Marie Delattre

Marie Monniaux

Catherine Kidner

Enrico Magnani

Samuel Knosp

Moritz Nowack

Norbert Bollier

Sophy Boeuf

Audrey Creff

Yen-Ting T Lu

Erica De Leau

Hinda Garance

Laurine Léa

Lise

Léo

J Loue-Manifel

A Euzet-Rodriguez

E Berenguer Peinado

Victor Girard

J Goodrich

G Ingram

N Dépège-Fargeix

J Just

S Grey-Switzman

M Erhardt

A Hoguin

F Simon

ANOVA test: ANalysis Of Variance test

Keywords: Triphosphate)-binding cassette ABCG: ATP-binding cassette subfamily Clustered Regularly Interspaced micropylar endosperm Mm, µm: milimeters, micrometers Nuclear Localization Sequence nm: nanometer NMR: Nuclear Magnetic Resonnance Spectroscopy PUTATIVE ASPARTATIC PIPES, EGTA, Magnesium

Chez les Angiospermes, le développement de la graine et l'accumulation des réserves imposent la mise en place d'une communication étroite entre l'embryon et l'albumen. Chez certaines Angiospermes, comme l'arabette des dames aussi appelée Arabidopsis thaliana, ce dialogue entre embryon et albumen est régulé par un complexe protéique composé de deux facteurs de transcription : ZHOUPI et INDUCER OF CBF EXPRESSION1 (ZOU/ICE). Plus précisément, ce complexe ZOU/ICE permet la dégradation de l'albumen et donc le bon développement de l'embryon et régule également la mise en place d'une interface complexe entre l'albumen et l'embryon composée d'une gaine extracuticulaire et de la cuticule embryonnaire. Dans une première partie, l'analyse de la régulation et de la fonction du complexe ZOU/ICE a permis de mettre en évidence une redondance fonctionnelle cryptique entre les gènes ICEs au cours du processus de développement de la graine mais aussi la régulation post traductionnelle de ces protéines ICEs, en particulier l'ubiquitination participant à leur dégradation par l'action de la protéine HOS1. Dans un deuxième temps, des approches de génétique, de biologie moléculaire et cellulaire chez la Bryophyte Marchantia polymorpha, dépourvue de graines et d'albumen, ont permis de mieux comprendre la fonction ancestrale du complexe ZOU/ICE. Ce complexe s'est révélé avoir un rôle clé d'une part dans le contrôle du développement des rhizoïdes, impliqués dans le transport de l'eau, et d'autre part dans la biogénèse de la cuticule. L'étude de la composition de la cuticule de M. polymorpha, et des cibles transcriptionnelles du complexe ZOU/ICE impliquées dans sa mise en place, ont permis d'émettre des hypothèses novatrices sur l'importance de la modification de la composition de la paroi, et la production des espèces réactives de l'oxygène dans la biogenèse de la cuticule des plantes.

1 Land plant evolution and key innovations: from the green algae to the angiosperms

Description of the different groups of plants

Colonization of the terrestrial environment by plants was one of the key transitions that made the Earth the unique planet that we inhabit today. Land plants emerged from the green algae, one of the oldest eukaryotic lineage. They are complex multicellular algae which exist as haploid individuals. Although, as in land plants, diploid cells (zygotes) are formed through the fusion of haploid cells (gametes) in charophyte algae, they generally undergo meiosis without prior cell division [START_REF] Delwiche | The Evolutionary Origin of a Terrestrial Flora[END_REF]. Land plants, otherwise known as embryophytes, emerged from aquatic environments around 470 million years ago (Mya) [START_REF] Kenrick | The origin and early evolution of plants on land[END_REF]. As their name suggests, they undergo embryogenesis, which is the developmental process through which the zygote (the produce of gamete fusion) becomes a multicellular embryo [START_REF] Rensing | Why) Does Evolution Favour Embryogenesis?[END_REF]. The embryophyte life cycle thus alternates between multicellular haploid (gametophyte) and multicellular diploid (sporophyte) phases.

Embryophytes are also characterised by the production of a hydrophobic, protective cuticle, that covers their surface, and is thought to have been a key innovation in conquering the terrestrial environment.

The group of living embryophytes that first evolved on land was the bryophytes (liverworts, mosses and hornworts), which have a haploid-dominant life cycle and are classified as "non-vascular" plants. Until recently, early land plant relationships were very uncertain, and because of their relative morphological complexity liverworts were assumed to be the "ancestral" group giving rise to all other embryophytes, probably because early fossilised spores are most similar to those of liverworts. However, the recent work of [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF] strongly supports that the three groups forming the bryophytes (liverworts, mosses and hornworts) are monophyletic and thus all share the same common ancestor with the tracheophytes (vascular plants) (Figure 1). The simplicity of some liverworts is likely to reflect loss of structural diversity during the course of evolution.

The tracheophytes are thought to have arisen about 420 Mya, and are characterized by the presence of a lignified vasculature with identifiable xylem and phloem [START_REF] Steemans | Origin and radiation of the earliest vascular land plants[END_REF]. Approximately at the same time, plants developed a new body part: the root (Figure 1).

In contrast to tracheophytes, bryophytes do not possess efficient sap-conducting vessels allowing them to transport solutes over long distances, nor specialized multicellular structures capable of extracting nutrients and water from the soil (see Introduction, § 2.2).

Tracheophytes are composed of three groups, the lycophytes which were the first extant tracheophyte group to emerge, the monilophytes which include ferns and horsetails and the Spermatophytes (seed plants) which is the most recent group to have emerged (Figure 1). A lack of vascular tissue in the bryophytes is thought to have limited plant size. However

Lycophytes, equipped with a vascular system were the dominant group during the Carboniferous period (300 Mya-360 Mya) and could grew into massive 30 meters high trees, which dominated the landscape [START_REF] Kenrick | The origin and early evolution of plants on land[END_REF]. Spermatophytes, which include gymnosperms and angiosperms appeared around 360 Mya (Figure 1). They are characterized by their ability to produce seeds and are the last group of plants which appeared on Earth [START_REF] Kenrick | The origin and early evolution of plants on land[END_REF]. The first gymnosperms appeared at the end of the Carboniferous period 360 Mya and reached their peak in the Jurassic period, at the beginning of the Cretaceous period , before slowly declining during the angiosperm radiation.

Angiosperms are thought to have first appeared about 125 Mya, and in addition to seeds, possess a further reproductive innovation: the flower [START_REF] Friis | Cretaceous angiosperm flowers: Innovation and evolution in plant reproduction[END_REF] (Figure 1).

Plant Life cycles: the rise of the sporophyte

A characteristic shared by all land plants is the alternation between multicellular haploid and diploid phases. This contrasts with the situation in most charophyte algae where the zygote is formed of only one diploid cell [START_REF] Bowman | Evolution in the Cycles of Life[END_REF][START_REF] Niklas | The evolutionary development of plant body plans[END_REF], although a similar situation is observed in other lineages, such as brown algae.

Although the earliest land plants, like extant bryophytes, almost certainly had a haploid (gametophyte dominant) life cycle, the course of plant evolutionary history of plants, has been marked by a reduction in the dominance of the gametophytic phase (both in terms of size and life span) in favour of the sporophytic phase. Thus, in bryophytes, the gametophyte is dominant and autotrophic, whereas the existence of the sporophyte generation is reduced to a very short time window, and sporophytes are almost always nutritionally dependant on the gametophyte.

The emergence of the tracheophytes marked the inversion of this dominance (Figure 1) [START_REF] Friedman | One Genome, Two Ontogenies[END_REF][START_REF] Graham | The origin of plants: Body plan changes contributing to a major evolutionary radiation[END_REF].

Two land plant models and their life cycles: Marchantia polymorpha and Arabidopsis thaliana

What is a model plant?

The term model plant was coined in the nineteen fifties, at the very beginning of the rise of A. thaliana as a key plant model. A. thaliana coexisted with other key models such as Antirrhinum (Anthirrhinum majus), Petunia (Petunia x hybrida) and Maize (Zea mays), which had been extensively studied over decades, and for which impressive banks of mutants (usually induced by the activity of endogenous transposons) and natural accessions were available [START_REF] Meyerowitz | Prehistory and history of Arabidopsis research[END_REF][START_REF] Somerville | A fortunate choice: the history of Arabidopsis as a model plant[END_REF]. A. thaliana rose to prominence however, due to its relatively short life cycle, small size, and ease of propagation. In addition, likely in part because it doesn't have a large endogenous transposon population, A. thaliana also has a small and quite simple genome (135 Mb). However, the point that underlay the truly meteoric rise of A. thaliana as a model plant, and which still makes it incredibly useful today, is its ease of Agrobacterium-mediated transformation and the associated capacity to generate both transgenic plants and stable mutants. A. thaliana as a model system for plant development was initiated in 1943 and became accepted by the scientific community in the eighties because of the different features which make an organism a convenient model to study [START_REF] Meinke | The preservation of plant genetic resources. Experiences with Arabidopsis[END_REF]. Numerous mutants are available, such as ethyl methane-sulfonate (EMS) mutants [START_REF] Qu | Generation and Identification of Arabidopsis EMS Mutants[END_REF] and T-DNA insertion mutants (Qu and Qin, 2014b) in several stock centers such as the Arabidopsis Biological Resource Center (ABRC) or the Nottingham Arabidopsis Stock Center (NASC) and can also be very easily produced using CRISPR-Cas9 technology (Lee et al., 2018b). Chosen from an outset of plants with small genome size, A. thaliana is also the first plant organism with a fully sequenced genome published in 2000 [START_REF] Meyerowitz | Prehistory and history of Arabidopsis research[END_REF][START_REF] Somerville | A fortunate choice: the history of Arabidopsis as a model plant[END_REF]The Arabidopsis Genome Initiative, 2000).

Since the A. thaliana whole-genome sequencing in 2000, many plant genomes have been sequenced. However during almost 10 years most plant genomes sequenced were angiosperm's and only in 2008 was the first bryophyte genome (Physcomitrella patens) sequenced, [START_REF] Rensing | The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants[END_REF]. Perhaps because the P. patens genome was the first to be sequenced this raised P. paten to become the main bryophyte model plant. M. polymorpha was a very popular model plant in the early 1900s but fell into oblivion until the beginning of 21 st century when genomic and genetic tools started to be available for plant to study evolution and development (Evo-Devo) [START_REF] Ishizaki | Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology[END_REF][START_REF] Ishizaki | Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha[END_REF][START_REF] Sugano | CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L[END_REF]. Although the complete chloroplast genome of M. polymorpha was one of the first sequenced [START_REF] Ohyama | Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA[END_REF], the whole genome took much longer to be sequenced. M. polymorpha was eventually chosen for the community sequencing program at the Joint Genome Institute, Department of Energy, USA and the first version was published in 2017 [START_REF] Bowman | Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome[END_REF].

Since then, newer versions have appeared [START_REF] Montgomery | Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin[END_REF] and gene annotations have been upgraded. CRISPR-Cas9 technology [START_REF] Sugano | CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L[END_REF] and homologous recombination [START_REF] Ishizaki | Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha[END_REF] has been developed, and sporeling and thallus can be transformed using Agrobacterium with very high efficiency [START_REF] Ishizaki | Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology[END_REF]Takenaka et al., 2000). The M. polymorpha genome is relatively small (approximately 280Mb), its life cycle is quite short and the plants are very easy to propagate (see below) and cross which are very good reason to use M. polymorpha as a model plant to study Evo-Devo (Bowman et al., 2016b[START_REF] Bowman | Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. Compared to other transformable Bryophytes such as the moss Physcomitrella patens [START_REF] Schaefer | Efficient gene targeting in the moss Physcomitrel la patens[END_REF] or the hornwort Anthoceros agrestis [START_REF] Frangedakis | An Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis[END_REF], M. polymorpha has fewer gene duplications facilitating genetic analysis. However, many biological processes in this species appear to have been "simplified" over the course of evolution (Bowman et al., 2016b).

Finally, it is important to bear in mind that whatever the model plant, neither A. thaliana or M. polymorpha are fully representative of their respective family or clade.

In the following paragraphs, I present the two plant models which have been used during my PhD: Arabidopsis thaliana and Marchantia polymorpha.

M. polymorpha.

M. polymorpha belongs to the liverwort order in the bryophyte clade and is thought to have emerged about 450Mya. As for other bryophytes, the autotrophic plant body of M. polymorpha is produced in the multicellular haploid gametophyte generation. The sporophyte is very highly reduced (more so than in many mosses for example), is only present for a very short window of time and is composed of a single sporangium. The gametophyte initiates development either from a unicellular haploid spore, the result of sexual reproduction followed by meiosis within the sporangium, or through vegetative propagation via the production of haploid multicellular propagules called gemmae (singular: gemma) which represent a form of asexual reproduction [START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF] (Figure 2).

Gemmae are produced in "gemma cups" which are bowl-shaped structures on the dorsal surface of the leaf-like structures formed by the adult gametophyte, the thalli (singular: thallus). They appear once in each bifurcation of the thallus. They are formed from single cells at the base of the cup which undergo repeated round of mitosis before detaching from the base of the cup and being pushed upwards by the developing gemmae beneath. More than a hundred gemmae can be produced by a single gemma cup [START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. Gemmae, which measure at maturity 1 to 2 mm, have a discoid shape and two apical notches (which have latent) meristematic activity are situated at opposite points on the extremity of the disk.

Mature gemmae do not show dorso-ventral differentiation, and are dormant whilst in the cup.

However once gemmae have been dispersed, a process that involves their propulsion from the thallus surface by rain splashes [START_REF] Brodie | The splash-cup dispersal mechanism in plants[END_REF], they will "germinate" upon contact with the ground. This process involves the rapid acquisition of dorso-ventral polarity, visible as the ventral development of smooth rhizoids from rhizoid precursor cells, which are initially present on both surfaces of the dormant gemma [START_REF] Kato | Gemma cup and gemma development in Marchantia polymorpha[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF][START_REF] Tarén | Factors Regulating the Initial Development of Gemmae in Marchantia polymorpha[END_REF].

Rhizoids are unicellular filament cells which are found on the ventral surface of the thallus either growing perpendicular to the thallus, the smooth rhizoids, or growing parallel to the thallus, the pegged rhizoids (see Introduction, §2-2) [START_REF] Jones | The evolution of root hairs and rhizoids[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF].

M. polymorpha is a dioecious plant producing male and female reproductive organs on different individuals. Sex is determined by the transmission of heteromorphic sex chromosomes, X in female individuals and Y in males [START_REF] Ishizaki | Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha[END_REF][START_REF] Okada | Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. Upon induction by environmental stimuli (long days and far red light), thalli develop umbrella-like structures: gametophores called respectively antheridiophores or archenoniophores in male and female individuals (Figure 2). The stalks of these reproductive structures are a prolongation of the thallus and form an inrolled structure. Archegoniophores are composed of a stalk topped by a receptacle that is divided into finger-like projections (Figure 2). A row of eggs, each surrounded by gametophytic tissues, forms between each finger [START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. Antheridiophores have a similar morphology but with a flatter receptacle that is undivided and produces antheridia containing bi-flagellated mobile sperm (Figure 2). On contact with a drop of water, the antheridiophore receptacle releases the sperm which will find its way (by swimming and/or capillarity) to the eggs. Once a sperm has reached an egg, fertilization takes place, resulting in the development of a zygote that then divides mitotically to form a sporophyte embryo. The embryo develops surrounded by a maternal gametophytic tissue divided into three parts (Figure 2). The most inner is the calypthra, a protective structure covering the sporophyte, which is itself gathered by the pseudoperianth, which is itself gathered with the other developing embryos, surrounded by the involucre, a fluffy and transparent protective structure (Figure 2). At the junction between the sporophyte and the gametophyte, specialized transfer cells with wall ingrowth develop forming a nutrient transfer zone called the placenta [START_REF] Ligrone | The Gametophyte-Sporophyte Junction in Land Plants[END_REF]. When the embryo becomes a mature sporophyte, it is retained under the receptacle of the archegoniophore, and is composed of three diploid tissues. The foot, forms a proximal diploid interface between the maternal gametophore tissues and a second structure, the seta. The seta elongates as a stalk which suspends and then pushes the sporangium out of the involucre. The sporangium is the third and most distal tissue structure of the sporophyte. The sporangium produces two cell types: elaters (elongated sporophytic cells that die and become very hygroscopic, and which are responsible of the dispersal of the spores) and spores, the products of meiosis (Figure 2).

Once dispersed, spores germinate by producing smooth rhizoids and form a new thallus, closing the loop/completing the life cycle [START_REF] O'hanlon | Germination of Spores and Early Stages in Development of Gametophyte of Marchantia polymorpha[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF].

It is thought that, although ancestral Bryophytes could probably produce stomata (air pores formed within the epidermis of most Embryophytes), this capacity was lost in liverworts [START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF][START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF]. Instead M. polymorpha produces stomata-like structures, called air pores, which play a similar role to that the stomata in permitting gas exchange but with no movement and less effective than stomata. They are composed of stack of four cells which form the "chimney" of the air pores. Beneath this chimney, an air chamber is present in which multiple photosynthetic filament cells are visible [START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF].

Finally, another feature of M. polymorpha (but also Bryophytes and Lycophytes) is the absence of true meristems [START_REF] Harrison | Development and genetics in the evolution of land plant body plans[END_REF]. The meristem-like is composed of only one cell, the apical cell and thalli grow from a region of very small cells called the apical notch.

Apical notches multiply as thalli expand and bifurcate [START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF].

A. thaliana.

A. thaliana belongs to the taxonomic family of the Brassicae in the eudicotyledonous group of angiosperm vascular plants. It is not a crop species, but belongs to the same family as rapeseed, cabbages, turnips, mustards etc… making it a very interesting model plant.

In contrast to M. polymorpha, but like most vascular plants, the dominant autotrophic part of the A. thaliana life cycle is produced in the multicellular diploid sporophyte generation, with the production of vegetative structures (in the case of A. thaliana a rosette of leaves), and reproductive structures (in the case of A. thaliana an erect inflorescence). As noted above, the A. thaliana life cycle is short, and plants can go from seed to seed in 3 months, or even less, under laboratory conditions. As in M. polymorpha, the A. thaliana reproductive phase is triggered by appropriate environmental conditions. The main plant body of A. thaliana is the rosette of leaves which at the appropriate time produce the inflorescence, a long stem where at the top of it, flowers are produced (Figure 2). The A. thaliana inflorescence bears flowers that are hermaphrodite and composed two external whorls of sterile organs (four sepals and 4 white petals), and two inner whorls of sexual organs comprising 6 stamens (male organs) and one pistil composed of two fused carpels (female organs) (Figure 2). The stamens and carpels produce very small multicellular gametophytes (Krämer, 2015).

The female gametophytes, also called embryo sacs (Figure 2), are produced in ovules that emerge from the internal carpel walls. Each carpel can produce 20 to 30 ovules (meaning that up to 60 ovules can be produced per flower). Within each ovule primordium, a specific cell called the megaspore mother cell undergo meiosis to give four megaspores, of which three degenerate and one undergoes limited mitosis to give the female gametophyte. Each female gametophyte is surrounded by maternal sporophytic tissues (the integuments and nucellus) and is composed of four specialized cell types: the egg cell (haploid) and the central cell (containing two haploid nuclei), two haploid synergids flanking the egg cell, and necessary for fertilization, and three haploid antipodal cells of unclear function (Figure 2). As in all angiosperms, both the egg cell and the central cell are fertilization competent and can thus be considered as female gametes (Krämer, 2015).

On the male side, the anther (at the tip of the stamen) produces male gametophytes in the form of pollens grain (Figure 2). Each pollen grain is derived from a microspore generated by meiotic divisions occurring within the developing anther. Post meiosis, each microspore develops a thick protective coat called the pollen wall. Each undergoes one round of mitosis to give a large vegetative and a smaller generative cell, that is engulfed by the vegetative cell.

The generative cell then undergoes a further round of division to give two sperm cells (Krämer, 2015).

Angiosperms, and thus A. thaliana, are characterized by a unique double fertilization event, in which the two sperm cells, delivered to the ovules by the pollen tube after pollen grain arrival and germination on the receptive carpel surface, fuse with the egg cell and the central cell of the female gametophyte. This event triggers seed development, which will be described in detail in section I-4 of the Introduction.

2 The move to land: key innovations

Life on land

During their emergence onto land, the ancestors of land plants were confronted with a dramatically new environment that imposed severe constraints on their growth and development. In order to adapt, they innovated, both morphologically and physiologically One of the most major challenges faced by land-colonizing plants was the low humidity of the Earth's atmosphere. This may have been an underlying factor in changes as profound as the transition from a gametophyte dominant to a sporophyte dominant life cycle, since unlike the dispersal of motile male gametes, the dissemination of spores and of seeds is not dependent on water [START_REF] Delaux | Molecular and biochemical aspects of plant terrestrialization[END_REF]. The development of a waxy, hydrophobic cuticle on the plant surface also became very important to prevent desiccation, and may have gone hand in hand with the evolution of air pores and ultimately stomata, which became necessary for the controlled exchange of gases at the surface of photosynthetic organs. In addition to preventing desiccation, the cuticle probably also protected plants against UV radiation and pathogen attack [START_REF] Yeats | The formation and function of plant cuticles[END_REF][START_REF] Ziv | Multifunctional Roles of Plant Cuticle During Plant-Pathogen Interactions[END_REF]. Cuticle structure, development and functions are explained further in section 2.3 of the Introduction. Another adaptation necessary for land life was the production of phenolic polymers (such as lignin and suberin) which, similarly to the cuticle (which contains the largely aliphatic polymer cutin), are used to form barriers against pathogen attack and spread, particularly upon wounding [START_REF] Serrano | The cuticle and plant defense to pathogens[END_REF][START_REF] Ziv | Multifunctional Roles of Plant Cuticle During Plant-Pathogen Interactions[END_REF] but also contribute to the structural properties of many land plants through the formation of the vascular tissue.

Water is critical to all forms of life, but particularly so to plants, whose tissues grow though the generation of intracellular hydrostatic pressure, and whose energy production depends upon water-cleaving photosynthetic reactions. Water can move through plant tissues by diffusion and/or by capillarity, the later occurring mainly within the fibrous structures provided by plant cell walls, and both being constrained by physical parameters including gravity. Thus, in situations where water is obtained from the substrate (as on land), neither diffusion nor capillarity can provide sufficient water to support large plant body plans. Vascular tissues, facilitating the rapid long-distance movement of water through plant tissues, were therefore a critical innovation underlying the colonization of land by plants. Their formation depended upon hydrophobic polymers, particularly lignin, which reinforces the cell walls of specialized cell types called xylem vessels. Xylem vessels develop as continuous cell files, and ultimately die, forming "pipes". The reinforcement and "waterproofing" of the cell walls of these structures through the inclusion of lignin within their cell walls gives them unique properties, including the capacity to resist negative pressures within the water columns that they can maintain intact thanks to their water-resistant properties. The negative pressure in these columns is caused by their being dragged upwards by controlled evaporation from the mesophyll cells of aerial organs in a process called transpiration [START_REF] Růžička | Xylem developmentfrom the cradle to the grave[END_REF].

The maintenance of efficient water movement is dependent upon two other factors, efficient water uptake from the soil, and a mechanism allowing the control of water evaporation from aerial surfaces. The former is provided, in vascular plants, by roots, and the later, by the combination of the waterproof cuticle, and the acquisition of stomata which act as valves to control the transpiration stream. Stomatal opening thus promotes the transport of both water and nutrients from the root to the shoot in the transpiration stream. Roots also participate in the anchorage of the plants to the substrate and the uptake of nutrients and water [START_REF] Raven | Selection pressures on stomatal evolution[END_REF].

As mentioned in the previous section, stomata are not restricted to tracheophytes. Some bryophytes (mosses and hornworts) also develop stomata but only on their sporophyte (which is not their dominant life-cycle phase) where they contribute to its dehiscence [START_REF] Chater | Origins and Evolution of Stomatal Development1[OPEN[END_REF][START_REF] Duckett | Exploding a myth: the capsule dehiscence mechanism and the function of pseudostomata in Sphagnum[END_REF][START_REF] Pressel | Hornwort stomata do not respond actively to exogenous and environmental cues[END_REF][START_REF] Rich | Plant Evolution: When Arabidopsis Is More Ancestral Than Marchantia[END_REF]. Many Bryophytes also develop air pores, which serve the same function as stomata, by permitting gas exchange between underlying mesophyll cells and the environment. However, in contrast to stomata, and despite some structural similarities the apertures of these structures do not open or close [START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF].

Bryophytes do not produce roots but developed functionally analogous structures called rhizoids which are uni (liverworts) or multi (mosses) cellular [START_REF] Delaux | Molecular and biochemical aspects of plant terrestrialization[END_REF][START_REF] Jones | The evolution of root hairs and rhizoids[END_REF][START_REF] Kenrick | The origin and early evolution of roots[END_REF]. Early during land plant evolution, the acquisition of mineral nutrients, and indeed water, by these root-like structures is thought to have been dramatically increased by the development of symbioses with microorganisms present in the soil. These interactions appear to have been critical for terrestrialization [START_REF] Delaux | Molecular and biochemical aspects of plant terrestrialization[END_REF][START_REF] Delaux | Reconstructing trait evolution in plant evo-devo studies[END_REF][START_REF] Humphreys | Mutualistic mycorrhiza-like symbiosis in the most ancient group of land plants[END_REF].

Rhizoids: the 2 in 1 structure of liverworts, which combine the uptake of nutrients and water and their transport

Once they had arrived on the land, plants were not fully submerged by water and nutrients and thus developed novel adaptations to uptake water and nutrients and transport them to the whole plant body: rhizoids, roots and vasculature. [START_REF] Kenrick | The origin and early evolution of roots[END_REF] define the root as "a multicellular organ characterized by special features including gravitropic response, endogenous branching, root hairs, and a protective root cap". Roots are thought to have evolved independently in several clades between 416 to 360 million years ago, and in combination with efficient vascular systems, were clearly a major factor in allowing plant life to flourish [START_REF] Hetherington | Networks of highly branched stigmarian rootlets developed on the first giant trees[END_REF][START_REF] Kenrick | The origin and early evolution of roots[END_REF][START_REF] Strullu-Derrien | The origin and evolution of mycorrhizal symbioses: from palaeomycology to phylogenomics[END_REF].

Rhizoids, roots and vasculature: three important innovations

Roots "sensu stricto" as seen, for example in the model plant A. thaliana, are therefore composed of three main structures : primary roots, lateral roots (the meristems of which are protected by the root cap evoked above) and root-hairs [START_REF] Hopmans | Current Capabilities and Future Needs of Root Water and Nutrient Uptake Modeling[END_REF]. Roothairs are tubular cell extensions which grow out from the epidermal cells of primary and lateral roots [START_REF] Scheres | Root Development. Arab. Book[END_REF]. By increasing the effective root surface area, root hairs are critical factors in water and nutrient uptake.

Bryophytes did not evolve roots, but instead they developed rhizoids [START_REF] Carol | Building a hair: tip growth in Arabidopsis thaliana root hairs[END_REF][START_REF] Pressel | Cellular Differentiation in Moss Protonemata: A Morphological and Experimental Study[END_REF]. Rhizoids are not restricted to land plants they also developed on the surface of the gametophyte of some aquatic streptophyte algae such as Chara [START_REF] Lewis | Green algae and the origin of land plants[END_REF] where they are either multicellular or unicellular. In the early land plants with a gametophyte dominant phase, rhizoids are unicellular in liverworts (except for Haplomitrium [START_REF] Bartholomew-Began | A morphogenetic re-evaluation of Haplomitrium Nees -(Hepatophyta) (Schweizerbart'sche Verlagsbuchhandlung)[END_REF] and hornworts, but are multicellular and can be highly branched in mosses [START_REF] Jones | The evolution of root hairs and rhizoids[END_REF][START_REF] Motte | Molecular and Environmental Regulation of Root Development[END_REF][START_REF] Tam | Conserved regulatory mechanism controls the development of cells with rooting functions in land plants[END_REF].

In the liverwort M. polymorpha, two types of rhizoids can be distinguished: pegged rhizoids and smooth rhizoids (Figure 3). Smooth rhizoids are living cells with a diameter of 18 to 24 µm for maximum 30 mm length which grow perpendicular to the thallus (gametophytic photosynthetic tissue) and appear to function mainly to anchor plants to the substrate and it seems that they could also participate to water transport [START_REF] Clee | The Morphology and Anatomy of Fegatella conica in relation to the Mechanism of Absorption and Conduction of Water[END_REF][START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF][START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. In contrast, pegged rhizoids are dead cells at maturity, and show extensive cell wall thickening, including the production of internal cell wall extensions called pegs which may prevent the cell body from collapsing [START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF].

They generally grow parallel to the thallus all along the midrib and they tend to be narrower ( 6 to 24 µm of diameter) and shorter (8 to 20 mm) than smooth rhizoids [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF].

In mature thallus, they form bundles enclosed by flaps of thallus tissue called ventral scales.

Interestingly not all liverworts produce both types of rhizoids [START_REF] Jones | The evolution of root hairs and rhizoids[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF], pegged rhizoids are restricted to complex thalloid liverwort suggesting they are an important evolutionary innovation [START_REF] Villarreal | Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate[END_REF]. Pegged rhizoids are almost undistinguishable from smooth rhizoids before peg development but after peg development and subsequent programmed cell death, they become very easily distinguishable from smooth rhizoids. During their differentiation the tips of nascent pegged rhizoids can still be peg-less, whereas the basal domain can be already completely full of pegs. Mainly thanks to their collection in bundles and dye uptake experiments, pegged rhizoids have been proposed to play a key role in water conduction [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF][START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF]. Bundles of pegged rhizoids are also present in the stalk of the gametophores where they are thought to transport water by capillarity [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF][START_REF] Mcconaha | Ventral Structures Effecting Capillarity in the Marchantiales[END_REF]. As discussed above, the evolution of true vascular tissues during land plant evolution contributed significantly to the emergence of higher plants by permitting the long distance transport of water and nutrients over long distances though stomatally controlled transpiration. [START_REF] Bonfante | Plants and arbuscular mycorrhizal fungi: an evolutionarydevelopmental perspective[END_REF][START_REF] Delaux | Molecular and biochemical aspects of plant terrestrialization[END_REF][START_REF] Fukuda | Vascular tissue development in plants[END_REF]. The higher plant vasculature system is composed of xylem, phloem and cambium, all of which derive from the procambium. Xylem is composed of tracheary elements of which the most important are:

the tracheids and vessel elements (Figure 3). They both have secondary cell wall thickenings, are dead at maturity and undergo programmed cell death as part of their differentiation programme [START_REF] Ligrone | Conducting tissues and phyletic relationships of bryophytes[END_REF][START_REF] Růžička | Xylem developmentfrom the cradle to the grave[END_REF]. These lignified thickenings provide the mechanical support for the entire plant. Despite their similarities, tracheid and vessel elements differ in their assembly and morphology. Tracheid elements are elongated overlap one another and are linked with each other by several pits (region without secondary cell wall) in their lateral wall. A pit is typically located at an opposing pit of a neighbouring tracheid forming pit pairs. In contrast, vessel elements arrange with each other by their end which are open wall forming a vertical file called vessel. Like tracheid elements, vessel elements also have pits on their lateral wall, allowing them to communicate with each other laterally [START_REF] Růžička | Xylem developmentfrom the cradle to the grave[END_REF].

Similarities between rhizoids and root hairs

Filamentous cells develop at the plant surface interface in almost all land plants during their life cycle. Depending on the plants, two types of filamentous cells can be found: root hairs are found on the sporophyte of vascular plants whereas rhizoids develop on the rootless sporophyte or gametophyte of non-vascular and some vascular plants, the monilophytes and lycophytes [START_REF] Banks | GAMETOPHYTE DEVELOPMENT IN FERNS[END_REF][START_REF] Banks | Selaginella and 400 Million Years of Separation[END_REF]. Both rhizoids and root hairs [START_REF] Carol | Building a hair: tip growth in Arabidopsis thaliana root hairs[END_REF][START_REF] Pressel | Cellular Differentiation in Moss Protonemata: A Morphological and Experimental Study[END_REF] elongate by tip growth but have slightly different diameter and length. Whereas the diameter of rhizoids varies from 6 to almost 30µm (depending of the type of rhizoids) in complex thalloid liverworts like M. polymorpha, root hairs in A. thaliana have approximately a dozen microns across and 1 mm length [START_REF] De Baets | Micro-scale interactions between Arabidopsis root hairs and soil particles influence soil erosion[END_REF][START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF].

In contrast to root hairs which are thought to play role predominantly in nutrient uptake, rhizoid function was first shown to be involved in the anchorage of the plant to the substrate [START_REF] Jones | The evolution of root hairs and rhizoids[END_REF]. However, pegged rhizoids are also involved in the uptake of water and in nutrient transport. Moreover aqueous dyes are very quickly transported by capillarity between the pegged rhizoids of M. polymorpha supporting that in addition to classical transport of water within the pegged rhizoid, capillarity between the pegged rhizoids is also possible to transport water [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF]. In addition, water can also be transported inside smooth rhizoids (even though these ones collapse easily) and pegged rhizoids [START_REF] Clee | The Morphology and Anatomy of Fegatella conica in relation to the Mechanism of Absorption and Conduction of Water[END_REF][START_REF] Jones | The evolution of root hairs and rhizoids[END_REF]. From an evolution point of view, since Mprsl1 mutant lack both type of rhizoids combined with the fact that smooth and pegged rhizoids are hard to distinguish early on suggested that pegged rhizoid evolved from smooth rhizoids [START_REF] Honkanen | Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants[END_REF][START_REF] Villarreal | Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate[END_REF]. In addition, the acquisition of pegged rhizoids has been proposed to be correlated with the ability of certain bryophytes to develop taller gametophores, as in the case in the complex thalloid liverwort, M. polymorpha [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF]. Together these data support that pegged rhizoids are likely an important innovation for early land plants.

In addition to their functional similarities, the development of rhizoids and root-hairs which both developed from a single epidermal cell, has also been found to be regulated in a similar manner. Indeed, both are under the control of basic helix loop helix transcription factors (bHLH TF). The Lotus japonicus ROOTHAIRLESS LIKE (LRL) transcription factor, which is encoded by 5 homologous genes in A. thaliana, has been found to control the elongation of root hairs [START_REF] Karas | Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti Involves the Nodulation Factor-Dependent Induction of Root Hairs[END_REF]. Only one gene encoding this transcription factor is present in M. polymorpha, and has been shown to play a key role in rhizoid development [START_REF] Breuninger | Diversification of a Transcription Factor Family Led to the Evolution of Antagonistically Acting Genetic Regulators of Root Hair Growth[END_REF]. ROOT HAIR DEFECTIVE6 (AtRHD6) and RHD SIX-LIKE1 (AtRSL1) are required for root hair development in A. thaliana [START_REF] Lin | GLABRA2 Directly Suppresses Basic Helix-Loop-Helix Transcription Factor Genes with Diverse Functions in Root Hair Development[END_REF][START_REF] Menand | An ancient mechanism controls the development of cells with a rooting function in land plants[END_REF]. Although, these genes are specific to land plants, members of the bHLH TF family to which the proteins that they encode can be found in green algae, suggesting the recruitment of these TF to rhizoid/root hair formation during terrestrialization [START_REF] Jones | The evolution of root hairs and rhizoids[END_REF]. In the moss P. patens, PpRSL1 and PpRSL2 are closely related to AtRHD6 and AtRSL1 respectively and are required for rhizoid growth and development. Indeed, the double mutant Pprsl1 Pprsl2 is defective in rhizoid development but not in the development of other tip growing cells [START_REF] Jang | Auxin promotes the transition from chloronema to caulonema in moss protonema by positively regulating PpRSL1and PpRSL2 in Physcomitrella patens[END_REF][START_REF] Menand | An ancient mechanism controls the development of cells with a rooting function in land plants[END_REF]. Complementation of Atrh6 mutant with PpRSL1 rescues the roothair phenotype supporting a conservation of protein function [START_REF] Menand | An ancient mechanism controls the development of cells with a rooting function in land plants[END_REF]. Similarly, forward genetic screens in M. polymorpha supported the role of RSL genes in rhizoid development. Loss of function of MpRSL1, the M. polymorpha orthologue of the A. thaliana RSLs, showed absence of rhizoids, but also, in this case, of other tip growing cells (such as papillae and gemmae) [START_REF] Proust | RSL Class I Genes Controlled the Development of Epidermal Structures in the Common Ancestor of Land Plants[END_REF]. In contrast, ectopic expression of MpRSL1 leads to only an excessive production of rhizoids including formation of rhizoids on both the ventral and the dorsal surface of the thallus. Together these data suggest that MpRSL1 controls rhizoid fate within epidermal cells. More recently, FEW RHIZOIDS1 (MpFRH1), which encoding a microRNA targetting the MpRSL1 transcript, was shown to act as a negative regulator of rhizoid development. Consequently, gain of function of MpFRH1 causes a severe reduction in rhizoid numbers [START_REF] Honkanen | Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants[END_REF][START_REF] Thamm | MpFEW RHIZOIDS1 miRNA-Mediated Lateral Inhibition Controls Rhizoid Cell Patterning in Marchantia polymorpha[END_REF].

Pegged rhizoids also show similarity to xylem vessels

Although rhizoids can be easily compared to root hairs, rhizoids, and especially pegged rhizoids, also show many striking similarities with the internal water-conducting cells in mosses called hydroids and xylem vessels in vascular plants [START_REF] Nakashima | NAC transcription factors in plant abiotic stress responses[END_REF][START_REF] Xu | Contribution of NAC Transcription Factors to Plant Adaptation to Land[END_REF].

All these structures are involved in water/nutrient transport and can be assimilated to vasculature-like and undergo programme cell death during their differentiation process.

Interestingly, the largest group of plant-specific TFs, the NAM, ATAF and CUC (NAC) domain proteins (or VASCULAR-RELATED NAC-DOMAIN (AtVND) in A. thaliana), control late development of moss and Angiosperm water conducting cells, including cell wall thickening but also associated traits such as salt and drought tolerance [START_REF] Lv | Overexpression of a Novel NAC Domain-Containing Transcription Factor Gene (AaNAC1) Enhances the Content of Artemisinin and Increases Tolerance to Drought and Botrytis cinerea in Artemisia annua[END_REF][START_REF] Nakashima | NAC transcription factors in plant abiotic stress responses[END_REF][START_REF] Xu | Contribution of NAC Transcription Factors to Plant Adaptation to Land[END_REF]. In A. thaliana VND6 and VND7 activate genes involved in secondary cell wall biosynthesis of the xylem vessels and regulate xylem-specific programmed cell death [START_REF] Fukuda | Vascular tissue development in plants[END_REF][START_REF] Kubo | Transcription switches for protoxylem and metaxylem vessel formation[END_REF][START_REF] Yamaguchi | VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system[END_REF]Yamaguchi et al., , 2011;;[START_REF] Zhong | Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis[END_REF]. In the moss P. patens, three out of eight NAC TF also controls cell wall modification and programmed cell death of hydroids (filamentous cells with a similar functions to rhizoids) [START_REF] Xu | Contribution of NAC Transcription Factors to Plant Adaptation to Land[END_REF]. The homology between moss and angiosperm gene regulatory network for xylem/hydroids is striking and could mean it was present in a common ancestor of land plants but lost in liverworts. This would depend whether NACs have a role in M. polymorpha pegged rhizoids which remains an opened question.

Until recently, the functional parallel between pegged rhizoids or hydroids and the xylem of vascular plants had not been demonstrated. However, recent work from [START_REF] Brodribb | Advanced vascular function discovered in a widespread moss[END_REF] showed, for the first time, functional similarities between xylem in vascular plants and hydroids, in the moss Polytrichum commune. These parallels include vascular conducting tissue which are collapse-resistant while transporting water under tension and efficient regulation of leaves transpiration allowing photosynthetic gas exchange without cavitation inside the vascular tissue. Frustratingly few studies in M. polymorpha have been published to support this [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF]. However, although the similar morphological structure of rhizoids strongly suggests that a parallel structure/function relationship between M.

polymorpha pegged rhizoids and angiosperm xylem vessels is very likely since i) they both share a role in water transport ii) they both are collected together in bundles, iii) they both develop patterned secondary thickenings and developmental programed cell death, they are distinct and likely evolved independently. Indeed, pegged rhizoids arose late in liverwort evolution (they are found only complex thalloid liverworts) suggesting they are a derived and not an ancestral feature. They also showed a very different developmental origin since they are externalised and not internalised as the xylem or the hydroids and do not join end to end unlike xylem vessels.

Plant cuticle, the waterproofing layer of the plant surface, is one of the first innovations of land plants

Terrestrialization necessitates the need to control the movement of water within plant tissues. This need underlay the evolution of roots, rhizoids and vasculature as we discussed in the previous sections. However, these innovations do not limit the drying effect of the terrestrial environment. The colonization of land therefore necessitated adaptations allowing plants to prevent uncontrolled water loss. Plants addressed this problem by "waterproofing" their surfaces.

What is a plant cuticle?

The structure of the cuticle

The primary interface between land plants and their aerial environment is provided by the hydrophobic barrier, the cuticle. The cuticle, which is synthesized by epidermal cells [START_REF] Kurdyukov | The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis[END_REF][START_REF] Samuels | Sealing plant surfaces: cuticular wax formation by epidermal cells[END_REF] and sometimes also covers the cell walls bordering substomatal chambers [START_REF] Osborn | Morphological and Ultrastructural Studies of Plant Cuticular Membranes. I. Sun and Shade Leaves of Quercus velutina (Fagaceae)[END_REF] is present in all land plants, from bryophytes [START_REF] Buda | An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens[W][END_REF][START_REF] Budke | The cuticle on the gametophyte calyptra matures before the sporophyte cuticle in the moss Funaria hygrometrica (Funariaceae)[END_REF][START_REF] Lee | Occurrence of land-plant-specific glycerol-3-phosphate acyltransferases is essential for cuticle formation and gametophore development in Physcomitrella patens[END_REF][START_REF] Renault | A phenol-enriched cuticle is ancestral to lignin evolution in land plants[END_REF] to angiosperms.

The concept of the cuticle was first proposed around two centuries ago [START_REF] Von Mohl | Ueber die cuticula der gewächse[END_REF][START_REF] Von Mohl | Untersuchungen der Frage: bildet die cellulose die grundlage sammtlicher vegetabilischen membranen[END_REF]. Since this time scientists have used diverse techniques to define and identify the cuticle structure, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), confocal microscopy, and spectroscopic methods [START_REF] Fernández | Effect of wheat phosphorus status on leaf surface properties and permeability to foliarapplied phosphorus[END_REF][START_REF] Heredia-Guerrero | Infrared and Raman spectroscopic features of plant cuticles: a review[END_REF][START_REF] Yeats | The formation and function of plant cuticles[END_REF]. These studies have revealed a very heterogeneous structure. The structure and heterogeneity of the cuticle seems also to depend of the preparation of the samples [START_REF] Guzmán | Ultrastructure of Plant Leaf Cuticles in relation to Sample Preparation as Observed by Transmission Electron Microscopy[END_REF] and the interpretation of the authors [START_REF] Jeffree | The Fine Structure of the Plant Cuticle[END_REF]. The cuticle can differ dramatically in thickness from the nanometre to the micrometres scale [START_REF] Jeffree | The Fine Structure of the Plant Cuticle[END_REF]. Cuticles are incredibly diverse in their structure, composition and properties, which themselves may vary between species, organs, genotypes, developmental stages, and environmental conditions etc [START_REF] Fernández | Cuticle Structure in Relation to Chemical Composition: Re-assessing the Prevailing Model[END_REF][START_REF] Guzmán-Delgado | The presence of cutan limits the interpretation of cuticular chemistry and structure: Ficus elastica leaf as an example[END_REF][START_REF] Knoche | Changes in strain and deposition of cuticle in developing sweet cherry fruit[END_REF][START_REF] Nawrath | Apoplastic diffusion barriers in Arabidopsis[END_REF][START_REF] Szakiel | Fruit cuticular waxes as a source of biologically active triterpenoids[END_REF]… In an extreme case [START_REF] Guzmán | Ultrastructure of Plant Leaf Cuticles in relation to Sample Preparation as Observed by Transmission Electron Microscopy[END_REF] showed that structural differences may be observed even within the same cuticle section observed by TEM, confirming strong heterogeneity of the structure of the plant cuticle even at nanometric scale.

Despite this heterogeneity, most cuticles contain of a lipophilic matrix, called cutin which is a cross-linked matrix of insoluble monomers of long chain hydroxyl fatty acids, and hydrophobic compounds, the waxes [START_REF] Jeffree | The Fine Structure of the Plant Cuticle[END_REF]. Waxes can be intracuticular, within the cutin-matrix, or epicuticular wax crystals or wax films on the external surface of the cuticle.

Structurally, the cuticle in seed plants can be divided into two parts: the cuticle proper forms the outer layer of the cuticle, is enriched in waxes and is sometimes is covered by additional wax structures such as epicuticular films and/or epicuticular wax crystals. The cuticle proper covers an inner zone, which is enriched in cutin with embedded polysaccharides and can be referred to as the cuticular layer (Figure 4) [START_REF] Jeffree | The Fine Structure of the Plant Cuticle[END_REF]. However, this description probably oversimplifies a very complex picture since different plants, organs, and even fixation techniques can provide highly divergent images. In general, TEM observed cuticles are composed of two or more, more or less well defined layers with different electron-density distributions (after heavy metal staining) corresponding to the different parts of the cuticle. The intensity of these layers are very dependent on both the cuticle composition and sample preparation. Cuticles may also sometimes show lamella within their layers [START_REF] Jeffree | The Fine Structure of the Plant Cuticle[END_REF][START_REF] Jeffree | Ultrastructure and Recrystallization of Plant Epicuticular Waxes[END_REF].

Another approach to studying cuticle structure which might, at first sight, seem less complex than studying the structure of intact cuticles in planta, has been to focus the analysis of the structure on some components or groups of components in a semi-purified form. For example, some studies focused their research on isolated waxes [START_REF] Dragota | Epicuticular Wax Crystals of Wollemia nobilis: Morphology and Chemical Composition[END_REF][START_REF] Jeffree | Ultrastructure and Recrystallization of Plant Epicuticular Waxes[END_REF], cutin monomers [START_REF] Graça | Linear and branched poly(omega-hydroxyacid) esters in plant cutins[END_REF][START_REF] Ray | Isolation and molecular structure of an oligomer produced enzymatically from the cuticle of lime fruit[END_REF], polysaccharides [START_REF] Guzman-Puyol | Pectin-Lipid Self-Assembly: Influence on the Formation of Polyhydroxy Fatty Acids Nanoparticles[END_REF] or flavonoids [START_REF] Domínguez | Biomechanics of isolated tomato (Solanum lycopersicum) fruit cuticles during ripening: the role of flavonoids[END_REF]. It has been suggested that different macromolecular arrangements of cutin components may be responsible for the variable ultrastructural cuticle patterns observed by TEM [START_REF] Graça | Linear and branched poly(omega-hydroxyacid) esters in plant cutins[END_REF]. However, separating out specific cuticle components is a complex undertaking and likely disrupts inter-molecular associations. In addition some components (such as "cutan" polymer found in the cuticles of some plant species, are highly resistant to extraction procedures [START_REF] Mckinney | Tetramethylammonium hydroxide (TMAH) thermochemolysis of the aliphatic biopolymer cutan: insights into the chemical structure[END_REF].

In summary, due to the problems highlighted above, structure and chemical composition of cuticles are often difficult to link [START_REF] Fernández | Cuticle Structure in Relation to Chemical Composition: Re-assessing the Prevailing Model[END_REF].

The Composition of the cuticle

As previously mentioned, cuticles are incredibly diverse in their structure, composition and properties, in this section we will see that a lipophilic matrix and hydrophobic compounds, cutin and wax respectively are present in many cuticles. However, these polymers are themselves very diverse in chemical composition and present in highly variable proportions.

Other components may include polysaccharides, phenolics and sometimes cutan (Fernández et al., 2016;Tegelaar et al., 1991). The complexity of the composition of each of them are overviewed below.

Waxes are a mixture of diverse very-long-chain aliphatic compounds containing between 22 and 48 carbon atoms that may carry an oxygen-containing functional group such as primary alcohols, esters, aldehydes, odd-chain numbered alkanes and ketones (Figure 4) [START_REF] Jetter | Composition of Plant Cuticular Waxes[END_REF]. The proportion between the different wax aliphatic compounds often varies within the different organs of the same plant and between species. In addition to these aliphatic chains, secondary metabolites as sterols, triterpenoids and phenylpropanoids may also be associated with cuticular waxes [START_REF] Jetter | Composition of Plant Cuticular Waxes[END_REF][START_REF] Li-Beisson | Acyl-Lipid Metabolism[END_REF]. Waxes can be extracted from intact cuticles using solvents and its monomers analysed, for example by mass spectroscopy.

Although the fine structure of cutin polymers remains largely unknown, their composition has started to be elucidated. Cutin is a polyester composed mainly of C16-C18 hydroxy or hydroxyl epoxy fatty acid monomers (Figure 4) [START_REF] Walton | Determination of the structures of cutin monomers by a novel depolymerization procedure and combined gas chromatography and mass spectrometry[END_REF].

These are esterified, mainly via their mid-chain hydroxyl groups into branched networks.

Glycerol is also found as a monomer up to 14% of total monomer mass in cutin polymers [START_REF] Pollard | Building lipid barriers: biosynthesis of cutin and suberin[END_REF]. Cutin cannot be extracted using solvents, but can be hydrolysed after solvent extraction, and analysed, by mass spectroscopy.

Others compounds can also be found in the cuticle such as polysaccharides, which are incorporated into the cutin matrix in which they may play a role in stiffness (Guzmán et al., 2014b). Phenolics can also be found in cuticle in the form of coumaric acid for example where they are often present in less than 1% in the cuticles (Figure 4) [START_REF] Graça | Glycerol and glyceryl esters of omega-hydroxyacids in cutins[END_REF]. They likely also contribute to cutin rigidity [START_REF] López-Casado | Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides[END_REF] but may also be important for UV protection. In addition to coumaric acid, in A. thaliana leaf tissue, ferulic acid has also been identified in the cutin polymer [START_REF] Rautengarten | Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester[END_REF]. Phenolics can be released during cutin hydrolysis, but due to the potential presence of oxidative crosslinks between phenolic molecules, their abundance may be underestimated during such procedures.

The proportions of some compounds vary more than others in the different cuticle compartments, tissues and organs. For example, in waxes extracted from A. thaliana secondary alcohols and ketones which are almost absent from rosette leaves but respectively reach 23% in inflorescence stems and 14% in flowers [START_REF] Bourdenx | Overexpression of Arabidopsis ECERIFERUM1 Promotes Wax Very-Long-Chain Alkane Biosynthesis and Influences Plant Response to Biotic and Abiotic Stresses[END_REF]. As another example of variability, the cutin monomer α-ω-dicarboxylic acid which is usually found at less than 5% in most plant cutins, is much more abundant in A. thaliana where it represents more than 50% of total monomer mass [START_REF] Pollard | Building lipid barriers: biosynthesis of cutin and suberin[END_REF]. Finally, cutan may also be an important content of the cuticle, sometimes being present in similar quantities to cutin [START_REF] Schmidt | Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves[END_REF][START_REF] Villena | Characterization and biosynthesis of non-degradable polymers in plant cuticles[END_REF]. It is a highly aliphatic matrix linked by ether bonds which is resistant to both solvent extraction and hydrolysis.

2.3.2

How to make a cuticle?

Cuticle biosynthesis within epidermal cells

Although they have different final chemical compositions, the biosynthesis of both cutin and wax starts with fatty acids. The main fatty acids used for cuticle biosynthesis have chain lengths of 16 or 18 carbons (C16/C18), although the actual ratio is unknown and is likely to vary between organs. They are synthesized in the plastids by the Fatty Acid Synthase complex and distributed to the endoplasmic reticulum (ER). The mechanism of intracellular trafficking of Fatty Acids from the plastids to the ER is still unknown. Once they have reached the ER membrane, acyl chains are activated to coenzyme-A (CoA) by long-chain acyl-CoA synthetases (LACS) [START_REF] Haslam | Extending the story of very-long-chain fatty acid elongation[END_REF][START_REF] Jessen | Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis[END_REF][START_REF] Lü | Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis[END_REF]. The acylactivation is common to both the wax and cutin biosynthesis pathways, and attributing LACS to specific pathways seems to be complicated. All nine LACS-encoding genes in the A. thaliana genome work redundantly or partially redundantly in lipid metabolism. In in vitro enzymatic assays most LACS proteins show higher enzymatic activity with saturated and monounsaturated 16 carbons fatty acids and mono and polyunsaturated 18-carbons fatty acids than with other fatty acids. [START_REF] Lü | Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis[END_REF][START_REF] Pulsifer | Arabidopsis LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast[END_REF][START_REF] Shockey | Arabidopsis Contains Nine Long-Chain Acyl-Coenzyme A Synthetase Genes That Participate in Fatty Acid and Glycerolipid Metabolism[END_REF]. Three of the nine LACS are involved in cuticle biosynthesis, and are specifically localized in the endoplasmic reticulum (Figure 5). All three have been shown to activate fatty acids for production of cuticular lipids. LACS1/ECERIFERUM8(CER8) acts redundantly with LACS2 to activate fatty acids for production of wax and for the incorporation of C16-C18 acyls groups into cutin [START_REF] Lü | Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis[END_REF]Weng et al., 2010). LACS4 is also partially redundant with LACS1 in providing substrates for cuticular wax biosynthesis [START_REF] Jessen | Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis[END_REF]. More recently, Zhao et al., 2019 and colleagues showed that the cuticle of the triple mutant lacs1lacs2lacs4 is permeable and has a severe decrease in wax and cutin loads confirming again the redundancy between these LACS-encoding genes, and the commonality between the substrates necessary for cutin and wax biosynthesis. Interestingly LACS4 also functions partially redundantly with LACS1 in pollen coat formation [START_REF] Jessen | Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis[END_REF]. Although all LACS enzymes appear to function in lipid metabolism, they are not all ER localized or involved in the synthesis of extracellular lipids. For example, LACS9 is specifically associated with the outer envelope of plastids and is involved in the activation and export of plastid-derived fatty acids for triacylglycerol assembly. Moreover, seven of the nine LACS appear to be involved in seed 29 oil production: LACS1, LACS2, LACS4, LACS6, LACS7, LACS8 and LACS9 have overlapping roles in fatty acid beta-oxidation [START_REF] Fulda | Peroxisomal Acyl-CoA Synthetase Activity Is Essential for Seedling Development in Arabidopsis thaliana[END_REF][START_REF] Jessen | Two activities of long-chain acylcoenzyme A synthetase are involved in lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis[END_REF][START_REF] Shockey | Arabidopsis Contains Nine Long-Chain Acyl-Coenzyme A Synthetase Genes That Participate in Fatty Acid and Glycerolipid Metabolism[END_REF][START_REF] Zhao | Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis[END_REF].

WAX.

Much of the wax biosynthetic process occurs within the endoplasmic reticulum and is composed of three majors steps. Once imported into the ER, C16-C18 acyl-CoA become substrates for the fatty acid elongase complex (FAE) (Figure 5). 5).

In A. thaliana, twenty-one KCS-encoding genes have been annotated [START_REF] Joubès | The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling[END_REF] including some with a wax synthesis activity. To date, CER6 is the only KCS which appears to have a strictly wax-specific function based on phenotypic and expression pattern analysis [START_REF] Hooker | Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis[END_REF][START_REF] Millar | CUT1, an Arabidopsis Gene Required for Cuticular Wax Biosynthesis and Pollen Fertility, Encodes a Very-Long-Chain Fatty Acid Condensing Enzyme[END_REF]. Likewise, several KCR-encoding genes have been annotated in the A. thaliana genome but only KCR1 has been reported to reduce βketoacyl-CoA to β-hydroxyacyl-CoA [START_REF] Beaudoin | Functional characterization of the Arabidopsis beta-ketoacyl-coenzyme A reductase candidates of the fatty acid elongase[END_REF]. The PASTICCINO2 (PAS2)

protein, has been shown to dehydrate 3-hydroxyacyl-CoAs into enoyl-CoAs [START_REF] Bach | The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development[END_REF] but also (confusingly) contributes to cell division and cell differentiation, possibly through an antiphosphatase activity involved in the regulation of cyclin-dependant kinase A [START_REF] Bellec | Pasticcino2 is a protein tyrosine phosphatase-like involved in cell proliferation and differentiation in Arabidopsis[END_REF][START_REF] Da Costa | Arabidopsis PASTICCINO2 is an antiphosphatase involved in regulation of cyclin-dependent kinase A[END_REF][START_REF] Harrar | Hormonal Control of Cell Proliferation Requires PASTICCINO Genes[END_REF]. Whether these two activities are linked remains a subject of debate. Some ER-localized enzymes involved in lipids synthesis are thought to form clusters on specific ER subdomains. The PAS1 protein has been proposed to act as a scaffold to recruit the FAE complex enzymes to such ER subdomains during wax biosynthesis [START_REF] Roudier | Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis[W][END_REF]. Finally, [START_REF] Zheng | Disruptions of the Arabidopsis Enoyl-CoA Reductase Gene Reveal an Essential Role for Very-Long-Chain Fatty Acid Synthesis in Cell Expansion during Plant Morphogenesis[END_REF] identified an enoyl reductase, CER10, responsible for the production of the elongated acyl-CoA (Figure 5).

Loss or partial loss of activity of the three last enzymes of the FAE complex (CER10, PAS2 or KCR1) results in a dramatic reduction in all major classes of very-long-chain-acyl lipids on the plant surface, and to severe developmental and cellular defects. These pleiotropic phenotypes probably reflect that fact that these enzymes are involved in other metabolic pathways, including those involved in the production of sphingolipids which are necessary for basic cellular functions [START_REF] Zheng | Disruptions of the Arabidopsis Enoyl-CoA Reductase Gene Reveal an Essential Role for Very-Long-Chain Fatty Acid Synthesis in Cell Expansion during Plant Morphogenesis[END_REF].

Carbon chain elongation by the FAE cycle can end by the conversion of the VLCFA-CoA to free VLCFA by thioesterases. The resulting free VLCFA is then be incorporated directly into the cuticle by an unknown process. The FAE elongation products can also be used in the alcohol forming pathway to produce VLC-primary alcohols through the action of alcohol fatty acyl CoA reductases (FAR), such as ECERIFERUM4 (CER4) in A. thaliana (Figure 5). CER4

only seems to be able to reduce C24 or C26 VLCFA-CoA into primary alcohols in the yeast heterologous system [START_REF] Jenks | Leaf Epicuticular Waxes of the Eceriferum Mutants in Arabidopsis[END_REF][START_REF] Rowland | CER4 Encodes an Alcohol-Forming Fatty Acyl-Coenzyme A Reductase Involved in Cuticular Wax Production in Arabidopsis[END_REF]. Free primary alcohols can be incorporated into waxes or they can be esterified by a wax synthase to create wax esters. The only wax synthase (WS) functionally identified to date, and probably the main one in A.

thaliana, is provided by the wax ester synthase/diacylglycerol acyltransferase 1 (WSD1) (Li et al., 2008). The wsd1 mutant shows a severe reduction in alkyl esters and an absence of detectable wax esters in stems and leaves. More recently, WSD1 has also been shown to contribute to drought tolerance [START_REF] Patwari | Surface wax esters contribute to drought tolerance in Arabidopsis[END_REF]. WSD1 is part of a large protein family.

The functions of many of the members of this family have not been fully characterised but the genes encoding these proteins show diverse expression patterns. Several other family members could also be implicated in wax biosynthesis including WSD11 which is thought to be involved in petal growth by providing waxes to the petal surface where they may act as a lubricant [START_REF] Takeda | Physical interaction of floral organs controls petal morphogenesis in Arabidopsis[END_REF]. WDS6, WSD7 and WSD1 have also been shown to contribute to water, salt and abscisic acid (ABA) stress tolerance [START_REF] Patwari | Surface wax esters contribute to drought tolerance in Arabidopsis[END_REF].

Another possible destination for VLCFA-CoA produced by the FAE complex is the production of aldehydes, odd-chain numbered alkanes, secondary alcohols or ketones via the alkane-forming pathway. Alkanes can be produced from VLCFA-CoA via the formation of intermediate aldehydes. However, to date the enzyme responsible has not been identified.

Nonetheless, [START_REF] Bernard | Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex[END_REF] proposed a model in which CER3 (a VLCFA-CoA reductase) and CER1 (an alkane forming enzyme) form an enzymatic complex catalysing the formation of VLC-alkanes from VLC-acyl-CoA. Although they are, themselves, very abundant wax compounds, VLC-alkanes can also undergo further modifications to form secondary alcohols and ketones. In A. thaliana, MAH1, a mid-chain alkane hydroxylase from the cytochrome P450 family catalyses the production of VLC-secondary alcohols which can be further oxidized, by the same enzyme, into VLC-ketones [START_REF] Greer | The cytochrome P450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis[END_REF].

CUTIN.

Similarly to wax biosynthesis, cutin biosynthesis occurs principally in the ER using C16-C18 fatty-acids as substrates. Once the C16-18 fatty-acids are activated by the transfer of a

CoA group, the resulting product, C16/C18 acyl-CoA, undergoes oxidation of the terminal and/or mid-chain carbon followed by the transfer of the fatty acid from CoA to glycerol.

The oxidation step is catalysed by members of the CYP86 cytochrome P450 family such as ATT1/CYP86A2, CYP86A4, CYP86A8 and probably CYP86A7 [START_REF] Li-Beisson | Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester[END_REF][START_REF] Molina | Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana[END_REF][START_REF] Voisin | Dissection of the Complex Phenotype in Cuticular Mutants of Arabidopsis Reveals a Role of SERRATE as a Mediator[END_REF][START_REF] Xiao | Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development[END_REF]. ω-hydroxy fatty acyl-CoA are hydroxyl fatty acyl-CoA with a carboxylic acid group and a primary hydroxyl group, and often carry one or two additional secondary hydroxyl groups. They are oxidised either on the terminal carbons, (mainly by members of the CYP86A family), or on a mid-chain carbon, (mainly by enzymes from the CYP77A family) (Figure 5) [START_REF] Pollard | Building lipid barriers: biosynthesis of cutin and suberin[END_REF].

Finally, the mature monoacylglycerol cutin monomers are generated by the enzymes of the Glycerol 3-Phosphate Acyltransferase or GPAT family [START_REF] Fawke | Glycerol-3-phosphate acyltransferase 6 controls filamentous pathogen interactions and cell wall properties of the tomato and Nicotiana benthamiana leaf epidermis[END_REF]Fich et al., 2016a;Li et al., 2007a). A. thaliana possesses ten genes annotated as GPATs but only eight of them have been shown to be potentially involved in the biosynthesis of apoplastic barriers.

GPAT10 and GPAT9 appear to be required for membrane or storage lipid biosynthesis [START_REF] Gidda | Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: functional divergence of the dilysine ER retrieval motif in plant cells[END_REF][START_REF] Nishida | The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana[END_REF]. The other GPATs cluster in a subfamily and have functions that affect the composition and/or quantity of cutin or suberin [START_REF] Beisson | Solving the puzzles of cutin and suberin polymer biosynthesis[END_REF]. Suberin is another major polymer found in plants with similar functions and properties as cutin but is found in the roots, seed coats and abscission zones where it has a protective role [START_REF] Franke | Suberina biopolyester forming apoplastic plant interfaces[END_REF][START_REF] Philippe | Cutin and suberin: assembly and origins of specialized lipidic cell wall scaffolds[END_REF][START_REF] Vishwanath | Suberin: biosynthesis, regulation, and polymer assembly of a protective extracellular barrier[END_REF]. GPAT4, GPAT6 and GPAT8 are required for cutin biosynthesis where they have an sn-2 acyltransferase activity. In addition to acyltransferase activity, these three enzymes catalyse a dephosphorylation reaction during the condensation between glycerol and fatty-acyl CoA chains [START_REF] Yang | A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol[END_REF][START_REF] Yang | A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution[END_REF]. The genes encoding these proteins are differentially expressed. GPAT4 and GPAT8 have been found to be expressed mainly in stems and leaves where they are required for the accumulation of C16 and C18 ω-hydroxy fatty acid and α,ω-dicarboxylic acid cutin monomers [START_REF] Li | Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers[END_REF]. GPAT6 is mainly expressed in flowers and catalyzes the production of three C16 monomers: 10,16-dihydroxypalmitate, hexadecane-1,16-dioic acid and 16hydroxypalmitate [START_REF] Li-Beisson | Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester[END_REF]. Interestingly, these three GPATs seem to preferentially catalyse the reaction of C16 and C18 fatty acids with hydroxyl or carboxyl groups suggesting there are probably undiscovered acyltransferases for the other cutin precursors such as the FA-CoA C20-C28 which are the main FA-CoA found in cutin polymers [START_REF] Li-Beisson | Acyl-Lipid Metabolism[END_REF][START_REF] Yeats | The formation and function of plant cuticles[END_REF]. GPAT5 and GPAT7 are involved in suberin precursor synthesis and do not have phosphatase activity [START_REF] Pollard | Building lipid barriers: biosynthesis of cutin and suberin[END_REF][START_REF] Yang | A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution[END_REF]. Finally, GPAT1/2/3 are more divergent, in terms of protein sequence, compared to GPAT4/6/8 and GPAT5/7. Although GPAT1 seems to be required for tapetum and anther development [START_REF] Zheng | Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility[END_REF] no acyltransferase activity have been detected for this enzyme [START_REF] Yang | A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution[END_REF].

GPAT2 and GPAT3 functions remain uncertain as mutants are not defective in lipid production and assays have revealed no acyltransferase activity with basic substrates for these enzymes.

However, modelling and sequence similarities suggest that both proteins could possess a potential sn-2 acyltransferase activity [START_REF] Zheng | Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility[END_REF].

OTHER COMPONENTS.

In addition to aliphatic compounds, the cuticle also contains phenolic monomers synthesized via the phenylpropanoid pathway, such as coumaric acid or ferulic acid [START_REF] Graça | Glycerol and glyceryl esters of omega-hydroxyacids in cutins[END_REF][START_REF] Rautengarten | Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester[END_REF]. Incorporation of phenylpropanoid compounds into cutin requires BAHD acyltransferases such as DEFICIENT IN CUTIN FERULATE (DCF) which transfers ferulic acid from CoA to the terminal hydroxyl of omega-hydroxy-fatty acids [START_REF] Rautengarten | Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester[END_REF]. Loss of DCF function does not cause any major alteration in cuticle permeability or cutin lipid monomer composition but a 90% decrease in ferulic acid content in A. thaliana non-suberized leaf tissue. This suggests that contrary to suberin, in which phenolic compounds play an important role in ensuring impermeability, this is not that case for the cuticle. [START_REF] Gou | A hydroxycinnamoyltransferase responsible for synthesizing suberin aromatics in Arabidopsis[END_REF][START_REF] Rautengarten | Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester[END_REF]. In contrast with wax and cutin biosynthesis, the enzymatic reaction catalysed by DCF occurs in the cytosol (Figure 5) [START_REF] Rautengarten | Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester[END_REF].

How phenolic compounds are exported, how they are integrated into the cuticle and what roles they play during cuticle development are questions with very few concrete answers, although this is a fertile field of ongoing research [START_REF] Biała | The Phenylpropanoid Case -It Is Transport That Matters[END_REF].

Export of cuticle components from epidermal cells

After production of wax components and monoacylglycerols in the endoplasmic reticulum, cutin and wax precursors must be exported from the epidermal cell and transported to the cell surface. This transport involves two key steps: From the ER through the plasma membrane (PM) and from the PM through the polysaccharide cell wall (PCW) to the plant surface.

To reach the plasma membrane, cutin and wax precursors must cross the cytosol. Although no direct evidence for this trafficking mechanism of the cuticular precursors has been shown, such close contacts are known to exist in plants and animals and to be involved in the transport of other hydrophobic molecules [START_REF] Larsson | LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membraneassociated endoplasmic reticulum[END_REF][START_REF] Samuels | Plant cell wall secretion and lipid traffic at membrane contact sites of the cell cortex[END_REF].

Very few more recent papers have been published addressing the theory that vesicle trafficking could be involved in the export of cutin/wax monomers. It is likely that this is due to the fact that mutants in these processes are lethal or show severe developmental phenotypes.

In the future the application of inducible knock-down approaches could be used to address this question.

Once at the plasma membrane, monoacylglycerols and wax precursors have been shown to exit the cell via polarly localized membrane transporters from the ATP-binding cassette subfamily G (ABCG). Full length transporters contain two nucleotide binding domains which hydrolyse ATP to provide energy for transport, and two multi-span transmembrane domains which allow substrate translocation across the membrane. However, some ABCG transporters exist as "half" transporters that must form homodimers or heterodimers to be functional. For example, the half transporter ABCG11 dimerizes with ABCG12/CER5 to mediate wax export. The need for both proteins in the functional complex is demonstrated by the fact that abcg11 and abcg11abcg12 mutants show the same decrease in extracellular wax quantity [START_REF] Bird | Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion[END_REF][START_REF] Pighin | Plant cuticular lipid export requires an ABC transporter[END_REF]. ABCG11 is also involved in cutin export since abcg11 mutants also shows a reduced cutin load. Moreover, ABCG11 can homodimerize.

This, together with the fact that abcg12 mutants show no decrease in cutin load, suggesting that the homodimer ABCG11/ABCG11 may have specific functions in cutin export. Importantly 50% of total waxes are exported apparently normally in abcg11abcg12 mutants, suggesting that other members of the ABCG family might be involved in wax transport [START_REF] Bird | Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion[END_REF].

Consistent with this idea, a transporter closely related to ABCG11 and ABCG12, ABCG13, has been shown to contribute to cutin export in flowers [START_REF] Panikashvili | The Arabidopsis ABCG13 transporter is required for flower cuticle secretion and patterning of the petal epidermis[END_REF]. Unlike the previously described ABC transporters, ABCG32 also called PERMEABLE CUTICLE 1(PEC1) is a full length transporter and is required for cutin precursors exportation since abcg32 mutants display a reduction of 40% in some cutin precursors (Figure 5) [START_REF] Bessire | A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis[END_REF].

Cuticle assembly at the epidermal surface

How the compounds discussed in the previous sections assemble at the plant surface and form the cuticle is still little known. However, some studies suggested that cutin is likely to be the primary component deposited into the cuticle (during embryogenesis or organogenesis)

whereas the wax deposition occurs later, and tends to be conditioned by environmental factors [START_REF] Delude | Plant Surface Lipids and Epidermis Development[END_REF][START_REF] Kannangara | The Transcription Factor WIN1/SHN1 Regulates Cutin Biosynthesis in Arabidopsis thaliana[END_REF].

CUTIN POLYMERIZATION.

The identities and proportions of cutin monomers and their loads in diverse plants, organs, and tissues have been extensively studied. However, the information is almost always based on hydrolysed cuticles, making understanding how these different monomers are linked together in vivo difficult to ascertain. Several techniques based on spectrometry and Nuclear magnetic Resonance Spectroscopy (NMR) have been applied to try and decipher the structure and linkages within the cutin polymer. They revealed after partial depolymerisation primary and secondary ester linkages between the cutin monomers and covalent linkages between some cutin hydroxyl fatty acids and oligosaccharides [START_REF] Graça | Glycerol and glyceryl esters of omega-hydroxyacids in cutins[END_REF][START_REF] Tian | Isolation and identification of oligomers from partial degradation of lime fruit cutin[END_REF]. However, they might not be the only options for the assembly of the different monomers, these later can also be linked on the primary OH by a linear or on a secondary OH by a branched pattern as has been shown within tomato cutin but the relative proportion of the types of cross-links remain uncertain [START_REF] Deas | The Intermolecular Structure of Some Plant Cutins[END_REF][START_REF] Deshmukh | Evidence for cross-linking in tomato cutin using HR-MAS NMR spectroscopy[END_REF][START_REF] Graça | Linear and branched poly(omega-hydroxyacid) esters in plant cutins[END_REF][START_REF] Kolattukudy | Lipid Polymers and Associated Phenols, Their Chemistry, Biosynthesis, and Role in Pathogenesis[END_REF]. To date, the ester links of glycerol OH groups remains unresolved. In vitro assays with the tomato (Solanum lycopersicum) cutin synthase SlCUS1 and a putative precursor of cutin polymer, 2-monoacylglycerol (2-MAG) in A. thaliana [START_REF] Yang | A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol[END_REF], showed a production of linear oligomers in aqueous solution by transesterification of the endchain hydroxyls groups [START_REF] Girard | Tomato GDSL1 is required for cutin deposition in the fruit cuticle[END_REF][START_REF] Yeats | The identification of cutin synthase: formation of the plant polyester cutin[END_REF][START_REF] Yeats | Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants[END_REF]. This CUS1 was the first enzyme identified in the polymerisation of the monomer [START_REF] Girard | Tomato GDSL1 is required for cutin deposition in the fruit cuticle[END_REF][START_REF] Yeats | The identification of cutin synthase: formation of the plant polyester cutin[END_REF][START_REF] Yeats | Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants[END_REF]. The CUS1 mutant exhibits a severe reduction of polymerised cutin in the tomato fruit cuticle [START_REF] Girard | Tomato GDSL1 is required for cutin deposition in the fruit cuticle[END_REF][START_REF] Yeats | The identification of cutin synthase: formation of the plant polyester cutin[END_REF], in both the cuticular layer and cuticle proper.

More precisely, the esterification mediated by CUS1 involves mostly primary hydroxyl groups in vitro [START_REF] Yeats | Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants[END_REF] but probably also the secondary hydroxyl group of 9(10),16dihydroxyhexadecanoic acid and both the primary and secondary hydroxyl groups of glycerol [START_REF] Philippe | Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization1[END_REF]. [START_REF] Girard | Tomato GDSL1 is required for cutin deposition in the fruit cuticle[END_REF][START_REF] Yeats | The identification of cutin synthase: formation of the plant polyester cutin[END_REF] also showed that the polymerisation of the cutin monomers by CUS1 is extracellular and occurs where the cuticle is deposited.

Interestingly, although the sicus1 mutant shows a polymerisation activity in vitro and in vivo the single mutant still has a very thin cuticle meaning that one or more additional enzymes are involved in the polymerisation of cutin.

It is possible that several other enzymes polymerise cutin monomers in various different ways, possibly via different groups, but CUS1 is a key component. These enzymes could belong to the same glycine-aspartic acid-serine-leucine (GDSL) motif lipase subfamily as CUS1.

Another possibility suggested in a few articles, is the auto-assembly of cutin monomers via a non-enzymatic mechanism involving cutinsomes; nanoparticles of partially polymerized cutin which are proposed to traverse the epidermal cell wall and self-assemble at its surface [START_REF] Domínguez | Self-assembly of supramolecular lipid nanoparticles in the formation of plant biopolyester cutin[END_REF][START_REF] Segado | Cutinsomes and CUTIN SYNTHASE1 Function Sequentially in Tomato Fruit Cutin Deposition[END_REF][START_REF] Stępiński | Cutinsomes as building-blocks of Arabidopsis thaliana embryo cuticle[END_REF]. It remains unclear if this process happened in planta or not. In addition, enzymes involved in polymerisation still need to be evoked for this mechanism to result in the production of a cuticle that cannot be extracted using solutes.

CUTIN LINKS TO CELL WALL POLYMERS.

Deposition of cutin and waxes occurs on the cell wall polysaccharide matrix. A gradient is formed in most plant surfaces where the most outer layer (the cuticle proper) is composed of waxes and cutin whereas the inner surface contains cutin and waxes but also polysaccharides [START_REF] Jeffree | The Fine Structure of the Plant Cuticle[END_REF]. Treatments for cuticle isolation revealed the strong link between cuticle proper and cell wall, since pectinase treatment or pectinase and cellulose treatments have been found to be necessary to release the cuticle from the leaves of several plant species and from conifer needles respectively [START_REF] Lendzian | Isolation of cuticular membranes from various conifer needles[END_REF][START_REF] Orgell | The Isolation of Plant Cuticle with Pectic Enzymes[END_REF].

In addition, several loss of function mutants affected in cuticle formation also show defect in cell wall organization, suggesting a coordination/cross-talk between the deposition of cell wall, including the cuticle layer, and the cuticle proper. An example of the cross talk between cell wall and cutin is the loss of function gpat6 mutant in A. thaliana where not only a defect in cutin synthesis is detectable [START_REF] Philippe | Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization1[END_REF] but also significant changes in cell walls [START_REF] Philippe | Cutin and suberin: assembly and origins of specialized lipidic cell wall scaffolds[END_REF]. Similar correlations have also been observed in the tomato fruit [START_REF] Philippe | Cutin and suberin: assembly and origins of specialized lipidic cell wall scaffolds[END_REF]. In A. thaliana another enzyme called DEFECTIVE IN CUTICULAR RIDGES (DCR), an acyltransferase from the BADH family, has been shown to have a key function in cuticle organization independently of cutin monomer composition and structure.

Indeed, the corresponding mutant dcr showed a thinner cuticle proper as well as a thinner cell wall suggesting a feedback regulation between the formation of the cuticle and the cell wall, which could be necessary to equilibrate the cuticle-cell wall continuum [START_REF] Mazurek | Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis[END_REF].

More recently, the first details of the physical relationship between cutin deposition and associated polysaccharides has been published. They revealed that cellulose, pectin and hemicellulose are present in cutin embedded polysaccharides and strongly suggest that pectins play an important role in the cutin and polysaccharide continuum, at least in tomato fruit. Moreover, CUS1 function in the polymerisation of cutin monomers appears to be correlated with the deposition of methyl-esters within pectin [START_REF] Philippe | Cutin and suberin: assembly and origins of specialized lipidic cell wall scaffolds[END_REF]. This is coherent with other published data such as the observed role of the transcription factor SHINE (SHN) in the regulation of cutin synthesis but also in the regulation of cell wall polysaccharides through enzymes involved in pectin degradation and modifications [START_REF] Shi | SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs[END_REF].

From the point of view of plant growth, since both cutin and the underlying cell wall have to extend in a coordinated fashion, some level of cross talk between them seems indispensable.

WAX DEPOSITION.

Some studies suggested that cutin is likely to be the first component deposited into the cuticle whereas the wax deposition occurred later [START_REF] Kannangara | The Transcription Factor WIN1/SHN1 Regulates Cutin Biosynthesis in Arabidopsis thaliana[END_REF]. However, to date the comprehension of wax deposition remains unclear.

In order to reach the cuticle, wax monomers have to cross the hydrophilic cell wall.

Lipid Transfer Proteins were initially defined by their capacity to transfer phospholipids between membranes [START_REF] Kader | LIPID-TRANSFER PROTEINS IN PLANTS[END_REF]. They are extracellular proteins with a hydrophobic cavity, able to bind fatty acids and small enough to cross the cell wall pores [START_REF] Beisson | Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database[END_REF][START_REF] Debono | Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface[END_REF][START_REF] Kader | LIPID-TRANSFER PROTEINS IN PLANTS[END_REF][START_REF] Kunst | Plant cuticles shine: advances in wax biosynthesis and export[END_REF][START_REF] Zachowski | Characterisation of acyl binding by a plant lipid-transfer protein[END_REF]. In A.

thaliana, the glycosylphosphatidylinositol-anchored LTP (LTPG1) is involved in cuticular lipid composition. The loss-of-function mutant, ltpg1, showed defect in cuticular wax composition with a decrease of C29 alkanes but no changes on total wax and total cutin monomers. The authors suggested that the decrease in C29 alkanes is counterbalanced by an increase in other C29 waxes (secondary alcohols and ketones precisely) [START_REF] Lee | Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola[END_REF].

How is the cuticle production controlled post germination?

The cuticle is deposited and directly embedded in the cell wall of all aerial epidermis cells, suggesting that epidermal cell fate and cuticle deposition may share common regulatory pathways. The leaf surface is covered by different epidermal cell types. In many species, the pavement cells cover the majority of the leaf surface whereas some epidermal cells undergo specific differentiation processes which lead, for example, to the formation of stomata or trichomes. Interesting the distribution and development of these cells can also be influenced by cuticular properties. For example, cer1, cer6 or even wax2 which are all cuticle defective mutants involved in wax production, showed abnormal stomatal indices [START_REF] Chen | Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production[END_REF][START_REF] Gray | The HIC signalling pathway links CO2 perception to stomatal development[END_REF]. However, it remains unclear how these factors are correlated. One possibility is that a defective cuticle could lead to changes in environmental perception by the epidermal cells and thus deregulate the stomatal density. Alternatively, the apoplastic signalling between cells involved in stomata patterning may be disturbed in cuticle-defective mutants, possibly due to changes in cell wall composition or structure as discussed above [START_REF] Javelle | Epidermis: the formation and functions of a fundamental plant tissue[END_REF].

Within the context of the regulation of cuticle formation, two major families of transcription factors need to be mentioned. The first one is the homeodomain-leucine zipper IV (HD-ZIP IV) transcription factor family comprising 16 members in A. thaliana, of which several have been shown to be involved in both differentiation and maintenance of epidermal cell fate and cuticle biogenesis [START_REF] Abe | Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis[END_REF][START_REF] Ariel | The true story of the HD-Zip family[END_REF][START_REF] Depège-Fargeix | Functional characterization of the HD-ZIP IV transcription factor OCL1 from maize[END_REF][START_REF] Javelle | Overexpression of the Epidermis-Specific Homeodomain-Leucine Zipper IV Transcription Factor OUTER CELL LAYER1 in Maize Identifies Target Genes Involved in Lipid Metabolism and Cuticle Biosynthesis[END_REF][START_REF] Peterson | Arabidopsis homeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis[END_REF]San-Bento et al., 2014a;[START_REF] Takada | ATML1 promotes epidermal cell differentiation in Arabidopsis shoots[END_REF][START_REF] Vernoud | The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize[END_REF]. An overexpression of the HD-ZIP IV protein OUTER CELL LAYER11

in Zea mays (ZmOCL11) gave plants with quantitative and qualitative changes in cuticular wax compounds [START_REF] Javelle | Overexpression of the Epidermis-Specific Homeodomain-Leucine Zipper IV Transcription Factor OUTER CELL LAYER1 in Maize Identifies Target Genes Involved in Lipid Metabolism and Cuticle Biosynthesis[END_REF] and showed that ZmOCL11 direct and indirect target genes are involved in cuticle formation including genes encoding ABCG transporters. The cognate binding site of HD-ZIP IV proteins is called the L1 box [START_REF] Abe | Identification of a cis-regulatory element for L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain protein[END_REF], and these sequences were found to be enriched in the promoters of ZmOCL11 targets. Similarly, the BODYGUARD (BDG) and FIDDLEHEAD(FDH) genes, both of which are required for normal cuticle formation in A. thaliana, contain L1 boxes upstream of their transcription start sites, which are bound by the HD-ZIP IV protein HDG1, suggesting a potential regulation by HD-ZIP IV proteins [START_REF] Abe | Identification of a cis-regulatory element for L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain protein[END_REF][START_REF] Abe | Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis[END_REF][START_REF] Nakamura | Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis[END_REF][START_REF] Wu | CFL1, a WW Domain Protein, Regulates Cuticle Development by Modulating the Function of HDG1, a Class IV Homeodomain Transcription Factor, in Rice and Arabidopsis[END_REF].

The second family of proteins is the SHINE transcription factor family in A. thaliana, which contains proteins from the APETALA2 (AP2/EREBP) clade, and includes SHINE1/WIN1, SHINE2 and SHINE3 [START_REF] Aharoni | The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis[END_REF][START_REF] Broun | WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis[END_REF]. Silencing of the three SHN genes leads to organ fusions and defects in the expression of cuticle biosynthesis genes but also of genes involved in pectin production and cell elongation, suggesting that these proteins, like some members of the HD-ZIP IV protein family, could have effects on epidermal cell differentiation [START_REF] Shi | SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs[END_REF]. Alternatively, they could act as hubs mediating the crosstalk between cuticle deposition and cell wall composition evoked previously. In support of the first hypothesis, a positive regulator of genes encoding SHN1 transcription factors, the MIXTA-like MYB transcription factor MYB106, regulates the formation of trichomes [START_REF] Oshima | The MIXTA-like Transcription factor MYB16 is a major regulator of cuticle formation in vegetative organs[END_REF]. However, trichome formation also involves rapid cell expansion and cell wall modifications, meaning that the second hypothesis cannot be excluded.

In summary, the formation of a functional cuticle appears to be tightly linked to the acquisition and maintenance of epidermal identity and morphology [START_REF] Delude | Plant Surface Lipids and Epidermis Development[END_REF][START_REF] Javelle | Epidermis: the formation and functions of a fundamental plant tissue[END_REF]. This fact can render the targeted analysis of either process very complex.

Although cuticle production is almost always correlated with epidermal cell identity, a few exceptions exist. The first one is the cuticle covering the embryonic root cap and the emerging lateral root. The root cap is formed at the tip of the root in order among other things to protect the root apical meristem [START_REF] Barlow | The root cap: Cell dynamics, cell differentiation and cap function[END_REF]. The embryonic root cap cuticle is composed of an TEM electron-opaque layer surrounded the root cap tip resembling the ultrastructure of aerial cuticle although its composition seems to slightly differ, possibly accounting for its relatively high permeability. Interestingly, this structure was not detected in the mutants of cutin biosynthesis genes such as gpat4, lcr or bdg [START_REF] Berhin | The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation[END_REF]. Although the presence of a root cap cuticle was initially considered surprising, consistent with the expression of genes such as BDG, both lateral roots and the embryonic root pole appear to have epidermal identity similar to the aerial organs developing cuticle, at least based on the expression of HD-ZIP IV protein-encoding genes [START_REF] Nakamura | Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis[END_REF].

The functions of the cuticle post germination

The cuticle was a major innovation allowing the colonization of land by plants. It appears logical that, as hinted at above, the cuticle has a high diversity of functions within land plants.

Limiting water loss

One of the primary roles attributed to the cuticle is in limiting water loss. The chemical properties of the compounds of which cuticles are composed (notably hydrophobicity), are associated with the water barrier effect of the cuticle. The compounds that mediate this affect seems to be predominantly the cuticular waxes based on correlative studies [START_REF] Bourgault | Constructing functional cuticles: analysis of relationships between cuticle lipid composition, ultrastructure and water barrier function in developing adult maize leaves[END_REF]. A positive correlation between an increase in wax amount and tolerance to drought stress has been reported in A. thaliana [START_REF] Aharoni | The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis[END_REF][START_REF] Castorina | Drought-Responsive ZmFDL1/MYB94 Regulates Cuticle Biosynthesis and Cuticle-Dependent Leaf Permeability[END_REF][START_REF] Cui | Dissecting Abscisic Acid Signaling Pathways Involved in Cuticle Formation[END_REF][START_REF] Kosma | The Impact of Water Deficiency on Leaf Cuticle Lipids of Arabidopsis[END_REF]. In contrast, cutin seems to create a more selectively permeable barrier than wax. In general, permeability increased by two to four orders of magnitude when the waxes were removed by organic solvents [START_REF] Schmidt | Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves[END_REF]. Alkanes are particularly non polar wax components and tend to be associated with decrease of water permeability of the cuticle. In contrast, triterpenoids which are nonaliphatic wax components are a less effective water permeability barrier (Leide et al., 2007;Buschhaus and Jetter, 2012).

Thus the proportion of alkanes within the cuticle and not the total amount of waxes may have the most significant effect on the water permeability of the cuticle. This has been clearly illustrated with two pepper species with contrasting water loss rates (Parsons et al., 2012) and in Prunus laurocerasus leaves and Pyrus communis leaves in which cuticular waxes are responsible for respectively 96% and 99,5% of the total resistance of water diffusion (Burghardt and Riederer, 2006). A correlation is likely to exist between the chain length of cuticular waxes and the water permeability since the cuticle of plants in arid regions tend to have a longer chain length than the ones from plants in temperate regions (Bush and McInerney, 2015).

An interesting point linked to the cuticle permeability is the so called "lotus effect" which is the ability of some plants to form a water bead on the surface of the leaf. This water "repellent" effect has been clearly linked to the presence of epicuticular wax crystals which allow the formation of a pocket of air under the water droplets (Barthlott and Neinhuis, 1997).

It seems that this mechanism allows the cleaning of the surface to prevent dust from filtering sunlight and thus preventing photosynthesis. It could also play an important role in washing away pathogen spores before they germinate.

Finally, triterpenoids found in waxes, particularly oleananes, lupines and ursanes are also thought to impact the permeability of cuticles to water vapour [START_REF] Jetter | Composition of Plant Cuticular Waxes[END_REF][START_REF] Schuster | The ecophysiology of leaf cuticular transpiration: are cuticular water permeabilities adapted to ecological conditions?[END_REF].

Protection against pathogens

Several studies have shown that the cuticle is an important element in providing protection against pathogen invasion. In contrast with the critical role of waxes in creating an efficient water barrier, it appears that the cutin polymer is likely more important in providing protection against pathogens. Highly counterintuitively, several mutants showed an increase in both leaf surface permeability and resistance to fungal pathogens, including mutants lacking the function of LCS2 [START_REF] Bessire | A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea[END_REF], BDG [START_REF] Chassot | Cuticular defects lead to full immunity to a major plant pathogen[END_REF] or DEWAX [START_REF] Ju | DEWAX Transcription Factor Is Involved in Resistance to Botrytis cinerea in Arabidopsis thaliana and Camelina sativa[END_REF]. Several potential mechanisms may explain why plant cuticle permeability is associated with increased resistance to fungal pathogens such as Botrytis [START_REF] Ziv | Multifunctional Roles of Plant Cuticle During Plant-Pathogen Interactions[END_REF]. Firstly, there is relatively little evidence that cutin acts as a physical barrier against pathogens. Instead it appears to be a source of signals that warn the plant of pathogen attack. Fungal pathogen ingress often involves cell wall digestion that can release cutin monomers and cell wall oligosaccharides [START_REF] Aragón | The intimate talk between plants and microorganisms at the leaf surface[END_REF]. These are perceived by the plants as Damage Associated Molecular Patterns (DAMPS) and trigger the plant to release antifungal diffusates and Reactive Oxygen Species (ROS) which inhibit the pathogen growth and infection [START_REF] L'haridon | A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity[END_REF][START_REF] Sela | Overexpression of AtSHN1/WIN1 Provokes Unique Defense Responses[END_REF]. These elicitors also trigger basic plant defence responses and cell wall repair responses [START_REF] Aragón | The intimate talk between plants and microorganisms at the leaf surface[END_REF][START_REF] Gilbert | Chemical signals responsible for appressorium formation in the rice blast fungusMagnaporthe grisea[END_REF]. It has been proposed that mutants in cuticle production could have increased basal levels of cutin monomers, and this show increased basal disease resistance (priming). Furthermore, increased cuticle permeability would facilitate the movement of anti-fungal components across the cell wall, enhancing their effects. Finally, pathogen-derived elicitors could also diffuse more rapidly into the epidermal cells and accelerate more classical pathogen activated responses.

Interestingly however, not all cuticle mutants improve plant resistance to all pathogens. Indeed susceptibility to some bacterial pathogens may be enhanced in the same mutants that show reduced fungal pathogen susceptibility as demonstrated in the A. thaliana shine mutant [START_REF] Sela | Overexpression of AtSHN1/WIN1 Provokes Unique Defense Responses[END_REF].

In addition to protection against pathogens, the cuticle is also likely involved in protection against insects. This is again very dependent on the insect species. As an example, reproduction and feeding of aphids on A. thaliana leaves does not seem to be correlated with wax load and composition but are potentially affected by the defective cutin of cer1 mutants [START_REF] Eigenbrode | Attachment to Plant Surface Waxes by an Insect Predator1[END_REF][START_REF] Negin | Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins[END_REF]. In general, glossy phenotypes with reduced epicuticular waxes have been shown to be correlated with greater resistance to insect attack [START_REF] Shepherd | idaeus L.)[END_REF] possibly because insects find it harder to "stick" to glossy leaves.

Finally, a more recent paper in Nicotiana glauca showed that phloem feeding insects are only affected by cutin disruption, while oviposition was affected by wax crystal structure but not its chemical composition [START_REF] Negin | Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins[END_REF].

Protection against UV radiation

Cuticles can play an important role in UV light protection. Although the protein complexes involved in photosynthesis complex are designed to deal with moderate UV irradiation in many species, they can be damaged by excessive UV light. The cuticle can provide a protective layer with dense epicuticular wax crystals which will increase the reflection of UV irradiation and thus contribute to protecting the sensitive inner tissues preventing damage both to the chloroplasts and, indeed, to nuclear DNA [START_REF] Krauss | Attenuation of UV radiation by plant cuticles from woody species[END_REF].

Specific compounds enhance UV protection, including hydroxycinnamic acid and related phenolics. Phenolics, covalently bound to the fatty acids of cutin, are thought to be responsible for the absorption of UV-B [START_REF] Baur | Absorption of UV-B to blue light radiation by leaf cuticles of selected crop plants[END_REF][START_REF] Ju | 373 Phenolics and Lipid-soluble Antioxidants in Fruit Cuticle of Apples and Their Antioxidant Activities in Model Systems[END_REF]. Moreover, experiments on apple fruits confirm the protective role of phenolics as UV-A and UV-B filters, which can trap a massive proportion of these radiations before they reach the epidermal cells. [START_REF] Solovchenko | Optical properties and contribution of cuticle to UV protection in plants: Experiments with apple fruit[END_REF].

Waxes are also important in protecting against UV radiation. The reflection of UV and visible light by waxes and epicuticular wax crystals has been quite extensively studied. In some species such as Dudleya brittonnii, a plant living in arid condition, up to 83% of UV-B radiation is reflected by epicuticular waxes [START_REF] Mulroy | Spectral properties of heavily glaucous and non-glaucous leaves of a succulent rosette-plant[END_REF]. The epicuticular waxes of this plant but also of other species, contains high levels of triterpenoids and alkanes [START_REF] Manheim | Interspecific variation in leaf wax of Dudleya[END_REF].

Consistent with the need to reconcile ongoing photosynthesis with UV protection, waxes have been shown to be better at reflecting UV light than visible lights in some cases [START_REF] Holmes | Effects of pubescence and waxes on the reflectance of leaves in the ultraviolet and photosynthetic wavebands: a comparison of a range of species[END_REF].

Other, more esoteric roles for the cuticle include roles in insect attraction through the generation of petal iridescence. This phenomenon has been described in Hibiscus trionum and in Eschscholzia californica (California poppy) where the cuticle on iridescent petals forms regular folds which diffract sunlight. This pattern is recognized by bees much faster than noniridescent flowers [START_REF] Moyroud | Disorder in convergent floral nanostructures enhances signalling to bees[END_REF][START_REF] Wilts | Ultrastructure and optics of the prism-like petal epidermal cells of Eschscholzia californica (California poppy)[END_REF].

Preventing adhesion and fusion of organs

Finally the cuticle plays key roles in plant development by preventing adhesion and fusion of developing organs [START_REF] Ingram | The roles of the cuticle in plant development: organ adhesions and beyond[END_REF]. Besides an increase of water loss, mutants with cuticle defect show morphological abnormalities. Numerous mutants have been reported during the development of vegetative and floral organs as well as during seed development, to combine cuticle defects and organ fusion. Interestingly, the adhesion and fusion of organs are mainly seen in cutin-defective mutants rather than wax-defective mutants.

Good examples are loss of function alleles of LCR and BDG in leaves, and ABCG11 and FDH in vegetative organs and inflorescences [START_REF] Bird | Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion[END_REF][START_REF] Jakobson | BODYGUARD is required for the biosynthesis of cutin in Arabidopsis[END_REF][START_REF] Kurdyukov | The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis[END_REF][START_REF] Panikashvili | The Arabidopsis DSO/ABCG11 transporter affects cutin metabolism in reproductive organs and suberin in roots[END_REF][START_REF] Wellesen | Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega -hydroxylation in development[END_REF]. Consistent with these results, expression and secretion of fungal and plant cutinases in A. thaliana leads to organ fusion (Sieber et al., 2000;Takahashi et al., 2010).

However, it is important to keep in mind that not all cutin-defective mutants show organ fusions as it is the case in A. thaliana with the gpat4gpat8 mutant, which affects enzymes involved in cutin monomer biosynthesis. This mutant only shows a reduction in growth [START_REF] Li | Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers[END_REF].

Such differences may reflect different degrees of defect severity since the triple mutants combination of gpat4gpat8 with another cutin-defective mutant, lacking the ABCG32 transporter shows organ fusion as well as strong dwarfism [START_REF] Fabre | The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis[END_REF].

Key innovations in seed plants

The seed is a structure produced by all spermatophytes (gymnosperms and angiosperms). They are vital for plant survival since they ensure that the future plant, the embryo (usually the product of sexual reproduction), is nourished, protected and successfully dispersed by the mother plant.

Spermatophytes, which are inherently sessile have, over the course of evolution, innovated multiple means of seed dispersal. These include harnessing the movement of external agents such as air (for example in dandelion, with its flying akene [START_REF] Seale | From passive to informed: mechanical mechanisms of seed dispersal[END_REF], or pine trees with their winged seeds), water (the floating coconut seed), and animals (primates [START_REF] Lambert | Evolutionary and ecological implications of primate seed dispersal[END_REF] and birds which disperse the seeds within the fleshy angiosperm fruits or the "pseudo fruits" of gymnosperms such as yew, or insects such as hornets and ants which disperse Stemona tuberosa [START_REF] Chen | Seed dispersal by hornets: An unusual insect-plant mutualism[END_REF]). Some angiosperms have even developed elegant mechanical tricks to generate their own kinetic enery and "fling" their seeds away (for example Cardamine hirsute with the explosive dispersal strategy of the siliques [START_REF] Hofhuis | Morphomechanical Innovation Drives Explosive Seed Dispersal[END_REF]. This dissemination is critically important for the conquest of new environments by sessile plants. However, for this dispersal to be really successful seeds also require further qualities, such as the ability to survive in a quiescent state for long periods of time. Perhaps the most impressive example of this is a 30 000 yearold silene seed found in the Siberian permafrost that germinated and gave rise to a viable plant [START_REF] Yashina | Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost[END_REF]. The ability of some seeds to remain viable, sometimes in extreme environments, over long time periods, is the culmination of multiple physiological and physical innovations that vary between species. They generally include the ability to delay germination until the environment is propitious (dormancy, which is conditioned by both physiological and physical factors), as well as the ability to maintain cell viability whilst in a highly dehydrated and biochemically inactive state. However, these factors would count for nothing without the presence of significant amounts of nutrients within most seeds, which serve to support the embryo when it does eventually make the decision to germinate, and which allow it to grow until it reaches metabolic autonomy (through photosynthesis and efficient water and mineral uptake). These energy supplies, which can be impressive (there are about 1400 kcal in a medium sized coconut), make seeds the primary source of energy for most humans and agriculturally produced animals. How did these remarkable structures evolve?

The seed

The transition to seeds: from the early seed-like structures to the seed

The transition to seeds is likely to have appeared in the progymnosperms, the sister group of Streptophytes, in the late Devonian (370Mya). These progymnosperms had a vegetative structure typical of seed plants combined with a mode of reproduction via spores that is more typical of Ferns. Three major steps likely led to the transition from the progymnosperms to the Streptophytes [START_REF] Doyle | Seed ferns and the origin of angiosperms[END_REF][START_REF] Linkies | The evolution of seeds[END_REF][START_REF] Taylor | Seed ferns from the late Paleozoic and Mesozioc: Any angiosperm ancestors lurking there?[END_REF]. The first was the evolution of heterospory which is the production of morphologically distinct products of meiosis within separate organs considered as female (producing megaspores, and subsequently female gametophytes) and male (producing microspores, and subsequently male gametophytes (pollen). Heterospory is observed in some fern species. The evolution of the integuments, specialized organs that enclose the megaspore/megagametophyte-producing tissue (the nucellus) to give compound structures called ovules, is considered to be another key innovation. Finally the evolution of specialized structures associated with the ovule, that are capable of receiving pollen is considered to be a critical third innovation. Together these innovations underly the transition to water-independent plant reproduction. The discovery of several fossils from extinct progymnosperms has led to a better understanding of this transition to seed production. Some examples are explained below.

Archaeopteris, an extinct progymnosperm, may be a good example of the first step during the transition to seeds. This extinct progymnosperm was a tree that produced structurally distinct megaspores and microspores from separate sporangia. Over time, preovules and ovules appeared, as seen in Runcaria, the oldest pre-ovule fossil. The female gametophyte is retained in the maternal sporophytic tissue of the ovule [START_REF] Gerrienne | The proto-ovule Runcaria heinzelinii Stockmans 1968 emend[END_REF]Gerrienne et al., 2004). The oldest known fossil seed-bearing plant is Elksinia polymorpha, a so called "seed fern" belonging to the extinct Pteridospermatophyta, from the Late Devonian [START_REF] Dimichele | Paleoecology of Late Paleozoic pteridosperms from tropical Euramerica 1[END_REF][START_REF] Linkies | The evolution of seeds[END_REF][START_REF] Niklas | The Evolutionary Biology of Plants[END_REF]. In this species the ovule (or pre-ovule) is partially enclosed in a cup-like structure called cupule. To date, the role of these cupules remain unknown although scientists hypothesized a role in seed protection and/or seed dispersal [START_REF] Soltis | Ancient seeds spill secrets about the evolution of flowering plants[END_REF]. The cupule was then replaced in the later medulossan seed ferns by a true testa (also called a seed coat). The testa is derived from the integuments of the ovules), is composed of three layers, and encloses the female gametophyte and, after fertilization, the embryo [START_REF] Doyle | Seed ferns and the origin of angiosperms[END_REF][START_REF] Taylor | Seed ferns from the late Paleozoic and Mesozioc: Any angiosperm ancestors lurking there?[END_REF].

To summarize during the emergence of seed plants the sporophyte generation became dominant and in parallel the gametophyte became smaller but still nourished and protected the embryo. Ultimately the female gametophyte was fully retained within sporophytic tissues, and the seed was born. Seeds contributed to the evolutionary success of Streptophytes, as illustrated by the dominance of gymnosperms for around 300My [START_REF] Willis | The Evolution of Plants[END_REF].

Much more recently further reproductive innovations, including the development of the ovary, and importantly, double fertilization with the associated development of a gametophytic derived tissue, the endosperm (described below), are thought to have contributed to the explosive radiation of the angiosperms.

What is a seed?

To summarise; two of the three compartments of the seeds are the product of fertilisation in gymnosperms and angiosperms: the embryo and the endosperm. Seed are composed of three genetically distinct tissues: 1) the sporophytic embryo nourished by 2) the female gametophyte (or, in angiosperms the female gametophyte-derived endosperm), which is surrounded by 3) a coat derived from sporophytic tissues. Seeds develop from ovules which consist of one (gymnosperms) or two (most angiosperms) integuments surrounding the nucellus (the megasporangium). From a developmental point of view, the ovule can thus be considered as an unfertilized seed, the immature seed precursor [START_REF] Gasser | Genetic Analysis of Ovule Development[END_REF] and from an evolutionary point of view, as a megasporangium surrounded by integument(s) [START_REF] Linkies | The evolution of seeds[END_REF]. The integument(s) ultimately give rise to the testa or seed coat, usually after pollination.

GYMNOSPERM SEEDS.

The word gymnosperm comes from the Greek words: gymnos which means naked, and sperma, the seed. Gymnosperms form a monophyletic group within the spermaphyte, and, as their name suggests, produce naked seeds. Most gymnosperm seeds are produced on reproductive structures called cones, and can be found directly on the scales (megasporophylls) of these cones. Gymnosperm ovules contain single multicellular female gametophytes that can produce several archegonia, each of which usually contains one egg cell (for example in pine, 2-3 archegonia are formed). Although some proliferation and nutrient storage can occur within the female gametophyte prior to pollination, the majority of seed development occurs after the fertilization of the ovule by sperm cells which can be motile or non motile, and are usually delivered by pollen grains. Pollination occurs when the male gametophyte i.e. the pollen grain, is deposited on the ovule. After pollination in most gymnosperms, one or more pollen tubes grow and transport the sperm cells to fertilize the egg cells which leads to development of embryos. In contrast, Ginkgo and Cycads release multiflagellated sperm from the pollen grains, which are captured in droplets of liquid extruded from the ovule, thus allowing the sperm to swim to the egg cell [START_REF] Doyle | Seed ferns and the origin of angiosperms[END_REF]. Several embryos may initiate development, but usually one "wins" and the others degenerate. A typical gymnosperm seed is thus composed of a single embryo covered of two other tissues (Figure 6). The first of these is the haploid maternal megagametophyte which absorbs nutrients from surrounding maternal tissues and nourishes the embryo, which in turn grows invasively into, and consumes, the megagametophyte tissues. The second is the diploid integument tissue which develops into the testa. Interestingly, most of the nucellus is replaced by the female gametophyte which is the main storage tissue in the Gymnosperm seed [START_REF] Linkies | The evolution of seeds[END_REF].

Angiosperm seeds.

Angiosperms and gymnosperms show several morphological differences both in terms of the whole plant body and the seed. However, angiosperms arose from the gymnosperm lineage, and the seeds of gymnosperms therefore also likely share characteristics with those of the angiosperm ancestor. Nonetheless angiosperms, in terms of their reproductive development, developed three major innovations not found in gymnosperms: the flower, a complex of reproductive organs promoting pollination and fertilisation ii) the pollen-receptive ovary that encloses the ovules and develops into a fruit after fertilization and iii) double fertilization, which results in the production of the endosperm.

The angiosperm flower, is a complex of reproductive organs composed of a perianth surrounding and protecting the male reproductive organs (called stamens) and the female reproductive organs (called carpels) [START_REF] Bateman | Morphological and molecular phylogenetic context of the angiosperms: contrasting the 'top-down' and 'bottom-up' approaches used to infer the likely characteristics of the first flowers[END_REF]. The female reproductive organs comprise one or more ovaries (thought to derive, like the organs of the perianth, from leaf-like structures). The ovules are attached by stalk-like structures to the inner surface of the ovary, which plays roles in pollen reception, selection and transmission, the transmission of nutrients from the maternal plant to the seed, and seed protection and dispersal (through fruit formation).

Fruits come in numerous shapes and forms and can sub-classed into multiple different categories but these will not be described here (Łangowski et al., 2016). As described above, the angiosperm ovule is composed of diploid maternal tissue, in the form of the nucellus (megasporangium) surrounded by the integuments. Most angiosperms produce ovules with two integuments, the inner and the outer, which are morphologically and functionally distinct, and whose development is largely mutually independent [START_REF] Endress | Angiosperm ovules: diversity, development, evolution[END_REF][START_REF] Gasser | Chapter Fourteen -Development and evolution of the unique ovules of flowering plants[END_REF].

Within the nucellus, during ovule development, a single maternal cell (MMC) is specified as the megaspore mother cell, and becomes morphologically distinct. This cell undergoes meiosis to give four daughter cells, of which one is selected to survive (and goes on to give the megaspore), whilst the other three usually degenerate. The megaspore then undergoes a series of mitotic divisions to give the female gametophyte. These steps are very similar to the situation in gymnosperm ovules. However, whilst in gymnosperms the female gametophyte can become quite large prior to fertilisation, and produce several archegonia, each containing an egg cell, in angiosperms the unfertilised female gametophyte is highly reduced. In most angiosperms only 3 rounds of mitosis occur, to give 8 nuclei, whilst in some more ancestral angiosperms, the mature female gametophyte contains only 4 nuclei [START_REF] Friedman | Reconstructing the ancestral female gametophyte of angiosperms: Insights from Amborella and other ancient lineages of flowering plants[END_REF]. The cells of the female gametophyte undergo cellularization and differentiate to give, at the distal pole (where the tips of the integuments meet to enclose the nucellus), an egg cell and 2 accessory cells called synergids, which are required for successful fertilization. More proximally a central cell is formed, which in 8 celled gametophytes is binucleate (double haploid), and in 4 celled gametophytes is mononucleate (haploid). 8 celled gametophytes also form "antipodal cells" (3) at the extreme proximal pole, adjacent to the "stalk" (funiculus) which attaches the ovule to the maternal tissues.

Understanding this organization is critical when describing the third particularity for angiosperm seeds, which is the development of the endosperm, the product of the unique, angiosperm-specific double fertilization event, which is described in more detail below, but which involves the fusion of the two genetically identical sperm cells carried to the ovule via the pollen tube with the egg cell (to give the embryo) and with the central cell, respectively (Figure 6). The endosperm is the result of central cell fertilization and, in 8 celled gametophytebearing species is triploid (the fusion of the two haploid central cell nuclei and one haploid sperm cell nucleus). In species with a 4 celled female gametophyte the resulting endosperm is thus diploid. Fertilization triggers the growth of the endosperm, and the embryo that it surrounds, a process that requires nutrients furnished by the mother plant through the nucellus and/or integuments. Endosperm characteristics and functions are described in the following section §2.4.3.1, Introduction. Angiosperm seeds are thus composed of three genetically distinct compartments organized like Russian dolls: i) the sporophytic embryo which gives the new plant, surrounded by ii) the (usually triploid) endosperm, which is considered to be analogous to the nutrient storing female gametophyte tissues found in gymnosperms, and which nourishes the embryo and iii) the sporophytic maternal tissue, the seed coat which surrounds the endosperm (Figure 6).

Double fertilization in angiosperms has been proposed to be a major factor in their dramatic radiation. It provides a coordinated trigger for endosperm and embryo development, thus potentially minimising investment by the mother plant in unfertilized ovules. Furthermore, the role of the endosperm in absorbing maternal nutrients, combined with the genetic contribution of both parents to this tissue, also appears to have led to "parental conflicts" within the endosperm, similar to those described in the mammalian placenta. To anthropomorphise, in outcrossing species the male genome is geared to maximise maternal contributions to the development of each of his offspring, whilst the maternal genome(s) aims to minimise/equilibrate nutrient distribution to give the maximum number of viable seeds per plant. Since this conflict is played out at the developmental/physiological level within the endosperm, changes in genome dosage in the endosperm (for example if a tetraploid mother encounters pollen from a diploid father) can lead to severe defects in endosperm function and lethality, providing an instantaneous block to successful reproduction (reproductive isolation) between the two plants; the basis of speciation. Since plants can double their genomes rather easily, this mechanism has been evoked as a potential driver of the rapid angiosperm radiation [START_REF] De | Hamilton's rule, imprinting and parent-offspring conflict over seed mass in partially selfing plants[END_REF][START_REF] Haig | Parent-Specific Gene Expression and the Triploid Endosperm[END_REF][START_REF] Lafon-Placette | Endosperm genome dosage, hybrid seed failure, and parental imprinting: sexual selection as an alternative to parental conflict[END_REF][START_REF] Lafon-Placette | Endosperm-based postzygotic hybridization barriers: developmental mechanisms and evolutionary drivers[END_REF].

Seed development in

Angiosperms requires double fertilization.

Generalities in seed development

As described above the development of the embryo and the endosperm is initiated by double fertilization which takes place in the ovule. The ovules develop within the ovaries which are located below pollen receiving and transmitting tissues called style and the stigma. The whole structure is called the carpel. As already mentioned, within each ovule the female gametophyte forms within the nucellus (which plays a role in nutrient provision) and the nucellus is enclose within the integuments. The female gametophyte is generally composed of 4 cell types derived from mitotic divisions: the egg cell, the synergids, the central cell and the antipodal cells [START_REF] Dresselhaus | Fertilization Mechanisms in Flowering Plants[END_REF].

The male gametophyte, in the form of the pollen grain, is produced within a separate floral organ, the stamen, which is composed of the anthers (where the pollen grains develop), and the filament which raises the anther out of the flower. Pollen grains are again formed from the products of meiosis (microspores). Microspores undergo one round of mitosis to form a vegetative cell and a generative cell. The later then undergoes a second mitotic division to producing the two haploid sperm cells (gametes). The male gametophyte is thus even more highly reduced than the female gametophyte. Mature pollen is released from the anther and once it lands on a receptive stigma, it germinates to produce a pollen tube which grows towards the ovules guided by molecular and physical cues within in the tissues of the stigma, style and ovaries. Ultimately pollen attractants emitted by the ovules themselves guide the pollen tubes towards the distal opening (the micropyle) of the ovule [START_REF] Mizuta | Chemical signaling for pollen tube guidance at a glance[END_REF][START_REF] Zhang | Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction[END_REF].

Once within the ovule the pollen tube bursts within one of the two synergids cells flanking the egg cell, releasing the two sperm cells, one of which fuses with the egg cell, generating the zygote, while the other fuses with the central cell, generating the endosperm [START_REF] Baroux | Evolutionary origins of the endosperm in flowering plants[END_REF][START_REF] Berger | Double-fertilization, from myths to reality[END_REF]. As noted above, since the sperm cells are generated by post meiotic mitoses, they are genetically identical as it is the case for the cells of the embryo sac. Thus the endosperm and the embryo usually carry the same genetic information, although they have a distinct fates [START_REF] Dresselhaus | Fertilization Mechanisms in Flowering Plants[END_REF].

Fertilization of the endosperm and embryo trigger their development, apparently largely independently. However endosperm fertilization has also been shown to be necessary to non cell-autonomously trigger the differentiation and expansion of the seed coat, probably via the production of auxin within the endosperm, and its transport to the seed coat tissues [START_REF] Figueiredo | Auxin: a molecular trigger of seed development[END_REF].

Seed Development in Arabidopsis thaliana

Much of the information provided above, although important for providing context, is rather general and can be applied to many species. However, to introduce the seed development work specifically carried out in my thesis, I will now concentrate on providing a more detailed overview of this process in the eudicotyledon angiosperm, A. thaliana.

THE EMBRYO.

The embryo develops through successive mitoses of the unicellular zygote during embryogenesis. This process can be divided into three parts : cell and tissue specification, organ growth and filling and dehydration [START_REF] Dumas | Fertilization and early seed formation[END_REF]. Embryogenesis starts directly after double fertilization. The zygote has an inherent basal (distal) apical (proximal) polarity, that it inherits from the polarity of the egg cell, and elongates along the apical/basal axis in the first few hours after fertilization. The first cytokinesis is asymmetric and gives a large basal and a small apical cell [START_REF] Bayer | Early plant embryogenesis-dark ages or dark matter?[END_REF][START_REF] Ten Hove | Building a plant: cell fate specification in the early Arabidopsis embryo[END_REF]. The basal cell subsequently undergoes several divisions to become the suspensor. The suspensor is a largely transient structure which is involved in both taking up nutrients and hormones from the endosperm, and the integuments, during early embryo development and in anchoring the embryo within the seed [START_REF] Kawashima | The suspensor: not just suspending the embryo[END_REF]. Although most of the suspensor undergoes programmed cell death relatively early during seed development, the upper cell of the suspensor contributes to the root apical meristem (RAM) of the embryo proper.

The apical cell of A. thaliana zygote divides with a rather stereotypical pattern to give the embryo proper. During this process, cells progressively acquire specific fates. One of the first to be acquired is protodermal fate, which is acquired by cells at the surface of the embryo, which will later give rise to the embryonic epidermis. Vasculature and ground tissue specificity is specified later as inner cells are produced through divisions. The upper tier of the ground tissue gives rise to the shoot apical meristem (SAM). During the early stages of embryogenesis, the embryo proper is more or less spherical (globular) (Figure 6). However, an important shape change transition occurs with the growth of the two nascent cotyledon primordia, which emerge on the flanks of the SAM to give a triangular and then a heart shaped embryo. [START_REF] Ten Hove | Building a plant: cell fate specification in the early Arabidopsis embryo[END_REF]. Subsequently the basal pole of the embryo differentiates into the root primordium. A massive amount is known about the early patterning of the embryo, which is regulated by cascades of transcription factors, signalling molecules, and, importantly, dynamic and complex auxin fluxes. This process has been very extensively reviewed [START_REF] Bayer | Early plant embryogenesis-dark ages or dark matter?[END_REF][START_REF] Dumas | Fertilization and early seed formation[END_REF][START_REF] Ten Hove | Building a plant: cell fate specification in the early Arabidopsis embryo[END_REF][START_REF] Kawashima | The suspensor: not just suspending the embryo[END_REF], and will not be discussed in detail here. The first stage of embryogenesis ends at around the heart stage, at which point all the main tissues and cell types that are found in the mature embryo have been specified and can be easily distinguished [START_REF] Ten Hove | Building a plant: cell fate specification in the early Arabidopsis embryo[END_REF].

The second phase of embryogenesis involves embryo growth and the initiation of nutrient uptake and storage from the endosperm. The embryo grows through cell division and expansion, which force it to invade the endosperm tissues that surround it (see below). As the embryo grows its shape continues to change, and the progression through the torpedo, walking stick and bend cotyledon phases is used as a measure of the progression of seed development (Figure 6). The torpedo stage; occurs approximately 7-8 days after pollination (depending strongly on growth conditions). During this phase the embryo elongates from the base to the "tip" of the seed. However, once the cotyledons reach the tip of the seed, they are forced to bend over, effectively folding the embryo in two (walking stick and bent cotyledon stages).

Ultimately, continued embryo expansion and elongation forces the tips of the cotyledons down the proximal domain of the seed (next to the seed stalk/funiculus) (Figure 6). At this stage (mature green (approximately 13 days after pollination)) the embryo fills the entire seed, replacing most of the endosperm and is thus the biggest compartment within the seed. The mature embryo is composed of the two cotyledons (enclosing the SAM) and the hypocotyl (tipped by the nascent root meristem) [START_REF] Le | Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors[END_REF].

THE ENDOSPERM.

A. thaliana endosperm development can be divided into three temporally overlapping phases: coenocytic endosperm development (expansion), cellularization of the endosperm and elimination (Figure 6). Once the second sperm cell has fertilized the central cell, the endosperm rapidly develops into a multinucleate coenocyte through nuclear division in the absence of cytokinesis. This is accompanied by very rapid expansion of the endosperm and surrounding maternal tissues, which determines the final size of the seed. At the end of this phase, corresponding to the late globular-early heart stage of embryogenesis, a large vacuole occupies the centre of the endosperm cavity, with the endosperm cytoplasm and nuclei distributed over the endosperm periphery [START_REF] Berger | Endosperm: the crossroad of seed development[END_REF][START_REF] Olsen | ENDOSPERM DEVELOPMENT: Cellularization and Cell Fate Specification[END_REF].

Cellularization is initiated at the micropylar pole, around the embryo at the late globular stage-early heart stage and progresses until the late torpedo/walkingstick stage. The endosperm region near the chalazal (proximal) endosperm (CE) never cellularizes [START_REF] Brown | Development of endosperm in Arabidopsis thaliana[END_REF]. The process of cellularization is rather mysterious and appears to be linked, to some extent, to ongoing nuclear proliferation [START_REF] Sørensen | Cellularisation in the endosperm of Arabidopsis thaliana is coupled to mitosis and shares multiple components with cytokinesis[END_REF]. It presumably involves fragmentation of the central vacuole, although how this is achieved mechanistically remains unclear. After cellularisation three populations of endosperm cells exist 1) a peripheral cell layer, 2) internal cells (the central endosperm) and 3) the uncellularized chalazal endosperm (Figure 6).

The third stage of endosperm development initiates at around the heart stage around the embryo and progresses in a wave, closely following the wave of endosperm cellularization.

During this phase, the central endosperm is rapidly eliminated make space for the growing embryo. This step involves the death of the endosperm, but also its physical separation from the embryo (see below). Unlike the central endosperm, the outer endosperm layer (the peripheral endosperm, PE) is persistent at maturity in A. thaliana. It develops as morphologically distinct cuboid cells. This layer plays roles in dormancy, germination and lipid storage [START_REF] Bethke | Rebirth and death: Nitric oxide and reactive oxygen species in seeds[END_REF][START_REF] Bryant | ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 Are Responsible for Making Omega-7 Fatty Acids in the Arabidopsis Aleurone[END_REF][START_REF] Penfield | Reserve Mobilization in the Arabidopsis Endosperm Fuels Hypocotyl Elongation in the Dark, Is Independent of Abscisic Acid, and Requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1[END_REF].

In summary, at maturity in A. thaliana, only one layer of endosperm remains surrounding the surface of the embryo. However endosperm development varies dramatically across the Angiosperms [START_REF] Dumas | Fertilization and early seed formation[END_REF].

THE MATERNAL TISSUE.

The maternal tissues of the A. thaliana seed are composed of the integuments and the nucellus. The two integuments enclose the nucellus and the zygotic tissues. The nucellus is a largely transient structure. Initially, during megaspore selection and early embryo sac formation, it encloses the entire embryo sac, separating it from the integuments. However, the distal nucellus is eliminated well before ovule maturity, leaving only a small population of proximal nucellus cells that separates the embryo sac from the chalazal zone of the ovule into which nutrients are unloaded from the funiculus [START_REF] Ingram | Dying to live: cell elimination as a developmental strategy in angiosperm seeds[END_REF]. Consistent with this position, the function of the nucellus in A. thaliana appears to be the control of the nutrient transfer from maternal tissues to the embryo sac (prior to fertilization) and to the endosperm during early seed development [START_REF] Lu | Seed tissue and nutrient partitioning, a case for the nucellus[END_REF][START_REF] Lu | The nucellus: between cell elimination and sugar transport[END_REF]. After fertilization the unfertilized antipodal cells of the female gametophyte (which lie adjacent to the nucellus) degenerate, and this is followed by the degeneration of the distal part of the remaining nucellus [START_REF] Ingram | Dying to live: cell elimination as a developmental strategy in angiosperm seeds[END_REF][START_REF] Maruyama | Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism[END_REF][START_REF] Xu | Endosperm and Nucellus Develop Antagonistically in Arabidopsis Seeds[END_REF].

The different fates and morphologies of the seed tissues and compartments (such as the nucellus or the endosperm) appear to be determined by their role as a storage/transport compartment for nutrients. The timing and importance of these roles in different tissues is highly species specific, although three types of seeds have been classified based of their nutrient storage compartment. In perispermic seeds, such as Amaranthus and quinoa, the main nutrient storage compartment is, in fact the nucellus, which forms a persistent storage tissue called the perisperm, which is mobilized at germination. In these species the endosperm is very much reduced [START_REF] Burrieza | Analogous reserve distribution and tissue characteristics in quinoa and grass seeds suggest convergent evolution[END_REF][START_REF] Lu | Seed tissue and nutrient partitioning, a case for the nucellus[END_REF][START_REF] Magnani | Seed Evolution, A "Simpler[END_REF]. The so called "endospermic seeds" such as those of maize or other cereals, contain a largely persistent endosperm, which stores large quantities of nutrients until germination, when they are remobilized to support the development of the germinating seedling [START_REF] Sreenivasulu | Seed-Development Programs: A Systems Biology-Based Comparison Between Dicots and Monocots[END_REF]. By contrast, in seeds with "transient" endosperm (such as A. thaliana or many legumes) the endosperm is completely or almost completely consumed during seed development by the embryo which thus become the main storage compartment [START_REF] Weber | MOLECULAR PHYSIOLOGY OF LEGUME SEED DEVELOPMENT[END_REF]. In reality these classifications are rather artificial. For example, in many cereals the nucellus forms a very transient "perisperm-like" storage tissue prior to fertilization that is rapidly eliminated and replaced by the endosperm after fertilization. In addition, some limited elimination does occur around the embryo in cereals such as maize, whereas some endosperm does persist in A. thaliana at seed maturity (Doll et al., 2020;[START_REF] Grimault | ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development[END_REF].

The reality therefore appears to be a continuum in which nutrients pass through/are stored in the nucellus, and/or the endosperm prior to remobilization towards the embryo at different developmental time-points in different species. Since seed storage strategies also vary across basal Angiosperm, it is difficult to establish which strategy can be considered as "ancestral" [START_REF] Friedman | Seed development in Trimenia (Trimeniaceae) and its bearing on the evolution of embryo-nourishing strategies in early flowering plant lineages[END_REF].

Coordination and regulation of the embryo/endosperm interface

During seed development the Russian doll organization of the three main seed compartments (maternal tissues, endosperm and embryo), and the controlled transfer of nutrients between them, necessitate very tight coordination and communication to ensure a successful developmental and metabolic outcome. How is this achieved?

The endosperm plays key roles in coordinating seed development THE EVOLUTION AND FUNCTION OF THE ENDOSPERM.

Despite the importance of the endosperm during seed development but also in human and animal nutrition (80% of maize kernel is the endosperm), the evolutionary origins of the endosperm remain unclear. To date, two hypotheses as to the evolution of the endosperm persist but neither of them has tipped the balance in its favour. The first one has been suggested in 1900 by [START_REF] Sargant | Recent Work on the Results of Fertilization in Angiosperms[END_REF]. It suggests that in seed-like structures the central cell was an additional egg cell. Thus, after fertilization two embryos would have developed simultaneously, with one adopting an "altruistic" function, and nourishing the second [START_REF] Friedman | Evidence of a Pre-Angiosperm Origin of Endosperm: Implications for the Evolution of Flowering Plants[END_REF][START_REF] Sargant | Recent Work on the Results of Fertilization in Angiosperms[END_REF]. This hypothesis was supported by the observation that two embryos are sometime found in some basal gymnosperms (Gnetales, such as Ephedra),

as a result from a double fertilization event [START_REF] Carmichael | Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte clade[END_REF][START_REF] Friedman | Double Fertilization in Ephedra, a Nonflowering Seed Plant: Its Bearing on the Origin of Angiosperms[END_REF].

However, Gnetales are now considered not to be closely related to the direct ancestors of the Angiosperms, which has led to reduced support for this first hypothesis [START_REF] Berger | Endosperm: the crossroad of seed development[END_REF][START_REF] Pires | Seed evolution: parental conflicts in a multi-generational household[END_REF]. The second hypothesis for the origin of the endosperm suggests that the endosperm derived from gametophytic structures which later acquired fertilization competence, tethering endosperm development to that of the embryo. The first factor that supports this hypothesis is the invasive growth of the embryo into the endosperm of angiosperms which is very similar to the observed interaction between the embryo and the nutritive female gametophytic tissues of gymnosperms. As evoked above, tethering endosperm development to fertilization, and thus embryo development could save energy by preventing the investment of maternal resources on unfertilized gametophytes. The fertilization of the central cell, implying the input of a paternal genome, has also been proposed to provide a form of hybrid vigor to this tissue [START_REF] Baroux | Evolutionary origins of the endosperm in flowering plants[END_REF][START_REF] Linkies | The evolution of seeds[END_REF][START_REF] Pires | Seed evolution: parental conflicts in a multi-generational household[END_REF].

Although the evolutionary origin of endosperm may remain unclear, it has both a central position and a central role in seed biology, which is often underestimated or even completely ignored. First and foremost, it serves as a nutrient source for the embryo during seed development and at germination. In most monocotyledons species, the majority of the endosperm is persistent and will only be broken down during seed germination to release the nutrients it stored during seed filling [START_REF] Ingram | Dying to live: cell elimination as a developmental strategy in angiosperm seeds[END_REF]. Studies showed that during germination, hydrolytic enzymes are secreted from the outermost layer (aleurone layer) of the cereal endosperm during seed germination, and that these diffuse into the starchy central endosperm reserves, allowing them to be catabolised and thus provide energy that can be absorbed via a specialised embryonic structure called the scutellum, thought to be analogous to a single cotyledon, to allow the germination of the embryo and seedling establishment (Yan et al., 2014). Similarly, in A. thaliana, the single endosperm layer that remains at seed maturity accumulates lipids that are then catabolized into sucrose during germination [START_REF] Penfield | Reserve Mobilization in the Arabidopsis Endosperm Fuels Hypocotyl Elongation in the Dark, Is Independent of Abscisic Acid, and Requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1[END_REF]. However, in species such as A. thaliana, the main compartment used to store nutrients acquired from the mother plant during seed development is the embryo (Berger, 1999).

The role of the endosperm in supplying nutrients to the embryo is not restricted to germination but is also observed during seed development. Nutrients are absorbed from the maternal tissues of the plant by the endosperm at specialized interfaces such as the chalazal endosperm in A. thaliana which is characterized by the presence of many organelles, and is situated close to the residual nucellus and the zone into which phloem vessels are unloaded in the chalazal seed coat. Several studies suggest that this chalazal endosperm transfers minerals but also sugars which are themselves used as chalazal endosperm regulators [START_REF] Lu | The nucellus: between cell elimination and sugar transport[END_REF][START_REF] Otegui | Developing Seeds of Arabidopsis Store Different Minerals in Two Types of Vacuoles and in the Endoplasmic Reticulum[END_REF]. Sugars have also been shown to enter endosperm at other sites than the chalazal endosperm in some Brassica species [START_REF] Montgomery | Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin[END_REF]. It seems likely that some sugars diffuse symplastically through the integuments to the micropyle and are then actively transported into the micropylar endosperm (ME), and from there to the embryo. This process likely involves the transporter SUCROSE TRANSPORTER5 (SUC5), which is strongly expressed in the micropylar endosperm [START_REF] Baroux | Chapter Twenty-One -Seeds-An evolutionary innovation underlying reproductive success in flowering plants[END_REF][START_REF] Baud | The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis[END_REF], as well as a cascade of sugar transporters from the SWEET family [START_REF] Chen | A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo[OPEN[END_REF]. Similar pathways appear to be taken by amino acids, which are also unloaded into the chalazal seed coat [START_REF] Karmann | The long and winding road: transport pathways for amino acids in Arabidopsis seeds[END_REF]. In cereals, the transport of sugars, and other nutrients into the endosperm appears to be predominantly achieved within highly specialized transport zones, showing specific cellular modifications such as the transfer cells observed within the Basal Endosperm Transfer Layer (BETL) of maize. Again molecular players such as sweet transporters are involved [START_REF] Sosso | Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport[END_REF]. Once within the endosperm the path of sugars and other nutrients to the endosperm has not been extensively studied, but both sugars and amino acids appear to be exported out of the endosperm at the interface with the embryo, and are presumably absorbed into embryonic tissues (Doll et al., 2020). Intriguingly, although it is tempting to think that because of its very close location to the embryo, the Embryo Surrounding Region (ESR) of the maize endosperm, which envelopes the very early maize embryo, like the micropylar endosperm in A. thaliana, might provide nutrition to the embryo, some studies suggested otherwise [START_REF] Olsen | Nuclear endosperm development in cereals and Arabidopsis thaliana[END_REF][START_REF] Sabelli | The Development of Endosperm in Grasses[END_REF].

The endosperm is also very important for allowing embryo growth. In many species it is the expansion of the endosperm which drives early seed growth and creates the space into which the embryo will subsequently expand. This is the case in A. thaliana [START_REF] Berger | Endosperm: an integrator of seed growth and development[END_REF][START_REF] Li | Endosperm: food for humankind and fodder for scientific discoveries[END_REF], where a balance between the growth-driving expansion of the endosperm and the growth restricting properties of the seed coat appear to condition the final size of the embryo [START_REF] Garcia | Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis[END_REF]. Furthermore, a recent study in Brassica has elegantly shown that physical constriction of the seed during growth, not only limits seed size, but may also set the timing for the switch to seed maturation programmes [START_REF] Rolletschek | The process of seed maturation is influenced by mechanical constraints[END_REF].

Finally, in addition to its roles in embryo nutrition during germination, the endosperm plays other key role in promoting seedling establishment post germination. In A. thaliana, a complex cross-talk between the embryo and endosperm is involved in ensuring that the decision to germinate is made at the correct time [START_REF] Lee | A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds[END_REF]. Several studies showed that endosperm softening is required for the growth of the embryonic root during germination [START_REF] Dutta | Endo-[beta]-Mannanase Activity Present in Cell Wall Extracts of Lettuce Endosperm prior to Radicle Emergence[END_REF]Lee et al., 2012b;[START_REF] Steinbrecher | The biomechanics of seed germination[END_REF][START_REF] Zhang | Involvement of reactive oxygen species in endosperm cap weakening and embryo elongation growth during lettuce seed germination[END_REF]. Moreover, mutants in specific cell wall modifying enzymes showed defects during the germination process (Iglesias- [START_REF] Iglesias-Fernández | Three endo-β-mannanase genes expressed in the micropylar endosperm and in the radicle influence germination of Arabidopsis thaliana seeds[END_REF][START_REF] Iglesias-Fernández | Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase-encoding gene AtMAN7[END_REF][START_REF] Sechet | Xyloglucan Metabolism Differentially Impacts the Cell Wall Characteristics of the Endosperm and Embryo during Arabidopsis Seed Germination[END_REF][START_REF] Shigeyama | α-Xylosidase plays essential roles in xyloglucan remodelling, maintenance of cell wall integrity, and seed germination in Arabidopsis thaliana[END_REF]Yan et al., 2014). Hormones directly affect the mechanical properties of the endosperm as revealed by analysis of the control of the expression of the wall softening Furthermore, during developmental endosperm elimination, the endosperm surrounding the embryo releases components rich in epitopes specifically detected by antiextensin antibodies used in the production of the sheath, an extra-cuticular layer deposited on the outer surface of the embryo as it develops (see section 2.4.3.2). Recently, the embryonic sheath was shown to act as a lubricant facilitating cotyledon emergence from the seed coat during germination (Doll et al., 2020). In addition, very recent work even suggests that endosperm-derived peptides may be involved in reinforcing the embryonic cuticle itself during germination, via a pathway similar to that used in integrity monitoring during casparian strip development (discussed in more detail below) [START_REF] De Giorgi | The Mature Endosperm Governs Early Seedling Development: A Focus on Cuticle Formation[END_REF].

IMPORTANCE OF COORDINATING ENDOSPERM DEVELOPMENT WITH EMBRYO DEVELOPMENT.

The endosperm occupies a central position in the seed. It separates the embryo from the maternal nutrient source close to the chalazal pole and from the integuments. Thus, irrespective of its persistence in the mature seed, the endosperm is likely to assume a pivotal role during seed development. Consistent with this, analysis of rice mutants with altered embryo size has shown that a number of mutations specifically affect the endosperm, highlighting the role of the endosperm in embryo development [START_REF] Hong | How is embryo size genetically regulated in rice?[END_REF]. Some mutants present larger embryos than normal with reduced endosperm, whereas in others an enlarged endosperm occupies the space left by an embryo of reduced size. Thus, the endosperm likely exerts both positive and negative influences on embryo development.

The coordination between embryo and endosperm development necessitates a high level of communication between compartments. Symplastic connections (plasmodesmata)

between the embryo proper and endosperm appear to be lost soon after fertilization [START_REF] Ingram | Family life at close quarters: communication and constraint in angiosperm seed development[END_REF]. As a result, communication must either involve the diffusion of signals across the apoplast between the two compartments, or mechanical cues. Mutants in genes expressed specifically in one compartment give information about compartment interdependence. In A.

thaliana an outstanding example for this interdependence is shown in mutants that have lost the function of the endosperm-specific ZHOUPI (ZOU) protein. Loss of function zou mutants, instead of having a largely transient endosperm, have a completely persistent endosperm, apparently due to a defect in cell wall modifications that are usually required for endosperm cell wall softening and cell separation. The embryo cannot grow through the persistent endosperm and remains small, although it seems to mature normally since it undergoes desiccation and is capable of germinating to give morphologically normal plants [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF][START_REF] Kondou | RETARDED GROWTH OF EMBRYO1, a new basic helix-loop-helix protein, expresses in endosperm to control embryo growth[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. The opposite is also true, embryo defects also affect endosperm development as can be in maize and A. thaliana with embryo-less mutants. In these mutants, the ESR, and later the EAS (Endosperm Adjacent to the Scutellum)

does not develop properly in maize whereas in A. thaliana when the embryo dies at early stages, endosperm elimination does not occur normally, possibly due a lack of "crushing" by the growing embryo, or potentially due to a lack of embryonic nutrient uptake (Doll et al., 2020;[START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF]Ogawa et al., 2015a;[START_REF] Opsahl-Ferstad | ZmEsr, a novel endosperm-specific gene expressed in a restricted region around the maize embryo[END_REF] . These examples illustrated the interdependence between embryo and endosperm development.

In addition, to nutritional transfer (discussed above) and peptide-mediated dialogues (of which an example is discussed in § 2.4.3.2), mechanical interactions between the embryo and the endosperm have been proposed to regulate not only seed germination (discussed above), but also seed development.

CELL ELIMINATION DURING ENDOSPERM DEVELOPMENT.

We have previously seen that part of the endosperm is usually eliminated during seed development. This process may be dramatic, as in A. thaliana or in legumes, or very modest as in maize [START_REF] Ingram | Dying to live: cell elimination as a developmental strategy in angiosperm seeds[END_REF]. During this process, the endosperm cell walls are modified and progressively eliminated allowing embryo growth, which also appears to be required for the elimination process to proceed). The bHLH transcription factor ZHOUPI (ZOU) is required for A. thaliana endosperm elimination (Figure 8). A second bHLH transcription factor, INDUCER OF CBF EXPRESSION1 (ICE1), which has shown to act in a complex with ZHOUPI (ZOU) is also critical for endosperm breakdown [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF][START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF]. Because ZOU and ICE1 form an obligate heterodimer, endosperm cell elimination is absent in zou and ice1 mutants.

Interestingly, transcriptome analysis has shown that, instead of cells generally accepted to be markers of developmental cell death in A. thaliana [START_REF] Daneva | Functions and Regulation of Programmed Cell Death in Plant Development[END_REF], the primary, and early targets (based on the strength and timing of their expression in developing seeds, and their total lack of expression in zou mutants) are annotated as involved in cell wall modifications, and include genes encoding proteins such as Pectin Lyases and REVERSIBLY GLYCOSYLATED POLYPEPTIDE 3 (RGP3), a direct target of the ZOU/ICE complex [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF][START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. Cell death marker genes, such as the early cell death marker PUTATIVE ASPARTATIC PROTEINASE A3 (PASPA3) which has no known function, and the nuclease encoding BIFUNCTIONAL NUCLEASE1 (BFN1), which plays a role in nuclear corpse clearance in roots [START_REF] Fendrych | Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis[END_REF] have only been found to be expressed later in endosperm development, although they are also downregulated in zou mutants. Furthermore, although ZOU and ZOU targets are expressed in seeds in which embryo growth arrests early, and endosperm cell walls undergo modification in these mutants, endosperm elimination and the expression of cell death marker genes is dramatically retarded [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF]. These data strongly support that ZOU/ICE complex does not act by directly triggering endosperm cell death, but rather that it acts to mediate cell wall modification in the endosperm in order to prepare the endosperm to be eliminated in a process that involves mechanical crushing by the invasively growing embryo. [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF].

Embryonic surface formation necessitates cross talk between endosperm and embryo THE EMBRYONIC CUTICLE.

In comparison with the cuticle on the surface of the aerial organs, cuticles found in the seeds are poorly understood, largely because they are very thin, and also mainly situated internally, in complex tissues, making compositional analysis very complicated. In A. thaliana seeds, five cuticles are present. The first cuticle is external, and associated with the outer cell layer of the seed coat. It is likely that this cuticle both prevents the fusion between seeds as they develop, and has special properties allowing efficient mucilage release and seed hydration at germination [START_REF] Panikashvili | The Arabidopsis DCR Encoding a Soluble BAHD Acyltransferase Is Required for Cutin Polyester Formation and Seed Hydration Properties[END_REF]. The second cuticle found in seed is a discontinuous cuticle visible between the inner epidermis of the outer integument and the outer epidermis of the inner integuments, at their zone of fusion. Little is known about this cuticle, although some biosynthesis genes are expressed in the integuments and cutin-like material has been identified during early seed coat development [START_REF] Creff | A mechanically sensitive cell layer regulates the physical properties of the Arabidopsis seed coat[END_REF][START_REF] Molina | The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds[END_REF][START_REF] Molina | Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana[END_REF]. During seed development the cell wall housing this cuticle becomes progressively suberized to impermeabilize the mature seed, and traces of the original cuticle are lost [START_REF] Fedi | Awake1, an ABC-Type Transporter, Reveals an Essential Role for Suberin in the Control of Seed Dormancy[END_REF]. There is also an additional cuticle layer made by the nucellus (O Cohen et al., 2019). The endosperm is also surrounded by a cuticle that is maternal in origin (produced by the inner cell layer of the inner integument). This endosperm-associated cuticle is considerably thicker than most of the other cuticles in the seed (approximately 10 times ticker) and appears to be involved in the barrier regulating water and oxygen entrance in order to avoid oxidation [START_REF] Coen | Deposition of a cutin apoplastic barrier separating seed maternal and zygotic tissues[END_REF][START_REF] Loubéry | A Maternally Deposited Endosperm Cuticle Contributes to the Physiological Defects of transparent testa Seeds1[OPEN[END_REF]. The fourth, and innermost cuticle of the seed is the embryonic cuticle, and is found at the interface between the embryo and endosperm.

In A. thaliana, the embryonic cuticle is specific to the embryo proper and thus is absent from the suspensor. Moreover, in contrast to the cuticle of the aerial organs which is inherited during growth, the embryonic cuticle must form de novo [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF][START_REF] Delude | Plant Surface Lipids and Epidermis Development[END_REF].

TEM analysis shows that the embryonic cuticle is deposited as patches during the globular stage of embryo development, and seems be continuous by the late-globular -early heart stage [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF]. As previously mentioned (Introduction, §2.3), cuticle formation and epidermal cell fate are difficult to separate, since cuticle formation appears to be an inherent property of the epidermis. Few, if any example of de novo epidermal specification and thus cuticle formation occurs during post-embryonic development (with the possible example of [START_REF] Lee | A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in Arabidopsis[END_REF]). Genes involved in epidermal specification such as ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) are expressed in the apical cell of the two celled embryo, suggesting that epidermal identity already exist at this stage, and may be a 'ground" state within the embryo proper [START_REF] Lu | Identification of a meristem L1 layerspecific gene in Arabidopsis that is expressed during embryonic pattern formation and defines a new class of homeobox genes[END_REF]. The timing of embryonic cuticle formation, suggests that, as expected, epidermal specification precedes embryonic cuticle formation.

Consistent with this genes involved in the biosynthesis of the cuticle are expressed in embryo epidermal cells from around the 16 celled (dermatogen) stage in embryo development [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF]. De novo epidermal fate specification, and thus production of the embryonic cuticle occurs when the embryo is surrounded by the endosperm, and to date very little is known about how this proximity affects either event [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. The extremely protected environment of the developing embryo also opens important questions regarding how the properties, and in particular the integrity, of the cuticle can be assured prior to embryo germination, when defects in the cuticle could lead to rapid water loss and death.

In A. thaliana, molecular and genetic analysis have revealed a central role for the two previously described basic helix-loop-helix transcription factor (bHLH TF), ZOU and ICE1 and their (indirect) targets in the establishment of embryonic cuticle integrity (Figure 8) [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. Interestingly, and consistent with the endosperm-specific expression pattern of ZOU, these genes are only required for embryonic cuticle integrity during seed development but not for cuticle formation in organs formed after germination. Recent work has started to elucidate the mechanisms via which the activity of the ZOU/ICE1 complex contribute to cuticle integrity. Cuticle integrity has also been found to depend on a (likely indirect) target of the ZOU/ICE complex, the subtilisin serine protease ABNORMAL LEAF SHAPE1 (ALE1) [START_REF] Tanaka | A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF], but also the two GASSHO (GSO)family leucine Rich Repeat receptor-like kinases (GSO1/SHENGEN3 and GSO2) (Figure 7).

Consistent with its being a ZOU target, ALE1 expression is restricted to the developing endosperm and strongly down-regulated in zou mutants. However GSO1 and GSO2 are expressed in the embryonic epidermis [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF]. Genetic analysis show that these factors act together in the same pathway to regulate cuticle integrity, but not cuticle biosynthesis [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF][START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. Interestingly, GSO1 is also known to be involved in ensuring the integrity of another apoplastic barrier, the Casparian strip [START_REF] Pfister | A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects[END_REF].

A recent study (Doll et al., 2020) has revealed that a two-way molecular dialogue between the embryo and the endosperm is necessary in order to ensure embryonic cuticle integrity. The missing factor in the pathway was a peptide related to the sulphated CASPARIAN STRIP INTEGRITY FACTORs (CIFs) that had previously been shown to bind to the GSO1 receptor during a unidirectional signalling process ensuring Casparian strip pathway integrity in the root [START_REF] Doblas | Root diffusion barrier control by a vasculature-derived peptide binding to the SGN3 receptor[END_REF]. In seeds, the CIF-related TWISTED SEED1 (TWS1) peptide is produced in an inactive form (with an inhibitory C-terminal extension that prevents receptor binding) in the embryo. To become active, the TWS1 peptide which is produced in the embryo, needs to be cleaved by ALE1, which removes the inhibitory C-terminal extension.

This cleavage can only occur in the presence of gaps in the cuticle, that allow the TWS1 precursor to encounter its endosperm localized activator. Once activated, TWS1 can diffuse back across cuticular gaps and bind its receptors, GSO1 and GSO2. This triggers a poorly understood signalling event that ensures the closure of cuticle gaps. One this has been achieved, the intact cuticle separates the subtilase ALE1 from its substrate TWS1, turning off signalling (Doll et al., 2020). Interestingly, ale1 shows a weaker cuticle phenotype than the gso1 gso2 or tws1 mutants, likely suggesting the presence of redundant subtilases in the endosperm (Figure 7).

How GSO-mediated signalling stimulated by TWS1 binding ensures cuticle gap closure remains unclear. However, in the root, CIF binding to GSO1 has been shown to generate apoplastic ROS, which are necessary for the oxidative crosslinking of Lignin monomers present around the nascent Casparian strip [START_REF] Fujita | SCHENGEN receptor module drives localized ROS production and lignification in plant roots[END_REF] . It therefore seems likely that apoplastic ROS production may be a key factor acting downstream of GSO signalling in seeds.

Although its composition remains unclear, staining of cotyledons with phenolic dyes (unpublished data) suggested an enrichment of the embryonic cuticle with phenolic compounds, which could be the targets of oxidative crosslinking. One potential hypothesis is that these phenolics could be crosslinked by apoplastic ROS and may act as a scaffold for the deposition of cutin and wax polymers to create an intact cuticle.

Interestingly, loss of GSO1 and GOS2 function also leads to a reduction in the phosphorylation of the MITOGEN ACTIVATED PROTEIN KINASE6 (MPK6) protein in seeds.

MPK6 has also been shown to be required to be necessary for normal embryonic cuticle integrity. However how exactly MPK6 is involved in GSO signalling remains a mystery. The mpk6 single mutants show several pleiotropic seed phenotypes which do not seem correlated.

Indeed, in addition to a fragmented embryonic cuticle, mpk6 mutants show bursting seed and abnormal seed size which are likely due to an independent role for MPK6 in the testa [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF].

ROLES OF THE EMBRYONIC CUTICLE.

We have seen in the previous section that the embryo cuticle can restrict the apoplastic movement of peptides, a property that is important for ensuring its integrity. Other studies have (A) ZOU/ICE complex promote the nutrient storage and transport in the endosperm (in blue), the cell wall modifications of the endosperm (in green) whose the only direct target of ZOU/ICE complex is known, RGP3. ZOU/ICE complex also controls the formation of the embryo surface. The embryonic cuticle integrity (in red) which involves the GSO1/GSO2 receptors, the subtilisase ALE1, the peptide TWS1 and the TPST (for more informations see Figure 7 in the Introduction). The embryo sheath production and deposition (in yellow) involve the peptide KRS and GSO1/GSO2 receptors. (B) Post-translational regulators and their modification sites on ICE1. The modified amino-acid is indicated in the brakets with its corresponding regulator, except for the KRAAM motif, the binding site of MP3/MPK6, where the amino acid substitution and the name of the correpsonding gain of function mutant are mentioned in the brackets. ensuring the stability of embryonic patterning and cell differentiation [START_REF] Povilus | Evidence for parent-of-origin effects and interparental conflict in seeds of an ancient flowering plant lineage[END_REF][START_REF] San-Bento | Epidermal identity is maintained by cell-cell communication via a universally active feedback loop in Arabidopsis thaliana[END_REF]. How early cuticle formation could affect other processes such as nutrient uptake from the surrounding endosperm remains unclear. However, the cuticle of the embryo remains rather permeable to small hydrophilic molecules throughout much of its development, particularly at the root pole [START_REF] Berhin | The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation[END_REF][START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF], presumably permitting nutrient uptake from the surrounding endosperm even after the death of the suspensor at the heart stage. Although it is tempting to suggest that nutrient uptake occurs predominantly at the embryo root pole it seems unlikely since expression of transporters in other species suggest that nutrients can transit over the entire embryo surface [START_REF] Weber | A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds[END_REF]. The A.

thaliana embryonic cuticle is thus likely to be more of a molecular filter than a hermetic barrier to nutrients (Doll and Ingram, 2021, in press).

THE SHEATH SURROUNDING THE EMBRYO.

The ZOU/ICE complex and the GSO-dependant signalling pathway are also involved in the formation of the embryo sheath. This structure is deposited on the cuticle surface, at the interface of the embryonic cuticle and the endosperm. It is thus the primary interface of the embryo with its environment. To date the exact composition of the embryo sheath remains unknown although it is enriched in components that are recognised by the anti-extensin antibody α-JIM12 [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. Embryo sheath deposition is not affected in mutants involved in cuticle biosynthesis, and unlike the cuticle, its components are produced by the endosperm (Doll et al., 2020). During germination, Doll et al., 2020 showed that the sheath renders the embryo surface less adhesive, facilitating the escape of the cotyledons from the seed coat. This anti-adhesive property may also partially explain the role of the embryo sheath during seed development in permitting the embryo to separate from, and grow through the endosperm.

To date, little is known about the mechanisms underlying the production, deposition, and composition of the embryo sheath. ZOU activity is critical for sheath development, as is clearly visible from the complete absence of α-JIM12 immunolocalisation signal in zou mutant.

The role of ZOU in sheath production appears to be mediated in part by a cysteine-rich peptide of STIGMA-SPECIFIC PROTEIN1 (STIG1) family, KERBEROS (KRS) (Figure 8). Although it belongs to the STIG1 family and thus show a cysteine-rich STIG1 domain and a signal peptide domain, the KRS peptide is distinguished from the other 5 members of this family in Arabidopsis by the presence of a non-conserved C-terminal region not found in other family members which is enriched in basic residues followed by a proline-enriched sequence [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. Loss of KRS function also leads to a complete loss of α-JIM12 immunolocalisation signal in the developing endosperm. Like Loss of ZOU function, loss of KRS function also leads to a strong adhesion between the embryo and surrounding endosperm. However, unlike the situation in zou mutants, endosperm softening and elimination appear to occur normally in krs mutants. As a result, the embryos of krs usually expand normally, but occasionally "twist" due to adhesion between the embryo and residual endosperm [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. Toluidine blue assay on krs mutant also showed a potential embryonic cuticle defect. However, how the process of cuticle deposition affects or is affected by sheath development remains unclear. Finally, timing of endosperm cell death does not seem to be affected in krs mutants [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF].

Interestingly, GSO1 and GSO2 -mediated signalling required for cuticle integrity, is also required for sheath formation. Unlike the situation in zou and krs mutants however, where α-JIM12 epitopes are never produced, defects in the GSO signalling pathway affect only the deposition of the sheath material on the cuticle surface, whilst the production of α-JIM12 epitopes occurs apparently normally. Like zou and krs mutants, mutants in the GSO signalling pathway show adhesion of the embryo to surrounding tissues, but this appears to be exacerbated by cuticle defects [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. Extensins are known to be the targets of oxidative crosslinking via their tyrosine residues (Mishler-Elmore et al., 2021) and one hypothesis currently being investigated in the laboratory is that ROS produced at the embryo surface by GSO signalling could be involved in sheath deposition. It is also possible that sheath components could have a scaffolding function from the embryonic cuticle, however cuticle defects in sheath mutants (such as krs) are relatively weak [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF].

ZOU/ICE1 form a key hub coordinating embryo surface formation and endosperm development WHAT DO WE KNOW ABOUT THE ZOU/ICE1 COMPLEX?

We have previously described the main functions of the ZOU/ICE complex in A.

thaliana (Figure 8). However, studies suggested that ZOU is present in most other angiosperms [START_REF] Catarino | The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land[END_REF][START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF].

ZOU orthologues have been reported in both Monocotyledons Zea mays (maize) and

Orysa sativa (rice) and in the leguminous Glycine max (soybean) [START_REF] Dou | Identification of ZHOUPI Orthologs in Rice Involved in Endosperm Development and Cuticle Formation[END_REF]Feng et al., 2018;Grimault et al., 2015a;[START_REF] Zhang | Identification of ZOUPI Orthologs in Soybean Potentially Involved in Endosperm Breakdown and Embryogenic Development[END_REF]. In species with a more persistent endosperm such as Z. mays, the embryo invasion previously described is limited to a few cell layers surrounded the expanding embryo: the ESR and the endosperm associated with scutellum (EAS), (the endosperm surrounded the scutellum (part of the embryo)). Interestingly, the ZOU orthologue in maize, ZmZOU (also known as OPAQUE11), seems to only be involved in ESR elimination (Feng and Song, 2018;Feng et al., 2018;[START_REF] Grimault | ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development[END_REF], although detailed studies of the EAS in loss of function mutants for ZmZOU have not been carried out.

A.thaliana encodes two ICE proteins, ICE1 and ICE2. Based on the few functional studies that exist, it seems that the central role of ZOU in seed development is likely to be conserved through angiosperms. However, ZOU and ICE orthologues are also found in non-seed plants, from bryophytes to monilophytes. However, except one study which investigated the ZOU-independent role of ICE in stomatal development in Physcomitrella patens [START_REF] Chater | Origin and function of stomata in the moss Physcomitrella patens[END_REF], no studies have looked at the functions of the ZOU/ICE complex in non-seed plants. Moreover, although seeds are fascinating, they can be challenging study systems and thus deciphering the role of ZOU/ICE complex in an early diverging plant might help to shed some light on basal function and regulation of the complex in plants. This question will be addressed in Chapter 2 and Chapter 3 of this thesis using the early diverging plant model, Marchantia polymorpha.

ICE1 IS POST-TRANSLATIONALLY REGULATED DURING STOMATA DEVELOPMENT AND COLD TOLERANCE.

In A. thaliana but also in other species, ICE1 also known as SCREAM1, has multiple functions in plants including regulation of cold tolerance and stomatal development (Chinnusamy et al., 2003;[START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF]. ICE1 is involved in the production of stomatal air pores in the surfaces of aerial organs. In this pathway, it acts partially redundantly with its homologue ICE2 (SCREAM2), through interaction with the well-known class Ia bHLH transcription factors SPEECHLESS, MUTE and FAMA (Kanaoka et al., 2008;[START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF]. In contrast to their strong seed phenotypes, ice1-2 mutants show only very weak stomatal phenotypes due to redundancy with ICE2. The double mutant ice1 ice2 does not produce any stomata, and is thus seedling lethal. Conversely, the dominant gain-of-function ice1-D mutant produces too many stomata on cotyledons and leaf surfaces, which seriously compromises plant viability [START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF].

ICE1 was originally described for its role in cold responses, and loss of function of ICE1 leads to increased cold sensitivity and a failure to induce cold response genes. However, recently this role in cold tolerance has been questioned by Kidokoro and colleagues which showed that CBF genes are repressed by transgene-induced DNA methylation of ice1 [START_REF] Kidokoro | DREB1A/CBF3 Is Repressed by Transgene-Induced DNA Methylation in the Arabidopsis ice1-1 Mutant[OPEN][END_REF][START_REF] Thomashow | SCREAMing Twist on the Role of ICE1 in Freezing Tolerance[OPEN[END_REF]. Several proteins involved in posttranslational regulation of ICE1 protein stability have been identified. In particular, ICE1 is posttranslationally regulated during cold stress by HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1), OPEN-STOMATA1 (OST1), SIZ1, and the MITOGEN ACTIVATED PROTEIN KINASEs, MPK3 and MPK6 [START_REF] Ding | OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis[END_REF][START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF]Li et al., 2017;[START_REF] Miura | ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance[END_REF][START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF][START_REF] Zhao | MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability[END_REF]. HOS1 encodes a RING finger ubiquitin E3 ligase which directly interacts with AtICE1 and promotes its degradation via the 26S-proteasome. hos1 loss-of-function mutants show increased ICE1 stability and a more sustained cold response whereas transgenic plants overexpressing HOS1 show increased ICE1 degradation in cold and thus increased sensitivity to cold [START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF]. In addition, a Ser-403 to alanine substitution (within the MPK6 phosphorylation site and HOS1 ubiquitination site) leads to increased ICE1 stability and improved freezing tolerance in A.

thaliana (Figure 8) [START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF][START_REF] Miura | ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance[END_REF].

More recently, [START_REF] Li | MPK3-and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis[END_REF][START_REF] Zhao | MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability[END_REF] have shown that the MKK5-MPK3/MPK6 cascade negatively regulates cold stress by leading to increased association with E3 ligases, promoting the ubiquitination of ICE1 and thus mediating its degradation via the 26S-proteasome [START_REF] Li | MPK3-and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis[END_REF][START_REF] Zhao | MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability[END_REF]. This MAP kinase cascade promotes the phosphorylation of ICE1 at Ser94, Thr366 and Ser403. In contrast, cold response is positively regulated by another MAP kinase cascade involving MEKK1-MKK2-MPK4, which also The ZOU/ICE1 complex plays a central role during seed development as described above. Although our understanding of ZOU/ICE1 regulated processes is becoming clearer, the regulation of the activity of the ZOU/ICE1 complex itself remains a mystery. However, several lines of evidence converge to suggest that, although the tissue specificity of complex action is conferred by ZOU expression, it is the stability of the more broadly expressed ICE1 protein that acts to regulate ZOU/ICE1 complex activity in seeds. The observation that significantly altering levels of ZOU activity in seeds leads to little, if any phenotype (except when levels are very low), taken together with the observation that ice1-D mutants show early endosperm lysis [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF]Feng et al., 2018;[START_REF] Grimault | ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development[END_REF], support a hypothesis in which ICE1 levels, rather than ZOU levels are limiting for complex function in the endosperm.

Unpublished confocal microscopy data on plant expressing an ICE1-D:GFP fusion under an endosperm promoter, suggest that, consistent with observations in other tissues, ICE1-D is stabilized in the developing endosperm compared to a simple ICE1:GFP fusion support this hypothesis.

Based on the results above, we hypothesize that the regulation of ICE1 stability is key in regulating ZOU/ICE1 complex activity in the endosperm. In addition, expression data and yeast two hybrid assay suggest that ZOU and ICE2 could form a heterodimer in vivo and that ICE2 might therefore also be involved in seed development. How ZOU/ICE1 activity is regulated in the endosperm, and the potential role of ICE2 in seeds, are questions that will be addressed in Chapter 1 of this thesis.

Aim of the thesis

The work carried out during my thesis aims to drive forward our knowledge about the functions of ZOU/ICE1 complex in seed development and more globally in land plants. To do this several subjects were addressed. In a first chapter, I will present a study aiming to elucidate the control of ZOU/ICE complex activity in Arabidopsis thaliana seeds. The second and a third chapters will focus on the potential ancestral functions of ZOU/ICE complex in the early emerging land plant, Marchantia polymorpha. In the second chapter, the characterization of M. polymorpha cuticle and an investigation the function of ZOU/ICE complex in cuticle development will be presented. In the third chapter I will describe the role of this complex in the formation of a novel water conducting tissue. In the third chapter, the characterization of M. polymorpha cuticle and an investigation the function of ZOU/ICE complex in cuticle development will be presented.

Bibliography

See general bibliography at the end of the thesis.

CHAPTER 1:

DOES DECIPHERING ZOU/ICE COMPLEX REGULATION SHED LIGHT ON ZOU/ICE FUNCTION DURING SEED DEVELOPMENT? Preamble

As seen in the introduction, in A. thaliana, ICE proteins are strongly post-translationally regulated during cold tolerance and stomatal differentiation (Chinnusamy et al., 2003;[START_REF] Ding | OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis[END_REF][START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF][START_REF] Miura | SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis[END_REF][START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF]. These regulations either stabilize or destabilize ICE proteins and are very diverse, from phosphorylation to ubiquitination or even sumoylation. However, although the function of ZOU/ICE1 complex in seeds has started to be elucidated, nothing is known about the regulation of ZOU/ICE1 complex activity in the endosperm (Creff et al., 2019a;Denay et al., 2014a;Doll et al., 2020c;Moussu et al., 2017a). Since ICE proteins are post-translationally regulated in other tissues and, in contrast to the situation for ZOU, for which changes in protein levels (with the exception of severe losses) appear not to affect function, ICE1 stabilization (gain of function) causes a precocious endosperm lysis (Denay et al., 2014a). This fact combined with the endospermspecific expression of ZOU, has driven the hypothesis that ZOU and ICE1 affect complex activity differently. Whereas ZOU provides tissue specificity, ICE1 could be the main factor via which levels of complex activity are regulated. Based on information from other tissues, this regulation likely occurs through post-translational modifications. During my PhD I focused my attention on one of the known post-translational regulators of ICE1 the E3 ubiquitin ligase HOS1, already known to be involved in the destabilisation of the ICE proteins in other contexts (Dong et al., 2006a;[START_REF] Li | MPK3-and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis[END_REF]. I also initiated experiments exploring the role of conserved KRAAM motif, recently discovered to be the binding site of two other negative regulators of ICE, the MAP kinases MPK3 and MPK6 (Putarjunan et al., 2019a). However, relatively few results have been obtained to date and this work will therefore not be presented in this thesis.

A second important point in this chapter was to understand why, contrary to the situation in stomatal development, ICE1 does not appear to show significant functional redundancy with ICE2 during seed development, despite the fact that in silico resources suggest that ICE2 is co-expressed with ICE1 in the endosperm.

Based on these questions, this chapter describes a study that uncovers the reason for the apparent lack of genetic redundancy between ICE1 and ICE2 during seed development.

This section is written as an article although some final validations are required before submission. We showed that, although at first sight ICE2 does not seem to play a role in seed development, ICE2 is not only co-expressed, but also interacts with ZOU. Functional redundancy has been revealed using a background lacking the E3 ubiquitin ligase HOS1, a known negative regulator of ICE activity in other tissues. Our data suggest that the E3 ubiquitin ligase HOS1 is likely implicated in the post-translational regulation of the activity of both ZOU/ICE1 and ZOU/ICE2 complexes and that HOS1-mediated ubiquitination likely regulates the stability of both ICE proteins during seed development. Apparent lack of redundancy is caused by an apparently seed-specific phenomenon whereby ZOU/ICE1 heterodimers (and potentially ZOU/ICE2 heterodimers) are required to activate ICE2 expression. This finding provides a cautionary example of how dangerous inferring genetic redundancy between different transcription factors based solely on genetic analysis of single and multiple mutants can be.

In a second section of this chapter, the genetic redundancy between ICE1 and ICE2 during seed development and in the other pathways is discussed in more detail as well as other putative regulators of the ZOU/ICE complex during seed development. This section describes the state of ongoing work and future directions for the project.

INTRODUCTION

Genetic redundancy describes a situation where two or more genes perform the same biochemical function or can partially or fully substitute for the function of the other. In cases of full redundancy, loss-of-function of one redundant element has no apparent effect on the phenotype being monitored. An example of this phenomenon is provided by the HD-ZIP IV Transcription Factors (TFs) ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) and PROTODERMAL FACTOR 2 (PDF2) which are necessary for epidermal specification during embryogenesis in Arabidopsis thaliana. Although loss of function of either protein does not significantly affect embryo development, loss of both proteins simultaneously leads to early embryo lethality due to severe epidermal defects (Ogawa et al., 2015b;San-Bento et al., 2014b). In backgrounds lacking ATML1, PDF2 shows haploinsufficiency, revealing dosage dependency and highlighting the functional overlap between the two proteins (San-Bento et al., 2014b). Loss of function of ATML1 alone leads to an almost complete loss of giant cell formation in the sepal, illustrating a non-redundant role for this protein, likely linked to differences in gene expression/protein dosage between ATML1 and PDF2 in sepals [START_REF] Meyer | Fluctuations of the transcription factor ATML1 generate the pattern of giant cells in the Arabidopsis sepal[END_REF].

A second example of functional redundancy is provided by the GASSHO1 and 2 (GSO1 and GSO2) receptor kinases, which act fully redundantly to ensure cuticle integrity maintenance in the developing seed (Creff et al., 2019b;Doll et al., 2020d;[START_REF] Tsuwamoto | GASSHO1 and GASSHO2 encoding a putative leucine-rich repeat transmembrane-type receptor kinase are essential for the normal development of the epidermal surface in Arabidopsis embryos[END_REF]. In contrast, GSO1 shows no genetic redundancy with GSO2 during integrity monitoring at a second apoplastic barrier, the Casparian strip (Pfister et al., 2014b). The lack of functional redundancy in Casparian strip integrity monitoring is due to the fact that unlike the situation in developing embryos, where GSO1 and GSO2 both bind the implicated ligand TWISTED SEED1 (TWS1) (Doll et al., 2020d), GSO2 is unable to bind the ligands implicated in Casparian strip monitoring (CIF1 and CIF2) [START_REF] Okuda | Molecular mechanism for the recognition of sequencedivergent CIF peptides by the plant receptor kinases GSO1/SGN3 and GSO2[END_REF]. Thus, tissue and/or process dependent changes in genetic redundancy between protein pairs can be caused by multiple factors including differences in relative gene expression between different tissues, or divergent interaction capacities with partner proteins. In backgrounds lacking ICE1, ICE2 shows haploinsufficiency (Kanaoka et al., 2008b). Similar redundancy has been demonstrated between ICE1 and ICE2 regarding their roles during the acquisition of freezing tolerance (Chinnusamy et al., 2003b;Kim et al., 2015b). ICE1 has more recently been shown to play a key role in regulating seed development via its interaction with the bHLH TF ZHOUPI (ZOU) (Denay et al., 2014b). This function was assumed to be nonredundant, due to the fact that loss of ICE1 function appear to phenocopy loss of ZOU function.

However subtle modifications of the zou loss of function phenotype are difficult to quantify and, furthermore, ice1 ice2 double mutants never produce seeds. As a result the role of ICE2, which is thought to have arisen from a duplication event within the Brassicaceae [START_REF] Kurbidaeva | Arabidopsis thaliana ICE2 gene: phylogeny, structural evolution and functional diversification from ICE1[END_REF], in seed development has not been studied.

The ZOU-ICE1 heterodimer plays two main roles in seeds development. The first is to regulate the elimination of the endosperm, the nutritive tissue surrounding the developing embryo in most angiosperm seeds. In A. thaliana, the endosperm is a transient tissue, that is progressively eliminated as the embryo grows, ultimately becoming restricted to a single and highly specialized peripheral cell layer that surrounds the embryo at seed maturity. The elimination of the so called "central" endosperm requires the activity of the ZOU-ICE1 complex, and seeds lacking either component contain a fully persistent endosperm and an embryo that is viable, but much reduced in size (Denay et al., 2014b;Kondou et al., 2008b;Yang et al., 2008b). In parallel, the ZOU-ICE1 complex is necessary for the formation of the embryonic surface. Cuticle integrity is defective in both mutants, likely due to the fact that the expression of a key endosperm-specific component of a recently identified surface integrity-signalling pathway, ABBNORMAL LEAF-SHAPE1 (ALE1) (Creff et al., 2019b;Doll et al., 2020d;Tanaka et al., 2001b) is dependent upon the ZOU-ICE1 complex (Denay et al., 2014b;Grimault et al., 2015b;Yang et al., 2008b). Finally, ZOU function has been shown to be required for the formation of a glycoprotein-rich extra-cuticular structure, the sheath, which is necessary for embryo-endosperm separation during seed development and seedling germination (Doll et al., 2020e;Moussu et al., 2017b). The implication of ICE1 in sheath formation has not been studied to date.

Although ZOU is expressed exclusively in the endosperm, and thus likely gives spatial specificity to complex activity, previous studies in which levels of ZOU expression and/or ICE1 expression or activity have been manipulated, suggest that the level of complex activity may be regulated through modulation of ICE1 levels (Denay et al., 2014b;Grimault et al., 2015b).

The activity and stability of the ICE1 protein is known to be regulated by both protein-protein interactions [START_REF] Hu | Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis[END_REF][START_REF] Hu | The Transcription Factor INDUCER OF CBF EXPRESSION1 Interacts with ABSCISIC ACID INSENSITIVE5 and DELLA Proteins to Fine-tune Abscisic Acid Signaling during Seed Germination in Arabidopsis[END_REF] and by a wide range of post-translational modifications such as sumoylation by the SUMO E3 Ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (SIZ1) (Miura et al., 2007b) ubiquitination by the ubiquitin E3 ligases HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) (Dong et al., 2006b), and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) (Lee et al., 2017a), phosphorylation by the SNF1-RELATED PROTEIN KINASE OPEN STOMATA 1 (OST1) and by the MITOGEN-ACTIVATED PROTEIN KINASES 3 and 6 (MPK6 and MPK3) (Ding et al., 2015b;Putarjunan et al., 2019b). Although less well studied, ICE2 is also known to be a target of HOS1 (Kim et al., 2015b), COP1 (Lee et al., 2017a) and likely of MPK3/6 (Putarjunan et al., 2019b).

Ubiquitination by HOS1 destabilizes both ICE1 and ICE2 during the acquisition of cold tolerance (Dong et al., 2006b;Kim et al., 2015b).

Here we have used gene expression, protein interaction and genetic analysis of the regulation of endosperm elimination, cuticle formation and sheath formation to explore the apparent lack of functional redundancy between ICE1 and ICE2 in seed development. We reveal a cryptic genetic redundancy between ICE1 and ICE2 in the regulation of all three processes.

RESULTS

APPARENT LACK OF REDUNDANCY IS NOT DUE TO A LACK OF EXPRESSION OF ICE2 IN THE

ENDOSPERM

In order to analyze potential roles played by ICE2 in the developing seed, we first asked whether ICE2 is expressed in seed tissues. Inspection of available in silico data revealed that like ICE1, ICE2 is expressed in developing seed tissues, and particularly in the developing embryo and endosperm (Le et al., 2010b). Endosperm transcript levels of ICE2 are considerably lower than those of ICE1 at the linear cotyledon stage (244 vs 1679). In situ hybridization analysis was used to confirm the expression pattern of ICE2 in developing seeds, and results broadly confirmed the pattern suggested by in silico data (Figure 1). Differences in the intensity of in situ hybridization signals in A. thaliana seed tissues compared with transcriptomic data have been previously highlighted [START_REF] Francoz | Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics[END_REF], and likely explain the apparently stronger expression in embryos in our experiments.

Mining of available transcriptomic data from whole silique samples (Xing et al., 2013b), revealed that at the linear cotyledon stage, ICE2 expression showed a 6-fold reduction in gene expression levels in zou mutants compared to the situation in wild-type plants, suggesting that ICE2 may be either a direct or indirect target of positive transcriptional regulation by the ZOUcontaining TF complexes. This result was confirmed by multiple Q-PCR analyses (Figure 1).

APPARENT LACK OF REDUNDANCY IS NOT DUE TO AN INABILITY OF ICE2 TO INTERACT WITH ZOU

Apparent lack of functional redundancy between ICE1 and ICE2 in the endosperm could be explained by an inability of ICE2 to interact with the confirmed ICE1 binding partner ZOU. To exclude this possibility, yeast two hybrid analyses were carried out to assess the potential interaction of these two proteins "in vitro" (Figure 2). We conclude that not only are ICE2 and ZOU co-expressed in the endosperm, but that, in addition, they are capable of physically interacting.

LOSS OF HOS1 FUNCTION LEADS TO A PARTIAL RESCUE OF THE ICE1 LOSS OF FUNCTION

PHENOTYPE

In order to assess the potential role of ICE2 in seed development the ice2-1 loss of function mutant was obtained (Kanaoka et al., 2008b). This single mutant showed no quantifiable defects in seed development or seedling cuticle development (Figure 3 and previously (Kanaoka et al., 2008b), that ice1-2 ice2-1 double mutants die as young seedlings due to a lack of stomatal development. However, in these segregating populations, it was not possible either to distinguish seeds containing double mutant endosperms through seed shape analysis, or double mutant seedlings, using qualitative toluidine blue staining (Creff et al., 2019b).

Our results support the idea that the apparent lack of functional redundancy between ICE1 and ICE2 as gauged by the phenotype of ice1 mutants, is caused by the fact that that ICE2 expression is partially ZOU, and thus ICE1 dependent. To test this hypothesis, we devised a strategy based on the idea that stabilizing the residual ICE2 protein present in an ice1 mutant background could reveal a role for ICE2 during seed development. Because loss of function of negative regulators such as COP1 and MPK6/MPK3 leads to strong pleiotropic defects in developing seeds and seedlings (Creff et al., 2019b;[START_REF] Deng | cop1: a regulatory locus involved in lightcontrolled development and gene expression in Arabidopsis[END_REF], we focused our attention on the E3-ubiquitin ligase HOS1 which is known to destabilize both ICE1 and ICE2 in vegetative tissues. In silico data show that transcripts encoding HOS1 accumulate, albeit at relatively low levels, in the developing endosperm, as well as in other seed tissues.

This ubiquitous expression in seeds was confirmed by in situ hybridization (Figure S1).

Single loss of function hos1-3 mutants (MacGregor et al., 2013) and double ice2-1 hos1-3 mutants produce seeds that are morphologically indistinguishable from those of wildtype plants, and, furthermore, show no defects in seedling cuticle permeability (Figure 3 and Figure 4). Intriguingly however, double ice1-2 hos1-3 mutants produced seeds that were visually "plumper" than those of ice1-2 plants, and which contained larger embryos. Thus, loss of HOS1 function appears to partially rescue the endosperm persistence phenotype of ice1-2 single mutants. Furthermore, the seedlings of ice1-2 hos1-3 mutants were both qualitatively, and quantifiably less permeable to toluidine blue than those of ice1-2 single mutants (Figure 3 and Figure 4).

RESCUE OF THE ICE1 LOSS OF FUNCTION PHENOTYPE BY LOSS OF HOS1 FUNCTION IS DEPENDENT UPON THE PRESENCE OF ICE2

In order to test the hypothesis that this partial rescue could be due to the stabilization of ICE2 due to loss of HOS1 activity, ice1-2 hos1-3 ice2-1+/-plants were generated and their progeny were analyzed. The seeds produced by these plants produced around 25% of seeds that had a very small embryo (similar to ice1-2) seeds, whilst the remaining seeds exhibited an ice1-2 hos1-3 phenotype, or in some cases an intermediate phenotype. Our results support the hypothesis that the apparent rescue of the ice1 mutant phenotype by loss of HOS1 function

ICE1 IS NECESSARY FOR SHEATH FORMATION

Our results support the fact that ZOU-ICE2 heterodimers may play a significant role in wild-type seed development, but that this role is masked by both genetic redundancies with, and transcriptional regulation by, ZOU-ICE1 heterodimers. In order to reveal the role of ICE2 in the presence of the ICE1 protein we sought to identify an ICE-dependent phenotype that might provide a more quantifiable readout than either endosperm persistence or cuticle permeability. Loss of ZOU function leads to a complete elimination of the embryo sheath, and extensive adhesion of the embryo and surrounding endosperm (Moussu et al., 2017b). We therefore investigated whether loss of ICE1 function also affects sheath formation, by carrying out immunolocalizations with the α-JIM12 antibody on imbibed seeds. As previously described (Doll et al., 2020;Lee et al., 2012), this antibody detects a continuous extra-cuticular structure on the surface of mature wild-type embryos. However, as has previously been shown for loss of ZOU function [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF], loss of ICE1 function leads to a complete elimination of JIM12 signal at the embryo surface, although signal in other tissues, such as the testa, is not affected (Figure 5). As for cuticle and endosperm phenotypes, a weak but distinct complementation of the sheath phenotype of the ice1-2 mutant was observed in ice1-2 hos1-3 mutant seeds in which patches of α-JIM12 labelling on the embryo surface, never observed in ice1-2 single mutants, were detected.

LOSS OF HOS1 FUNCTION LEADS TO AN APPARENT INCREASE IN SHEATH PRODUCTION WHICH IS ATTENUATED BY LOSS OF ICE2.

No visible difference in α-JIM12 signal intensity was observed between wild-type and ice2-1 mutants. However, in contrast to the situation for endosperm elimination and cuticle integrity phenotypes, for which loss of HOS1 function does not generate an easily detectable "gain of ICE function" phenotype, loss of HOS1 leads to a significant increase in the intensity of sheath labelling with the α-JIM12 antibody compared to wild-type (Figure 5). This suggests that sheath biogenesis may provide a more quantifiable output for the ZOU/ICE1 pathway than either endosperm elimination or cuticle permeability. We therefore analyzed sheath production in ice2-1 hos1-3 mutants, and found that loss of ICE2 function led to a reduction of the excess signal observed in hos1-3 mutants to near wild-type levels, thus revealing the ICE2dependency of embryo sheath formation during seed development.

MATERIALS AND METHODS

PLANT GROWTH.

Arabidopsis thaliana seeds were gas sterilized with chlorine (3mL HCl (33%) in 150mL bleach) for 2 hours and then sown on Murashige and Skoog (MS) medium with 0,5% sucrose in sterile condition, stratified for 2 days at 4°C and grown for 7 days in short-day conditions (8h light, 19°C). Seedlings were then transferred to compost (Argile 10 (favorit) and grown under short day conditions (8h light, 19°C) for two to three weeks before transferred to long day conditions (16h light, 21°C YEAST TWO HYBRID ASSAY.

The Dual Hunter split ubiquitin-based system from Dualsystems Biotech was used to carry out yeast two-hybrid experiments [START_REF] Möckli | Yeast split-ubiquitin-based cytosolic screening system to detect interactions between transcriptionally active proteins[END_REF]. Yeast protocols were performed as described by the manufacturer. Single bait and prey constructs were cloned by PCRamplified cDNA fragments into pCR BluntII-TOPO vector (Invitrogen), and then transferred into the appropriate yeast vector.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF TOLUIDINE BLUE PERMEABILITY.

To assess cuticle integrity, toluidine blue staining assays were performed [START_REF] Tanaka | A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis[END_REF]. For qualitative toluidine blue staining tests, etiolated seedlings were used. Seedlings were stratified in the dark, exposed to a 6h light flash and then grown for 4 days in darkness.

They were then stained for 2 min in 0.05% w/v Toluidine blue solution with 0.1% v/v Tween 20 and washed abundantly under tap water. Pictures were taken with a Keyence VHX900F digital microscope.

For quantitative toluidine blue staining test, 7-day old light-grown seedlings were stained in the same way. In order to extract toluidine blue and chlorophyll, the cotyledons of ten seedlings were collected and placed in 1mL of 80% ethanol for at least 3 hours in darkness.

Toluidine blue content of the resulting solution was measured at 626nm with a spectrophotometer. This absorbance was then normalized using chlorophyll absorbance at 430nm, a wavelength not absorbed by toluidine blue. 6 repetitions were performed per condition (Xing et al., 2013a).

IN SITU HYBRIDIZATION ON SILIQUES.

In situ hybridization on siliques was performed as described in Yang et al 2008.

Templates for antisense probe synthesis were PCR-amplified from pCRII vectors (ThermoFisher) containing full-length ORFs amplified from silique cDNA with primers containing 5'-extensions with T7 or SP6 RNA polymerase promoters (HOS1-HIS-F and HOS1-HIS-R for HOS1 and ICE2-geno-F and ICE2R for ICE2). Antisense RNA probes were then made using T7 or SP6 RNA polymerase as described in [START_REF] Chanvivattana | Interaction of Polycomb-group proteins controlling flowering in Arabidopsis[END_REF]. Siliques were infiltrated with ice cold 4% paraformaldehyde (pH=7 adjusted with sulfuric acid) in PBS for three cycles of 20 minutes under vacuum. To increasing fixation efficiency, the two top corners of the siliques were cut. Siliques were dehydrated through an ethanol series, then into histoclear via an ethanol/histoclear series and finally embedded in Paraplast Plus (MC Cormick Scientific) Jackson, 1991. Sections of 6µm were cut with a HM355S microtome and stretched onto Superfrost Ultra plus TM (ThermoFisher Scientific) slides and then dewaxed and hydrated.

Sections were then treated with 2x saline sodium citrate (20min) and digested with proteinase K in 50mM Tris-HCl, pH 7.5, 5mM EDTA for 15min at 37°C. Treatment for 2min with 0.2% glycine in PBS followed by a post-fixation with 4%% paraformaldehyde in PBS (10min, 4°C) were performed. Sections were then treated with 0.25% w/v acetic anhydride in 100mM triethanolamine (pH 8.0) for 10 min and finally rinsed and dehydrated through an ethanol series. An overnight hybridization was then carried out at 50°C with RNA probes (produced using DIG RNA labelling kit from Roche) diluted in DIG easy Hyb solution (Roche) following the manufacturer's instructions. Slides were washed in a solution containing 0.1% saline sodium citrate and 0.5% SDS, at 50°C, and then blocked for 1H in blocking solution (Roche)

in TBS and for 30min in a BSA solution (1% BSA, 0.3% Triton-X-100, 100mM NaCl, 50mM

MgCl2). Finally, 2h incubation at room temperature in a 1/3000 dilution of alkaline phosphatase-conjugated antidigoxigenin antibody (Roche) in BSA solution.

Alkaline phosphatase activity was detected in a reaction overnight in darkness in a revelation buffer (0.5 g/L of nitroblue tetrazolium (NBT) and O.2 g/L of 5-Bromo-4-chloro-3-indolyl phosphate (BCIP). Slides were washed 4 times in water to stop the reaction and were mounted in Entellan (Sigma). Pictures were taken with Axio Imager 2 microscope (Zeiss).

IMMUNOLABELLING ON SECTIONS.

Mature and desiccated seeds were imbibed for 2-3 hours in water and punctured three times with a needle before being fixed as follow. Seeds were fixed in ice-cold PEM buffer (50mM PIPES, 5mM EGTA and 5mM MgSO4, pH6.9) containing 4% (w/v) paraformaldehyde under vacuum on ice (2 x 30 min) and overnight at 4°C. Samples were washed twice in PEM buffer and dehydrated by incubation in increasing concentrations of ethanol (50%, 70%, 80%, 90% and three times in 100%), for an hour each on ice under vacuum followed by an overnight incubation at 4°C in 100% Ethanol. Samples were then infiltrated with increasing concentrations of LR WHITE medium resin in absolute ethanol (30%, 50%, 70%, and three times in 100%), for an hour on ice under vacuum followed by an overnight incubation at 4°C

for each bath. Seeds were then transferred into small Beam capsules (Electron Microscopy Sciences) and incubated overnight at 60°C for polymerisation. 1µm sections were cut on a Leica RM6626 microtome and floated onto adhesive slides (Superfrost Ultra plus TM (ThermoFisher Scientific)). For immunolabelling, sections were first incubated for 45 minutes in PBS containing 2%(w/v) Bovine Serum Albumin (BSA). They were then incubated with a 10 fold dilution of primary antibody in PBS containing 0.1% BSA overnight at 4°C followed by one hour at room temperature. Sections were then washed three times in PBS for 5 minutes, then in PBS containing 0.1% Tween 20 (w/v) for 10 minutes and finally in PBS for 5 minutes. Incubation with a 100-fold dilution of an appropriate anti-rat secondary antibody (Alexa Fluor 488 conjugate, Invitrogen) in PBS with 0.1% BSA for one hour in the dark, in a wet chamber was then performed. The sections were washed three times in PBS for 5 minutes and counter-stained with a solution of 0.25ng/L of calcofluor (Fluorescent Brighter 28, Sigma Aldrich) in PBS for 5 minutes. Finally, sections were washed rapidly three times in PBS and mounted in Vectashield (Eurobio) (Moussu et al., 2017b). Observations were made using an Axio Imager 2 microscope (Zeiss).

RNA EXTRACTION AND QPCR ANALYSIS.

Replums were frozen in liquid nitrogen and total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma). Total RNAs were digested with Turbo DNA-free DNase I (Ambion) according to the manufacturer's instructions. RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol.

PCR reactions were performed in an optical 96-well plate using FastStart Universal SYBR Green Master Rox (Roche), in a final volume of 10 μl, according to the manufacturer's instructions. The following standard thermal profile was used for all PCR reactions: 95˚C for 10 min, 40 cycles of 95˚C for 10 s, and 60˚C for 30 s. As a reference, primers for the EIF4 and AP2M cDNA were used. PCR efficiency (E) was estimated from the data obtained from standard curve amplification using the equation E = 10 -1/slope . Expression levels are presented as E -ΔCt , where ΔCt = Ct GOI -Ct HKG . GOI, Gene of Interest; HKG: Housekeeping genes.

TOLUIDINE BLUE PERMEABILITY.

To assess the cuticle integrity, toluidine blue staining assays were performed [START_REF] Tanaka | A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis[END_REF]). For qualitative toluidine blue staining test, etiolated seedlings were used [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF]. Seedlings were stratified in the dark, exposed to 6h light flash and then grown for 4 days in darkness. They are then stained for 2min in 0.05% w/v Toluidine blue solution with 0.1% v/v Tween 20 and washed abundantly under tap water. Pictures were taken with a Keyence VHX900F digital microscope.

For quantitative assay of toluidine blue staining, 7-day old seedling were stained in the same way. In order to extract the toluidine blue stain and the chlorophyll, the cotyledons of ten seedlings were collected and placed in 1mL of 80% ethanol for at least 3hours in darkness.

Toluidine blue staining of the resulting absorbance was measured at 626nm with a spectrophotometer. This resulting absorbance was then normalized with the chlorophyll absorbance at 430nm, a wavelength not absorbed by toluidine blue. 6 repetitions were performed per condition.

MICROSCOPY.

Mature and desiccated seeds were photographed with a Keyence VHX900F digital microscope. Light and fluorescent microscopy imaging was performed using a Zeiss AxioImager2. 6).

DISCUSSION

Despite the apparent dependence of ICE2 upon ZOU complexes, we were able to show that loss of HOS1 function can lead to an ICE2-dependent partial rescue of the ice1-2 mutant phenotype, suggesting that stabilization of residual ICE2 protein in the hos1-3 mutant background is sufficient to partially compensate for loss of ICE1 function. This result highlights a previously undocumented role for HOS1 in seed development. However, how or even if the activity of HOS1 is regulated during seed development (for example at the level of expression or subcellular localization (MacGregor and [START_REF] Macgregor | Exploring the pleiotropy of hos1[END_REF] in seeds), remains to be elucidated.

Furthermore, we show that loss of HOS1 function alone does not lead to observable changes in seed morphology or seedling cuticle permeability, suggesting that the proposed stabilization of ICE1 and ICE2 in this background does not cause a dramatic deregulation in seed development. The topology of the cuticle integrity monitoring pathway means that an increase in cuticle impermeability would not be a predicted result of excessive production of ZOUdependent elements in the endosperm (Doll et al., 2020d). Nonetheless, previous work showed that plants expressing the gain of function ICE1 variant, ICE1-D show precocious endosperm elimination [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF]. One explanation for the discrepancy between this finding and the phenotype of hos1-3 mutants could be the timing of HOS1 expression. Based on in silico data and in situ hybridization (Figure S1) HOS1 expression is strongest at early time points in endosperm development. In addition, it appears likely that other negative regulators of ICE activity, including the MPK6 and MPK3 proteins which are unable to bind the ICE1-D variant (Putarjunan et al., 2019b) act in parallel with HOS1 in the endosperm.

Nonetheless, we have shown here that for one ICE-dependent process in the developing seed, namely embryo sheath deposition, the loss of HOS1 function generates a phenotype that is the inverse of that caused by loss of ICE function. Importantly the strong accumulation of HOS1 destabilizes the ZOU/ICE1 and ZOU/ICE2 complexes via its interaction and regulation of the ICE proteins. ZOU/ICE1 complex promotes seed development but also positively egulates ICE2 transcription. ZOU/ICE2 complex also promotes seed development in a more sublte way but whether this complex also influences ICE2 transcription remains to be confirmed.

sheath material observed in hos1-3 mutants is attenuated in hos1-3 ice2-1 mutants, again underlining the fact that ICE2 can make an important contribution to seed development, even in the presence of ICE1.

Taken together our results show that the hos1-3 mutant background can this be used to reveal the functional relevance of ICE2 during seed development. Our work highlights the importance of understanding the molecular mechanisms underlying genetic redundancy (or apparent lack of genetic redundancy) before drawing mechanistic conclusions regarding gene/protein function.

ICE1-qPCR-F GCTTTGATTTGATCAGGCAGT qPCR ICE1 ICE1-qPCR-R GTTTGCCTTGGATGTTTTCC ICE2-qPCR-F CTCCTTGCCAAGTCCTAAAG qPCR ICE2 ICE2-qPCR-R CTAATCACCGCTTGTTGAAC EIF4-F TTCGCTCTTCTCTTTGGCTCTC qPCR EIF4 EIF4-R GAACTCATCTTGTCCCTCAAGTA At5g46630-Fbis TCAGGTGCCAATGTTCACAGC qPCR AP2M At5g46630-Rbis ACCGCTCTTCTCCCAAACCTTG HOS1-HIS-F TACGAGAGAAATCAACGGT ISH HOS1 HOS1-HIS-R CTGCGAATCTACGTCTCCT ICE2-geno-F ATGAACAGCGACGGTGTTTGG ISH ICE2 ICE2-R CAAACCAGCGTAACCTGCTG ICE1 RP SALK TTTGTAGGGCCTTTGTTGTTG genotyping ice1-2 ICE1 LP salk ATTCTTTGCTCTGCCTCTTCC LbB1-3 SALK ATTTTGCCGATTTCGGAAC HOS1genoL TGGACCGCATATTTCAGGTAG genotyping ice2-1 HOS1genoR AAGGCACGATAATGGTCTTCC LbB1-3 SALK ATTTTGCCGATTTCGGAAC ICE2genoF TGCATCCGACATCTTTTATCC genotyping hos1-3 ICE2genoR TAACTTCCGGAGATTCACCG Lb3 TAGCATCTGAATTTCATAACCAATCTCGATACAC
Table1 : Primers used in this study.

AtICE1 and thus mediating its degradation via the 26S-proteasome [START_REF] Li | MPK3-and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis[END_REF][START_REF] Zhao | MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability[END_REF]. This MAP kinase cascade promotes the phosphorylation of ICE1 at Ser94, Thr366 and Ser403. In contrast, cold response is positively regulated by another MAP kinase cascade involving MEKK1-MKK2-MPK4, which also suppresses the kinase activities of MPK3 and MPK6. Recently, (Putarjunan et al., 2019a) deciphered the role of the KRAAM motif during stomatal development. Together with an upstream MPK docking motif, the KRAAM motif acts as a specific binding and phosphorylation site for MPK3 and MPK6. Under dark condition, the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) also interacts with and degrades ICE1 proteins through ubiquitination pathways during stomatal development, leading also to its destabilization (Lee et al., 2017b). Whether this pathway also acts to regulate ICE stability, and thus ZOU/ICE activity in the endosperm remains unresolved. Although it is clear that mutants in MPK6 affect embryonic cuticle development, this has been proposed to be due to their activity downstream of the ALE/GSO mediated embryonic cuticle integrity pathway (Creff et al., 2019a). Interestingly however, unpublished results from Audrey Creff, show that the expression of a constitutively active form of MPK6 specifically in the endosperm, causes the production of misshapen seeds. Whether this is a consequence of precocious endosperm breakdown remains to be resolved.

During cold tolerance acquisition, ICE1 can be phosphorylated by OPEN STOMATA1

(OST1) at Ser-278 increasing the activity of ICE1 in activating the transcription of CBF3 (Ding et al., 2015a). (Ding et al., 2015a) have shown that when S278 is substituted with an aspartic acid this mimics a constitutive phosphorylation whereas S278 substituted with an alanine prevents phosphorylation. Similarly to OST1-mediated phosphorylation of ICE1, the sumoylation of ICE1 on Lys-393, mediated by the SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (SIZ1), leads to its stabilization [START_REF] Miura | SIZ1, a small ubiquitin-related modifier ligase, controls cold signaling through regulation of salicylic acid accumulation[END_REF][START_REF] Miura | ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance[END_REF]. According to in silico data, all these genes are expressed during seed development and thus might also be involved in the stabilisation of the ZOU/ICE complex. In order to investigate the post-translational regulation of ICE1 during seed development, truncated and mutated versions of ICE1 targeting the different regulator modification sites will be introduced into different backgrounds and this, combined with genetic analysis should clarify potential roles for these regulators in the seed.

Finally, it is important to highlight the fact that identifying post-translational regulators of ZOU/ICE complexes in the endosperm, although providing valuable genetic tools, does not really answer the important question of why/how complex activity is regulated. Does post-translational regulation by HOS1 or MPK6 for example occur downstream of specific developmental cues to coordinate seed development? Is complex activity altered by metabolic shifts and switches during seed development, for example reporting embryonic or maternal nutrient status? Although these possibilities are all plausible, their reality, and potential mechanistic basis remain totally unexplored.

Bibliography

See general bibliography at the end of the thesis.

95

CHAPTER 2

ARE THE ZOU/ICE FUNCTIONS IN EMBRYONIC SURFACE FORMATION IN ARABIDOPSIS THALIANA CONSERVED IN MARCHANTIA POLYMORPHA?

Preamble

As seen in the Introduction, in A. thaliana, the ZOU/ICE1 complex is involved in three genetically distinct pathways: ensuring embryonic cuticle integrity, orchestrating embryo sheath deposition and production and finally triggering endosperm lysis. The embryonic cuticle integrity pathway involves several proteins included the peptide TWS1, which after having been produced and sulfated in the embryo, can diffuse across gaps in the cuticle to be activated by cleavage at the embryo/endosperm interface by the subtilase ALE1. ALE1 production depends on the activity of the ZOU/ICE complex in the endosperm. Activated TWS1 can then diffuse back into the embryonic apoplast and bind GSO receptors at the surface of the embryo, ensuring embryonic cuticle integrity via a poorly understood mechanism that, by analogy with related pathways, likely involves the release of apoplstic ROS at the embryo surface (Doll et al., 2020;[START_REF] Fujita | SCHENGEN receptor module drives localized ROS production and lignification in plant roots[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. The production of the embryo sheath involves another peptide, KRS, whose production also depends upon the activity of ZOU/ICE1 [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF] , and which belongs to a family of peptides previously proposed to be involved in ROS sensing [START_REF] Wrzaczek | Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS[END_REF]. However, relatively little is known about sheath production and deposition. Anti-extensin antibodies such as α-JIM12 and α-LM1 label epitopes that are produced by the endosperm and accumulate in a continuous layer on the embryo surface where the sheath is formed. The production of these epitopes is eliminated in zou, ice1 and krs mutants, whereas in mutants lacking components of the cuticle integrity signalling pathway it is only their deposition at the embryo surface that is defective [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. Finally, endosperm lysis and elimination in the endosperm is also controlled by the ZOU/ICE1 complex. This process seems to involve cell wall softening and cell separation, followed by a physical crushing to the softened endosperm by the expanding embryo [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF]. This process appears to be largely independently of embryo cuticle development, and likely depends upon a distinct set of ZOU/ICE1 target genes [START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF]. All these observations leave us with a complex picture of the function of the ZOU/ICE1 complex, in which it is difficult to ascertain what the overarching function of these proteins might be. This complexity is compounded by tha fact that, although seeds are fascinating, they can be challenging study systems due to their complex structural and genetic composition. Because the endosperm, where ZOU/ICE1 complexes act is derived from the female gametophyte, which in turn forms the majority of the plant body in early diverging land plants, we reasoned that deciphering the role of ZOU/ICE complex in these plants might help to shed some light on the ancestral function of this complex, and its regulation in plants. In this chapter a focus will therefore be placed on deciphering the ancestral role of ZOU/ICE complexes in M. polymorpha.

The first section of this chapter involves a study deciphering the existence and the role of ZOU/ICE complexes in M. polymorpha. We show the existence of two ICE proteins and two ZOU proteins in this species. We show that the interaction between ZOU and ICE proteins is conserved in M. polymorpha and that both MpZOU proteins and both MpICE proteins can complement the seed phenotypes of respectively A. thaliana zou and ice1 mutants. We go on to demonstrate that two complexes, MpZOU2/MpICE2 and possibly MpZOU2/MpICE1 are involved the formation of the cuticle of gemmae, young dormant clones of the mother plant produced by asexual reproduction in M. polymorpha. Using dyes and microscopy techniques, as well as Mass Spectroscopy analysis, we confirmed that M. polymorpha cuticle contains both abundant phenolic compounds and some more classical aliphatic cuticle components and that MpZOU2 and both MpICE proteins are involved in the deposition of the cuticle but potentially also in determining its final composition. Finally, transcriptomic analysis revealed potential targets of ZOU/ICE complex function in M. polymorpha, supporting a hypothesis in which ZOU/ICE may be involved in ensuring the structural establishment of the cuticle rather than in regulating cuticle biosynthesis in M. polymorpha gemma.

The first section of this chapter is written as an article although some final validations are required before submission.

In a second section of this chapter, I focus on other potential roles of MpICE2 and MpICE1 during M. polymorpha development. Early expression of the genes encoding these proteins will be discussed. Parallels between the roles of ZOU/ICE in embryo sheath production and in ensuring embryonic cuticle integrity in A. thaliana and the role of ZOU/ICE in M. polymorpha will be addressed. The role of MpICE2 in the gamete-bearing tissue will be presented.

Introduction

Early land plants are thought to have evolved from charophyte freshwater green algae around 450 million years ago. This colonisation of land included the development of various key innovations necessary for survival in drier conditions [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF][START_REF] Wickett | Phylotranscriptomic analysis of the origin and early diversification of land plants[END_REF]. One of them was the development of components involved in water transport and permeability control. These include the biosynthesis and deposition of hydrophobic extracellular biopolymers like i) cutin, one of the main component of cuticle, ii) lignin, synthesized from phenolic precursors and deposited in secondary cell walls to provide structural support and facilitate long-distance water transport (the xylem in angiosperms) iii) and suberin, one of the main component of cork resulting from the polymerisation of lipids and phenolics [START_REF] Beisson | Solving the puzzles of cutin and suberin polymer biosynthesis[END_REF][START_REF] Fich | The Plant Polyester Cutin: Biosynthesis, Structure, and Biological Roles[END_REF][START_REF] Weng | The origin and evolution of lignin biosynthesis[END_REF]. In addition to cutin, which is a fatty acid derived polymer, the plant cuticle often also contains phenolic compounds such as ferulic and cinnamic acid derivatives, and cuticular waxes, a mixture of very-long-chain aliphatic compounds that render the cuticle highly impermeable to water [START_REF] Yeats | The formation and function of plant cuticles[END_REF].

In contrast to angiosperms which synthesise both cutin and lignin, bryophytes synthesise only cutin. However, their genomes encode elements of the lignin-biosynthesis machinery. It was recently shown that these components play an essential role in both plant growth and cuticle deposition and function in mosses [START_REF] Kriegshauser | Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes[END_REF][START_REF] Renault | A phenol-enriched cuticle is ancestral to lignin evolution in land plants[END_REF]. [START_REF] Buda | An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens[W][END_REF] also previously described the presence of phenolic residues in the cutin of moss cuticles. Recent phylogeny analysis strongly support that liverworts and mosses are sister groups [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF], and it therefore seems likely that the pre-lignin pathways highlighted in the moss P. patens could be also conserved in the liverwort M. polymorpha, as could be the distinctive phenolic-enriched cuticle.

In A. thaliana, during seed development the basic Helix-Loop-Helix transcription factor heterodimer ZHOUPI/INDUCER OF CBF EXPRESSION1 (ZOU/ICE1) has been shown to be involved in two genetically distinct processes: endosperm breakdown and embryo surface formation involving both the formation of the embryonic cuticle, and the production of the extracuticular embryo sheath [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF][START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. Although both AtZOU and AtICE1 proteins can form homodimers in yeast, it has been shown that the heterodimerization of AtICE1 and AtZOU is required to activate target gene expression and is thus responsible for complex activity. Consistent with this, loss of AtICE1 function, loss of AtZOU function and loss of function of both proteins simultaneously give very similar seed and embryo phenotypes [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF]. Although AtICE1 has a broad expression pattern, and functions in multiple processes outside the seed including stomatal development and cold-tolerance (Chinnusamy et al., 2003;[START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF], AtZOU expression, and thus ZOU/ICE1 activity, is restricted to the developing endosperm.

The role of the ZOU/ICE1 complex in ensuring the formation of the embryo cuticle appears to be achieved at least partly through the transcriptional regulation (possibly indirect) of the subtilisin serine protease-encoding ABNORMAL LEAF-SHAPE1 (AtALE1) gene.

AtALE1 acts in a complex dialogue between the embryo and the endosperm which ensures embryonic cuticle integrity and involves the two receptor-like kinases GASSHO1 and GASSHO2 (AtGSO1 and AtGSO2) and their ligand AtTWISTED SEED1 (AtTWS1) (Doll et al., 2020;[START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. Like Atale1 mutants, Atzou and Atice1 mutants show embryo cuticle defects [START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF]. In addition to regulating cuticle formation AtZOU, likely together with AtICE1, is also involved in the embryo sheath [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. The embryo sheath is composed of a glycoprotein matrix that forms on the surface of the embryo outside the embryonic cuticle. Its deposition requires the coordinated production of material in the endosperm and signalling in the embryo. Molecular players involved in this process include the cysteine-rich peptide AtKERBEROS (KRS) whose expression is AtZOU dependent.

Sheath deposition also requires the embryo expressed receptor like kinase AtGSO1 and AtGSO2, as well as AtALE1, suggesting functional links between the cuticle integrity pathway and sheath deposition [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF].

In A. thaliana, Atzou and Atice1 also show a persistency of the endosperm due to a lack of cell wall modifications and lysis of the endosperm. This problem in endosperm degradation prevents the embryo to grow through the endosperm leading to a collapse of the water filled endosperm cells during desiccation stage of seed development and thus shrivelling seed phenotype to Atzou and Atice1 mutants [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. ZOU and ICE-encoding genes are conserved throughout land plants, in all of which at least one copy of each gene can be found. Given the extremely specific expression pattern of AtZOU in the endosperm, an Angiosperm-innovation, this observation raises the interesting question of whether the ZOU/ICE complex is conserved, and, if so the nature of its ancestral role [START_REF] Catarino | The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land[END_REF][START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF].

In contrast to seed plants, in bryophytes, autotrophic photosynthetic structures are predominantly formed by the haploid gametophytes, which release gametes that fuse to form reduced sporophytic structures that are often nutritionally dependent upon the parental gametophyte. In seed plants this situation is inverted, with the production of dramatically reduced gamotophytes, that develop within the sporophytic tissues. The endosperm, which together with the embryo is a product of the unique double fertilization process observed in angiosperms, appears to have arisen through the acquisition of fertilization competence within a non-egg cell of the much reduced female gametophyte [START_REF] Buda | An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens[W][END_REF]. We propose that the functions of the ZOU/ICE1 complex in the endosperm, could be derived from ancestral gametophytic functions (for example in supporting the development of the sporophyte generation) in early land plants. Studying these functions may therefore provide valuable insights into the functions and regulation of ZOU/ICE1 in the endosperm, which are otherwise, very difficult to pinpoint precisely due to pleiotropic phenotypes of mutants, and the relative inaccessibility of this tissue.

To this end, we have carried out a study of the ZOU/ICE complex in the liverwort Marchantia polymorpha. We have used histology, genetics and biochemistry to show that M. polymorpha cuticle is composed of aliphatic and phenolic compounds. Moreover, we show that ZOU/ICE function in cuticle development is ancient since MpZOU2 and both MpICE share functions in cuticle formation/polymerisation/composition of M. polymorpha gemmae.

RESULTS THE M. POLYMORPHA GENOME CONTAINS TWO ZOU-ENCODING AND TWO ICE-ENCODING GENES.

Previous results in A. thaliana have shown that AtZOU and AtICE1, acting as heterodimers, are necessary to activate target promoter activity and mediate endosperm breakdown and embryo cuticle formation [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF]. Both proteins are ancient within land plant and at least one copy of each gene can be found in all land plants, raising the question of the ancestral role of ZOU and ICE [START_REF] Catarino | The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land[END_REF][START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF]. BLAST searches and subsequent phylogenetic analysis revealed one M. polymorpha orthologue of AtZOU which was named MpZOU1 (Mapoly0126s0029) and a paralogue, which was named MpZOU2 (Mapoly0012s0202). Sequence comparisons showed that MpZOU1 was more similar to AtZOU in the bHLH domain (69,8%) and in the C-terminal region (28,4%) than MpZOU2 which shows 50,9% and 22,4% similarity respectively. Whereas P. patens lack orthologues of AtZOU and gymnosperms such as Pinus has both orthologues of MpZOU1 and MpZOU2, all Angiosperms (including A. thaliana) lack MpZOU2, and only those of MpZOU1 are present. Interestingly, despite these losses, all land plant groups have at least one copy of ZOU suggesting that this protein retains functions that are indispensable for land plant biology (Figure S1).

AtICE1 and AtICE2 are thought to have arisen from a duplication within the Brassica lineage [START_REF] Kurbidaeva | Arabidopsis thaliana ICE2 gene: phylogeny, structural evolution and functional diversification from ICE1[END_REF]. We found two M. polymorpha co-orthologues of AtICE1/AtICE2 which were named MpICE1 (Mapoly0150s0015) and MpICE2 (Mapoly0150s0016). Sequence similarity in the bHLH domain and the C-terminal between MpICE2 and AtICE1 are respectively 85,9% and 60,4% whereas the similarity between MpICE1 and AtICE1 are 74,4% and 47,2% for the bHLH domain and the C-terminal region respectively. Phylogenetic analysis revealed that most land plants have several copies of the ICE gene, as is the case for Zea mays, where two co-orthologues and one potential paralogue of the A. thaliana genes have been described (Figure S2) [START_REF] Grimault | ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development[END_REF] . These results globally confirm the relevance of cross-species interactions demonstrated by yeast-2-hybrid experiments, and suggest that the properties of ZOU and ICE proteins have been conserved between bryophytes and angiosperms.

MPZOU2 AND MPICE2 ARE EXPRESSED IN THE DORSAL GAMETOPHYTE EPIDERMIS

In order to analyse potential roles played by MpZOU1, MpZOU2, MpICE1 and MpICE2 in M. polymorpha development, we first asked where they are expressed in the gametophyte.

Transcriptional reporter lines containing 5kb of regulatory sequences upstream of the start codon of the MpZOU1 and the MPZOU2 genes fused with either nuclear localised TdTomato fluorescent protein or to the GUS gene encoding β-glucoronidase were generated. Similarly, GUS and TdTomato transcriptional reporter lines were generated using 6.7kb and 5.3kb of regulatory sequences upstream of the start codon of MpICE1 and MpICE2 respectively.

The proMpZOU1:GUS and proMpICE1:GUS reporters showed a very similar expression pattern in 10-day-old thallus, predominantly on the ventral surface along the midrib, in the scales, the parenchyma and the rhizoids (Figure 2). Weaker expression of proMpZOU1:GUS and proMpICE1:GUS lines was also detectable in the dorsal epidermal cell layer, overlapping with the expression pattern of proMpZOU2:GUS and proMpICE2:GUS reporters (see below).

In contrast to the predominantly ventral expression of MpZOU1 and MpICE1, the proMpZOU2:GUS and proMpICE2:GUS reporters were predominantly expressed in the dorsal epidermis of 10-day-old thallus (Fig GUS). However, weaker, but detectable expression was also detected in the ventral epidermis of proMpZOU2:GUS thallus, overlapping with the expression pattern of proMpZOU1:GUS. No detectable GUS staining was visible on the dorsal surface of the thallus of proMpICE1:GUS reporter lines. Early expression of the genes of interest was also investigated. Interestingly, GUS staining was detected in the epidermis of developing gemmae of proMpZOU2:GUS lines revealing an early expression of MpZOU2. 15 day old thalli of nuclear TdTomato transcriptional reporter lines were also analysed and results were consistent with expression patterns observed in the GUS reporter lines.

Expression of proMpZOU1:TdTomato-NLS and proMpICE1:TdTomato-NLS was easily detectable in rhizoids but could also be detected in air pore chamber cells and epidermal cells on the dorsal thallus surface (Figure 2). In contrast, proMpZOU2:TdTomato-NLS and proMpICE2:TdTomato-NLS lines confirmed a strong expression of MpZOU2 and MpICE2 in dorsal epidermal cells and air pore chambers. Despite the apparent absence of detectable signal in the ventral regions and rhizoids of proMpICE2:GUS lines, expression in the rhizoids was detected for proMpICE2:TdTomato-NLS. MpZOU2 was again found to be expressed in rhizoids based on proMpZOU2:TdTomato-NLS lines.

Expression data suggest, firstly, that both MpZOU1, MpZOU2, MpICE1 and MpICE2 are all expressed in both dorsal and ventral thallus tissues. However, the levels of expression of these genes differ, with MpZOU1 and MpICE1 expressed predominantly in the ventral surface and rhizoids and thus potentially involved in ventral parenchyma and rhizoid development, and MpZOU2 and MpICE2 predominantly expressed in the dorsal epidermis, and thus potentially sharing epidermal functions.

It should be borne in mind that the ICE proteins in A. thaliana are strongly regulated at the post-translational level [START_REF] Ding | OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis[END_REF][START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF][START_REF] Miura | SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis[END_REF][START_REF] Miura | ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance[END_REF][START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF] and that gene transcription does not necessarily reflect protein levels.

Translational reporters are under construction to address this issue.

M. POLYMORPHA GEMMA CUTICLE IS COMPOSED OF BOTH ALIPHATIC AND PHENOLIC MOLECULES

Plant cuticles contain a highly variable array of aliphatic molecules such as waxes and cutin [START_REF] Yeats | The formation and function of plant cuticles[END_REF]. To date very little is known about cuticle composition in M.

polymorpha. Thus, we used the lipophilic dye Nile Red, originally used to stain the suberin-rich endodermis in A. thaliana roots [START_REF] Greenspan | Nile red: a selective fluorescent stain for intracellular lipid droplets[END_REF][START_REF] King | Some oil soluble dyes which stain suberized deposits in orange vesicles[END_REF], to see whether we could stain the cuticles of wild-type M. polymorpha gemmae. Subsequent analysis by confocal microscopy strongly suggests that the cuticle of M. polymorpha gemmae also contains aliphatic molecules (Figure 3A), as indicated by a very thin layer of Nile Red staining covering the surface of gemmae, which appears to be reinforced near the apical notch. Although bryophytes synthesize cutin, the main component of the cuticle, a recent study has shown that proteins associated with the phenolic-based lignin-biosynthesis machinery of Angiosperms play an essential role in ensuring normal cuticle structure/deposition in mosses [START_REF] Renault | A phenol-enriched cuticle is ancestral to lignin evolution in land plants[END_REF]. Consistent with this finding [START_REF] Buda | An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens[W][END_REF]) also described the presence of phenolic residues in the cutin of moss cuticles. Recent phylogenetic analysis strongly supports the idea that liverworts and mosses are sister groups [START_REF] Puttick | The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte[END_REF], and we therefore hypothesize that the pre-lignin pathways highlighted in P. patens could be also conserved in M. polymorpha, as could the distinctive dependence upon phenolic compounds for coordinated cuticle assembly. We therefore tested different staining techniques to assess the presence of phenolic compounds in the M. polymorpha cuticle. We concentrated on dyes previously reported to stain lignin, which is composed of phenolic compounds. We found that Diphenylborinic acid, which stains A. thaliana lignin [START_REF] Corona-Carrillo | Peroxidase activity in scutella of maize in association with anatomical changes during germination and grain storage[END_REF][START_REF] Reich | HPTLC Methods for Identification of Green Tea and Green Tea Extract[END_REF] stains the cuticle of wild-type M. polymorpha gemmae (Figure 3B). Consistent with the staining results, mass spectrometry analysis of cuticles from wild-type M. polymorpha gemma demonstrated that several types of phenolics are present in relatively high quantity in the cuticle, in particular p-coumarate and ferulate (Figure 4).

BOTH MPICE AND MPZOU2 GENES PROMOTE GEMMA CUTICLE FORMATION

To assess the potential role of the M. polymorpha ZOU and ICE proteins in cuticle development, CRISPR-Cas9 single loss of function mutants were generated for MpZOU1, MpZOU2, MpICE1 and MpICE2. In addition, the double mutants, Mpzou1 Mpzou2 and Mpice1

Mpice2 were generated (Figure S4 andS5).

In A. thaliana, the cuticle is exclusively synthesized by epidermal cells [START_REF] Ingram | The roles of the cuticle in plant development: organ adhesions and beyond[END_REF][START_REF] Javelle | Overexpression of the Epidermis-Specific Homeodomain-Leucine Zipper IV Transcription Factor OUTER CELL LAYER1 in Maize Identifies Target Genes Involved in Lipid Metabolism and Cuticle Biosynthesis[END_REF] In M. polymorpha, nothing is as clear as it is in A. thaliana however some clues suggest that although epidermal cells are on both side of the thallus, the cuticle might be more pronounced on the dorsal surface. Indeed, the dorsal surface is the surface in direct contact with the air and environmental hazards while the ventral surface is in contact with the earth and therefore more protected than the dorsal surface from drying out.

Moreover, cuticle has already been reported on the dorsal surface on the thallus, i.e. on air pore ledges [START_REF] Schönherr | Hydrophobic cuticular ledges prevent water entering the air pores of liverwort thalli[END_REF][START_REF] Xu | The land plant-specific MIXTA-MYB lineage is implicated in the early evolution of the plant cuticle and the colonization of land[END_REF]. In addition to that, since MpZOU2 and MpICE2 show expression predominantly in the dorsal epidermis of the thallus, similarly to the expression of MpSBG9, a R2R3 MYB transcription factor involved in cuticle biosynthesis in M. polymorpha [START_REF] Xu | The land plant-specific MIXTA-MYB lineage is implicated in the early evolution of the plant cuticle and the colonization of land[END_REF], we investigated whether M. polymorpha ZOU/ICE complex might be involved in cuticle development.

Confocal microscopy analysis after Nile Red staining of gemmae from the different backgrounds was carried out. Consistent with the results of expression analysis, Nile Red In order to understand more about alterations in cuticle composition in mutants lacking MpZOU/MpICE complexes, Mass Spectrometry analysis of the cuticles of gemmae directly harvested from gemmae cups was carried out on wild type (Tak2xTak1), Mpzou2, Mpice2, Mpzou1Mpzou2 and Mpice1Mpice2 plants. Several aliphatic molecules were found to be less abundant in some backgrounds. Statistically significant differences were only observed specificially in the Mpzou1Mpzou2-db5 background for C18:3 Δ9,12,15, C18:1 Δ9 and C16:1 Δ7. By contrast, C14:0, C16:0, C17:1 Δ10, C16:2 Δ7,10, C16:3 Δ7,10,13 and C9 DCA (dichloroacetate) were statistically less abundant in both Mpzou2-3 and Mpzou1Mpzou2-db5 backgrounds compared to wild-type (Figure 4). Among the phenolics detected in WT gemma cuticle, only one, p-coumarate, showed a significant difference compared to wild-type. This compound was found to be more abundant specifically in Mpzou2-3 gemma cuticle. None of the Mpice mutants tested showed statistically significant differences in the amount of any of the molecules identified. However, when interpreting this data, it is important to bear in mind that some exotic/unexpected fatty acids or other molecules were not identified in the analysis and that important changes were therefore not detected. In addition, delipidation may have removed unpolymerized components, which would have masked potential defects in cuticle assembly.

WHAT ARE THE DIRECT/INDIRECT TARGETS OF MPZOU2?

To identify genes acting downstream of MpZOU2 as effectors of cuticle biogenesis, we carried out transcriptional profiling (RNAseq) of gemmae of wild type (Tak2xTak1), Mpzou2 and Mpzou1Mpzou2 mutants.

Lists of genes differentially regulated in Mpzou2 and Mpzou1 Mpzou2 double mutants were established. Because the Mpzou2 single mutant used in our analysis was in the opposite gender type to our wild-type and Mpzou1 Mpzou2 double mutant alleles, and to increase rigour, the two lists were then combined and only genes misregulated in both mutant genotypes were retained. Since co-expression of ZOU and ICE1 genes activates transcription in several in planta assays in moss and tobacco [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF]Feng et al., 2018) we concentrated on transcripts that were less abundant in mutants compared to wild type. Within this set of 565 differentially expressed genes (DEG), we identified numerous genes encoding proteins involved in cell wall modification and/or structure, including expansins, xyloglucan endotransglycosylases (XETs), and pectate lyases (Figure 5). At the level of cuticle biosynthesis, three genes identified by [START_REF] Bowman | Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome[END_REF] as orthologues of key genes in A.

thaliana cuticle biogenesis were present in our dataset, encoding orthologue of glycerol-3- phosphate acyltransferase 6 (GPAT6) (Mp7g03880), a Cutin Synthase (CUS) (Mp8g13940), and an ABC transporter (Mp8g12130) (Figure 5) [START_REF] Bowman | Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome[END_REF].

Among the 565 DEGs, 339 DEGs have GO annotation and 81 GO terms were enriched.

(http://plantregmap.gao-lab.org/) (Figure 5). Loss of function of Mpzou2 and Mpzou1Mpzou2 significantly affected cell wall biogenesis and structure.

The dataset was also strikingly enriched for gene encoding proteins encoding REDOX associated proteins. Of particular note were peroxidases, with 9 downregulated, of which 8 are predicted to encode class III peroxidases, Class III peroxidases have various roles, and are often associated with cell wall remodelling, including the polymerisation of phenolic units to form lignin, or the polymerisation of extensins [START_REF] Mishler-Elmore | Extensins: Self-Assembly, Crosslinking, and the Role of Peroxidases[END_REF][START_REF] Rojas-Murcia | High-order mutants reveal an essential requirement for peroxidases but not laccases in Casparian strip lignification[END_REF] (Table S1). In addition, we found that a large number of genes encoding REDOX associated proteins were downregulated in our dataset, including genes encoding Aquaporins (associated with the transport of H2O2 (3), Tyrosinases (6), Polyphenol oxidases (4) and Dirigent proteins (2) (Figure 5). Despite the fact that analyses were carried out in different tissues, more than 50% of the genes belonging to this composite class have previously been identified as upregulated in plants lacking function of the MpTCP1 protein, which has been shown to be a key regulator of REDOX associated proteins [START_REF] Busch | MpTCP1 controls cell proliferation and redox processes in Marchantia polymorpha[END_REF]. In addition a large number [START_REF] Fich | The Plant Polyester Cutin: Biosynthesis, Structure, and Biological Roles[END_REF] of genes encoding Phytocyanin-domain proteins, also thought to be involved in ROS reactions [START_REF] Negin | Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins[END_REF][START_REF] Reyt | Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips[END_REF] were also downregulated in our dataset. Finally, we were interested to find that a gene encoding a STIG1-domain (cysteinerich peptide) protein (Mp3g18230), as well as two EXTENSIN-encoding genes were downregulated in our dataset. Comparison with the A. thaliana showed that this protein was most closely related to KERBEROS, a key target of the ZOU complex in the A. thaliana endosperm, and necessary for the formation of the EXTENSIN-rich embryo sheath [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. The STIG domain of the related protein GRIM REAPER (GRI) has been proposed to be involved in ROS sensing (Figure S6) [START_REF] Wrzaczek | Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS[END_REF].

Material and Methods

PLANT GROWTH

All wild-type plants used in the project were Marchantia polymorpha Takaragaike-1 (Tak-1) for the male or Takaragaike-2 (Tak-2) for the female [START_REF] Ishizaki | Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology[END_REF]. Spores for transformation were the progeny of Tak2 x Tak1 [START_REF] Ishizaki | Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology[END_REF]. Lines were asexually maintained and propagated through gemmae growth as described in [START_REF] Takenaka | Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L[END_REF][START_REF] Okada | Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha[END_REF] except that we used Johnson media (see details in Annexes) complemented with 1.4% agar. Gemmae were transferred on plate to long-day conditions (16h, 21°C) for approximately 3 to 5 weeks, before harvesting the new generation of gemmae for further analysis.

Arabidopsis thaliana seeds were gas sterilized with chlorine (3mL HCl (33%) in 150mL bleach) for 2 hours and then sown on Murashige and Skoog (MS) medium with 0,5% sucrose in sterile condition, stratified for 2 days at 4°C and grown for 7 days in short-day conditions (8h light, 19°C). Seedlings were then transferred to compost (Argile 10 (favorit)) and grown under short day conditions (8h light, 19°C) for two to three weeks before transferred to long day conditions (16h light, 21°C). All A. thaliana mutants used in this study were previously published: the loss of function mutant Atzou-4 allele [START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF] was used for all complementation experiments as was the loss of function Atice1-2 allele [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF]. The ecotype Columbia-0 (Col-0) was used as a wild-type control.

GENERATION OF CRISPR-CAS9 KNOCK OUT MUTANTS

CRISPR-Cas9 mutants were generated as described in [START_REF] Sugano | CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L[END_REF]. Two gRNAs for each gene were designed to generate single mutants (Figure S3). For ZOU constructs, the oligonucleotides encoding the sgRNA were first cloned into pGE_EN03 (Table 1). For both Mpzou1 and Mpzou2 single mutants, the sgRNA constructs were then introduced into pMpGE010 or pMpGWB401 destination vectors (containing the CAS9 protein). For double Mpzou1 Mpzou2 mutants, two separate constructs were introduced by co-cultivation with two Agrobacterium GV3101 strains. For ICE constructs, the oligonucleotides encoding the sgRNA were also first cloned into pGE_EN03 and then introduced into pMpGE010 or pMpGWB401 destination vectors. For Mpice1 Mpice2 double mutants, the two sgRNAs were cloned into a single destination vector, pMpGE_EN04 and pBC-GE14 (Table 1) [START_REF] Sugano | CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L[END_REF][START_REF] Sugano | Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha[END_REF].

YEAST-TWO HYBRID ASSAYS

The full length of M. polymorpha and A. thaliana ZOU and ICE1 proteins were expressed as fusions to the GAL4 DNA binding domain (bait constructs) or with the GAL4 transcriptional activation domain (prey constructs) and cloned into pGBKT7-GW or pGADT7-GW (Lu et al. 2010). The truncation of AtICE1, MpICE1 and MpICE2 was respectively residues 263-494, 443-710 and 389-692, based on Denay et al. 2014 for AtICE1 and on the alignment of AtICE1-T, MpICE1 and MpICE2. Matchmaker Y2HGold Yeast was co-transformed with bait and prey plasmids and transformants were selected on media lacking leucine and tryptophan (-LW). Interaction of bait and prey constructs resulted in activation of reporter genes for histidine and adenine synthesis (-LWHA).

CLONING

Promoter regions of MpZOU1, MpZOU2, MpICE1 and MpICE2 were cloned using Gateway technology (Invitrogen). 5kb of regulatory sequences upstream of the start codon of the MpZOU2 genes was PCR-amplified using MpZOU2_Prom_F and MpZOU2_Prom_3R primers for MpZOU2, cloned into pENTRDTOPO. From the resulting vector, MpZOU2 promoter was introduced into pDONR221 using Topoisomerase I mediated ligation. 5kb of regulatory sequences upstream of the start codon of the MpZOU1 genes was PCR-amplified using YT5F and YT5R primers with the Gateway extensions attB1-B2 and cloned by BP reaction into pDONR221 vector (Table1). 6.7kb and 5.3kb of regulatory sequences upstream of the start codon of MpICE1 and MpICE2 respectively genes were PCR-amplified using the pMpICE1b-F and pMpICE1b-R primers for MpICE1 or the pMpICE1a-F and pMpICE1a-R primers for MpICE2 with the Gateway extensions attB1-B2 and cloned by BP reaction into pDONR221 vector (Table1). Plasmids were purified and the expression vectors containing the transcriptional constructs were obtained by recombination with the destination vector pMpGWB104 or pMpGWB116 [START_REF] Ishizaki | Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha[END_REF] in LR clonase mediated reaction following the manufacturer's instructions.

For Mpzou2 complementation, MpZOU2 promoter and MpZOU2 CDS PCR-amplified with and recombined in pMpGWB301 [START_REF] Ishizaki | Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha[END_REF] using Gibson Assembly mix (Biolab). Primers were designed with NEBuilder assembly tool: YT67F and YT67R for MpZOU2 promoter and YT68F and YT68R for MpZOU2 CDS (Table 1).

M. POLYMORPHA SPORELING TRANSFORMATION AND A.THALIANA FLORAL DIPPING

TRANSFORMATION.

M. polymorpha spores from Tak-2 x Tak-1 crosses, (approximatively 5 sporangia per transformation) were sterilised for 15minutes with 0.1% sodium hypochlorite (NaDCC) solution, spun for 1 minute at maximum speed in a microcentrifuge, resuspended in 500µL of water and grown 7 days into 50mL of OM51C (Takenaka 2000) liquid media under 130 rpm. agitation at 22°C, 16 hours light. Agrobacterium-mediated transformation of 7 day-old germinated spores was performed as previously described in Ishizaki 2008 unless otherwise described. Resulting cells were incubated under 16 hours light, 22°C. Gemmae from the second generation were subjected to further analysis. Sporeling transformation was performed for all constructs except for generating the Mpzou2 complementation lines which required thallus transformation as described in [START_REF] Kubota | Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli[END_REF].

Constructs s of interest for the complementation of MpICE and MpZOU genes in A.

thaliana, were electroporated into Agrobacterium tumefaciens strain GV3101 mp90. Bacteria were plated at high density onto Yeast Extract Broth (5g/L Beef extract, 1g/L Yeast extract, 5g/L Peptone, 5g/L Sucrose, 15g/L Bacto Agar) plates with the suitable antibiotics and incubated 2 days at 28°C. Bacteria were scraped off plates, suspended in 30mL of infiltration solution (MgCl2 10mM, sucrose 5% and Silwet-L-77 0.03%) and further diluted with 120mL of infiltration solution. Atice1-2 or Atzou-4 flower buds were dipped in the bacterial solution for 10s, with gentle agitation, covered with plastic bags for 24h and allowed to set seeds. Primary transformants were selected on solid MS medium containing suitable antibiotics and then transferred to soil. T1 and T2 generation plants were selected on agar-solidified MS with antibiotics. T2 lines were genotyped and subjected to phenotypic and molecular analyses.

SEED-SHAPE ANALYSIS

Percentages of misshapen seeds were counted from images taken with the VHX900F digital microscope (Keyence), using the cell counter extension of the ImageJ software. For each genotype, seeds from 4 distinct mother plants were analysed with approximately 1000 seeds analysed per mother plant.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF TOLUIDINE BLUE PERMEABILITY

To assess cuticle integrity, toluidine blue staining assays were performed [START_REF] Tanaka | A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis[END_REF]. For qualitative toluidine blue staining tests, etiolated seedlings were used. Seedlings were stratified in the dark, exposed to a 6h light flash and then grown for 4 days in darkness.

They were then stained for 2 min in 0.05% w/v Toluidine blue solution with 0.1% v/v Tween 20 and washed abundantly under tap water. Pictures were taken with a Keyence VHX900F digital microscope.

For quantitative toluidine blue staining test, 7-day old light-grown seedlings were stained in the same way. In order to extract toluidine blue and chlorophyll, the cotyledons of ten seedlings were collected and placed in 1mL of 80% ethanol for at least 3 hours in darkness.

Toluidine blue content of the resulting solution was measured at 626nm with a spectrophotometer. This absorbance was then normalized using chlorophyll absorbance at 430nm, a wavelength not absorbed by toluidine blue. 6 repetitions were performed per condition [START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF].

HISTOCHEMICAL STAINING FOR GUS ACTIVITY OF WHOLE MOUNT THALLUS

MpZOU1, MpZOU2, MpICE1 and MpICE2 promoters were cloned into the pMpGWB104 vector and subsequently Tak2xTak2 spores were transformed [START_REF] Ishizaki | Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha[END_REF]. Histochemical assays for GUS activity were performed according to [START_REF] Jefferson | GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF] with some minor modifications. Briefly, after three 20 minute vacuum infiltrations with the GUS staining solution (50mM sodium phosphate buffer, pH 7.2 ; 0.5mM potassiumferrocyanide; 0.5mM potassium-ferricyanide; 10mM EDTA; 0.01% Triton ; 1mM 5-bromo-4cloro-3-indolyl-Beta-glucuronic acid (Duchefa Biochemie, CAS number 114162-64-0), plants were incubated overnight at 37°C. They were then dehydrated by incubation in increasing concentrations of Ethanol (15%, 30%, 50%), for 20 minutes (per bath) at room temperature.

Plants were then fixed in FAA fixative solution (10% formaldehyde, 5% acetic acid, 50% ethanol) for 45 minutes under vacuum followed by either 2 hours at room temperature or overnight at 4°C. Dehydration steps to remove the chlorophyll were then performed by incubation in further increasing concentrations in Ethanol (50%, 70%, 100%), for 20 minutes per bath at room temperature. Observations of GUS activity were made using a Keyence VHX900F digital microscope.

HISTOCHEMICAL STAINING FOR GUS ACTIVITY OF VIBRATOME-SECTIONED THALLUS

15 to 20 day-old transcriptional reporter lines (described in the previous section) were harvested and placed into a small plate. 5% Phytagel (Sigma CAS : 71010-52-1 ; P8169) at 50°-60°C was then poured onto the plants. The plate was then stored for 30 min to 1hour at 4°C to cool down before mounted onto the Leica VT1000 S Microtome. Approximately 10µm sections were performed and placed into 1%PBS solution before staining as described above.

Observations of GUS activity were made using a Keyence VHX900F digital microscope.

CONFOCAL MICROSCOPY

Expression of proMpZOU1:TdTomato, proMpZOU2:TdTomato, proMpICE1:TdTomato and proMpICE2:TdTomato 10-day-old thallus were analysed by confocal microscopy using the SP8 Leica confocal microscope (Emission 581nm, Excitation 554nm).

HISTOCHEMICAL STAINING WITH NILE RED AND DIPHENYLBORINIC ACID

Mature gemmae were harvested directly from the gemmae cups and placed in filter baskets. Nile Red staining solution was prepared to contain 2µg/mL Nile Red (CAS number 7385-67-3) diluted in 50mM PIPES (SIGMA, CAS number 100037-69-2) at pH 7.0. Gemmae were incubated in this solution for 20 minutes at room temperature and then washed three times with water. Gemmae were mounted on slides and imaged with a LSM700 microscope (Leica) or SP8 confocal microscope (Leica) (553nm for excitation and 637nm for emission).

A similar staining procedure was used for Diphenylborinic acid as described in [START_REF] Corona-Carrillo | Peroxidase activity in scutella of maize in association with anatomical changes during germination and grain storage[END_REF] The staining solution was prepared to contain 0.5% of Diphenylborinic acid (CAS number 524-95-8 ) diluted in Ethyl acetate (CAS number 141-78-6 ). Gemmae were treated 10 minutes in ethyl acetate and the staining solution was then poured onto the gemmae for 10 minutes at room temperature and then washed three times successively with water.

Gemmae were mounted on slides and imaged with a LSM700 microscope (Leica) or SP8 confocal microscope (Leica) (365nm for excitation and 418nm for emission) [START_REF] Corona-Carrillo | Peroxidase activity in scutella of maize in association with anatomical changes during germination and grain storage[END_REF][START_REF] Dimakos | Boronic acid/Brønsted acid co-catalyst systems for the synthesis of 2H-chromenes from phenols and α,β-unsaturated carbonyls[END_REF][START_REF] Reich | HPTLC Methods for Identification of Green Tea and Green Tea Extract[END_REF].

TRANSMISSION ELECTRON MICROSCOPY

Gemmae collected from 1month old mother plants of Mpzou1 Mpzou2-db5, Mpice1

Mpice2-db1 and Tak2xTak1 progeny were directly immerged into the fixative solution composed of 3% glutaraldéhyde and 1% PFA. Gemmae was then fixed a second time following the OTO-fixation described in [START_REF] Deerinck | NCMIR methods for 3D EM: A new protocol for preparation of biological specimens for serial block face scanning electron microscopy[END_REF]. EPON resin was used to embed the samples which were then sectioned by the serial-block-face module and imaged by scanning with the Zeiss Sigma VP300 (Gatan inc.) microscope at 4kV.

RNA EXTRACTION AND RNA-SEQUENCING

Gemmae from approximately 10 gemmae cups of Tak2xTak1 progeny, Mpzou2-3, Mpzou1Mpzou2-5 thallus were harvested and frozen in liquid nitrogen before RNA extraction.

In each case, three independent RNA samples were extracted for each genotype (1 RNA sample equals gemmae from approximately 10 gemmae cups). Gemmae were ground with two beads using the TissueLyser (1min, frequency 30/sec). Total RNAs were extracted in TRIzol. Briefly, 1mL of TRIzol were added in each tubes before the samples thawed and then were vortexed. After centrifugation (12,000 rpm, 10min, 4°C) ethanol was added to the supernatant to a final concentration of 35%. RNAs were purified using silica spin columns from Qiagen Plant RNA extraction kits. The rest of the RNA extraction was done according to the manufacturer's instructions. Genomic DNA contamination was removed from total RNA samples using the Turbo DNA-free DNase I (Ambion) according to the manufacturer's instructions. RNA integrity and quantity were checked using an Agilent Bioanalyzer 2100.

Sequencing of the 9 samples was carried out on an Illumina NextSeq 500 sequencer, using single end mode, leading to 50-54 million reads per sample, of length 75 bp.

Current reference genome sequence for Marchantia polymorpha, MpTak1v5.1 [START_REF] Montgomery | Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin[END_REF] has been built from a male individual. To allow a correct mapping of RNA-seq reads from both female and male individuals, we added to this genome sequence the scaffolds described to be specific to females from a previous version of the reference genome sequence (JGI v3.1, (Bowman et al., 2017), scaffolds 17, 18, 210, 227, 240, 250 and 497).

Reads were checked for quality and absence of adapters and contaminants with FastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). They were then mapped on the genome sequence using HISAT2 [START_REF] Kim | The unified ICE-CBF pathway provides a transcriptional feedback control of freezing tolerance during cold acclimation in Arabidopsis[END_REF], in two-pass mode, using the reference genome annotation: a first mapping was performed with the complete dataset to discover putative new splice junction, before the per-sample mapping. Reads per gene were counted using FeatureCounts v1.5.1 (Liao et al., 2014). DESeq2 version 1.28.1 (Love et al., 2014) was used with R version 4.0.2 to perform the principal component analysis and the differential gene expression analysis. Genes having less than 6 reads in the sum of all samples were considered as non-expressed and discarded.

Mapping reads on a genome sequence containing both chromosomes U and V could theoretically lead to irrelevant results, for example in the presence of a pseudo-autosomal region or of scattered homologous genes [START_REF] Bowman | Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome[END_REF] having high sequence similarities between female and male genomes. To assess the specificity of reads matching on sex-determining chromosomes, we ran HISAT2 1000 times with a random seed and counted reads matching equally well U and V chromosomes. We observed an average of 45 of those reads in each sample, while at the same time an average of 125,000 reads was specific to chromosomes U or V, so we considered they would have a neglectable effect on the differential analysis.

CUTIN COMPOSITIONAL ANALYSIS

Protocol for cutin monomers analysis was adapted from [START_REF] Elejalde-Palmett | ABCG transporters export cutin precursors for the formation of the plant cuticle[END_REF][START_REF] Li-Beisson | Acyl-Lipid Metabolism[END_REF][START_REF] Renault | A phenol-enriched cuticle is ancestral to lignin evolution in land plants[END_REF]. Gemmae from Tak2xTak1 progeny, Mpzou2-3, Mpzou1Mpzou2-db5, Mpice2-6, Mpice1Mpice2-db1 mutants were harvested from the cups of 4-week-old thallus to have a minimum 10mg of lyophilised material per genotype. For each sample, about 10 mg of ground, lyophilized gemmae tissue was added to a pre-weighed 12 ml glass tube for processing. The tissue was delipidated by extensive washing with a series of solvents, as follows. A total of 5 ml of isopropanol pre-heated to 85 °C was added to each sample and incubated at 85 °C for 10 min, then after cooling to room temperature, the samples were shaken at 1000 r.p.m. for 1h and centrifuged at 1,500g for 2 min to pellet most of the tissue. The supernatant was removed with a glass Pasteur pipette. The tissue was then washed with the following series of solvents: 2:1 chloroform: methanol, 1:1 chloroform: methanol, 1:2 chloroform: methanol, pure methanol. The samples were agitated with each of these solvents at 1000 r.p.m. for at least 1h and up to overnight, with centrifugation (as described above) after each wash. The tissue was then dried in SpeedVac at 40°C for 1h. The glass tube was weighed to determine the dry, delipidated tissue weight. A total of 1 ml of reaction medium (12:3:5 methanol: methyl acetate: 25% sodium methoxide) and 5 µg heptadecanoic acid and 10 µg pentadecalactone (internal standards) were added to each sample, and the mixture was then heated at 60 °C for 2h to depolymerize the cutin. The samples were then cooled to room temperature and 2 ml dichloromethane, 0.25 ml glacial acetic acid and 0.5 ml 0.9% NaCl in 100 mM Tris (pH 8.0) was added to each and vortexed to mix. Two phases were separated by centrifugation for 2 min at 1,500g. The lower, organic phase was transferred to a new vial and 1 ml of 0.9% NaCl in 100 mM Tris (pH 8.0) was added and mixed by vortexing. Two phases were again separated by centrifugation (2 min at 1,500g).

Lower phase was recovered and dried on 0.5 g anhydrous sodium into a clean vial to remove any residual water. Each cutin sample was dried under a stream of nitrogen, then derivatized with 50 µl each of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide for 10 min at 90 °C.

The samples were analysed by gas chromatography (GC) using an Agilent GC8890 coupled to a Leco Pegasus BT time-of-flight mass spectrometer. Data were analyzed with the ChromaTOF software, which was used to identify compounds in NIST chemical libray based on both retention index and fragmentation spectrum, and to calculate peak area from compound-specific extracted ion chromatogram. Peak areas were normalized to the internal standards and the dry, delipidated tissue weights.

Discussion

Our work has revealed a potentially very ancient and conserved role for ZOU/ICE complexes in cuticle development in land plants. Our results suggest that both the MpZOU2/MpICE1 and MpZOU2/MpICE2 complexes control the formation of the phenolicenriched cuticle of gemmae in M. polymorpha.

123

REDUNDANCY OF ICE PROTEINS

Our results show that both MpICE proteins can interact with both MpZOU proteins in yeast. We show that the MpICE1 and MpICE2 expression patterns overlap in the dorsal epidermis of M. polymorpha thallus. Consistent with functional redundancy between the MpICE proteins, the Mpice1 Mpice2 double mutant shows a stronger cuticle phenotype in gemmae (as shown by staining) than either single mutant. A similar situation has been observed in A.

thaliana, where AtICE1 and AtICE2 act redundantly during stomatal development (Kanaoka et al., 2008c). In this developmental pathway, both AtICE1 and AtICE2 form heterodimers sequentially with the key transcription factors SPEECHLESS, MUTE and FAMA. Similarly, to our observations in M. polymorpha for cuticle production in Mpice1 and Mpice2, both Atice1 and Atice2 single mutants still produce functional stomata although Atice1 has a stronger phenotype than Atice2. However, the double mutant Atice1 Atice2 does not produce any stomata. Brachypodium, another Brassicae, encodes two ICE proteins which, similar to A. thaliana also result from the Brassicae-specific duplication. However, unlike in A. thaliana, the loss of only BdICE1 results in the total loss of stomata. BdICE2 is nevertheless required for stomatal developmat but at a late stage of stomatal differentiation and function [START_REF] Raissig | Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity[END_REF].

The redundancy of ICE1 and ICE2 during seed development in A. thaliana has recently been studied (Loue-Manifel et al. in preparation, Chapter 3). The results of this work highlight the importance of understanding the tissue specific regulatory relationships between related transcription factors when drawing conclusions regarding the presence or absence of functional redundancy between family members. Finally, in a context of significant potential post-translational regulation [START_REF] Ding | OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis[END_REF][START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF][START_REF] Li | MPK3-and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis[END_REF][START_REF] Miura | SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis[END_REF][START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF] it is also important to bear in mind that the overlaps in gene expression highlighted in this article only reflect the RNAs levels due to the use of transcriptional reporter lines.

In M. polymorpha, the apparent redundancy observed for MpICE proteins does not appear to exist for MpZOU proteins. Despite the fact that the expression pattern of MpZOU1 and MpZOU2 overlap and that, furthermore, both genes could complement the Atzou mutant, suggesting that their molecular mechanism of action could be similar, we found that the cuticle defects of Mpzou1 Mpzou2 double mutant appears very similar to those of Mpzou2 single mutants. Our transcriptomics suggest that this apparent redundancy is not due to transcriptional regulation of MpZOU1 by MpZOU2, since no reduction of expression of MpZOU1 is observed in Mpzou2 mutants. However, such a regulation cannot be completely excluded since the strong ventral expression of MpZOU1 might mask changes in gene expression in the dorsal epidermis.

As is the case in M. polymorpha, two AtZOU orthologues have been found both in rice and in soybean. However, in both cases, only one of the two genes (OsZOU-1 in rice and GmZOU-1 in soybean) is capable of (partial) complementation of the Atzou mutants, suggesting that the molecular mechanisms underlying ZOU protein functions may diverge extensively in the Angiosperm lineage [START_REF] Dou | Identification of ZHOUPI Orthologs in Rice Involved in Endosperm Development and Cuticle Formation[END_REF][START_REF] Zhang | Identification of ZOUPI Orthologs in Soybean Potentially Involved in Endosperm Breakdown and Embryogenic Development[END_REF]. However, double mutants lacking these genes have not yet been characterized.

HOW DOES THE MPZOU2/ICE COMPLEX REGULATE CUTICLE DEVELOPMENT?

In A. thaliana, and in other species where ZOU and/or ICE function has been studied, little evidence exists to suggest that ZOU/ICE complexes directly regulate the biosynthesis of aliphatic cuticle components [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF][START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. Indeed, in A. thaliana it appears that one of the main functions of ZOU/ICE1 in regulating cuticle development is to ensure the correct production of the ALE1 protein, which is, in turn, necessary for cuticle integrity monitoring [START_REF] Creff | A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis[END_REF]Doll et al., 2020).

Although three genes identified by Bowman and colleagues as orthologues of key genes in A. thaliana cuticle biogenesis were present in our transcriptomic dataset [START_REF] Bowman | Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome[END_REF], only one (an orthologue of A. thaliana glycerol-3-phosphate acyltransferase 6 (GPAT6)) can be truly considered to be potentially involved in cutin biosynthesis. The other two encode an ABC transporter and a Cutin Synthase (CUS), potentially involved in cutin monomer export and cuticle assembly respectively. Interestingly many more genes potentially involved in cell wall modification and/or the polymerization of phenolic monomers are downregulated in Mpzou mutants, than genes involved in aliphatic cuticle biosynthesis.

In terms of cell wall modification, a large number of genes encoding proteins with potential Xyloglucan Endo-transglycosylase activities, as well as pectin remodelling enzymes were identified as down regulated in mutants. Recent research has shown that pectins, xylans and xyloglucans may be intimately associated with the cutin of angiosperms, and that this association may be critical for the coordination of organ growth and cuticle deposition. ZOU/ICE1 could therefore play important regulatory roles in this process, potentially explaining the fragmented appearance of mutant cuticles [START_REF] Hama | Probing the Molecular Structure and Orientation of the Leaf Surface of Brassica oleracea L. by Polarization Modulation-Infrared Reflection-Absorption Spectroscopy[END_REF][START_REF] Philippe | Cutin and suberin: assembly and origins of specialized lipidic cell wall scaffolds[END_REF][START_REF] Xin | Cutin:xyloglucan transacylase (CXT) activity covalently links cutin to a plant cell-wall polysaccharide[END_REF].

The second major group of genes down-regulated in plants lacking MpZOU2 are genes encoding proteins that have been associated with defects in the deposition and/or oxidative polymerization of the Lignin-rich Casparian strip in the A. thaliana root. These include several members of the UCLACYCLIN-like protein family (Plastocyanin domain-containing) , required in A. thaliana for the deposition of a specific lignified Casparian strip zone [START_REF] Reyt | Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips[END_REF], dirigent proteins [START_REF] Hosmani | Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root[END_REF] and secreted peroxidases similar to ones involved in the Reactive Oxygen Species (ROS)-mediated polymerization of lignin in the early Casparian strip [START_REF] Fujita | SCHENGEN receptor module drives localized ROS production and lignification in plant roots[END_REF][START_REF] Lee | A mechanism for localized lignin deposition in the endodermis[END_REF][START_REF] Rojas-Murcia | High-order mutants reveal an essential requirement for peroxidases but not laccases in Casparian strip lignification[END_REF].

In addition to these peroxidases, which should, through their activity also act to detoxify apoplastic ROS, other proteins implicated in apoplastic ROS homeostasis including a catalase and several polyphenol-oxidases and tyrosinases and well as many other genes involved in the biosynthesis of secondary metabolites such as flavonoids, potentially implicated in ROS scavenging, particularly in Bryophytes, were downregulated [START_REF] Chapman | RBOH-Dependent ROS Synthesis and ROS Scavenging by Plant Specialized Metabolites To Modulate Plant Development and Stress Responses[END_REF][START_REF] Davies | The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective[END_REF].

These findings could suggest that altering ZOU/ICE1 activity alters apoplastic REDOX homeostasis, and potentially the polymerization of aromatic components within the cell wall. This possibility is exciting since, in A.thaliana, the GASSHO1 receptor kinase implicated in embryo cuticle monitoring together with ALE1 is known, in the root endodermis, to act in lignin polymerization through the production of apoplastic ROS [START_REF] Fujita | SCHENGEN receptor module drives localized ROS production and lignification in plant roots[END_REF].

The fact that our results suggest that the M. polymorpha cuticle, like that of P. patens, is enriched in phenolic components therefore opens the exciting possibility that ROS production could be a key function of MpZOU2/MpICE activity, and that ROS are subsequently implicated in the polymerization of phenolic cuticle components. The formation of a phenolic "scaffold" could, in turn, be necessary for directing the deposition of aliphatic components, similar to the situation in the root endodermis of A. thaliana, in which suberin deposition appears, to some extent, to depend upon lignin polymerization [START_REF] Lee | A mechanism for localized lignin deposition in the endodermis[END_REF]. It is important to note that the biochemical analysis carried out in the context of this work involves extensive delipidation and hydrolysis steps optimised to release monomers from polymerized plant cuticles. It is not clear to what extent a defect in polymerization might be revealed using this technique, since non-incorporated components are likely to be lost before analysis. In addition, some polymerization products involving phenolic components, or, potentially, cell wall proteins such as extensins, would not necessarily be detected in the analysis.

Finally, it is interesting to note that one of the genes down-regulated in the Mpzou2 and Mpzou1 Mpzou2 double mutant encodes a protein closely related to AtKRS, a cysteine rich peptide implicated in embryo sheath formation in the seed [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. AtKRS expression is completely eliminated in Atzou mutants. The STIG domain of the related protein AtGRIM REAPER has been proposed to be involved in ROS sensing [START_REF] Wrzaczek | Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS[END_REF], 126 further underlining links to ROS homeostasis. The embryo sheath is a structure enriched in epitopes specifically identified by anti-extensin antibodies, and two genes encoding EXTENSIN-like proteins also appear in our list of down regulated genes. Since extensins are known to undergo peroxidase-mediated oxidative polymerisation [START_REF] Mishler-Elmore | Extensins: Self-Assembly, Crosslinking, and the Role of Peroxidases[END_REF], it is therefore possible that these proteins are also targets of proposed ROS production downstream of ZOU/ICE activity. Further studies of extensin deposition in M. polymorpha gemmae and cuticles will be required to address this question such as immunolocalization with anti-extensins and ROS staining on the different mutants. CRISPR-Cas9 mutant of the closed homologue of AtKRS will also be generated as corresponding reporter lines.

Bibliography

See general bibliography at the end of the thesis. AAAATCTCA--------------------------------TGGCCGAGCGCAGG-3 II.1 Gene expression patterns.

In the work described above, we showed that the expression patterns of MpZOU1 and MpICE1 (mainly on the ventral surface of the thallus) differ from those of MpZOU2 and MpICE2

(seen mainly on the dorsal surface of the thallus). Nonetheless, these expression patterns seem to overlap slightly. The early post-gemma expression of MpZOU1, MpZOU2, MpICE1

and MpICE2 was also investigated using the transcriptional reporter lines described above during this thesis. This analysis showed that MpZOU2 and MpICE2 expression initiates around the apical notch about 3 days after germination and then spreads out across the dorsal surface of the thallus. MpICE1 expression starts slightly later than that of MpICE2 and MpZOU2, 5 days after germination around the apical notch and in the rhizoid primordia cells, shortly before pegged rhizoids start to be visible. Interestingly, although the early expression pattern of MpZOU1 is very similar to that of MpICE1, the exact point at which MpZOU1 expression initiates is less clear (Figure 1) (3 days after germination based on the data of Yen-Ting Lu, previous PhD student with J. Goodrich and 5 days after germination based on the data of Jeanne Loue-Manifel). These experiments were carried out under similar environmental conditions, but nonetheless, one possible explanation could be that MpZOU1 expression is environmentally controlled. In order to confirm the exact expression of both MpZOU and MpICE genes, qRTPCR at different time points should be carried out.

II.2 Roles in surface formation.

In A. thaliana, the ZOU/ICE complex controls embryonic sheath production, likely via control of the production of the peptide KRS, and sheath deposition via control of the production of ALE1 [START_REF] Moussu | ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface[END_REF]. The sheath is deposited outside the embryonic cuticle and incorporates endosperm-derived material rich in extensin-like molecules. Because of the conserved role of ZOU/ICE in suface development of M. polymorpha gemma, and that fact that a KRS-like peptide-encoding gene, as well as two extension-n-encoding genes are transcriptionally misregulated in mutants lacking MpZOU function, we tested whether immunolabelling of gemmae with anti-extensin antibodies might shed light on a putative KRSrelated function in M. polymorpha. Immubolabelling with three anti-extensin antibodies, α-LM1, α-JIM12 and α-JIM19 (Lee et al., 2012;[START_REF] Knox | Identification of novel cell surface epitopes using a leaf epidermal-strip assay system[END_REF][START_REF] Smallwood | Localization of cell wall proteins in relation to the developmental anatomy of the carrot root apex[END_REF][START_REF] Smallwood | Localization of cell wall proteins in relation to the developmental anatomy of the carrot root apex[END_REF] was been carried out on the double mutants Mpzou1Mpzou2 and Mpice1Mpice2 and on wildtype. Although no obvious labelling was observed with α-JIM12 and α-JIM19, α-LM1 anti- extensin labelling was visible surrounding WT gemmae (Figure 2). A subtle difference between the mutants and the WT was visible with this antibody. α-LM1 labelling tends to be slightly fuzzier in the double Mpzou1Mpzou2 mutant than in the wild-type and the double Mpice1Mpice2 mutant. The fact that α-LM1, α-JIM12 and α-JIM19 can all detect the embryonic sheath in A. thaliana, but that only α-LM1 labelling was detectable in M. polymorpha gemmae, could suggest that carbohydrate decorations of extensins are different in the two species.

Similar differences in sheath antigenicity were observed even between angiosperms, so this would not be suprising. However, it would be interesting to test more anti-extensin antibodies to see whether a more prominent structure is detectable and/or a more obvious phenotype is visible in M. polymorpha mutants laching ZOU and ICE proteins.

As with anti-extensin labelling, a stronger phenotype in Mpzou1 Mpzou2 mutants compared to the double Mpice1 Mpice2 mutant was also observed during with Nile red staining of the cuticle. However, no other ICE-encoding genes are present in the M. polymorpha genome. One possible explanation for this phenomenon, is that ZOU proteins from M.

polymorpha can form functional, or partially functional homodimers in the absence of ICE. A.

thaliana ZOU proteins have been shown to be capable of homodimerization [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF], but functions for such homodimers have not been revealed. This possibility deserves further exploration in the future Since the formation of the plant cuticle is thought to have been an innovation primarily to protect against uncontrolled water loss, we also decided to test whether desiccation tests could help us to shed light on the function of MpZOU2, MpICE1 and MpICE2. Gemmae from the different genotypes, together with wild-type gemmae, were directly put on soil in order to avoid stress-related phenotypes caused by the transfer from a humid environment (in vitro) to a much drier environment (soil). After 15 days, watering was stopped and plants were kept without watering for 18, 21 or 24 days, before being rewatered. The first obvious result of this study was the finding that wild-type M. polymorpha has a remarkable capacity to recover after a long period of desiccation (Figure 3). Mpice2 single mutants and Mpice1 Mpice2 double mutants were compared to wild-type plants and this revealed that plants lacking the function of both MpICE genes struggle to recover after 24 days without watering (Figure 3). This phenotype is also visible in the Mpice2 loss-of-function mutant but it tends to be less severe than in the double mutant. A parallel experiment comparing Mpice1 with the double mutants Mpice1Mpice2 and Mpzou1Mpzou2 and the wild-type showed similar results. Double mutant plants were very small and brownish after treatment, whereas a less severe phenotype was observed in the single Mpice1 mutant. More data are required to draw conclusions from these observations. However, it is interesting to note that both Mpice1 and Mpice2 single mutants show similar recovery phenotypes, which are less severe than either double mutant. This To address this question, crosses were carried out using Mpice2 and Mpzou2 mutants.

Sporangia from both Mpice2 x Tak1 (male wild-type) and Mpice2 x Mpice2 crosses show defects in spore dispersal (the sporangium does not burst and the spores are not released).

Since this defect was only observed when Mpice2 was crossed as female suggests this might be due to a defect in maternal tissues surrounding the sporangia (i.e. calypthra or pseudoperianth, see Reciprocal crosses of Mpice2 (Mpice2-6 as female or Mpice2-5 as male) with either the wild-type (Tak1 or Tak2) or Mpzou2 male (Mpzou2-3) revealed a maternally controlled defect of Mpice2.

The photographs of the second rows are higher magnification photographs of the white rectangle in the first row photographs. Scale bar, 500µm.

is therefore possible that other bHLH TFs might interact with MpICE2 to control sporangium development. Although, M. polymorpha does not have stomata, its genome contains a gene encoding a FAMA like protein [START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF] 5) suggesting a potential diminution of the number of air pores. However, although air pores in Mpice1 Mpice2 mutant tend to appear bigger, less neatly formed and have a "crushed" aspect compared to wild-type, no evidence proved conclusively that this phenotype is due to a specific function of MpICE in air pore development (Figure 5). Indeed it seems possible that this phenotype could be cuticle-related, since air-pores in M. polymorpha are surrounded by prominent cuticular extensions, or ledges, which are thought to be important for their function [START_REF] Schönherr | Hydrophobic cuticular ledges prevent water entering the air pores of liverwort thalli[END_REF]. 1-month old thallus were transferred on soil (soil:perlite 1:1) and keep for adaptation for 2 weeks under 75% humidity, 21°C, long day conditions before induction of gametophore production with far red light (730 nm). Once gametophores were produced and were mature enough to be crossed, 20 archegoniophores were crossed in order to confirm the sterility and the observed phenotypes. Sperm was harvested from males by placing drops of water on the surface of the male receptacles, waiting until the water was cloudy with released sperm, then collecting and placing onto the female archegoniophores. After 4 weeks, sporangia were fully matured. To confirm the observed phenotype was not due to growth delay, extra sporangia were kept 4 extra weeks in observation. Pictures were taken with a Keyence VHX900F digital microscope.4 repetitions have been performed and 1 repetition is equivalent to 20 crossedarchegoniophores. This has been performed with the help of a L3 student, Louis Valay I supervised.

Air pores analysis.

A piece of sixteen-day old thallus grown in vitro was sectioned just after the apical notch, taped on and imaged at 5kV and -15°C using an Environmental scanning electron microscope (Hirox 3000). 2 month-old thallus grown in vitro on Johnsons media (see Annexe)

was photographed near the midrib with a Keyence VHX900F digital microscope.

Bibliography

See general bibliography at the end of the thesis.

Preamble

During angiosperm seed development, the endosperm is totally or partially degraded.

This developmental process involves endosperm cell elimination followed by developmental cell death. In A. thaliana, ZOU/ICE complex activity in the endosperm promotes cell wall modifications that lead to thinning and cell separation. Previous transcriptomic studies showed that several genes involved in the modification of cell wall proteins and polysaccharides are downregulated in zou mutant, including Reversibly Glycosylated Protein3 [START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF].

RGP3 is the only direct target of ZOU/ICE complex conclusively identified to date, and encodes a UDP-arabinose mutase participating in the plant cell wall biosynthesis/modification through arabinose metabolism [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Rautengarten | Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester[END_REF]. ZOU-mediated cell wall modifications have been proposed to allow the deformation of endosperm cells under the pressure exerted by the expanding embryo, thereby priming them for pressure-induced cell death (bursting). This in turn facilitates the growth of the embryo [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF].

Another excellent example in which cell wall modification and cell death are tightly linked, is the differentiation of xylem vessels, discussed in Introduction, § 2.2.

Link between cell wall modifications and the conditioning of cells for developmental cell death is not an Angiosperm, or indeed a vascular-plant-specific phenomenon but is also seen in Bryophytes such as M. polymorpha, in which at least two tissue maturation programmes associate cell wall modifications and cell death. A good example is provided by M. polymorpha elaters which are dead, hygroscopic cells involved in spore release and found in sporangia.

Another example is pegged rhizoids, which are dead cells with cell wall ingrowths involved in water transport. Again, pegged rhizoids are dead at maturity, and cell death is preceded by the development of complex cell wall modifications.

Finally, it is interesting to note that even in animal systems, extracellular matrix remodelling and cell death (in this case apoptosis) are often intricately linked [START_REF] Shi | Regulation of apoptosis during development: input from the extracellular matrix (review)[END_REF].

In and also with A. thaliana AtICE1 and AtZOU, respectively (Figure S3). Further, the full-length ICE proteins had transcriptional activation activity in yeast and a C-terminal portion that included the bHLH domain was sufficient for interaction with ZOU proteins. We next tested whether the M. polymorpha genes could rescue the seed phenotypes of their corresponding A. thaliana mutants when expressed in transgenic seed from the A. thaliana ZOU or ICE promoters. Both MpZOU1 and MpICE1 rescued the cuticle defects of Atzou-4 and Atice1-2 mutants as measured by toluidine blue staining of etiolated seedlings (Figure 1A and1B).

The seed of Atzou and Atice mutants contains a small embryo which does not fill the seed, due to the presence of a persistent endosperm which impedes embryo growth. In addition, the seed is shrivelled, due to the collapse of the watery endosperm cells during seed maturation and desiccation. In Atzou-4 mutants, expression of either the AtZOU or MpZOU1 coding sequences from the AtZOU promoter gave a full rescue, with a large embryo filling the oval shaped seed and no shrivelling (Figure 1C and1D). In Atice1-2 mutants, expression of AtICE1 gave a full rescue, whereas MpICE1 gave a partial rescue such that many seed had larger embryos than in Atice1-2 and less shrivelling but were misshapen relative to wild type (Figure 1C and1D). Collectively these data support that the ZOU ICE partnership arose early in land plant evolution and the properties of the proteins have been broadly conserved in bryophyte and angiosperm lineages thought to have diverged about 430 million years ago.

To determine the developmental function of MpZOU1 and MpICE1 in M. polymorpha, we first examined their expression using transcriptional reporters comprising 5 and 6.6kb of regulatory sequences upstream of the start codons, respectively, fused either to the GUS gene encoding ß-glucoronidase or nuclear localised TdTomato fluorescent protein. The proMpZOU1:GUS and proMpICE1:GUS reporters were expressed strongly in 20 day old thallus tissue and with similar patterns, in ventral tissues around the midrib that included the parenchyma, scales and rhizoids (Figure 2A). We also examined proMpZOU1:TdTomato-NLS and proMpICE1:TdTomato-NLS reporters in two week old thallus and observed strong signals in the thallus, emerging rhizoids and developing pegged rhizoids where pegs were present at the base of the rhizoid but not yet at the tips (Figure 2). Overall these results indicated that MpZOU1 and MpICE1 might share common functions, including roles in ventral parenchyma and or rhizoid development

We next created multiple independent loss of function alleles of MpZOU1 using genome editing to create frameshift mutations in the region encoding the bHLH domains (Figure S4). The alleles are likely to be null as they are predicted to cause premature termination of translation, truncating the proteins and removing the highly conserved bHLH domain. We first examined whole mount, cleared thallus tissue of 12 day old plants using differential interference contrast microscopy, as this allowed us to check large number of rhizoids. Wild type plants had a mixture of smooth and pegged rhizoids, whereas the mutants had smooth rhizoids but lacked any pegged rhizoids (Figure 3). To confirm that this phenotype was due to disruption of MpZOU1, we complemented the mutant phenotype by introducing a transgene expressing the MpZOU1 coding sequence (proMpZOU1::MpZOU1 CDS) into the Mpzou1-1 mutant background and observed that pegged rhizoids were present (Figure 3).

We next performed genome editing of MpICE1 and identified independent mutant alleles (Figure S4). The Mpice1 mutants had a very similar phenotype to Mpzou1 (Figure 3), consistent with MpZOU1 and MpICE1 acting together and being required to promote pegged rhizoid differentiation in thallus tissue. To test the effects of over-expression, we made transgenic plants that expressed MpZOU1 or MpICE1 coding sequences under the control of the constitutive MpEF1 promoter [START_REF] Althoff | Comparison of the MpEF1α and CaMV35 promoters for application in Marchantia polymorpha overexpression studies[END_REF].

In plants that expressed proMpEF1:MpZOU1 CDS, all rhizoids differentiated as pegged rhizoids and no smooth rhizoids were present (Figure 3). In addition, the development of pegged rhizoids was accelerated. In wild type, no pegged rhizoids were visible on 3-day old gemmae and they did not become morphologically discernible, via presence of pegs until day 5-6. By contrast, in three day old transgenic plants most rhizoids were already pegged rhizoids. However, unlike plants that misexpress MpRSL1, in which rhizoids are produced from the dorsal epidermis as well as ventrally [START_REF] Proust | RSL Class I Genes Controlled the Development of Epidermal Structures in the Common Ancestor of Land Plants[END_REF], rhizoids were only produced ventrally (Figure 3) suggesting that MpZOU1 expression was not sufficient to cause epidermal cells to develop as rhizoids but rather to promote differentiation in existing rhizoids. We also generated proMpEF1::MpICE1 transgenic plants, however rhizoid development appeared normal in these lines (Figure 3), suggesting that MpZOU1 is the factor giving spatial specificity or limiting the activity of the MpZOU1/MpICE1 heterodimer. Since ICE is known to be strongly post-translationally regulated in A. thaliana during stomata development and cold tolerance (Chinnusamy et al., 2003;[START_REF] Ding | OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis[END_REF][START_REF] Dong | The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[END_REF][START_REF] Lang | OST1 phosphorylates ICE1 to enhance plant cold tolerance[END_REF][START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF], the absence of phenotype in proMpEF1::MpICE1 might also suggest that the post-translational regulation of MpICE1 is conserved limiting the activity of MpZOU1//MpICE1 complex.

To identify genes acting downstream of MpZOU1/MpICE1 as effectors of pegged rhizoid differentiation, we carried out transcriptional profiling (RNAseq) of 12-day old gemmae of wild type, Mpzou1 and Mpice1 mutants. Since MpICE1 activated transcription in yeast assays (Figure S3), and co-expression of ZOU and ICE1 genes activates transcription in several in planta assays in moss and tobacco [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF]Feng et al., 2018), we looked for transcripts that were less abundant in mutants compared to wild type. Consistent with their shared phenotype, a large and statistically significant fraction of the down regulated genes were common to the two mutants (Figure S5). Within this set of 119 genes, we identified numerous genes involved in cell wall modification and potentially linked to peg production S1). In addition, there were also several genes linked to developmental programmed cell death (dPCD). Notably, Mp8g07700 encoding a type II metacaspase was strongly downregulated. In A. thaliana there are six type II metacaspases (MC4-9), cysteine proteases with various roles in cell death and immunity [START_REF] Bollhöner | Post mortem function of AtMC9 in xylem vessel elements[END_REF][START_REF] Bollhöner | The function of two type II metacaspases in woody tissues of Populus trees[END_REF], with Metacaspase9 (MC9) associated with cell death during xylem differentiation [START_REF] Olvera-Carrillo | A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants[END_REF]. Phylogenetic analysis indicates that there are four type II metacaspases in M. polymorpha, and that the duplications occurred since the divergence of Bryophyte lineages so they are equally related to AtMC4-9. In addition, five genes encoding [START_REF] Rojas-Murcia | High-order mutants reveal an essential requirement for peroxidases but not laccases in Casparian strip lignification[END_REF]. We also compared the 119 genes with a single cell RNA seq dataset from 4 day old thallus tissue and found that a statistically significant fraction (35 genes, p= 8.8 X 10 - 13 , hypergeometric test) were expressed in 2 cells that were likely pegged rhizoids and notably included the metacaspase and 7 DMP4 like genes.

Pegged rhizoids are an innovation found in complex thalloid liverworts and their presence is correlated with species in which the gametangiophores are elevated above the thallus on stalks, facilitating spore dispersal via wind [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF][START_REF] Villarreal | Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate[END_REF]. In M. polymorpha, two grooves run up the stalk and are densely packed with pegged rhizoids, implicated in water transport to the receptacles [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. In the stalks of Mpzou1 and Mpice1 mutants, transverse sections showed that normal pegged rhizoids were absent from the grooves and instead smooth rhizoids lacking pegs and larger in diameter were present. Occasionally rhizoids developed pegs, but these were smaller and extended less far into the lumen of the rhizoid, suggesting that they were not fully differentiated (Figure 3). Strikingly, the gametangiophores of mutants were typically shorter than wild type and did not survive for long, shrivelling and drying out even when grown in humid conditions. Most likely as a result the mutants were not fertile either as males or females.

These results are consistent with the presence of pegged rhizoids in stalks being important for hydration and viability of the aerial gametangiophores.

The pegged rhizoids of M. polymorpha, unlike xylem, are external to the thallus and have become internalised in the stalk due to inrolling of thallus tissues to form the stalk [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF][START_REF] Shimamura | Marchantia polymorpha : Taxonomy, Phylogeny and Morphology of a Model System[END_REF]. They are believed to have evolved a role in water transport independently of xylem via convergent evolution [START_REF] Duckett | Of mosses and vascular plants[END_REF]. Notably they have acquired structural reinforcement, via pegs, which help to prevent collapse during desiccation, and dPCD to clear cellular contents. Consistent with this evolutionary convergence, we find that the ZOU/ICE partnership has been recruited as a master transcriptional regulator in M. polymorpha and promotes expression of cell wall modifying genes and dPCD genes. In A. thaliana the ZOU/ICE heterodimer is required for the breakdown of endosperm that permits growth of the embryo during seed development. This involves programmed cell death of endosperm cells and is marked by expression of dPCD genes such as PASPA3 and DMP4 [START_REF] Olvera-Carrillo | A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants[END_REF], but differs in that cell walls and debris are eliminated post mortem and expansion of the embryo is required to trigger cell death [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF]. Nonetheless, it raises the possibility that ZOU/ICE genes played a role in dPCD in early land plants and that this has been recruited to different roles in the M. polymorpha gametophyte and the angiosperm endosperm. Given that the pegged rhizoids are a derived feature of complex thalloid liverworts it will be interesting to know whether ZOU/ICE have a more general role in dPCD processes. For example, the elaters in the sporophyte are involved in spore dispersal and are also dead at maturity, with distinctive secondary thickenings.

Materials and Methods

YEAST-TWO HYBRID ASSAYS.

The full length of M. polymorpha and A. thaliana ZOU and ICE1 proteins were M. polymorpha spores from Tak-2 x Tak-1 crosses, (approximatively 5 sporangia per transformation) were sterilised for 15minutes with 0.1% sodium hypochlorite solution, spun for 1 minute at maximum speed in a microcentrifuge, resuspended in 500µL of water and grown 7 days into 50mL of OM51C (Takenaka 2000) 10s, with gentle agitation, covered with plastic bags for 24h and allowed to set seeds. Primary transformants were selected on solid MS medium containing suitable antibiotics and then transferred to soil. T1 and T2 generation plants were selected on agar-solidified MS with antibiotics. T2 lines were genotyped and subjected to phenotypic and molecular analyses.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF TOLUIDINE BLUE PERMEABILITY.

To assess cuticle integrity, toluidine blue staining assays were performed [START_REF] Tanaka | A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis[END_REF]. For qualitative toluidine blue staining tests, etiolated seedlings were used. Seedlings were stratified in the dark, exposed to a 6h light flash and then grown for 4 days in darkness.

They were then stained for 2 min in 0.05% w/v Toluidine blue solution with 0.1% v/v Tween 20 and washed abundantly under tap water. Pictures were taken with a Keyence VHX900F digital microscope.

For quantitative toluidine blue staining test, 7-day old light-grown seedlings were stained in the same way. In order to extract toluidine blue and chlorophyll, the cotyledons of ten seedlings were collected and placed in 1mL of 80% ethanol for at least 3 hours in darkness.

Toluidine blue content of the resulting solution was measured at 626nm with a spectrophotometer. This absorbance was then normalized using chlorophyll absorbance at 430nm, a wavelength not absorbed by toluidine blue. 6 repetitions were performed per condition [START_REF] Xing | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2[END_REF]. Data were subjected to an ANOVA test, and where differences were detected, a Scheffe test was used to whether significant differences existed between specific genotypes.

SEED-SHAPE ANALYSIS.

Percentages of misshapen seeds were counted from images taken with the VHX900F digital microscope (Keyence), using the cell counter extension of the ImageJ software. For each genotype, seeds from 4 distinct mother plants were analysed with approximately 1000 seeds analysed per mother plant. Data were subjected to an ANOVA test, and where differences were detected, a Scheffe test was used to whether significant differences existed between specific genotypes.

HISTOCHEMICAL STAINING FOR GUS ACTIVITY OF WHOLE MOUNT THALLUS.

MpZOU1 and MpICE1 promoters were cloned into the pMpGWB104 vector and subsequently Tak2xTak2 spores were transformed [START_REF] Ishizaki | Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha[END_REF]. Histochemical assays for GUS activity were performed according to [START_REF] Jefferson | GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF] with some minor modifications. Briefly, after three 20 minute vacuum infiltrations with the GUS staining solution (50mM sodium phosphate buffer, pH 7.2 ; 0.5mM potassium-ferrocyanide; 0.5mM potassiumferricyanide; 10mM EDTA; 0.01% Triton ; 1mM 5-bromo-4-cloro-3-indolyl-Beta-glucuronic acid (DuchefaBiochemie, CAS number 114162-64-0), plants were incubated overnight at 37°C.

They were then dehydrated by incubation in increasing concentrations of Ethanol (15%, 30%, 50%), for 20 minutes (per bath) at room temperature. Plants were then fixed in FAA fixative solution (10% formaldehyde, 5% acetic acid, 50% ethanol) for 45 minutes under vacuum followed by either 2 hours at room temperature or overnight at 4°C. Dehydration steps to remove the chlorophyll were then performed by incubation in further increasing concentrations in Ethanol (50%, 70%, 100%), for 20 minutes per bath at room temperature. Observations of GUS activity were made using a Keyence VHX900F digital microscope.

HISTOCHEMICAL STAINING FOR GUS ACTIVITY OF VIBRATOME-SECTIONED THALLUS.

15 to 20 day-old transcriptional reporter lines (described in the previous section) were harvested and placed into a small plate. 5% Phytagel (Sigma CAS : 71010-52-1 ; P8169) at 50°-60°C was then poured onto the plants. The plate was then stored for 30 min to 1hour at 4°C to cool down before mounted onto the Leica VT1000 S Microtome. Approximately 10µm sections were performed and placed into 1%PBS solution before staining as described above.

Observations of GUS activity were made using a Keyence VHX900F digital microscope.

CRISPR-CAS9 GENE EDITING.

CRISPR-Cas9 mutants were generated as described in [START_REF] Sugano | CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L[END_REF]. Two gRNAs for each gene were designed to generate single mutants. The oligonucleotides encoding the sgRNA were first cloned into pGE_EN03. For Mpzou1 and Mpice1 single mutant, the sgRNA constructs were then introduced into pMpGE010 destination vectors (containing the CAS9 protein) and sporelings were transformed using Agrobacterium GV3101 strain. [START_REF] Sugano | CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L[END_REF][START_REF] Sugano | Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha[END_REF].

MICROSCOPY.

12-day old thallus were placed between the slide and slip cover with a drop of chloral hydrate: glycerol (7:1) solution overnight and analysed using a Zeiss axioimager 2 with Differential Interference contrast (DIC).

To image pMpZOU1::TdTomato and pMpICE1b::Tdtomato lines, gemmae were directly grown in chamber slides Nunc™Lab-Tek™II filled with 5mL of B5 Gamborg medium.

Chamber slides were placed in a 21°C growth chambers under continuous light from SpectraluxPlus NL36W/840 Plus (Radium Lampenwerk) fluorescent bulbs during 7

days. Images were acquired on a Leica SP8X spectral confocal microscope inverted system equipped with a 460-670 nm super continuum white light laser. Imaging was conducted using either a 10× air objective (HC PL APO 10×/0.40 CS2), a 20× air objective (HC PL APO 20×/0.75 CS2) or a 40× water immersion objective (HC PL APO 40×/1.10 W CORR CS2).

Excitation laser wavelength and captured emitted fluorescence wavelength window were 561 nm, 570-625nm for TdTomato and 488nm, 650-700nm for Chlorophyll autofluorescence. To limit chlorophyll signal in the tdTomato channel time-gating was used.

RNA EXTRACTION AND RNASEQ.

12-day old thallus were harvested, frozen in liquid nitrogen and grounded with a pestle and a mortar to have minimum 100mg of ground powder. In each case, three independent RNA samples were extracted for each genotype. Total RNAs were extracted in TRIzol. Briefly, 1mL of TRIzol were added in each tubes before the samples thawed and then were vortexed.

After centrifugation (12,000 rpm, 10min, 4°C) ethanol was added to the supernatant to a final concentration of 35%. RNAs using silica spin columns from NEB biologicals (catalogue NES-5-008). After centrifugation ( 11,000 g, 30sec, RT) the flowtrough was discarded and 700µL of RW1 buffer (Qiagen) was added before centrifugation ( 11 000 g, 30sec, RT). The flow through was discarded and 500µL of RPE buffer (Qiagen) was added before centrifugation (11 000 g, 2min, RT). The column was finally dried out with another 2 minutes centrifugation at RT before elution of the RNAs with 40µL RNAse-free water. RNA quality was checked by running the samples on agarose gel and directly used for either RNASeq analysis (see section RNASeq)

or reverse transcripted for qPCR expression analysis as follow.

Total RNAs were extracted as described in RNA extraction section and sent to Edinburgh Genomics for quality control, cDNA preparation, library preparation and sequencing with Illumina HiSeq. Sequencing data were analysed using Salmon and then DESeq2 system.

PLANT GROWTH

All wild-type plants used in the project were Marchantia polymorpha Takaragaike-1 (Tak-1) for the male or Takaragaike-2 (Tak-2) for the female [START_REF] Ishizaki | Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology[END_REF]. Spores for transformation were the progeny of Tak2 x Tak1 [START_REF] Ishizaki | Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology[END_REF]. Lines were asexually maintained and propagated through gemmae growth as described in [START_REF] Takenaka | Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L[END_REF][START_REF] Okada | Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha[END_REF] except that we used Johnson media (see Annexe) complemented with 1.4% agar. Gemmae were transferred on plate to long-day conditions (16h, 21°C) for approximately 3 to 5 weeks, before harvesting the new generation of gemmae for further analysis.

Arabidopsis thaliana seeds were gas sterilized with chlorine (3mL HCl (33%) in 150mL bleach) for 2 hours and then sown on Murashige and Skoog (MS) medium with 0,5% sucrose in sterile condition, stratified for 2 days at 4°C and grown for 7 days in short-day conditions (8h light, 19°C). Seedlings were then transferred to compost (Argile 10 (favorit)) and grown under short day conditions (8h light, 19°C) for two to three weeks before transferred to long day conditions (16h light, 21°C). All A. thaliana mutants used in this study were previously published: the loss of function mutant Atzou-4 allele (Yang et al., 2008a) was used for all complementation experiments as was the loss of function Atice1-2 allele (Denay et al., 2014a;Kanaoka et al., 2008a). The ecotype Columbia-0 (Col-0) was used as a wild-type control. MpICE2 apopear to be involved in cuticle formation. The slightly overlapping expression patterns of the two ZOU-protein encoding genes, and the two ICE encoding genes in the ventral epidermis of M. polymorpha raised the question of whether functional redundancy also exists between the different complex components with respect to pegged rhizoid development.

To answer this question, pegged rhizoid development was investigated in the gametophytes and gametophores of the following CRISPR-Cas9 mutants: Mpzou2, Mpice2, Mpzou1 Mpzou2 and Mpice1 Mpice2 (Figure 1 and 2). Mpzou2 and Mpice2 showed no defect in pegged rhizoid development either in their gametophyte or in the stalk of their gametophores, suggesting that MpZOU2 and MpICE2 play either minimal or highly redundant roles in the control of pegged rhizoid formation. This observation was confirmed by the phenotypes of both double mutants Mpzou1Mpzou2 and Mpice1Mpice2 which resembled the single mutants, Mpzou1 and Mpice1 in terms of pegged rhizoid formation. This is contrary to the augmentation of cuticle phenotypes observed in double mutants compared to Mpzou2 and Mpice2. This discrepancy regarding the functional redundancy of the MpZOU/MpICE complexes deserves to be investigated in more detail. One possibility is that our assays for pegged rhizoid formation are not accurate enough to detect subtle differences in the timing of rhizoid differentiation of, indeed the number of pegged rhizoids formed. It would also be interesting to investigate whether proMpZOU2::MpZOU1-CDS or proMpZOU1::MpZOU2-CDS can complement the cuticle phenotype of Mpzou2 or the pegged rhizoid phenotype of Mpzou1 mutants, in order to test whether complexes show functional specificity that is independent of their expression patterns. Similar experiments could also be carried out using the MpICE genes.

Finally MpZOU1/MpICE1 complex activity involves extensive cell wall remodelling to thicken the rhizoid walls and to generate pegs, which are outgrowths composed of cell wall GENERAL DISCUSSION AND PERSPECTIVES signalling pathway. However the MPK6 loss-of-function mutant is also affected in seed size and seed bursting, phenotypes that are unlikely related to the ALE1/GSO pathway.

In M. polymorpha ICE proteins the KRAAM motif is only partially conserved in MpICE1 Interestingly, no interaction in yeast between PpICE1 and AtMPK3/6 has been highlighted

although MPKs are present in Physcomitrella genome. This absence of interaction might suggest that the MPK3/6-mediated post-translational regulation of ICE is not an ancient mechanism, and may have appeared later during land plant evolution. This is supported by the fact that A. thaliana MPK3 and MPK6 orthologues are not found in either Physcomitrella patens or M. polymorpha [START_REF] González-Coronel | A phylogenetic study of the members of the MAPK and MEK families across Viridiplantae[END_REF]. However, other MPKs could functionally replace MPK3 or MPK6 in the regulation of ICE in Bryophytes. HOS1 is also known to negatively regulate ICE1 and ICE2 in A. thaliana during stomatal development, cold tolerance and more recently seed development. Interestingly, M. polymorpha also seems to have one HOS1 protein (Mapoly0088s0017). However, to date more tools need to be generated to address divergence in the ZOU/ICE complex at the level of its regulation. In addition to posttranslational regulation, it would be particularly interesting to assess whether transcriptional regulation, such as that that I have revealed for ICE2 in seeds, is a feature in lower land plants.

My results in M. polymorpha suggest that this may not be the case, however this possibility, and the molecular basis for my observations in A. thaliana seeds, again require further study.

bHLH transcription factors are the second largest transcription factors family in Angiosperms. They are involved in many developmental processes and cellular differentiation events. Twenty subfamilies of bHLH transcription factors found in Angiosperms (out of a total of 26) are also present in early land plants suggesting that bHLH transcription factors appeared before. [START_REF] Catarino | The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land[END_REF][START_REF] Pires | Origin and diversification of basic-helix-loop-helix proteins in plants[END_REF]. Most characterised plant bHLH transcription factors bind to the conserved G-box (5'-CACGTG-3') and G-box related DNA sequences [START_REF] Giuliano | An evolutionarily conserved protein binding sequence upstream of a plant lightregulated gene[END_REF][START_REF] Hao | Basic Helix-Loop-Helix (bHLH) Transcription Factors Regulate a Wide Range of Functions in Arabidopsis[END_REF]. Since ZOU and ICE are bHLH transcription factors they likely bind to G-box sequences. Detailed comparative analysis of genes acting downstream of ZOU/ICE complexes (which could be identified by combining omics approaches such as by ChIP seq and RNASeq) would help to identify whether the direct targets of ZOU/ICE complexes are conserved in A. thaliana and M. polymorpha.

Transactivation in the heterologous system Physcomittrella patens already confirmed genetic data showing the necessity for the A. thaliana ZOU and ICE proteins to form heterodimers in order to activate RGP3 gene promoter via its G-box sequence (Denay et al., 2014a). It would be interesting to carry out parallel omics experiments, combined with similar transactivation approaches using the M. polymorpha ZOU and ICE proteins to determine how target specificity of the M. polymorpha ZOU/ICE complex is defined.

II The ZOU/ICE complex in the control of M. polymorpha gemma cuticle and A. thaliana embryonic cuticle formation

In A. thaliana, ZOU/ICE1 heterodimers are required for the formation of a functional embryonic cuticle. Interestingly this function appears not to involve the regulation of the biosynthesis of cognate cuticle components such as cutin monomers, but rather an implication in pathways involved in a pathway involved in cuticle integrity monitoring. Although the main molecular actors of this pathway have been elucidated, how pathway activation leads to the production of an itact embryonic cuticle remains a mystery, potentially because the composition of the embryonic cuticle is also enigmatic. Published and unpublished data from the group have suggested the potential importance of phenolic components in the A. thaliana embryonic cuticle. Several dyes generally used to stain phenolic-enriched polymers (such as lignin and suberin) have been tested and can stain the embryonic cuticle, including Fluorol Yellow dye which is mostly used to stain cutin and suberin, but also Auramine O which is specifically used for suberin and lignin staining (unpublished data from N. Doll and AC Marsollier and (Creff et al., 2019a)). Interestingly, comparative analysis of embryonic cuticle staining of zou mutant and wild-type revealed a decrease of cuticle staining in zou suggesting that phenolic compounds are reduced in the zou embryonic cuticle or at least that phenolic compounds within the embryonic cuticle are less easily detectable by dyes in this mutant. The presence of phenolic compounds (particularly Sinapic acid) in the embryonic root cuticle was confirmed by Berhin and colleagues [START_REF] Berhin | The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation[END_REF]. The origin of these phenolic componds in unclear, although the expression of genes encoding several enzymes involved in the biosynthesis of specialized phenolics, including SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE (SHT) [START_REF] Grienenberger | A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines[END_REF] is strong in the developing endosperm of Arabidopsis, and almost completely lost in zou mutants (unpublished results). Thus some components of the embryo cuticle, as well as the extensin enriched extracuticular sheath could be derived from the endosperm and produced under the control of ZOU/ICE.

Interestingly, the cuticles found at the surface of Bryophyte gametophytes also appear to be enriched in phenolic compounds [START_REF] Kong | Origins and Evolution of Cuticle Biosynthetic Machinery in Land Plants[END_REF][START_REF] Renault | A phenol-enriched cuticle is ancestral to lignin evolution in land plants[END_REF]Loue-Manifel et al., in preparation). In this context it is important to remember that the endosperm is derived from the female gametophyte in Angiosperms, albeit as a fertilized derivative. Taken together these results could support a tentative but tantalising hypothesis in which the control of embryonic cuticle formation in angiosperms by ZOU/ICE complexes may have evolved from an ancestral role in the formation of the cuticle of the gametophyte of the ancestor of early diverging land plants such as M. polymorpha. One possibility is that phenolics and/or crosslinkable cell wall proteins such as extensins could serve as a scaffold to allow the correct deposition and polymerisation of cutin and waxes in both contexts. The common denominator might be a dependence upon ROS-mediated cross-linking, mediated, in the case of the embryo at least, by the activity of the cuticle integrity signalling pathway. However ROS staining of the embryos of zou mutants has not revealed conclusive defects in ROS production (Nicolas Doll, unpublished data), probably due to the technical constraints of such experiments. Likewise, preliminary results in M. polymorpha did not show any conclusive differences between Mpzou1Mpzou2 and Mpice1Mpice2 double mutants, Mpzou2, Mpice2 and the WT. However, only one type of ROS (H202) has so far been assayed, and differences in cuticle permeability likely make results very difficult to interpret due to differential access to reagents. Other dyes exist and would need to be tested in M. polymorpha.

Specific p-coumarate antibodies have been developed to label cell walls in grasses [START_REF] Tranquet | Monoclonal antibodies to p-coumarate[END_REF]. INRA-COU1 antibody can recognize free p-coumaric acid and p-coumarate esters whereas esterified p-coumarates are recognized by INRA-COU2 [START_REF] Tranquet | Monoclonal antibodies to p-coumarate[END_REF].

Since we hypothesized that cuticles of A. thaliana embryo and M. polymorpha gemma might have a similar phenolic enriched composition and p-coumarate is one of the most abundant phenolic compounds in A. thaliana aerial organ cuticles, these two antibodies have also been tested on both species. Unfortunately, neither INRA-COU1 nor INRA-COU2 labelled cuticles in M. polymorpha gemma (unpublished data from J. Loue-Manifel and AC. Marsollier)) Gemma cuticle composition also confirms an enrichement in phenolics compounds. However, among the phenolics detected in WT gemma cuticle, only one, p-coumarate, showed a significant difference compared to wild-type. This compound was found to be more abundant specifically in Mpzou2-3 gemma cuticle. None of the Mpice mutants tested showed statistically significant differences in the amount of any of the molecules identified. However, when interpreting this data, it is important to bear in mind that some exotic/unexpected fatty acids or other molecules were not identified in the analysis and that important changes were therefore not detected. In addition, delipidation may have removed unpolymerized components, which would have masked potential defects in cuticle assembly. However, when interpreting this data, it is important to bear in mind that some exotic/unexpected fatty acids or other molecules were not identified in the analysis and that important changes were therefore not detected. In addition, delipidation may have removed unpolymerized components, which would have masked potential defects in cuticle assembly. pegs which seem to have a specific signature under UV lights for example [START_REF] Duckett | Pegged and smooth rhizoids in complex thalloid liverworts (Marchantiopsida): structure, function and evolution[END_REF]. This might indicate the presence of large quantities of ROS-crosslinked phenolics. A lack of ROS production in mutants lacking ZOU/ICE complexes could, via an unknown mechanism, be behind the observed regulation of peroxidases in our datasets. Bryophyte cuticles are also enriched in phenolic compounds which might also be crosslinked by ROSdependent peroxidases. In the future it would be fascinating to assess whether growing M. polymorpha in the presence of ROS scavengers can lead to similar transcriptomic changes to those seen in mutants lacking ZOU/ICE complexes, or, indeed, to phenotypes reminiscent of those observed in such mutants.

III

One particularly interesting discovery from my transcriptomic analysis was the finding that the expression of the closest M. polymorpha homologue of the ZOU-regulated AtKRS gene, which is required for the production of the extensin-rich embryo sheath in A. thaliana, is also significantly reduced in Mpzou2 and Mpzou1Mpzou2 mutants. The fact that AtKRS belongs to the family of potential ROS sensing/regulated STIG peptides could prime a hypothesis whereby ROS-dependent extensin production, modification and/or polymeration could be a common factor involved in scaffolding cell wall modifications involved in both cuticle deposition and pegged rhizoid formation. Interestingly in the Mpzou1, Mpice1 transcriptomic analysis, another predicted STIG-encoding genes is downregulated, strengthening this possibility (Figure S6, Chapter 2). It is interesting, in this context, to note that elicitor induced stress-lignin in Spruce appears to contain large quantities of an extensin-like protein that has been proposed to play a scaffolding function [START_REF] Lange | Elicitor-Induced Spruce Stress Lignin (Structural Similarity to Early Developmental Lignins)[END_REF].

How cuticle formation, pegged rhizoid formation and endosperm elimination are linked mechanistically is less clear. There are obvious superficial parallels between pegged rhizoid formation and endosperm elimination since both processes ultimately involve cell death.

However it is interesting to note that while, at least in dicotyledonous plants, endosperm elimination, even if only partial, usually involves, or even depends upon the almost complete dismantling of cell wall, and presumably their recycling, the opposite is true in pegged rhizoids, where death is associated with extreme cell wall thickening. From this point of view pegged rhizoid formation superficially resembles xylem vessel formation much more strongly than it resembles endosperm elimination. Consistent with this view, transcriptomic analysis of zou mutants (Xing et al., 2013a) showed that genes whose expression was reduced or lost in zou mutants could globally be divided into two classes. The first contained genes whose expression was also reduced in ale1 mutants, and included genes implicated in stress response, including oxidative stress and ROS responses (Creff et al., 2019a). The second contained genes whose expression was completely unaffected in ale1 mutants, and whose function was associated either with sheath formation (KRS, being a rare example of a gene in this later class with a link to ROS) or, predominantly, with cell wall degradation (for example pectin lyases). Several genes involved in simple sugar metabolism also fell into this second category. Based on this is seems plausible that the role of ZOU/ICE complexes in endosperm elimination is relatively "new" (or at least highly derived), and associated with nutrient recycling, and the switch of the

Figure 1 :

 1 Figure 1 : Key innovations in land plant evolution. Phylogenetic relationships between the major groups of land plants. Key innovations that occured during land plant terrestrialisation are indicated by coloured dots. Dates are indicated in million years ago (Mya).

Figure 2 :

 2 Figure 2 : Life cycles of Marchantia polymorpha and Arabidopsis thaliana. M. polymorpha life cycle (A) is gametophytic dominant whereas A. thaliana life cycle (B) is sporophytic dominant. Alternation of the haploid gametophyte and the diploid sporophyte are represented in blue and pink respectively. M. polymorpha photos of germinated spores and spores and elaters are from Shimamura et al., 2016. A. thaliana photos of rosette, siliquees and flower are from Krämer et al., 2015.

Figure 3 :

 3 Figure 3: Description and structure of M. polymorpha rhizoids, root hairs and xylem in vascular plants. (A-B) Root hairs are filament cells formed on the root of tracheophytes visible on a light microscopy photograph of A. thalina roots from Vissenberg et al., 2020. (A) The main zone of roots : at the tip the root cap which protects the root apical meristem (RAM) and further in the tissue, root hairs develop all around the root. Scale bar, 200µm. (B) Confocal microscopy photograph of a specific root-hair promoter (green) showing the unicellular root hair. Scale bar, 50µm. (C) Schematic reprensentation of the four differerent xylems found in Angiosperms: Tracheary elements (protoxylem (Px) and metaxylem (Mx)), fiber (Fib) and xylem parenchyma (Xp) from Schuetz et al., 2012. (D-G) The two types of rhizoids in M. polymorpha : pegged rhizoids and smooth rhizoids. (D) Underneath de thallus the bundles of pegged rhizoids and the beard of smooth rhizoids are growing respectively parallel and perpendicular to the thallus. Scale bar, 100µm. (E) DIC microscopy photograph of smooth rhizoid and pegged rhizoid. Scale bar, 50 µm. (F-G) Transverse section of thallus showing difference tissues. the air pores are on the dorsal surface whereas the bundle of pegged rhizoids with their pegs are growing inside the scales to form bundles. (G) is a higher magnification of the bundle of pegged rhizoids visible in the box in (F). Pegs are visible in the transverse section of pegged rhizoid. Scale bar, 100µm.

Figure 4 :

 4 Figure 4: Schematic representation of the structure and composition of the cuticle of land plants. (A) Examples of cutin monomers and monomer functional groups, from Fich et al., 2016. (B) Examples of phenolic compounds found in cuticle. (C)The cuticle is composed of three zones. The zone closest to the cell wall is the cuticular layer. It is mainly composed of cutin and wax polymers, is closely linked to the cell wall and thus enriched in pectins. The middle zone is the cuticle proper, mainly composed of cutin polymers and intracellular waxes. Phenolics are also present in these two layers. Finally, the outer layer is the epicuticular waxes in which wax crystals cover wax films. (D) Example of cutin polymer. Diacids (orange) allow for cross-linking between cutin chains (arrow), as well as incorporation of glycerol (red) into the polymer, fromFich et al., 2016. (D) Examples of hypothetical cutin attachment to polysaccharides, using the pectin xylogalacturonan as an example. They may be joined through the single carboxyl end of the cutin dendrimer (left side of the dashed line) or its many hydroxyl ends (right of the dashed line). Arrows indicate possible covalent linkages between these two polymers: ester linkages (red) and ether linkages (blue), from[START_REF] Fich | The Plant Polyester Cutin: Biosynthesis, Structure, and Biological Roles[END_REF]

Figure 5 :

 5 Figure 5: Cutin and wax biosynthesis pathways and cuticle formation on the surface of epidermal cells. The different steps of the two pathways are explained in the section INTRODUCTION I-3. Enzyme names are indicated in orange and their corresponding genes in green.

Figure 6 :

 6 Figure 6: Seed development. (A) Seed architecture of pine (gymnosperm, top left), rice (angiosperm endospermic seed, top right, Arabidopsis (angiosperm endospermic seed, bottom left) and quinoa (angiosperm, persipermic seed). Female gametophyte, nucellus, endosperm and embryo are represented in purple, orange, blue and yellow, respectively. Fertilization event is reprensented by the white arrow. From Lu and Magnani, 2018. (B) Schematic representation of the different stages of A. thaliana seed development. Maternal tissue are in red, endosperm in blue and embryo in yellow. The main phases of endosperm development (in blue) and embryo development (in yellow) are labelled. Different endosperms are represented : peripherical endosperm (PE), micropylar endosperm (ME) and the uncellularized chalazal endosperm (CE).

  EXPANSIN2 (EXP2) gene. Embryo expansion-induced mechanical cues in the endosperm have been proposed to play a key role in in triggering Gibberellin-dependent gene expression in the endosperm triggering changes in the physical properties of this later[START_REF] Sánchez-Montesino | A Regulatory Module Controlling GA-Mediated Endosperm Cell Expansion Is Critical for Seed Germination in Arabidopsis[END_REF].

Figure 7 :

 7 Figure 7: The different steps required for the embryonic cuticle formation in A.thaliana. For each step, the main molecular actors and their corresponding pathways are represented. As a first step, the most external cell layer of the embryo proper differentiates into an epidermis. Then, the epidermal cells start to produce cuticle components that are secreted into the apoplast to form a discontinuous "patchy" cuticle. A bidirectional exchange of signals between the embryo and the endosperm allows the detection of the gaps in this structure, mediates their closure, and thus ensures cuticle integrity. After the completion of cuticle gap closure, the pathway is shut down unless new gaps appear during embryo expansion. FromDoll and Ingram., 2021, in press.

Figure 8 :

 8 Figure 8 : ZOU/ICE-dependant signalling pathways and ICE1 post-translational regulators in other tissue.(A) ZOU/ICE complex promote the nutrient storage and transport in the endosperm (in blue), the cell wall modifications of the endosperm (in green) whose the only direct target of ZOU/ICE complex is known, RGP3. ZOU/ICE complex also controls the formation of the embryo surface. The embryonic cuticle integrity (in red) which involves the GSO1/GSO2 receptors, the subtilisase ALE1, the peptide TWS1 and the TPST (for more informations see Figure7in the Introduction). The embryo sheath production and deposition (in yellow) involve the peptide KRS and GSO1/GSO2 receptors. (B) Post-translational regulators and their modification sites on ICE1. The modified amino-acid is indicated in the brakets with its corresponding regulator, except for the KRAAM motif, the binding site of MP3/MPK6, where the amino acid substitution and the name of the correpsonding gain of function mutant are mentioned in the brackets.

  of the cuticle to prevent the leakage of developmentally important signals out of the developing embryo and into surrounding tissues could be an important factor in

  suppresses the kinase activities of MPK3 and MPK6. Recently,[START_REF] Putarjunan | Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS[END_REF] have deciphered the role of the KRAAM motif during stomatal development. Together with an upstream MPK docking motif, the KRAAM motif acts as a specific binding and phosphorylation site for MPK3 and MPK6. Thus, ICE1 ubiquitination via HOS1 and phosphorylation via the MKK5-MPK3/MPK6 cascade both act to decrease ICE1 protein stability post germination. These studies of the post-translational regulation of ICE1 have been carried out on seedlings and vegetative leaves during cold stress and stomatal development. During seed development much less is known, although ice1-D mutants have been shown to cause precocious endosperm elimination, suggestion that KRAAM domain-mediated regulation occurs in the seed context (Denay et al., 2014), However, although public transcriptomes showed that known ICE1 regulators are expressed in seed, it remains unclear whether they play roles during endosperm development. THE REGULATION OF THE ACTIVITY OF THE COMPLEX ZOU/ICE DURING SEED DEVELOPMENT : AN OPEN QUESTION!

  Genetic redundancy is also observed between the basic helix-loop-helix (bHLH) transcription TFs SCREAM/ INDUCER OF CBF EXPRESSION 1 (ICE1) and ICE2, which act together during the development of the stomatal lineage through their hetero-dimerisation with the sequentially acting bHLH TFs SPEECHLESS (SPCH),MUTE and FAMA (Kanaoka et al., 2008b). Loss of ICE2 function does not affect stomatal development, and only very mild defects in stomatal differentiation are observed in backgrounds lacking ICE1. However, loss of function of both proteins leads to a complete lack of formation of stomata, leading to seedling lethality.

  emb

Figure 1 :

 1 Figure 1 : Expression pattern of ICE2 during seed development. (A-F) Images of in situ hybridizations of digoxigenin-labelled ICE2 antisense (A-E) and sense probe (F), visualised by DIC microscopy. Four developmental stages are presented : globular stage embryo (A), old heart stage embryo (B), torpedo stage embryo (C), bent stage embryo (D) and mature stage embryo (E). Signal appears purple in the embryo, endosperm and seed coat. The endothelium stained in dark-brown is an artefact. emb is for embryo; end is for endosperm. Scale bar, 50µm. (F) In silico data from EFPbrowser showing the strong expression pattern of ICE1 in the endosperm. (G) In silico data from EFPbrowser broadly confirms the expression pattern of ICE2 in the endosperm. (H) Expression of ICE1 relative to that of EIF4 in the wildtype (Col-0) and zou-4 siliques ; error bars reprensent standard errors. Five biological replicates were used. (I) Expression of ICE2 relative to that of EIF4 in the wild-type (Col-0) and zou-4 siliques ; error bars reprensent standard errors. Five biological replicates were used.

Figure 4 ).

 4 Figure4). Analysis of the progeny of ice1-2 ice2-1 +/-plants allowed us to confirm, as reported

Figure 2 :Figure 3 :

 23 Figure 2 : ZOU and ICE2 interact in yeast. Interactions tested in yeast. Empty boxes indicate empty vector controls. Growth on minimal non selective (-LT) and selective with low stringency (-LTH) and selective with high stringency (-LTHA) media is shown. the three dots are from the left to the right, no dilution, 1/10 dilution and 1/100 dilution.

Figure 4 :

 4 Figure 4 : Embryonic cuticle defect is partially rescued in hos1-3 ice1-2 double mutants. (A) Toluidine blue assays on etiolated cotyledons of wild-type (Col-0), ice1-2 mutant, ice2-1 mutant, hos1-3 mutant, hos1-3 ice1-2 mutant and hos1-3 ice2-1 mutant. Scale bars 200µm. (B) Quantification of toluidine blue uptakes by the cotyledons of young seedlings, normalized to chlorophyll content. N= 18, 10 seedlings per repetition. * indicates statistical differences with ANOVA followed by Sheffe tests between hos1-3 ice1-2 and the other genotypes. ** indicates statistical differences with ANOVA followed by Sheffe tests between ice1-2 and the other genotypes. Error bars indicate standard deviations.

Figure 5 :

 5 Figure 5 : Comparison of the immunohistochemical properties of the sheath in dessicated and imbibed seeds. Seeds from Col-0, ice1-2, ice2-1, hos1-3, hos1-3 ice1-2 and hos1-3 ice2-1 subjected to immunolocalization with JIM12 rat monoclonat antibody (green) and post-stained with calcofluor white (magenta). Arrowheads indicate the sheath. c, cotyledon. Scale bars, 50µm.

Figure 6 :

 6 Figure6: Schematic summary of the role and the HOS1-mediated regulation of ZOU/ICE1 and ZOU/ICE2 complexes in seed development highlighted in this study. HOS1 destabilizes the ZOU/ICE1 and ZOU/ICE2 complexes via its interaction and regulation of the ICE proteins. ZOU/ICE1 complex promotes seed development but also positively egulates ICE2 transcription. ZOU/ICE2 complex also promotes seed development in a more sublte way but whether this complex also influences ICE2 transcription remains to be confirmed.

Figure S1 :

 S1 Figure S1 : Expression pattern of HOS1 during seed development. (A-F) Images of in situ hybridizations of digoxigenin-labelled HOS1 antisense (A-E) and sense probe (F), visualised by DIC microscopy. Four developmental stages are presented : globular stage embryo (A), heart stage embryo (B), torpedo stage embryo (C), bent stage embryo (D) and mature stage embryo (E). Signal appears purple in the embryo, endosperm and seed coat. The endothelium stained in dark-brown is an artefact. emb is for embryo; end is for endosperm. Scale bar, 50µm. (G) In silico data from EFPbrowser broadly confirms the expression pattern of HOS1 in the endosperm.

Figure 1 :

 1 Figure 1 : MpZOU and MpICE complement seed and cuticle phenotypes of Arabidopsis Atzou-4 and Atice1-2 mutants. (A) Dry seeds of wild-type (Col-0), Atzou-4 mutant, Atice1-2 mutant, pAtZOU::AtZOU, pAtZOU::MpZOU1 or pAtZOU::MpZOU2 in Atzou-4 mutant and pAtICE1::AtICE1, pAtICE1::MpICE1 or pAtICE1::MpICE2 in Atice1-2 mutant. Partially complemented seeds are indicated by an asterisk. Scale bar, 200µm. (B) Quantification of misshappen dry seeds in different background. N=5, approximately 1000 seeds per repetition. *** indicates statistical differences with ANOVA. Error bars indicate standard deviation. (C) Toluidine blue assays on etiolated cotyledons of wild-type (Col-0), Atzou-4 mutant, Atice1-2 mutant, pAtZOU::AtZOU, pAtZOU::MpZOU1 or pAtZOU::MpZOU2 in Atzou-4 mutant and pAtICE1::AtICE1, pAtICE1::MpICE1 or pAtICE1::MpICE2 in Atice1-2 mutant. Scale bars 500µm. (D) Quantification of toluidine blue uptakes by the cotyledons of young seedlings, normalized to chlorophyll content. N= 6, 10 seedlings per repetition.. *** indicates statistical differences with ANOVA. Error bars indicate standard deviations.

Figure 2 :

 2 Figure 2 : Transcriptional reporter lines of MpZOU1, MpZOU2, MpICE1 and MpICE2 show an overlap of expression pattern. (A) MpZOU2 and MpICE2 promoter activity are restricted to the epidermis whereas the activity of MpZOU1 and MpICE1 promoters are limites to the ventral midrib. Transverse section of thallus of WT (Tak-1) and GUS reporter fusion of MpZOU1, MpZOU2, MpICE1 and MpICE2 promoters are the main photographes. Respective whole mount thallus are shown in the bottom left side of the transverse section picture. Black bar indicate the orientation of the section. Transverse section in the gemmae cup of pMpZOU2:GUS thallus show strong expression in developping gemmae (black and white arrowheads) and weaker expression in mature gemmae (white arrowheads). d, dorsale side of the thallus ; v, ventral side of the thallus ; e, epidermis ; s, scale where bundles of pegged rhizoids are found. Scale bar, 500µm. (B) Confocal microscopy images of MpZOU1, MpZOU2, MpICE1 and MpICE2 promoters fused to TdTomato fluorescent protein. Photographes of the dorsal surface of the thallus (first row) and ventral surface of the thallus with a focus on rhizoids (second row) show an overlap of expression pattern MpZOU and MpICE. Air pores are indicated with an asterisk. r, rhizoids. Scale bar, 50µm. Photographs of pMpZOU1::Tdtomato on the dorsal surface and pMpZOU2::TdTomato on the ventral surface are from Yen-Ting LU.

  MpICE2 was not consistently detected in gemmae. Expression of MpZOU1 and MpICE1 was also detected in gemmae, predominantly in the rhizoid precursor cells (discussed in Chapter 3, §II, Figure1).

Figure 3 :

 3 Figure 3 : Cuticle staining of M. polymorpha gemmae with the lipophylic dye, Nile Red and the phenolic compounds dye, Diphenylborinic acid showed a critical role for MpZOU2 and both MpICE in cuticle formation of gemmae. (A) Nile Red staining of the gemmae cuticle indicates a cuticle defect in the Mpzou2-3, Mpzou1zou2-db5, Mpice2-6 and Mpice1ice2-db1 mutants. *, apical notch ; arrow indicates intact cuticle. Scale bar, 10µm. (B) Diphenylborinic acid stains the cuticle of wild-type (Tak-1) gemmae and indicates cuticle defect in Mpzou2-3, Mpzou1zou2-db5; Mpice1-5, Mpice1ice2-db1 mutants. *, apical notch ; arrow indicates intact cuticle. Scale bar, 10µm.

Figure 4 :

 4 Figure 4: MpZOU2 (and MpICE2 to a lesser extent) are critical for cuticle formation in M. polymorpha gemmae. (A)Transmission electron micrographs of the gemmae cuticle showing alteration in Mpzou1Mpzou2-db5 and Mpice1Mpice2-db1 mutant cuticle layer compare to the WT (Tak-1). cw, cell wall; cut, cuticle; v, vesicles; mat?, putative material coming for deposition. Scale bar, 1µm. (B) Cutin compositional analysis of the gemma cuticle of Mpice2-6, Mpzou2-3, Mpice1Mpice2-db1 and Mpzou1Mpzou2-db5 revealed gemma cuticle composition differences in Mpzou2-6 and Mpzou1Mpzou2-db5 compared to WT. DCA; dicarboxylic fatty acid. Compounds with statistical differences between the different backgrounds are highlighted in red. * indicates statistical differences with two-tailed t-test (p<0.01). Error bars indicate standard deviations, n=3.

Figure 5 :

 5 Figure 5 : RNA Sequencing analysis. (A) Principal Component Analysis (PCA) show distinct gene expression profiles across WT (Tak2 xTak1 progeny) in red, Mpzou2-3 mutant in blue and Mpzou1Mpzou2-db5 mutant in green. (B-C) Enriched Gene Ontology terms in Biological process (B) and Molecular Functions (C) for differential expresion genesdown regulated in Mpzou2-3 single mutant and Mpzou1Mpzou2-db5 double mutant. The top 10 significantly enriched GO categories (p<0.01)for each analysis. Significant enrichment is observed for cell wall (CW) structure and biogenesis (in bold) (B) and ROS production (in bold) (C). (http://plantregmap.gao-lab.org)

Figure S2 :Figure S4 :Figure S5 :

 S2S4S5 Figure S2 : Phylogeny of land plant ICE genes. Bayesian phylogeny, rooted on MpbHLH032, with percent probabilities indicated on branches. The clade containing the AtICE1 and AtICE2 orthologues is highlighted in cerise. Species abbreviations are At Arabidopsis thaliana, Mg Mimulus guttifera, Ac Aquilegia coerula, Os, Oryza sativa, Nc Nymphae colorata, Amtr Amborella trichopada, Pa Picea abies, Sc Salvinia cucullata, Sm Selaginella moellendorffii, Aa Anthoceros punctatus, Pp Physcomitrella patens, Mp Marchantia polymorpha. Different taxonomic groupings coloured as shown in box on left.

Figure S6 :

 S6 Figure S6: Alignment of STIG1 domain of the 20 M. polymorpha STIG1-like proteins with the two A. thaliana STIG1 domains of KERBEROS and GRIM REAPER. Alignment of the STIG1 sequence domains (highlighted in green) of the 20 MpSTIG1-like proteins with the two STIG1 domains of AtKERBEROS (AT1G50650-KRS) and AtGRIM REAPER (AT1G53130-GRI). The conserved cysteine residues in the STIG1 domains are highlighted in yellow. Red text is one of the genes down-regulated in the Mpzou2 and Mpzou1 Mpzou2 double mutant encodes a protein closely related to AtKERBEROS. Blue text is one STIG1-like protein found down-regulated in the Mpzou1 mutant.

Figure 1 :

 1 Figure 1: Transcriptional reporter lines of pMpZOU1::GUS, pMpZOU2::GUS, pMpICE1::GUS and pMpICE2::GUS show different expression pattern in gemma cup and gemma. (A) Transverse section in a gemma cup of pMpZOU1:GUS and pMpICE1: GUS showed expression in primary rhizoid cells. Gemmae are surrounded by dashed white lines and the photograph in the left bottom corner is a higher magnification of a gemma; arrowhead indicates primary rhizoid cell. Scale bar, 100µm. (B-C) 3-day old (B) and 5-day old (C) young gemmae of GUS transcriptional reporter lines for MpZOU1, MpZOU2 , MpICE1 and MpICE2 genes. In MpZOU2 and MpICE2 reporter lines, the GUS staining is limited to the apical notch region and detected in 3-day old gemmae. MpICE1 promoter activity is also restricted to the region of the apical notch but starts at day 5. The low level of MpZOU1 promoter activity does not allow to have a clear understanding and temporal expression pattern. v: ventral surface; d: dorsal surface; * apical notch. Scale bar 250µm

Figure 2 :

 2 Figure 2: Analysis of mature gemma surface by immunolocalization with several different antibodies. Antibody signal is green and calcofluor signal is red. Immunolocalization with anti-extensin antibodies, JIM12 (first row), JIM19 (second row) and LM1 (third and fourth rows) are tested on wild-type (WT) gemmae, Mpice1Mpice2-db4 and Mpzou1Mpzou2-db5, respectively 1st column, second column and thirsd column. Only LM1 antibody labeled gemma surface. Scale bar, 50µm.

Figure 3 :

 3 Figure 3: Mpice1, Mpice2, Mpice1Mpice2 and Mpzou1Mpzou2 mutants are more sensitive to water stress.Constant watering is used as a control (first row) to compared with 18 days, 21 days and 24 days with water deficiency between WT (first two columns), Mpice1-6, Mpice2-3, Mpice1Mpice2-db4 and Mpzou1Mpzou2-db5. These experiments show difficulties for both Mpice single mutants and both double mutants Mpice1Mpice2 and Mpzou1Mpzou2 have difficulties of recovery from water stress. Plants were 15 days old when the dessication test starts. Experiment 1 and experiment 2 correspond to two independent experiments carried out under the same environnemental conditions.

Figure 2 ,Figure 4 :

 24 Figure 4: Defect in spore release occured only when Mpice2 is crossed as female.Reciprocal crosses of Mpice2 (Mpice2-6 as female or Mpice2-5 as male) with either the wild-type (Tak1 or Tak2) or Mpzou2 male (Mpzou2-3) revealed a maternally controlled defect of Mpice2. The photographs of the second rows are higher magnification photographs of the white rectangle in the first row photographs. Scale bar, 500µm.

Figure 5 :

 5 Figure 5 : Mpice1Mpice2 double mutants have defective air pores. (A) Photographs of 2-month old thallus of WT and Mpice1Mpice2-db5 mutant showing a drastic decrease in the number of air pores around the midrib. Air pores are indicated by arrowheads. Scale bar, 200µm. (B) SEM micrographs of wild-type, Mpice1-5, Mpice2-2 and Mpice1Mpice2-db5 on the dorsal surface of 16-day old thallus. Mpice1Mpice2-db5 show bigger and crushed air pores compared to WT and Mpice2-2 thallus surface. Mpice1-5 air pores seem to be slightly bigger and flatter than the WT. Scale bar, 50µm.

  Chapter 2, I explored the putative parallels between the role of the ZOU/ICE complexes in Marchantia polymorpha surface formation (cuticle biogenesis) with their roles in embryo surface formation in A. thaliana. In this Chapter, I investigate another putative role for M. polymorpha ZOU/ICE complexes; the production of pegged rhizoids. This work was initiated by a previous PhD student in Goodrich group, Yen-Ting Lu, before I arrived in the group, but I carried out many of the experiments necessary to complete this study, and as I result I appear as co-first author on the resulting manuscript in preparation.

Figure 1 :

 1 Figure 1 : MpZOU and MpICE complement seed and cuticle phenotypes of Arabidopsis Atzou-4 and Atice1-2 mutants. (A) Dry seeds of wild-type (Col-0), Atzou-4 mutant, Atice1-2 mutant, pAtZOU::AtZOU, pAtZOU::MpZOU1 or pAtZOU::MpZOU2 in Atzou-4 mutant and pAtICE1::AtICE1, pAtICE1::MpICE1 or pAtICE1::MpICE2 in Atice1-2 mutant. Partially complemented seeds are indicated by an asterisk. Scale bar, 200µm. (B) Quantification of misshappen dry seeds in different background. N=5, approximately 1000 seeds per repetition. *** indicates statistical differences with ANOVA and Sheffe tests (p-value 0.01). Error bars indicate standard deviation. (C) Toluidine blue assays on etiolated cotyledons of wild-type (Col-0), Atzou-4 mutant, Atice1-2 mutant, pAtZOU::AtZOU, pAtZOU::MpZOU1 or pAtZOU::MpZOU2 in Atzou-4 mutant and pAtICE1::AtICE1, pAtICE1::MpICE1 or pAtICE1::MpICE2 in Atice1-2 mutant. Scale bars 500µm. (D) Quantification of toluidine blue uptakes by the cotyledons of young seedlings, normalized to chlorophyll content. N= 6, 10 seedlings per repetition.. *** indicates statistical differences with ANOVA. Error bars indicate standard deviations.

Figure 2 :

 2 Figure 2 : Transcriptional reporter lines of MpZOU1 and MpICE1 show expression in rhizoids. (A) MpZOU1 and MpICE1 promoter activity are restricted to the ventral surface of the thallus where the pegged rhizoids are formed. Transverse section of thallus of WT (Tak1) and GUS reporters fusion of MpZOU1 and MpICE1 promoters are the main photographs. Respective whole mount thallus are shown in the bottom left side of the transverse section photographs. Scale bar, 500µm (whole mount) and 100µm (vibratome secions). (B) Confocal microscopye images of MpZOU1 and MpICE1 promoters fused to TdTomato-NLS fluorescent protein. Photographs of the whole mount thallus and of the ongoing pegged rhizoids are shown in the first and second panel. Scale bar, 1mm (whole mount) and 200µm (rhizoids).

Figure 3 :

 3 Figure 3: MpZOU1 and MpICE1 promote pegged rhizoids differentiation and MpZOU1 over expression causes excess pegged rhizoid formation. (A) Clearing of 10-day old thallus. Mpzou1-1 and Mpice1-5 are missing pegged rhizoids compared to the WT (wild-type), the complementation pMpZOU1::MpZOU1 in Mpzou1-1 and pMpEF1::MpICE1. In contrast the ectopically expressed MpZOU1, pMpEF1::MpZOU1 shows an overproduction of pegged rhizoids. Arrowheads indicate pegged rhizoids. Scale bar, 100µm. (B) The two first columns show clearing of 3-day-old and 6-day-old young plants. Arrowheads indicate pegged rhizoids. Scale bar, 50µm. The third column show absence of rhizoids on the dorsal surface of pMpEF1::MpZOU1 in WT. Scale bar, 1mm. Pictures from Yen-Ting LU. (C) Gametophores of Mpzou1-1 and Mpice1-5 are defective in pegged rhizoids and very short and dry. First row are photographs of Tak1 (wild-type male), Mpzou1-1 and Mpice1-5. Scale bar, 500µm. White line indicates transverse section realised in the embedded stalk showed in the second and third rows. Black square in the second row indicate the zoom in visualised in the third row. Scale bar for the second and third rows are 50µm.

  expressed as fusions to the GAL4 DNA binding domain (bait constructs) or with the GAL4 transcriptional activation domain (prey constructs) and cloned into pGBKT7-GW or pGADT7-GW(Lu et al. 2010). The truncation of AtICE1, MpICE1 was respectively residues 263-494 and 443-710 based on[START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF] for AtICE1 and on the alignment of AtICE1-T and MpICE1. Matchmaker Y2HGold Yeast was co-transformed with bait and prey plasmids and transformants were selected on media lacking leucine and tryptophan (-LW). Interaction of bait and prey constructs resulted in activation of reporter genes for histidine and adenine synthesis (-LWHA).M. POLYMORPHA SPORELING TRANSFORMATION AND A. THALIANA FLORAL DIPPINGTRANSFORMATION.

  h y l o g e n y o f l a n d p l a n t Z OU g e n e s . B a y e s i a n p h y l o g e n B a y e s i a n p h y l o g e n y , r o o t e d o n Mp b HL H3 6 , wi t h p e r c e n t p r o b a b i l i t i e s i n d i c a t e d o n b r a n c h e s . T h e c l a d e c o n t a i n i n g t h e A t Z OU o r t h o l o g u e s i s h i g h l i g h t e d i n l i g h t b l u e , t h e Z OU2 c l a d e c o n t a i n i n g a n a n c i e n t p a r a l o g u e i n c e r i s e . Z OU2 i s n o t c o n s e r v e d i n a n g i o s p e r ms , wh e r e a s mo s s e s a n d f e r n s l a c k Z OU1 . Wh e r e a v a i l a b l e t h e n u mb e r i n g o f b HL Hs f o l l o ws t h a t o f P i r e s e t a l . S p e c i e s a b b r ev i a t i o n s a r e A t A r a b i d o p s i s t h a l i a n a , Mg Mi mu l u s g u t t i f e r a , A c A q u i l e g i a c o e r u l a , Nc Ny mp h a e c o l o r a t a , A mt r A mb o r e l l a t r i c h o p a d a , P a P i c e a a b i e s , S c S a l v i n i a c u c u l l a t a , S m S e l a g i n e l l a mo e l l e n d o r f f i i , A a A n t h o c e r o s p u n c t a t u s , P p P h y s c o mi t r e l l a p a t e n s , Mp Ma r c h a n t i a p o l y mo r p h a p o l y mor p h a . Di f f e r e n t t a x o n o mi c g r o u p i n g s c o l o u r e d a s s h o wn i n b o x o n l e f t .

Figure S2 :Figure S4 :

 S2S4 Figure S2 : Phylogeny of land plant ICE genes. Bayesian phylogeny, rooted on MpbHLH032, with percent probabilities indicated on branches. The clade containing the AtICE1 and AtICE2 orthologues is highlighted in cerise. Species abbreviations are At Arabidopsis thaliana, Mg Mimulus guttifera, Ac Aquilegia coerula, Os, Oryza sativa, Nc Nymphae colorata, Amtr Amborella trichopada, Pa Picea abies, Sc Salvinia cucullata, Sm Selaginella moellendorffii, Aa Anthoceros punctatus, Pp Physcomitrella patens, Mp Marchantia polymorpha. Different taxonomic groupings coloured as shown in box on left.

Figure S5 :

 S5 Figure S5: Transcriptional profiling of 12 day old thallus tissue and overlap with scRNAseq data. (A) Number of transcripts with reduced abundance (log2 fold change <-2, FDR < 0.05) relative to wild type in 12 day old thallus. (B) Overlap of scRNAseq data from presumed pegged rhizoid cells with RNAseq dataset (orange) with average expression level in pegged rhizoid (x axis) and expression relative to average expression in other cell types. The 35 genes downregulated in zou and ice mutants are also highly expressed in pegged rhizoids relative to other cell types.

(

  KRGGT motif and not KRAAM). MpICE1 only partially rescues the A. thaliana ice1-2 seed phenotype potentially due to altered interactions with putative regulators, such as AtMPK6 in the complemented line. Interestingly, during an unpublished study carried out during my Master degree, I showed that ZmICEa, a very divergent ICE variant from maize that lacks a KRAAM domain, fully complements the ice1-2 seed phenotype and, moreover gives stomatal phenotypes similar to those of ICE1-D when expressed in Arabidopsis. It is therefore possible that the KRGGT motif in MpICE1 leads to excessive destabilization of the MpICE1 protein, at least in A. thaliana. However, partial complementation could also be due to other factors, and merits further exploration. MpICE2 which gives a full rescue of the seed phenotype in A.thaliana has a more conserved KRAAM motif since only one amino acid changed from a methionine to a leucine (KRAAL motif). This raises the question of a possible difference in the regulation of MpZOU1/MpICE1 and MpZOU2/MpICE2 complexes in M. polymorpha.

  What is the link between pegged rhizoid formation, cuticle biogenesis and endosperm cell death? My work has elucidated two functions of M. polymorpha ZOU/ICE complexes. Whereas MpZOU1/MpICE1 predominantly controls pegged rhizoid fate, MpZOU2/MpICE2 and potentially MpZOU2/MpICE1 also control cuticle formation in gemmae. Complex specific function appears likely to be determined by expression patterns. Extensive RNASeq analysis from different mutant backgrounds in two different tissues revealed that ZOU/ICE complexes in M. polymorpha appear to activate the expression of a large number of peroxidase-encoding genes, as well as numerous genes involved in cell wall modification and genes linked to apoplastic ROS responses and metabolism. The similarities between these datasets suggest potential mechanistic connections between the role of ZOU/ICE1 complexes in cuticle formation, discussed above, and in pegged rhizoid development. Indeed, like cuticle development, pegged rhizoid development involves remodelling of the cell wall to create the

  endosperm from a sink to a source tissue during seed development. Linking this function to the reinforcement of the embryo cuticle may have been an innovation in seed plants aimed at coordinating nutrient fluxes in the seed. This intriguing hypothesis is currently being tested in the Ingram group. Finally, I would like to point out that although during my PhD I managed to show tantalising similarities between two evolutionary very distant plant models, A. thaliana and M. polymorpha, Annexe 1: Composition of Johnson's medium.

  To date only loss of ICE1 function has been shown to affect seed development, although ICE2 acts redundantly with ICE1 in other tissues, an observation that I will investigate more fully in Chapter 1. Three ICE1 orthologues (a, b and c) exist in maize, two of which are expressed in seed and interact with ZmZOU in heterologous systems (unpublished data). A previous study in the laboratory showed no obvious macroscopic endosperm persistence in the Zmicea loss-of-function mutant. Analysis of the other Zmice mutants has not been carried out so far but the absence of endosperm phenotype in Zmicea might be explained by redundancy between the ICE proteins(Feng et al., 2018;[START_REF] Grimault | ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development[END_REF]. To date no embryonic cuticle defect associated with loss of function of ZmZOU or ZmICE have been identified. In rice, where the endosperm is similar to that in maize, two ZOU orthologues have been found. However, only one shows some functional conservation regarding endosperm lysis and embryonic cuticle

formation compared with A. thaliana ZOU

[START_REF] Dou | Identification of ZHOUPI Orthologs in Rice Involved in Endosperm Development and Cuticle Formation[END_REF]

. Interestingly, although developmentally soybean and A. thaliana endosperms are much more similar to each other than to Monocotyledon endosperms, only one of the A. thaliana ZOU orthologues in soybean partially rescues the A. thaliana zou seed phenotypes (embryonic cuticle and endosperm breakdown)

[START_REF] Zhang | Identification of ZOUPI Orthologs in Soybean Potentially Involved in Endosperm Breakdown and Embryogenic Development[END_REF]

. To date, no studies have investigated the role of ICE orthologues during seed development in other species.

  Our work has revealed a previously undocumented role for ICE2 during A. thaliana seed development, where it acts redundantly, and likely as a complex with ZOU, to regulate endosperm elimination and embryo surface formation. This redundancy is in line with that

	observed between ICE1 and ICE2 function in other tissues. The fact that ICE2 is expressed at
	lower levels than ICE1 during wild-type seed development likely explains the fact that ICE1
	can completely compensate for loss of ICE2 activity during normal seed development. ICE2
	expression levels during wild-type seed development would nonetheless, be expected to
	partially compensate for loss of ICE1 function. However, we have shown that ICE2
	transcription appears to depend, either directly or indirectly, upon the activity of ZOU and thus,
	since ZOU acts as an obligate heterodimer, on ICE1 and potentially ICE2. The reduction of
	ICE2 transcription engendered by a loss of ICE1-ZOU heterodimers may thus explain the very
	strong phenotype exhibited by ice1 single mutants (Figure

Table S1 : List of gene dowregulated in Mpzou2 and/or Mpzou1/Mpzou2 double mutants.

 S1

	L2FC means Log 2 FoldChange

  . In A. thaliana FAMA is involved in the last differentiation step of stomata development, and it interacts with both AtICE1 and AtICE2[START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF]. It is thus possible that MpFAMA interacts with MpICE proteins to control

	sporangium development. Another possibility, is that the phenotype observed when Mpice2 is
	used as a female is an attenuated version of the phenomenon that renders Mpzou2 mutants
	sterile, and is thus ZOU dependent. To test these hypotheses, additional experiments are
	necessary, including further histological analysis of the abnormal sporangium development
	observed in Mpice2xTak1 progeny. However, an ongoing collaboration between Jeanne Loue-
	Manifel, Justin Goodrich, Tomoo Shimada and Kenta Moriya suggests that the first explanation
	is correct.
	2017), and both MpICE and MpZOU2 showed strong expression pattern in air pores (data not
	shown), an analysis of air pores morphology in the different mutant lines produced in this thesis
	was carried out. Disappointingly, no drastic phenotype such as complete absence of air pores
	or a drastic diminution of air pore number were revealed either histologically or by SEM
	analysis in Mpice1, Mpice2 or Mpice1Mpice2 mutants, although Mpice1 Mpice2 mutants
	sometimes show a lighter green patchy thallus phenotype (Figure

Finally, although the regulation of cold tolerance acquisition by the ICE proteins has been proven many times in A. thaliana, cold tolerance experiments with double Mpice1 Mpice2 mutants compared to wild-type plants have not revealed any conclusive results (data not shown). Thus whether or not ICE-mediated cold tolerance is conserved through land plants is unclear. Similarly A. thaliana ICE proteins are involved in stomatal differentiation

[START_REF] Kameriing | SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation[END_REF]

. Since air pores in M. polymorpha have a similar function to stomata

(Chater et al.,

For 5 Litres of 1X modified Johnson's medium

	Stock A	100 mL
	Stock B	100 mL
	Inositol (10mg/mL)	50 mL
	Stock C	5 mL
	Stock D	5 mL
	(NH4)2SO4	2 mL
	NH4H2PO4	3 mL
	MES	2.665 g
	Sucrose	50g
	Sigma agar	1.4%
	Adjust pH to 5.6 with 1M KOH and autoclave 120°C for 20min.
	Stock solutions ( for 1L each):
	100X Stock A		Final concentration
	KNO3	30.3g	6mM
	MgSO4	6.15g	1mM
	100X Stock B		Final concentration
	Ca(NO3)2 4H2O	47.2g	4mM
	1000X Stock C		Final concentration
	KCl	1.864g	25µM
	H3BO4	0.773g	10µM
	MnSO4 4H20	0.223g	1µM
	ZnSO4 7H20	0.288G	1µM
	CuSO4 5H20	0.0624g	0.25µM
	(NH4)6Mo7O24 4H20	0.309g	0.25µM
	1000X Stock D		Final concentration
	FeSO4 7H20	6.95g	25µM
	Na2EDTA	9.365g	25µM
	NH4H2PO4 (1mol/L)	5.75g for 50mL	0.6mM
	(NH4)2SO4 (1mol/L)	6.69g for 50mL	0.4mM

Acknowledgements

Annotations

L2FC Mpzou2/WT L2FC Mpzou1Mpzou2/WT Top HIT Mp3g18230 STIG1 domain -1,679850021 -4,294301965 Mp2g10330 JA-Ile deactivating enzyme (CYP94B3) -1,327924162 -0,940423885 Mp3g23170 MYB transcription factor -2,707818682 -3,571246967 Mp3g07510 MYB transcription factor -2,549523808 -3,185515099 Mp3g04970 Myb-like domain -0,728540782 -0,879602916 Cytochrome P450 related Mp6g19120 -3,657957976 -6,353718462 Mp6g11710 Germacradienol synthase / Germacradienol/germacrene-D synthase -6,204110866 -6,053683434 Mp3g18510 (2Z,6Z)-farnesyl diphosphate synthase / ZFPS -6,626048223 -5,281864582 Mp2g24750 -2,67491704 -4,685058499 Mp3g18500 -5,313832586 -4,634722289 Mp2g20490 5-epiaristolochene 1,3-dihydroxylase / EAH -2,579323246 -4,269675605 Mp6g04630 -2,435226268 -3,75907174 Mp6g04600 -1,621785791 -3,460551583 Mp4g05010 Germacradienol synthase -2,973629418 -3,198944876 Mp6g04320 -2,40527229 -3,085369455 Mp3g03420 AT2G26710.1 (BAS1,CYP72B1,CYP734A1) -3,990297352 -2,693133848 Mp1g14660 Delta(24)-sterol reductase / Lanosterol Delta(24)-reductase -1,255666607 -2,263541235 Mp8g14150 -2,914011212 -2,256330317 Mp7g00020 -1,682831136 -1,432359542 Mp1g04680 -0,771255534 -0,93503808 Mp1g25410 Potential ent-kaurenoic acid hydroxylase (KAO) -Gibberellin -0,669700785 -0,660037592 Mp5g09870 -0,574703374 -0,497470976 Peroxidases Mp5g17590 peroxidase 3 involved in casparian strip liginigcation RCI3 -4,144982045 -6,884261076 Mp5g13790 peroxidase 3 involved in casparian strip liginigcation RCI3 -2,284265005 -5,723168557 Mp7g19380 PRX53,(ascorbate peroxidase) -3,097263959 -3,883401881 Mp5g04010 -3,604857165 -3,717339956 Mp4g14580 PER56, close to RCI3 -1,88302968 -3,362657017 Mp3g05740 -0,853313689 -3,004393956 Mp5g04000 -2,757875913 -2,881948515 Mp3g16820 -1,39306771 -2,597819369 Mp7g11550 -1,233857366 -1,724011788 Cuticle related Mp8g13940 Cutin synthase -1,555501731 -3,72514311 Mp8g12130 ABC transporter -1,492170298 -1,268143618 Mp7g03880 GPAT6 -1,118071118 -2,064602519 Cell wall modifications Mp3g09650 expansin -5,534844879 -6,338062134 Mp2g18610 expansin -3,822336091 -6,115673799 Mp3g09760 expansin -3,429680852 -4,965898663 Mp5g20420 expansin -2,084121598 -4,408311971 Mp8g18860 expansin -2,404274208 -4,010961724 Mp4g06560 expansin -1,56646211 -2,465096368 Mp1g26350 expansin -1,071819623 -1,415005489 Mp4g01200 xyloglucan -2,549715885 -4,556374471 Mp8g10190 pectate lyase -2,080119452 -4,997598966 Mp8g00770 pectate lyase -2,116247907 -1,343337421 Mp2g08500 EXT -1,891224815 -3,912913768 Flavonoids related Mp8g12170 Flavonoid 3',5'-hydroxylase -2,441426329 -4,33476539 Mp8g09770 -3,730585551 -3,990691331 Mp8g10910 Anthocyanidin 3-O-glucosyltransferase -1,086944081 -3,27859408 Mp5g06850 Flavone 3'-O-methyltransferase -2,364954888 -2,774292226 Mp5g10800 Flavonoid 3'-monooxygenase -1,682827538 -2,373857094 Mp5g06840 Flavone 3'-O-methyltransferase -2,197659759 -2,33355098 Mp3g03440 UDP-GLYCOSYLTRANSFERASE 74C1-RELATED -1,275111368 -2,294823317 Mp5g00520 Flavonol synthase -2,979408361 -1,484288026 Mp4g14100 chalcone synthase (CHS) -1,304809318 -0,753649336 Mp2g23890 Flavonol 3-O-glucosyltransferase -1,311408514 -0,720219622 Mp5g20040 -2,9247462 -4,247452008 Mp6g17890 -1,810559503 -2,718923901 Mp5g21450 -2,832378901 -2,52059368 Mp6g17910 -2,054544579 -2,471884996 Mp5g14440 -2,410991923 -5,156006114 Mp5g14430 -2,674724977 -5,101156671 Mp5g19990 -2,804233277 -4,222960359 Mp5g20020 -2,733601262 -3,074832776 Mp5g15320 -2,018907212 -2,924461921 Mp8g06340 -1,478012301 -2,410671869 Mp5g21460 -2,244741873 -1,880548245 Mp6g17920 -0,814533533 -0,94227911 Mp6g10200 -1,18691394 -1,343674751 Mp7g01630 -1,226028973 -0,927557394 Mp2g18520 -0,760359516 -0,628892246 UCLACYCLIN-like protein family Mp1g07620 UCC1 -3,356450435 -9,225060402 Mp3g20270 WAK type RLK -2,479732009 -7,129624909 Mp3g17490 Another UCC -1,583639863 -5,883546029 Mp2g14890 UCC1-like -2,936340194 -4,724312999

Mp4g03640 D-mannose binding lectin protein -7,308547264 -8,689340194 Mp5g06310 Peroxidase -9,881484643 -6,51345322 Mp3g06900 Expansin -3,334748823 -6,165767015 Mp5g17080 Peroxidase -9,128887961 -9,197099693 Mp5g22020 FASCICLIN-like arabinogalactan 2 -5,381568334 -2,686180513 Mp5g17030 Peroxidase -8,904455447 -3,423555011 Mp8g02360 Proline rich extensin signature -3,190385544 -5,652239957 Mp5g02520 Peroxidase -8,185049792 -3,406338144 Mp3g03160 Rhamnogalacturonan endolyase -8,8 -8,1 Mp5g17120 Peroxidase -8,164674098 -8,23288664 Mp2g13580 Rhamnogalacturonan endolyase -8,6 -8,7 Mp5g17130 Peroxidase -8,105389317 -8,891553292 Mp2g13570 Rhamnogalacturonan endolyase -8,2 -7,8 Mp5g17110 Peroxidase -7,062941802 -7,062087351 Mp5g20220 Rhamnogalacturonan endolyase -5,885335982 -7,252274609 Mp2g24530 Peroxidase -6,58548836 -6,24048508 Mp7g11130 Rhamnogalacturonan endolyase -3,84641169 -2,264821661 Mp6g21140 Peroxidase -5,785625602 -5,853838371 Mp5g20220 Rhamnogalacturonan endolyase -5,885335982 -7,252274609 Mp5g19410 Peroxidase -5,585295307 -8,852405877 Mp4g00700 Xyloglucan endotransglycosylase -9,8 -10,6 Mpzg01380 Peroxidase -5,236155709 -6,026463768 Mp4g00680 Xyloglucan endotransglycosylase -7,839091308 -7,907304139 Mp4g14270 Peroxidase -5,033286376 -5,105498111 Mp4g00670 Xyloglucan endotransglycosylase -6,051564703 -5,409005562 Mp5g09230 Peroxidase -4,505291281 -4,338896425 Mp4g00690 Xyloglucan endotransglycosylase -5,685791453 -5,754003905 Mp5g02600 Peroxidase -3,203014465 -2,607079395 Mp2g02380 Xyloglucan endotransglycosylase -5,328890025 -5,397103167 Mp1g22970 Peroxidase -3,021317617 -4,255561426 Cell Death Mp5g10750 Peroxidase -2,99377374 -6,845421615 Mp8g07700 Metacaspase -5,98687609 -4,337532352 Mp5g06280 Peroxidase -2,474439263 -3,115963437 Mp6g12240 DUF679 containing DMP-like -8,53321704 -9,328491709 Other Mp5g12180 DUF679 containing DMP-like -6,547208129 -7,533705979 Mp4g03020 Aquaporin -7,214844725 -5,358732309 Mp5g12170 DUF679 containing DMP-like -4,56976316 -5,50409815 Mp1g29040 Aquaporin -4,750690852 -3,029208179 Mp4g17600 DUF679 containing DMP-like -3,708551749 -5,019035685 Mp4g17620 DUF679 containing DMP-like -2,173907669 -2,324182094 Protease Mp5g16420 Aspartyl protease -10,79466634 -9,565979272 Mp5g16430 Aspartyl protease -9,366918598 -8,215863957 Mp5g16410 Aspartyl protease -6,990028668 -7,058242037 Mp5g12100 Aspartyl protease -5,922008559 -7,22629013 Mp5g16350 Aspartyl protease -4,965458929 -5,22836089 Mp5g12090 Aspartyl protease -4,263418673 -2,942451302

General preamble

My thesis is divided in four main parts: Introduction, Chapter 1, Chapter 2, Chapter 3.

The first part is an introduction divided in two sections: the first one presents briefly the two model plants used during this thesis Arabidopsis thaliana and Marchantia polymorpha and the second focuses more specifically on three innovations acquired during the evolution of plants on land: the rhizoids, the cuticle and the seed.

Then three chapters of results and discussion are presented, one on A. thaliana and two on M. polymorpha. My contribution is defined at the beginning of each Chapter.

A general bibliography is presented at the end of the thesis.

AUTHOR CONTRIBUTIONS

Jeanne Loue-Manifel carried out the genetic analysis presented in the paper and much of the phenotypic analysis. Some of the analyses of embryonic cuticle permeability and immunolocalizations were performed by Anais Euzet-Rodriguez, a master student that Jeanne Loue-Manifel supervised during the last year of her PhD. Yeast-two-hybrid experiments were carried out by Victor Girard, a master student supervised by Nathalie Dépège-Fargeix. Q-RT-PCR analysis to confirm the original observation made by Audrey Creff that ICE2 expression is reduced in the zou mutant background were performed by Eduardo Berenguer Peinado. In situ hybridization was carried out by Sophy Boeuf. The article was written by Gwyneth Ingram with inputs from Jeanne Loue-Manifel, from Nathalie Dépège-Fargeix, and from Justin Goodrich. Jeanne Loue-Manifel prepared the figures.

ABSTRACT

The bHLH transcription factors INDUCER OF CBF EXPRESSION 1 and 2 (ICE1 and ICE2) act redundantly to regulate stomatal development and cold acclimation in Arabidopsis thaliana. In contrast, previous studies in seeds suggest that INDUCER OF CBF EXPRESSION 1, acting as a heterodimer with ZHOUPI (ZOU), plays an apparently non-redundant role in endosperm elimination and embryo surface formation. Here we have investigated this apparent lack of redundancy by studying the role of ICE2 in seed development. We show that not only is this gene co-expressed with ICE1 and ZOU in the endosperm, but that ICE2 can physically interact with ZOU. We demonstrate that ICE2 expression is dependent upon ZOU activity, which masks functional redundancy between ICE1 and ICE2. We show that this redundancy can nonetheless be revealed in a background lacking the ubiquitin E3 ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1), a known negative regulator of the activity of ICE1 and ICE2 in other tissues. Our study thus demonstrates a previously undocumented role for ICE2 in A. thaliana seed development and the dependency of the expression of this gene upon ZOU. In addition, we demonstrate that the E3 ubiquitin ligase HOS1 is likely implicated in the post-translational regulation of the activity of ICE1 and ICE2 in seeds.

II -Further considerations

We have shown that HOS1 likely regulates the stability of both ICE1 and ICE2 during seed development and that this regulation can be used to reveal the usually cryptic role of ICE2 during seed development. However very little is known about the post-translational regulation of A. thaliana ICE2 protein. Similarly to ICE1, ICE2 is thought to be phosphorylated by HOS1. Although the phosphorylation site has not been conclusively confirmed yet it seems to be conserved between ICE1 and ICE2 proteins (Kim et al., 2015c). Tools have been generated during this PhD to investigate the stability of the ICE1 and ICE2 proteins. Thus ICE1 and ICE2 variants mutated either in the KRAAM motif (R236H) or in the HOS1-ubiquitination site (S403A) to increased transactivation of ICE1 or ICE2, have been generated, introduced into wild-type plants and ice1 mutants, and are currently being analysed. Phylogenetic analysis suggests that the ICE2 gene in A. thaliana arose from a duplication of ICE1. However, whether this duplication has resulted in a neofunctionalization or subfunctionalization remains globally unclear. Several articles illustrated that ICE1 and ICE2 regulate the expression of C-REPEAT BINDING FACTORS (CBF) genes, involved in cold tolerance, and show unequal functional redundancy in inducing freezing tolerance [START_REF] Fursova | Identification of ICE2, a gene involved in cold acclimation which determines freezing tolerance in Arabidopsis thaliana[END_REF]Kim et al., 2015c). This unequal genetic redundancy of ICE1 and ICE2 is similar to that observed during seed development, where the, ice2 seed phenotype resembles that of wild-type seeds, whereas the ice1 seed phenotype is almost as severe as that of the ice1ice2 double mutant. Unequal genetic redundancy is frequently observed in homologous gene pairs that have evolved through gene duplication in plants [START_REF] Briggs | Unequal genetic redundancies in Arabidopsisa neglected phenomenon[END_REF].

Interestingly, in support of the idea that ICE1 and ICE2 may have functionally diverged since ICE2 arose, it has been noted that a cathelicidin-like domain, a domain involved in antimicrobial resistance, is present only in the ICE2 protein [START_REF] Kościuczuk | Cathelicidins: family of antimicrobial peptides. A review[END_REF]Kurbidaeva et al., 2014b;[START_REF] Tarasov | The Atlg12860 gene of Arabidopsis thaliana determines cathelicidin-like antimicrobial activity[END_REF]. This domain may be responsible for the resistance to bacterial infection observed in plants overexpressing ICE2 [START_REF] Tarasov | The Atlg12860 gene of Arabidopsis thaliana determines cathelicidin-like antimicrobial activity[END_REF].

However, although a gain of function mutant of ICE1 also exhibits enhanced disease resistance [START_REF] Miura | SIZ1, a small ubiquitin-related modifier ligase, controls cold signaling through regulation of salicylic acid accumulation[END_REF][START_REF] Zhu | Induction of BAP1 by a moderate decrease in temperature is mediated by ICE1 in Arabidopsis[END_REF], it is unclear whether ICE1 has antimicrobial activity or not.

HOS1 is not the only known post translation regulator of ICE1. Li and colleagues and Zhao and colleagues have shown that the MKK5-MPK3/MPK6 cascade negatively regulates cold stress by leading to increased association with E3 ligases, promoting the ubiquitination of

Author contributions

Studies of the permeability of the embryonic cuticle and seed shape quantifications have been performed by Jeanne Loue-Manifel. Yeast-two-hybrid experiments have been carried out by Yen-Ting Lu, Justin Goodrich and Antoine Hoguin, an L3 student supervised by Yen-Ting Lu.

Abstract

The evolution of land plants from aquatic ancestors involved several important innovations, including the production of a diffusion-limiting cuticle. Cuticles are found on the surfaces of all land plants, and are composed of complex polymers, of widely varying composition, but usually containing a significant proportion of aliphatic molecules. The formation of an intact cuticular membrane provide protection against multiple environmental stresses, but particularly against water loss to the dry terrestrial environment. In A. thaliana, two basic Helix-Loop Helix transcription factors, ZHOUPI (ZOU) and ICE1, have been shown to act non-cell autonomously in the endosperm of the seed to ensure the formation of an intact cuticle at the surface of the neighbouring embryo. ZOU and ICE1 act as a heterodimer, an association that we show is conserved in Marchantia polymorpha, an early land plant encoding two ZOU isoforms (MpZOU1 and MpZOU2) and two ICE1-like proteins (MpICE1 and MpICE2).

Using genetic approaches, we show that, consistent with its expression in epidermal tissues, MpZOU2, in association with either MpICE1 or MpICE2, plays a role in the biogenesis of cuticle of young M. polymorpha propagules (gemmae). Transcriptomic analysis reveals that the loss of MpZOU2-containing complexes causes in a reduction principally in the expression of genes involved in cell wall modification and the homeostasis of apoplastic Reactive Oxygen Species.

Biochemical analyses show that that the M. polymorpha gemma cuticle is composed of both aliphatic and phenolic compounds. Although loss of MpZOU function causes some changes in cuticle composition, these do not support a major defect in cuticle biosynthesis. Taken together our results point to a scenario in which, similar to their role at the embryonic surface in A. thaliana, ZOU/ICE complexes are required for the formation of a structurally intact cuticle in M. polymorpha, possibly though ensuring organised cuticle polymerisation, rather than directly in the regulation of the biosynthesis of cuticle monomers.

THE ZOU/ICE COMPLEX IS CONSERVED IN M. POLYMORPHA

To test potential interactions between MpZOU and MpICE proteins, yeast-two-hybrid assays were carried out (Figure S3). Since the interaction between AtZOU and AtICE1 proteins has been shown to be mediated by their bHLH and C-terminal regions [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF], constructs encoding both full length and N-terminally truncated (-T) versions were assembled and used to assess interactions. As indicated by growth on selective media, both MpZOU2 and MpZOU1 interact with both MpICE2-T and MpICE1-T. These results suggest that the ZOU/ICE interactions are likely conserved in all land plant and four potential heterodimers may form in planta: MpZOU1/MpICE1, MpZOU1/MpICE2, MpZOU2/MpICE1 and MpZOU2/MpICE2. Furthermore, we found that both MpICE1-T proteins could interact with AtZOU and that AtICE1-T could interact with both MpZOU proteins.

MPZOU AND MPICE COMPLEMENT RESPECTIVELY A. THALIANA ATZOU AND ATICE1 MUTANTS

Since the yeast-two-hybrid experiment suggested that cross-species interactions are possible between A. thaliana and M. polymorpha ZOU and ICE proteins, we tested whether MpZOU1 and MpZOU2, could complement loss of AtZOU function and whether MpICE1 and MpICE2 could complement loss of AtICE1 function. Homozygous Atzou-4 mutants [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF] were transformed with full length MpZOU1 or MpZOU2 cDNAs under the control of the AtZOU promoter and a proAtZOU::AtZOU construct was used as a positive control. In parallel, full-length MpICE1 or MpICE2 cDNAs under the control of the AtICE1 promoter were introduced into homozygous Atice1-2 mutants [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF].

Mature dry seeds from multiple Atzou-4 lines transformed with either proAtZOU::MpZOU1, proAtZOU::MpZOU2 or proAtZOU::AtZOU were compared to the seeds of Atzou-4 mutants, and wild-type plants. All three constructs were capable of fully rescuing the shrivelled seed phenotype of Atzou-4 mutants. Similarly seeds from independent Atice1-2 lines transformed with proAtICE1::MpICE1, proAtICE1::MpICE2 or proAtICE1::AtICE1 were compared to wildtype and Atice1-2 seeds. A full rescue of the Atice1-2 shrivelled seed phenotype was observed for proAtICE1::MpICE2 and proAtICE1::AtICE1 in Atice1-2. However proAtICE1::MpICE1 showed partial but not complete complementation of the Atice1-2 phenotype.

Quantification of misshapen seeds of transgenic lines containing pAtZOU::AtZOU, pAtZOU::MpZOU1 and pAtZOU::MpZOU2 in the Atzou-4 background and pAtICE1::AtICE1 and pAtICE1::MpICE2 in the Atice1-2 background showed no significantly different from wildtype seeds. However, consistent with visual observations, 30% of transgenic seeds from lines stained gemmae fell into two categories. Whereas loss of function of MpZOU1 and MpICE1, which are mainly expressed ventrally show WT-like cuticle staining, in Mpzou2, Mpice2, Mpzou1Mpzou2 and Mpice1Mpice2 gemmae, the cuticle was much less strongly stained by Nile Red. Interestingly, the cuticle staining of Mpice2 mutants was stronger than that of Mpice1Mpice2 double mutants suggesting either some functional redundancy (consistent with expression analysis), or reciprocal regulation between these genes. However, the potential redundancy observed for the MpICE proteins, does not seem to occur for the MpZOU proteins since staining of the Mpzou2 mutant cuticle with Nile Red resembled that of the double mutant Mpzou1 Mpzou2.

Based on our results it seems likely that MpZOU2 interacts in planta with MpICE2 and MpICE1 and the function of MpZOU2/MpICE heterodimers is to regulate cuticle biogenesis.

The role of MpZOU2 in cuticle development was confirmed by the phenotypic rescue of Mpzou2 with MpZOU2 cDNA driven by the endogenous MpZOU2 promoter, as shown by Nile red staining (Figure 3A). Similarly to our findings with Nile Red staining, the cuticle of Mpzou1 gemmae was found to stain strongly with diphenylborinic acid whereas Mpzou1Mpzou2, Mpice1Mpice2, Mpzou2 gemmae showed very weak cuticle staining (Figure 3B). Surprisingly, Mpice1 mutants stain less strongly than Mpice2 mutants. This observation again suggests a role for both MpICE proteins in the formation of the cuticle of M. polymorpha gemmae, but could indicate that these proteins act differently in the regulation of the deposition of phenolic and aliphatic components.

Transmission electron microscopy analysis of gemmae was applied to further explore the defects in gemma cuticle in Mpzou2, Mpice2, Mpzou1Mpzou2 and Mpice1Mpice2. Using this approach, the cuticle structure of M. polymorpha wild-type gemma was found to be very similar to P.patens' [START_REF] Renault | A phenol-enriched cuticle is ancestral to lignin evolution in land plants[END_REF]. It is composed of three visibly distinguishable layers: an inner grey layer, associated within the cell wall, a middle white layer probably corresponding to cutin and intracuticular waxes, and a dark outer-layer. These three layers were found to be fragmented in some cases and/or totally absent in the two double mutants Mpzou1Mpzou2 and Mpice1Mpice2 (Figure 4). Thus, it sometimes appears that the cell wall, (which does not seem to be defective in any of the mutants), is the most outer layer, directly in contact with the environment. Vesicles wrapped in electron-dense component and electron-dense components at the interface between the cell wall and the cuticle are also visible in the wild-type. This might be explained by there being materials coming via vesicles for deposition within the cuticle. In the two double mutants, Mpzou1Mpzou2 and Mpice1Mpice2 these electron-dense materials appear thicker and stuck around the vesicles suggesting a potential problem either in the transport (which is unlikely) or in the deposition of the materials within the cuticle.

II.4 Material and Methods

Histochemical staining for GUS activity of vibratome-sectioned thallus.

15 to 20 day-old transcriptional reporter lines (described in Chapter 2, § I) were harvested and placed into a small plate. 5% Phytagel (Sigma CAS : 71010-52-1 ; P8169) at 50°-60°C was then poured onto the plants. The plate was then stored for 30 min to 1hour at 4°C to cool down before being mounted onto the Leica VT1000 S Microtome. Approximately 10µm sections were performed and placed into 1%PBS solution before staining as described above. Observations of GUS activity were made using a Keyence VHX900F digital microscope.

Histochemical staining for GUS activity in whole mount thallus.

MpZOU1, MpZOU2, MpICE1 and MpICE2 promoters were cloned as fusions to the GUS reporter via Gateway recombination using the pMpGWB104 destination vector and subsequently Tak2xTak2 sporelings were transformed [START_REF] Ishizaki | Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha[END_REF]. Histochemical assays for GUS activity were performed according to [START_REF] Jefferson | GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF] with some minor modifications. Briefly, after three 20 minute vacuum infiltrations with the GUS staining solution (50mM sodium phosphate buffer, pH 7.2 ; 0.5mM potassium-ferrocyanide; 0.5mM potassiumferricyanide; 10mM EDTA; 0.01% Triton ; 1mM 5-bromo-4-cloro-3-indolyl-Beta-glucuronic acid (Duchefa Biochemie, CAS number 114162-64-0), plants were incubated overnight at 37°C. They were then dehydrated by incubation in increasing concentrations of Ethanol (15%, 30%, 50%), for 20 minutes (per bath at each step) at room temperature. Plants were then fixed in FAA fixative solution (10% formaldehyde, 5% acetic acid, 50% ethanol) for 45 minutes under vacuum followed by either 2 hours at room temperature or overnight at 4°C. Dehydration steps to remove the chlorophyll were then performed by incubation in further increasing concentrations of ethanol (50%, 70%, 100%), for 20 minutes per bath at each step at room temperature. Observations of GUS activity were made using a Keyence VHX900F digital microscope.

Immunolabelling on sections.

Mature gemmae were directly harvested from the gemma cup and fixed in ice-cold PEM buffer (50mM PIPES, 5mM EGTA and 5mM MgSO4, pH6.9) containing 4% (w/v) paraformaldehyde and 1% (w/v) glutaraldehyde under vacuum on ice (2 x 30 min) and overnight at 4°C. Samples were washed twice in PEM buffer and dehydrated by incubation in increasing concentrations of ethanol (50%, 70%, 80%, 90% and three times in 100%), for an hour each on ice under vacuum followed by an overnight incubation at 4°C in 100% Ethanol.

Samples were then infiltrated with increasing concentrations of LR WHITE medium resin in absolute ethanol (30%, 50%, 70%, and three times in 100%), for an hour on ice under vacuum followed by an overnight incubation at 4°C for each bath. Seeds were then transferred into small Beam capsules (Electron Microscopy Sciences) and incubated overnight at 60°C for polymerisation. 1µm sections were cut on a Leica RM6626 microtome and floated onto adhesive slides (Superfrost Ultra plus TM (ThermoFisher Scientific)). For immunolabelling, sections were first incubated for 45 minutes in PBS containing 2%(w/v) Bovine Serum Albumin (BSA). They were then incubated with a 10 fold dilution of primary antibody in PBS containing 0.1% BSA overnight at 4°C followed by one hour at room temperature. Sections were then washed three times in PBS for 5 minutes, then in PBS containing 0.1% Tween 20 (w/v) for 10 minutes and finally in PBS for 5 minutes. Incubation with a 100-fold dilution of an appropriate anti-rat secondary antibody (Alexa Fluor 488 conjugate, Invitrogen) in PBS with 0.1% BSA for one hour in the dark, in a wet chamber was then performed. The sections were washed three times in PBS for 5 minutes and counter-stained with a solution of 0.25ng/L of calcofluor (Fluorescent Brighter 28, Sigma Aldrich) in PBS for 5 minutes. Finally, sections were washed rapidly three times in PBS and mounted in Vectashield (Eurobio). Observations were made using an Axio Imager 2 microscope (Zeiss).

Desiccation test.

32 gemmae were sowed directly on soil (soil:perlite 1:1) under long-day conditions (16h, 21°C) (e.g. 4 gemmae per pot) to grow for 15 days under normal conditions. 8 plants e.g. 2 pots were then placed under one watering condition: continuous watering, 18 days without watering, 21 days without watering or 24 days without watering. Plants pictures after 24 days were obtained with a CANON EOS 450D camera equipped with objectives SIGMA 18-50mm.

Cold tolerance test.

8 gemmae per genotype were placed on a plate with Johnson media under short day conditions at 6°C. Plants grew 4 days or 17 days before being photographed with a CANON EOS 450D camera equipped with objectives SIGMA 18-50mm.

CHAPTER 3

Is the ZOU/ICE function in endosperm cell elimination in Arabidopsis thaliana conserved in Marchantia polymorpha?

In a first section of this Chapter, presented as an article, we show that the MpZOU1/MpICE1 complex controls the development of a novel water conducting tissue, the pegged rhizoids.

Data are presented showing that cell wall modifications and cell death are both key events occurring during this process, and parallels are discussed between the ZOU/ICE-mediated endosperm elimination process observed in A. thaliana, and the differentiation of pegged rhizoids.

The second section of this Chapter contains a more general discussion of open questions that our research has revealed, and describes potential future research perspectives.

AUTHOR CONTRIBUTIONS

Yen formation of an intact embryonic cuticle [START_REF] Denay | Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1[END_REF][START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF][START_REF] Yang | The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development[END_REF]. Both proteins are conserved throughout land plants [START_REF] Catarino | The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land[END_REF][START_REF] Harris | Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata[END_REF], raising the question of their role in plants lacking seed or endosperm. To address this, we characterised their orthologues in the model liverwort M. polymorpha. Phylogenetic analysis indicates that M. polymorpha has a single orthologue of A. thaliana ZOU, Mp4g19650

here designated MpZOU1 and a second paralogous gene MpZOU2 (Mp8g04130) that derives from an ancient duplication likely present in the common ancestor of land plants (Figure S1).

The position of the two introns in the M. polymorpha and A. thaliana genes is conserved, also consistent with a common evolutionary origin. There are also two M. polymorpha coorthologues of the A. thaliana ICE genes, MpICE1 (Mp4g04920) and MpICE2 (Mp4g04910)

which derive from a more recent duplication, after the divergence of liverworts and mosses (Figure S2). We did not find ICE or ZOU genes in any green algae genomes, suggesting that they arose early in evolution of land plants. Here we characterize MpZOU1 and MpICE1 which we found to share discrete functions in rhizoid development.

We first tested whether the M. polymorpha proteins could interact with one another using a yeast two hybrid assay. Indeed, MpZOU1 and MpICE1 interact both with each other, L2FC corresponds to Log2 Fold Change material. These modifications are followed by cell death, although the functional link between the two processes remains unclear. Does cell wall remodelling lead to cell death, or is cell death triggered independently of cell wall remodelling by the MpZOU1/MpICE1 complex. This question is relevent given the superficial similarity between the functions of MpZOU1/MpICE1 in rhizoids and AtZOU/AtICE1 in the breakdown of the A. thaliana endosperm. In the endosperm it has been shown that cell wall modification (in this case thinning and softening) precedes and is a prerequisite for endosperm cell death, which is also promoted by the physical crushing of the softened endosperm by the growing embryo [START_REF] Fourquin | Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds[END_REF].

Whether cell death in the pegged rhizoids of M. polymorpha can truly be functionally equated to the cell elimination process observed in the developing endosperm remains an open question.

I The regulation of the activity of the ZOU/ICE complex: Conservation and divergence.

The work presented in this thesis has confirmed that not only are ZOU and ICE proteins conserved in M. polymorpha, but that the ZOU/ICE complex is also conserved and is thus likely to have been an early and important factor for gene regulation in land plants. My work has also underlined intriguing parallels between the function of the ZOU/ICE complexes in M. polymorpha and those in seed plants, relating to the regulation of cell elimination/death, and the formation of the cuticle. However, despite this apparent molecular and functional conservation, my work has also pinpointed interesting points of divergence.

One of the main questions that I hoped to address during my PhD was how the activity of the ZOU/ICE complex in seeds is regulated, and whether regulatory mechanisms might have already been recruited in early land plants, and could potentially help in the eludidation of complex function. Previous work in A. thaliana had suggested that ZOU/ICE complex in the endosperm activity is likely controlled at the level of ICE1 stability. My work has confirmed that this is the case, by revealing the role of HOS1 in the endosperm. I have also revealed an additional role for ZOU/ICE1 in regulating the transcription of ICE2, highlighting the likely complex nature of regulatory interactions in this tissue.

Complementation experiments using MpICE1 and MpICE2 suggests that MpICE1 posttranslational regulation could be divergent from that of AtICE1 and AtICE2. This difference might be explained in terms of the conservation of the important KRAAM motif in MpICE1. In A. thaliana, both ICE1 and ICE2 proteins interact with other bHLH class Ia transcription factors SPEECHLESS MUTE AND FAMA to specify three cell-state transitional steps leading to stomata differentiation. During this developmental process, ICE1 serves as a scaffold to recruit SPCH and MPK3/6. The association of MPK3/6 and ICE1 leads to phosphorylation and degradation of ICE1 protein by an unknown proteosomal pathway. In addition to the phosphorylation of ICE1 and through its interaction with SPCH, ICE1 brings SPCH into proximity with MPK6 which thus phosphorylates SPCH leading to its dowregulation. Together, this inhibits stomatal cell fate (Putarjanan 2019). To date, we do not know if this mechanism is conserved in seed development through the ZOU/ICE complex However, similarly to the overproduction of stomata when ICE1 is stabilized in ICE1-D (mutated in the KRAAM domain), endosperm lysis is precocious suggesting again a stabilisation and gain of activity of ICE1 (Denay et al., 2014a). My preliminary results on the embryo sheath also support this. Published data also confirm a role of MPK6 in embryonic cuticle formation through the ALE1/GSO these two models remain distinct points on a very wide and diverse evolutionary tree. They do not necessarily reflect "the norm" in plants belonging to the same clade, group or even family.

A. thaliana and M. polymorpha therefore cannot be considered to be representative of the angiosperms or the bryophytes respectively. As a result, comparing gene functions and/or regulatory machanisms between these two species, although it may provide glimpses of potential commonalities and divergence, cannot be considered to provide conclusive proof of functional conservation at any level.