpSMXLD is the most temperature-responsive gene out of the four PpSMXL: it is repressed by heat (42°C, and nonsignificantly at 37°C) and induced by cold (4°C, and non-significantly at 8°C).

  patens, non-vasculaire), par comparaison avec ce qui est connu chez les plantes vasculaires, permet de questionner l'évolution des voies cellulaires associées aux SL chez les plantes terrestres. Chez les plantes vasculaires, les voies de biosynthèse et de perception des SL sont assez bien décrites. La voie de signalisation des SL commence par la perception de la molécule par le récepteur D14 dans le cytosol, qui la clive et reste associé à une partie de la SL. Ce complexe interagit dans le noyau avec deux partenaires : la protéine à boîte F MAX2, capable de recruter un complexe d'ubiquitination, et certaines protéines SMXL. Ces protéines agissent comme des répresseurs de la réponse aux SL et vont être ubiquitinées sous l'action du complexe recruté par MAX2, puis rapidement dégradées par le protéasome. Chez P. patens, la plupart des gènes de biosynthèse et de signalisation des SL sont retrouvés, parfois en nombres différents comparés aux plantes vasculaires. Seules des approches de génétique inverse permettent de définir précisément leur fonction. La précédente caractérisation du mutant de biosynthèse Ppccd8 chez P. patens a montré que les SL sont synthétisées via une voie similaire à celle des plantes vasculaires. En outre, la fonction des SL dans la régulation de l'architecture est conservée chez P. patens. Au contraire, en accord avec la découverte précédente que l'unique homologue de MAX2 chez P. patens n'est pas impliqué dans la signalisation des SL, les résultats ici présentés indiquent que la signalisation des SL n'est que partiellement conservée entre P. patens et les plantes vasculaires. En effet, seuls 2 homologues PpKAI2L de D14, sur les 13 possédés par P. patens, sont impliqués dans la perception des SL, d'après l'étude de mutants perte-de-fonction et les analyses de biochimie. Par ailleurs, les protéines PpKAI2L sont phylogénétiquement plus proches d'une autre protéine appelée KAI2 que de D14. Or KAI2 ne perçoit pas les SL chez les plantes vasculaires. De plus, la caractérisation des mutants perte-defonction Ppsmxl, obtenus par l'utilisation de la technologie CRISPR, et les analyses de liaison génétique avec PpMAX2 et PpCCD8 montrent que les quatre protéines PpSMXL ne jouent pas un rôle majeur dans la réponse aux SL. Cependant, les protéines PpSMXL les plus proches phylogénétiquement des SMXL ancestrales apparaissent comme des régulateurs importants de la croissance, ce qui pourrait constituer le rôle ancestral des protéines SMXL, en accord avec des études phylogénétiques récentes. Cette régulation de la croissance constituerait la réponse à un autre signal endogène, plus ancestral que les SL, le KL (ligand de KAI2). La transduction du signal KL serait conservée au moins chez les plantes terrestres et impliquerait chez P. patens certaines protéines PpKAI2L et les protéines PpMAX2 et PpSMXL. L'identité moléculaire du KL n'a pas encore été élucidée. N'étant pas conservée chez P. patens, la signalisation des SL résulte possiblement d'une innovation spécifique de la lignée des plantes vasculaires. Ainsi, la voie de réponse aux SL présente chez la mousse résulterait d'une évolution convergente vers la perception des SL. Il reste donc à élucider comment le signal SL est transduit chez P. patens, en aval de sa perception par certaines protéines PpKAI2L. Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France

AtKAI2 (B); PpKAI2L-D (C); PpKAI2L-E ; (D) PpKAI2L-F (E) ; PpKAI2L-K (F) and PpKAI2L-H (G) was monitored (see Supplementary FigureIV-4) and used to determine the apparent Kd values. The plots represent the mean of two replicates and the experiments were repeated at least three times. The analysis was performed with GraphPad Prism 8.0 Software.

of first shown amino-acid (aa) and the predicted secondary structure are indicated above the WT protein sequence. The sgRNA sequence is shown in blue, with the PAM site underlined. Deletions are shown as dashes, insertions are noted with orange letters. The mutation type is shown on the right. Premature STOP codons are noted in bold, and with a red star on the aa sequence. On protein sequences, the number of not shown aa is noted between slashes. For PpKAI2L-E, the serine (S) of the catalytic triad is noted in bold blue. For PpKAI2L-H, a deletion of the full coding sequence between ATG and STOP was obtained through homologous recombination; the use of CRE recombination led to 46 residual nucleotides, (not shown) corresponding to the LoxP site (see Methods). See Supplemental TableIV-2 for the list of mutants carrying one or several of the shown mutations.

Supplemental Figure VI-3 -Expression of PpSMXL genes in P. patens tissues according to the eFP Browser database. Tissular expression pattern for PpSMXLA, PpSMXLB, PpSMXLC and PpSMXLD. Results taken from the http://bar.utoronto.ca/efp_physcomitrella/cgi-bin/efpWeb.cgi website, in absolute mode.

Acknowledgments

Acknowledgments

The authors thank Adrian Scaffidi (University of Western Australia, Perth, Australia) for the gift of carlactone, and Mark Waters (The University of Western Australia, Perth, Australia) for Arabidopsis kai2-2 and d14-1 kai2-2 mutants.

We are grateful to Jean-Paul Pillot (IJPB) for precious help with Arabidopsis branching assays, and to Fabien Nogué (IJPB) for stimulating discussions.

Acknowledgements. We would like to thank Fabien Nogué for his helpful advice and for giving us guide RNAs targeting the Pp108 non-coding locus. We also give special thanks to François-Didier Boyer for readily supplying (±)-GR24 and its enantiomers, and Gladys Cloarec for her assistance with confocal microscopy observations. We thank Martine Pastuglia for the gift of BiFC control constructs and Michael J. Prigge for nicely sending us the pMP destination vectors.

ancestral role. To address this second question, we investigated the function of the 4 SMXL genes found in the genome This chapter is presented as a research paper that will be submitted, in a less detailed form, to The Plant Cell. The method chapter we refer to in methods of the present paper is provided as an annex to the thesis manuscript and is to be published in the Methods in Molecular Biology book series.

Fundings. The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). The collaboration with VIB Ghent was supported by a Programme Hubert Curien (PHC) Tournesol grant.

were loaded at 5 μL/min with 0.05% TFA in 5% acetonitrile and peptides separation was performed at a flow rate of 300 nl.min -1 with a 5 to 35% solvent B gradient in 40 min. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 100% acetonitrile. NanoLC-MS/MS experiments were conducted in a Data Dependent acquisition method by selecting the 20 most intense precursors for CID fragmentation with Q1 quadrupole set at low resolution for better sensitivity. Raw data were processed with MS Data Converter tool (AB Sciex) for generating .mgf data files and protein identification was performed using the MASCOT search engine (Matrix Science, London, UK) against the PrKAI2 sequence with oxidation of methionines and ligand-histidine adduct as variable modifications. Peptide and fragment tolerance were respectively set at 20 ppm and 0.05 Da. Only peptides with a mascot ion score above identity threshold (25) calculated at 1% FDR Homology model. Superimposition model figure were prepared by using PyMOL (DeLano Scientific) with the crystal structure of AtD14 (PDB ID 4IH4) and PpKAI2L-H (PDB ID 6AZD) Phylogenetic analysis. Phylogenic analysis was conducted in MEGA5 [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Protein sequences were aligned using CLUSTALX. The Maximum Likelihood method based on the Dayhoff matrix-based model was used (Schwarz and Dayhoff, 1979). Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used.

Supplementary table IV-1.

Oligonucleotides used in this study.

Supplementary table IV-2.

Mutants used in the study. PpKAI2L-H (I-L), PpKAI2L-F (M-P), PpKAI2L-K (M-P), AtKAI2L-K (U-X) and AtD14 (Y-AB) proteins in the presence of SL analogs. Changes in intrinsic fluorescence emission spectra of PpKAI2L proteins, in the presence of various concentrations of (+)-GR24 (A;E;I;M;Q;U;Y), (-)-GR24 (B;F;J;N;R;V;Z), (+)-2'-epi-GR24 (C;G;K;O;S;W;AA), (-)-2'-epi-GR24 (D;H;L;P;T;X;AB). Proteins (10 µM)

Supplementary table IV-

123

SMXL are strictly nuclear-localized proteins [START_REF] Yang | Nuclear-localized AtHSPR links abscisic acid-dependent salt tolerance and antioxidant defense in Arabidopsis[END_REF][START_REF] Wallner | Strigolactone-and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation[END_REF][START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF], similarly to MAX2 [START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF][START_REF] Shen | The F-Box Protein MAX2 Functions as a Positive Regulator of Photomorphogenesis in Arabidopsis[END_REF], while D14 [START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF][START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF] and KAI2 [START_REF] Lee | A missense allele of KARRIKIN-INSENSITIVE2 impairs ligand-binding and downstream signaling in Arabidopsis thaliana[END_REF] can be found in both the nucleus and the cytosol. D14 can even be found outside of cells, as evidenced by its transport through the phloem in rice and in pea [START_REF] Kameoka | Phloem Transport of the Receptor DWARF14 Protein Is Required for Full Function of Strigolactones[END_REF]. SMXL7 and SMAX1 have been shown to localize more specifically to speckles inside the nucleus [START_REF] Soundappan | SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis[END_REF][START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF], as well as D53 [START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF], but the identity and functional relevance of these structures have not been further investigated. D14 is apparently recruited to these speckles upon SL signaling (like TPR2), while SL-induced interaction between D14 and MAX2 occurs across the whole nucleus [START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF]. These speckles might reveal SMXL7 association with active transcription sites where its repressive activity is needed. Liang and colleagues also reported that SMXL6 and SMXL7 in Arabidopsis can interact together, hinting at the formation of oligomeric structures, reminiscent of other Clp-ATPases characteristics [START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF] (see previous paragraphs V-C and V-E).

The first studies investigating SMXL proteins were published in 2013 [START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF][START_REF] Jiang | DWARF 53 acts as a repressor of strigolactone signalling in rice[END_REF][START_REF] Stanga | SUPPRESSOR OF MORE AXILLARY GROWTH2 1 Controls Seed Germination and Seedling Development in Arabidopsis[END_REF]. However smxl mutations had been evidenced much earlier in 1977, when Iwata and colleagues obtained the d53 (Ossmxl7) gain of function mutation in rice, in a forward genetics mutagenesis screen for dwarf mutants [START_REF] Iwata | Linkage studies in rice (Oryza sativa L.). Linkage groups for six genes newly described[END_REF]. When this same mutant was extensively characterized in 2013, it was discovered to encode a stabilized version of the D53 protein (where the RGKT degron motif is deleted), leading to several phenotypic defects, notably hyper branching [START_REF] Zhou | D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling[END_REF]. This mutant was then linked to SL since d53 overexpresses D10/OsCCD8 and overproduces SL, while it has a decreased FC1/OsBRC1 expression, suggesting SL signaling is hindered. The WT D53 protein is degraded by the proteasome in response to SL in a D3/OsMAX2 and D14 dependent fashion, while the mutant d53 protein remains stable. As a RNAi knock down of D53 expression in d3 and d14 backgrounds restores almost completely the hyper branching phenotype, it seems that D53 is the major target (if not the only target) of D3 in rice.

The same year (2013), the smax1 loss of function mutation was identified in Arabidopsis, in an suppressor screen of max2 elongated hypocotyl and delayed germination phenotypes [START_REF] Stanga | SUPPRESSOR OF MORE AXILLARY GROWTH2 1 Controls Seed Germination and Seedling Development in Arabidopsis[END_REF]. However, a similar KAR/KLinduced degradation of SMAX1 was only experimentally evidenced this year, in rice [START_REF] Paszkowski | SD-The negative regulator SMAX1 controls mycorrhizal symbiosis and strigolactone biosynthesis in rice[END_REF][START_REF] Zheng | Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness[END_REF] and in Arabidopsis [START_REF] Khosla | Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2[END_REF]. The association of KAI2, MAX2 and SMAX1/SMXL2, in a KAR/KL-dependent way, was also demonstrated recently in Arabidopsis (Wang et al., 2020a) (chapter III).

Authors contributions:

A.G is the main author of the manuscript and performed most of the experiments. Some steps in genotyping mutant and transgenic lines and support with molecular cloning was given by P.L.B, and Y2H experiments were done by S.S. Plasmids used for Ppsmxl mutants generation in the first strategy, as well as higher order Ppsmxl mutants were obtained by M.L.O. Preparation of protein extracts for GFP-trap pull-down was done with the kind help of A.J. Acquisition of GFP-trap pull-down data was done by the VIB proteomics platform in Ghent, and MS/MS data analysis was carried out by D.C. S.B and M.L.O supervised the project. S.G and S.L collaborated in the investigation of PpSMXL interactions. S.B, A.S.G and C.R contributed to experimental design and to the argumentation developed herein.

Expression of PpSMXL genes is slightly affected by (±)-GR24 enantiomers but very sensitive to light

In Angiosperms, AtSMXL7 and OsD53 are upregulated in response to the synthetic compound (±)-GR24 treatment. (±)-GR24 is known to stimulate both the SL and the KAI2-dependent pathway (putative KL pathway) in Angiosperms, as the enantiomers contained in this racemic mix have different target pathways: (+)-GR24 stimulates the SL pathway, (-)-GR24 stimulates both pathways [START_REF] Scaffidi | Strigolactone Hormones and Their Stereoisomers Signal through Two Related Receptor Proteins to Induce Different Physiological Responses in Arabidopsis[END_REF]Wang et al., 2020a). Hence, we tested separately the effect of the two (±)-GR24 enantiomers on PpSMXL genes expression to check if these genes are transcriptionally regulated through the SL and putative KL pathways. We assessed this response in the dark and in control light conditions (long day regimen), as we found previously that light could modify the effect of these molecules on transcript levels of putative SL and KL-related genes (Lopez-Obando et al., 2016a). In dark conditions, we found that PpSMXLA is slightly induced by (+)-GR24 but not impacted by (-)-GR24 (Figure VI-3, grey panels). On the opposite, PpSMXLC transcript levels are mildly increased by (-)-GR24 but not impacted by (+)-GR24. PpSMXLB behaves as PpSMXLA, although its induction by (+)-GR24 is even higher. On the other hand, PpSMXLD does not appear to be regulated by either enantiomer in the dark. In the light, the response to (-)-GR24 was abolished for PpSMXLC, similarly to the response to (+)-GR24 of PpSMXLA, which even tended to become inverted (Figure VI-3, white panels). Again, PpSMXLB followed the same trend as PpSMXLA. On the contrary, PpSMXLD was upregulated by (-)-GR24 in the light. These moderate effects of (±)-GR24 enantiomers are in the same amplitude range as those reported for Angiosperms SMXL genes Finally, we noted that all four genes were significantly more expressed in the dark than in the light: If we compare levels of expression in control samples, PpSMXLA is decreased more than 10 fold, PpSMXLB approximately 4 fold, PpSMXLC In order to determine whether endogenous compounds produced via PpCCD8 activity (probably carlactone and derived non-canonical SL) have equivalent effects as (+)-GR24 on PpSMXL expression, we replicated the previous experiment in Ppccd8 mutant plants (Proust et al., 2011). In dark conditions, all four PpSMXL genes had similar response profiles to (+)-GR24 and (-)-GR24 in Ppccd8 as in WT and their expression levels were little affected by the loss of PpCCD8 function. Thus, endogenous SL-like compounds produced via PpCCD8 enzymatic activity have little effect on PpSMXL expression in the dark (Supplemental Figure VI-4, grey panels). In the light, there was virtually no change again for PpSMXLA, PpSMXLB and PpSMXLC. However, PpSMXLD control levels were increased and the gene became unresponsive to (-)-GR24, suggesting that PpCCD8-derived compounds inhibit PpSMXLD expression and that these compounds are somehow necessary for (-)-GR24-mediated PpSMXLD induction (Supplemental Figure VI-4, white panels). Taken as a whole, PpSMXL expression analysis in the Ppccd8 mutant reveals no significant effect of endogenous PpCCD8-derived compounds, except on PpSMXLD in light conditions. Treatments with (±)-GR24 enantiomers were also carried out in the Ppmax2-1 loss of function mutant [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF], to investigate the role of PpMAX2 in PpSMXL transcriptional regulation. In dark conditions (Supplemental Figure VI-4, grey panels), all four PpSMXL genes tended to be less expressed in the Ppmax2-1 mutant than in WT and this difference was significant for PpSMXLA and PpSMXLC. Furthermore, PpSMXLA transcript level was not induced by (+)-GR24 anymore, while it became significantly repressed by (-)-GR24, reinforcing a trend that was already seen in WT.

Likewise, PpSMXLB expression profile changed in a comparable manner. On the contrary, PpSMXLC became responsive (increase) to (+)-GR24 and ceased to respond to (-)-GR24, the same trend of inversion was observed for PpSMXLD (Supplemental Figure VI-4, grey panels). Hence, in dark conditions, PpMAX2 likely induces PpSMXL expression in the absence of exogenous compounds, while it is needed to regulate response to (±)-GR24 enantiomers, in an opposite way for the PpSMXLA/B and the PpSMXLC/D clades. In light conditions (Supplemental Figure VI-4, white panels), PpMAX2 loss of function resulted in a switch towards dark conditions' behavior for PpSMXLA and PpSMXLB, with PpSMXLB even being repressed by (-)-GR24 treatment. On the other hand, PpSMXLC and PpSMXLD became responsive to (+)-GR24 (increase) white panels). Therefore, in the light also, lack of PpMAX2 results in a switch of responsiveness to (±)-GR24 enantiomers. Considered together, these results show that PpSMXL genes' transcription is mainly regulated by light and responds in opposite trends to (±)-GR24 enantiomers, both regulations being permitted at least partly via PpMAX2, with again a split of behavior between the two PpSMXL clades. It is also important to note that, at least regarding PpSMXL expression, the Ppmax2-1 loss of function mutant remains responsive to (-)-GR24, further confirming our previous conclusion that (-)-GR24 is not a perfect mimic of the signal perceived through PpMAX2 (likely KL) (see Chapter IV).

135 Transcript levels of the four PpSMXL genes, relative to the two reference genes PpElig2 (Pp3c14_21480) and PpAPT (Pp3c8_16590). Two-week-old plants were incubated in the dark for one week and then treated with 1µM (+)-GR24 (diagonal hatches) or (-)-GR24 (horizontal hatches) or DMSO (control, solid color) for 6 hours, also in the dark (grey background). The same experiment was repeated in white light (white background). For each treatment, six biological replicates and two technical replicates were used. Points are the mean of the two technical replicates, coloured bars represent medians. 2-fold differences in median values are estimated as significant (DE).

Expression of PpSMXL genes is regulated by light in a complex way

Wanting to clarify the effect of light on PpSMXL expression, we checked whether this expression was also impacted by shorter exposure times and specifically by red light, as was shown for PpMAX2 [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. First, to assess the validity of our experimental setup, we measured the expression of known light responsive genes PpPOR1 and PpHY5a. These two control genes were elevated by red light in the medium and short term (respectively), showing that our light treatments worked . Examining PpSMXL response to these light conditions, we found first that all four genes indeed tend to be repressed by white light in WT plants . The amplitude of white light effect was dimmer than noted before, consistently with the shorter time of exposure (24 hours here, instead of one week of long days white light regimen in Figure . Unexpectedly, continuous red light for 24h did not have the same effect as white light for PpSMXLA and PpSMXLC, suggesting blue light might also play an important role in regulating these two genes expression. On shorter term, all except PpSMXLD were induced transiently by red light, the most early responsive one being PpSMXLC. In the absence of PpCCD8 (Ppccd8 mutant, Supplemental

PpSMXL proteins are localized mainly in the nucleus

Functional nuclear localization signals (NLS) were found in AtSMXL7 [START_REF] Liang | SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[END_REF] andOsSMAX1 proteins [START_REF] Choi | The negative regulator SMAX1 controls mycorrhizal symbiosis and strigolactone biosynthesis in rice[END_REF]. This N-terminal region is broadly conserved in the four PpSMXL proteins, suggesting they are also located in the nucleus (Supplemental

Ppsmxl loss-of-function mutants do not display a constitutive SL response phenotype

In order to clarify the function of the four PpSMXL genes in P. patens, we employed two CRISPR-Cas9 mutagenesis strategies. In the first one, we used only one guide RNA targeted against the most upstream possible CDS region. However, since the functional relevance of most of SMXL proteins domains is barely known, we ensured obtaining knock-out mutants by carrying out another CRISPR mutagenesis strategy in parallel with the former. In this second strategy, we used multiple guides, with one in each untranslated region (UTR) (Figure VI-1 andsupplementary table). Mutations giving rise to a complete deletion of the CDS are noted as Ppsmxl∆, while those causing a frameshift are simply noted as Ppsmxl . Reasoning that the four PpSMXL proteins group into two clades, from our previous observations and from literature [START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF], we first sought double mutants for each clade, e.g. Ppsmxl(∆)ab and Ppsmxl(∆)cd mutants. Our starting hypothesis was that P. patens SMXL proteins could be functionally analogous to the SMXL7/D53 clade of Angiosperms, e.g. they would be acting as negative regulators of SL signaling. Under this hypothesis, PpSMXL loss of function should give rise to a growth phenotype opposite to that of the SL biosynthesis mutant Ppccd8, e.g. restricted protonema extension. We noted that all mutants solely disturbed in the AB clade (both double and single mutants) were highly similar to the WT when grown in white 2014), is probably cell division (this is further discussed in chapter VII). Accelerated cell division rate and filaments branching still must be confirmed in Ppsmxl mutants, however it is supported by the observation that double Ppsmxl mutants of both clades (tend to) develop more caulonema filaments than WT in the dark . Furthermore, the observation that phenotypic response to (+)-GR24 cannot occur in the absence of PpSMXLC/D also suggest that SL act at least partially via the PpMAX2-PpSMXL pathway. However, as we have seen herein (and in chapter IV), loss of PpMAX2 function or loss of function in (almost) all clade (A-E) PpKAI2-L does not abolish response to (+)-GR24.

Hence, it is evident that SL signaling does not solely rely on the PpMAX2-dependent pathway, and we can hypothesize that perception of SL by PpKAI2-L proteins of the (JGM) clade does not act via a regulation of the PpMAX2-dependent pathway but nonetheless requires functional PpSMXLC/D.

PpSMXLC likely has a specific role among PpSMXL proteins

We noted that higher order Ppsmxl mutants display highly disturbed growth of gametophores, loss of fertility and rapid browning of the protonema, suggesting that extreme activation of the PpMAX2-dependent pathway is very Puzzlingly, we found that the amount of pulled down PpSMXLC was apparently decreased by (±)-GR24, suggesting that the PpSMXLC protein might actually be less accumulated in response to this treatment, potentially degraded in response to the (-) component (but this might take longer than the 20 minutes we tested earlier, here 6h).

However, this potential degradation could be independent of proteasomal activity, as we did not find ubiquitin to be an interactor of PpSMXLC, unlike what was shown for AtSMXL7 [START_REF] Struk | Quantitative Tandem Affinity Purification, an Effective Tool to Investigate Protein Complex Composition in Plant Hormone Signaling: Strigolactones in the Spotlight[END_REF]. Alternatively, the treatment might have not been sufficient to enable recovery of ubiquitin at levels above the identification threshold. This might be due to this experiment having been carried out in light conditions, in which we have noticed many times that response to (±)-GR24 enantiomers was decreased (namely caulonema growth assays and transcriptional response assays). Despite the possible technical shortcoming of this experiment, we can note that the interactors found for PpSMXLC clearly support the major implication of this protein in general repression of growth, probably by repressing its interactors' activity. We found that (±)-GR24 treatment had virtually no impact on PpSMXLC interactions (supplemental Figure VI-26), which is strikingly different from what was shown for AtSMXL7 [START_REF] Struk | Quantitative Tandem Affinity Purification, an Effective Tool to Investigate Protein Complex Composition in Plant Hormone Signaling: Strigolactones in the Spotlight[END_REF], for which more than 30 interactions were promoted by a 1µM (±)-GR24 treatment in the dark. Another notable difference with AtSMXL7 interactome, in the absence of (±)-GR24, is that no protein involved in transcription, especially TPL, was found among PpSMXLC interactors (supplemental Figure VI-25), despite it having a canonical EAR motif and being found in the nucleus. Other interactors are similar between PpSMXLC and AtSMXL7 and might represent more common and possibly ancestral interactors of SMXL proteins, such as interactors associated with cytoskeleton, translation, energetic metabolism and photosynthesis. Interestingly, (±)-GR24 prevents salt stress damage in rice, by limiting chlorophyll degradation and decrease of the photosynthetic rate, and increasing POD (peroxidase) and SOD (superoxide dismutase) activities [START_REF] Ling | Effects of strigolactone on photosynthetic and physiological characteristics in salt-stressed rice seedlings[END_REF]. Thus, we could hypothesize that either D53 or OsSMAX1 (or even both) activity would have the opposite outcome, e.g. favoring decrease of photosynthetic ability and accumulating ROS (reactive oxygen species). These two processes are also highlighted by PpSMXLC interactome, therefore proteins involved in these processes might likewise be ancestral interactors of SMXL proteins.

Methods

Cultivation of Physcomitrium patens Gransden. Unless otherwise explicitly stated in legends, experiments were always carried out on PpNO3 medium (corresponds to the minimal medium described by Ashton et al., 1979), in the following control conditions: 25°C during daytime and 23°C at night, 50% humidity, long days conditions with 16 hours of day and 8 hours of night (quantum irradiance of ~80 µmol/m 2 /s). Multiplication of tissues from young protonema fragments prior to every experiment is done in the same conditions but using medium with higher nitrogen content (PpNH4 medium, PpNO3 medium supplemented with 2.7 mM NH4 tartrate). For red light experiments, plants were grown on PpNO3 medium in Magenta pots at 25°C, in continuous red-light (~45 µmol µmol/m 2 /s). Cellophanes of appropriate sizes were used for monitoring of protonema extension kinetics, as well as for the cultures launched in 6well plates for gene expression studies (see a detailed protocol in our methods chapter titled "Methods for medium-scale study of the biological effects of strigolactone-like molecules on the moss Physcomitrella patens"). Analysis of caulonema growth in the dark was performed in 24-well plates, with ~2 weeks of growth in control conditions before incubation (± treatment) in the dark and placed vertically for ~10 days (see the same method chapter).

Gene expression analyses by qPCR. Total P. patens RNA were extracted and rid of contaminant genomic DNA using RNeasy Plant Mini Kit and on-column DNAse I treatment (Qiagen), following supplier's indications. cDNA were obtained using the Maxima TM H Minus retrotranscriptase (ThermoFisher), from 50-250 ng of total RNA. cDNA extracts were diluted at 1/5-1/8 before use. RT qPCR was performed in a 384 well thermocycler (QuantStudioTM 5 , ThermoFisher), using SsoAdvanced Universal SYBR Green Supermix (BioRad) and appropriate primers. The thermocycler was programmed to run for 3 min at 95°C, followed by 40-45 cycles of 10 sec at 95°C and 30 sec at 60°C. Each biological replicate was run twice to assess technical variation. Expression of genes of interest was normalized by two reference genes among PpElig2 (Pp3c14_21480), PpAPT (Pp3c8_16590) and PpACT3 (Pp3c10_17080) (all three are expressed at similar levels (Le Bail et al., 2013)). Relative expression was calculated as RE = 2-CTgene/2-CTref where CTref is the mean value of the two reference genes. For the study of PpSMXL genes' expression across development (figure 2), WT P. patens was cultivated in petri dishes from fragmented tissues for 6, 11 or 15 days, or in Magenta pots for 35 days. Four biological replicates were used for each timepoint. For the "response to GR24" experiment (figure 3 and supplemental to figure 3), WT, Ppccd8 and Ppmax2-1 were cultivated from fragmented protonema in 6-well plates for 2 weeks in control conditions, then transferred in the dark for one week, and treated with 1 µM (+)-GR24, 1 µM (-)-GR24 or 0.01% DMSO in the dark for 6 hours. Six biological repeats were used for each genotype and treatment. For the "response to light" experiment, 2-week-old WT, Ppccd8 and Ppmax2-1 were similarly transferred in the dark for 5 days, and then either kept in the dark for 24 hours, kept in control white light conditions for 24 hours, or placed under constant red-light for 1, 6 or 24 hours. Six biological repeats were used for each genotype and treatment. Generation of BiFC constructs. Gateway cloned inserts of genes of interest were integrated into pbiFP vectors using LR Clonase II mix (ThermoFisher). Inserts containing a STOP codon were cloned in pbiFP2 and pbiFP3, those not containing a STOP were cloned in pbiFP1 and pbiFP4 (when possible, all four vectors were obtained for a given gene).

Resulting vectors were electroporated into Escherichia coli DH10B cells and clones were selected on spectinomycin.

In phase integration of the coding sequence relative to the half eYFP tag was checked by sequencing insert's ends. Yeast two hybrid (Y2H) assays. The DupLEXA yeast two-hybrid system (OriGene Technologies) plasmids pEG202 (LexA-bait fusion), pJG4-5 (B42-HA-prey fusion) and pSH18-34 (high sensitivity LacZ reporter). pEG202 plasmids were transformed into the RFY206 yeast strain, together with pSH18-34. pJG4-5 plasmids were transformed into the EGY48 yeast strain. Transformed strains were mated according to interaction of interest, on yeast extract peptone dextrose non-selective medium (YPD: 2% peptone, 1% yeast extract, 2% agar, 2% D-glucose, pH adjusted to 7.0 with NaOH). Mated cells were then selected by two rounds of growth on YNB glucose (0.43g/L YNB, 5g/L NH4SO4, 0.6 g/L dropout, 1% D-glucose, 30 mg/L L-leucine, 2% agar). For assays, selective YNB galactose X-gal medium was prepared from 2X concentrated YNB galactose medium (2.125g/L commercial YNB, 6.25 g/L NH4SO4, 0.75 g/L dropout (-His-Ura-Leu-Trp)) by mixing 175 mL of 2X YNB galactose, 2X agar solution (4%), 50 mL 20% galactose, 50 mL 10% raffinose, 3 mL L-leucine, 400 µL X-gal (100 mg/mL) and 50 mL of 10X phosphate buffer (7% Na2HPO4;7H20 and 3% NaH2PO4). Selected mated cells were pinned on selective YNB galactose X-gal plates, with addition of either 500 µL of 50 mM (±)-GR24 or 500 µL of DMSO, and grown for 72 hours at 30°C. GFP-trap experiment. 200 mL liquid PpNH4 cultures of WT, proZmUbi:flag-GFP and proZmUbi:GFP-PpSMXLC#1 lines were launched from fragmented 7 day-old protonema tissues in 500 mL Erlenmeyer flasks. After 3 weeks of constant gentle agitation in control conditions, 200 µL of 3 mM (±)-GR24 or 200 µL of 0.1% DMSO were added to cultures. For each genotype, three biological replicates were used per treatment. After 6 hours, tissues were harvested by filtering cultures (140 µm filters) and were immediately flash frozen in liquid nitrogen and grinded with a mortar and pestle. Protein extraction was carried out with solutions described in -and following the-GFP-trap protocol by Chromotek (RIPA lysis buffer was supplemented with protease inhibitors (cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail from Roche) and 1mg/mL DNAse I). Cell lysates (volume adjusted to 1 mL with dilution buffer) were kept at -80°C until pull-down. 50 µL of lysate was mixed with 50 µL 2X SDS sample buffer to proceed to SDS page and immunoblot analysis to check for proteins integrity and GFP expression (3H9 rat monoclonal anti-GFP antibody, goat anti-rat antibody). Dilution buffer was removed from cell lysates and the extracts were incubated with GFP-trap_A agarose beads covalently coupled with alpaca anti-GFP-nano-antibody (Chromotek), for 2 hours at 4°C under constant gentle agitation (beads were previously washed twice with ice cold dilution buffer). Then, beads were recovered by centrifugation at 500 g for 2 minutes (4°C), washed twice with ice cold dilution buffer, and bound proteins were eluted by mixing the beads with 200 µL of MilliQ water and 50 µL of pure formic acid. Eluted fractions were recovered three times and pooled, then dried in a speedvac. Dry extracts were dissolved in 50 µL of urea buffer (20 mM HEPES pH8.0, 8M urea), DTT was added to the final concentration of 15 mM, and samples were incubated at 55°C for 30 minutes under agitation. Then, IAA was added to the final concentration of 30 mM and the samples were incubated at room temperature for 15 minutes in the dark and under agitation. Samples were diluted two-fold with 20 mM HEPES (pH8.0) and predigested by endo-proteinase LysC (1:100 v/v) for 4 hours at 37°C under agitation. Samples were again diluted two-fold in 20 mM HEPES and digested with trypsin (1:100 v/v) overnight at 37°C under agitation, then acidified with TFA (to 1% final concentration). Peptides were captured using C18 OMIX tips (Agilent), following providers' instructions. Peptide extracts were then characterized by MS/MS analyses.

Agroinfiltration of

Graphics generation and statistical analyses.

Supplementary table VI

Cloning of PpSMXL promoters Primer name

Sequence (recombination/restriction site)

GCTAGATCGATGGGTCCAGGCGG Guide RNAs used to ease integration of constructs at the Pp108 locus Guide name Sequence of crRNA + PAM Pp108-gRNA1 AATCTAATTCTGACTAGTGGTGG Pp108-gRNA2 ATTACTAGTAAAAGCACATAAGG As we noticed opposite effects of (+)-GR24 and (-)-GR24 on the number of caulonema filaments grown in the dark, suggesting an opposite role of the two enantiomers on cell division, we tested the effect of these molecules on regeneration. Regeneration can broadly be defined as the development of specialized tissues from a few (or even a single) totipotent cell(s), either pre-existing (stem cell(s)) or obtained by dedifferentiation of (a) specialized cell(s) [START_REF] Birnbaum | Slicing across Kingdoms: Regeneration in Plants and Animals[END_REF]. (±)-GR24 effect as an inhibitor of protonema regeneration from protoplasts has already been shown [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF], however the role of separate enantiomers had yet to be clarified. To have a clearer view of the expected effects of endogenous compounds mimicked by (+)-GR24 (SL) and (-)-GR24 (mainly KL)

on regeneration, we also included the Ppccd8 and the Ppmax2-1 mutants in our tests, the former not producing SL and the latter putatively displaying a KL-insensitive phenotype. We started by carrying out regeneration assays on dissected phyllids, as we expected a very poor regeneration of protoplasts in Ppmax2-1 and therefore potential masking of (±)-GR24 enantiomers effects (figure VII-1).

In this new experimental setup, the repressive effect of (±)-GR24 on regeneration was replicated as seen previously on protoplasts, as regeneration of WT phyllids was affected even by only 30 nM (±)-GR24, at 48h and 96h

(figure VII-1A). Additionally, concordantly to the more rapid germination of Ppccd8 spores (Proust et al., 2011), this SL-deficient mutant has a significantly higher regeneration ability than WT in the absence of (±)-GR24 treatment. On the contrary, the Ppmax2-1 mutant's regeneration is dramatically decreased compared to WT. Regeneration abilities of these two mutants in the absence of (±)-GR24 treatment hint at PpCCD8-derived compounds (putative SL) having a negative impact on regeneration, while the PpMAX2-transduced signal(s) (putative KL) would have a positive role in regeneration. Interestingly, by testing three concentrations of (±)-GR24, we noticed that the effect was not clearly dosedependent except for Ppmax2-1, which was particularly blatant at the 72h time-point for WT. We thought that this phenomenon could stem from the more limited effect of the (-)-GR24 enantiomer present in the mixture, which would become more visible when high concentrations of (±)-GR24 are used. Moreover, another surprising observation was that Ppccd8 regeneration did not appear more affected by (±)-GR24 treatments than WT, unlike in caulonema growth assays in the dark (see chapters IV and VI).

Reasoning that this non dose-dependent effect could be explained by the use of the racemic mixture, we carried out a similar experiment using (+)-GR24 (figure VII-1B). Furthermore, we let phyllids regenerate in the dark, to separate the effects of light from that of (+)-GR24 addition. In this second experiment, we could observe a significant negative effect of 300 nM (+)-GR24 on WT regeneration, which suggests that phyllids are more responsive to (+)-GR24 in the dark (in the light, the effect of (±)-GR24) was not significant even at 3 µM). Here again, regeneration of Ppccd8 in control conditions was higher than that of WT, while Ppmax2-1 regeneration was almost null. Ppccd8 responded better than WT, while Ppmax2-1 response was only revealed by a tendency here, owing to its very low level of regeneration even in control conditions (a later observation at 96h might have enabled this observation). These results confirm again that (+)-GR24 mimics PpCCD8-derived compounds and has a dose-dependent negative impact on regeneration from phyllids.

Methods specific to chapter VII:

Phyllids regeneration assay. In the experiment presented in figure VII-1A, a variation of the protocol detailed in our Methods chapter (see annex 1) was employed, where phyllids were regenerated in long days conditions instead of in the dark. For the experiment presented in figure VII-1B, this protocol was followed without modifications.

Protoplasts regeneration assay.

A variation of the protocol used by [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF] Cold stress assay. Small fragments of protonema were grown in control conditions (16h day/8h of night, ~90µmol m -2 s -1 white light, ~55% hygrometry, 25°C during the day and 23°C at night) for two weeks, on low nitrogen content medium (PpNO3) without underlying cellophane (initiation period). Half of the plants were transferred to a growth cabinet with the same growth conditions except for the temperature (10°C in days and nights) and the half remained at 25°C, for two more weeks (stress period). Finally, all plants were grown at 25°C for an additional week (recovery period). Diameters were monitored at 4 timepoints: at the end of the initiation period, after one week of stress, at the end of the stress period, and finally at the end of the recovery period. The effect of stress and recovery was assessed for each genotype as the normalized difference between the mean diameter of plants incubated at 10°C and the mean diameter of plants kept at 25°C throughout the experiment (control groups).

Supplemental figures of chapter VII

Promoter + upstream gene 3'/5' untranslated region (for A and B only) according to [START_REF] Nakamura | Photosynthesis nuclear genes generally lack TATA-boxes: A tobacco photosystem I gene responds to light through an initiator[END_REF] Putative TATA boxes were searched for in ~50pb upstream of the most upstream starting transcrit found on Phytozome, or anywhere in the 5'UTR (looking for possible alternative TSS). Enhancer cisacting elements such as G-Boxes (CACGTG consensus) and HSE were also found. HSE (heat shock elements) have been searched for using the core consensus sequence GAANNTTC (according to [START_REF] Schöffl | The DNA sequence analysis of soybean heat-shock genes and identification of possible regulatory promoter elements[END_REF]. Motifs corresponding to this consensus with only one mismatch are taken as HSE-likes. Intensity of coloration reveals the level of activity of the pathway or of given regulations. Dotted lines show effects that

have not yet been proven experimentally, plain lines show effects backed up by evidences given in chapters IV, VI and VII. For better clarity, transcriptional regulation of genes acting in these pathways are not included. Our hypothesis that the SL pathway acts by stabilizing/activating PpSMXLC/D is illustrated by the dotted green arrows. In the upper panel, balance between the SL and KL pathways leads to a WT phenotype. In Ppmax2 mutants, the PpKAI2L(A-E)-PpMAX2 pathway is shut down, leading to a collective increase of PpSMXL protein levels/activity. Since PpSMXL limit protonema growth in favor of gametophore development, these mutants prematurely develop gametophores. In the Angiosperms [START_REF] Conn | Convergent evolution of strigolactone perception enabled host detection in parasitic plants[END_REF]Bythell-Douglas et al., 2017), thereby giving rise to KAI2L receptors with switched ligand specificity towards SL, at least for P. patens. This was confirmed by our findings, presented herein in chapter IV.

Following the hypothesis of convergent evolution, the presence of an additional SMXL clade in bryopsids mosses could likewise hint at a neo-functionalization event, enabling some SMXL to act in SL signaling while retaining the ancestral ability to interact with MAX2. Following this hypothesis, SL signaling in P. patens would follow the same core pathway as in Angiosperms. This hypothesis of SMXL convergent evolution was recently challenged by the discovery that the MAX2 homolog of P. patens is not necessary for response to SL [START_REF] Lopez-Obando | Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling[END_REF]. While it cannot be excluded that another F-box protein can undertake the role of MAX2 in SL signaling in P. patens, it suggests that either the ancestral role of MAX2 was not in SL signaling, or that MAX2 lost this function in P. patens evolutive history.

However, as phylogenetic studies conclude that KAI2 and SMAX1 functions are ancestral in the respective KAI2/DDK and SMXL protein families, and as the MAX2 is conserved as a single copy gene in land plants, the most supported hypothesis is that the ancestral KAI2-MAX2-SMAX1 pathway was involved in KL signaling, and that this (Li et al., 2020a), should be studied to determine whether SL response ability is actually specific to mosses amongst Bryophytes.

VIII-C) About the role(s) of SL in extant mosses

A remaining question is why the ability to respond to SL was selected along P. patens evolution: What was the significant advantage for fitness that response to SL granted to P. patens ancestors?

In the past decade, it has been suggested that SL are used by bryopsids mosses as Quorum Sensing (QS) molecules instead of hormones per se, and that the ancestral function of SL was communication with the rhizosphere microbiota (AMF) (Proust et al., 2011;[START_REF] Walker | Strigolactone synthesis is ancestral in land plants, but canonical strigolactone signalling is a flowering plant innovation[END_REF]. We can oppose to this view the observation that SL in P.

patens repress their own biosynthesis (Proust et al., 2011) (as in Angiosperms), while for QS signals a positive feedback re-seal it and carefully swirl it to spread the treatment across the well. Transfer your plates vertically in the dark container and put them in the culture chamber for ~10 days. The instance provided here shows the Ppccd8 SL-deficient mutant response to (+)-GR24 at 0.1 µM (central picture) and wells for each genotype and treatment. So, in one given plate (corresponding to one genotype), you are able to test up to 4 treatments: a negative control (diluted solvent), a positive control ((+)-GR24) and two SL-like molecules. Do not prepare these plates too much in advance, as the medium dries out very quickly in such small wells (see Note 9).

3.

Prepare treatments: under the hood, mix your stock solution of SL (or pure DMSO for the control treatment)

with sterile MilliQ water to your chosen working concentration. If you cannot proceed to step 4 immediately, keep your working solutions in the fridge. Remember that you will need 50 µL per well for the treatment and that this assay is more sensitive than caulonema growth (for (+)-GR24, effects have been observed starting at the minute concentration of 3 nM).

4.

Distribute the treatments in the wells (figure 1234). You have to do so before starting P. patens dissection, for two reasons: firstly, the overlay of liquid treatment will ease tissue deposition into the wells. Secondly, if you treat the tissues after deposition you risk introducing further variability in treatment duration (from the time of dissection)

between your different samples. In all cases where the CRISPR-Cas9 tool was used in the present thesis work, transformation was carried out on P. patens protoplasts obtained from young protonema (5-7 day-old), so as to limit the risk of mutating diploid/polyploid cells, as endoreduplication is more prevalent in older filaments [START_REF] Lang | The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution[END_REF]. The classical PEG/ heat-shock mediated protoplast transformation method was used, with minor modifications. Transformed protoplasts were left to regenerate their cell walls overnight in the dark, then cultivated for 5-7 days on non-selective solid medium with controlled osmolarity (8.5% mannitol), in an alginate mixture polymerized by adding CaCl2 to ensure proper mechanical support for regeneration of protoplasts, in control long days conditions. When regeneration is ongoing, the alginate layer containing the regenerants is transferred on classical PpNH4 medium containing the geneticin antibiotic (G418, 50µM) for one week. The regenerants that survived this selection step are then cultivated on non-selective PpNH4 medium and genotyped. In our experiments, we used an independent plasmid containing the selection gene for resistance to G418, along with the plasmid permitting expression of a nuclear-targeted (SV40 NLS), codon-optimized version of Cas9 from Streptococcus pyogenes (Lopez-Obando et al., 2016b). Other plasmids targeting specific loci in P. patens genome were added, depending on the aim of the transformation.

2-A) Use of the CRISPR-Cas9 system for mutagenesis:

The first strategy we employed to generate mutations in the PpSMXL genes was a classical one using only one guide RNA. This guide was designed specifically for each gene, to target the uppermost possible locus in the CDS: at the beginning of the fourth exon for PpSMXLA and PpSMXLB, at the start of the second exon for PpSMXLC and only 80bp downstream of the START codon for PpSMXLD (see figure VII-1). Plasmids containing the resulting sgRNAs were used in combinations of one to four, to target from one to four loci at once (along with plasmids expressing Cas9 and the selection marker, Figure 2-1). This first strategy is analogous to the one employed to generate mutations in the PpKAI2-L genes (see chapter IV).

The second strategy was a test to replicate the effects of classical HR (homologous recombination) strategy without the need for removal of a selection cassette inserted in the genome in place of the knocked-out gene. This also prevents the risk of insertion of the cassette in several copies at the targeted locus [START_REF] Cove | The Moss Physcomitrella patens[END_REF]. This use of the CRISPR system was devised following the advice from Fabien Nogué, a colleague at the IJPB and expert of the CRISPR system in moss. Moreover, it was later supported by publications from independent research teams working on this system in P. patens, concluding that targeting multiple sites within a single region can produce larger deletions [START_REF] Mallett | Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens[END_REF]. For this purpose, guide RNAs were designed in the ~600bp of UTR closest to the START/STOP codon, for each Chimeric nature of some transformants was also noted using the first mutagenesis strategy. However, it did not prove to be an issue as enough mutants were obtained (at least 2 mutants for each desired combination: ab, cd). In the case of the second strategy, the issue was somehow more prevalent, either because of chimerism (mix of WT and mutated cells) or aneuploidy. To isolate mutated cells, these regenerants were submitted to another round of protoplastization. However, no "pure" PpsmxlΔaΔb, PpsmxlΔa and PpsmxlΔd mutants could be recovered after this "isolation" step (WT copies of the PpSMXL genes were still present thus they were probably aneuploids). Moreover, in the case of original PpsmxlΔaΔb lines, recombination between the PpSMXLA and PpSMXLB loci was sometimes observed, undoubtedly linked to their very similar DNA sequences (homeology).

2-B) Use of the CRISPR-Cas9 system for knock-in facilitation:

As the HR process is initiated by double strand breaks in the DNA, we reasoned that facilitation of these breaks at a specific locus in P. patens genome, using the CRISPR system, could ease insertion of a construct at this locus by HR (knock in) (Figure 2-3). Hence, guide RNA targeting the Pp108 homology regions, outside of the sequence contained 252 in the insert, were employed along with the previously described Cas9 and selection plasmids and with a plasmid containing the desired construct (Figure 2-1, case 2B). This method was employed to generate transgenic lines described in chapter VI (proZmUbi:GFP-PpSMXL, proZmUbi:flag-GFP and proPpSMXL:GUS), as well as proZmUbi:PpSMXL-GFP, proZmUbi:GFP-PpSMXLmut and proZmUbi:PpSMXLmut-GFP lines (expressing PpSMXL proteins where the degron/degron-like motif is replace by LVGI).

Theoretically, the use of the selection plasmid carrying the geneticin resistance gene was not necessary, as the pMP vectors contain a kanamycin section marker, inserted at the Pp108 locus along with the transgenic construct.

However, geneticin selection often gives more clean-cut results, perhaps because WT P. patens Gransden laboratory strains readily develop Kanamycin resistance themselves. Hence, we kept the same method of transformants selection as used for mutagenesis.