Sana Dieudonné

Fabrice Besnard
email: fabrice.besnard@ens-lyon.fr

Revisiting bract inhibition with natural population of Arabidopsis thaliana

Inhibition de la bractée lors de la transition florale chez

Arabidopsis thaliana

Résumé de la thèse

Le phytomère constitue l'élément de base du développement continu des plantes. Il se compose d'un noeud constitué d'une feuille et d'un méristème à la base de la feuille, ainsi que d'un entrenoeud, segment de tige séparant deux noeuds. Le passage du stade végétatif au stade reproductif se traduit par un remodelage du phytomère. Chez les Brassicaceae, comprenant la plante modèle Arabidopsis thaliana, cela s'illustre notamment par la perte de la feuille, appelée bractée. Cependant, de précédentes études ont rapporté la présence de bractées à l'aisselle des premières fleurs de certaines accessions naturelles de A. thaliana, lorsqu'elles sont cultivées dans des conditions environnementales spécifiques. Ces fleurs dites "chimeriques" résulteraient d'un conversion de branche en fleur. Au cours de ma thèse, nous montrons que la présence de bractées au niveau des premières fleurs est fréquente chez certaines accessions naturelles d'A. thaliana, mais pas chez l'accession de référence Col-0. Chez Tsu-0, notamment, la présence de ces bractées ne dépend pas des conditions environnementales. De plus, elles ne résultent pas d'une conversion de branches en fleurs, contrairement à ce qui avait été proposé par les études précédentes. Afin d'établir les bases moléculaires à l'origine du developpement de ces bractées, nous avons procédé à l'analyse transcriptomique de méristèmes apicaux caulinaires au cours de la transition florale chez les deux accessions naturelles. Ces transcriptomes révèlent des hétérochronies dans la dynamique d'expression des gènes. Leur lien avec la présence de bractées reste à établir. Notre étude décrit pour la première fois les différences de dynamique d'expression de gènes au cours de la transition florale à l'échelle de la population d'A. thaliana. v Nous avons pu montrer que la différence d'expression des gènes entre ces deux accessions culmine au moment précis de la transition florale, faisant echo à ce qui avait été montré à l'échelle de l'espèce. De plus, nous avons cartographié les variants génétiques responsables de la présence de bractées chez Tsu-0 en utilisant deux approches indépendantes, l'analyse génétique d'individus F2 segrégants en masse et l'utilisation de lignées recombinantes ségrégantes. Nous avons ainsi pu identifier deux régions situées dans le chromosome 1 et associées au phénotype. Nous avons donc recoupé les éléments des analyses génétiques et ceux des analyses transcriptomiques afin de proposer de potentiels gènes candidats. Ainsi, cette thèse revisite l'étude des mécanismes qui contrôlent l'inhibition de la bractée au moment de la transition florale chez A. thaliana, en se basant sur une comparaison entre deux populations naturelles. Par conséquent, ces résultats peuvent également nous permettre de mieux comprendre l'origine évolutive de la perte des bractées chez les Brassicaceae. vi

Il y a, avant cette page, des dizaines de versions effacées. Des remerciements vains, fades, lâches, dont je ne me sens plus l'auteure. Ma gratitude se retrouve en effet prisonnière d'une cage de mots. Comment remercier chacune et chacun d'entre les membres passés et présents du RDP, de l'Appart, de la famille, des amis? Sans oublier celles et ceux qui ont croisé cette thèse pour y apporter un peu du leur? Et le tout, avec des mots aussi banals qu'un merci, et une place aussi glauque que celle d'un manuscrit de thèse! Alors derrière mes mercis, voyiez-y mes larmes, entendez ma voix tremblante, et sentez mon étreinte. La place me manque pour tous vous nommer, mais vous êtes bien toutes et tous entre ces lignes! Avant tout, Fabfab, merci pour tout. Merci pour ton dynamisme, ta tolérance, ta disponibilité, ta générosité, ta patience... et surtout merci mille fois de ne jamais m'avoir mis la pression, et de toujours m'avoir donné les pleins droits à l'erreur. Merci aussi pour toutes les choses que tu m'as fait découvrir : les plantes, les bractées, les méristèmes, le MEB, les lignes de codes sur R (qui l'eut cru!), et le RDP et son peuple! Et dans ce peuple, je remercie aussi mon directeur de thèse officiel / Vice Président Recherche de l'ENS : Teva (oui, c'est la classe!). Merci d'avoir cru en moi dés le début, et de m'avoir montré ce visage que peu de gens te connaissent. Non, pas celui avec les cheveux, je parle de ce visage paternel, adorable, souriant, bon vivant, ce visage qui donne du baume au coeur! Et puis je pense aux mamans de ce peuple : Gégé et Steph, vous m'avez tant appris, soutenue, encouragée! Rappelée quand j'oubliais des plantes, aidée quand je galèrais et été là quand j'avais besoin de fondre en larmes! Et surtout Steph : merci pour tout ce que tu as fait pendant la rédaction!!! Vous êtes toutes les deux les piliers de notre équipe. Alors quand on me demande ce que je veux faire après ma thèse : que répondre d'autre que "comme Gégé"? Alors merci à Olivier, de me permettre de tester ça après la thèse! Merci à tout mon Comité de Suivi de Thèse, pour m'avoir guidée, et aidée dans les moments les plus difficiles. Surtout Benoît, merci pour ton soutien! Merci à celles et ceux qui m'ont fait rire sur Slack (Arthur, Julie, Raquel), ont partagé mes hauts et mes bas émotionnels (oh Julie! Qu'aurai-je fais sans toi!!), m'ont fait rêvé sur la terrasse d'un rooftop (Coco), m'ont fait découvrir tout plein de restos/ partagés plein d'apéros (Léa, i Yannick, Juan, Jack, Ana, Julie, Raquel, Alex, Adri, Coco, Lucie, .... et j'en oublie tant!).

Merci à toutes et tous, d'avoir partagé ce verre (de ice tea zero) pendant les innombrables pots qui rythmaient la vie du labo (et ça reviendra!). Et merci à toutes celles et ceux qui ont partagé mon bureau (et supporté mes bavardages incessants): Mohammad, Leti, Yvon, Thierry, Christine, Arthur, Justine, et surtout Aurélie et Florian, merci de me permettre de me défouler! Mais j'avais la chance de ne pas avoir qu'un cercle de scientifiques à Lyon! D'avoir rencontré des gens pour qui la journée commence à 6h du matin (si ce n'est avant!)! Merci JB, Olivier, Antony, Ricardo, David, Jean-Paul, Philippe, Momo, Cardin, Yann, Fred! Merci de m'avoir appris la méthode MDF, de m'avoir montrée que l'homme parfait existe (et c'est un Chavanon!), que les meilleures années se vivent après 50 ans, et que la vie appartient vraiment à celles et ceux qui se lèvent tôt ! Et avant ma vie Lyonnaise, j'avais pris mes racines dans une terre fertile, (celle de Massy), et je voulais aussi remercier du fond du coeur mes fondations, mes piliers. Merci Thibault, d'avoir supporté mes (loooongues) études, la distance, mon linge, mes morales écolos, mes gâteaux, ma dictature, mes caprices : 11 ans que tu me supportes, me soutiens, me fait rire! Merci papa, maman, pour les taboulés et les kebbes, les leçons de vie et le soutient inconditionnel que j'ai toujours reçu. Merci Sadim pour avoir fait de moi une scientifique dés mon plus jeune âge, et pour être toujours dispo quand l'ordinateur fait des siennes. Merci Dima ma moitié, de partager ma vie depuis son stade le plus embryonnaire, et de me permettre de vivre le bonheur le plus simple, le plus pur, et le plus grand: celui d'avoir une soeur jumelle. Et puis Anousone, merci d'être plus qu'un ami, plus qu'un frère, plus qu'un ange gardien. D'être celui qui m'accompagne dans mes montagnes russes émotionnelles les plus intenses. De connaître mes années les plus obscures, mes parts les plus sombres, et, depuis le collège, de

Preamble

Many organisms develop through several phases in which they respond to different environmental conditions and stimuli. The switch from one phase to the other often involves various changes. For instance, the juvenile-to-adult transition generally comprises behavioural and physiological modifications, as well as a morphological remodelling. Insect metamorphosis well illustrates the abrupt transition from a juvenile larva to an adult imago. In fact, insect metamorphosis usually includes intense morphological changes (apparition of new organs, exoskeleton, etc.), physiological remodelling (change in the respiratory and hormonal system, etc.), and behavioural adaptation (mating, migration, predation, etc.). Although this developmental switch can be influenced by external stimuli, it is an irreversible and robust toggle: once the adult phase is reached, the organism does not return to its juvenile phase, whatever the environmental conditions. How developmental switches are controlled is an intriguing question in biology.

Modifications in the control of developmental switches can explain the diversification of species. In insects, the timing of ecdyzosteroids peak, the metamorphosis hormone, is responsible for different types of life cycle (Truman & Riddiford 1999). When the ecdyzosteroids peak happens before hatching, juvenile insects are born with a similar-to-adult shape: they are hemimetabolous. When the ecdyzosteroids peak occurs after hatching, juvenile insects are born with a larval form, and will metamorphose afterwards into their adult imago shape: they are holometabolous. Hence, the regulation of reproductive transition controls essential aspects of post-embryonic development, and its variations can participate to phenotypic diversification between species. How robust and irreversible developmental switches can also accommodate modifications is an emerging question.

Flowering plants provide a striking example of morphological remodelling at reproductive transition. In angiosperms, reproductive transition leads, in the first place, to flower production.

In some species, other changes appear with floral transition. In the model plant Arabidopsis thaliana, bracts, the leaves that subtend flowers, disappear. However, a natural accession of A. thaliana, Tsushima-0, presents bracts under the first flowers, but not above. Hence, bract inhibition is still functional in Tsu-0, but temporarily disturbed at floral transition. What is the nature of such "bract-flower"? How does floral transition induce different developmental 1 trajectories at a population scale? What kind of genetic determinant(s) in Tsu-0 can explain the temporal release of bract inhibition at floral transition?

In my thesis research, I use bract-flowers in Tsu-0 to explore how floral transition can display morphological differences at a population scale, while remaining robust and functional.

In this document, I first describe the presence of bract-flowers in some Brassicaceae species and in different natural populations of A. thaliana, including Tsu-0 (Chapter 1). I then focused on Tsu-0 's bract-flowers and highlighted essential distinctions with previously described bracts Contrary to most animals, plants final shape and form do not entirely rely on the embryonic establishment of a predictive body plan. Instead, plant embryogenesis only results in the formation of the essential features of the mature plant. This comprises the apical-basal axis, with at each tips an apical meristem (the shoot apical meristem -SAM -and the root apical meristem), the cotyledons, and the main tissue types. This "body plan" become more complex after germination, when the SAM start operating by producing phytomers. Phytomers are the basic unit of plant shoot development [START_REF] Barlow | metamers and modules and the development of shoot and root systems[END_REF][START_REF] Sussex | Developmental programming of the shoot meristem[END_REF][START_REF] Weigel | A developmental switch sufficient for flower initiation in diverse plants. Nature 377[END_REF]. Phytomers are composed of a leaf, subtending one to several axillary meristems (aM, or buds), and attached together to the stem at the node. The phytomer also includes the stem portion between two consecutive nodes, called internode. Each aM can reiterate this basic developmental unit, by becoming a SAM of a new growth axis and producing phytomers (Fig. 1A).

The structure of the phytomer is highly conserved among plant kingdom [START_REF] Mcsteen | Shoot Branching[END_REF]. Hence, in mosses, like in A. thaliana or poplar, the aM develops at the axil of a leaf, and nodes are separated by a small stem portion (fig. 1B). However, each of the phytomers' components has undergone structural and functional modifications along with plant diversification. For example, 480 million years of architecture divergence between moss and seed plant groups can be mainly explained by small changes at the phytomer level. These changes concern the identity of the aM (that can be flowers in angiosperms), and the length and width of the internode (can be rigid). But the general structure of the phytomer have been maintained (fig. 1).

Plant general architecture results then from the geometrical interrelationships between phytomer elements, and from phytomer organisation along each stem [START_REF] Barlow | metamers and modules and the development of shoot and root systems[END_REF][START_REF] Barthélémy | Plant Architecture: A Dynamic, Multilevel and Comprehensive Approach to Plant Form, Structure and Ontogeny[END_REF]. In fact, as described in Barthelemy et al. the entire diversity of plant architecture relies on three major "fundamental morphogenetic processes" affecting the phytomer: growth, branching, and reiteration [START_REF] Barthélémy | Plant Architecture: A Dynamic, Multilevel and Comprehensive Approach to Plant Form, Structure and Ontogeny[END_REF]. According to Barthelemy et al, the three morphogenetic processes concern different part of the phytomer. The "growth" concerns the internode and will determine the length and thickness of the plant axis. "Branching" allows to establish new growth axis from the meristems, and thus determines the general plant architecture. In fact, the "branching" of a determinate meristems, the flower, will stop the growth axis, while the branching of an indeterminate meristems will reiterate the growth axis. Hence, the third mophogenetic process, the reiteration, is specific to aM. Because phytomers are produced continuously, one individual plant architecture varies constantly through a modulation of these three morphogenetic phenomena. This explains how a young vegetative plant displays distinct phenotype from its adult hundred-year-old version. Moreover, phytomer production in plants is highly variable depending on environmental conditions. Understanding the huge diversity of plant architecture thus requires to consider the formation and the plasticity of its basic developmental brick, the phytomer.

Before addressing how changes of phytomer elements participate to the diversity of plant architecture, I will briefly review how phytomers are continuously produced by the SAM. To avoid confusion, the rest will only consider angiosperms, and is based on the knowledge mainly obtained using the model plant A. thaliana.

Production of phytomers is ensured by the Shoot Apical Meristems

Plant development is characterized by a continuous production of new organs and tissues by the meristems. Apical meristems are a group of stem cells located in the two apices of the plant.

The SAM produces all the aerial part of the plant by ensuring both the maintenance of the stem cell niche and the differentiation of cells into organs. The capacity to divide and self-renew can be conserved within the meristems during the entire plant life, and this characterizes their continuous development. Meristems have been described and studied for centuries. The SAM can be divided into several functional regions: the pool of meristematic cells is maintained within the central zone (CZ), the differentiation of organs happens in the peripheral zone (PZ), while the rib zone (RZ) contains the cells forming the inner part of the stem. In addition, the meristem can be divided into 3 main layers of cells with specific cell division plane (Carles & J. C. Fletcher 2003, fig. 2). In the first two layers (called L1 and L2), cells only undergo division anticline to the surface plane, which establishes layer-specific cell lineages. In fact, the L1 layer mostly contributes to the epidermis establishment, while the L2 layer derives into the germinal cells and the ground tissue. The L3 layer undergoes division in all planes, and generates the most internal plant tissues, including the vascular system. The balance between stem cell pool maintenance and cell differentiation is tightly regulated and well understood.

The pool of undifferentiated meristematic cells is located in the central part of the meristem, into the CZ (fig. 2A). Cells have a slow rate of division that is tightly controlled, which ensures the constant renewal of stem cells and controls meristem size [START_REF] Grandjean | In Vivo Analysis of Cell Division, Cell Growth, and Differentiation at the Shoot Apical Meristem in Arabidopsis. en[END_REF]). The control of the stem cell niche maintenance within the central zone is ensured by a genetic feedback loop relying on both cell-cell interactions and mobile protein signaling (fig. 2C). CLAVATA 3 CLV3 and WUS constitute a negative feedback loop that maintains the size of the WUS expression zone. WUS encodes a transcription factor that regulates gene expression in the SAM (see fig. 2C), notably by activating cytokinin pathway, that maintains undifferentiated cells.

WUS can also move trough plasmodesmata into apical neighbouring cells, where it activates CLV3, a stem cell fate gene, in a dosage dependant manner (J. Fletcher 2018). CLV3 gene encodes a peptide that binds, in part, to the CLV1 receptor, and transduces a WUS inhibition signal. Hence, by restricting WUS expression domain, the WUS-CLV signaling dynamically regulates the pool of stem cells. Besides, WUS inhibits KAN1, that promotes differentiation in the PZ, ensuring the undifferentiated state of the pool of stem cells.

KAN1 is however expressed in the PZ, where cells divide more rapidly and differentiate.

Cell differentiation in this PZ leads to primordia initiation at a specific position and timing. The arrangement of organs around the stem is called phyllotaxy and is usually a characteristic of the species. In A. thaliana, organs initiate one after the other at a 137,5°angle on average, allowing us to visually trace back the sequence of organ initiation from a meristem top view (fig. 2B).

The timing of organ initiation is also regulated, and the time between the initiation of two consecutive organs is called the plastochron. The regulation of the position and the timing of Cells at the flank of the meristem then divide more rapidly than their neighbour and form a dome called the leaf primordium: the first constituent of the phytomer is specified. Development of the leaf primordium is not dissociable from the genetic specification of the leaf axis: the adaxial-abaxial (upper-lower), apical-basal, and lateral axis. The adaxial-abaxial axis is genetically established before any morphological distinction of such axis (F. [START_REF] Zhao | Stable establishment of organ polarity several plastochrons before primordium outgrowth in Arabidopsis[END_REF].

Hence, leaf identity markers such as ASYMETRIC LEAVES 1 (AS1 ) is inseparable from its role in establishing the adaxial-abaxial axis (Machida et al. 2015). In fact, the adaxial AS1-AS2 complex promotes leaf development and inhibits the SAM Class 1 KNOX genes such as KNOTTED-LIKE FROM ARABIDOPSIS THALIANA (KNAT1 ) [START_REF] Guo | Direct Repression of KNOX Loci by the ASYMMETRIC LEAVES1 Complex of Arabidopsis[END_REF], see fig. 3). This allows the separation between the SAM and the organ. This complex also restricts abaxial markers YABBY genes, while promoting the adaxial REVOLUTA (REV) gene (Machida et al. 2015), and thus allows the separation between the two leaf faces: abaxial and adaxial.

In addition to leaf-meristem separation, the early leaf partitioning guides cells differentiation within the leaf. For instance, the adaxial (upper) part of the leaf becomes specialized in light absorption, while the abaxial (lower) part becomes specialized in gas exchanges (Manuela & Xu 2020b). Disturbing the positional information leads to early malformation of the leaf with a radial symmetry (Machida et al. 2015). Hence, the leaf is the first element of a phytomer to be specified at the SAM and its identity is indiscernible from its polarity markers.

The establishment of the adaxial-abaxial axis is critical for the proper localisation of the axillary meristem initiation, as it appears at the axil (adaxial part) of the leaf (fig. 3).

Axillary meristem organogenesis

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). Hence, the spatial control of axillary meristem initiation relies on polarity and boundary information given by the previously formed leaf (fig. 4).

At the leaf axil, the initiation of aM involves a complex gene regulatory network to establish meristematic features. Cells originating from the SAM continue to express a low level of SHOOT MERISTEMLESS (STM), that maintains the competence for meristem identity thanks to a STM -ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) feed-forward loop (Cao AMs are thus the second phytomer element to be produced. It requires the former production of a leaf and is regulated by environmental, hormonal, metabolic and genetic cues (fig. 4).

Internode growth Internode is the last phytomer element that becomes visible in A. thaliana.

Unlike aM or leaves, very little is known about internode development. Again, boundary genes are essential for the formation of the rib meristem, which ensures internode growth. The boundary between the differentiating meristematic cells and the elongating stem cells is built by Shoot meristems are the place of a continuous organ production: leaf, aM and internode, which together form the phytomer. This production relies on positional and hormonal information. Moreover, modifications of the shape, size, or growth of the phytomer elements can arise both at an individual and at a species level. I will describe how changes in the relationship between each element can impact the general plant architecture.

1.1.4 Modifications of the phytomer structure impact the diversity of plant architectures Despite the high conservation of phytomer among plant kingdom, its structure can diverge, explaining the diversity of plant architectures. The macroscopic divergence in the overall archi-axillary meristem. Most palm trees (Arecaceae) rarely produce branches (but they still can!), while in Solanaceae species (potato and nightshade family), almost every aM develops (fig. 6).

In addition to its relative growth, the meristem itself has undergone intense modifications in seed plants, as they can become a determinate meristem: the flower.

The flower: a determinate meristem Flowering plants are characterized by the presence of a specialized meristem, that can be in axillary or apical position that compacts reproductive system: the flower. Hence, in addition to a vegetative indeterminate axillary meristem, angiosperms can produce a determinate meristem: the floral meristem. The first allows to reiterate plant growth through the development of a new shoot, while the second produces the flower and thus stops its own growth axis, impacting plant architecture [START_REF] Prenner | The key role of morphology in modelling inflorescence architecture[END_REF]). Flower has undergone intense and diverse structural changes, allowing various reproductive strategies. Many examples can illustrate the diversity of flowers (fig. 7), in term of shape, colors, size, and floral organ number.

Floral meristems produce a determinate number of floral organs, whose number and category vary among species [START_REF] Endress | Patterns of floral construction in ontogeny and phylogeny[END_REF]. Simultaneous mutations of all floral organ identity genes lead to homeotic conversion of all flower organs into leaf-like organs [START_REF] Bowman | Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes[END_REF]. It is now well accepted that floral organs are derived from leaves. Floral organs are then considered as "modules" (Klingenberg 2014), produced by the flower meristem, like phytomers.

Their internodes and aM would then remain dormant. Still, in some mutants, the meristematic features at the axil of flower organs can re-emerge (Xu, Hu, McKim, et al. 2010).

Therefore flowers in angiosperms illustrate well how changes in the shape/size of the leaf, the axillary meristem and the internode, can lead to a complete novel phenotype.

The bract: a special leaf Most probably because of the particular status of flower, the leaf subtending it received a specific botanic term: bract. In some species, bracts have also undergone intense structural changes allowing distinct functions, from attracting pollinator with smells and colors (Araceae), to protecting the fruit (chestnut bur), or the flowers (artichoke) (fig. 7).

So far, I very quickly illustrated how the tremendous variety of plant architecture across species can be in part explained by countless modifications of the phytomer. Nevertheless, phytomers can undergo modifications during plant life thus changing plant architecture through different developmental stages. Next, I will focus on phytomer modifications following floral transition, showcasing the knowledge acquired from the model plant A. thaliana.

Floral transition, a developmental switch that remodels phytomers

If the SAM generates all the phytomer components, their respective growth is separated in time, making phytomers a spatial more than a temporal developmental unit. For instance, in A. thaliana, leaf cells divide, differentiate and grow in a determinate manner during a certain time after their initiation in the SAM, thanks to the activity of the leaf meristem [START_REF] Ichihashi | Behavior of Leaf Meristems and Their Modification[END_REF]. Internode development occurs in another temporality. When they initiate, internodes consist of a thin layer of cells produced by the rib zone; their elongation occurs after a dormant stage and is triggered by flowering signals, that cause plant "bolting" (Serrano-Mislata & Sablowski 2018). The dynamics of axillary meristems depend on their nature. Floral meristem develops during a certain time just after its initiation at the SAM, while the indeterminate axillary meristem will develop after a dormant stage, sparked by flowering signals [START_REF] Bencivenga | Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis[END_REF]. Therefore, phytomer structure depends on the plant species but can also be remodeled across time within the same species.

I will first rapidly describe the different developmental stages of A. thaliana, and then focus on the floral transition.

Developmental phase changes and architecture remodelling

Like many organisms, plant development also goes through different stages, in which they can have distinct architectures that serve specific functions. The phytomers are often remodelled during the switch from one phase to another. Several developmental stages have been defined for A. thaliana's life cycle. The seed germination is considered as a developmental phase change because of the important remodelling it triggers. Like other developmental transition, seed germination is tightly controlled by both environmental, and genetic signals, as well as by the carbon content and hormonal signaling (Carrera-Castaño et al. 2020). The transition from a dormant state to a germinating plant marks the separation between the embryogenesis and the continuous development of the plant. After germination, A. thaliana plants will enter a vegetative stage, itself divided into juvenile and adult stage. Vegetative stage is characterized by an important growth of the leaf, while the internode and the axillary meristem remains dormant [START_REF] Bencivenga | Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis[END_REF]. This allows the formation of the rosette, a condensed set of leaves arranged in a circle from the neck of the plant, that characterizes some Brassicaceae species.

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). In fact, juvenile leaves have a rounded shape, that becomes bigger and more serrated in the adult phase. In addition, the distribution of trichome changes across the switch to the adult phase, in which trichomes become produced in both adaxial and abaxial sides of the leaf [START_REF] Telfer | Phase change and the regulation of trichome distribution in Arabidopsis thaliana[END_REF]. The juvenile-to-adult switch is mainly regulated by the MIR156/SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) age pathway, hormonal signaling, light and sugar content (Manuela & Xu 2020a). Finally, the annual plant A. thaliana undergoes a senescent phase in which plant looses its leaves, and stores the energy into developing seeds, that will initiate a new life cycle (J. [START_REF] Kim | New insights into the regulation of leaf senescence in Arabidopsis[END_REF].

Hence, biologists have distinguished several developmental stages that describe A. thaliana's life cycle. Each transition is marked by major changes affecting morphological and physiological features, and are tightly regulated by multiple factors. I have merely provided a brief overview of these developmental switches, and I will now go deeper into the description of the floral transition.

Floral transition remodels both new and old phytomers

Following floral transition, plant architecture is drastically transformed. The flat ground-level rosette will become the pedestal of an aerial branched plant with flower-bearing axis, that optimizes fertilization and seed dispersion. This major transformation involves both alterations of pre-existing vegetative phytomers and a new layout for the de novo reproductive phytomers (fig. 9).

From vegetative to inflorescence SAM At floral transition, the shape of the SAM first changes by "doming", a typical morphology that allows to distinguish a vegetative SAM from an inflorescence SAM at the tissue scale. This phenomenon occurs early during the floral transition. It is under the control of the same key genes that regulate the global process of floral transition in a complex gene regulatory network [START_REF] Kinoshita | Regulation of shoot meristem shape by photoperiodic signaling and phytohormones during floral induction of Arabidopsis[END_REF]. Also, regulation of local GA signaling was recently shown to be essential [START_REF] Kinoshita | Regulation of shoot meristem shape by photoperiodic signaling and phytohormones during floral induction of Arabidopsis[END_REF]). The doming is the result of an increase in both cell number and cell size.

Basipetal modification of pre-existing phytomers Following the reproductive switch, the inflorescence SAM starts to produce a novel meristem, the flower. The switch from vegetative meristem to floral meristem is abrupt: no intermediate branch-flower are typically produced at floral transition. Since flowers have a determinate growth, lateral branching is no longer possible, which draws typical linear inflorescence units (also called "a raceme"). Furthermore, in A. thaliana, the floral transition also induces modifications of pre-existing phytomers, that were produced during the vegetative stage, before the floral switch. Indeed, when plant flowers, the aMs initiated at the vegetative stage in the axil of leaves quit their dormancy state and initiate branches. Besides, internode elongates, driving plant "bolting" [START_REF] Bencivenga | Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis[END_REF]).

Flowering-induced phytomer modifications are not synchronous: in A. thaliana, the development of the aM can be observed at the time the first flowers are produced. Internode elongation, and thus plant "bolting", is observed later (around 1-2 weeks later) (personal observations, see results section for description and timing of aM emergence). Plus, these modifications appear in a basipetal manner: the youngest phytomers are modified before the oldest, so that highest aM develops before lowest (Hempel & Feldman 1994). Interestingly, some of the oldest phytomers will never undergo internode elongation but develop buds at flowering. This results in lateral branches emerging from leaf rosette. This phenomena may be the result of a different level of sensibility to flowering signal (Périlleux et al. 2019).

Because of its huge agronomic relevance, floral transition and flower development have been extensively studied, and reviewed. The various effects of the environment as well as many flowering time genes have been identified. A comprehensive knowledge has been built about the genetic regulatory network (GRN) accompanying floral transition in A. thaliana [START_REF] Wils | Gene-regulatory networks controlling inflorescence and flower development in Arabidopsis thaliana. en[END_REF]. Because this biological process just precedes the phytomer remodeling, I will briefly summarize some of its important points, focusing on the GRN, to sketch a relevant context in which the developmental switch that affects phytomer occurs. Even when exposed to inductive photoperiod, young plants will not flower. Indeed, the competence to flower also depends on plant age as well as sugar resources. Plant senses its age thanks to the MIR156/SPL pathway. MIR156 is a micro RNA expressed during juvenile phase, that inhibit SPLs genes, activators of floral transition [START_REF] Schmid | Dissection of floral induction pathways using global expression analysis[END_REF]). I resumed how environmental signal can be integrated and can rewire plant GRN, that will trigger the formation of a reproductive phytomer. I will now address the genetic pathways that ensure the proper development of a reproductive phytomer. Because very little is known about internode elongation, I will focus only on the control of the flower meristem. I will later address the leaf part of the reproductive phytomer.

Regulation of Floral Transition

The genetic control of flower development

Flower production involves a complex GRN that have been extensively studied. This process can be divided into three key developmental steps: floral meristem specification, floral organs patterning and development, and floral meristem termination. We will briefly describe the main regulators of the two first steps in A. thaliana. Specification of floral meristem is the first step that mark the divergence between vegetative and reproductive phytomer. Floral organ patterning and termination ensure a proper development of the flower. When disrupted, flowers often show sign of vegetative phenotypes. In annual plant like A. thaliana, reproductive stage specializes in fertilization and seeds dispersion, and leads to phytomer remodelling. The function of hundreds of genes in the regulation of the key developmental steps of flower formation have been uncovered, for which we have merely provided a brief overview. Contrary to flower, the development of its subtending leaf, the bract, have not received much attention. However, in the model plant A. thaliana, like most Brassicaceae, floral transition is also characterized by bract inhibition. Absence of bract on floral phytomers is common to several clades, but the mechanism underlying its inhibition remains unclear. This next part will be dedicated to our current knowledge about bract development and inhibition, and will consider not only A. thaliana, but also several Poaceae species.

Developmental regulation of the bract in the reproductive phytomer

Bracts are very common among Angiosperms. In many cases, the term bract is used in its broad sense and refers to either leaf subtending the flower or the inflorescence. Here, we will define bract stricto sensu as the leaf composing the reproductive phytomer of Angiosperms.

Hence, bracts are leaves subtending the flower within the phytomer unit (Flower Subtending Bract, FSB in fig. 14). Therefore, bracts are not produced by the floral meristem but rather by the inflorescence meristem. They are thus distinct from leaf-like sepals that precede the other floral organs (Flower Preceding Prophyll, FPP fig. 14). In the literature on Poaceae's, bracts often refer to leaves subtending an inflorescence (bracts lato sensu, see fig. 14). Still, these studies can give insights on the mechanisms behind leaf suppression. Hence, bracts can be used to describe structure from different developmental origin. In this manuscript, we will precise three different terms depending on the developmental origin: bract stricto sensu (s.s.) defines a leaf subtending a flower (FSB in [START_REF] Prenner | The key role of morphology in modelling inflorescence architecture[END_REF]), bract lato sensu (l.s.) defines a leaf subtending an inflorescence, and bract fpp defines modified perianth preceding the flower (see fig. 14). When the developmental origin is not known, we will refer as to "bract" or bract-like structure.

Convergent evolution of bract loss

Bracts are not visible in several angiosperm clades, some of which include model species like in Brassicaceae or Poaceae. Despite the lack of precise functional knowledge, it seems that several independent genetic events occur to converge to bract loss in these different clades [START_REF] Mach | Different Words, Same Message: How Grasses and Arabidopsis Say "Hold the Bract[END_REF]).

Bract loss is therefore an open question, both in term of their evolutionary and developmental origin.

Before addressing the developmental origins in the following, I would like to briefly address the possible evolutionary forces that could have favored such a recurrent scenario of bract loss.

Well developed bracts can achieve various functions, as shown in fig. 7: colored bracts can attract pollinators, while a spiny cupule protects young floral buds. However in grasses, like maize, rice, or wheat, and also in Brassicaceae, bract loss seems to follow a flower size reduction.

Cryptic bract in bractless species

As already mentioned, A. thaliana flowers usually do not bear bract, at least when observing the plant with the naked eye. However, microscopic observations of the first steps of flower development suggest the presence of a cryptic bract, that is rapidly inhibited.

Morphological evidence in A. thaliana

Monitoring of variations of the SAM surface growth during flower development using the sequential replica method [START_REF] Kwiatkowska | Flowering and apical meristem growth dynamics[END_REF] shows that the first steps of flower development are marked by the presence of a shallow crease associated to the first lateral bulge of organogenesis.

The spherical flower meristem develops later just at the position of this crease (Kwiatkowska 2006). This crease is proposed to be the axil of a cryptic bract, whose growth stops just when floral meristem emerges (fig. 12). In this article, the authors speculate that the inhibition of bract growth is triggered by the floral meristem itself.

Molecular evidence in A. thaliana

The cryptic bract domain is also associated with several molecular markers. On its first steps, the primordium anlagen of an inflorescence SAM looses STM expression, which specifies meristematic cells (fig. 13A-B). STM expression re-appears later when floral meristem reaches a certain size, emphasizing Kwiatkowska's morphological observations [START_REF] Long | Initiation of Axillary and Floral Meristems in Arabidopsis[END_REF]. In addition of a lack of STM (fig. 13B), the abaxial region of the primordia shows expression of lateral organ identity genes such as ANT (Long & Barton 2000; fig. 13C-D). Also, the abaxial region of the primordium shows specific expression patterns of knwon polarity genes such as YAB3 and FILAMENTOUS FLOWER (FIL) [START_REF] Goldshmidt | Signals Derived from YABBY Gene Activities in Organ Primordia Regulate Growth and Partitioning of Arabidopsis Shoot Apical Meristems[END_REF], fig. 13E-F). This cryptic bract domain also shows expression of genes involved in boundary establishment like CUC3 [START_REF] Hibara | Arabidopsis CUP-SHAPED COTYLEDON3 Regulates Postembryonic Shoot Meristem and Organ Boundary Formation[END_REF]), which marks a first boundary between the cryptic bract and the floral meristem, and a second boundary between the floral meristem and the SAM (fig. 13G).

Besides, genes involved in developmental transition such as SPL9 are also expressed in the cryptic bract domain [START_REF] Hyun | Competence to Flower: Age-Controlled Sensitivity to Environmental Cues[END_REF]. Altogether, these data support the existence of a specific region in the abaxial part of the emerging flower, with morphological and gene expression parameters that reassemble the leaf. This cryptic bract is transient during flower emergence, and its function (if any) remains unaddressed. Nonetheless, the cryptic bract can develop into a full bract in particular conditions that I will discuss below.

Cryptic bracts in other species

Apart from Brassicaceae, bract loss appears in different group, which the most studied are grasses (Poaceae), and especially the model plant maize. In maize, inflorescence meristems (IM) are divided into two different types: the tassel IM, located in the terminal position at the top of the plant and producing male flowers, while the ear IM, are axillary axis subtended by a leaf and producing female flowers. Both IM produce spikelet pair meristem (SPM), which in turn will produce spikelet meristems (SM). The spikelet meristem starts by producing a pair of leaf-like structures called the glumes and can then produce several florets in the spikelet axis.

Each floret is enclosed in a pair of other leaf-like structure (called the palea and the lemma).

The floret is very peculiar because it contains almost only reproductive organs (stamens and ovary), the petals being reduced to tiny specific structures, the lodicules (fig. 14).

In maize, the monitoring of floral meristem development spotted the presence of a small bump on its axil, associated to a cryptic bracts l.s. (fig. 15C). As the flower grows, the cryptic bract l.s. disappears fast. In addition, TASSELSHEATH4 (TSH4 ), an homolog of SPL15 and SPL9, is expressed in the cryptic bract domain, in a mutually exclusive manner from the meristematic markers RAMOSA2 (RA2 ) (Chuck et al. 2010). Hence, in both A. thaliana and Maize, bract inhibition leaves remnants structure transiently visible on microscopic observations. This domain is also characterized in both species by a lack of meristematic features, and the presence of specific markers.

2.3 Basal bract emergence in bractless species: experimental and natural conditions

Experimental restoration of bracts involves defects in flower development

In both bractless Poaceae and Brassicaceae, bract development has been investigated using mutants that fail to inhibit bracts, revealing interesting genes whose alteration induces bract development.

In maize, bract l.s. loss seems to be triggered by TSH4 gene (Chuck et al. 2010). TSH4 is a protein involved in lateral organ inhibition, and marks the boundary between leaf and lateral meristem. In tsh4 mutant, bracts l.s. are de-repressed, while the size and number of axillary meristems and internodes are reduced, and the shape and size of the grains and the phyllotactic pattern is perturbed (fig. 15A-B

2014

), but these plants also show severe sign of flower impairment (fig. 16).

Thus, it appears that in all these mutants, bract inhibition and floral identity seems strongly related. The link between flower identity and bract suppression has also been investigated by It is noteworthy that in several studies, bract has been taken in a lato sensu or fpp sense, rather that stricto sensu. In fact, the diversity of flower shapes in Angiosperms shows that leaves can precede flowers (i.e, be located in the same axis), and that leaf-like sepal exists which are sometimes called "bracts" (here, they will be referred as bract fpp). For instance, in strong ap1 mutants, the 1st flower whorls can be transformed into bract-like structure ( At the precise time of floral transition, it is possible to see both the first flowers and phytomers of the future lateral branches underneath. While axillary meristems are not really visible before the floral transition (see Results -chapter 2 fig. 4, and (Hempel and Feldman, 1994)), they rapidly bulge out at the leaf axil in a basipetal wave once the first flower has been produced. (fig. 3A). On the contrary, in T. hassleriana, the emergence of the round flower meristem starts later, on the axil of an already developed leaf: the bract (fig. 3B). Interestingly, in the bract-making mutant puchi1, bop1, bop2, floral meristem also initiate at the axil of a sharp leaf (fig. 3C), even though, as discussed previously, this meristem may have a mixed shoot-flower identity. In Tsu-0 plants, we can observe the presence of bracts at the axils of the first flowers, but never after the 4th (fig. 3D). We did not manage to observe in live the full developmental sequence of a "bract-flower", which is extremely challenging. Hence, we cannot be sure that the bract develops before the flower. Still, our images show that the bract does not develop later, protruding from the floral meristem for example. Taken together, a bract develops before the flower in bracteate Cleomaceae. In the ebracteate A. thaliana, restoration of a bract in mutants or wild-type Tsu-0 accession involves a similar developmental event, in coherence with the evidence supporting the presence of a cryptic bract (see introduction). Hence, the basal bracts observed in Tsu-0 can be considered as bracts stricto sensu, homologous to bracts in bracteate species. Interestingly, these first flowers are no longer able to prevent bract growth.

Basal bracts emerges at floral transition in the main and the lateral shoots

In Tsu-0, bracts were always present on the first few flowers, never exceeding the 4th (data not shown). Tsu-0 produced on average one bract per cauline branch, regardless of the branch position along the main stem (fig. 4C). On the contrary, in Col-0, bract production seems correlated with cauline branch position, with the higher branches producing slightly more bracts that the bottom branches (fig. 4C). Besides, the number of cauline branches in Col-0 is reduced compared to Tsu-0 (fig. 4B). Col-0 shows on average 8 cauline branches when cultured 20 days in SD before switch to LD, while they are on average 11 in Tsu-0. Hence, in Col-0, cauline branches exceeding the 10th position shows 0,25 bract on average, but their frequency is rare, so that high branches bearing bracts are still rare. We next defined the bract mean frequency as the average number of bracts per cauline branches per plant for a given genotype (see methods).

Basal bracts are frequent in natural populations of A. thaliana, and may be linked to flowering time

In order to evaluate to what extend basal bracts are widespread in A. thaliana, we screened the presence of bracts in different natural accessions (fig. 5A). Bracts have been observed on the first flowers of several accessions such as Sha, Star-8, or Bor-4 (fig. 5A1). Several natural accessions present a similar bract mean frequency as Tsu-0, such as Mc-1, Bik-1, or Berg-1 (fig. 5A2), even though statistical analysis cannot be meaningful with such small number of plants. Still, this first rapid screen indicated for the first time that unexpectedly (see Brassibase), basal bracts are not rare in A. thaliana natural populations grown in laboratory conditions.

The basal position of bracts indicates that their formation is restricted to the floral transition. To test whether the flowering time is linked with basal bract production, we determined bract mean frequency with respect to flowering time in five different natural accessions (fig. 5B). Flowering time, assessed as the day when the first flower opens, ranged from day 22 to day 44. Isogenic Tsu-0 plants show a negative correlation between bract mean frequency and flowering time (5. Surprisingly, the trend was different in the other natural accessions: in Kn-0, Ler-0 and Col-0, bract emergence did not depend on flowering time; and in Wu-0, the later a plant flowers, the more it bears bracts. Thus, the link between bract formation and flowering time is complex and distinct mechanisms could explain the formation of basal bracts in different accessions.

Discussion

Leaves are a basic component of the phytomers, but they are not present at the reproductive phytomers of several clades such as Brassicaceae, or Poaceae (see introduction). The mechanisms of bract inhibition have not been fully resolved. So far, studies investigating bracts inhibition relies on mutants of A. thaliana, maize or rice, that also presented flower impairment, making bract inhibition mechanism difficult to isolate. We describe a new system to address the question of bract inhibition using natural populations of A. thaliana that make bracts with normal flowers. Bracts in wild-type plants were produced only at the base of inflorescence, during floral transition.

Floral transition offers a specific developmental context in which bract can emerge in some genotypes. The timing of floral transition is linked with bract production. However, the relationship between flowering time and bract emergence seems dependent on the genetic background.

Various environmental cues could explain the variability of the phenotype observed even within the same genetic background. Assessing some of them could be interesting: in particular, we will test in the next chapter 2 the role of the photoperiod in bract production.

The spatial and temporal restriction of bract formation suggest a particular developmental signature associated with floral transition, where the regulation of gene expression could be less canalized. It has been described that the first flower presents higher morphological variations ((Monniaux et al., 2016)). Moreover, the phenotypes of bract-mutants flowers were often gradual from the base to the top, with the most severe phenotypes at the base. This illustrates that remodeling of the GRN controlling floral transition may not be sharp, but rather presents a high variability at this precise developmental transition. Examples of the particular status of the floral transition step have been also studied at an inter-species level. Transcriptomes of several nightshades described higher divergence at the floral transition too, which could participate to architecture evo-

lution ((Lemmon et al., 2016)).

A transcriptomic analysis over floral transition in Tsu-0 and Col-0 is described in the next chapter 2.

Affordability of DNA sequencing can now facilitate the investigation of the genetic determinants of bract production in natural populations. Beside, as bract is produced only at floral transition, transcriptome related to its formation can be circumscribed. In chapter 3, we will describe the complex genetic determinants of basal bract in Tsu-0. However, as bracts are present in a lot of different genetic backgrounds, we can address whether the genetic determinants of bract production is identical in each accession. In fact, bracts have been suppressed independently in several species, and this can be the result of convergent or divergent mechanisms. On the other hand, bracts are observed in different mutants, showing that several genes participate to its suppression in A. thaliana. It will not be surprising that (re)emergence of bracts at the base of inflorescence could result from different genetic variants, that would have yet in common to decanalize the control of bract inhibition at floral transition. The investigation of a broad panel of natural accessions could thus teach us about the convergence/divergence of bract inhibition within A. thaliana.

Methods and Materials

Please refer to the "Methods and Material" section. However, several studies have now reported the coexistence of proper bracts with wild-type flowers in A. thaliana. These bract-flowers have only been observed on the first flowers at the basis of the inflorescence, that is on flowers produced at floral transition. Floral transition thus offers a temporal window where the mechanisms of bract suppression can be decoupled from flower development. In chapter 1, we described that several A. thaliana natural accessions make "basal" bract. Such basal bracts are also observed in many other Brassicaceae species, sometimes with a much higher frequency ( (Salariato et al., 2015)). In a series of seminal studies, Hempel and Feldman investigated the production of basal bracts appearing in particular accessions cultured under strong photo-inductive conditions ( (Hempel and Feldman, 1995;Hempel et al., 1998)). Using precise time course observations following photo-induction, they concluded that bract-flowers belong to phytomers produced before floral transition. They were likely indeterminate axillary meristem converted to floral fate after floral transition, thus keeping their subtending leaf. The authors proposed a temporal mechanism to explain the bypass of bract suppression by floral meristems. Before floral transition, leaves are normally initiated with a vegetative axillary meristem. After floral transition, the initiated leaf is not suppressed. However, its associated axillary meristem can still be converted into floral fate depending on the timing and the strength of floral induction. Their hypothesis is supported by the observations of a gradation of indeterminate shoot-flower phenotypes, interpreted as partial conversions of vegetative axillary meristems into flowers. Since these works, though, no further studies have elucidated the precise developmental pathways involved, such as those controlling the conversion of axillary meristem fates. It is also unclear whether the observations described in this particular context are applicable to other A. thaliana accessions or to other culture conditions. In this chapter, we present our data comparing floral transitions between two accessions, one making basal bracts, Tsu-0, versus the bractless laboratory reference accession Col-0. We quantified various parameters that could explain bract development. We investigated shoot apical meristems undergoing floral transition at different scales and profiled their associated transcriptomes. We showed that bracts in Tsu-0 did not result neither from a strong photoinduction, nor from a conversion of branches into flowers. We rather spotted subtle changes in the transcriptome of Tsu-0 during floral transition, and especially during the emergence of the first flower. Altogether, this study helps to better understand the GRN and the developmental context associated with basal bract production in A. thaliana.

Bract formation in

Results

Basal bracts in Tsu-0 does not recapitulate previous studies

In Tsu-0, bract emergence is not impacted by the photoperiodic condition

The chimeric phytomers described by Hempel and Feldman at the floral transition were obtained with plants of either Ler-0 and Nössen accessions, submitted to a strong photo-induction: they were germinated in Short Day (SD) and switched to intense Continuous Light (CL) conditions after 30 days ((Hempel and Feldman, 1995)). To test whether bract production in Tsu-0 results from a strong photo-induction, we cultured plants in different photoperiodic conditions. We quantified bract mean frequency in Continuous Light (CL), in Long Day (LD), and in biphasic condition where plants were first exposed to Short Day (SD) for 20, 28, or 35 days before switch to LD or CL. We also tested the impact of restoring short days environment after an inductive LD treatment of 20 days.

Increasing photoperiod did not induce an increase in bract mean frequency neither in Tsu-0 nor in Col-0. Besides, none of the different photoperiodic condition was linked to a significant change in bract mean frequency in neither Col-0 nor Tsu-0 plants (fig. 1A). In all conditions, Tsu-0 plants presented more bracts than Col-0 plants, highlighting a strong genetic determinant invariant to a wide range of photoperiodic conditions. Hence, contrary to previous studies, basal bracts in Tsu-0 are independent from photo-induction, even though the light intensity we used was different from these studies.

Because none of the condition we tested increased the level of bracts, we choose the 20SD -> LD condition for the rest of this chapter as it allows to synchronize plants and to get plants suitable for dissection. literature. We next described molecular and transcriptomic characteristics of floral transition in Tsu-0 that could be linked with basal bract production.

Acquisition of floral identity in Tsu-0 and Col-0 during floral transition revealed by live confocal imaging attempt

To address the molecular characteristics of Tsu-0's first flower, we next attempted to explore the expression pattern of LFY, a marker of floral identity, during the emergence of the first flowers in the two natural accessions. We dissected transitioning meristems of pLFY::2mCherry-N7 lines in Col-0 and Tsu-0 backgrounds. However, dissection of meristems before bolting, in the rosette stage, leads to important tissue dammage that hamper the acquisition of good quality images. Besides, PI stainning is hardly suitable for damaged tissues, and 2mCherry fluorescent dye and PI cannot be separated. Hence, despite three replicates with > 30 dissected plants/ replicates, very few images were exploitable. Moreover, LFY signal was weaker at transition stage than at bolting stage when reporter lines plant were initially screened (fig. 3). Taken together, the following image analysis needs to be taken with a lot of reserve. Our images suggest that pLFY::2mCherry-N7 is expressed in bract-flower in Tsu-0, but not in axillary meristems, confirming the floral identity of bract-flowers (fig. 3). The lack of good quality images did not allow any quantification. Nevertheless, no visible difference has been observed so far in the expression pattern of LFY between the two accessions. Similarly, visual comparison between the first and the second flower did not show any change in the expression of LFY. Hence, the expression pattern, as well as the expression level of LFY in the first flower of Tsu-0 did not show obvious visible discrepancies. Moreover, the sequence of the pLFY promoter in Tsu-0 and Col-0 did not show any variation. It can thus be suggested that production of basal bract in Tsu-0 seems to be LFY-independent. Despite the absence of quantification, low pLFY::2mCherry signal suggests that LFY expression continues to increase even after floral transition, making the first flowers a "LFY-reduced" flower. Moreover, the expression of LFY seemed highly variable in the first flowers, with number of apices that present no visible signal (data not shown). Taken together, these results insinuate that LFY expression level and pattern did not seem to distinguish Tsu-0 from Col-0 flowers, at least not visually. It also could hint that flowers dissected at T stage express a lower level of LFY than flowers dissected at bolting, that might indicate an incomplete floral fate commitment. Nevertheless, confocal analysis only shows expression and spatial data for targeted cloned genes. We then carried a global transcriptomic analysis of apices throughout floral transition in the two accessions to catch molecular hints that might lead to basal bract formation.

Transcriptional divergence culminates at floral transition in Tsu-0 where meristems show contrasting hallmarks of immaturity and maturity

Matching developmental stages in Tsu-0 and Col-0 with morphological clues indicate a similar but delayed profile In our culture conditions, floral transition of Tsu-0 plants is delayed by 2-3 days compared to Col-0 (fig. 4)). To see how this affects meristem development at a microscopic scale, we monitored its morphological evolution across floral transition. We dissected and imaged meristems from both accessions before, during and just after floral transition. Despite their different flowering time, four identical morphological stages can be easily identified in both Col-0 and Tsu-0. The Vegetative stage (V stage) corresponds to a small and flat meristem, only surrounded by leaf primordia, that plants harbor before they are transferred to long day conditions (day 1). The Late Vegetative stage (L stage) corresponds to a bigger and domed vegetative stage (5 days after switching to inductive conditions). At the Transitioning stage (T stage), the first flower primordia is produced, and the axillary meristem starts to expand at the axil of grown leaves. The Floral Stage (F stage) corresponds to big and domed meristems surrounding by flower primordia, clearly recognizable with their round shape. T and F stages occurred later in Tsu-0 plants than in Col-0: in Tsu-0, the T stage happened around day 13, while in Col-0, T stage was observed around day 9. These stages are similar to the stages described previously ((Klepikova et al., 2015), (Kinoshita et

al., 2020))

Matching similar morphological stages in the the two accessions allow us to compare functionally equivalent developmental stages although plants differ in absolute age. Notably, important molecular parameters related to the control or the consequences of these developmental transformations should be synchronized and display similar levels between matched stages. Conversely, variations of some molecular parameters between matched stages could point to the cause of why similar developmental stages end up in different outcomes (here the production or inhibition of a bract), as nicely shown in nightshades meristems within and across species (Lemmon et al., 2016) A de-correlation between morphological and transcriptomic signature of floral transition in Tsu-0 and Col-0

We performed RNA-sequencing on the shoot apices of Col-0 and Tsu-0 for the four developmental stages described above. A Principal Component Analysis (PCA) shows that developmental stages constitutes the main driver of transcriptomic dynamic differences between the two accessions (fig. 5A). Developmental stages well recapitulate floral transition as shown by the expression of key regulators of floral transition (fig. 1). Besides, all replicates are grouped together despite sampling time difference (fig. 5A), showing that the morphological staging is robust to at least 4 days of sampling disparity (see Appendix). Genotypes constituted the second main driver of transcriptomic divergence. Notably, the developmental stages of the two accessions displayed transcriptomic shifts despite their similar morphological features. In particular, the T stage, that corresponds to the expected moment for basal bract emergence, displays the highest transcriptomic shift: T stage in Tsu-0 appears older than the T stage in Col-0 (fig. 5A). However, if a basal bract made of cells expressing a leaf-like transcriptome emerges at the stage T Tsu-0, one could expected to detect a more juvenile transcriptome in Tsu-0 compared to Col-0. In order to better characterize the dynamic of gene expression in both accessions, we selected a subset of genes that are differentially expressed between at least two stages in at least one of the two accessions (fig. 5B). This subset represents 7498 genes that we coined hereafter "dynamical genes" (fig. 5C). In a quite expected manner, a Gene Ontology (GO) term enrichment analysis indicates that this pool is enriched for genes involved at the molecular scale in the regulation of transcription and whose function is associated to responses to stimuli and hormones and post-embryonic development. A PCA on these dynamical genes shows similar results as with the whole dataset: the main driver of variation is the developmental stages, while the second driver is the genotype (fig. 5D). Because sampling dates of T and F stages were different between Col-0 and Tsu-0 (fig. 4 and Supplemental data), we established the relation between the sampling date and the "transcriptomic age", defined as the projection of the different samples in the first component found by the PCA. Fig. 5E shows that the older transcriptome of T stage in Tsu-0 is not only due to an older sampling date. In fact, Tsu-0 is delayed on both axis: the sampling date and the transcriptomic age. In Tsu-0, the transition from L to T stage is as divergent as the transition from L to F stage in Col-0. Reaching T stage involves the highest change in gene expression, and it is more pronounced in Tsu-0 (fig. 5E and fig. 2). On the other hand, few transcriptomic divergences exist between the T and F stage in Tsu-0, contrary to Col-0. Hence, comparing development between Col-0 and Tsu-0 reveals a complex heterochrony at stage T: while meristems look alike (doming), Tsu-0 plants both have juvenile traits (bracts) and older transcriptome.

To better understand the gap between morphological and transcriptomic characteristics in Tsu-0, we next investigated the dynamics of gene expression in both accessions.

A reduced down-regulation of genes in Tsu-0's floral transition

To better assess which genes participate to this heterochrony, we carried a clustering analysis based on the dynamics of genes across floral transition in both accessions. To group genes based on the variations of expression levels rather than their absolute levels, the expression levels of each

Discussion

Bract loss has happened several times independently in angiosperms, like Brassicaceae or Poaceae.

Attempts have been made to investigate bract loss mechanisms using mutants. However, the isolation of bract inhibition mechanisms have been challenged by the pleiotropic effect of the mutants, questioning whether mutant bracts could be qualified as bracts stricto sensus (s.s.). Nevertheless, the presence of bracts s.s. associated with wild-type flowers in some natural accessions of A. thaliana (like Tsu-0) offers an new opportunity to study bract formation and decipher the mechanism of its inhibition. Their exclusively basal position on the inflorescence suggests a strong link with floral transition. In this chapter, we then used bract-making Tsu-0 plants in comparison with non-bract making Col-0 plants and focused precisely on floral transition to understand the developmental features that characterize basal bract formation.

In Tsu-0, the absence of floral indeterminacy and the range of bract phenotypes in bract-flowers suggest a perturbation of bract inhibition only, independently of floral determinism. This is further confirmed by the very similar transcriptomic profiles of floral determinism genes, both globally Lemmon et al. showed that the divergence of transcriptomes peaks at floral transition between different species, despite an identical meristem morphology. Besides, they show how heterochronic shifts, i.e. desynchronization of gene expression dynamics with respect to the matched similar phenotype, can be responsible for the evolution of plant branching system. Here, we provide the first example of such model at an intra-specific level. In this case, our results suggest that transcriptomic divergence during T stage could also lead to phenotypic evolution, here the presence or absence of basal bracts. Besides, like in nightshades, we also depicted the distortion between morphological and transcriptomic status of the meristem: Tsu-0's T stage transcriptome was closer to Col-0's F stage, but morphologically identical to Col-0 T stage.

Observing this older transcriptome in Tsu-0, was surprising as bracts could have been seen as the remnants of a vegetative phase. So far, we could not however identify particular gene expression characteristics, such as leaf GO term enrichment, associated to such vegetativness at T stage. But identifying a GRN responsible for basal bracts formation in our transcriptomic dataset is of course very challenging. Bract development or inhibition occurs in a very narrow domain. As the transcriptomes have been sampled from the whole inflorescence meristems, it is uncertain to isolate differential gene expressions related to bract formation from the numerous unrelated variations expected in two different natural genetic backgrounds. A single cell RNA sequencing could allow to isolate, based on the expression of abaxial "cryptic bract" genes (see Introduction), the specific transcriptome of bract domain between the two accessions. As this technology is getting more affordable, we will be able to consider using scRNAseq to precise bract-related transcriptome. Finally, this transcriptomic approach is based on the assumption that bract formation in Tsu-0 results from a difference in the transcription of a set of genes, but this can be wrong, and other scenarii could be investigated, that will be discussed in Chapter 3.

One of the possible mechanisms of bract formation is a mis-localization of some genes specifying bract versus floral domain. Even if LFY expression level was identical between Col-0 and Tsu-0 all throughout the four stages, it was interesting to address its spatial localization in the first flowers of Tsu-0 and Col-0. However, observing the emergence of the first flowers requires to dissect and image vegetative meristems, which is a challenging task. Besides, LFY signal in transgenic reporter lines were selected on bolted meristems, but it appears that the LFY signal of vegetative meristem was way below the one observed after bolting. For theses reasons, as well as time constraints, we had not enough good quality pictures to clearly describe the expression pattern of LFY during the emergence of the first flower in Tsu-0 and Col-0. Apart from LFY imaging, it would also be of great interest to image identity markers of the cryptic bract domain such as FIL, or SPL 10. In fact, as shown in maize, a mis-patterning of cryptic bracts versus flower domain can lead to bract formation (Chuck et al., 2010). To this end, generating appropriate transgenic bract domain reporter lines is crucial. Identifying genes with more bract/leaf specificity and less pleiotropic expressions (such as in flowers for instance) will be helpful. To this end, pinpointing the causal gene(s) responsible for basal bract formation in Tsu-0 can thus not only inform on the mechanisms of bracts suppression, but might provide good candidate reporters to monitor and/or perturb bract development.

Conclusions and perspectives

Previous studies of bract development in A. thaliana relied on phenotypes showing signs of floral indeterminism. Here, we explored the formation of basal bracts in Tsu-0, a natural accession that does not show signs of floral indetermination. It appears that the mechanism of bract production in this context is different from the one previously described, as bracts are not a conversion of branches into flower after strong photo-induction. Because bracts are restricted to the first flowers, we captured the transcriptome related to floral transition in Tsu-0 that depicted sign of heterochrony at T stage, with an older transcriptome. Taken together, this chapter exposes the first study of transcriptomic variations over floral transition within the same species. Interestingly, it shows that hundreds of genes display a significant change in their expression dynamics, and most of these differences are moderate (changes in expression levels or heterochronic shifts) rather than opposite trends. Moreover, despite a macroscopic phenotypic output (basal bract formation), most of the known major regulator genes previously involved in bract formation and floral determinism show an astonishing similar expression profile. Although this needs to be confirmed, this intermediate result suggests that other unexpected genes (and possibly pathways) would explain basal bract polymorphism in natural A. thaliana populations. In line with this hypothesis, the importance of overlooked genes in population-scale micro-evolution in nature has been showed for flowering time in A. thaliana ((Brachi et al., 2010)), although our experimental set-up does not mimic natural growth conditions. As we will detail in the next chapter, transcriptomic dataset can also help for the identification of the causal gene of basal bract production in Tsu-0. These results

Introduction

Phenotypic evolution proceeds through a variety of changes, including transformations, gains, losses or reversions. The nature of the heritable genomic changes that produce such phenotypic evolution is a central question in biology. Given the high gene conservation among even distantly related organisms, trait gains or losses are mostly not caused by simple gene gains or losses. As observed in many mutagenesis experiments, a loss of gene function do not simply results in a loss of trait, but rather in pleiotropic effects. Therefore, we need more examples of how precise phenotypic changes occur in nature to characterize their causal genomic variation and their underlying developmental regulatory network. One surprising phenotypic change that participate to the evolution of forms are the complete or partial loss of a morphological trait. However, existence of atavism, i.e the resurgence of an ancestral trait, suggests the maintenance of the genetic information required to build the trait. It appears that small changes in gene activity could channel the development of a structure into its complete, partial or lost form. Sometimes, cryptic structures are maintained. Bract development in the model plant Arabidopsis thaliana offers a case study to investigate the nature of such small heritable genetic changes. Bracts, the leaves subtending flowers, have been lost in Brassicaceae clade. Nevertheless, at least in A. thaliana, a bract starts to form just before each flower grows out, but its development stops early and the bract remains cryptic (Penin, 2008;Kwiatkowska, 2006). However, many Brassicaceae species keep making a few bracts at the basis of inflorescences (see Chapter 1). Although the mechanism of floral induction is very well established, the inhibition of bract is still poorly understood. Moreover, the mechanisms of its resurgence at the basis of some inflorescences are completely unexplored. Like in previous studies, (Hempel and Feldman, 1994;Hempel et al., 1998;Hempel and Feldman, 1995), we have reported the presence of fully developed bracts in some natural populations of A. thaliana. Bracts were only subtending the first flowers at the basis of the racemes, as it was observed in some other Brassicaceae species, and were called "basal bracts" (see chapter 1). In A. thaliana, such bracts can be triggered by specific photo-induction (Hempel andFeldman, 1994, 1995), or observed in standard laboratory culture conditions for many natural accessions (see chapter 1). Hence, in A. thaliana as in many Brassicaceae species, we propose that basal bracts are an atavic structure. That is to say, the developmental program of bracts production beyond the cryptic stage are retained. However, except for such rare bracts, this developmental program is usually not triggered, and/or inhibited.

Comparing bracteate and ebracteate accessions of A. thaliana offer a way to identify natural genetic variations that trigger this developmental program, at least during floral transition. The nature of such genetic variation is so far totally unknown.

In this chapter, we present how using the natural accessions Tsu-0 and Col-0 enabled us to map the genetic variations associated with transient bract formation at the floral transition. Our genomic mapping identified several QTL controlling bract formation, revealing its complex genetic determinism. We re-analyzed genomic data to comprehensively annotate the putative functional effects of natural polymorphisms in Tsu-0, especially in the genetically mapped intervals. We also used transcriptome profiles during floral transition to capture differential gene expression correlating with the production of bract in Tsu-0 and some bract-mutants. By crossing all these data, we propose a list of candidate genes that could be at the origin of atavic bracts at floral transition in the A. thaliana natural accession Tsu-0.

Results

Bulk Segregant Analysis (BSA) identifies a complex genetic determinism governed by several candidate loci in Chrom 1 and Chrom 5

To map the gene(s) responsible for atavic bract formation at floral transition using bulk segregant analysis (BSA), we first generated a mapping population (Col-0 x Tsu-0). We measured the bract mean frequency (defined as the mean number of bracts per branch for each individuals) of the parents (N= 90/genotype), the F1 (N= 176) and the F2 (N= 684). For practical reasons, we split this quantification into four replicates including all genotypes (fig. 1). As mentioned earlier (see Chapter 1), bract formation is variable even in a fixed isogenic background: mean bract per branch in a plant ranges from 0 up to 0.5 in Col-0 and from 0 to 1.9 in Tsu-0. This stochastic variability diminishes the statistical power of associative mapping. Except for the first replicate, the distribution of bracts per branch was normal in Tsu-0, so we calculated its mean value for each genotype. The phenotype of the F1 population was intermediate between Col-0 and Tsu-0, as F1 bore on average 0.44 (+/-0.28) bracts/branch, Col-0 0.11 (+/-0.13) and Tsu-0 1.0 (+/-0.40) (fig. 1A). This indicates that at least one co-dominant allele is involved in bract formation. No transgressive phenotype was observed in the F2 as bract distribution ranges within the parental extrema. F2 bract mean frequency did not mark any peaks around parental and F1 means, incompatible with a mandelian segregation of a single locus. Instead, F2 distribution was close to the F1: F2 bore on average 0.42 bract/branch (+/-0.31) (fig. 1A). Thus, several genes may be involved in the formation of basal bracts. We then performed a BSA by selecting F2 plants presenting high and low bract frequency (named hereafter Tsu-like and Col-like plants, respectively), measured as the mean bract number per branch in a plant. We defined Tsu-like plants as plants showing higher bract frequency than the highest F1 plant of the same replicate, resulting in a selection of only 17 individuals over the 684 scored F2 plants. We defined Col-like as plants showing no bracts in replicates 1, 2 and 4, representing 56 individuals (fig. 1A). After bulk sequencing of the DNA extracted from Col-like and Tsu-like pools, we scanned the variations in Col-0 versus Tsu-0 allele frequency along the genome using the deltaSNPindex method [START_REF] Takagi | QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations[END_REF]. To generate an accurate list of natural genomic variations between the parental strains of our two bulks, we re-sequenced the genome of the Col-0 and Tsu-0 parental strains used in our experiments and performed a genomic variant analysis. The loci linked with mapping in chromosome 1 (Ia was located at 12 Mb while I at 5,6 Mb, and Ib was located at 17 Mb while I was at 22 Mb). These results clearly point to the existence of two principal additive QTLs in chromosome I controlling transient bract formation in Tsu-0. We decided to focus on chromosome 1 as it resulted from both BSA and RILs analysis. Within the chromosome 1, we will keep the interval coordinates from both techniques to provide a transparent and well-informed analysis.

On the main stem, the presence of bracts is genetically linked to cauline branch number.

We next investigated the association of QTLs I and I with different pleiotropic phenotypes that could provide interesting insights on the underlying allelic variations responsible for basal bracts.

In the RILs dataset, we noticed a positive correlation between the number of bracts and the number of cauline branches on the main stem (3A). Yet, the latest generally increases with the flowering time (supplemental data), which is, as we showed in Chapter 1, also associated with bract frequency. To investigate whether these different traits can be linked with bract frequency, we extracted from our phenotyping data the percentage of plants having bracts specifically on the main stem as well as the mean number of cauline branches in RIL assays. We treated these two new phenotypes as co-variables in the R/qtl analysis. Interestingly, the number of cauline branches and the percentage of plants having bracts on the main shoot are both linked with peak I (fig. 3B) but not QTL I . This suggests that, in Tsu-0, the two QTLs in chromosome I contribute to transient bract formation through distinct mechanisms, and that one of these mechanisms would be linked to the control of cauline branch number.

Transgressive traits in some RILs evoke bract mutants phenotypes

Despite the RIL analysis indicates an additive contribution of QTL I and I for bract frequency, we noticed other interesting transgressive phenotypes that could be linked with bracts. However, they were not systematically scored in all RILs and cannot be analyzed as co-variables and mapped in a quantitative manner. The first transgressive phenotype is flower indeterminacy. Two indeterminacy phenotypes were observed, including branched flowers (4A) and prolonged organogenesis inside carpels, and neither of them were never observed in the control parental strains Col-0 and Tsu-0 (N>7310). As mentioned earlier, most of the known bract-making mutants also display defects in floral indeterminacy. Such transgression in RILs could indicate that in Tsu-0, a similar pleiotropic allele exists. Nevertheless, Tsu-0 may not present floral inderterminancy because of a particular allelic combination that favors basal bract formation while compensating floral indeterminacy. Genomic recombination in RILs would break this epistatic inhibition. However, the incomplete scoring of these traits preclude their genetic mapping: we could not determine whether they are associated with one or several QTLs and whether they overlap with I and I controlling bract frequency.

The second transgressive phenotype is a shift of bract position on the flower pedicel, that was noticed in at least 13 RILs in our dataset. As pictured in 4B-C, shifted bracts are not attached to the stem at the node, but rather upstream on the flower pedicel. Interestingly, similar shifted bracts have been observed in bract-bearing mutants like bop-1, bop-2 or jag-5D (Dinneny, 2004;Ohno, 2004;Norberg, 2005) (see also Chapter 1). In some RILs (470, 165, 008) almost all bracts are shifted, while in other RILs this occurs only sporadically. Here again, precise mapping and relations of this trait with QTL I and I could not be investigated. However, no obvious correlation with bract frequency was detected, as RIL 008 had 0.2 bract/branch, while RIL 165 1.52 bracts/branch. Altogether, the observation of the transgressive phenotypes can be used as additional clues to identify and prioritize the best candidate genes.

Fine mapping the QTLs of chromosome I using Near Isogenic Lines (NIL)

BSA and RIL genetic mapping were not precise enough to isolate the genes responsible for basal bract in the Tsu-0 natural accession, so we generated Near Isogenic Lines to further reduce our Altogether, BSA and RIL strategies point to a major role of the chromosome 1 with several QTLs. BSA identified two QTLs: one QTL of 2MB between the position of 11Mb and 13Mb, and one QTL of 1Mb between the position of 17Mb and 18Mb. RILs analysis identified 2 peaks associated with the markers at 5.6 and 22.2 Mb. We thus allocate the surrounding markers at 4.2 and 7.2 Mb and at 20.4 and 23.4, leading to two target regions of 3Mb. The construction of NILs that segregates at each of these loci will allow to better precise the relevance of each QTL identified. In addition, BSA identified also a large QTL in chromosome 5, that was not confirmed with the RILs analysis, so we decided to exclude the later from further analysis. In fact, the Va and Vb QTL probably result from a lack of power due to a low amount of individuals per bulk (see Discussion).

Taken together, and considering all the chromosome 1 QTLs, our strategy retrieve intervals that correspond to 3743 potential candidate annotated genes, suitable for further researches. While waiting for the establishment of the NILs population to precise the QTL, we studied our potential candidate genes through additional dataset to isolate some interesting genes that could be tested rapidly.

Data-mining of candidate genes involved in bract formation by transcriptomics

Coupling BSA and RILs phenotyping was not sufficient to reduce the interval to an individually testable number of candidate genes. We then aggregated to the genetic evidence other data that are the following: -genetic intervals from BSA (QTLSeqR software) and from RILs (r/qtl2 software) -prediction of the functional impacts of natural genomic Tsu-0 polymorphisms on every mapped genes -functional prediction of the mapped gene based on GO annotations -expression profiles of the mapped genes in Tsu-0 and Col-0 meristems before, during and after floral transition (see chapter 2) -expression profiles of the mapped genes in meristem of "bract-making" mutants -expression profiles of the mapped genes in published public dataset ( [START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF]).

We propose three different scenarii concerning the genes responsible for basal bracts in Tsu-0 (hereafter called "bract-gene"). Depending on the scenario, the diverse data require to meet different expectations. We first assumed that bract-gene are located in chromosome 1, within one of the 4 QTLs described earlier.

Scenario 1: bract-genes may be differentially expressed during the emergence of the first flower in Tsu-0 compared to Col-0

We first hypothesized that a confident bract-gene candidate may be differentially expressed during the production of the first flower in Tsu-0 compared to Col-0. We have previously carried a RNA-sequencing of Tsu-0 and Col-0 shoot apex across floral transition, and in particular during the formation of the first flower. We thus identified the genes located in the mapping region and differentially expressed during T stage, that corresponded to the emergence of the first flower. 474 genes were retained. Among them, 448 genes bear polymorphism(s) that could explain their differential expression level in Tsu-0 genetic background and constitute Candidate Gene Set 1 (CGS1). We investigated the dynamics of those gene expression across floral transition as described in chapter 2. 54 genes are dynamical during floral transition (their expression level varies across the 4 developmental stages) that belong to a different dynamic group in Col-0 compared to Tsu-0, including NAC019 or ASL9. Interestingly, ASL9 is also downregulated in lfy-12 and jag5-D mutants, that are "bract-bearing" mutants. Expression pattern of ASL9 is mainly located in plant hypocotyl, vascular tissues and mark boundary regions ((Klepikova et al., 2016)). Its homologous ASL2 is known for its role in leaf development and polarity (Machida et al., 2015). Altogether, ASL9 gather interesting characteristics and could constitute a good candidate bract-gene. Scenario 2: Genes responsible for bracts may have protein coding differences in Tsu-0

We also explored the hypothesis that bract gene(s) code for a protein which present amino acid differences in Tsu-0, inducing bract formation at its first flower. According to our transcriptomic dataset, 2082 genes are expressed in the SAM and located in the mapping region, but do not show differential expression with Col-0 at T stage (contrary to GeneSet1). From this first set, 1134 genes bear genomic variation within the open reading frame (ORF) that could cause plausible effects on the gene (and/or protein) function (see Methods): we group these genes into the Candidate Gene Set 2 (CGS2). We focused on the genes presenting high impact coding variation (see Methods), that

Discussion

In bractless species belonging to the Brassicaceae or Poaceae families, the formation of transient bracts offers the opportunity to study the genomic basis underlying the development of such atavic traits. Bracts have been described in a certain number of mutants in A. thaliana or maize but the mechanisms of bract inhibition and/or atavic reformation remain elusive. In these examples, the pleiotropic effects of mutated genes, which also affect flower development, preclude the study of how a wild type flower inhibit bract development in a normal context. Natural variation captures phenotypes that are often not uncovered by mutant screens. In Tsu-0, a bract develops on the first flower without affecting flower development. In that sense, this genetic background provides an example of a developmental variation that breaks pleiotropy and avoid deleterious additional effects. Such natural variation represents a plausible evolutionary path for a trait reversion and, by symmetry, for its loss.

In this chapter, we presented all the work performed in order to identify the genetic determinants that allow plants with a Tsu-0 genotype to develop transient atavic bracts at the floral transition. We showed that coupling BSA and RILs analysis allow to circumscribe the mapping region into several loci in chromosome 1 representing a total of 9Mb, but not to precisely identify bract-gene variations.

Contrary to mutant strains originated from laboratory mutagenesis, two random natural accessions bear a huge amount of genomic polymorphisms which hurdle the identification of the causal variations responsible for the phenotypic difference. This challenge is particularly important when it comes to subtle and quantitative phenotypes, like bract mean frequency. Here, we combined two genomic approaches, BSA and RILs, to map bract gene(s). The BSA allows to first determine the genetic architecture of the trait and estimate the mapping interval(s). Because of their length, we used a set of RILs to fine map the previous intervals and reduce the amount of candidate genes.

By coupling both approaches, we mapped bract genes into at most a 9Mb intervals spread into several smaller QTLs within chromosome 1, but failed to shorten them. We noticed a major discrepancy between BSA and RIL approaches. While both methods pinpoint 2 loci in chromosome 1 (although with different coordinates), the BSA outputs a 17-Mb signal in chromosome 5, which is absent from RIL analysis. Several hypotheses could explain this. First, some experimental details may have biased our BSA results. Indeed, we took less than 20 F2 for the Tsu-like pool, when generally BSA mapping strategies relies on at least 40 F2 individuals per pools [START_REF] James | User guide for mapping-by-sequencing in Arabidopsis[END_REF].

Pooling a low number of individuals increases the risk of selecting confounding false positive unrelated phenotypes. While a stringent pool selection minimizes the chance to have heterozygotes in Tsu-like bulks, it might reduce the precision of the mapping. On the contrary, the Col-like bulk contains individuals with no bracts at all: it may includes heterozygous as many F1 individuals had no bracts. Finally, because of technical issues at the DNA extraction step, we could not ensure precisely an equal amount of DNA for each individuals (see methods), that may add some inaccuracies.

On the other hand, the RIL approach may also have one caveat: the RIL set we used segregates a mutation in the beginning of chromosome 5, derived from the parental Col-0 strain used to generate the RILs. The mutated gene, SG1 (SHOOTGROWTH1) [START_REF] Coustham | SHOOT GROWTH1 Maintains Arabidopsis Epigenomes by Regulating IBM1[END_REF], resulted in drastic shoot growth phenotypes, not suitable for bract counting. We thus needed to discard all the RILs with Col-0 genotype between markers c5_02900 and c5_04011 to avoid the mutated sg1 allele. This restricted the choice of possible RILs that recombined into the Va QTL identified with the BSA. However, we still were able to investigate RILs bearing Tsu-0 haplotype between markers c5_02900 and c5_04011, that were not linked to an increase in bract levels. Hence, bract phenotype was mostly explained by the genotype in chromosome 1, rather than in chromosome 5. However, we cannot exclude that Chromosome 5 contains epistatic variations with minor effect that increase the bract mean frequency in Tsu-0. In fact, our Tsu-like bulk displayed a high bract mean frequency. It is possible that F2 plants in this "high" bulk were selected on both chromosomes: the major effect of the variation in chromosome 1, and minor effect a the variation in chromosome 5. We also have shown that bract mean frequency and floral transition were linked, and the QTL in chromosome 5 could contain flowering time genes that could indirectly promote the presence of bract. The existence of (one or several) QTLs for transient bract formation on chromosome 5 clearly needs more experiments to be settled down. However, our genetic data demonstrates the polygenic determinism of the trait and the major role played by chromosome 1 that most likely bears two QTLs. While both BSA and RILs approaches captured two QTLs within chromosome 1, their coordinates were not overlapping: the two BSA QTL (coined Ia and Ib) are located in-between the two RIL's QTLs (I and I ).

To better map the gene, we are now trying to generate NILs. We look for recombination events to shorten the intervals while limiting any epistatic relation elsewhere. Such NILs can give precision on which of the QTLs identified are relevant. We planned to establish lines that are Col-0, Tsu-0 or heterozygotes at the loci I , Ia, Ib, and I . Other genomic approaches can be used to better map the bract genes. As bracts are common in natural accession of A. thaliana, it is suitable for GWAS studies. GWAS approaches can help to identify common variations linked to bract formation in a wild range of natural accessions. It can also identify other variations that participate in the establishment of basal bracts, and how much each of these genomic variants contribute to the frequency of the trait at the species level. Notably, it can be interesting to investigate to what extend bracts can be linked with other traits (cauline branch number, flowering time, flower indeterminacy or shifted bracts) as is was observed in some genotypes.

In order to better investigate the candidate genes contained in the current interval, we used a transcriptomic analysis of the SAM across floral transition in both natural accessions and used it to explore the set of mapped genes. We also used a transcriptomic analysis of three bract-bearing mutants, and online datasets such as expression profile or GO terms. We established three different hypothesis to guide our data mining across the transcriptional dataset. The first was to hypothesize that bract-gene is differentially expressed during T stage in Tsu-0 versus Col-0. This leads to 2 main interesting genes: ASL9 and NAC019. The second hypothesis was to consider that the causal polymorphism would affect only post-transcriptional steps in Tsu-0: ARF1 is such a potential candidate gene. Finally, we investigated the miRNA lying in the QTL, but we did not retain interesting candidate gene. However, none of the clues we used to better characterize the candidate genes corresponding to each scenario are necessary nor sufficient to identify the exact gene responsible for bract emergence in Tsu-0. First, most of the variations have unclear functional effects: for example, it is very hard to predict the real effect of SNP or short indels lying in the promoter of regulating sequence of a coding gene. It must also be noted that we have not looked specifically for large structural variations and that we might have missed such genomic variations in our mapped intervals. Such analysis is now undergoing. A last caveat of variant prediction in natural accessions is a frequent rate of false positive functional impacts, due to the fact that the reference genome and annotation has been optimized on one particular accession ((Gan et al., 2011)). Second, avalaible expression pattern data [START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF] do not concern the precise localization in time and space within the meristem, so it is difficult to target the domain of cryptic bract formation. Finally, Tsu-0 transcriptome present a number of genes with a different dynamical group than Col-0: very likely, most of these genes have nothing to do with bract emergence. Hence, the clues are indicators for further explorations, rather than definite proofs for the candidate genes. Moreover, this data mining approach is certainly biased by our literature knowledge. In fact, among number of genes responding to each scenarii, we picked the ones that seemed interesting candidate for us. As developmental biologists, we considered genes involved in the developmental processes we know the best. However, number of mutants developmental phenotypes can results from unexpected genetic pathways. Finally, more than a third of the genes contains in our interval have unknown functions. It is highly possible that the causal Tsu-0 variation we look for affects such an unknown gene. It is thus necessary to first reduce at maximum the number of potential candidate genes, so the length of the QTLs. For the Bulk Segregant Analysis, plants have been cultured 20 days in SD before switching to LD. For the RILs, plants have been directly cultured in LD.

Plant materials

The natural accessions came from the Versailles Arabidopsis Stock Centre (Col-0 : 186AV, Tsu-0 : 91AV), as were the RILs (RIL set name: 3RV). with syringes (23G, 0.6x25mm), under the binocular loupe. In both cases, after dissection, plants were immediately put on an Apex Culture Medium (1/2 MS medium supplemented with 1% sucrose, 0.8% agarose, 1x vitamin solution (myo-Inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, thiamine hydrochloride 10mg/L, glycine 2mg/L) and 100nM N6-Benzyladenine (BAP)) waiting for imaging.

Microscopic meristem imaging

Meristems were imaged using a Scanning Electron Microscope (Hirox 3000 SEM), or with a confocal microscope Zeiss 700 LSM, according to the manufacturers instruction and without prior fixation. For plants expressing pLFY::2mCherry-N7, imaging, meristem cell walls have been stained with 100 µg/ml propidium iodide solution.

3 Plants phenotyping

Bract quantification and flowering time measurement

Bract mean frequency was defined as the mean number of bracts per shoot per plant in the part derived from the main shoot (so we excluded secondary rosette branches). Practically, we counted the number of bracts visible to the naked eye on the main stem and the secondary cauline branches. To ensure that bracts were visible, phenotyping was performed after flowers of the last cauline branch were opened and internodes elongated.

We also used two other metrics to quantify the presence of bracts in genetic lines: bract median frequency was defined as the median number of bracts per shoot per plant, and the percentage of uniquely mapped reads: for the thirty three samples, 88 to 97% of the reads were mapped to a simple locus. Altogether, these quality checks showed that the sequencing and the alignment were of very good quality. Raw reads quantification was performed using the reference transcriptome (ENA Browser 2021) and the STAR software [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF].

More than 80% of the reads were affected. Normalization of read counting was performed using the R Bioconductor packages DESeq [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. en[END_REF], with the following parameters: genes for which the total number of reads was below 10 were discarded, and data were transformed for Col-0 and Tsu-0 between the different stages (see results -Chapter 2). Multiple DEG analysis were corrected using Benjamin-Hochberg correction, and genes with a p-value < 0.5 were retained.

Analyzing and comparing the global dynamics of the transcriptomes

From the matrices of DEG between developmental stages in both Col-0 and Tsu-0, we first computed the set of "dynamical genes" (DynG), defined as the union of genes differentially expressed in at least one stage out of four in both accessions. PCA on the DynG was performed and visualized using FactoMineR [START_REF] Lê | FactoMineR: A Package for Multivariate Analysis[END_REF]) and factoextra [START_REF] Kassambara | factoextra: Extract and Visualize the Results of Multivariate Data Analyses[END_REF] on R. PCA using the set of DynG produces similar sample separations as using all genes, in-variations falling 500 bp downstream the ORF. Additional gene information (Aliases, names, curator summary and computational description) was retrieved from ThaleMine (Pasha et al. 2020) using AGI code. Finally, associated GO terms (including compartment, function and process) were also retrieved with AGI code using TAIR bulk data retrieval tool (TAIR -Bulk Download -GO 2021) and formatted in the table using custom script. The three different scenarii for causal gene identification (see chapter 3) resulted in three different files listing the relevant genes corresponding to each scenario in the genetic mapping intervals. The different information fields gathered per gene were used in a table file to further filter the most interesting candidates. These three files are provided in the appendix.

On the other hand, natural populations result from several genetic variations, that together enrich the pool of phenotypic variations. So far, bract emergence with absence of floral defects have only been observed in natural population. In addition, natural variations are by definition viable mutations. Because they are present in naturally occurring Arabidopsis thaliana plants around the world, they can participate to species diversification. Hence, natural variations can facilitate the access to a plausible evolutionary path leading to architecture evolution, shedding light on its underlying genetic basis. It constitutes a good step before questioning bract loss at a larger evolutionary scale, between two distantly related species. Inhibition of bract constitutes an interesting step in the evolution of Brassicaceae's architecture, because they affect the composition of the well conserved phytomer. Still, presence of basal bract in natural populations have been observed from the early 90's, but affordable sequencing tools were missing to deeper explore the genetic basis of such phenotypes. Now, both the emergence of next generation sequencing and the availability of many seeds derived from natural population crosses, allow us to explore the GRN of basal bract emergence. Besides, natural variations can been used to precise the new functions of genes, as it has been the case for FRIGIDA (Zhang & Jiménez-Gómez 2020). Basal bract phenotype may be linked with genes that have not been studied yet, and can thus reveal, or precise, some genes functions. Natural populations make the perfect link between classical genetics and evolutionary studies. Their use constitute then a fantastic tool to question evolutionary developmental biology. To this end, we used the natural accession Tsu-0, that present frequent basal bract formation, to map their underlying genetic determinant(s).

However, catching the exact genetic divergence(s) that is (are) responsible for basal bract formation appeared quite challenging. First, because bract frequency is gradual and varies extensively even among a same batch of isogenic plants grown in the same conditions: Tsu-0 plants can present no bract per branch as well as 2 bracts per branch. Second, because the trait is polygenic, which increases the quantitative spread of the trait value in crossing panels: for example, the F2 distribution spreads in between the two parental lines without any visible subclasses. Third, because the phenotype is subtle, and could be triggered by any genetic variation (affecting regulatory sequences, as well as protein coding sequences), contrary to strong impact mutation easily detectable in mutants. Because of the large QTL obtained not at V and L stage, and the MIR157D targets SPL3 and SPL5 show an enhanced toggle in Tsu-0 compared to Col-0 (less expressed at L stage, and over expressed at T stage). A first glance at the implication of both genes have been assayed by phenotyping natural populations bearing the exact same variations as Tsu-0. However, no genotype-phenotype correlation have been observed for the 8 natural accessions tested, although the number of scored plants per accession was extremely reduced (n=6) in this first exploratory experiment.

We have no doubt about finding the candidate genes after a better QTL length reduction.

The validation of a candidate gene will require to couple the use of natural population and mutants. Natural populations can be used to confirm the link between the candidate variants and basal bracts formation. In a second time, CRISPR technology can be used in Col-0 at the target locus to validate the candidate genes. Such tool will then be crucial to investigate the role of the gene in uncoupling bract inhibition from flower emergence.

Natural accessions have been classically used to study flowering time [START_REF] Brachi | Linkage and Association Mapping of Arabidopsis thaliana Flowering Time in Nature[END_REF], or the environmental control of flowering (Lempe et al. 2005). However, the floral transition properly have not been investigated through the lens of natural variations. Because basal bract is temporally restricted to the first flowers, we investigated the dynamics of the transcriptome at the moment of floral transition between the two natural accessions.

2 Developmental switch at a population level

Heterochrony at a population level

The floral transition (FT) has been investigated in the two natural accessions by a morphological description of the SAM, by monitoring gene reporter lines for LFY expression, and by a transcriptomic analysis of the SAM over FT. Both the morphological features associated with FT, as well as the expression pattern of LFY, show no visible distinction. Hence, as the timing of FT was different, we were able to use the morphological feature to define 4 identical stages of FT, that were used for the transciptomic analysis. The RNA-seq analysis revealed a morphotemporal distortion between the two accessions at the time of FT. That is, a discrepancy between SAM morphology and transcriptomic signature. Such distortion was the highest at T stage, that corresponds to the emergence of the first flower. At T stage, Tsu-0 trancriptomes

INTRODUCTION 1 DISCUSSION 1

 11 Phytomers govern plant shoot architecture throughout life time 1.1 Phytomers are the basic units of plant development . . . . . . . . . . . . . . . . . . 1.1.1 Conservation of the phytomer general structure among angiosperm . . . . . 1.1.2 Production of phytomers is ensured by the Shoot Apical Meristem . . . . . . 1.1.3 Control of phytomer production within the Shoot Apical Meristem . . . . . 1.1.4 Modifications of the phytomer structure impact the diversity of plant architectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Floral transition, a developmental switch that remodels phytomers . . . . . . . . . . 1.2.1 Developmental phase changes and architecture remodelling . . . . . . . . . . 1.2.2 Floral transition remodels both new and old phytomers . . . . . . . . . . . . 1.2.3 Regulation of Floral transition . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 The genetic control of flower develoment . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 Specification of a new type of meristem: the flower . . . . . . . . . . . . . . 1.3.2 Proper patterning and termination of flowers are necessary to avoid shoot features in flowers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Developmental regulation of the bract in the reproductive phytomer 2.1 Convergent evolution of bract loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Cryptic bract in bractless species . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Morphological evidences in A. thaliana . . . . . . . . . . . . . . . . . . . . . 2.2.1 Molecular evidences in A. thaliana . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Cryptic bracts in other species . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Basal bract emergence in bractless species: experimental and natural conditions . . ix 2.3.1 Experimental restoration of bracts involved defects in flower development . . 2.3.2 Natural bract development without floral defects . . . . . . . . . . . . . . . 3 Objectives of this thesis References RESULTS 1 Revisiting bract inhibition with natural population of Arabidopsis thaliana 2 Bract formation in Arabidopsis thaliana reveals divergent developmental trajectories at floral transition between two natural accessions 3 Genetic determinants of bract transient formation in a natural accession of Arabidopsis thaliana METHODS AND MATERIALS 1 Plant materials and culture condition 2 Meristem dissections and imaging 3 Plant phenotyping 4 DNA extraction and sequencing for Bulk Segregant Analysis (BSA) 5 Time course RNA-sequencing over flowering 6 Bio-informatic analysis x References Natural population to study plant development 2 Developmental switches at a population level 3 Bract loss: an evo-devo perspective References APPENDIX xi

(

  Chapter 2). In Chapter 2, I also investigated what characterizes the floral transition in Tsu-0 from the reference Col-0, and identified a climax of gene expression divergence at the time of bract emergence. Finally, I assessed the complex genetic regulation of bract emergence in Tsu-0, and mapped its genetic determinants into several main Quantitative Trait Locus (QTLs) in chromosome 1, suitable for further research (Chapter 3). This work allows a better understanding of bract inhibition in A. thaliana, and brings insights into the mechanisms of regulation and evolution of developmental switches. I will introduce my research by describing flowering plant architecture and its remodelling during floral transition, and then review our current knowledge on bract inhibition.

INTRODUCTION 1

 1 Phytomers govern plant shoot architecture throughout life time 1.1 Phytomers are the basic units of plant growth 1.1.1 Conservation of the phytomer general structure among angiosperms

(

  CLV3 ), WUSCHEL (WUS ) and KANADI 1 (KAN1 ) are the major regulators of the maintenance of the CZ (Yadav et al. 2014; Truskina & Vernoux 2018; Carles & J. C. Fletcher 2003).

  primordia initiation mainly rely on hormonal signaling, including auxin and cytokinin (Truskina & Vernoux 2018; Galvan-Ampudia, Chaumeret, et al. 2016; Bhatia & Heisler 2018). 1.1.3 Control of phytomer production by the Shoot Apical Meristem Leaf organogenesis Leaf organogenesis starts with a local and transient auxin maximum.

  Axillary meristems (aM) emerge at the flank of the SAM, at the junction between the adaxial part of the leaf and the growth axis. Position of the aM is ensured by the promotion of WUS activity by adaxial markers such as REV, while abaxial markers like YABBY inhibit WUS (Sarojam et al. 2010). The aM is similar to a SAM, as it will reiterate plant growth in the same fashion (Xin et al. 2017). However, aMs are formed post-embryonically from meristematic cells originating from the SAM, that will later acquire similar SAM features (C. Zhang et al. 2018). The development of axillary branches includes two major steps: initiation and outgrowth (Y. Wang & Jiao 2018). These two steps can be eventually separated in time by a period of dormancy. The localisation of the axillary stem cell niche is tightly regulated and depends on leaf boundary and polarity markers (McConnell & Barton 1998). aM only initiates at the adaxial part of the leaf, and at the boundary region: both REV (adaxial gene) and BLADE-ON-PETIOLE (BOP, boundary gene) regulate aM positioning (Hepworth & Pautot 2015; Q. Wang et al. 2016, see fig.4). Interestingly, ectopic meristems can develop in the leaf margin of compound leaves, that gather boundary and adaxial markers (Hagemann & Gleissberg 1996). Plus, disturbing leaf polarity in Medicago truncatula as well as in A. thaliana can lead to ectopic organ formation on the abaxial (and not adaxial) part of the leaf (McConnell & Barton 1998; Zhou et al.

  et al. 2020, fig.4). Expression of REV at the adaxial part of the leaf (around p10 stages) boosts STM expression, that reaches higher levels and induces the formation of the axillary meristem dome (Shi et al. 2016). WUS and CLV3, are expressed de novo following axillary meristem initiation (Xin et al. 2017) triggered by cytokinin signaling (J. Wang et al. 2017) in a pattern that recapitulates the one found in the SAM (fig.4). However, the aM are regulated by other transcription factors that do not participate to SAM regulation, like LATERAL SUPPRESSOR (LAS ) and REGULATOR OF AXILLARY MERISTEM 1 (RAX1) genes (Xue et al. 2020). The first is expressed in the leaf adaxial part (Yadav et al. 2014), and regulates REV ; while RAX1 is expressed in the boundary regions and is necessary for aM formation (Müller et al. 2006). Interestingly, RAX1 is activated by LEAFY (LFY) transcription factor, a major determinant of floral meristem identity (Chahtane et al. 2013). First, this highlights a common feature between flowers and vegetative aMs, but also explains why aMs grow following floral initiation. Hence, aM initiation strongly relies on the presence of a leaf which provides essential landmarks for the subsequent development of a stem cell niche (fig.4). The outgrowth of aM mainly relies on hormonal signaling (Y. Wang & J. Li 2008) and sugar content (Mason et al. 2014; Rameau et al. 2015), but also on environmental signals. Auxin has been for long associated with bud outgrowth in a process called apical dominance: cutting the plant apex would remove auxin basipetal transport from the SAM, and would therefore induce aM growth. It is not clear how differences in auxin concentrations and timing of actions can lead both to inhibition or the activation of axillary buds (Xue et al. 2020). Nevertheless, auxin acts with different hormones, and through different signal integrators. Auxin regulates cytokinin and strigolactone levels, which respectively activates and inhibits aM outgrowth (Dun et al. 2012). Integration of all hormonal information allows a precise regulation of bud outgrowth (Xue et al. 2020). In addition, sugar content and signaling have been shown to strongly contribute to aM development. Apex decapitation induces rapid redistribution of sugar towards new sinks -the aMs -, and promotes their growth (Barbier et al. 2015). Sugar metabolism with light signaling, are also key players of aM development (Wahl et al. 2013; Leduc et al. 2014). Hence, like flower development which I will discuss later, aM development involves a plethora of pathways, but also an important balance between source-sink status.

  PENNYWISE (PNY ) genes (Bencivenga et al. 2016), which are essential for proper internode elongation. Before flowering, ATH1 acts at the boundary region between stem and meristem and inhibits stem growth probably by promoting Gibberellin (GA) catabolism (Gómez-Mena & Sablowski 2008; L. Zhang, Sun, et al. 2018). We will see later that flowering triggers intense remodelling of the phytomers, including the internodes. In A. thaliana, internodes elongate from the rib zone, under the control of both hormonal and light signals that trigger floral transition (fig.5). GA plays an important role in internode elongation, by triggering cells growth and division. GA level is tightly controlled by photo signaling through photo-receptors (Peng & Harberd 1997; Mazzella et al. 2000). Moreover, flowering signals may inhibit ATH1 locally but also through mobile signals (McKim 2020), derepressing stem growth. In fact, transport of the florigen FLOWERING LOCUS T (FT) has been shown to promote stem elongation (Shalit-Kaneh et al. 2019). Contrary to monocotyledon plants, in dicotyledons like A. thaliana, internode growth follows a basipetal gradient: the highest internodes elongate first. Nevertheless, very little is known about the establishment, maintenance and elongation of the internode, it is noteworthy that internode elongation highly depends on flowering signals (fig.5).

  Vegetative stage thus allows plants to optimize energy storage. During the juvenile stage, plant produce leaves thanks to the activity of the meristems, but they are not competent to flowering signals. The transition to an adult phase is marked by the acquisition of flowering competence, physiological modifications and leaf morphological changes (Yang et al. 2013; Périlleux et al.

Flowering

  is controlled by numerous external and internal factors, including photoperiod, ambient temperature, vernalization, sugars and hormones content, and age (Adrian et al. 2009; Conti 2017; Johansson & Staiger 2015; L. Li et al. 2016). Because different plant species respond differently to those signals, we will only focus on A. thaliana. Floral transition results in the activation of the inflorescence identity within the SAM, and the expression of floral meristem genes in the lateral zone. This is controlled by a complex but well known GRN that integrates the different flowering signals. All signaling pathways lead to the essential integrator FLOWERING LOCUS T (FT ) (Koornneef et al. 1991) and TWEEN SISTER OF FT (TSF ) (A. Yamaguchi et al. 2005), that moves from the leaves to the shoot apex (Corbesier et al. 2007) where it combines with FD (Abe et al. 2005) to activate the floral meristem genes together with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1 ) (fig.9). Mutants of FT, FD or SOC1 are late flowering (Koornneef et al. 1991; Abe et al. 2005; Samach et al. 2000), but can still produce flowers thanks to genetic redundancy. Indeed, several mutations are needed to completely inhibit flowering (Reeves & Coupland 2001), suggesting the evolutionary importance of flowering control. The key integrators of flowering are activated by different partially redundant pathways depending on the flowering signals. Photoperiod, i.e. the period of lighting time within a 24-h period, is captured by both phytochromes and circadian clocks genes. CONSTANS (CO) is expressed on a rhythmical fashion regardless of light input (Yanovsky & Kay 2002). Under dark, CO protein are degraded, while it is stabilized in long days afternoon by light signalling (Soltis et al. 2006). Hence, photoperiod is sensed by the coincidence between light exposure and afternoon, that stabilized CO protein. CO is a transcription factor that activates FT expression (Yoo et al. 2005, fig.9).

  SPL9 and SPL10, especially, induce MIR172 expression that remove SCHLAFMUTZE (SMZ ) -TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1 ) mediated floral transition inhibition (Aukerman suggesting that plants competence to flower is conditioned by an adequate energy storage. In addition to age, sugar and photoperiod, plant flowering is regulated by other factors such as by ambient temperature or cold exposure (vernalization). Although we are not going to detail those pathways, it is noteworthy that their understanding have been greatly enlightened by studies on natural populations of A. thaliana (Lempe et al. 2005; Brachi et al. 2010; L. Zhang & Jiménez-Gómez 2020; Weigel 2012).

1. 3 . 1

 31 Specification of a new type of meristem: the flower The floral meristem initiates in the PZ of the inflorescence shoot apical meristem. This step is extremely studied because flower emergence can be accessible after plant bolting, that makes the dissection easier. Like for leaves during the vegetative phase, the position of the floral meristem is dictated by phyllotaxis that emerges from the dynamical auxin transport from cell to cell (Smith et al. 2006; de Reuille et al. 2006). A local auxin maxima at 137,5°from the i organ precedes the emergence of the i+1 floral primordium. This auxin maximum is transduced to an intracellular signal notably by the action of MONOPTEROS/AUXIN RESPONSE FAC-TOR 5 (MP/ARF5 ), a transcription factor that will modify gene expression (Aida et al. 2002; Reinhardt et al. 2003; Przemeck et al. 1996). Among others, MP up-regulates its own expression and reinforces local convergence of auxin fluxes, creating a positive feed-back loop driving organogenesis (Bhatia & Heisler 2018). MP also stabilizes the plastochron by activating ARA-BIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6 ), a negative regulator of cytokinin signaling. This small protein moves between cells through plasmodesmata, generating an inhibitory field that slows down the activation of neighbouring flower initia, whose sites are already getting specified by auxin accumulation (Besnard et al. 2014; Galvan-Ampudia, Cerutti, et al. 2020). Besides, within the anlagen, MP will also activate both LEAFY (LFY ) and AINTEGU-MENTA (ANT ) genes family, essential for the acquisition of floral meristem identity (N. Yamaguchi, Wu, et al. 2013). Because of its predominant role in establishing flower meristem identity, LFY have been intensively studied. Its main upstream and downstream regulators have been identified (Winter et al. 2015). In addition to MP, LFY is activated by other pathways, involving the DORNRÖSCHEN-LIKE (DRN/DRNL), the PUCHI/BLADE-ON-PETIOLE (BOP ) genes (Chandler & Werr 2017) and by genes involved in the floral transition pathway (Abe et al. 2005). LFY regulates hormonal status within the floral meristem, notably cytokinin, auxin and GA, resulting in a local maximum of cytokinin and auxin, and a local minimum of GA (Denay et al. 2017). Cytokinin and auxin maxima will drive the meristematic identity of the cells, while the minimum of GA will activate DELLA proteins, that together with SPL activate many MADS-box transcription factors (N. Yamaguchi, Winter, et al. 2014). The activation of APETALA 1 (AP1 ), a key MADS-box transcription factor, is also ensured directly by LFY (Wagner et al. 1999). The combined activity of AP1 and its closely related CAULIFLOWER (CAL), and LFY, with other essential MADS-box genes such as AGAMOUS-LIKE 24 (AGL24 ) and SHORT VEGETATIVE PHASE (SVP ), will trigger the acquisition of floral meristem identity (Denay et al. 2017). This is achieved by the inhibition of TERMINAL FLOWER 1 (TFL1 ) that ensures the inflorescence state (Bradley 1997), and the activation of floral identity genes (Kaufmann et al. 2010) (fig. 10). TFL1 inhibition does not affect the SAM, that stays indeterminate, in part thanks to its activation by AGL14 (Pérez-Ruiz et al. 2015). However, some divergence in the activity of these transcription factors is noteworthy. Notably, it has been suggested that LFY and AP1/CAL have antagonistic consequences on TFL1 expression: LFY activates TFL1, while AP1 inhibits it (Goslin et al. 2017; Serrano-Mislata, Goslin, et al. 2017). It is hypothesized that this antagonistic interplay ensures that the SAM remains indeterminate after floral transition (Serrano-Mislata, Goslin, et al. 2017). The aM, on the other hand, are prevented from the flowering signals thanks to the activity of BRANCHED1 (BRC1 ), that will delay floral transition in the buds (Niwa et al. 2013) (fig.10).

1. 3 . 2

 32 Proper patterning and termination of flowers are necessary to avoid shoot features in flowers Once the floral meristem is initiated, a combination of transcription factors ensure the expression of each floral organs (sepals, petals, stamens and carpel) in a sequential mode referred to as the ABC model (Coen & Meyerowitz 1991). Since then, increasing research on A. thaliana but also other species have precised the original ABC model, that can slightly diverge across angiosperms. Briefly, in the ABC model, for each floral organ corresponds a combination of MADS-box transcription factors that give the organ identity (fig.11). The spatial and temporal regulation of each MADS-box gene is however complex and involve a lot of actors (Krizek & J. C. Fletcher 2005). The establishment of boundary between each floral organ is essential for the proper whorls patterning, similarly to leaf and organ development. This is ensured by general "boundary genes" that also act in the leaf boundary establishment such as CUP-SHAPED COTYLEDON 3 (CUC3 ) ( Vroemen et al. 2003), but also genes that are specific to the separation between two whorls. For instance, SUPERMAN ensures the boundary between carpel and stamens and thus the proper number and position of both whorls (Prunet et al. 2017). Hence, production of floral whorls by the floral meristem can remind the production of organs by the SAM or the aM. The overlapping functions of genes in leaves and flowers organs suggest their homology and is in line with the modular/ phytomer-based plant growth. Hence, leaf and floral organs developmental program share similarities, and a misregulation of flower patterning often leads to vegetative branch-like features (fig.11).

  Besides, as most Poaceae and Brassicaceae are annual plants, floral production marks the end of the individual plant life cycle. Bract loss could have resulted from a different partitioning of cell fate within the nascent primordium. It could then be the results of a different sourcesink energy distribution in favor of the reproductive organ. Such mechanism that favors flower growth could have been selected in plants with an annual life cycle. Thus, one evolutionary explanation for bract loss is its hypothetical way to optimize energy distribution in favor of a rapid and numerous flower organs initiation (Chuck et al. 2010; Hodgson et al. 2017; Midgley & Bond 1989). While this remains very hypothetical, understanding the mechanism of bract inhibition could help address the evolutionary significance of bract loss.

  ). Altogether, these phenotypes suggest a role in partitioning cells fate within the phytomer. Moreover, TSH4 is a target of MIR156, a micro-RNA involved in floral transition(Xu, Hu, J. Zhao, et al. 2016) and expressed in the floral meristem, antagonistic to TSH4 expression pattern (fig.15). Sequestration of TSH4 in this boundary region by MIR156 during Spikelet Pair Meristem emergence could hence be responsible for bract inhibition (Chuck et al. 2010). Bract l.s. loss has been studied in other Poaceae species, including barley, wheat, or rice. Different pathways have been involved in bract loss (fig.16). Several of these genes are also involved in floral transition pathways, such as SPL and NECK LEAF 1 (NL1 ) in rice (L. Wang, Ming, et al. 2021; L. Wang, Yin, et al. 2009), TSH4 in maize (Chuck et al. 2010), THIRD OUTER GLUME 1 (TRD1 ) in barley (Houston et al. 2012), or VERNALIZATION PROTEIN 1 (VRN1 ) in wheat (C. Li et al. 2019). Other pathways can be involved in bract suppression in Poaceae, such as leaf initiation (through PLASTOCHRON genes in rice (Kawakatsu, Taramino, et al. 2009; Kawakatsu, Itoh, et al. 2006)), or meristem maintenance (through RIL1 in rice (Ikeda et al. 2019)).In A. thaliana, the homologous of TSH4, SPL9 and SPL15, are still involved in floral transition with MIR156, but do not seem to inhibit bract, as double mutant does not show any bract phenotype[START_REF] Schwarz | The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis[END_REF]).In A. thaliana, several mutants bearing "bracts" (s.s., l.s or fpp) have been described in the literature. The pathways involved here mostly refer to the acquisition of flower identity or the specification of floral organs. Hence, the mutants also show defects of the flower identity itself. Indeed, 35S:LFY(Weigel & Nilsson 1995), UNUSUAL FLOWER (UFO) KO mutants (ufo) (Hepworth, Klenz, et al. 2006), BLADE-ON-PETIOLE (BOP) KO mutants (bop1, bop2 ) (Hepworth 2005), PUCHI KO mutants (puchi1 ) (Karim et al. 2009), JAGGED dominant (jag-5D) (Dinneny 2004; Ohno 2004), agl6 OE (Koo et al. 2010) are described as having "bracts", even though the nature of the leaf is difficult to address, as flower identity is itself strongly impaired (fig.17, and fig.16). However, mutants in floral transition pathways can also lead to bract emergence, as it is the case for agl24, svp, soc1 (Liu et al. 2009), ft (Müller-Xing et al.

  Nilsson et al. After specific destruction of floral cells, using a diphteria toxin A under the control of the LEAFY promoter, bract-like structures replaced flowers, suggesting that absence of flower removes bract inhibition (Nilsson et al. 1998). Still, disturbing floral transition pathways, in A.thaliana, can trigger bract emergence. In fact, fd mutants plants with pFD::MIR156 construct produced more branches, and present flowers with "bracts", but with inflorescence defects (J.-W.Wang et al. 2009)

  Xu, Hu, McKim, et al. 2010). Mutants converting flower whorls into leaf-like shape have been already described in the literature[START_REF] Bowman | Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes[END_REF][START_REF] Bowman | a regulator of floral homeotic genes in Arabidopsis[END_REF]), which highlights the leaf homology of the floral organs. Still, such leaves do not correspond to bracts s.s., as they are produced by the floral meristem and not subtending it within the phytomer module. Besides, some genes of the ABC family participate to the establishment of bracts fpp (S.[START_REF] Kim | Sequence and Expression Studies of A-, B-, and E-Class MADS-Box Homologues in Eupomatia (Eupomatiaceae): Support for the Bracteate Origin of the Calyptra[END_REF]. Taken together, these data suggest that bracts described in the literature can have several origins: those resulting from a modification of floral whorls (fpp), From 1995, Hempel and Feldman observed that WT plants can bear bract on their first flowers and they call the corresponding phytomers as chimeric ones. The frequency of chimeric phytomers has been shown to increase when plants were exposed to a strong continuous light condition(Hempel, Zambryski, et al. 1998. Time lapse dissection of vegetative meristem shows that the first flower is produced immediately after the transfer to a flower-inductive condition, and does not bear bract(Hempel & Feldman 1994; Hempel & Feldman 1995). So they concluded that bract-flowers are the result of a conversion into flower of aM initiated before the floral induction. The phytomer would keep a "developmental bias" with a vegetative identity, hence keeping a leaf as branches do, but the fate of the aM could still be reoriented to a floral identity(Hempel, Zambryski, et al. 1998). To support their hypothesis of a branch-to-flower conversion, they report the frequent occurrence of floral indetermination, with chimeric shootflower phenotypes (14% of bracts-flowers have chimeric shoot-flower phenotypes)(Hempel & Feldman 1995; Hempel, Zambryski, et al. 1998). The frequency of shoot-flower phenotypes increased when plants were exposed to a strong light condition (up to 50% of plants with chimeric shoot-flower phenotype). Their results extend the relationship between bract inhibition and flower identity to wild type population. Nevertheless, observing bracts on the first flowers of WT plants is frequent. In A. thaliana, a number of natural accessions display basal bracts on the first flowers of the raceme, without being exposed to a particular intense light condition (personal observation), questioning how widespread such chimeric phytomers are.2.3.2 Natural bract development without floral defectsAbsence of bract is a specificity of Brassicaceae. However, upon closer inspection, it appears that some genera of Brassicaceae present bracts, sometimes in the entire inflorescence (in Onuris clade, Salariato et al. 2015) but mostly only in the lowermost flowers of the inflorescence (see Results, Chapter 1). The latter is very common and can easily be observed in Europe. In addition, basal flowers with bracts lack visible floral defects (personal observations, see Results -Chapter 1).In this introduction, I have reviewed how the concept of phytomer can enlighten the diversity of angiosperm architecture, both between species and along the different plant developmental stages. In other terms, the study of phytomer can be useful to unravel the species-specific determinants of plant architecture. It also addresses the control of plant developmental plasticity through the phytomer remodeling that accompanies developmental transitions. Understanding the mechanisms controlling this essential module are thus of tremendous importance in plant biology. However, our knowledge is very unbalanced: the literature is abundant on flower development, but much less is known about the remodelling affecting internodes or leaves. The leaf however undergoes exceptional remodelling at floral transition in A. thaliana, as it does not grow. Attempts to study bract inhibition have relied on mutants or natural accessions bearing bracts. Such bract-flowers have been observed in various environmental and genetic contexts.Several pathways may converge to this phenotype, triggered by one or a combination of different factors such as a specific environment, a disrupted developmental switch, or a specific genetic background. Mechanisms leading to bract inhibition are not always related to flower identity, as it can been deduced from the observation of wild type plants. This genetic context can thus help to isolate the mechanisms of bract loss, in order to better understand the developmental and evolutionary pathway(s) leading to bract loss.3 Objectives of this thesisUnlike flowers, bract development has received very little attention. Yet, convergent bract loss with the maintenance of a cryptic form in several angiosperms constitute a relevant biological context to address trait loss during evolution. How do different species converge to loose bracts? And why do distantly related species maintain a cryptic bract (cb)? To date, studies on bract inhibition relied on mutants producing ectopic bracts in bractless species. However, such mutants also display malformed flowers with indeterminate shoot-like phenotype. Do the corresponding genes really participate to bract inhibition, or do the presence of bract on these mutants indirectly result from the indeterminate branch-like flower? Hempel and Feldman have addressed the question of bract loss by studying wild-type plants under specific conditions. Still, the bract-flowers observed here resulted from a photo-induced conversion of branches into flowers with frequent branch-like flower phenotypes. Thus, it was commonly accepted that bracts and flowers development are not compatible in A. thaliana. Nevertheless, botanists have for long reported bract-flowers in basal flowers of Brassicaceae inflorescences, including A. thaliana. In fact, upon close inspection, it appears that several populations of A. thaliana present bract-flowers at the base of the inflorescence, that are not affected by indeterminate branch-like phenotype, like Tsu-0. What are the mechanisms of bract inhibition at floral transition? And how can bract re-emerge specifically at floral transition? Thanks to the increasing affordability of next generation sequencing technologies, it is now possible to newly question the mechanisms of bract loss in A. thaliana. I used the natural accession Tsu-0 as a genetic tool to revisit the genetic and developmental mechanisms underlying bract inhibition at floral transition in A. thaliana. I first explored the bract-flowers phenotype in Tsu-0 as well as in other accessions and different Brassicaceae species (Results -Chapter 1). I also compared bract-flower in Tsu-0 from those described in the literature. Because bractflowers affected the first flowers produced at floral transition, I tried to capture the differences in the GRN of floral transition between Tsu-0 and Col-0, and how theses differences could explain the bract-flower phenotype of Tsu-0 plants (Results -Chapter 2). I finally mapped the genetic basis of bract-flower phenotype. By coupling genomic and transcriptomic approaches, I tried to reduce the list of relevant genetic candidates in the mapped interval, that would be 21. Chahtane, H. et al. A variant of LEAFY reveals its capacity to stimulate meristem development by inducing RAX1. en. The Plant Journal 74, 678-689. issn: 1365-313X (2013). 22. Chandler, J. & Werr, W. DORNRÖSCHEN, DORNRÖSCHEN-LIKE, and PUCHI redundantly control floral meristem identity and organ initiation in Arabidopsis. en. Journal of Experimental Botany 68, 3457-3472. issn: 0022-0957, 1460-2431 (June 2017). 23. Chuck, G., Whipple, C., Jackson, D. & Hake, S. The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries. en. Development 137, 1585-1585. issn: 0950-1991, 1477-9129 (May 2010). 24. Coen, E. S. & Meyerowitz, E. M. The war of the whorls: genetic interactions controlling flower development. en. Nature 353, 31-37. issn: 0028-0836, 1476-4687 (Sept. 1991). 25. Conti, L. Hormonal control of the floral transition: Can one catch them all? en. Developmental Biology 430, 288-301. issn: 00121606 (Oct. 2017). 26. Corbesier, L. et al. FT Protein Movement Contributes to Long-Distance Signaling in Floral Induction of Arabidopsis. en. Science 316. Publisher: American Association for the Advancement of Science Section: Report, 1030-1033. issn: 0036-8075, 1095-9203 (May 2007). 27. Coudert, Y. et al. Three ancient hormonal cues co-ordinate shoot branching in a moss. en. eLife 4, e06808. issn: 2050-084X (Mar. 2015). 28. Denay, G., Chahtane, H., Tichtinsky, G. & Parcy, F. A flower is born: an update on Arabidopsis floral meristem formation. en. Current Opinion in Plant Biology 35, 15-22. issn: 13695266 (Feb. 2017). 29. De Reuille, P. B. et al. Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis. en. PLANT BIOLOGY, 6 (2006). 30. Dinneny, J. R. The role of JAGGED in shaping lateral organs. en. Development 131, 1101-1110. issn: 0950-1991, 1477-9129 (Mar. 2004). 92. Ohno, C. K. The Arabidopsis JAGGED gene encodes a zinc finger protein that promotes leaf tissue development. en. Development 131, 1111-1122. issn: 0950-1991, 1477-9129 (Mar. 2004). 93. Peng, J. & Harberd, N. P. Gibberellin Deficiency and Response Mutations Suppress the Stem Elongation Phenotype of Phytochrome-Deficient Mutants of Arabidopsis. en. Plant Physiology 113, 1051-1058. issn: 1532-2548, 0032-0889 (Apr. 1997). 94. Pérez-Ruiz, R. V. et al. 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IntroductionA

  plethora of studies investigated the mechanisms of flower development. However, in the model plant A. thaliana, flower development is also accompanied with the inhibition of its subtending leaf: the bract. Unlike flower, little is known about the mechanisms of bract development. Bract suppression happened independently in different clades, but to what extend they share common features remains elusive. Some mutants bearing bracts have been generated in A. thaliana, as well as in maize or rice, but did not allow to really understand bract loss. In Arabidopsis thaliana, mutants bearing bracts display pleiotropic phenotypes that also affect flower development. Flower impairment in these mutants made it difficult to uncouple the mechanisms of bract loss from flower emergence. Bract emergence has also been observed in wild type plants, when exposed to a continuous and strong light condition ((Hempel et al., 1998)). In this context, some bract-flowers displayed signs of indeterminate shoot-flower phenotypes: the authors suggested it resulted from a conversion of branches into flowers. So far, no study has focused on phenotypes where bract inhibition was uncoupled from a proper flower development. However, botanists are for long familiar with the presence of bracts in some Brassicaceae species, under normally developing flowers often located at the basis of inflorescence. Hence, in some Brassicaceae, flowers can develop normally despite the presence of bract. In this chapter, we describe the existence of bract-flowers in some natural populations of the model plant A. thaliana. We propose this developmental context as a new standard to understand the mechanism of bract loss and its evolutionary origin.

  transition, determinate floral meristems are produced, which stop the growth axis, impacting angiosperm's architecture ((Prenner et al., 2009)). In addition, in some groups like Brassicaceae, including the model plant A. thaliana, floral transition leads to bract inhibition. The study of mutants bearing "bracts" allowed to identify several genes involved in bract inhibition ((Dinneny, 2004; Ohno, 2004; Norberg, 2005; Penin, 2008)): lfy ((Weigel and Nilsson, 1995)), ufo ((Hepworth et al., 2006)), puchi ((Karim et al., 2009)), bop ((Hepworth, 2005)), jagged ((Dinneny, 2004; Ohno, 2004)). However, those mutants displayed pleiotropic phenotypes, especially with defects in flower development. Observing that proper bract and flower developments are somehow incompatible in A. thaliana, it has been suggested that the mechanism of bract inhibition is tightly coupled to the flower developmental program.

  and for canonical flowering genes such as AP1 or LFY in Tsu-0 and Col-0. Hence, acquisition of floral identity is highly conserved between the two natural accessions. Besides, bract-flowers are impacted neither by varying photoperiodic conditions, contrary to previous observations ((Hempel et al., 1998)), nor by the plastochron. The Tsu-0 accession offers a robust genetic context in which bract inhibition is compromised, at least during the short time window of floral transition. Like Tsu-0, a number of other natural accessions bear bracts on their first flowers (see Results -Chapter 1). To what extent bract-flowers in other natural accessions is governed by the same mechanisms as in Tsu-0 is still an open question. Of course, the question is even more relevant at the levels of Brassicaceae, or of Angiosperms. The main specificity of bracts in wild type A. thaliana plants is their position, exclusively restricted to the first flowers. Bract-flowers have not been observed after the fourth flower in Tsu-0. This indicates the existence of a particular context during floral transition that perturb bract inhibition in Tsu-0. To study floral transition, we identified four developmental stages that have identical meristem morphology. Transcriptome profiling over floral transition in Tsu-0 and Col-0 reveals insightful characteristics of Tsu-O's floral transition. First and foremost, the four developmental stages very well depicted the floral transition process in both accessions. Despite their different sampling time, they display a similar global profile. This is expected from two natural genetic backgrounds within the same species: the gene regulatory network that triggers floral transition is conserved. Notably, as described previously in different species ((Lemmon et al., 2016)), the T stage, that corresponds to the emergence of the first flowers, is characterized by a climax of gene expression change when compared to other stages. Most of the differential gene expression between Col-0 and Tsu-0 happened during T stage. This higher transcriptomic change at T stage in Tsu-0 could maybe favor a faster adaptation to flowering signals: the transcriptome associated to flowering is reached before the visible production of flower. How far these characteristics can be correlated with basal bract production needs to be addressed by adding new natural accessions transcriptomes at floral transition. Interestingly, our result at the level of two A. thaliana natural accessions recapitulates previous study of floral transition in nightshades (Lemmon et al., 2016).

  represented 110 genes. One of them is ARF1, a repressive transcription factor mediating the auxin transcriptional responses (Tiwari et al. (2003)), that present a splice donor variant at c.1163G>C, and thus could become an interesting candidate gene to test. Scenario 3: miRNA misregulation in Tsu-0 during floral transition Because miRNA play a major role in floral transition, we next considered all the miRNA present in the mapping intervals. On the 24 miRNAs present in the mapping region, none of them have known function in floral transition or flower determinism. Still, they could highlight unexpected roles in bract inhibition.

Figure 3 -

 3 Figure 3-Figure supplement 1. Relation between the flowering time and the number of branches in a set of RILs, Tsu-0 and Col-0. Globally, the number of branches increase with flowering time

  F1 and F2 plants used for the BSA were generated by crossing the parental lines Tsu-0 and Col-0 on both sides. ufo-1 seeds came from the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info) (N16361, Col-2 background), bop-1, bop-2, and puchi-1, bop-1, bop-2 were published in Karim et al. 2009 (Col-0 background), lfy-12 was published in Maizel & Weigel 2004 (Col-0 background). Tarenaya hassleriana seeds were kindly provided by Pr Eric Schranz and Frank Becker, from Wageningen University (WUR, Holland). Allaria petiolata were found in the wild in Massy.

3. 1

 1 Macroscopic imagingMacroscopic pictures in Chapter 1, Fig.1 H, I were taken using the Keyence VHX-900F according to the manufacturer's instructions. Pictures in Chapter 1, Fig. A, C, D, F and G were taken by the Canon EOS 450D camera, according to the manufacturer instructions. The camera device of a Samsung Galaxy A10 was used to take the pictures in Chapter 1, Fig.1 B and E.

with

  Variant Stabilizing Transformation (VST) function (Anders & Huber 2010), to harmonize the variance. To check the consistency between biological replicates, a Principal Component Analysis on all samples with VST transformed data was generated using the same package. Biological triplicates of all conditions were very close to each other, allowing relevant comparisons per developmental stages and genotypes. Differential analysis was made using the R Bioconductor package edgeR (McCarthy et al. 2012). Reads were first normalized using TMM (Trimmed mean of M-values) to reduce libraryspecific biases. Normalization factors were between 0.94 and 1.049 in our samples, showing that few genes were over represented in one sample compared to another. A generalized linear model was applied for the analysis of differentially expressed genes (DEG). 2 types of DEG were performed in this study: DEG at each stage between the different genotypes, and DEG

  toujours me tirer vers le haut.

	List of abbreviations used RAX : REGULATOR OF AXILLARY MERISTEM
	REV : REVOLUTA AGL24 : AGAMOUS LIKE 24 RIL: Recombinant Inbred Line AHP6 : ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 RZ : Rib Zone aM : Axillary Meristem SAM : Shoot Apical Meristem ANT : AINTEGUMENTA SNP : Single Nucleotide Polymorphism AP1 : APETALA1 SOC1 : SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ARF : Auxin Response factor SPL : SQUAMOSA PROMOTER BINDING PROTEIN AS1 : ASYMETRIC LEAVES 1 STM : SHOOT MERISTEMLESS A. thaliana : Arabidopsis thaliana SVP : SHORT VEGETATIVE PHASE ATH1 : ARABIDOPSIS THALIANA HOMEOBAX GENE1 T. hassleriana : Tarenaya hassleriana BOP : BLADE-ON-PETIOLE TFL1 : TERMINAL FLOWER 1 BRC1 : BRANCHED1 TSF : TWIN SISTER OF FT CAL : CAULIFLOWER TSH4 : TASSELSHEATH4
	CB : Cryptic Bract Tsu-0 : Tsushima-0 ecotype WUS : WUSCHEL
	CC : Creative Common CLV3 : CLAVATA 3 YAB : YABBY
	CO : CONSTANS
	Col-0 : Columbia-0 ecotype
	CUC : CUP-SHAPED COTYLEDON
	CZ : Central Zone
	DRNL : DORNRÖSCHEN-LIKE
	FIL : FILAMENTOUS FLOWER
	FMs: Floral meristem
	FPP : Flower preceding prophyll, equivalent to bract fpp.
	FSB : Flower Subtending Bract, equivalent to bract stricto sensu
	FT: FLOWERING LOCUS T
	GRN : Gene Regulatory Network
	GA : Gibberellic Acid GWAS: Genome Wide Association Study
	HIF: Heterogeneous Inbred Family
	IM: Inflorescence shoot apical Meristem
	KAN1 : KANADI 1
	KNAT1 : Knotted-like from Arabidopsis thaliana 1
	KNOX : Knotted-like homeobox
	LAS : LATERAL SUPPRESSOR
	LFY : LEAFY
	LOF : LATERAL ORGAN FUSION
	MIR : Micro RNA
	MP : MONOPTEROS
	NL1 : NECK LEAF 1
	OE : Over Expressor
	PNY : PENNYWISE
	PZ : Peripheral Zone
	QTL : Quantitative Trait Locus
	RA2 : RAMOSA 2
	ii iii viii

ix

Table of contents

Remerciements

Acknowledgments

We thank José Jimenez Gomez for interesting advice. We thank Pr Eric Schranz and Frank Becker, from Wageningen University (WUR, Holland) for kindly providing the Tarenaya hassleriana seeds.

We are very grateful to our culture team for taking great care of the plants. The research leading to these results received funding from the phD program of ENS Paris-Saclay.

Acknowledgments

We thank the HELIXIO platform for carrying the RNA-sequencing despite important stock shortages due to the COVID-19 pandemic. We thank Hicham Chahtane for kindly providing seeds of the lfy-12 mutants ; Géraldine Brunoud and Carlos Galvan for cloning advices ; and Claire Lionnet for helping with the imaging. A deep thank to Florian Ingels for his help with the plastochron script analysis.

We are very grateful to our culture team for taking great care of the plants. S. D.'s PhD position was funded by the phD program of ENS Paris-Saclay. This work was supported by an INRAE grant (BAP departement, project PHYTOCHIMERA 2019). This manuscript has been written in Overleaf, using the eLife free template.

Acknowledgments

We thank the BGI platform for carrying the DNA sequencing. We thank José Jimenez Gomez for interesting insights on BSA and RILs analysis. We are very grateful to our culture team for taking great care of the plants. The research leading to these results received funding from the phD program of ENS Paris-Saclay. This work has also been supported by an INRAE grant for young researchers (BAP departement, project PHYTOCHIMERA 2019). This manuscript has been written in Overleaf, using the eLife free template.

Authors contributions

S.D. carried out all the experiments and the analysis. S.D. and F.B. designed the experiments and interpreted the results. S.D. wrote the first draft of the manuscript, and F.B. helped edit it.

will bring new insights on bract inhibition in A. thaliana.

Methods and Materials

Please refer to the general section "Methods and Materials".

Authors contributions

S.D. carried out all the experiments. S.D. and F.B. designed the experiments, proposed the computer analysis, analyzed and interpreted the results. B.J. and F.B. carried out the RNA-sequencing analysis. S.D. wrote the first draft of the manuscript, and F.B. helped edit it.

Genetic determinants of bract transient formation in a natural accession of Arabidopsis thaliana

Sana Dieudonné 1 , Stéphanie Hallet 1 , Fabrice Besnard 1* *For correspondence: fabrice.besnard@ens-lyon.fr ( ) Present address: † Reproduction et Développement des Plantes, ENS de Lyon, France 1 Reproduction et Développement des Plantes, ENS de Lyon, France

Abstract

While the model plant Arabidopsis thaliana has lost bracts under its flower, we identified a natural accession, Tsu-0, that frequently bears bracts but only on its first flowers produced at the basis of flowering branches. We therefore used this accession, in association with the bractless reference Col-0 accession, to map the genetic determinants controlling the formation of "basal" bracts that appear at floral transition. By using both bulked F2 segregant populations and Recombinants Inbred Lines, we established that the genetic determinism of this trait is complex and located in two major loci in chromosome 1. We then crossed evidence from different datasets to select the best candidates within the mapping region. These includes the effects of natural polymorphisms, the dynamics of gene expression over floral transition and expression data from bract mutants.

We propose interesting candidate genes that could give possible insight on the mechanisms and evolution of bract loss in A. thaliana.

The transcriptomic data allowed us to explore the genes within the mapping region with a reading criterion. However, it did not allow to unbiasedly detect the candidate gene responsible for bracts in Tsu-0, that would need further analysis. Taken together, the data mining approach we used allows only to highlight some interesting candidates that could deserve more attention. For example, a rapid confirmation of the importance of a candidate variation can be investigated by phenotyping natural accessions possessing the same variation. This work is ongoing.

We identified several QTLs that may be linked with basal bract formation in Tsu-0. The identification of the precise gene(s) controlling this process will need more investigations. In fact, several characteristics of this trait may explain this challenge: the phenotype is subtle (a few cells in a restricted time-window) harnessing its direct targeting (microscopy, RNAseq), it is also polygenic (potentially several genes with single limited effect) and a bit stochastic (high variability in an isogenic population grown in the same culture condition) . In addition, the drawback of working with natural variation is the high load of polymorphisms, increasing the number of potential candidate genomic variations even in small genetic intervals. Adding transcriptomic data did not bypass this challenge. Hopefully, we can make a better use of our RNAseq data using more elaborated analysis to detect hetechronic shifts and infer modification in gene networks linked to functional developmental modules (see chapter 2). But this chapter illustrates how key is the initial precision of the mapping, to concentrate the effort of cross-data mining on a smaller number of candidates. To this end, we are currently establishing NILs populations to reduce the length of the QTLs.

This study highlight the remaining challenge to identify natural variations responsible for subtle phenotyes. Nevertheless, tackling the challenge will be extremely useful to benefits all the opportunities of natural variations in developmental and evolutionary biology.

Authors contributions

Meristem dissection and imaging

Meristem dissection

Meristems were dissected from A. thaliana plants after 20 days of synchronization in SD conditions, at different times after transfer to LD depending on the experiment. Dissections have all been performed from 9:00 a.m and noon, by alternating between Tsu-0 and Col-0. When dissected after bolting (for pictures in Chapter 1, Fig. 3B, and Chapter 2, Fig. 3), plant stems were cut at the base of the inflorescence and put in 2% agarose medium plates: flowers were removed with thin tweezers under a binocular loupe (Leica M125). When dissected from the rosette, before bolting, plants were completely removed from the soil, washed carefully with water, and big leaves were removed with thin tweezers. Then, remaining rosette axis and roots were carefully planted in a 5% agarose medium, and the smallest leaves were removed percentage of branch with bract was defined as the average percentage of branch presenting at least one bract, per plant. Flowering time was measured in number of days after transferring plants in growth chambers when the first flower blooms (open petals).

4 DNA extraction and sequencing for Bulk Segregant Analysis (BSA)

The mapping population has been generated by crossing (Col-0 x Tsu-0 ) at both sides. As no difference in bract frequency has been observed (data not shown), F1 plants coming from both side of the cross have been used. For practical reasons, the bulk segregant analysis have been split into four replicates. After stratification at 4°C for 2-3 days, plants have been grown 20 days in SD, then transferred in LD. A 1cm² leaf have been sampled for each F2 individuals at around 10 leaves rosette stage, and kept at -20°C for DNA extraction when the plant was selected in one of the bulk. To re-sequence the genome of the parental lines, DNA was extracted from a bulk of Tsu-0 and Col-0 seedlings respectively. All DNA was extracted and purified using a CTAB-based protocol (cetyl trimethylammonium bromide), following instructions as in [START_REF] Healey | Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species[END_REF]. To prepare each bulks, purified DNA from individual samples were then pooled in order to limit the variation of representation of each plants in the bulk sequencing.

The final concentration was between 13 and 25 ng/µl. Pooled DNA bulks and parental DNA were then sent to BGI for library preparation and sequencing on BGISEQ-500WGS sequencing system to generate 5 Go data of 100bp paired-end reads per library (target coverage of 40X).

5 Time course RNA-sequencing over flowering

Tissue sampling and RNA extraction

Tsu-0 and Col-0 meristems were dissected every day in LD condition after 20 days in SD, in order to capture the right developmental stages. The mutants lfy-12, puchi1, bop1, bop2, and jag5-D meristems have been dissected every days starting from 1 week after LD transfer, because only the F stage was targeted. Three different independent replicates have been used.

The number of meristems in each replicates can be find in the Appendix.

Morphological stages have been first established on a blank experiment, where Col-0 and

Tsu-0 were dissected every days to monitor morphological clues happening before floral transition. The V stage has been defined as the starting stage, and dissected at the days plants were transferred to LD, so that plants have not experienced any LD condition at the time of the dissection. The L stage was sampled the same day for both Col-0 and Tsu-0 as they had the same meristem shape. The T stage has been defined as the stage in which the first flower emerges (the emergence of a round (rather that triangular) primordium can be guessed), and the emergence of axillary meristem starts to become visible at the axils of young leaf primordia.

The F stage has been defined as the stage in which the flowers are clearly recognizable, with the differentiation of the first whorls on the first flower. For the mutants, the F stage was defined when several rounded primordia becomes visible at the SAM (that will become branch-like flowers, or only branch). Note that for the leafy mutant, homozygous lfy-12 plants were easily distinguishable from a WT SAM (like in Chapter 1, fig. 3C).

Meristem dissections were performed as described previously, with the difference that as soon as the meristems were dissected, they were put on micro tubes corresponding to the right stage into liquid nitrogen and stored at -80°C until RNA extraction. The speed of meristem dissection have been optimized to avoid the induction of stress-related gene expression: meristem dissection did not exceed 3min between the first organ removal and liquid nitrogen disposal.

Meristem tissues were grounded with a RockyII tissue lyser according to the manufacturer's instructions. RNA extraction of each sample was performed with the PicoPure RNA Isolation Kit (Arcturus, Catalog KIT0202) according to the protocol. The final RNA concentration of each samples can be found in the Appendix. RNA concentration ranged from 3 to 64 ng/µl (average 23ng/µl), with a RIN value comprised between 5,6 and 7,6 (average 6,8).

RNA-sequencing

The RNA sequencing was performed by Helixio company. Library preparation was made using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs); NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs); and NEB-Next® Multiplex Oligos for Illumina (Index Primers Sets 1, 2 et 3), from 40ng of ARN, in order to obtain amplified cDNA. cDNA was sequenced using NextSeq500 from Illumina. Sequence quality was controlled using the Sequencing Analysis Viewer and sequences that did not pass the quality filter were removed. Remaining reads were on average 43 million sequences per sample.

6 Bio-informatic analysis 6.1 Computing environment R (v3+ or v4+) (R Core Team 2013) was used for several bio-informatic analysis using specific R libraries and packages, as described below. Most plots were drawn using ggplot2 package (Wickham 2016). This file was used to select a list of specific SNPs and small indels of Tsu-0 (91AV stock) versus Col-0 (186AV stock), filtering for positions with coverage metric DP>10. This reference list of Tsu-0 polymorphisms was then used as the -dbsnp option in a second pass of joint genotyping using all four gVCF as input (two parental samples plus the two bulks) to emit a common vcf file. Finally, relevant polymorphic positions from the reference list in the two bulks were selected after filtering for a depth >= 3, and the resulting single vcf was converted to a table file (GATK's tool VariantsToTable), giving for each polymorphic site the global depth and the precise depth of each parental allele in the four samples. Subsequent analysis of read ratio for BSA was performed using QTLseqr (see below)

DNA sequencing analysis and genomic variant analysis

6.3 QTL identification from Bulk Segregant Analysis using QTLseqR QTL identification were carried using the QTLseqr package (Mansfeld & Grumet 2018) according to the instructions. After filtering data with the following parameters (refAlleleFreq = 0, minTotalDepth= 10, maxTotalDepth = 90, minSampleDepth = 10, minGQ= 99, depthDifference = 15), the deltaSNP index [START_REF] Takagi | QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations[END_REF]) was generated and loci that reached the 95% confidence interval were retained and formed the Ia, Ib, Va and Vb QTLs.

QTL identification using Recombinant Imbred Lines analysis

RILs Col-0 x Tsu-0, F7 generation, were publicly available in the Versailles Stock Centre (http://publiclines.versailles.inra.fr/rils/index), as well as their genotyping data for 79 markers throughout the genome (including 19 within chromosome 1). The RILs analyzed were initially selected depending on their known genotypes within chromosome 1 and 5 using GGT 2.0 software (van Berloo 2008). The detailed genotypes of each line are available in supplemental data. We counted their bract frequency as described above. The correlation between genotype and phenotype was done using R/qtl software according to the instructions Broman et al. 2019, with the following modifications on parameters: number of permutations was set up at 1000; for all the tests, error prob was set at 1x10-5. We also used gCIM software Zhang et al. 2020 to compare the two approaches. For the gCIM analysis, the following parameters were used: analysis was performed in the RIL mode, using random effects and a walk speed of 6 cM.

Interaction between bract mean and percentage of plants with at least a bract on the main stem were assessed with R/qtl using MQM models according to the instructions.

RNA-sequencing analysis

Sequenced reads were filtered based on their quality using Trimmomatic software (Trimmomatic).

The percentage of reads that did not satisfy the filter criteria is less than 4% for the thirtythree samples. Filtered reads were then aligned to the reference genome (TAIR10) using STAR [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]) with the following parameters: non canonical splice junctions were removed, multi-mapping reads was limited to 10, and only reads mapped once were considered to determine splicing junctions. A post-alignment quality check was performed, assessing the dicating that most the meaningful variations of the dataset is driven by these DynG. We then restricted our next analysis to this subset. To group genes with similar dynamics irrespective of their absolute read number in the data (because differences in CPM values between genes are not functionally relevant), we choose to use a k-mean clustering on vegetative-stage normalized CPM values. To normalize CPM values in each accession, we substracted for each gene at each stage the expression value at stage V (thus, normalized expression at stage V is 0 for all genes). K-mean clustering was performed using kmeans function from the 'stats' R core package with the following parameters: centers=3 and nstart=100. The number of clusters ('centers') was estimated from heuristic exploration of visual plots (drawn with 'fviz-cluster') and mathematical estimation using 'fviz-nbclust' and 'fviz-gap-stat' functions (last three functions from factoextra package). We used agriGO (v2.0, Tian et al. 2017) to detect Go term enrichments in each of these clusters with the Singular Enrichment Analysis (SEA) tool against the appropriate pre-calculated background (Arabidopsis genemodel TAIR9) and automatic default parameters (Fisher or Hypergeometric test, Yekutielli adjustment, significance level=0.05, minimum number of mapping entries=5).

Gene selection inside genetic mapping intervals

The R library 'GenomicFeatures' [START_REF] Lawrence | Software for Computing and Annotating Genomic Ranges[END_REF]) was used to import gene information from the most recent annotation file of A. thaliana at the gff format [START_REF] Cheng | Araport11: a complete reannotation of the Arabidopsis thaliana reference genome[END_REF].

Custom R scripts were used to intersect all gene loci falling within genetic mapping data. For each gene, custom scripts were used to import the results from the RNA-seq analysis (differential expression, dynamics, etc.) in a single table. In addition, we aggregated data on polymorphisms discovered from the variant analysis described above. We used snpEff (v5.0d, Cingolani et al.

2012

) to predict the functional impacts of each of the genomic variant in the genetic mapping intervals. We next classified the different predicted impacts based on the variant ontology term attributed by snpEff, to define different categories (see classification of these ontology terms in the Appendix): all variations, variations with a putative functional effects that can be further split into HIGH/MODERATE/LOW/MODIFIER effects. Using custom scripts, we summed up the number of variations of different categories in the ORF and promoter (2 kbp upstream of the transcriptional start site) of each gene. For miRNA genes, we also consider DISCUSSION "Les mots ne désignent pas, ils questionnent. A chacun de nous, chaque mot, chaque fois, redemande son sens. Et nous lui donnons chaque fois un sens nouveau" Jean-René Huguenin Bract designates a leaf that subtends a flower. It is also often used in a broader sense to define any leaf appearing in reproductive parts of the plant: flowers or inflorescence, as it is the case for Poaceae. In this manuscript, we are only using bracts in its stricto sensu sense, i.e, as the leaf subtending the flower. Still, studies on bract loss in Poaceae are interesting regarding the mechanisms that control the suppression of leaf development (whatever organ this leaf is subtending). Because the basic composition of the phytomer is extremely well conserved among plant kingdom, addressing bract loss brings insights on both plant development and architecture evolution.

1 Natural population to study plant development

Advantages and drawbacks of natural variation studies

Many modifications of plant architecture roots at variations affecting the phytomer. Such variations can be observed in laboratory-generated mutants or in natural populations. Mutant screens are able to reveal severe phenotypes that could certainly not be maintained in natural conditions. The strong impact of the EMS mutagenesis often generates null alleles of genes, offering a direct way to investigate their function. Moreover, mutant approaches allow to focus on a single gene, avoiding uncontrolled epistasy except for carefully chosen other genes in controlled crosses. by BSA and RILs approaches, and because of the numerous genomic variations differentiating Tsu-0 from Col-0, it was hardly possible to came up with a single confident gene name. To face this challenge, we are considering different strategies. We are currently establishing HIFs from the heterozygous RILs that will allow to isolate one of the targeted QTL, as the genotype of the outside region will be identical. We hope this could not only precise the implication of each QTLs, but also produce recombinants that reduce the length of the QTL. Complementary, and because basal bracts are common in natural populations, we consider studying a broad sample of natural accessions to carry a GWAS study. This approach is both complementary to confirm at a larger extend the implication of a QTL, but can also spots new QTLs involved in basal bracts in other populations. GWAS study can thus address the convergence or divergence of basal bract phenotype within the scope of natural populations of A. thaliana.

Using both mutants and natural variations approaches to question evolution and development

To complement natural population approaches, we used mutants bearing bracts to help understand basal bract emergence in Tsu-0. Several pathways have been involved in the lack of bract inhibition in both A. thaliana and Poaceae. Interestingly, two main genetic pathways can be retained: floral identity genes pathways represented by mutants like lfy-12 (

2010) (see Introduction).

In Tsu-0, the absence of any floral defects, as well as the climax of gene expression divergence at T stage, suggest a floral transition-related genetic pathway. Hence the SPL genes can be interesting to investigate. Notably, SPL10 is located between the QTL Iα and the QTL Ia, and present a deletion in its promoter, and two SNP in the 5'UTR and 3'UTR regions. MIR157D is also located between the QTL Ib and Iβ, and present SNP on its coding sequence (but not the conserved target binding region). Plus, SPL10 is over expressed at T stage in Tsu-0, but appeared "older" than Col-0 ones, while they were closer before and after (at L and F stage). This result was surprising regarding the existence of bract in Tsu-0, because the presence of a leaf in a phytomer could be associated instead to a "more juvenile" (or "younger") stage. These data suggest that basal bract emergence does not result from a delay in the acquisition of floral identity, as confirmed also by the expression of LFY in flowers that do bear bracts. This reinforces the idea that flower identity and bract development can happen simultaneously in certain context. the timing of the ecdyzosteroid peak is associated with an important difference in insect life cycle (Truman & Riddiford 1999). In Solanaceae, the timing of certain gene expression is associated with an important architecture remodelling (Lemmon et al. 2016). Our RNA-seq analysis enlightens for the first time the existence of such heterchronic shifts in the GRN of FT at a population scale. Whether this is responsible for bract emergence is a question that still needs to be investigated. Increasing the number of bract-bearing versus non bract-bearing natural accessions could help answer the question. It makes no doubt that the increasing affordability of RNA-sequencing and analysis will facilitate such investigation. Thus, the study of the trancriptome associated with FT in a large panel of natural accessions will help to better understand the types of transcriptomic remodelling related to architecture divergence at a population scale. In addition, we could shift the expression of some candidate genes in Col-0 transgenic plants by putting them under the control of earlier/later expressed promoter, to see whether this might affect the presence of basal bract. To this end, we would need a better understanding on the genes that are shifted in Tsu-0 compared to Col-0. Recently, Calderwood et al., in the group of R. Morris, developed a curve registration method to better investigate time course transcriptomes [START_REF] Calderwood | Comparative transcriptomics reveals desynchronisation of gene expression during the floral transition between Arabidopsis and Brassica rapa cultivars[END_REF]. This method allows to study the complex multidimensional transformations between two global time courses and to identify meaningful underlying differences. We are currently building a collaboration with R. Morris and R. Kristianingsih in order to make the most out of our RNAseq data.

Evolution of gene expression at floral transition is observed at a population level

Developmental switches: perfect times for the emergence of phenotypic variations?

Developmental switches enable plasticity in both animal and plant life cycle, through the intense phenotypic remodelling it triggers. The contrasted phenotypes before and after the switch often rely on a different set of genes. Developmental switches can be considered as a complete rewiring of the GRN operating in the tissues of interest. But this rewiring must also be tightly regulated to avoid the emergence of detrimental phenotypes. Investigating developmental switches at different scales is indispensable to understand how such a transition self-organizes. Interestingly, our work reveal that the "control" can be loose, because transient "chimeric" phenotype can appear in some situations. So why is FT favorable to the emergence of new transient phenotypes?

Transient phenotypes could arise from a combination of atypical gene expression levels.

While transitioning from one state to the other, many genes are inevitably up or down-regulated. We could imagine that some genes with normally antagonistic effects would maintain intermediate expression levels for a short period of time, generating "chimeric" phenotypes. Such situation would not be stable in neither states of the transcriptome before and after the developmental switch, because of genetic feed back loops, and epigenetic marks. If the time to switch from one GRN to another is long relative to the time required to generate a phenotype, a third "transient" developmental state can be defined, which does produce macroscopic phenotypes.

Reciprocal inhibition of antagonistic genetic modules made of several genes can involve a complex network of interactions between genes. We could then imagine that transient phenotypes are promoted by small modifications of genetic interactions in the network which delays the reciprocal inhibition, thereby extending the time window of the transient transcriptomic state. Another possible explanation for transient phenotype could root at the dynamics of epigenetic marks during the GRN remodeling. In fact, Müller-Xing et al., showed the existence of a time window during which floral reversion can happen, in some Polycomb group mutants (Müller-Xing et al. 2014). It appears that the rewiring of a new GRN at developmental switch involved two main phases. First, after signal transduction, new gene regulations are established involving down regulation of genes important for the earlier stage, and up-regulation of genes important for the next stage. In a second time, epigenetic marks "locks" on the new regulatory network, which can persist despite the absence of the initial trigger (flowering signals). This is supposed to be at the origin of the absence of vegetative reversion, even though flowering signals may become nonexistent. We did not investigate the epigenetic landscape of Tsu-0 and Col-0 during floral transition. Yet, it would be interesting to monitor the speed at which new epigenetic marks are deposited, such as repressive marks on "bract/leaf" genes. A delay in the deposition of such marks could be responsible for the absence of bract inhibition at the first flowers. The abrupt transition observed in Tsu-0 transcriptome could then prevent from the rapid establishment of epigenetic marks.

This study presents a body of evidence that Tsu-0 's bracts are not the persistence of a vegetative signature, but instead result from a disturbed remodeling of GRN at floral transition.

Bracts and flowers development are thus ontogenetically compatible, probably because they both belong to a much older developmental unit: the phytomer.

3 Bract loss: an evo-devo perspective 3.1 Is leaf a dispensable part of the phytomer?

The three elements of a phytomer (internode, leaf, meristem), are extremely well conserved in the plant kingdom. Despite a huge modification of their morphology, the fundamental composition of the phytomer has stayed identical through million years of plant diversification. This conserved modular unit may dictate strong developmental constraints. Leaves are the first organ to be formed in a phytomer. The establishment of leaf polarity and proper boundaries with the meristem are crucial to the formation of the meristem at its axil. This could explain the maintenance of a cryptic bract domain in species that do not present a complete bract development. Bracts, and any leaf, could be seen as an organizing center for the formation of the meristem. Several data can support this hypothesis: the presence of ectopic meristems at the leaf margin of tomato compound leaf (that includes both boundary and polarity molecular Bract, and to a broader extent leaf loss appear independently in several clades, from which some are major plant models. Interestingly, both Paceaeand Arabidopsis have an annual life cycle, so the apparition of flowers also sign plant death. In other species, a lesser selection on photosynthetic performance could have instead allow the acquisition of new function, like protection, or attraction. In annual and pollinator-free plants, bract loss could have allow a better reallocation of energy into flower development, rather than leaf growth. This would not be surprising as the role of the source-sink status in plant architecture, notably in bud outgrowth, appear crucial (Barbier et al. 2015). Studying the role of strigolactone and sugar metabolism in the context of the flower versus bract development, as it was in the context of axillary bud versus stem and leaf, would need to be considered.

To have a first look at the relative growth of bract and flower in both Col-0 and Tsu-0, it would be necessary to live-image meristems at the moment of first flower growth. Then, we could compute the relative growth of the flower versus (cryptic) bract: in Tsu-0, we can expect (cryptic) bract to grow faster in the first flower, or floral meristem to grow slower. The delicate rosette dissection, as well as the short timing in which the first flower can be captured, made this experiment highly challenging. It appears then interesting to manipulate bract/flower growth rate in transgenic Col-0 and Tsu-0 lines, whether by using hormonal signaling, or cell cycle arrest. A reduction of bract frequency when increasing flower growth rate, and the other way around, would argue for a important role of the flower-bract resource competition. Such role can be rapidly investigated using non-model Brassicaceae species that bears bract on the lower half part of the inflorescence, because bract-flowers would then be easier to catch. A comparison of relative growth between (cryptic) bract and flower in such species, versus in species were bracts are totally absent would be interesting. This file contains the list of the SnpEff effects and their definition. It specify the one that were not considered for the establishment of the CGset2 (ie. the filter "unlikely post-transcriptionnal effect").

Appendix 7: List of genomic variants between Col-0 and Tsu-0.

This vcf file contains the list of the variants that were identified between Col-0 and Tsu-0 after re-sequencing analysis.