Célia Baroux

Sarah Cookson

Lieven De Veylder

Mohamed Zouine

Keywords: Endoreduplication, Tomato, Transcriptome, Growth, FisH UMR1332

J'aimerais remercier l'équipe Organogénèse du Fruit et endoreduplication (maintenant nommée : Floraison, Développement du Fruit et Contraintes Environnementales) de m'avoir accueilli comme doctorant et aux membres de l'équipe de m'avoir accepté.

Merci Nathalie (G) d'avoir acceptée de travailler avec moi pendant cette thèse, d'avoir fait preuve de tant de patience et de bienveillance avec moi (bienveillance, qui sera je pense le mot qui dictera cette expérience). Merci d'avoir rendu possible tout le travail que j'ai pu faire et de m'avoir laissé une certaine liberté pendant cette thèse qui a été une très bonne expérience pour moi. Merci aussi pour tout ce que j'ai pu apprendre durant cette thèse aussi bien mes compétences nouvelles (je pense à la bioinfo) que personnellement (je pense à la bienveillance). Je n'ai aucun regret d'avoir choisi cette thèse même si dans l'ensemble tout a été chamboulé. J'espère que de ton côté tu ne regrettes pas trop ton choix de doctorant des trois dernières années et que les autres seront moins chiant que moi.

Avant d'arriver en thèse, il y a eu la licence et le master qui ont été des éléments déterminant pour en arriver là et beaucoup de gens sont intervenus et leur aide m'a été précieuse et je les remercie. Pour cela je souhaiterais particulièrement remercier plusieurs personnes, Michel Hernould, Didier Thoraval, Fred Gévaudant et Fred Delmas. Michel, je veux te remercier pour tous nos échanges toujours enrichissants et aux pires moments de la journée (souvent proche du départ pour rentrer à la maison) mais aussi pour m'avoir accepté en master et m'avoir soutenu pendant celui lorsque c'était difficile. Merci à Didier, tes cours en licences étaient géniaux et m'ont donné envie de continuer (je m'excuse de ne pas avoir choisi la génétique) vers le master et ton soutien m'a été précieux. Bien sûr, merci aux deux Fred pour leurs expériences et connaissances qui ont toujours su prendre le temps de partager malgré leur emploi du temps overbooké. Merci encore à tous les deux d'avoir accepté lorsque l'on était en master de prendre le temps de faire plaisir à des gamins ayant envie de faire iGEM. Fred D, je te remercie de ta gentillesse et ta sympathie, c'était toujours un plaisir de faire « n'importe quoi » avec toi (je pense à la magnetofection entre autres). Merci à Fred G d'avoir toujours su me donner des conseils avisés peu importe le moment sur des manips ou des questions.

Merci à Norbert pour m'avoir formé, littéralement, pendant mon M2 et le début de ma thèse car je pense sincèrement avoir énormément appris aussi bien scientifiquement qu'humainement en travaillant avec toi. Merci d'avoir été présent aussi aux moments où j'en avais besoin. C'est toujours un plaisir de passé du temps avec toi et je suis très content qu'aujourd'hui nous soyons amis ! JP (Michael ou Jean-Pascal), on ne sait jamais avec toi, merci à toi aussi d'avoir été un super collègue en plus d'être un ami. Même si au départ tu faisais peur dans ton antre au bout ou avec tes centaines de boîte, finalement tu n'es pas un si mauvais gars (haha). Merci d'avoir été là pour le soutien pendant la thèse, la reprise du sport et les soirées, journées et moment passé ensemble, toujours du bon temps. Bon courage pour ta thèse, je serais là en soutient ! Bien sûr, merci aux autres doctorants de l'équipe, Arthur et Sabri, pour votre bonne humeur. Cela a toujours été plaisant de travailler avec vous et d'échanger sur le sport, la musique, les répliques de film… vous êtes tous les deux des gars bien et je croise fort les doigts pour que votre thèse se déroule bien et que vous ayez les best résultats ! Arthur tu n'as que le fardeau de 30 ans de recherche international à conclure (:p). Spécial dédicace à toi Gabriel qui vit bien loin de nous, on ne t'oublie pas ! Enfin je souhaitais remercier mes proches, ceux qui n'ont pas travaillé avec moi mais qui ont su être un soutien moral infaillible pendant ce parcours jusqu'aujourd'hui. Un énorme merci à mon meilleur ami, Quentin, avec qui je partage tout depuis plus de 12 ans (4518 jours pour être exact, Facebook 2021). Je voudrais remercier tout particulièrement ma moitié, Floriane, qui a été un soutien majeur sans qui je pense tout cela n'aurait pas été possible. Merci d'être la aujourd'hui et d'être la mère de ma fille, Freya ! Merci de toujours avoir été là, de me supporter à la maison quand je râle et que je suis de mauvaise humeur. Ton soutien est extrêmement important et je pense que je ne pourrais jamais te rendre l'équivalent.

Et pour finir, je remercie mes parents d'avoir permis que je choisisse ce qu'il me plait de faire et d'avoir soutenu mes choix quel qu'ils soient. Je dédie ce manuscrit à ma mère qui me manque et que j'aimerais toujours.

H) The integration of ploidy distribution with ploidy specific transcriptome reveals that ploidy specific gene expression is following specific spatio-temporal patterns. Discussion A) Endoreduplication in the pericarp induces differential gene expression. B) Endoreduplication during early fruit development is associated with cell specialization. C) Localization of ploidy levels in the pericarp can be inferred using ploidy transcriptome data and publicly available transcriptome. D) Ploidy level distribution is dynamic during early fruit development. Figure 34. Ploidy markers from a ploidy specific transcriptome of Arabidopsis thaliana root cortex [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF] compared to the pericarp ploidy transcriptome.

Chapter 1 : INTRODUCTION

Potential roles of endoreduplication in Plants What is endoreduplication? Some generalities and definitions

The cell cycle is a universal process, occurring in all living organisms, that results in cell division and the transfer of the genetic information (the whole copy of the genome) from a mother cell to two daughter cells (Figure 1A).

Endoreduplication is a modified cell cycle (also referred as endoreplication or endoploidization) during which cells increase their DNA content through successive rounds of nuclear genome duplication without cell division leading to increased ploidy level (Figure 1A). Ploidy is a relative measure of DNA content of a cell referring to the haploid DNA quantity of the genome, with 1C referring to 1 chromatid copy for a chromosome. Therefore, if DNA quantity of a diploid cell is 64C, this means that each chromosome has 64 copies of chromatids in this particular cell. Endoreduplication occurs without cytokinesis and the classical mitotic DNA replication events such as, DNA condensation/decondensation phases or spindle assembly [START_REF] Edgar | Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth[END_REF]. In endoreduplicated cells, duplicated DNA is still attached as sister chromatids by the centromeres forming a structure called polytene chromosomes [START_REF] Edgar | Endoreplication Cell Cycles: More for Less[END_REF]. These polytene chromosomes have been visualized for example in Drosophila salivary gland cells that serve as a model for the study of endoreduplication in insects (Figure 1B, [START_REF] Zhimulev | Polytene Chromosomes[END_REF] and their existence was demonstrated in Tomato fruit pericarp cells [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF].

Endoreduplication is a widespread phenomenon found in several clades in Eukaryotes. However, ploidy levels are highly variable between species or even between different tissues of the same individual. Moreover, it is not known if during endoreduplication DNA is always totally replicated in the different species or organs in which it occurs. In animals, endoreduplication is only found in some specific cell types such as in giant neurons of Gastropods (200000C), in salivary glands of Drosophila (2048C) and follicle cell epithelium and in Silkworm Bombyx mori (up to 20 rounds of DNA replication = 1 048 576C). In Mammals, endoreduplication also occurs in trophoblast giant cells (1024C), hepatocytes, cardiac myocytes, and cancer cells (D 'Amato and Durante 2001[START_REF] Orr-Weaver | When bigger is better: the role of polyploidy in organogenesis[END_REF][START_REF] Shu | Endoreplication: The Good, the Bad, and the Ugly[END_REF]. The role of endoreduplication in animals is not fully elucidated but has been proposed to support rapid growth and strong metabolism of specialized organs such as nurse cells and salivary glands of larva in Drosophila or trophoblast in Mice [START_REF] Fox | Endoreplication and polyploidy: insights into development and disease[END_REF][START_REF] Lee | Endoreplication: polyploidy with purpose[END_REF][START_REF] Orr-Weaver | When bigger is better: the role of polyploidy in organogenesis[END_REF]. Another role for endoreduplication corresponds to body size control in ants and nematodes [START_REF] Scholes | Can endopolyploidy explain body size variation within and between castes in ants?[END_REF].

In plants, endoreduplication is found in almost 90% of Angiosperms (Joubès and Chevalier, 2000). This phenomenon occurs in several plant families and does not seem to follow phylogenic linkage suggesting that endoreduplication results from separate evolution (homoplasy) because of specific strategies of life rather than common ancestry [START_REF] Barow | Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size[END_REF]. For example, annual or bi-annual plants tend, more than perennials, to perform endoreduplication as a common developmental process [START_REF] Barow | Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size[END_REF]. Thus, endoreduplication could support rapid growth needed to complete a life cycle from seed to seed in a season [START_REF] Barow | Endopolyploidy in seed plants[END_REF]Meister 2003, Bainard et al., 2012). Within the same organism, endoreduplication can occur in several organs of the plant, at variable levels such as in Arabidopsis thaliana (Arabidopsis) leaves and sepals (up to 16C), roots (up to 8-to 16C) and trichomes (32-to 64c) or as in Tomato fruit pericarp cells (up to 512C) and leaves (up to 8C) [START_REF] Cookson | Cell and leaf size plasticity in Arabidopsis: what is the role of endoreduplication?[END_REF][START_REF] Roeder | Variability in the Control of Cell Division Underlies Sepal Epidermal Patterning in Arabidopsis thaliana[END_REF][START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF][START_REF] Churchman | SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana[END_REF][START_REF] Joubès | Molecular and Biochemical Characterization of the Involvement of Cyclin-Dependent Kinase A during the Early Development of Tomato Fruit[END_REF]. Recent studies suggest that endoreduplication could be involved in response to environmental stresses allowing rapid cell differentiation and strong metabolic capacities to improve tolerance to stress [START_REF] Scholes | Plasticity in ploidy: a generalized response to stress[END_REF], Bhosale et al., 2019[START_REF] Lang | Endoreplication -a means to an end in cell growth and stress response[END_REF]. But endoreduplication is also involved in different plant developmental processes. A more detailed description of the different roles of endoreduplication during plant development and in particular in our model, the Tomato fruit, is given below.

Endoreduplication and plant development

Endoreduplication occurs throughout plant development in many different organs and tissues: in above ground organs such as hypocotyl, leaf, and in reproductive structures such as flowers, fruits and seeds; in below ground organs such as the roots but also specialized structures like nodules. Endoreduplication occurs more frequently in organs that develop a large structure rapidly and have a need for a strong metabolic activity and storage, such as Maize endosperm cells [START_REF] Leiva-Neto | A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm[END_REF] or Medicago nodules [START_REF] Foucher | Cell cycle regulation in the course of nodule organogenesis in Medicago[END_REF]. In general, DNA quantity in a cell is positively correlated with cell size (Figure 1A, [START_REF] Beaulieu | Genome size is a strong predictor of cell size and stomatal density in angiosperms[END_REF][START_REF] Bourdon | Endoreduplication and Growth of Fleshy Fruits[END_REF] and most of the described cases of endoreduplicated cells are larger than nonendoreduplicated ones in the same organ [START_REF] Breuer | BIN4, a Novel Component of the Plant DNA Topoisomerase VI Complex, Is Required for Endoreduplication in Arabidopsis[END_REF]. These observations led to link endoreduplication with a role in cell growth and organ size control [START_REF] Kondorosi | Plant cell-size control: growing by ploidy?[END_REF][START_REF] Larkins | Investigating the hows and whys of DNA endoreduplication[END_REF][START_REF] Barow | Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size[END_REF]. Endoreduplication also seems to be important for other developmental processes and hereafter, a series of organs and developmental events in which endoreduplication has a role are detailed.

Endoreduplication in the hypocotyl in Arabidopsis

In Arabidopsis, the hypocotyl is formed during embryogenesis and is composed of a fixed number of cells. Therefore, after germination most of the growth occurs through cell expansion [START_REF] Gendreau | Cellular Basis of Hypocotyl Growth in Arabidopsis thaliana[END_REF]. Endoreduplication takes place in the hypocotyl cells very early after germination and ploidy levels can reach up to 8C in light-grown plantlets [START_REF] Gendreau | Cellular Basis of Hypocotyl Growth in Arabidopsis thaliana[END_REF]. Interestingly, in this organ, light seems to inhibit endoreduplication since in the darkness, higher ploidy levels (16C) are observed (Figure 2A and 2B). The increased proportion of higher ploidy level are associated with an increase in cell length and hypocotyl length [START_REF] Gendreau | Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl[END_REF][START_REF] Amijima | The polar auxin transport inhibitor TIBA inhibits endoreduplication in dark grown spinach hypocotyls[END_REF]. In this organ, the hypothesized role for endoreduplication is thus to be a strategy to grow more in a short time, in dark environment, supporting etiolated growth in order to reach light rapidly. (B) Endoreduplication patterns in the hypocotyl of dark-grown Arabidopsis seedlings A and B, adapted and modified from [START_REF] Gendreau | Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl[END_REF] Endoreduplication in the Arabidopsis thaliana leaf a. Epidermal pavement cells In Arabidopsis, leaf development is divided in two phases, a cell division phase and a cell expansion phase that overlap in time. First, the leaf primordium, emerging from the shoot apical meristem, grows by cell proliferation, that occurs in the entire leaf and produces new cells (Figure 3A). In a second phase, cell proliferation progressively stops, and cells start to expand. This cell expansion phase progresses as a front from the tip to the base of the leaf (Gonzalez et al., 2012, Figure 3A). This second growth phase is associated with the occurrence of endoreduplication [START_REF] Melaragno | Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis[END_REF] with ploidy levels reaching up to 16C in the epidermal pavement cells (Figure 3B), the outermost cell layer of the leaf. To better understand how endoreduplication is related to increasing cell size in leaves, a mathematical model was built to simulate epidermal cell size distribution as a function of endoreduplication dynamics [START_REF] Kawade | Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue[END_REF]. This model, taking into account that endoreduplication dynamics is following a Poisson law and considering that cell size is determined according to ploidy level, recapitulated experimental data showing the Gaussian distribution of cell size. However, a meta-analysis of published data linking endoreduplication with cell growth in leaves of Arabidopsis mutants showed that in most cases endoreduplication is not the only factor explaining the changes in cell size [START_REF] Tsukaya | Has the impact of endoreduplication on cell size been overestimated?[END_REF]. Endoreduplication in the leaf is influenced by environmental factors such as day length since the transfer of Arabidopsis plants from short-day-period to long-day-period increases the proportion of 8C and 16C nuclei accompanied with an increase in leaf size (Del Prete et al., 2019). Interestingly, this increased proportions of 8C and 16C is associated with an accumulation of the floral transition markers FLOWERING LOCUS T, CONSTANS and APETALA suggesting that endoreduplication could be involved in signaling transition from vegetative phase to reproduction (Del Prete et al., 2019). In these leaves, the increase of ploidy levels has also been associated with a complex reprogramming of metabolism, including changes in sugar metabolism, cell wall synthesis and secondary metabolism (Del Prete et al., 2019). In Arabidopsis leaf, the potential roles of endoreduplication are thus to regulate at least partially cell-size and leaf size and is also associated with flowering after changes by light conditions and metabolic changes.

b. Leaf Epidermal specialized cells: the trichomes

In Arabidopsis leaves, the epidermal pavement cells are not the only ones to undergo endoreduplication. Trichomes, large cells that differentiate from pluripotent epidermal cells called protodermal cells in young leaves, also undergo endoreduplication (Figure 4A). In Arabidopsis, trichomes have a three branched morphology dependent on ploidy levels (Figure 4B). The early differentiation of trichomes requires on a positive transcriptional signaling cascade of a transcription factor complex composed of GLABRA1 (GL1), TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) [START_REF] Hülskamp | Genetic dissection of trichome cell development in Arabidopsis[END_REF][START_REF] Fambrini | The Dynamic Genetic-Hormonal Regulatory Network Controlling the Trichome Development in Leaves[END_REF]. One of the first consequence of this transcriptional activation is that the cell stops dividing and starts endoreduplicating and enlarging (Figure 4A). The cell expansion is mainly driven by the growth of the vacuole and occurs simultaneously with branching.

Figure 3. Endoreduplication during Arabidopsis leaves development

(A) Schematic representation of Arabidopsis developmental stages from 4 to 20 days after sowing, at rosette, leaf and cellular level. The developmental phases are represented in different colors: in green, the cell proliferation phase; in orange, the meristemoid division phase (not discussed here) and in yellow, the cell expansion phase. (B) Representation of an epidermal cell population colored by ploidy levels in the leaf. A from [START_REF] Gonzalez | Leaf size control: complex coordination of cell division and expansion[END_REF]. B adapted and modified from [START_REF] Melaragno | Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis[END_REF] When fully grown, the trichome has reached 32C DNA content and has three branches (Figure 4B and 4C).

Generally, size and branching of the trichome follow a positive correlation with DNA quantity and mutants with increased endoreduplication levels show increased trichome branching (Figure 4D, Kasili et al., 2011, Fambrini and[START_REF] Fambrini | The Dynamic Genetic-Hormonal Regulatory Network Controlling the Trichome Development in Leaves[END_REF]. Trichome overbranching is also observed after treatment with colchicine (Figure 4E, [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF], a drug able to disrupt the cell cycle, inducing whole genome duplication and thus polyploidy in plants [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF]. On the other hand, inhibition of endoreduplication during trichome initiation results in dedifferentiation of the cell that finally is integrated in the epithelium as an epithelial cell, suggesting a strong link between endoreduplication and the differentiation of trichome in the Arabidopsis leaf [START_REF] Bramsiepe | Endoreplication Controls Cell Fate Maintenance[END_REF]. In Arabidopsis, endoreduplication in the trichome is thus proposed to have a role in regulating cell size and controlling cell fate.

Endoreduplication in the root

Endoreduplication also occurs during root development. The primary root can be divided in three zones: the meristem zone where cells are mostly dividing, a transition zone in which cells shift from the cell cycle to the endocycle, and the elongation zone where cells expand and endoreduplicate reaching up to 16C. In Arabidopsis, proper root development depends on the correct maintenance of the meristem size and a fine balance between dividing and differentiating (expanding and endoreduplicating) cells (Figure 5, [START_REF] Heyman | Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL11[OPEN][END_REF][START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. In Arabidopsis root, the transition between division and endoreduplication is regulated by hormones, and more specifically by auxin and cytokinin. A gradient of auxin is present in the root as a maximum in the meristem zone to a minimum in the transition zone (Figure 5, [START_REF] Breuer | Endocycling in the path of plant development[END_REF][START_REF] Mambro | Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root[END_REF]. This auxin minimum is located at the place where cytokinin signaling takes place and where the transition between the cell cycle and endoreduplication is occurring (4C-8C boundary in Figure 5). The phytohormones auxin and cytokinin play an important role in this regulation. It has been shown that cytokinin controls the shift from the mitotic cycle to the endocycle in the root [START_REF] Takahashi | Cytokinins Control Endocycle Onset by Promoting the Expression of an APC/C Activator in Arabidopsis Roots[END_REF]. Plants with reduced response to cytokinin have lower endoreduplication activity in roots [START_REF] Takahashi | Cytokinins Control Endocycle Onset by Promoting the Expression of an APC/C Activator in Arabidopsis Roots[END_REF]. A screen of ethyl methanesulfonate for larger or smaller organs and change in ploidy levels in treated Arabidopsis seeds resulted in the identification of a mutant named high ploidy 2 (hpy2) [START_REF] Ishida | SUMO E3 Ligase HIGH PLOIDY2 Regulates Endocycle Onset and Meristem Maintenance in Arabidopsis[END_REF]. hpy2 seedlings have higher ploidy levels in the root, very short primary root and a reduced root meristem area composed of larger cells compared to WT [START_REF] Ishida | SUMO E3 Ligase HIGH PLOIDY2 Regulates Endocycle Onset and Meristem Maintenance in Arabidopsis[END_REF]. Thus, in the primary root, endoreduplication seems to regulate growth by allowing cell expansion after the transition zone. Some epidermal cells of the root can differentiate into root hairs cells that are long tubular shaped cells. Root hairs cells can be as long as 1 mm or more. They greatly increase the rooting area helping in nutrient acquisition, anchorage, and microbe interactions [START_REF] Grierson | Root Hairs. The Arabidopsis Book[END_REF]. It has been reported that root hair cells undergo several rounds of endoreduplication and reach 16C ploidy level when fully developed [START_REF] Sliwinska | Synchronously developing collet hairs in Arabidopsis thaliana provide an easily accessible system for studying nuclear movement and endoreduplication[END_REF]. (A) Development of a trichome of Arabidopsis wild type. Trichome development starts with the specification of single cells within the epidermal layer of the leaf (cell fate/initiation). After this stage, the cell will start doing three rounds of endoreduplication and enlarge (outgrowth), attaining ten-times the epidermal cell size. Then, branching initiates and simultaneously cell is continuing to enlarge and undergo one more endoreduplication round (Cell growth and expansion). Finally, the trichome will eventually continue to grow until being fully grown (Maturation).

(B) Scanning electron micrograph of wild-type (Col-0) trichome showing the three branches morphology. Bar = 50µm. Adapted from [START_REF] Kasili | BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes[END_REF] (C) Flow cytometry analysis representing the ploidy levels present in wild-type Col-0 leaves. Adapted from [START_REF] Heyman | Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL11[OPEN][END_REF] (D) Flow cytometry analysis representing ploidy levels present in mutant line uvi-4 having increased ploidy levels in leaves (left). Adapted from [START_REF] Heyman | Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL11[OPEN][END_REF] Scanning electron micrograph of uvi-4 mutant trichome showing over branched phenotype (right). Bar = 300µm. Adapted from Heyman et al., 2011 (E) Trichome phenotype observed in plants treated with colchicine. Colchicine is a drug perturbing the cell cycle and leading to the production of plants with whole genome duplication. [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF] It was shown that root hair cells undergo one more round of endoreduplication compared to their neighbor non hair epidermal cells (16C vs 8C, [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF] indicating a potential role of endoreduplication in the specification of these root hair cells. In the hyp7 mutant, mutated in the RHL1 protein having an essential role in the DNA topoisomerase VI complex, showing a decreased of endoreduplication results in leaf epidermal cells, trichomes and root hair cells that are smaller [START_REF] Sugimoto-Shirasu | RHL1 is an essential component of the plant DNA topoisomerase VI complex and is required for ploidy-dependent cell growth[END_REF]. However, in in several hair mutants, the differentiation of root hair cells in the collet and root regions is impaired when there is less endoreduplication while r the size of these cells is less or not affected by the decreased ploidy level, suggesting that endoreduplication might be necessary for the differentiation but that in these root hair cells, growth is independent of ploidy levels [START_REF] Sliwinska | On the relationship between endoreduplication and collet hair initiation and tip growth, as determined using six Arabidopsis thaliana root-hair mutants[END_REF]. Endoreduplication in root hair cells thus seems to play a role in the establishment of their identity [START_REF] Sliwinska | On the relationship between endoreduplication and collet hair initiation and tip growth, as determined using six Arabidopsis thaliana root-hair mutants[END_REF][START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF].

Other specialized root structures also undergo several rounds of endoreduplication such as the nodules in Medicago (Medicago truncatula) or Lotus (Lotus Japonica) [START_REF] Foucher | Cell cycle regulation in the course of nodule organogenesis in Medicago[END_REF]Kondorosi, 2000, Suzaki et al., 2014). Nodules are specialized structures formed after symbiotic infection of roots of leguminous plants by soil bacteria responsible of atmospheric nitrogen fixation. Nodules can be divided in four separated areas of different functions. First, a meristem zone produces new cells, through cell division, that will later be forming the different nodule cell layers (Figure 6A and6B). The invasion zone is composed of endoreduplicated cells that can reach 16C and that can be infected by rhizobia. The next area is an interzone that contains symbiotic cells that are larger than in the meristem area or the invasion zone. In the interzone cells are endoreduplicated and can reach 16C to 32C. The last zone is the nitrogen-fixing zone where symbiotic cells are the largest with large vacuoles and ploidy levels reaching 64C. The areas containing the largest cells display higher ploidy levels indicating a positive relation between endoreduplication and cell size (Figure 6B, [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF].It has been shown using plant with impaired endoreduplication process that a proper transition from the cell cycle to the endoreduplication is necessary for correct the differentiation and functionality of the nodule structures showing that endoreduplication is an important process for the differentiation of nitrogen fixing cells [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF][START_REF] Cebolla | The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants[END_REF].

Endoreduplication in the flower

Endoreduplication takes place in flowers of many different species such as Orchids (Phalaenopsis, [START_REF] Ho | Endoreduplication level affects flower size and development by increasing cell size in Phalaenopsis and Doritaenopsis[END_REF], Cabbage (Brassica oleracea L., [START_REF] Kudo | Nuclear DNA endoreduplication during petal development in cabbage: relationship between ploidy levels and cell size[END_REF] or Red Clover (Trifolium pratense, [START_REF] Kocová | Endoreduplication as a part of flower ontogeny in Trifolium pratense cultivars[END_REF].

In Arabidopsis developing flowers, endoreduplication is found in sepals that correspond to the green leaf like structures of the flower (Figure 7A). The role of sepals in is to be protective by enclosing the developing reproductive organs. Both abaxial and adaxial epidermis cells of the Arabidopsis sepals undergo endoreduplication reaching up to 16C and rarely 32C [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF]. These cells are referred to as giant cells because of their length that is nearly a fifth of the sepal length (360µm long, Figure 7B and 7C, [START_REF] Roeder | Cell cycle regulates cell type in the Arabidopsis sepal[END_REF]. In this organ, non endoreduplicated small cells (10µm long) co-exist with these endoreduplicated giant cells. Giant cell identity is determined before endoreduplication starts. However, it seems that endoreduplication is inhibiting small cell identity since transgenic plants with increased endoreduplication have a reduced number of cells expressing small cell identity markers [START_REF] Roeder | Cell cycle regulates cell type in the Arabidopsis sepal[END_REF]. Increase or decrease of the proportion of giant cells results in change of sepal curvature in an outward or inward manner, respectively, whereas wild type Arabidopsis sepals have a flat shape [START_REF] Roeder | Variability in the Control of Cell Division Underlies Sepal Epidermal Patterning in Arabidopsis thaliana[END_REF]. Thus, endoreduplication is influencing the morphology of sepals. Moreover, transgenic plants with increased ploidy levels in sepal epidermal cells results present an increased proportion and size of giant cells without any alteration of sepal length or growth rate [START_REF] Roeder | Cell cycle regulates cell type in the Arabidopsis sepal[END_REF]. In transgenics plants harboring more giant cells and thus higher ploidy levels in the sepals, due to the specific overexpression of the LGO/SMR1 gene in the sepal epidermis, an increased expression of defense genes has been observed including the up regulation of Glucosinolate biosynthesis genes [START_REF] Schwarz | Transcriptomic Effects of the Cell Cycle Regulator LGO in Arabidopsis Sepals[END_REF]. Glucosinolate is a secondary metabolite stored for being used as a repulsive compound against herbivores insects. The upregulation of glucosinolate biosynthesis is also upregulated in mature trichomes [START_REF] Jakoby | Transcriptional Profiling of Mature Arabidopsis Trichomes Reveals That NOECK Encodes the MIXTA-Like Transcriptional Regulator MYB106[END_REF], thus suggesting that endoreduplication could have a role in the regulation of secondary metabolism implicated in defense. Based on these data, in the Arabidopsis sepals, endoreduplication could have a role in regulating organ curvature, cell size, cell fate and maybe defense response pathways.

Endoreduplication in fruits and seeds

Regarding fruits and seeds, endoreduplication has also been reported in 28 species among 12 families of angiosperms for fruits and in 47 species among 27 families for seeds (D 'Amato, 1984;[START_REF] Barow | Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size[END_REF][START_REF] Bourdon | Endoreduplication and Growth of Fleshy Fruits[END_REF]. For example, in the capsule of the Foxtail Lily (Eremurus robustus), in cells from the pericarp tissue developing from the ovary wall, polyploid nuclei have been observed as well as polytene chromosome structures associated with increased nuclear size (Figure 8A and 8B, H Lauber 1946). Endoreduplicating cells have also been reported in fleshy fruit species such as cucurbits [START_REF] Coombe | The Development of Fleshy Fruits[END_REF][START_REF] Barow | Endopolyploidy in seed plants[END_REF] or Tomato (Joubès and Chevalier 2000). In several fruits, endoreduplication is negatively correlated with the duration of the growth period. Indeed, it was observed that fruits developing in less than 13 weeks undergo 3 to 8 rounds of endoreduplication whereas endoreduplication is absent in most of the studied fruit species with longer developmental timing (Figure 8C Bourdon et al., 2010). For example, tomato developing in less than 10 weeks performs 8 endocycles whereas apple developing in a bit more than 20 weeks is only composed of non endoreduplicated cells (Figure 8C) Endoreduplication has also been reported, for more than 60 species, in the endosperm, a differentiated structure of the seed specialized in the storage and nutrition of the developing embryo. This tissue presents a large panel of ploidy levels, up to 3072C in wild cucumber (Echinocystis lobata, D 'Amato 1984). Maize is the most studied species regarding endoreduplication as a potential regulator of cell size, kernel size and starch content [START_REF] Leiva-Neto | A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm[END_REF][START_REF] Sabelli | The contribution of cell cycle regulation to endosperm development[END_REF][START_REF] Sabelli | Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm[END_REF]. Endosperm is formed after the double fertilization. First, one of the sperm nuclei fuses to the egg cell nucleus and forms the embryo. The second sperm nucleus fuses to the polar nucleus of the binucleate central cell resulting in the formation of a 3C cell, the primary endosperm. The endosperm then develops by several divisions and undergo up to several rounds of endoreduplication until full development [START_REF] Sabelli | The contribution of cell cycle regulation to endosperm development[END_REF]. The onset of endoreduplication in the Maize endosperm is associated with the production of the zein protein that accumulates as a storage of nitrogen, and with a reduction of chromatin condensation [START_REF] Sabelli | The contribution of cell cycle regulation to endosperm development[END_REF].

In Teosinte, the Maize wild relative and ancestor, the endosperm has been studied to understand, at the cellular level, the domesticated characters that permitted to obtain a larger endosperm in Maize (Figure 8D). Teosinte produces highly endoreduplicated nuclei in the endosperm, till 96C (Figure 8E), whereas Maize is producing nuclei with one more endoreduplication round (192C, Figure 8F, [START_REF] Vilhar | Cytometrical Evidence That the Loss of Seed Weight in theminiature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm[END_REF]. However, the major difference in endoreduplication between Teosinte and Maize is the spatial distribution of highly endoreduplicated nuclei in the endosperm. In Maize, endoreduplicated nuclei are localized in the upper part of the endosperm while in Teosinte endoreduplicated nuclei are localized in all the central endosperm (Figure 8F). It was hypothesized that this modified distribution of highly endoreduplicated cells might have been selected by domestication and allowed a more efficient starch accumulation in Maize than in Teosinte (Figure 8F, Dermastia et al., 2009). (E) Ploidy map of teosinte caryopsis at 11 days after pollination (F) Ploidy map of maize caryopsis at 16 days after pollination A and B adapted from [START_REF] Lauber | Untersuchungen über das Wachstum der Früchte einiger Angiospermen unter endomitotischer Polyploidisierung[END_REF]. C from Chevalier et al., 2011. D and E from Dermastia et al., 2009. F adapted from Vilhar et al., 2002. In conclusion, endoreduplication in the endosperm is frequent and studies of Maize and its ancestor Teosinte shows that this process is important for kernel development and might be linked to starch accumulation in endosperm.

In conclusion, endoreduplication seems to have different roles in different tissues and organs. High endoreduplication levels are often associated with large cell size and organ size in leaves or fruits. The analysis of mutants with reduced endoreduplication allowed to observe that some mutants are able to compensate this reduction by increasing cell number to preserve the organ size [START_REF] Massonnet | New Insights into the Control of Endoreduplication: Endoreduplication Could Be Driven by Organ Growth in Arabidopsis Leaves1[W][END_REF]. Endoreduplication might have a role in enhancing/potentiating cell expansion rather than driving it because cell expansion can occur at a similar level even in absence of increased ploidy levels. Cell differentiation is also another event in which endoreduplication has a role because important impairment of endoreduplication limits differentiation in trichomes and nodules, for example. Finally, endoreduplication could have a role differentiation and regulation of metabolism in Maize kernel for starch and protein accumulation both occurring in large endoreduplicated cells.

Endoreduplication in the model of study during the PhD: the tomato fruit pericarp

Because Tomato fruit develops rapidly and can present large variation in size and form, it is used as a model of study to understand which roles endoreduplication and its spatial distribution could have for fleshy fruit development. Hereafter I will describe what is known about where and when endoreduplication is occurring during Tomato fruit development.

Endoreduplication during Tomato fruit development

In Tomato (Solanum lycopersicum), the fruit is a berry formed by the differentiation of the ovary. It is composed of a pericarp that derives from the walls of, at least, two fused carpels, of a jelly locular tissue surrounding the seeds and of a placenta maintaining seeds attached to a central columella (Figure 9A). At the end of Tomato fruit development, the pericarp, the locular tissue and columella to a lesser extend are composed of cells that have undergone several rounds of endoreduplication (Figure 9B, [START_REF] Joubès | Molecular and Biochemical Characterization of the Involvement of Cyclin-Dependent Kinase A during the Early Development of Tomato Fruit[END_REF] and the ploidy levels varies between cultivars suggesting that the number of endoreduplication rounds is influenced by the genotype [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF].

In Tomato, endoreduplication already occurs in the ovary wall during flower development [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF][START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF] that starts with the formation of the floral meristem and finishes with the anthesis that corresponds to the pollination and fertilization of the fully functional flower.

Before fertilization, the ovary wall grows mainly by cell division and is composed of seven to nine cell layers between stage 11 and 16 (4-and 8 mm Gillaspy et al., 1993 Figures modified andadapted from Joubès et al., 1999 (B) and Renaudin et al., 2017 (C and D). (E) from Jean-Philippe Mauxion (personal communication).

At stage 18, before anthesis, 43% of the nuclei are endoreduplicated, with around 2-3% of 8C and almost 40% of 4C, considered as a first round of endoreduplication due to the absence of division structures, such as condensed chromosome of metaphase to telophase and division planes [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. After fertilization and during the whole fruit development, the pericarp volume represents nearly half of total fruit volume and the growth rate of the fruit is the same as the growth rate of the pericarp [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. This is indicating that the pericarp is mainly representing the tomato fruit growth, thus the next paragraph will mostly be focusing on the cellular events and endoreduplication dynamic during pericarp development.

The ovary wall and the derived structure, the pericarp, is divided in three areas (Figure 9C and 9D). First, an outer-epidermis contains three cell layers named E1 (most outer layer), E2 and E3 (the two-sub outer-epidermal layers, Figure 9C). Second, an inner part, formed after anthesis is composed of multiple layers grouped under the name of M' layers. M' cells mostly originate from layers E2 and E3 after periclinal cell divisions (Figure 9D). Third, four cell layers formed before anthesis are composing the M layer (Figure 9C). The M and M' layers form the mesocarp. Finally, the inner part of the pericarp in contact with the locular tissue is corresponding to two cell layers with the inner-epidermis layer I1 and its sub-epidermal layer I2 (Figure 9C).

Fruit growth and development is divided in three phases according to [START_REF] Gillaspy | Fruits: A Developmental Perspective[END_REF]. The first phase corresponds to the fruit set during which internal signals, corresponding to hormonal signal of gibberellins, auxins and downstream regulators, are inducing growth [START_REF] Ariizumi | Genes that influence yield in tomato[END_REF]. This phase starts if the fertilization succeeded and lasts for nearly 20h (Figure 9D; [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. The following phase lasts between 7 to 10 days depending on the genotype [START_REF] Gillaspy | Fruits: A Developmental Perspective[END_REF]. During this period, the pericarp is characterized by the occurrence of high cell division activity. In the pericarp, cell divisions start the first day after anthesis and are mostly localized in the three epidermal layers (E1, E2 and E3, Figure 10A and 10B) and two inner-epidermal layers (I1 and I2, Figure 10E and10F). The mitotic activity in the outer epidermis generates new cell layers whose number varies in function of the genotype (Figure 10G). However, during this period, cell expansion occurs already and increases greatly the volume of the M layer cells by 100 times compared to anthesis (Figure 10C). The increase in M layers volume participates to half the increase of the fruit volume [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF][START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. Concomitantly, ploidy levels increase in the pericarp from a mean C value (sum of the C value weighted by their frequency) of 3.2 to 7 from anthesis to 10 days post fertilization. This change in mean ploidy levels is due to the disappearance of 2C nuclei and the appearance of new classes of ploidy (8C and 16C, Figure 10H) from anthesis to 10DPA (Days Post-Anthesis, [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. At the end of this phase, in the pericarp, only the outer epidermis continues to divide. Bourdon et al., 2011 (I) and Renaudin et al., 2017 (B to G) The last phase starts when the fruit growth is mostly due to cell expansion in the mesocarp. Pericarp cell number does not change much from this stage. At the end of this phase the pericarp volume is ten times more important than at the start of this phase. This growth is mainly driven by cell expansion of the M' cells participating to 50% of the increase in size [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. This cell expansion occurs simultaneously with a drastic increase in ploidy levels due to the occurrence of 2 to 3 rounds of endoreduplication (64C, 128C and 256C, Figure 10H). At the end of this phase corresponding to the mature green stage the fruit has reached its final size and the mean ploidy levels increased to 24 in cherry tomato and can be higher, up to 70, in other genotypes [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. After this period of growth, the fruit will start the ripening phase.

Ploidy distribution in the pericarp and potential role of endoreduplication

At mature green stage, the establishment of a ploidy map in the pericarp allowed to get a better view of the distribution of the ploidy levels inside this tissue (Figure 10I; [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. At this stage, the epidermal layers E1 and E2 are the only ones without endoreduplicated cells. In E1, 2C cells represent more than 60% of the cells whereas the other 40% are 4C cells (Figure 10I). In E2, 2C cells represent less than 10% of the cell population, 4C cells represent nearly 40% and the other 50% are half 8C and half 16C (Figure 10I). The last outer-epidermal layer, E3, is composed of 4C to 32C cells and 8C cells represent the most represented group. In the inner epidermis, I1 is composed of 8C to 32C cells and I2 of 16C to 256C cells (Figure 10I). The absence of 2C and 4C cells in the inner epidermis indicates that dividing activity has disappeared from these layers. Most of the cells in I1 layer are 16C whereas ploidy levels are nearly equally represented in I2 layer. Finally, M and M' layers are composed of the highest ploidy levels. In M', ploidy levels range from 8C to 256C but 8C cells are found only in the first M' layer and 16C only in the two first M' layers (Figure 10I). In addition, the measurement confirmed a positive correlation between ploidy and cell size [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Although this correlation is not perfect. It suggests that endoreduplication is more likely favoring the potential of growth by increasing the range of possible growth rather than driving cell growth [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF][START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF]. Endoreduplication supporting rapid growth is also suggested by the presence of 8C nuclei at anthesis and rapid increase of 8C cells during the first days of development [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. 8C cells before anthesis are hypothesized to be in the M layer because M layer attains higher ploidy levels at the end of the development [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. The role of endoreduplication in Tomato fruit thus seems to be an enhancer of cell expansion since it is present before cell expansion starts, at anthesis, and both cell expansion and ploidy rise rapidly after anthesis in the mesocarp.

Finally, we have seen that before anthesis some cells in the ovary are already endoreduplicated that might be supporting the rapid cell expansion of mesocarp cells.

In Tomato fruits, central mesocarp cells are large and highly endoreduplicated cells, are also accumulating a lot of soluble sugars and organic acids in vacuoles (Beauvoit et al.,2014), thus endoreduplication could have a role in regulating metabolism of mesocarp cells to increase cell expansion through sugar accumulation in vacuoles.

Genetic regulation of endoreduplication in Plants Dynamic of CDK-CYC complexes during cell cycle progression

The endocycle is a modified version of the classical mitotic cell cycle. The cell cycle in Eukaryotes is divided in four phases, Gap1 (G1), S (Synthesis), Gap2 (G2) and M (Mitosis) phases (Figure 1A). During the G1 phase the cell decides to progress into the cell cycle or not in function of environmental and internal signals. Favorable signals, such as temperature or water and hormones or sucrose allow the synthesis of the mRNA and proteins necessary for the subsequent cell cycle phases. After the G1 checkpoint and requirements are passed, the cell enters the S phase, during which the whole genome is duplicated through the production of an extra copy of each chromosome pair, thus passing from 2n to 4n (n is referring to the number of each chromosome). The S phase is followed by the G2 phase during which a verification of DNA integrity after duplication, named G2/M checkpoint, occurs and necessary components for the next step are produced. The M phase is the last step of the cell cycle and corresponds to the equivalent segregation of the chromosomes. The M phase ends with the cytokinesis leading to the formation of two daughter cells that will contain the same genetic information. The endocycle is only composed of a succession of G and S phases without M phase, resulting in an increase in cell ploidy levels and production of polytene chromosomes (Figure 1B, [START_REF] Vlieghe | Physiological Relevance and Molecular Control of the Endocycle in Plants[END_REF].

During the cell cycle, the correct progression of each phase requires the action of heterodimeric complexes composed of a catalytic subunit referred to as CYCLIN-DEPENDENT KINASE (CDK) and a regulatory subunit, the CYCLIN (CYC). In CDK-CYC complexes, the CDK assures phosphorylation activities whereas the CYC defines the substrate specificity and the localization within the cell [START_REF] Inzé | Cell Cycle Regulation in Plant Development[END_REF]. The high number of CDKs and CYCs proteins and the diversity of CDK-CYC complexes are crucial for the molecular regulation of the cell cycle and the endocycle. In Arabidopsis, the CDKs belong to a conserved family of serine/threonine protein kinases containing 12 members classified from A to F and CDKA and CDKB have been implicated to direct the regulation of the cell cycle [START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF] while CDKD and CDKF are CDK-activating kinases regulating small RNA processes and help to preserve mitotic activity in Arabidopsis [START_REF] Hajheidari | CDKF;1 and CDKD Protein Kinases Regulate Phosphorylation of Serine Residues in the C-Terminal Domain of Arabidopsis RNA Polymerase II[END_REF][START_REF] Takatsuka | Cyclin-dependent kinaseactivating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana[END_REF] and CDKG are regulating mRNA splicing [START_REF] Zheng | CDKG1 protein kinase is essential for synapsis and male meiosis at high ambient temperature in Arabidopsis thaliana[END_REF]. In Arabidopsis, the CYC family is a large family of around 30 members categorized in four sub-families, CYCLINS A, B, D and H, generally involved in G1/S and S phase of the cell cycle [START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF]) but the role of each cyclin has not clearly been established. Some have been associated to mitosis regulation such as CYCB1;1, CYCB1;2, CYCA2;3, CYCA3;1 and CYCD3;1 [START_REF] Veylder | Molecular control and function of endoreplication in development and physiology[END_REF][START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF] or to meiosis regulation such as for example CYCA1;2, CYCA2;1, CYCA3;2 and CYCB3;1 [START_REF] Bulankova | Identification of Arabidopsis Meiotic Cyclins Reveals Functional Diversification among Plant Cyclin Genes[END_REF].

CDK-CYC complexes have an essential role in the control and the maintenance of the cell cycle preventing endocycle. Consequently, the repression of their function in the cell is essential for the shift from the cell cycle to the endocycle [START_REF] Edgar | Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth[END_REF].

Transition from the cell cycle to the endocycle

The regulation of the CDK-CYC activities is done through transcriptional regulation, phosphorylation, the binding of protein inhibitors and proteolysis [START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF][START_REF] Edgar | Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth[END_REF]. In plants, the main studies about the regulation of endoreduplication have been done on Arabidopsis trichomes and roots and have identified several regulating actors. Some information regarding the molecular regulation of endoreduplication is also available for other plants such as Tomato and Medicago. Here, I will present the main molecular regulators of the endocycle starting with the transition from cell cycle to endocycle, then the maintenance of the endocycle and to finish the exit from the endocycle.

To undergo endoreduplication, the cells need to exit the mitotic cycle. This transition is permitted by a reduction of the CDK-CYC complexes activities.

Regulation of CDK-CYC complexes by proteolysis

The degradation of the CYC by the proteasome is a first level of control of the CDK-CYC complex. This degradation is done through the action of the Ubiquitin-Proteasome system (UPS). The UPS is composed of three enzymes, the Ubiquitin-activating enzyme E1 that activates ubiquitin to be transferred to the Ubiquitin-conjugating E2 to then bind with the Ubiquitin Ligase E3 that will finally transfer the ubiquitin to target the protein to the proteasome. The ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) is an E3-ubiquitin ligase complex that recruits E2 and an activator protein responsible for substrate specificity. Examples of these activators are the CELL CYCLE SWITCH 52 (CCS52) proteins (APC/C CCS52 abbreviation represents an APC/C complex loaded with a CCS52 protein) involved in cell cycle and endocycle regulation. Three CCS52 proteins have been identified in Arabidopsis, CCS52A1, CCS52A2 and CCS52B (Fülop et al., 2005[START_REF] Lima M Def | Genomic evolution and complexity of the Anaphase-promoting Complex (APC) in land plants[END_REF] and so far only CCS52A1 and CCS52B have been identified in Tomato [START_REF] Mathieu-Rivet | Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato[END_REF]. CCS52A are able to induce endoreduplication by targeting the mitotic CYCLINs, CYCB1, CYCB2 and CYCA2, for degradation by the UPS, as described in several plants such as Medicago, Tomato or Arabidopsis and different cell types as for example the trichomes of Arabidopsis, expanding root cells or the pericarp of tomato [START_REF] Cebolla | The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants[END_REF], De Veylder et al., 2011[START_REF] Baloban | Complementary and dose-dependent action of AtCCS52A isoforms in endoreduplication and plant size control[END_REF], Mathieu-Rivet et al., 2011). In Arabidopsis, loss of function of AtCCS52A1 results in reduced ploidy levels and accumulation of mitotic CYCLINs in non-dividing cells such as the trichomes and root hair cells. Overexpression of CCS52A in Medicago nodules (MtCCS52A) and in Tomato (SlCCS52A) results in increased ploidy levels and cell size, reduction of cell number and reduced organ size compared to wild type [START_REF] Larson-Rabin | FZR2/CCS52A1 Expression Is a Determinant of Endoreduplication and Cell Expansion in Arabidopsis[END_REF][START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF], Mathieu-Rivet et al., 2011). AtCCS52A1 activity may be inhibited by the activity of the Arabidopsis ULTRAVIOLET-B INSENSITIVE4 (UVI4) after forming a complex with the UBIQUITIN-SPECIFIC PROTEASE14 (UBP14) that is inhibiting endoreduplication by stabilizing the mitotic CDK/CYC complex [START_REF] Heyman | Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 Maintains Cell Division Activity by Temporal Inhibition of the Anaphase-Promoting Complex/Cyclosome[END_REF][START_REF] Xu | UBIQUITIN-SPECIFIC PROTEASE14 Interacts with ULTRAVIOLET-B INSENSITIVE4 to Regulate Endoreduplication and Cell and Organ Growth in Arabidopsis[END_REF]. Loss of function plants Atuvi4 and Atubp14 present similar phenotypes corresponding to reduced meristem size, increased ploidy levels in roots, reduced root length and increased ploidy levels in trichomes [START_REF] Heyman | Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 Maintains Cell Division Activity by Temporal Inhibition of the Anaphase-Promoting Complex/Cyclosome[END_REF][START_REF] Xu | UBIQUITIN-SPECIFIC PROTEASE14 Interacts with ULTRAVIOLET-B INSENSITIVE4 to Regulate Endoreduplication and Cell and Organ Growth in Arabidopsis[END_REF]. The exact mechanism of how UBP14 and UVI4 are inhibiting the APC/C activity is still unknown.

In mature leaves, AtCCS52A2 is supporting endoreduplication onset since ccs52a2 plants have reduced ploidy levels [START_REF] Lammens | Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset[END_REF]. However, the targets of CCS52A2 have not been described. In the root, CCS52A2 is not implicated in the transition into the endocycle and seems to be involved in the inhibition of cell division to assure the maintenance of the root quiescent center, a structure of mitotically inactive cells, which role is to maintain the stem cell fate of the surrounding cells (Verstraelen et al., 2009). Finally, among the three CCS52 proteins, CCS52B seems to be implicated in the regulation of cell division by promoting the degradation of cyclins just before cytokinesis allowing reentering a new cell cycle [START_REF] Yang | Cell Cycle Control by Nuclear Sequestration of CDC20 and CDH1 mRNA in Plant Stem Cells[END_REF].

Regulation of CDK-CYC by phosphorylation

A second level of control of the mitotic CDK-CYC complexes is phosphorylation. WEE1, a protein kinase implicated in the phosphorylation of the mitotic CDK/CYC at the G2/M checkpoint, favoring the transition from the cell cycle to the endocycle. In Tomato, SlWEE1 phosphorylates the CDKA of the mitotic CDK/CYC complexes after the S-phase [START_REF] Gonzalez | The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit[END_REF]. This regulation is important for Tomato plant development and its impairment results in overall plant and fruit size reduction as well as cell size and ploidy level reduction [START_REF] Gonzalez | The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit[END_REF]. However, AtWEE1-deficient plants grow normally under optimal conditions [START_REF] Schutter | Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA Integrity Checkpoint[END_REF]. In Arabidopsis, AtWEE1 is rather implicated in more specific responses, such as response to both abiotic and biotic stresses, including DNA damage or nematode infection [START_REF] Cabral | The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls[END_REF]. In late meristem identity 1 (lmi1) mutant plants, a transcription factor from the homeodomain zipper class I family (HD-ZIP) is mutated and plants are affected in leaf growth with failure to form serration. lmi1 plants also have cells with reduced endoreduplication and reduced expression of cell cycle related genes including WEE1 (Vuolo et al., 2019). Expression of genes favoring endoreduplication under the control of LMI1 promoter, such as CCS52A or WEE1, was sufficient to rescue the phenotype of lmi1. Moreover, LMI1 is able to induce WEE1 expression by binding the promoter region of WEE1 (Vuolo et al., 2019). These findings might be indicating that AtWEE1 also plays a role in leaf morphology in Arabidopsis through LMI1 regulation (Vuolo et al., 2019).

Binding of Cyclin kinase inhibitor proteins

Cyclin Kinase Inhibitors (CKI) mediate another control of the CDK-CYC activity by interacting with the CDKs thus inhibiting their ATP binding activity (Russo et al, 1998). So far, two families of CKI have been described in plants: the INTERACTOR/INHIBITOR OF CDK/KIP-RELATED PROTEINs (ICK/KRPs), homolog of the KIP proteins in Mammals (Torres [START_REF] Acosta | Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors[END_REF], that are involved in regulating the cell cycle (Nakayama et al., 1999), and the plant specific SIAMESE-RELATED PROTEINs (SMRs) [START_REF] Kumar | Why do plants need so many cyclin-dependent kinase inhibitors?[END_REF] involved in regulating G2 checkpoint. Both KRP and SMR are sharing a Cterminal motif that allows binding to cyclins [START_REF] Churchman | SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana[END_REF] and recently Nterminal motifs have been identified as CDK binding site and as potential phosphorylation site both in SIM and SMR1 [START_REF] Kumar | Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE[END_REF][START_REF] Dubois | SIAMESE-RELATED1 Is Regulated Posttranslationally and Participates in Repression of Leaf Growth under Moderate Drought[END_REF] KRP protein family is composed by 7 members in Arabidopsis and a kinematic analysis of cell division and cell expansion in the triple mutant krp4/6/7 revealed that KRPs' might be more associated to the regulation of the rate of cell cycle and endocycle than in the regulation of the transition between these processes (Sizani et al., 2019, see Chapter 1.II.3 Maintenance and ending of the endocycle). The SMR protein family is composed of 14 members in Arabidopsis. The first described member of the SMR family is SIM, which when mutated leads to the formation of multicellular and clusters of trichomes while in the wild type trichomes are large endoreduplicated cells distant from other trichomes. On the other hand, SIM overexpressing lines have a severe dwarfism phenotype with larger leaf epidermal cells than WT plants. sim plants also have reduced levels of endoreduplication in leaves, hypocotyl and trichomes compared to WT [START_REF] Walker | SIAMESE, a gene controlling the endoreduplication cell cycle in Arabidopsis thaliana trichomes[END_REF] while SIM overexpression resulted in increased of ploidy levels [START_REF] Churchman | SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana[END_REF]. Thus, SIM seems to promote endoreduplication and might be acting by inhibiting the cell cycle [START_REF] Churchman | SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana[END_REF][START_REF] Van Leene | Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana[END_REF][START_REF] Kumar | Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE[END_REF] through the CDKA-CYCD complex, responsible for the G1/S transition, (Gutierrez;[START_REF] Gutierrez | Coupling cell proliferation and development in plants[END_REF] or CDKB complexes activities, necessary for the G2/M transition (Boudolf et al., 2004).

Atsmr1 loss of function plants display reduced ploidy levels and cell sizes in leaves and sepals but not in trichomes where SIM is expressed, protecting against reduction of ploidy levels [START_REF] Roeder | Variability in the Control of Cell Division Underlies Sepal Epidermal Patterning in Arabidopsis thaliana[END_REF]. SMR1 is also named LGO (LOSS OF GIANT CELLS FROM ORGANS). Overexpression of LGO under the control of a sepal epidermis specific promoter triggers the production of sepals covered with endoreduplicated giant cells [START_REF] Schwarz | Transcriptomic Effects of the Cell Cycle Regulator LGO in Arabidopsis Sepals[END_REF]. Interestingly, LGO's effect on the entry into endocycles is dosage-dependent [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF]. AtSMR5 and AtSMR7 can also induce endoreduplication when overexpressed and are implicated in DNA damage response [START_REF] Yi | The Arabidopsis SIAMESE-RELATED Cyclin-Dependent Kinase Inhibitors SMR5 and SMR7 Regulate the DNA Damage Checkpoint in Response to Reactive Oxygen Species[END_REF].

Transcriptional regulation of CCS52

Lastly, the transition from the cell cycle to the endocycle is also transcriptionally regulated mainly through the control of CCS52 genes expression.

E2F proteins form a family of transcription factors with six members in Arabidopsis named E2Fa to E2Ff and are implicated in the regulation of cell cycle progression [START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF]). E2F family is divided in two groups with typical E2Fs, E2Fa to E2Fc, and atypical E2F proteins, E2Fd to E2Ff, also named DEL1 to DEL3, with DEL referring to DP-E2F-like [START_REF] Ramirez-Parra | E2F-DP Transcription Factors[END_REF]. Typical E2F proteins require heterodimerization with DP proteins to assure their function [START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF]) while atypical do not need dimerization with DP to regulate gene expression [START_REF] Lammens | Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset[END_REF]. E2Fa, E2Fb and E2Fc can be bound by the RETINOBLASTOMA RELATED 1 (RBR1) protein that modulates E2F activities. When RBR1 binds to E2Fa, the expression of both CCS52A1 and CCS52A2 is repressed thus inhibiting the entry into the endocycle [START_REF] Magyar | Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes[END_REF]. The atypical E2F protein, AtDEL1, can bind to the promoter of AtCCS52A2 and inhibit its expression resulting in the entry into the endocycle [START_REF] Lammens | Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset[END_REF]. AtDEL1 interacts with a mediator subunit, AtMED16, to inhibit AtCCS52A2 expression [START_REF] Liu | The Histone H3K4 Demethylase JMJ16 Represses Leaf Senescence in Arabidopsis[END_REF]. The role of mediator proteins is to be a bridge between transcription factors and the RNA polymerase II (RNA pol II) and work as a coactivator of RNA-pol II to bind DNA and perform transcription. AtMED16 does not only interact with AtDEL1 to regulate AtCCS52A2 expression but is also able to bind AtCCS52A1 promoter and repress its expression. However, the partner of AtMED16 in AtCCS52A1 regulation is unknown [START_REF] Liu | The Histone H3K4 Demethylase JMJ16 Represses Leaf Senescence in Arabidopsis[END_REF].

Interestingly, AtDEL1 is regulated in a light dependent manner in the hypocotyl by a chain of regulation that involves transcriptional regulation by other E2F proteins, AtE2Fb and AtE2Fcand by proteolysis by the action of the A.thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) an E3 ubiquitin ligase, that targets E2Fb to degradation in dark condition [START_REF] Gendreau | Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl[END_REF], Berckmans et al., 2011). In the dark, AtCOP1 triggers AtE2Fb degradation, thus permitting AtE2Fc to repress AtDEL1 transcription and consequently stimulate endoreduplication. In light conditions, AtE2Fb activates AtDEL1 that can thus limit endoreduplication [START_REF] Berckmans | Light-Dependent Regulation of DEL1 Is Determined by the Antagonistic Action of E2Fb and E2Fc[END_REF].

Transcriptional regulation is also important for the entry into endoreduplication in the transition zone in Arabidopsis roots. Cells in the transition zone, as described before, have a low auxin content that is due to regulation of auxin polar transport and the synthesis of auxin degradation proteins upon positive regulation of cytokinin (Di [START_REF] Mambro | Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root[END_REF]. In this transition zone, AtCCS52A1 gene expression is positively regulated by a type-B ARABIDOPSIS RESPONSE REGULATOR (ARR) transcription factor, AtARR2 that regulates transcription in response to cytokinin [START_REF] Takahashi | Cytokinins Control Endocycle Onset by Promoting the Expression of an APC/C Activator in Arabidopsis Roots[END_REF].

In Tomato, down-regulation of the transcriptional regulator SlIAA17 leads to an increase of the size and ploidy levels of the fruit pericarp cells resulting in increased final size of the fruit [START_REF] Su | The Auxin Sl-IAA17 Transcriptional Repressor Controls Fruit Size Via the Regulation of Endoreduplication-Related Cell Expansion[END_REF]. Aux/IAA proteins form heterodimers with AUXIN RESPONSE FACTOR (ARF) proteins that are transcription factors regulating auxin dependent genes expression. When auxin content is low, then Aux/IAA binds to ARF thus blocking auxin responsive gene expression. In high auxin concentration, ARF are released from Aux/IAA binding allowing gene expression and Aux/IAA are bound by an auxin inducible E3 ubiquitin ligase triggering Aux/IAA proteolysis (Li et al., 2016). The increase in mean ploidy level in pericarp cells in the SlIAA17 mutant indicates that auxin signaling could limit the entry into the endocycle in Tomato fruit but the molecular mechanisms by which SlIAA17 is regulating endoreduplication are unknown. [START_REF] Su | The Auxin Sl-IAA17 Transcriptional Repressor Controls Fruit Size Via the Regulation of Endoreduplication-Related Cell Expansion[END_REF].

In conclusion, altogether these data show that the exit from the cell cycle and entry into the endocycle is induced by several mechanisms of repression targeting CDK-CYC complexes. The degradation of the CDK-CYC complexes is also tightly regulated by transcriptional control of CCS52 genes and seems to be subjected to hormonal regulation as well.

Maintenance and ending of the endocycle.

The molecular regulation of the transition from the cell cycle to the endocycle has been studied more extensively than the progression and maintenance of this process.

The main hypothesis regarding the regulation of the maintenance of the endocycle is a cyclic regulation inducing oscillations of the CDK-CYC activity during the G/S phases (Verkest et al., 2003[START_REF] Lee | Endoreplication: polyploidy with purpose[END_REF][START_REF] Breuer | Endocycling in the path of plant development[END_REF]. These oscillations in CDK-CYC activity are hypothesized to be controlled by KRP proteins [START_REF] Veylder | Molecular control and function of endoreplication in development and physiology[END_REF]. A strong overexpression of AtKRP2 inhibits both cell division and endoreduplication while weaker overexpression results in an inhibition of mitosis only suggesting [START_REF] Verkest | The Cyclin-Dependent Kinase Inhibitor KRP2 Controls the Onset of the Endoreduplication Cycle during Arabidopsis Leaf Development through Inhibition of Mitotic CDKA;1 Kinase Complexes[END_REF]. Single krp knockout in Arabidopsis results in plants with no difference compared to wild type probably due to redundancy in the function of the KRP family members [START_REF] Cheng | Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis[END_REF][START_REF] Sizani | Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana[END_REF]. However, multiple krp knockout plants, krp4/6/7 and krp1/2/3/4/7, present an increased seed size, cotyledon size and leaf size due to an increase in the number of cells with higher number of endocycles [START_REF] Sizani | Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana[END_REF][START_REF] Cheng | Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis[END_REF]. CDK activity is upregulated in these multiple krp mutants while total amount of KRP transcripts in krp4/6/7 is around 35% less than WT indicating the importance of KRP in the cell cycle process [START_REF] Sizani | Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana[END_REF]. Taken together, these data led to hypothesize that KRP are global moderators of both cell cycle and endocycle process but not actors in the transition between cell division and endoreduplication [START_REF] Sizani | Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana[END_REF]. In Tomato, fruit specific overexpression of SlKRP1 results in a reduction of ploidy levels in the fruit showing that also in Tomato, KRP inhibits endoreduplication.

Overexpression of CYCA2 represses the progression of the endocycle by inhibiting the pre-replication complex during the S phase [START_REF] Yu | The Tobacco A-Type Cyclin, Nicta;CYCA3;2, at the Nexus of Cell Division and Differentiation[END_REF][START_REF] Imai | The A-Type Cyclin CYCA2;3 Is a Key Regulator of Ploidy Levels in Arabidopsis Endoreduplication[END_REF]. Interestingly, CYCA2 is repressed by ILP1 (INCREASE LEVEL OF POLYPLOIDY 1) a protein hypothesized to be implicated in the maintenance of the endocycle [START_REF] Yoshizumi | INCREASED LEVEL OF POLYPLOIDY1, a Conserved Repressor of CYCLINA2 Transcription, Controls Endoreduplication in Arabidopsis[END_REF]. Ilp1 plants have reduced ploidy levels whereas ILP1 overexpression induces more endoreduplication than in wild-type plants [START_REF] Yoshizumi | INCREASED LEVEL OF POLYPLOIDY1, a Conserved Repressor of CYCLINA2 Transcription, Controls Endoreduplication in Arabidopsis[END_REF]. In Arabidopsis trichomes, the end of the endoreduplication is controlled by the transcriptional regulation of CCS52A1 by a GT-2 like plant specific trihelix transcription factor (AtGTL1). Indeed, the loss of function of Atgtl1 results in the overexpression of AtCCS52A1 and AtCCS52A2, in increase ploidy levels [START_REF] Breuer | The Trihelix Transcription Factor GTL1 Regulates Ploidy-Dependent Cell Growth in the Arabidopsis Trichome[END_REF] and trichomes sizes while a trichome specific overexpression results in the absence of trichomes [START_REF] Breuer | Transcriptional repression of the APC/C activator CCS52A1 promotes active termination of cell growth[END_REF]. AtGTL1 is expressed in the late trichome development steps and limits trichome growth and endoreduplication [START_REF] Breuer | The Trihelix Transcription Factor GTL1 Regulates Ploidy-Dependent Cell Growth in the Arabidopsis Trichome[END_REF]2012). However, the endoreduplication and growth finally stops in the Atgtli1 mutant suggesting that other regulation steps assure the arrest of growth and endoreduplication processes.

In conclusion, the molecular regulation of endoreduplication is occurring at the gene expression and protein activity level from the onset to the end of the endocycle. CCS52 and SMR proteins seem to be central in the regulation of the entry into the endocycle, while KRP and transcriptional regulation of cyclins direct the maintenance of the endocycle. Finally, in trichome, the endocycle end is controlled by the transcriptional repression of CCS52 thus favoring the stabilization of CDK-CYC complexes that repress endocycle progression. As revealed by the previous results I presented, most of the molecular control of endoreduplication has been studied in Arabidopsis and is not yet well established in other plants.

Genetic manipulation of endoreduplication in other plant than Arabidopsis

In this part, I will present additional examples of genetic modifications of endoreduplication in different plants and tissues than Arabidopsis and their consequences on growth or other processes helping to better understand endoreduplication function.

In Maize endosperm, a decrease of ploidy levels obtained with a dominant negative approach overexpressing a CDKA unable to perform kinase activity, was not associated to any change in cell size or kernel size but to slightly decreasing zein accumulation and gene expression related to starch biosynthesis [START_REF] Leiva-Neto | A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm[END_REF]. In Maize endosperm, starch accumulation takes place in the endoreduplicated cells, but in transgenic plants with a reduced CDKA activity and lower endoreduplication levels, the accumulation of starch is unmodified. This might be due to the plasticity of the tissues and suggesting the possibility to uncouple endoreduplication and the sugar metabolism of endoreduplicated cells in the Maize endosperm [START_REF] Sabelli | The contribution of cell cycle regulation to endosperm development[END_REF]. Moreover, the increase of endoreduplication in Maize endosperm, by the overexpression of an antisense RNA targeting RBR1 (RBR1 AS ) does lead to change in a decrease in cell size and nuclear size as well as an increase in cell number suggesting a role of RBR1 in coupling cell size and ploidy level. But it does not lead to changes in kernel size and no changes of seed weight or Zein protein content despite a decrease in non zein protein content per DNA quantity [START_REF] Sabelli | Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm[END_REF] possibly suggesting that extra DNA copies are not transcribed. Moreover, increase in Programmed Cell Death (PCD) activity has been reported at 16DAP stage of the kernel development, when PCD is normally starting [START_REF] Sabelli | Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm[END_REF]. However, this observation could not be linked either to a regulatory role of RBR1 on this process or an endoreduplication related regulation. These observations led to conclude that CDKA and RBR1 can influence endoreduplication in Maize endosperm without influencing the phenotype of the endosperm suggesting the absence of link between endoreduplication and cell size or the increase sugar metabolism activity in endosperm cells. However, the authors hypothesized that the absence of phenotype in the RBR1 AS plant could be due to compensatory mechanisms by regulation of gene expression. It has been observed in RBR1AS plant that despite higher ploidy levels measured, less protein is produced compared to the DNA quantity that is found. This is suggesting that newly formed alleles are less transcribed and since they found DNA METHYLTRANSFERASE (MET1) to be up regulated in RBR1AS plant, it has been proposed that change in chromatin conformation could be responsible of reduction of transcription per allele [START_REF] Sabelli | Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm[END_REF].

In Tomato, the downregulation of Tomato ortholog of Arabidopsis regulators of endoreduplication, SlWEE1 and SlCCS52A1 results in overall plant and fruit size reduction with reduced endoreduplication levels [START_REF] Gonzalez | The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit[END_REF], Mathieu-Rivet et al., 2010). Moreover, in these plants, fruit pericarp is thinner due to the presence of smaller cells in the fruit of these plants [START_REF] Gonzalez | The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit[END_REF], Mathieu-Rivet et al., 2010). SlKRP1 overexpression (SlKRP1-OE) specifically in fruit expanding cells resulted in reduction of endoreduplication in the pericarp. No fruit size nor cell size nor metabolic differences have been observed probably because compensation processes took place [START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF]. The absence of changes of cell size shows that reduction of ploidy levels can be dissociated from cell expansion in the pericarp. In addition, endoreduplication is reduced in SlKRP1-OE plants but not abolished. This might be indicating that endoreduplication is necessary for fruit growth and that the number of remaining endoreduplication rounds might be sufficient to maintain fruit size, cell size and metabolism.

The genetic manipulation of endoreduplication in Maize and Tomato allowed to hypothesize that this process could be necessary to at least endosperm development and fruit pericarp development. Finally, in Tomato, SlKRP1 overexpression is able to uncouple endoreduplication and cell size because although endoreduplication is reduced the mean cell size seems unaffected [START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF]. This could be signifying that other regulation is affecting cell size not under the control of endoreduplication or that the reduced ploidy levels are sufficient to attain the necessary cell size for normal development of the pericarp.

Endoreduplication takes place in several tissues of multiple plant species and is often associated with specialized role of cells or enlarge cell size. These effects of endoreduplication, at cellular and organ level, could result from modifications of transcription upon genome duplication. In the following section, I will focus on what is known about the effect of endoreduplication on changes in gene expression.

Effect of endoreduplication on the regulation of transcription Global Effect on gene expression in polyploid plants

Endoreduplication corresponds to the duplication of chromatins in some cells of an organ whereas polyploidy is the heritable condition of possessing more than two complete sets of chromosomes. Polyploidy can occur after a cross between two plants of the same species (Autopolyploidy) or of two close species (Allopolyploidy). In both cases, the number of chromosomes in the cells is changed compared to the parentals and the polyploid plant contains additional copies of each chromosome pairs while endoreduplicated cells contain only one pair of each chromosome.

In general, in a diploid plant and non-polyploid cell, we can consider that each allele of a gene is equally contributing to the absolute quantity of the corresponding transcribed mRNA [START_REF] Coate | Quantifying Whole Transcriptome Size, a Prerequisite for Understanding Transcriptome Evolution Across Species: An Example from a Plant Allopolyploid[END_REF]. In autopolyploid and allopolyploid organisms each chromosome is duplicated while in endoreduplicated cells, each chromatid is duplicated thus increasing the number of alleles for each gene. This changes in chromosome or chromatid number raises several questions regarding transcription regulation. Are polyploid or endoreduplicated cells maintaining the same absolute mRNA quantity than diploid cells? Is each allele maintaining a diploid expression so is there a doubling of expression of genes by doubling allele number? For now, these questions have been more covered in the case of polyploidy (for review see [START_REF] Doyle | Polyploidy, the Nucleotype, and Novelty: The Impact of Genome Doubling on the Biology of the Cell[END_REF] than in endoreduplication and no clear answers can be found to these questions.

The allopolyploid Glycine dolichocarpa, and its diploid parents, Glycine tomentella and Glycine syndetika, are wild relative family of soybean have been studied for global expression level, transcriptome size, as a function of genome size. It was shown that transcriptome size of the tetraploid plant was 1.4x bigger than the parents but proportionally to the number of alleles, it was smaller by ~0.7x, thus corresponding to a 30% decrease of expression per diploid genome compared to the diploid parents [START_REF] Coate | Quantifying Whole Transcriptome Size, a Prerequisite for Understanding Transcriptome Evolution Across Species: An Example from a Plant Allopolyploid[END_REF]. The same range of results have been found in synthetic tetraploids of Arabidopsis with an increase of the transcriptome size of 1.4x to 1.7x in tetraploid compared to the diploid and a ≈30% decrease of the expression by diploid genome [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF], MJ.Song et al., 2020). In Arabidopsis female gametophyte the non endoreduplicated egg cells and central cells with around 10 to 30% of decrease of expression per diploid genome (Q. [START_REF] Song | Single-cell RNA-seq analysis reveals ploidy-dependent and cell-specific transcriptome changes in Arabidopsis female gametophytes[END_REF]. In polyploid plants, global transcriptome analyses thus showed that both total RNA and mRNA are increased with polyploidization and this increase tends to not follow a 1:1 ratio (doubling of expression, [START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF]. The mechanisms controlling the dosage of gene expression relative to genome duplication during polyploidization are not known.

Global effect on gene expression of endoreduplication

In the case of endoreduplication, the genome is duplicated through the increase of the number of chromatids per chromosome and thus the copy number of genes is increased triggering an increased transcriptional potential. Observations tend to favor the hypothesis that in the case of cell differentiation, endoreduplication is supporting an increase in metabolic activity by increasing transcription through duplication of DNA (Sabelli et al., 2013 Frawley and[START_REF] Orr-Weaver | When bigger is better: the role of polyploidy in organogenesis[END_REF] and that expression is likely to increase with ploidy levels but not follow a linear proportionality [START_REF] Doyle | Polyploidy, the Nucleotype, and Novelty: The Impact of Genome Doubling on the Biology of the Cell[END_REF]. However, even if endoreduplication has been studied for many years and often hypothesized to increase gene expression, to date only few studies have quantified how much gene expression is changing with this process.

In tomato pericarp endoreduplicated cells, transcription of a subset of genes has been positively correlated with endoreduplication. The quantification of gene expression of rRNA 5.8S,SlWEE1,SlCCS52A,and RNA pol II subunit (SlRPB) have shown that the relative expression of these genes increases when endoreduplication increases [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF]. Moreover, for RNA pol II, the protein quantity of both active and inactive forms, quantified by immunofluorescence, doubled with the doubling of the ploidy level [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF]. This increase in transcription and translation of the RNA pol II led to hypothesize that overall transcription and translation activities could be doubled with endoreduplication in Tomato pericarp cells since RNA pol II is responsible for transcription activities [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF]. Based on the results described above showing a potential doubling of expression with genome duplication [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF], a ploidy-based transcriptome of Tomato pericarp nuclei has been obtained using fluorescence assisted nucleus sorting and RNA sequencing in order to tackle the question of the proportional change of expression following increase of ploidy [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. In the study, the four main endoreduplication levels observed in 30DPA fully grown fruit before ripening, namely 4C, 8C, 16C and 32C, were studied [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. This study shows that the gene expression is globally increased between ploidy levels and this increase corresponds to a factor 2 between consecutives ploidy levels for most of the genes. This global shift of expression was found for 4694 genes having a proportionally increased expression between the four endoreduplication levels studied. Additional experiments implying other species or tissues could help understanding if the shift is a universal process or if it is specific to tomato pericarp.

A single cell RNA seq study of the whole Arabidopsis root was done to study developmental trajectories of cell lineages since whole roots contain mix of cells at varying developmental stages (Jean-Baptiste et al, 2019). The analysis of single cell transcriptomic data allows to place cells in "pseudotime" order along a trajectory that describes their maturity. This positioning could be done for root hair cells that at mature stage are endoreduplicated. The analysis showed that the median total mRNA quantity profile is decreasing while pseudotime thus differentiation and endoreduplication increases. On the other hand, this median profile increases with pseudotime, thus ploidy, for hair cell specific genes (Figure 11A). Thus, differentiating root hair cells have increased the expression of root hair cell specific genes and reduced expression of unspecific genes. In addition, the authors showed that transcription rate of mRNA specific for hair cells is increasing across pseudotime thus when ploidy increases (Figure 11B, Jean-Baptiste et al., 2019).

In conclusion, endoreduplication seems to be able to induce global shift of expression in Tomato and in Arabidopsis root hair cells. Endoreduplication could also be related to an increase in transcription rate. In both cases endoreduplication could influence mRNA transcription. However, mostly mRNA have been assayed while it is representing only 5% of total RNAs. It could be interesting to know much about the dynamic of other RNA pools (rRNA, tRNA and non-coding RNA) that could vary since 5.8S rRNA transcript abundance in Tomato pericarp is following the increase in ploidy levels [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF].

Ploidy specific gene expression in endoreduplicated cells

Endoreduplication generally occurs in specialized cells and can induce ploidy specific gene expression. Hereafter, I review examples in which increase expression in specialized genes might be necessary for differentiation and seem to be related to endoreduplication.

In Maize endosperm, endoreduplication occurs in the central part of the tissue. Endoreduplicated cells represent most of the endosperm cells at 10-12 DAP, corresponding to almost mid-time of endosperm development (Schweizer et al., 1995;[START_REF] Leiva-Neto | A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm[END_REF]. At this timepoint, more than 30% of the total transcripts correspond to around 100 of the most expressed genes, the ZEIN genes encoding the most important storage proteins in kernel (Figure 12A Chen et al., 2014). This observation suggests that this increase of ZEIN genes expression is related to the increase in ploidy levels [START_REF] Leiva-Neto | A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm[END_REF]. Moreover, ZEIN transcripts, represent almost 60% of all the transcripts in the endosperm between 10 to 20DAP, when ploidy levels are increasing and endoreduplicated cells represent most of the endosperm cells [START_REF] Chen | Dynamic Transcriptome Landscape of Maize Embryo and Endosperm Development[END_REF]. Between 10 to 20DAP, STARCH SYNTHASE genes are upregulated, reinforcing the potential the role of endoreduplicated cells in storage function [START_REF] Chen | Dynamic Transcriptome Landscape of Maize Embryo and Endosperm Development[END_REF][START_REF] Rymen | Cold Nights Impair Leaf Growth and Cell Cycle Progression in Maize through Transcriptional Changes of Cell Cycle Genes[END_REF].

Upregulation of genes implicated in specific metabolism in endoreduplicated cells is not occurring only in Maize endosperm but also in the Arabidopsis root. In this organ, ploidy specific transcriptome was obtained by RNA-seq and the data analysis has identified genes differentially expressed as a function of ploidy [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. Enrichment analysis of gene ontology terms related to biological processes on the identified differentially expressed genes revealed that genes highly expressed in endoreduplicated cells are related to chromosome organization and DNA methylation pattern, two processes known to influence gene expression (see further paragraphs, [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF] that might be responsible of a global shift of expression. The analysis of this root ploidy specific transcriptome has also shown specific ploidy expression of hormones and stress responsive genes. For example, genes related to auxin response or to cold response are more expressed in 2C cells while ethylene response and water deprivation response genes are more expressed in 8C and 16C cells (Figure 12B, [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. These findings suggest that tolerance to stresses and hormonal signaling might be regulated and programmed by endoreduplication during root development. Moreover, expression of cell wall synthesis genes have also been found to be specifically regulated by ploidy levels, thus endoreduplication might be an answer to high demand of cell wall materials for growing cells and in response to stresses [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF], Bhosale et al., 2019). In the transcriptome of microdissected areas of different part of Medicago nodules, characterized by composition of cells with different ploidy levels, it was shown that low endoreduplicated cells express genes related to positive regulation of endoreduplication and DNA replication [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF] and highly endoreduplicated cells express nodule-specific cysteine rich peptides (NCR) that are peptides implicated in nodule development and necessary for symbiotic cell differentiation [START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using lasercapture microdissection coupled to RNA sequencing[END_REF][START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF][START_REF] Montiel | Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides[END_REF]. Highly endoreduplicated cells express NCR and genes encoding ions, sugars and amino acids transporters [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. Mesembryanthemum crystallinum, a salt tolerant halophyte plant, possesses modified trichomes called epidermal bladder cells (EBC) that are specialized in salt accumulation and are highly endoreduplicated [START_REF] Barkla | Making Epidermal Bladder Cells Bigger: Developmental-and Salinity-Induced Endopolyploidy in a Model Halophyte[END_REF]. In these large cells, present on the whole areal tissues, developmentally programmed endoreduplication can reach levels of 128C to 1024C. In flower buds, ploidy levels can increase up to 65536C after salt stress, corresponding to 2 to 6 supplementary rounds of endoreduplication [START_REF] Barkla | Making Epidermal Bladder Cells Bigger: Developmental-and Salinity-Induced Endopolyploidy in a Model Halophyte[END_REF]. Compared to non endoreduplicated roots cells, EBC express genes related to endoreduplication progression and DNA replication such as F box-like17 (FBL17), Mini Chromosome Maintenance (MCM) and E2F target gene1 [START_REF] Barkla | Making Epidermal Bladder Cells Bigger: Developmental-and Salinity-Induced Endopolyploidy in a Model Halophyte[END_REF] and maintain low levels of transcripts of inhibitors of endoreduplication such as DEL1, UVI4 and TEOSINTE BRANCHED1/CYCLOIDEA/PCF 15 (TCP15) [START_REF] Barkla | Making Epidermal Bladder Cells Bigger: Developmental-and Salinity-Induced Endopolyploidy in a Model Halophyte[END_REF]. After salt stress, upregulation of genes implicated in ion transport, ABA signaling, inositol and proline metabolism and conversion from C3 to Crassulacean acid metabolism (CAM, [START_REF] Oh | Cell type-specific responses to salinity -the epidermal bladder cell transcriptome of Mesembryanthemum crystallinum[END_REF] was observed. This expression reprogramming is supporting the hypothesis that endoreduplication could be a general way to promote metabolism in response to environmental stress leading to production of mitigating stress metabolites. It has been shown that the increase in ploidy is often accompanied by the production of secondary metabolites in the case of stresses such as phenylpropanoid and antioxidant (phenolic compounds and NADPH, Scholes and Paige 2015) in response to stresses. Finally, in tomato pericarp, ploidy specific expression analysis showed that endoreduplicated cells in fully grown fruit, but not yet mature express genes related to carbohydrate metabolism, transport, and cell organization (Figure 12C, [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF].

A comparison of the transcriptome of nuclei of different ploidy level from tomato pericarp of fully grown fruit, revealed a total of 1211 differentially expressed genes have been found [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Those genes have expression profiles that are related to specific ploidy levels possibly revealing different cellular functions in the pericarp. In cells with low ploidy levels, differentially expressed genes are related to photosynthesis and flavonoids pathways whereas highly polyploid cells are expressing genes related to carbohydrate metabolism. These findings suggest that endoreduplication in tomato pericarp could be an important factor of cell differentiation (Figure 12C, [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. It has been shown that pericarp cell expansion is driven by the expansion of vacuole due to accumulation of sugars and organic acids that led to influx of water [START_REF] Beauvoit | Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[END_REF]. This could be suggesting that endoreduplication in highly endoreduplicated cells is accompanying cell expansion by increasing expression of genes implicated in carbohydrate metabolism.

In conclusion, it appears that through the doubling of all alleles at each cycle endoreduplication potentialize the increase of gene expression, allowing a global shift of transcriptome and the increase of expression of specific genes related to the differentiation of the cell: storage, symbiotic development, sugar metabolism, response to stress nodule peptides. Gene expression regulation mechanisms.

Endoreduplication is associated with a general increase of gene expression in some plants and with specific patterns of expression as observed in Arabidopsis, Medicago and Tomato [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF][START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF][START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF].

In Eukaryotes, genes expression can be regulated at the nucleus level by a specific spatial positioning of the chromosomes within the nucleus [START_REF] Guerreiro | Spatial chromatin organization and gene regulation at the nuclear lamina[END_REF], and at the chromatin level with changes of chromatin condensation [START_REF] Liu | The Histone H3K4 Demethylase JMJ16 Represses Leaf Senescence in Arabidopsis[END_REF], due to histones modification or nucleotide methylation [START_REF] Finnegan | DNA methylation, a key regulator of plant development and other processes[END_REF] impacting the binding of the final regulatory elements, the transcription factors.

During endoreduplication, chromosomes morphology is modified because the number of chromatids on a single chromosome increase from two to several hundred [START_REF] Nagl | DNA endoreduplication and polyteny understood as evolutionary strategies[END_REF]. This modification of chromosome morphology might be influencing their positioning in the nucleus, the condensation of the chromatin (methylation pattern, histone composition and modification) and at the end, gene expression. The following sections will give insight into these questioning by reviewing the different mechanisms known to influence gene expression.

Regulation of gene expression by chromosome positioning in the nucleus.

Chromosomes are separated DNA molecules carrying the genetic information and having a specific organization/localization/positioning in the nucleus called chromosomes territories. Chromosome territories have been shown in animals to be involved in the regulation of gene expression (Figure 13A; [START_REF] Orsztynowicz | Changes in chromosome territory position within the nucleus reflect alternations in gene expression related to embryonic lineage specification[END_REF]. In the next paragraphs, I will describe how chromosome positioning within the nucleus can influence gene expression and when possible link this information to endoreduplication.

In non-plant systems, genes localized at the nuclear periphery tend to exhibit low transcription while those localized more internally in the nucleus often have higher expression [START_REF] Van Steensel | Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression[END_REF]. By using a CRISPR based tool named CRSPR-GO (referring to CRISPR-genome organization) allowing to move parts of chromosomes to specific nuclear compartments, the expression of genes adjacent to the Cas9 targeted locus was repressed when the targeted region was the nuclear periphery (Figure 13 D, [START_REF] Wang | CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization[END_REF]. However, expression of chromatin located at the nuclear periphery is not always repressed. When Chromatin interacts with nuclear envelop proteins, transcription is in general repressed [START_REF] Shaklai | Gene silencing at the nuclear periphery[END_REF] while localization to nuclear pores through interaction with nucleoporin is associated with active transcription [START_REF] Liang | Functional interactions between nucleoporins and chromatin[END_REF] In general, chromosomes position within nuclei is not random, but some parts of the chromosomes can move outside of the chromosome territories to intersect with other chromosomes territories [START_REF] Cremer | Chromosome Territories[END_REF]. These interphase areas are named topologically associating domains (TADs) and are visible by super-resolution microscopy and sequencing technologies (Figure 14A, [START_REF] Szabo | TADs are 3D structural units of higher-order chromosome organization in Drosophila[END_REF][START_REF] Szabo | Principles of genome folding into topologically associating domains[END_REF]. TAD's have been shown to be related to transcriptional co-regulation of homologous or heterologous chromosome portions in Animals and Drosophila [START_REF] Pombo | Three-dimensional genome architecture: players and mechanisms[END_REF]Dillon 2015, Szabo et al., 2019). In plants, most studies about chromosome sub-organization within the nuclei were done on the model plant Arabidopsis and few studies exist in other plant species such as Maize, Wheat or Tomato. In Arabidopsis, chromosome territories exist in interphase nuclei but, contrary to Animals, they adopt a random positioning except for the ones carrying the Nucleolus Organizer region (NOR, [START_REF] Berr | Interphase Chromosome Arrangement in Arabidopsis thaliana Is Similar in Differentiated and Meristematic Tissues and Shows a Transient Mirror Symmetry After Nuclear Division[END_REF]. Telomeres are generally found around the NOR and telomeres of sister chromatids can rarely be separated (Figure 14B, Schubert et al., 2012). As in Animal, specific chromosome positioning can influence gene expression in Arabidopsis. For example, chromosomes regions associated with the nucleolus in Arabidopsis, named nucleolus associated domains (NAD) are preferentially non-expressed regions including telomeres, rDNA sequences, transposable elements and inactive coding sequences [START_REF] Pontvianne | Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome[END_REF]. Also, similarly to animals, the Arabidopsis nuclear periphery has been identified as a domain of transcriptional repression enriched in transposable elements and non-transcribed genes (Bi et al., 2015). At the nuclear periphery two specific chromatin regions can be found: first, the heterochromatin that corresponds to condensed chromatin generally transcriptionally inactive and second, the chromocenters that are domains of dense heterochromatin composed of centromeres repeats and pericentromeric areas of the chromosome [START_REF] Simon | Structure and Function of Centromeric and Pericentromeric Heterochromatin in Arabidopsis thaliana[END_REF]. On the other hand, it has been shown that light can induce the repositioning of light inducible genes loci to the nuclear periphery and that this repositioning is associated with transcription activation [START_REF] Feng | Light-regulated gene repositioning in Arabidopsis[END_REF]. In addition, artificial relocation of transgene to nuclear pores through the binding to nucleoporin increases its transcription [START_REF] Smith | Marker gene tethering by nucleoporins affects gene expression in plants[END_REF].

In Arabidopsis, the distribution and interaction of different chromosomal territories was analyzed in cells of different ploidy levels (Figure14 B and C, Schubert et al., 2012;Schubert et al, 2014). In highly endoreduplicated cells, chromosome territories are still present and similar chromosome territories are found as in diploid cells (Figure 14C, Kato andLam 2003, Berr and[START_REF] Berr | Interphase Chromosome Arrangement in Arabidopsis thaliana Is Similar in Differentiated and Meristematic Tissues and Shows a Transient Mirror Symmetry After Nuclear Division[END_REF]. Euchromatin that corresponds to the decondensed and transcriptionally active form of the chromosome is forming loops that anchor at the chromocenters [START_REF] Berr | Interphase Chromosome Arrangement in Arabidopsis thaliana Is Similar in Differentiated and Meristematic Tissues and Shows a Transient Mirror Symmetry After Nuclear Division[END_REF]Schubert 2007, Poulet et al., 2017). In endoreduplicated nuclei, chromocenters are internalized and less heterochromatic structures are observed indicating a decondensation of the chromatin [START_REF] Poulet | The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants[END_REF]. This might be indicating that specific structural arrangements exist in endoreduplicated cells and could be involved in the regulation of gene expression.

Hi-C seq is a method based on sequencing technologies in which 3D chromatin conformation is frozen due to chemical crosslinking of DNA, then DNA is fractionated and sequenced and this allows-to-allows identification of TAD. In Arabidopsis, the analysis of chromosome conformation by sequencing has shown that TADs are not commonly observed and that chromosomes interact with each other around the heterochromatin domains (pericentromeric and telomeres, [START_REF] Grob | Hi-C Analysis in Arabidopsis Identifies the KNOT, a Structure with Similarities to the flamenco Locus of Drosophila[END_REF][START_REF] Feng | Light-regulated gene repositioning in Arabidopsis[END_REF]. The absence of TAD could be related to the small genome size and low chromosome number of Arabidopsis [START_REF] Feng | Light-regulated gene repositioning in Arabidopsis[END_REF]. Arabidopsis could thus be an exception because TAD-like structures were observed in other plants with larger genomes and chromosome numbers such as Maize (n=10), Tomato (n=12), Sorghum (n=10), Foxtail millet (n=18), and Rice (n=12) [START_REF] Doğan | Three-dimensional chromatin packing and positioning of plant genomes[END_REF]. The borders of TAD-like structures are enriched in expressed genes, decondensed chromatin regions, and are depleted of transposable elements in Maize, Tomato, Sorghum, Foxtail millet and Rice [START_REF] Dong | 3D Chromatin Architecture of Large Plant Genomes Determined by Local A/B Compartments[END_REF]. Autotetraploid Arabidopsis plants have decreased intra-chromosomal interactions and higher interchromosomal interactions compared to diploid Arabidopsis [START_REF] Zhang | The effects of Arabidopsis genome duplication on the chromatin organization and transcriptional regulation[END_REF] and among the 743 differentially expressed genes between these plants (436 upregulated and 307 downregulated) 72% are located in the differential chromatin interaction domains [START_REF] Zhang | The effects of Arabidopsis genome duplication on the chromatin organization and transcriptional regulation[END_REF]. However, there is no data available about chromatin interactions in relation to endoreduplicated chromosomes.

In Maize and rice endosperm cells that are endoreduplicated, heterochromatin regions are less compact compared to mesophyll cells, mostly non endoreduplicated [START_REF] Rymen | Cold Nights Impair Leaf Growth and Cell Cycle Progression in Maize through Transcriptional Changes of Cell Cycle Genes[END_REF][START_REF] Dong | Tissue-specific Hi-C analyses of rice, foxtail millet and maize suggest non-canonical function of plant chromatin domains[END_REF]. This decreased compaction thus triggers an increased accessibility of the chromatin, reducing the local repression of some chromosomes. In addition, it has been shown that there are less repressive compartments in endoreduplicated tissues, endosperm, for example, compared to less endoreduplicated tissues such as mesophyll cells [START_REF] Dong | Tissue-specific Hi-C analyses of rice, foxtail millet and maize suggest non-canonical function of plant chromatin domains[END_REF]. Moreover, TADlike borders, enriched in groups of genes with an increased expression, are larger in endoreduplicated tissues (endosperm) [START_REF] Dong | Tissue-specific Hi-C analyses of rice, foxtail millet and maize suggest non-canonical function of plant chromatin domains[END_REF]. These observations suggest that TAD-like structures could be implicated in regulating general gene expression in plants by modifying accessibility of their borders for transcription and that endoreduplicated tissues tend to have a chromatin organization favoring transcription [START_REF] Dong | Tissue-specific Hi-C analyses of rice, foxtail millet and maize suggest non-canonical function of plant chromatin domains[END_REF].

Transcriptional regulation and chromatin accessibility

In Eukaryote nuclei, DNA is not free but is compacted by proteins under the form of chromatin (Figure 14A). The conformation of the chromatin is a key element for the regulation of gene expression through DNA accessibility [START_REF] Zentner | Regulation of nucleosome dynamics by histone modifications[END_REF]. DNA packaging in the nucleus is highly regulated and mediated by the nucleosomes composed of a protein octamer of core histones. The octamer is composed of a heterotetramer of HISTONE3 (H3) and HISTONE4 (H4) and a heterotetramer of HISTONE2A (H2A) and HISTONE2B (H2B) (Figure 15A; [START_REF] Klug | A low resolution structure for the histone core of the nucleosome[END_REF]. Posttranslational chemical modifications of the core histones include acetylation, phosphorylation and methylation and are sufficient to affect DNA compaction and thus accessibility of the transcriptional machinery. (Figure 15B, Zentner and Henikoff 2013). Several euchromatin related histone modifications have been found and are classified according to their repressing or activating role in transcription. Classical repressive marks are H3K27me3 and H2Aub1 (ubiquitination) and activating marks are H3 and H4 acetylation, H3K4me3, H3K36me3, H2Bub1 and H2AS9ph (phosphorylation, [START_REF] Zhao | Histone modifications and their regulatory roles in plant development and environmental memory[END_REF]. In Arabidopsis, for example, it has been shown that tri-methylation on lysine 27 of H3 (H3K27me3) is necessary to repress genes implicated in totipotency to maintain cell identity of guard cells forming the stomata and H3K4me3 is necessary to activate genes of aging in leaves [START_REF] Lee | Cell-type-specific transcriptome and histone modification dynamics during cellular reprogramming in the Arabidopsis stomatal lineage[END_REF], Liu et al., 2019). In Arabidopsis endoreduplicated cells, microscopic observations of chromatin linked fluorescent markers has shown that chromatin is occupying more space in the nuclei than in non-endoreduplicated ones, suggesting a chromatin decondensation (Kato andLam 2003, Poulet et al., 2017). Interestingly, this observed chromatin decondensation is coherent with data obtained by ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) a method using the capacity of a transposase enzyme to insert sequencing adapter between nucleosomes where chromatin is accessible giving then the sequence of accessible chromatin regions for transcriptional machinery (Buenrostro et al., 2015). Indeed, ploidy specific ATAC-seq analysis of Medicago nodules and single-cell ATAC seq (scATAC-seq) analysis of Arabidopsis roots have shown that chromatin in endoreduplicated cells is more accessible than in non endoreduplicated cells [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF][START_REF] Dorrity | The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution[END_REF].

Interestingly, in Medicago nodules, genes with a differential chromatin accessibility between different ploidy levels have been found differentially expressed in relation with these ploidy levels [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. For example, s genes found highly expressed in 16C and 32C nuclei were found in regions with higher accessibility in the ATAC-seq data [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. The observed increase of chromatin accessibility found by ATAC-seq in endoreduplicated cells might be indicating a reduction of nucleosome activity thus DNA packaging potentially through histone modifications [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. However, this correlation between dynamic accessibility and gene expression in single cells data from Arabidopsis root was poor [START_REF] Dorrity | The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution[END_REF].

As mentioned previously, DNA packaging is regulated by the nucleosomes and their post-translational modifications. Interestingly, some links between endoreduplication and histone modification have been described. For example, the mutants of CAF-1 (CHROMATIN ASSEMBLY FACTOR-1), encoding a protein responsible of the deposition of new H3 and H4 that are acetylated and HUB1 (HISTONE MONO-UBIQUITINATION 1), encoding a protein responsible of monoubiquitylation of H2B [START_REF] Desvoyes | Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development[END_REF] contain cells entering earlier in the endocycle (Ramirez-Para et al., 2007[START_REF] Fleury | The Arabidopsis thaliana Homolog of Yeast BRE1 Has a Function in Cell Cycle Regulation during Early Leaf and Root Growth[END_REF] suggesting that histone marks could influence gene expression controlling the transition to endoreduplication. In Arabidopsis, the overexpression of KRP5, an inhibitor of the CDK-CYC activity, results in an increase in endoreduplication. Interestingly, KRP5 protein is localizing to the chromocenter [START_REF] Jégu | Multiple functions of Kiprelated protein5 connect endoreduplication and cell elongation[END_REF] and the overexpression phenotype is amplified in the mutant atxr5/atxr6 (arabidopsis trithorax-related) mutated in genes implicated in the maintenance of repressive H3 methylation in heterochromatin [START_REF] Jégu | Multiple functions of Kiprelated protein5 connect endoreduplication and cell elongation[END_REF]. This finding is suggesting that KRP5 which regulates endoreduplication could also influence the maintenance of heterochromatin through ATRX proteins regulation. Although not always linked to endoreduplication, the co-occurrence of chromatin remodeling with endoreduplication in several mutants could indicate a necessary role of histone modification and chromatin remodeling during this process. A direct link between differential histone mark modifications and ploidy specific gene expression has been shown in only one study in Medicago nodules [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. ChIP-qPCR analysis (Chromatin Immuno-Precipitation) that uses the specificity of antibodies to recognize and pull down a specific protein with its associated DNA was used to specifically study modified histones H3K27me3 or H3K9ac at specific loci. This analysis has shown that NCR genes have a reduction in the repressive histone mark H3K27me3, an increase in the active histone mark H3K9ac in 8C, 16C and 32C nuclei compared to low ploidy level 4C and this is associated with an increase of expression of these genes [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. In Tomato, no link has been established between modification of histone marks and endoreduplication.

In conclusion, in the nodules of Medicago, specific histone modifications seem to change as a function of ploidy levels favoring ploidy specific gene expression or repression. However, more research needs to be done to study the link between ploidy specific gene expression and chromatin conformation regulation through specific histone marks.

Role of transcription factors in the ploidy specific regulation of expression

Increase of chromatin accessibility means that gene promoters become accessible for transcription factors to induce transcription. However, in an endoreduplication context, no study describes the involvement of transcription factors in the regulation of ploidy specific gene expression.

DNA methylation and gene expression regulation

Direct chemical modifications of DNA, such as the methylation of the pyrimidine base cytosine, can also influence gene expression. In coding genes, cytosine methylation occurs generally in the promoter sequences and also in coding sequence. Cytosine methylation of promoters generally has a repressing effect on gene expression [START_REF] Berdasco | Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells[END_REF].

In Arabidopsis, DNA methylation occurs in all organs [START_REF] Bartels | Dynamic DNA Methylation in Plant Growth and Development[END_REF] and is implicated in multiple developmental programs such as gametogenesis, fruit development and ripening or response to stresses [START_REF] Bartels | Dynamic DNA Methylation in Plant Growth and Development[END_REF]. In Tomato fruits, ripening genes promoters are actively demethylated to induce their expression at the onset of ripening (Lang et al., 2017, Tang et al., 2020).

Endosperms of Arabidopsis, Maize and Rice, that contain high proportion of endoreduplicated cells, have DNA globally hypomethylated and in Rice endosperm more pronounce hypomethylation is observed on specific genes related to starch accumulation [START_REF] Lauria | Extensive Maternal DNA Hypomethylation in the Endosperm of Zea mays[END_REF][START_REF] Hsieh | Genome-Wide Demethylation of Arabidopsis Endosperm[END_REF][START_REF] Zemach | Local DNA hypomethylation activates genes in rice endosperm[END_REF]. In Arabidopsis, plants cultivated in short day conditions and transferred to long day show a floral transition that induces an increase in ploidy levels in the leaves (Del Prete et al., 2019). This increase endoreduplication is accompanied by differential gene expression of numerous genes among which three are involved in DNA methylation modification, METHYLTRANSFERASE 1, CHROMOMETHYLASE 3, DEMETER-LIKE 2 (MET1, CMT3 and DML2, respectively, Del Prete et al., 2019). MET1 and CMT3 methylate cytosine whereas DML2 is removing methylation. The increase of expression of MET1, CMT3 and DML2 could indicate a global reprogramming of the chromosome methylation pattern in relation with endoreduplication and flowering. Lastly, in Medicago nodules, DNA demethylation by MtDME is necessary for the differentiation of symbiotic nitrogen fixing cells [START_REF] Satgé | Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula[END_REF]. Moreover, genes with high expression in endoreduplicated nuclei (32C) are significantly hypomethylated [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF]. These data are indicating that endoreduplication is associated with a specific hypomethylation of nodule related genes thus favoring their expression.

In conclusion, we have seen several examples in which endoreduplication is associated with modifications of nuclear genome organization, chromatin compaction or DNA methylation associated with changes in gene expression. Thus, endoreduplication can be implicated in defining ploidy specific pattern of gene expression supporting differentiation of cells through the action of different regulatory mechanisms. For now, the relation between transcriptional regulation and organization of endoreduplicated chromosomes is not clearly established.

Presentation of my PhD work

Tomato fruit growth is driven by two main processes, cell division and cell expansion. Cell expansion often occurs simultaneously to endoreduplication. Endoreduplication is a process resulting from the capacity of cells to modify their classical cell cycle into a partial cell cycle, or endocycle, where successive rounds of full genome replication occur in the absence of mitosis. In plants, endoreduplication is found in numerous organs and cell types and particularly in organs or tissues of agronomical high value, such as the fleshy fruit pericarp of tomato presenting high ploidy levels (Joubès and Chevalier, 2000).

As described in the introduction, endoreduplication is part of the normal development in several plants and multiple organs and is regulated through a complex genetic network organized around the CDK-CYC activity. The work of the hosting team showed that the pericarp is composed of a heterogeneous population of cells in term of ploidy levels and cell size. Endoreduplication in the pericarp is not randomly nor evenly distributed but rather distributed in a gradient toward the center of the pericarp. In addition, endoreduplication is able to induce a global shift of gene expression and specific set of genes are differentially regulated as a function of ploidy levels in mature green fruits.

Before the start of my PhD, only one developmental stage at the end of the growth of the fruit preceding ripening had been studied for ploidy distribution and ploidydependent gene expression. However, there was no information about the dynamic distribution of endoreduplication during early fruit development in the pericarp nor ploidy specific gene expression. My PhD work is part of a larger project aiming at understanding: i) how endoreduplication onset and progression are molecularly controlled during fruit growth, ii) why a heterogeneous population of cells co-exists within the pericarp tissue and iii) what is the functional role of endoreduplication during fruit growth. The first objective of my PhD was thus to obtain the distribution of ploidy levels during early pericarp development, while endoreduplication is taking place, and the PhD is integrating in a wider project with a long-term objective of defining potential rules of endoreduplication establishment and progression in the pericarp. The second objective was to identify, during early pericarp development, which genes are specifically expressed in relation to ploidy levels with the long-term goal of selecting genes that could be used as markers of ploidy levels. This was achieved by determining the ploidy distribution in situ and obtaining ploidy-specific transcriptional profiles during early pericarp development. First, I first determined the ploidy and cell size distribution in the pericarp of growing fruits by using the fluorescent in situ hybridization (FISH) method to image chromatin specific loci within the pericarp. By combining the average ploidy distribution and individual cell size during early fruit development, I have been able to study the relationships existing between ploidy levels, cell position and area and gained insight into tissue formation in relation to genome duplication. Second, to obtain a genome-wide ploidy-specific transcriptional information during fruit development, nuclei from pericarp of fruits harvested at three growth stages (6-, 9-and 12-days post anthesis), were sorted by Fluorescence Activated Cell Sorting according to their ploidy levels and used for RNA sequencing. The analysis of this datasets allowed identifying differentially expressed genes as a function of endoreduplication and developmental timing. The ploidy distribution and transcriptomic data obtained, and their integration are presented in chapter 2.

To obtain these data, I had to implement, adapt to our model or develop several methodologies. The FisH method was implemented and optimized for the quantification of ploidy levels in situ in the pericarp tissue. In parallel, I worked on the development of the CRISPR-imaging technique for ploidy level quantification. I also optimized the protocol for RNA-seq on sorted nuclei of different ploidy levels by the use of spike-in to normalize gene expression. The different steps of this technical optimization are presented in chapter 1.

Finally, the data obtained during my work are discussed in the discussion section.

Technological development for ploidy determination in situ and for ploidy dependent transcriptomic Introduction

In plants, the final size of an organ is determined by cell division that sets the number of cells and cell expansion often associated to endoreduplication increasing nuclei DNA content. In the course of Tomato fruit development, cells from the fleshy part of the fruit, the pericarp, undergo endoreduplication and can attain high ploidy levels, up to 512C [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. Ploidy levels are often correlated with cell sizes [START_REF] Melaragno | Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis[END_REF][START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF][START_REF] Robinson | Ploidy and Size at Multiple Scales in the Arabidopsis Sepal[END_REF] and endoreduplication in plants is involved in differentiation of cells in various organs but the control of the establishment and the molecular regulation of this process are still not fully known.

Ploidy levels are classically determined by flow cytometry. To do so, plant tissues are chopped in a nucleus extraction solution allowing to obtain nuclei with maintained structures and DNA is stained with a fluorescent dye such as 4',6-diamidino-2phénylindole (DAPI) in order to estimate DNA content [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF]. The flow cytometry approach is precise, reliable and allows to assay many samples, but this quantification of DNA content is representing the landscape of ploidy levels of the chopped tissue and not the spatial distribution of ploidy levels in the studied tissue. Thus, cytogenetic approaches are necessary to determine this spatial distribution. For example, fluorometry which is based on the use of fluorescent dyes binding to DNA, generally similar to the ones used in flow cytometry, has been used to determine ploidy levels by quantifying the total fluorescence in the nucleus after imaging with an epifluorescence microscope [START_REF] Melaragno | Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis[END_REF]. This method allows the determination of ploidy levels in young tissues with small cells and nuclei, for example in Arabidopsis hypocotyls, but is not appropriate for large cells and nuclei such as the ones found in tomato pericarp with high ploidy levels [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. A second method to quantify ploidy in situ corresponds to Fluorescence in situ Hybridization (FisH) and consists in the hybridization to DNA of a synthetic DNA sequence with fluorescently dyed nucleotides complementary to a target sequence from the nuclear genomic DNA. With fluorescence microscopy, dots in the nucleus representing the hybridized loci are observed and ploidy levels are determined by counting the number of these dots for an easy quantification, the synthetic DNA sequence only targets a single locus in the genome. This method was used to map ploidy levels in the pericarp of fully grown Tomato fruit pericarps [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. This analysis showed that ploidy levels are not evenly distributed within the tissue but follow a gradient toward the center of the pericarp [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF].

The fluorometry and FisH techniques require tissue fixation, so it could be interesting to be able to quantify ploidy levels in living cells. To do so, tools based on the CRISPR/CAS9 technology have emerged for imaging chromatin in living cells. These methods called CRISPR imaging can use a deficient dCas9, mutated in the two endonuclease domains and fused with fluorescent proteins (FP) to directly visualize the targeted loci of interest (Figure 16A; [START_REF] Chen | Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System[END_REF]. Genomic loci can also be visualized indirectly by targeting sgRNA using virus RNA stem-loops called aptamers that are specifically recognized and bound by an aptamer binding protein fused with FP (Figure 16B, [START_REF] Fu | CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci[END_REF]. A similar method of sgRNA recognition is done by adding repeats of 8 nucleotide long sequences at the end of the sgRNA called PUF binding sites that are specifically recognized by the Pumilio/fem-3 mRNA binding factor (PUF) proteins (Figure 16B, [START_REF] Cheng | Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling[END_REF]. Lastly, an indirect visualization of loci can be done using a dCas9 fused with peptides specifically recognized by a small protein called single-chain variable fragments (scFV) that is an artificial fusion protein based on variable chains of antibody (Figure 16C, [START_REF] Tanenbaum | A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging[END_REF]. In this case, scFV are fused to a FP and bind the peptides tail of the dCas9 in order to visualize the DNA. These CRISPR-imaging tools have been used multiple times in animal cells to visualize specific genomic loci (for review see [START_REF] Khosravi | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres[END_REF].

In plants, CRISPR imaging using a dCas9 coupled to a FP (Figure 16A) has been used in Nicotiana benthamiana with a transient transformation approach to visualize and quantify telomeres and study their regulation and dynamism in leaf interphase nuclei [START_REF] Dreissig | Live-cell CRISPR imaging in plants reveals dynamic telomere movements[END_REF]. However, plants stably expressing this system could not be obtained so far since stable transformation of Arabidopsis with the dCas9 fused to a FP targeting telomeres did not work, probably because the dCas9 bound to repeated sequences could interfere with the cell cycle progression in the plant (Fujimoto and Matsunaga 2017). Recently, a study showed that indirect visualization using CRISPR imaging using virus aptamers (Figure 16B) was efficient in transient transformation but did not result in any stably transformed plant when targeting the telomeres and only few plants were obtained when targeting the centromeres [START_REF] Khosravi | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres[END_REF]. However, the authors reported that in the stably transformed plants targeting the centromeres, the dot like pattern observed was not as expected for interphase nuclei [START_REF] Khosravi | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres[END_REF].

Endoreduplication has been proposed to increase the metabolic activity of cells by inducing a global increase in transcription [START_REF] Lee | Endoreplication: polyploidy with purpose[END_REF]. A transcriptomic analysis of sorted nuclei of tomato pericarp cells by Fluorescent Activated Nucleus Sorting [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF] showed that endoreduplication is inducing a global shift of gene expression and that specific sets of genes, including lipid and carbohydrate metabolism genes, are differentially expressed in nuclei with different ploidy levels [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. (C) Indirect methods using modified dCas9 with peptides epitopes. A tail of peptide epitopes is added to the dCas9 that recruits simplified antibody called scFV fused to fluorescent protein that allows localization of the binding.

During my PhD, we aimed at studying how endoreduplication distribution is evolving within the pericarp during early fruit development and obtain the transcriptome of the main ploidy levels found in early developmental stages. To determine in situ ploidy levels of the cells in the pericarp I used FisH methodology and I initiated the development of a method allowing ploidy quantification in living cells based on the CRISPR-Cas9 technology. For the transcriptomic analysis, I obtained RNA sequencing data from nuclei sorted by FACS. To obtain the data presented in Chapter 2.II.2.A, I had to implement, adapt to our model or develop several methodologies. In the following paragraphs, I will present the technical and methodological optimizations that we implemented after enlightening the difficulties we tackled. These paragraphs will describe the use of a set of synthetic RNAs to properly normalize the transcriptome analyses, the improvement of the FisH methodology in tomato pericarp combined with a semi-automated image acquisition and analyses for ploidy quantification and finally the development of a method based on the CRISPR-Cas9 technology to quantify ploidy.

Results and discussion

Use of spike-in to normalize gene expression in a global shift in expression context.

In Tomato pericarp cells, a global shift of expression has been found between cells of different and increasing ploidy levels [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF], see Chapter 1.III.2: Global effect on gene expression of endoreduplication). Knowing if a shift in expression exists between different samples studied by RNA-seq is important for proper analysis and interpretation of the data. Indeed, classical RNA-seq normalization approaches (Anders and Huber, 2010; Robinson and Oshlack, 2010;Maza et al., 2013) are inadequate in this case since they are generally based on the assumption that fewer than half of the genes are down-regulated or up-regulated between two conditions, which is a priori not the case with ploidy. To solve this issue, in Pirrello et al, 2018, a mathematical equation that is using a factor representing the fold change induced by the global shift of expression, in the case of endoreduplication a factor2, was applied and allowed to perform classical differential expression analysis and estimate the fold change of each gene. Another solution consists in adding a known number of artificial sequences, or spike-ins, to the RNA samples to provide a scaling factor used for the normalization. To obtain the transcriptome of nuclei of different ploidy levels during my PhD, I thus implemented the use of spike-ins in our experimental setup.

We did three cultures of thirty Tomato plants each to obtain three biological repeats and randomly harvested nine fruits per plants. A mix of 20 fruits for 6DPA, and 10 fruits for 9-and 12DPA was used for the ploidy-based nuclei sorting. We collected100k nuclei for each ploidy level from each stage. For 6DPA, we studied 2C, 4C, 8C and 16C nuclei and for 9 and 12 DPA we studied 4C, 8C, 16C and 32C nuclei. Total RNA was extracted from a fixed number of 100k nuclei for each ploidy level because RNA quantity extracted from different ploidy levels can be variable [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. Thus, using 100k nuclei allows to compare ploidy levels. Before RNA extraction, we added to each sample a commercial solution named "ERCC RNA Spike-In Control Mixes" which is a mix of 92 RNAs from the ERCC plasmid reference library for which the sequences and concentrations are known and variable allowing to assess the sensibility of the experiment. The same quantity of spike-in was added to each sample meaning that one quantity of spike-in corresponds to 100k nuclei indifferently to the ploidy level.

After sequencing and data analysis, we detected 21 of the 92 spike-in in all samples and those were among the more abundant synthetic RNAs. The lowest quantity for a spike RNA corresponded to around 70k copies in one sample indicating that theoretically we could be able to detect an RNA even if there was only one copy per nucleus since we used 100 k nuclei for the sequencing.

As in [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF] we tested if a global shift in expression existed in the samples studied and if it was similar to the two-fold observed at 30DPA. Using the 21 spike-ins detected in all samples, we measured the fold change of the ratio spike-ins expression/ global RNA expression between two ploidy levels at 6DPA, 9DPA and 12DPA. We found that the relative quantity of spike-ins changes between ploidy levels. For the comparisons 16C/4C, the fold changes varied between 2 and 8 at 6DPA or between 1.8 and 2.3 for 16C/8C at 9DPA (Figure 17A). If a global shift in expression between two ploidy levels correlates with the quantity of DNA, a fold change of 2 would be expected between 16C and 8C and 4 between 16C and 4C. The observed values are thus close to the expected values suggesting a shift in expression near 2-fold between consecutive ploidy levels (Figure 17A). To compare expression and visualize differences between ploidy levels, we represented the log2 fold change between two ploidy levels and the mean of normalized counts per gene on a MA plot. In a situation with no shift of expression between conditions, the expression of most genes is expected to remain constant between the conditions studied shaping the plot symmetrically around a log fold change value of 0. In our experiment, the fold changes of the spike-ins were used to visualize, in the MA plot, the expected symmetry in case of a shift in expression. Between 16C and 4C at 6DPA, for example, we observed that the symmetry of the plot was located a bit higher than a log2 fold change value of 2 corresponding to the expected global shift in expression of 4 (Figure 17B). These results are supporting the hypothesis that in tomato pericarp, whatever the developmental timepoint, a global shift of expression is following ploidy level increase [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. In conclusion, the use of spike-ins in the context of a global shift of expression allowed a reliable determination of gene expression in the different ploidy level samples and stages studied in the PhD. By analyzing the data with the assumption of a global shift in expression, we found 17484, 16751 and 18596 genes expressed in at least one sample across the samples with different ploidy levels at 6, 9 and 12 DPA respectively. After removing the genes not expressed in all samples, we found 16751, 16213 and 17300 genes that were declared as being non-DE, thus having an expression that is proportional to the ploidy level at 6, 9 and 12 DPA, respectively. We found among the 18318 genes declared as non-DE across all samples, 14843 genes in common between the three stages (Figure 18A). This overlap indicates that whatever the time point the expression of a large set of genes follows the level of ploidy. This list of genes was enriched in several GO terms related to general cell function such as "RNA processing", "cell cycle" and "primary metabolic process" (Figure 18B). This is in favor of hypothesize role of endoreduplication to boost general function of the cell and support growth.

In conclusion, the ploidy transcriptome with the use of spike-ins allowed to quantify that global gene expression is doubling with endoreduplication at early developmental stages and this is equivalent to the global shift observed in the later mature green stage [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF] suggesting that in the pericarp the doubling of gene expression is due to endoreduplication and not influenced by developmental stage. In our case spike-ins have helped to determine that gene expression is increasing with ploidy levels but did not allowed to determine an absolute value of increase, doubling is a likely value and to determine the exact value more sampling will be necessary to increase statistical force.

Optimizing the quantification of ploidy in situ from sample preparation to imaging using Fluorescence in situ Hybridization a. Optimization of the FisH protocol Previously, by using Fluorescence in situ Hybridization (FisH), the ploidy distribution in Tomato pericarp of mature green fruits, fully-grown fruits but not yet ripe, has been determined [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. This FisH method is based on the use of a fluorescently dyed DNA sequence complementary to a targeted nuclear DNA sequence. In the study from [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF], two bacterial artificial chromosome (BAC) clones were used to target chromosomes 7 and 12 and their hybridization resulted in a single signal per targeted locus. The numbers of signals or dots were then counted and corresponded to the ploidy of the cell analyzed. However, the use of the BAC FisH approach can be challenging since the quality of the labelled probe can be variable. In addition, the protocol for BAC FisH even if efficient, required that the samples were processed in Eppendorf tubes and imposed a slow pipetting of fluids in the tube at each step. This pipetting was critical to not disrupt the pericarp sections of 150 µm thick, containing very large and turgid mesocarp cells making the tissue very fragile, and thus limited the number of samples and increased experiment duration.

For the PhD work, we thus decided to optimize the FisH method to decrease the duration of the experiment and increase the number of samples at once. For the quantification of ploidy in situ, we thus changed the method from BAC FisH to Oligo-FisH that uses oligonucleotides as probes instead of BAC because oligonucleotides are smaller and easily diffuse through the cell walls and membranes. We also targeted another region of the genome because each oligo probe bears only one fluorophore, so Oligo-FisH needs to be used with a repeated sequence. To do so, we chose the 5S rDNA locus that is only located on the short arm of chromosome 1 (Figure 19) in Tomato, thus a 2C cell will have two dots so dot number will double with each endocycle. In addition, samples were handle in 24 well plates allowing preparation and analysis of more samples at once and reducing experiment duration. Pericarp sections were placed just after slicing in baskets and change of bath was performed by transferring baskets between wells thus limiting manipulation of the pericarp sections and also reducing experiment duration. At last, the final step corresponding to the hybridization step after the addition of the fluorescent probe was performed in 0.2µL PCR tubes and in a thermal cycler allowing a precise and programmed management of time and temperature. After the hybridization steps (see materials and methods), slides bearing the samples were prepared and were used for imaging with a confocal.

After this optimization, the protocol duration was two half-days compared to several days for the BAC-FisH. In addition, we increased the number of samples that could be processed at once.

b. Semi-automated imaging

For reliable and complete ploidy quantification by FisH spot quantification, several nuclei per cell layer need to be imaged and all layers of the pericarp have to be assayed (Figure 2). On a pericarp sections, the objective was to determine cell size, cell position, nucleus volume and number of FisH dots. To do so, cell size has been estimated by dying the cell wall with Calcofluor-White, nucleus volume is estimated using a DAPI staining of the DNA. If we would like to obtain all the information -cell size, nuclei size and spot number-from a single acquisition it would be necessary to image large pericarp areas with the best resolution possible since this is needed for reliable FisH spot detection. A pericarp section represents about ~0.6mm² (~600000µm², 1160µmx520µm) and around 50-60 nuclei are visible in such section, without considering the small and numerous epidermal cells (Figure 20). Cells in the pericarp are large and all nuclei cannot be acquired on the same image since they are distributed in a 35-40 µm thick region over the total 150µm thickness of the section and the FisH spots within the nuclei are also distributed in a 3D manner within the nuclei. This means that several Z acquisition of ~0.6mm² would be necessary with the minimal distance between two Z steps (140 nm) to not miss any spot. We estimated that 100 to 200 Z-acquisitions would be needed and the total acquisition time for such a pericarp sample with the best resolution would be around 48h. To reduce this acquisition time while keeping spatial information and the best resolution with x63 objective for visualizing the FiSH dots, we designed a semi-automated pipeline to scan only the nuclei to be studied (Figure 20). First, we started scanning the region of the pericarp to be studied (about 0.6mm²) by tile imaging with a medium resolution with the same x63 objective (Step 1 on Figure 20) allowing the identification of the nuclei and of the cell walls (Step 2 on Figure 20). For the 50 to 60 nuclei detected in the scanned regions, we defined groups of nuclei based on their relative Z positions and sizes (Step 3, Figure 20). The confocal was then programmed to use these defined groups of nuclei to image these multi-position series of nuclei in batches (Step 4 on Figure 20) with high resolution and automatically save acquisitions (Step 5 on Figure 20). All these steps allowed obtaining acquisition of DAPI and FiSH signals for all nuclei from one pericarp section with their position at best resolution in between 3-4 hours to 18 hours for 12DPA fruits that contain bigger nuclei.

c. Ploidy quantification pipeline

For ploidy quantification, the scans obtained with the confocal allowed obtaining a 3D representation of each nucleus and of the FisH dots. These dots were quantified manually to obtain ploidy levels. The next step was to map the nuclei on the pericarp sections and locate them into the different cell layers. To do so, we used the confocal stage coordinate of the center of the grid acquisition that is recorded in the metadata of the image files and these coordinates were converted to pixel coordinates that can be used to retrieve positions of nuclei obtained in the same way in each file. This operation was done automatically as well as the measurement of nucleus size (Steps 6 and 7 on Figure 20). Finally, the last step was to assign each nucleus to a cell layer for obtaining the spatial distribution of ploidy levels in the pericarp. Because identifying cell layers on thick pericarp sections was difficult, we determined the relative distances of each nucleus from the outermost cell wall of the exocarp and the endocarp to calculate a normalized distance value in the pericarp. Then, the normalized distance was compared to control normalized distances determined on previously identified cell layers from images of thin sections of the pericarp (Step 8 on Figure 20 and see Figure 20bis for example of comparison to standard images). In optimal conditions, images could be acquired in 8-18H (Steps 1 to 5, Figure 20), allowing to process 8 pericarp samples per week, and nuclei mapping and FisH quantification took 3-4H per pericarp (Steps 6 to 8, Figure 20). FisH dots counting, done manually, was thus the most time-consuming step for ploidy quantification. In collaboration with engineers from the Bordeaux imaging center, a plugin for ImageJ was developed to measure nuclear volume and determine the number of FisH dots automatically in order to reduce analysis duration allowing and perform batch analyses. The first step of the plugin corresponds to the design of an area defined by the nucleus segmentation step (Figure 21A). This segmentation was used to limit the region for the segmentation of the FisH spots only inside the nucleus. Then, the same types of filters used for nuclei, gaussian and median filters, were applied for spots segmentation. Filtering was combined with a rolling ball algorithm to homogenize background in order to facilitate the segmentation of the fish spots (Figure 21B). After, FisH spots were identified in two steps. First all pixel values on the original image were divided by the filtered pixel values combined with a rolling ball algorithm from Image J (Figure 21B) to reduce the uneven isolated high pixel values below a determined threshold inferior to the FisH spots size. Rolling ball algorithm can be image as a 3D surface with the Z value corresponding to grey value of pixels of the image, so a high grey value area corresponds to a peak with small radius. Then, a ball with a determined radius is rolled beneath the surface so that it touches the surface in one or more points and it evenly adjust grey value of background as it rolls over the surface. This first method resulted in a sharper separation of the pixels between FisH spots and the background (Figure 22A). The second method consisted in the use of pixel values histogram (Figure 22B) and an iterative method calculating the edges of the different classes of the histogram that can then be threshold to isolate each spot as different objects (Figure 22B).

Nucleus segmentation was easily performed by using different filtering steps corresponding to both gaussian and median filters, followed by a thresholding and allowed measurement of nuclei volume. However, during the development of this plugin (Figure 21A), the main difficulty to overcome was the correct detection of the spots and their segmentation. Indeed, the Fish spots can be very close to each other and it is sometime very difficult to clearly identify one or two dots even using human eye (Figure 23).

We observe that FisH spots are distributed within the nucleus in two groups corresponding to the two homologous chromosomes and it was often easier to count the spots in one of the two groups because the spots were better separated. Thus, we tried to use this characteristic to correct the counting after processing by having spot numbers for each group separately. For example, if 13 spots were counted in the group A and only 3 spots in the group B it is highly probable that the 3 spots in the second group results from incorrect segmentation of spots being too close. Thus, after identification of the spots, they were distributed into two groups using an iterative k-mean clustering approach in order to determine two clusters of dots that could be representing the two chromosomes. The actual version of the plugin is not efficient to count ploidy levels using a 5SrDNA probe.

With this pipeline, the segmentation of the FisH spots was not efficient for all images probably because of the variable quality of the observed spot signal between samples. Indeed, often, spots were so close that even for human eyes it was complicated to determine if there were multiple spots in a single area. This limit of correct separation of FisH spots could be due to the nature of the 5SrDNA composed of repeated sequences that could vary in chromatin condensation. In addition, the condensation of the locus could vary in function of the status of the cell, and thus the visualization as separated dots or as closer "merging" spots could differ if the DNA is less condense or if it is compacted.

During the PhD, we also tested another probe targeting the telomeres, but the manual quantification was too complicated because of the number of spots to count and the number of samples, for example for a 32C nuclei a number of 768 spots are expected. The spots observed with the telomere probe were clearly separated and distributed all around the nucleus, thus giving the possibility to use the developed plugin to estimate ploidy levels. This was not tried during my PhD but would be interesting to apply. Another potential solution to obtain clear dots could be to target a non-repeated sequence by using a span oligo approach that allows the production of probes for nonrepeated target sequences distributed on a smaller unique chromosomal region. Targeting this type of sequencing could be eventually less sensible of the "merging" spots issue.

In conclusion, ploidy quantification by oligo-FisH has been optimized for tomato pericarp sections by the development of a pipeline with semi-automated image acquisition, quantification and analyses. To visualize and quantify in situ ploidy in living cells, we initiated the development of a method based on CRISPR/Cas9 technology.

Figure 23. Example of plugin assisted FisH spot counting and issues.

Examples of an easy segmentation of a 2C nucleus with well separated spots and of a "difficult" sample with spots close to each other limiting correct identification and quantification of the spots by the plugin.

Visualization of genomic loci in living plant cells using CRISPR/Cas9 technology

To quantify ploidy in situ in Tomato, we developed a direct labelling approach based on the CRISPR/Cas9 technology by fusing the dCas9 to a FP and targeting two different genomic loci (Figure 16A). We chose two repeated loci, the telomeres and the 5S rDNA locus (Figure 19A, [START_REF] Chang | FISH mapping and molecular organization of the major repetitive sequences of tomato[END_REF], in order to increase the dCas9 concentration in a relatively small area thus increasing fluorescent signal. For telomeres, a unique sgRNA has been used targeting the repeated sequence TTTAGGG (sgRNA telo ), similarly to [START_REF] Dreissig | Live-cell CRISPR imaging in plants reveals dynamic telomere movements[END_REF]. By using this probe, the expected number of dots is at least 24 for a 2C cell because each chromosome has two telomeres loci, and the number of dots should double if telomeres are duplicated during the endocycle. We designed six sgRNAs for the 5SrDNA locus, targeting the sequence at different positions and both DNA strands (Figure 19B, sgRNA 5S 29/35/46/63/85/104) and we used the sgRNAs alone or in combination to see which sgRNA are the more efficient and if signal is increased with simultaneous use of multiple sgRNA. Each combination of dCas9 with sgRNA targeting telomeres or 5S rDNA locus were tested in transient transformation before performing stable transformation (Figure 24A), but no signal difference depending on the sgRNA used were observed in transient transformation. Fluorescence observed in transient transformation was localized in the nuclei with a strong signal in nucleoli and the dot observed was not following the same pattern if telomeres or 5SrDNA was targeted, lot of dots or few dots, respectively (Figure 24A). The signal obtained while targeting telomere was equivalent to the one reported in literature [START_REF] Dreissig | Live-cell CRISPR imaging in plants reveals dynamic telomere movements[END_REF].

No stably transformed plant could be obtained with the dCas9sgRNA telo and in general, only few transgenics were obtained. We obtained T1 transgenic plants for sgRNA 5S 35 (one plant), sgRNA 5S 46 (two plants), sgRNA 5S 85 (three plants) and the combination of four sgRNA 5S 35/46/85/104 (one plant). Among these T1 plants, only two individual sgRNA 5S constructs, the sgRNA 5S 46 (2 plants) and sgRNA 5S 85 (1 plant), successfully produced fruits with seeds and showed fluorescence that could be observed at any stages of development in leaves, but they did not have dot like patterns (Figure 24B). T2 and T3 lines were the obtained, but the fluorescence in these plants was difficult to image because the signal was strong in the nucleolus and weak, near to the background, in the rest of the nucleus. Despite this week signal, we could observe dots various types of cells: two dots in the diploid guard cells (upper panel for sgRNA5S 85 on Figure 24B) and more than two dots in the sepal or leaf epidermal cells that undergo endoreduplication (lower panel for sgRNA5S 85 and upper right panel for sgRNA 5S 46 Figure 24). Finally, we tried to visualize these dots in the fruit pericarp but the autofluorescence was too strong limiting the visualization of signal in nuclei.

________________________________________________________________ Figure 24. Illustration of CRISPR-imaging observations after transient and stable transformation.

(A) Fluorescence in agro-infiltrated three weeks old Nicotiana benthamiana plants.

Plants have been transformed with a construct bearing a dCAS9-GFP and a sgRNA targeting either telomeres or 5S rDNA. (B) Fluorescence in stably transformed Tomato plants with a dCAS9-GFP and one or four sgRNA targeting the 5S rDNA locus. The red cross is representing no transformant has been obtained for telomeres constructs while for the 5S rDNA three individual T3 lines were obtained with the sgRNA 5S 85 and sgRNA 5S 46. Red star: line transformed with a construct having four sgRNA targeting the 5S rDNA locus.

Finally, the CRISPR-imaging technique seems to be a promising method for in situ visualization of chromatin loci in living cells, however it seems that the use of the tool is not as straight forward as the gene editing tool. The direct labelling approach or targeting biologically important repeated sequences is working well in transient transformation as reported in literature [START_REF] Dreissig | Live-cell CRISPR imaging in plants reveals dynamic telomere movements[END_REF][START_REF] Khosravi | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres[END_REF] but targeting telomeres and 5S rDNA with a dCas9 limits plant fitness as reported in the literature [START_REF] Fujimoto | Chromatin Live Imaging with Genome Editing Techniques: Switching from Scissors to a Lamp[END_REF]Matsunaga 2017, Khosravi et al., 2020). To further develop the tool, it could be interesting to explore indirect labelling approaches that is based on the labelling of another protein that recognize a peptide tail bind to the Cas9 or a fluorescent protein recognized a specific nuclei sequence of the sgRNA (see figure 16B and 16C for more detailed examples). The indirect labelling approaches seem to increase signal to noise ratio [START_REF] Khosravi | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres[END_REF] and more importantly it is necessary to be able to target non repeated sequences not implicated in genome organization such as the rDNA loci or telomeres [START_REF] Simon | Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana[END_REF][START_REF] Khosravi | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres[END_REF]. To target non repeated sequences assembling polycistronic sgRNA that is processed into individual sgRNAs by the tRNA-processing system could be used. By doing so, a non-repeated sequence can be targeted by using a large number of sgRNA [START_REF] Xie | Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[END_REF] to span the sequence and visualize it with direct (Figure 16A) or indirect labelling approaches (Figure 16B and16C).

Dynamics of ploidy distribution and ploidy-dependent transcriptome during pericarp fruit development Introduction

In all multicellular organisms, the cell cycle occurs during development, results in cell division and is responsible for transmitting the whole copy of the genome, thus the genetic information, from one mother cell to two daughter cells. In many organisms, somatic cells undergo changes in ploidy levels resulting from endopolyploidy (Orr-Weaver, 2015). In plants, endopolyploidy is mostly the result of endoreduplication (D 'Amato, 1984) that occurs in several vegetative and reproductive tissues in numerous Angiosperms [START_REF] Nagl | DNA endoreduplication and polyteny understood as evolutionary strategies[END_REF][START_REF] D'amato | Role of Polyploidy in Reproductive Organs and Tissues[END_REF].

Endoreduplication causes the cells to undergo full genome duplication without cell division [START_REF] Edgar | Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth[END_REF] and without change in chromosome number and thus triggers an increase of ploidy levels solely by increasing the number of chromatids forming polytene chromosomes [START_REF] Edgar | Endoreplication Cell Cycles: More for Less[END_REF]. These polytene chromosomes have been observed in various plant species (Carvalheira, 2000) and for example in the pericarp of Tomato fruits [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF].

Endoreduplication is associated with several developmental processes and contributes to cell differentiation and morphogenesis (Hulskamp et al., 1999, Bransiepe et al., 2010[START_REF] Roeder | Variability in the Control of Cell Division Underlies Sepal Epidermal Patterning in Arabidopsis thaliana[END_REF]. For example, trichomes are large epidermal hair cells with a three-branches structure that require multiple rounds of endoreduplication to be functional (Hulskamp et al., 1999). In addition, a positive correlation has been shown between endoreduplication and cell size such as in tomato fruits, cells that display highly endoreduplicated nuclei, from 2C/4C to 256C [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. Although these observations indicate a role of endoreduplication in cell growth, the strength of the relation between endoreduplication and the increase of cell expansion is debated [START_REF] Tsukaya | Has the impact of endoreduplication on cell size been overestimated?[END_REF]. Endoreduplication has also been suggested to increase the metabolic activity of the cells (Inze and [START_REF] Inzé | Cell Cycle Regulation in Plant Development[END_REF] and to be involved in the response to environmental stresses allowing building a strong metabolic capacity to improve tolerance [START_REF] Scholes | Plasticity in ploidy: a generalized response to stress[END_REF], Bhosale et al., 2019[START_REF] Lang | Endoreplication -a means to an end in cell growth and stress response[END_REF]. Another possible role of endoreduplication is to increase gene expression since gene copy number is increased. In fully grown tomato fruits, endoreduplication leads to a global shift in expression but genes with ploidy-specific expression have also identified revealing that cells with different ploidy levels might have different function within the pericarp [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. In Arabidopsis, a root ploidy specific transcriptome also allowed the identification of ploidy specific gene expression [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF].

In fully grown tomato fruits, the pericarp tissue, corresponding to the fleshy part, is composed of a heterogenous population of cells, with cell sizes ranging from 300 µm2 to 45000 µm2 and ploidy levels ranging from 2C to 128/256C [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF]. The pericarp tissue can be divided in three parts: the exocarp that corresponds to the small cells at the external side, the mesocarp containing to the larger cells in the center and the endocarp corresponding to the two cell layers in the internal side [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. The mapping of endoreduplication in the tomato pericarp revealed that ploidy levels are distributed as a gradient toward the center of the pericarp and that all the cells inside a cell layer do not have the same ploidy level [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. However, the reason for this heterogeneity is not understood.

Endoreduplication in the pericarp of tomato fruit is thus not randomly nor evenly distributed. In addition, endoreduplication is able to induce a global shift of gene expression and specific set of genes are differentially regulated as a function of ploidy levels in mature green fruits. However, there is no information about the dynamic distribution of endoreduplication during early fruit development in the pericarp nor ploidy specific gene expression. Ploidy mapping and transcriptome study during fruit development could help understanding how endoreduplication progresses and determines subpopulations of cells.

In this study, we obtained and analyzed the transcriptome of nuclei sorted based on their ploidy level from tomato pericarp. We studied young fruits at three stages of development, when ploidy levels rise and endoreduplication is establishing. In parallel, and for the same developmental stages, we mapped ploidy levels in the pericarp using fluorescent in situ hybridization (FISH). Finally, we integrated these two datasets with the aim of mapping gene expression within the pericarp as a function of ploidy.

Results

Ploidy specific transcriptome during early Tomato fruit development: experimental setup and data-preprocessing

To explore the variation in gene expression based on ploidy levels of cells, we used pericarp from fruits at three developmental stages: a stage at which most cells are dividing, a stage corresponding to the end of the cell division period and finally a stage at which the pericarp cells enter endoreduplication [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. For these stages, corresponding to 6-, 9-and 12-day post anthesis (DPA), ploidy levels increased over time, and the proportion of ploidy classes also changed (Figure 25A). Indeed, ploidy levels ranged from 2C to 16C for 6DPA and from 2C to 64C for 9 and 12DPA fruits. Nuclei from the pericarp cells were collected by Fluorescent Activated Cytometry Sorting according to their DNA content [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF], see Chapter 3: Materials and methods). For each time-point, four populations of nuclei-2C, 4C, 8C and 16C for 6DPA and 4C, 8C, 16C and 32C for 9 and 12DPA corresponding to the 4 major classes of ploidy at these stages, were recovered and used for RNA extraction and sequencing [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF], see Chapter 3: Materials and methods).

After quality control and pre-processing [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF], see Chapter 2.I.2.A): Use of spike-in to normalize gene expression in a global shift in expression context, the reads were mapped to the Sly 1.0 Tomato reference genome. After mapping, the number of uniquely mapped reads corresponding to reads aligned at only one locus was almost similar between the samples and ranged between 13k and 20k transcripts. All transcripts annotated on the Tomato genome are not represented because the sequenced RNA is extracted from pericarp cells and all genes are not expressed all the time in all organs, but the number of transcripts correspond to transcript number found in [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF] To determine the variation in expression within our dataset, we assessed intra-and inter-group variability using Principal Component Analysis (PCA), with a group corresponding to the transcriptome of nuclei of a certain ploidy level at one time-point (Figure 25B). We first observed that the biological replicates clustered together in the PCA plot, indicating a good reproducibility of the expression values. The PCA analysis also showed that 80% of the variance between samples was explained by two components. The first component, PC1, explained 56% of the variance between the samples and corresponded to the changes in ploidy levels. The second component, PC2, explained 24% of the variance and corresponded to the developmental stages (Figure 25B). We noticed that 2C nuclei at 6DPA are really close to 4C at the same stage and it is also the case of a 32C sample at 9DPA that is close to a 16C. The PCA analysis thus showed that the samples could be used to study the expression changes in function of the developmental stages and in function of the changes in ploidy levels.

Gene expression is changing during early fruit development and based on ploidy levels.

To study the changes in gene expression dependent on ploidy or developmental timing, we searched for differentially expressed genes (DEG) between the samples of our dataset. For that, after normalization [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF], a pairwise comparison of the expression values was done and expression fold changes between two ploidy levels at a specific developmental stage were calculated. We found that 1938, 1921 and 1878 genes were differentially expressed in at least one comparison with a FDR < 0.01 and log2 FC >1 at 6DPA, 9DPA and 12DPA respectively (Figure 25C). For each time point and as previously shown by [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF] the number of DEG, up-or downregulated increased when comparing more distant ploidy levels. (D) Venn diagram representing the common genes found between ploidy specific DE genes at 6DPA, 9DPA and 12DPA.

For example, in 6DPA fruits, 34, 297 and 454 were up regulated when comparing 2C nuclei to 4C, 8C and 16C nuclei, respectively. Moreover, among the DEG from all developmental stages, there were 765 in common (Figure 25D) and 231, 394 and 192 specifically in between 6 vs 9DPA, 9 vs 12DPA and 6 vs 12DPA, respectively. The common DEG found between the different time points could indicate that some genes are specifically regulated in a ploidy specific manner during the whole development.

To identify potential specific expression profiles related to ploidy level changes, the 1938, 1921 and 1878 DE genes were clustered based on their co-expression at a specific time-point, by a hierarchical clustering method (R Core team, 2017).

In 6DPA fruit pericarps, genes with similar expression profiles were grouped into 9 clusters corresponding to expression profiles ranging from high expression in 2C nuclei to high expression in 16C nuclei (clusters 6-A to 6-I, Figure 26A). Four main expression trends were visible, corresponding to a peak of expression in 2C nuclei (Clusters A and B), in 2C and 4C nuclei (Clusters C, D), in 4C nuclei (cluster E) and in endoreduplicated (8C and 16C) nuclei (Clusters F to I).

In 9DPA and 12DPA fruit pericarps nuclei, for which expression profiles analysis was done on nuclei with ploidy levels ranging from 4C to 32C, DEG with similar expression profiles were grouped in 9 and 10 clusters (clusters 9-A to 9-I and clusters 12-A to 12-J, Figure 26B and 26C), respectively. Expression profiles in the clusters from 9 and 12DPA were very similar, and four major trends were observed. These four types of expression profiles corresponded to a peak of expression in 4C nuclei (Clusters 9-A/B and 12-A/B), in both 4C and 8C nuclei (clusters 9-C and 12-C/D), in 8C nuclei (clusters 9-D and 12-E) and finally, the last trend was represented by clusters of genes having high expression in polyploid nuclei (8C and 16C: clusters 9-E and 12-F; , 8C, 16C and 32C : clusters 9-F and 12-G; and higher ploidy levels,16C and 32C, clusters 9-G to 9-I and 12-H to 12-J). To facilitate gene set and statistical analysis, we applied a new clustering to obtain a minimal set of clusters reporting the major expression profiles. This new clustering yielded five clusters for each time-point (Figure 27A-C) in which the main expression trends previously observed were visible: peak of expression in 2C (clusters 6-1/6-2, Figure 27A), 4C (clusters 6-3, 9-1/9-2 and 12-1/12-2, Figure 27A-C), 8C (clusters 9-3 and 12-3, Figure 27B-C), 16C (cluster 6-5, Figure 27A) or 32C (clusters 9-5 and 12-5, Figure 27B-C) nuclei; high expression in low ploidy levels nuclei, 2C and 4C, (cluster 6-2, Figure 27A) and high expression in polyploid nuclei, 8C,Figure 27A-C).

For each time-point, these trends thus corresponded to specific expression at a particular ploidy level, in nuclei with low ploidy levels or with high ploidy levels. During fruit development, transcriptional dynamics are thus associated with changes in ploidy levels.

Figure 27. Clustered normalized expression profiles of differentially expressed genes in pericarp of 6-9-12DPA fruits

Five clustered expression profiles of DE genes obtained with an ascendent hierarchical clustering for 6DPA (A), 9DPA (B) and 12DPA (C). On a cluster plot, the blue line represents the median profile and top right number is the total number of genes.

Biological processes expressed as a function of ploidy.

To identify the biological processes associated to ploidy and developmental stage specific expression profiles, we estimated over-representation of functional categories by using the Gene Ontology (GO) enrichment tool from PLAZA 4.5 (Van Bel et al., 2017) combined with the REVIGO web tool that allows to compute GO term enrichment lists after removing redundant terms [START_REF] Supek | REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms[END_REF], Bonnot et al., 2017). This combined use of GO enrichment analysis and REVIGO tool permits to highlight enrichment in specific GO terms by considering the hierarchy of the GO terms, thus making data easier to interpret. Briefly, the GO enrichment tool is attributing to a set of genes, GO terms and an associated p-value resulting from an enrichment test assaying the representativity of the GO term in the sample compared to the total number of genes associated to this GO term in the genome. In REVIGO, the list of GO terms and their associated p-value are computed to determine for each term a redundancy value (dispensability) and a value representing how the term is an outlier compared to the input list (uniqueness). Then the computed REVIGO values can be used to reduce large GO lists containing redundant enriched GO terms.

By combining the GO terms enriched in all clusters from the three time points studied, we ended up with 250 unique terms reduced to 43 after the REVIGO analysis. We found 30, 19 and 15 significantly enriched GO terms in the different clusters from 6DPA, 9DPA and 12DPA fruits, respectively. With this analysis, we found that the number of enriched GO terms varied between clusters but was not following the number of genes within a cluster, which varied from 123 to 618 (Figures [START_REF] Bi | Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery[END_REF][START_REF] Biais | Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism[END_REF][START_REF] Bollier | Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR[END_REF]. For example, in cluster 6-3 containing 249 genes seven terms grouping 120 genes were enriched while in cluster 6-5 composed of 347 genes only 3 terms grouping 68 genes were found enriched.

a. Biological processes expressed in 6DPA fruits

In 6DPA fruit pericarps, cluster 6-1 containing genes highly expressed in 2C cells and cluster 6-3 composed of genes with specific expression in 4C (Figure 28) nuclei were enriched in GO terms mainly associated to the classical cell cycle process. For cluster 6-1, the terms "chromosome organization" (55 genes), "cell cycle" (42 genes), "DNA replication" (45 genes) were found and included RBR1 and E2Fb genes, encoding proteins important for the regulation of cell division by modulating the expression of many genes involved in cell cycle progression (Figure 28A, Gutierrez 2009[START_REF] Abraham | Ectopic Expression of E2FB, a Cell Cycle Transcription Factor, Accelerates Flowering and Increases Fruit Yield in Tomato[END_REF], Őszi et al., 2020). The target genes of RBR1 and E2Fb were also found in the term "DNA replication" and include the subunits of the ORIGIN RECOGNITION COMPLEX (ORC), ORC4/5/6 [START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF]). In addition, the MINI CHROMOSOME MAINTENANCE (MCM) genes, MCM2 to 7 were found in the term "chromosome organization". Together, ORC and MCM proteins are implicated in the initiation of DNA replication between the Gap1 (G1) phase at which the cell decides to continue into cell cycle or stops in function of environmental and internal signals, and the S phase, DNA replication phase of the cell cycle (DePamphilis 2005,Tujeta et al., 2012). In the cluster 6-3, the enriched terms corresponded to "microtubule-based movement" (25 genes), "mitotic cell cycle" (22 genes), "movement of cell or subcellular component" (25 genes) and "nuclear division" (11 genes). Related to these terms, CYCLIN genes such as CYCA1, CYCB2 having a role in the mitotic cycle and the G2/M transition [START_REF] Menges | Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control[END_REF][START_REF] Sabelli | Expression, regulation and activity of a B2-type cyclin in mitotic and endoreduplicating maize endosperm[END_REF], CYCB3 and the CYCLIN DEPENDENT KINASE, CDKB, were found in this cluster. CDKB is involved in the G2 to M transition in Tomato fruit [START_REF] Czerednik | Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes[END_REF]. Moreover, several kinesin genes (25 genes) were found in this cluster, with for example the centromeric kinesin PHRAGMOPLAST ORIENTATING KINASE, POK1, encoding a protein responsible for phragmoplast orientation during the M phase of the cell cycle [START_REF] Müller | Two Kinesins Are Involved in the Spatial Control of Cytokinesis in Arabidopsis thaliana[END_REF]. The phragmoplast is a plant specific structure that forms during the cytokinesis, division of the cell, and serves as a scaffold for the formation of the new cell wall that will separate the daughter cells. The genes found in clusters 6-1 and 6-3 thus suggest that at 6DPA; the 2C nuclei could correspond to cells in late G1 phase (G1/S transition) whereas the 4C nuclei would correspond to cells in late G2 phase, maybe at the G2/M transition.

In cluster 6-2 representing highly expressed genes in 2C and 4C nuclei, we found 11 enriched terms mostly associated with cuticle formation such as "lipid metabolic process" (47 genes), "fatty acid metabolic process" (29 genes), "fatty acid derivative metabolic process" (11 genes), "cuticle development" (17 genes), "cutin biosynthetic process" (9 genes), "wax metabolic process" (11 genes) and "plant type cell wall loosening" (8 genes) (Figure 28A). Cuticle is a protective layer localized at the surface of plant organs and produced by the first cell layers of the organ [START_REF] Isaacson | Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss[END_REF]. The role of cuticle is to protect organs primarily from water loss [START_REF] Kenrick | The origin and early evolution of plants on land[END_REF] but also from pathogens [START_REF] Zhao | Role of the tomato TAGL1 gene in regulating fruit metabolites elucidated using RNA sequence and metabolomics analyses[END_REF]. Cuticle is composed of a cutin membrane composed of insoluble polyesters covered by cuticular waxes that are a mainly very long chain fatty acid (Fernandez et al., 2016). In Tomato, the fruit is covered by a thick cuticle layer having an important protective role. Cuticle synthesis and deposition at the fruit surface represent important events occurring during fruit development and regulated by a complex set of genes [START_REF] Vogg | Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid β-ketoacyl-CoA synthase[END_REF][START_REF] Isaacson | Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss[END_REF][START_REF] Hen-Avivi | Scratching the surface: genetic regulation of cuticle assembly in fleshy fruit[END_REF]. Several genes associated to cuticle synthesis in plants (Figure 32) were thus found in the cluster 6-2 and include very long chain fatty acid synthesis genes such as β-KETOACYL-COA SYNTHASE (KCS 3/6/10), ECERIFERUM1 and 3 (CER 1 and CER3) and GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 4 (GPAT4) that encode enzymes responsible for fatty acid elongation, alkane formation (wax biosynthesis) and cutin polyester formation, respectively. Wax and cutin are the principal constituents of cuticle and need to be exported in the extracellular space [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], Bernard and Joubes 2013[START_REF] Lashbrooke | The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly[END_REF]. Transportation of cuticle polymers is performed by ATP BINDING CASSETTE G proteins, ATCG32 for cutin and ATCG11 for wax [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][START_REF] Lashbrooke | The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly[END_REF]. Interestingly, we found both ATCG32 and ATCG11 in cluster 6-2.

Clusters 6-4 and 6-5, containing genes with high expression in 8C and 16C nuclei (Figure 28B), had only 2 and 3 enriched GO terms, respectively. In cluster 6-4, 15 genes belonged to the GO category "carbohydrate metabolic process" that is associated with central metabolism and are particularly related to sucrose metabolism, glycolysis and pentose phosphate pathway (Figure 28A). Among these genes, the SUCROSE SYNTHASE (SUSY) and NEUTRAL INVERTASE (NI), responsible for fruit imported sucrose cleavage into fructose and glucose, were found. The PYRUVATE KINASE (PK) and the GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) involved in glycolysis and the GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PDH), involved in the first step of the pentose phosphate pathway, were also found. In cluster 6-5, terms related to catabolism and synthesis of complex sugars were enriched: "carbohydrate metabolic process" (35 genes), "cellular polysaccharide metabolic process" (15 genes). These terms correspond mainly to 15 genes among which 10 encode proteins involved in starch synthesis, secondary cell wall synthesis and modifications. For example, we found the GLUCOSE-1-PHOSPHATE ADENYLYLTRANSFERASE which encodes the large subunit of the ADP-GLUCOSE PYROPHOSPHORYLASE catalyzing the first rate-limiting step in starch synthesis (Balicora et al., 2004) and the 1-4-ALPHA-GLUCAN BRANCHING ENZYME encoding an enzyme responsible for starch branching. Finally, for cell wall modifications, we found four XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) and a GLYCOSYLTRANSFERASE participating in the modification of existing cell wall and linkage in the cell wall matrix (Terao et al., 2013, Amos andMohmen 2019).

In conclusion, at 6DPA, non endoreduplicated cells are actively dividing and involved in cuticle synthesis (clusters 6-1 to 6-3). Genes found in cluster 6-4, highly expressed in 8C and 16C cells, indicate that at 6DPA endoreduplicated cells are specialized in sugar metabolism and this might be related to metabolic fluxes in young Tomato fruit during cell proliferation phase. Moreover, in endoreduplicated cells at 6DPA, the intense cell wall related activity could be in relation with cell growth occurring in the mesocarp [START_REF] Terao | Changes in the distribution of cell wall polysaccharides in early fruit pericarp and ovule, from fruit set to early fruit development, in tomato (Solanum lycopersicum)[END_REF][START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF].

b. Biological processes expressed in 9DPA fruits

In 9DPA fruit pericarp, cluster 9-1 in which expression peaked in 4C nuclei (Figure 29B), was enriched with 5 GO terms (Figure 29A), including "carbohydrate catabolic process" (14 genes) and "wax biosynthetic process" (8 genes). Among these genes, several are associated with cuticular wax synthesis (CER1 and WAX ESTER SYNTHASE/ACYL-COA DIACYLGLYCEROL ACYLTRANSFERASE: WSD1) and glycolysis (ENOLASE and PK). In the "carbohydrate catabolic process" category, we found genes involved in sugar transportation and cell wall related genes. For example, we found a BETA-FRUCTOFURANOSIDASE responsible for sucrose import from the vascular tissues (Friedman andZamir 2003, Shen et al., 2019) and an ALPHA-L-ARABINOFURANOSIDASE. In Arabidopsis, the ALPHA-L-ARABINOFURANOSIDASE, by hydrolyzing arabinose branching of pectins, plays a role in secondary wall synthesis of vascular tissue cells [START_REF] Montes | Cell Wall Modifications in Arabidopsis Plants with Altered α-l-Arabinofuranosidase Activity[END_REF]. Arabinose modification is important for cell adhesion and is necessary for maintenance of stomatal closure [START_REF] Jones | Cell wall arabinan is essential for guard cell function[END_REF]. Arabinose modification has also been implicated in the fruit firmness of the mutant Cnr (colorless non-ripening) that stay green [START_REF] Orfila | Altered Middle Lamella Homogalacturonan and Disrupted Deposition of (1→5)-α-l-Arabinan in the Pericarp ofCnr, a Ripening Mutant of Tomato[END_REF]. In cluster 9-2, genes are also highly expressed in 4C cells, as in cluster 9-1, and 11 GO terms were found to be enriched (Figure 29). A large part of those terms are related to cuticle synthesis pathways such as for example "cuticle development" (12 genes), "cutin biosynthesis process" (5 genes) or "lipid metabolic process" (44 genes), but we also found a term associated to microtubule, "movement of cell or subcellular component". The "cuticle related terms" contained between 12 and 44 genes including CER1 and NON-SPECIFIC LIPID-TRANSFER PROTEIN (nsLTP) genes involved cutin and wax secretion in the apoplast for the formation of cuticle [START_REF] Liu | Non-specific lipid transfer proteins in plants: presenting new advances and an integrated functional analysis[END_REF][START_REF] Petit | Breeding for cuticle-associated traits in crop species: traits, targets, and strategies[END_REF]. The "microtubule related terms" contained 11 genes with myosin (2 genes) and kinesin (9 genes) genes, usually involved in microtubule motor transport of large structure in cells (Li et al., 2012).

Cluster 9-4 and 9-5 are clusters with high gene expression in endoreduplicated nuclei, 8C, 16C and 32C (Figure 29B). The cluster 9-4 was enriched in terms associated with hydroxylation of molecules such as "organic hydroxy compound metabolic process" (12 genes), "alcohol metabolic process" (8 genes) and "cellular lipid biosynthetic process" (3 genes) (Figure 29A). These terms corresponded to 12 genes with 4 related to the carotenoid pathway such as the PHYTOENE SYNTHASE 1/2 (PSY1/2), the ZEAXANTIN EPOXIDASE (ZEP) that encode genes synthesizing precursors of tomato pigments and abscisic acid [START_REF] Kachanovsky | Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids[END_REF][START_REF] Ariizumi | Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum)[END_REF][START_REF] Karniel | Development of zeaxanthin-rich tomato fruit through genetic manipulations of carotenoid biosynthesis[END_REF]. Moreover, we found an ALDEHYDE OXIDASE gene (AAO3) responsible for the last step of the synthesis of abscisic acid (ABA), important hormone implicated in Tomato fruit cell expansion [START_REF] Nitsch | ABA-deficiency results in reduced plant and fruit size in tomato[END_REF], that derives from the carotenoid pathway. In addition, two genes related to the inositol pathway, responsible for ascorbic acid (AsA) synthesis [START_REF] Munir | Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation[END_REF] were found. Finally, the term "DNA replication initiation" (5 genes) was also enriched in this cluster with genes encoding MINI CHROMOSOME MAINTENANCE proteins (MCM 2/4/6) and a CELL DIVISION CONTROL PROTEIN 6 HOMOLOG (CDC6). Some MCM proteins have been associated with endocycle in mammals and maize endosperm [START_REF] Edgar | Endoreplication Cell Cycles: More for Less[END_REF][START_REF] Sabelli | Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm[END_REF] whereas CDC6 proteins have been associated both with cell cycle and endocycle replication processes [START_REF] Castellano | Expression and Stability of Arabidopsis CDC6 Are Associated with Endoreplication[END_REF][START_REF] Gutierrez | The Arabidopsis Cell Division Cycle[END_REF]. Genes usually associated with DNA repair are also found under the enriched term "DNA metabolic process" such as a DNA REPAIR PROTEIN MUTS homolog and BREAST CANCER TYPE 1 (BARD1) and its partner BREAST CANCER SUSCEPTIBILITY1 (BRCA1). These expression data from cluster 9-4 and 9-5 might indicate that at 9DPA, endoreduplicated cells (8C, 16C and 32C) would be already engaged into secondary metabolism pathways responsible for color of Tomato fruits and in ABA production in the pericarp.

The last cluster, 9-5, was enriched in only two GO terms associated with metabolism of sugars and starch: "carbohydrate metabolic process" (32 genes) and "glycogen biosynthetic process" (4 genes). These two terms contain the same genes also found in cluster 6-5 that are related to secondary cell wall synthesis and starch synthesis. Although cluster 9-3 contained 243 genes, no enrichment in biological process terms was found. The terms related to metabolism of sugars and starch indicate that 16C and 32C cells at 9DPA contain genes that are related to sugar metabolism. The presence of G6PDH is indicating an activation of the pentose phosphate pathway for the biosynthesis of nucleotides, consistent with needs of endoreduplicated cells. Finally, sugar metabolism related terms are giving an insight at cellular level of the pericarp metabolism that is potentially driven by endoreduplicated cells [START_REF] Biais | Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism[END_REF][START_REF] Beauvoit | Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[END_REF]. c. Biological processes expressed in 12DPA fruits At 12DPA, clusters with genes showing decreasing expression with ploidy, clusters 12-1 and 12-2 (Figure 30B), were enriched in GO terms associated with cuticle development and microtubule dynamic for cluster 12-1 ("lipid metabolic process" (57 genes), "cutin biosynthetic process (8 genes)" and "microtubule-based movement" (20 genes)) and modification of protein activities and developmental process for cluster 12-2 ("lipid metabolic process" (35 genes), "protein phosphorylation" (46 genes) and "anatomical structure morphogenesis" (50 genes)) (Figure 30A). Lipid and cutin related terms in cluster 12-1 mostly corresponded to the cuticle biosynthetic pathway genes and microtubule associated terms corresponded to genes encoding kinesin proteins and kinesin proteins are implicated in several cellular event such as meiosis, mitosis movement of chloroplast or in the morphogenesis of trichomes (Li et al., 2012). In cluster 12-2, the lipid related term contained a set of genes associated with cuticle synthesis and inositol mediated signaling. The inositol mediated signaling is represented by three INOSITOL 1 4 5-TRISPHOSPHATE 5-PHOSPHATASE, one GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED WALL TRANSFER PROTEIN 1 and one PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C that have been described in Arabidopsis to be involved in ABA signaling [START_REF] Gunesekera | Inositol Polyphosphate 5-Phosphatases 1 and 2 Are Required for Regulating Seedling Growth[END_REF][START_REF] Sanchez | Arabidopsis PLC1 Is Required for Secondary Responses to Abscisic Acid Signals[END_REF][START_REF] Carland | COTYLEDON VASCULAR PATTERN2-Mediated Inositol (1,4,5) Triphosphate Signal Transduction Is Essential for Closed Venation Patterns of Arabidopsis Foliar Organs[END_REF]). In the cluster 12-2, we found terms related to organ morphogenesis and protein phosphorylation among which 29 genes corresponded to RECEPTOR LIKE KINASE (RLK). RLKs belong to a large protein family involved in various plant developmental processes such as growth, stress (abiotic or biotic) and cellular signaling and response [START_REF] Jose | Arabidopsis Transmembrane Receptor-Like Kinases (RLKs): A Bridge between Extracellular Signal and Intracellular Regulatory Machinery[END_REF]. The 50 genes associated to organ morphogenesis were mainly associated to hormones, mostly auxin. Seven genes related to auxin signaling and transport were found with for example, AUXIN RESPONSE FACTOR 9 involved in inhibiting cell division [START_REF] Wang | The Tomato Aux/IAA Transcription Factor IAA9 Is Involved in Fruit Development and Leaf Morphogenesis[END_REF], and auxin transporters AUXIN1/LIKE-AUX (SlLAX3) and polar transporter PIN FORMED 9 (SlPIN9). Six genes associated to organ morphogenesis belonged to class IV Homeodomain-Leucine Zipper Gene Family (HD-Zip IV) and include PROTODERMAL FACTOR2 PDF2 and GLABRA2, that are found in the six genes, both necessary for differentiation of epidermal cells and trichomes [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]. Since cluster 12-2contains genes with high expression in 4C nuclei, the enrichment in the described GO terms might indicate that the 4C cells analyzed would still be engaged in cell division and cuticle synthesis and could be localized in the outer-epidermis layers in 12DPA fruits. However, these 4C cells or a sub-population of these cells could be engaged in differentiation and integration of developmental signals through hormone signaling and RLK activity. The cluster 12-4 was only enriched in one metabolic related term, "carbohydrate metabolic process" (28 genes) with genes implicated in secondary cell wall synthesis modifications and sugar metabolism (Figure 30A). Cell wall related genes were for example a GLYCOSYLTRANSFERASE FAMILY GT8 PROTEIN (GAUT) responsible for the synthesis of homogalacturonan (HG), the most represented polysaccharide in pectin (Atmotjo et al., 2011) and one of the XTH found in cluster 6-5 and 9-5. Sugar metabolism genes were related to glycolysis and interestingly a gene encoding an enzyme responsible for the dephosphorylation of Trehalose-6-phosphate, the TREHALOSE-6-PHOSPHATE PHOSPHATASE was found. Trehalose-6-phosphate is able to inhibit SNF1-related/AMPK protein kinases activity andSnRK1 is responsible of sensing energy homeostasis to regulate pathways activities [START_REF] Hardie | AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy[END_REF], is a positive regulator of starch synthesis and promote cell and organ size growth [START_REF] Li | Dynamic and diverse sugar signaling[END_REF]. TREHALOSE-6-PHOSPHATE PHOSPHATASE turns T6P into a non-inhibiting form for snRK1 and might be responsible for removing the inhibition of starch synthesis in crop plants [START_REF] Zhang | Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate[END_REF][START_REF] Paul | The Role of Trehalose 6-Phosphate in Crop Yield and Resilience[END_REF]. In Tomato mechanisms of sugar sensing are still unknown, however transgene experiment expressing the Apple SnRK1 in tomato resulted in reducing the duration of fruit development and in an increased nitrogen assimilation [START_REF] Wang | Expression of a heterologous SnRK1 in tomato increases carbon assimilation, nitrogen uptake and modifies fruit development[END_REF]. The cluster 12-5 was only enriched in "steroid metabolic process" (9 genes) with three genes implicated in brassinosteroid synthesis or signaling (Figure 30A). Brassinosteroids are a group steroidal phytohormones with roles in plant development and stress responses [START_REF] Zhu | Brassinosteroid signalling[END_REF]. Moreover, Brassinosteroids are implicated in enhancing sugar content and carotenoid accumulation in Tomato and inducing parthenocarpy in cucumber fruit [START_REF] Nie | Enhancing Brassinosteroid Signaling via Overexpression of Tomato (Solanum lycopersicum) SlBRI1 Improves Major Agronomic Traits[END_REF][START_REF] Hu | Regulation of fruit ripening by the brassinosteroid biosynthetic gene SlCYP90B3 via an ethylene-dependent pathway in tomato[END_REF], Fu et al., 2008). Finally, the cluster 12-3 was not enriched in any biological process GO term.

To summarize, in 6-9-12DPA fruits cells with low ploidy levels (2-4C) are mainly associated with cuticle synthesis and transport and cell divisions and cells with higher ploidy levels (8-16-32C) are mainly associated to central metabolism (6-9-12DPA) and secondary metabolism (9-12DPA).

Dynamics of ploidy specific gene expression in the course of early fruit development

To study the evolution of the genetic programs related to ploidy levels in the course of fruit development, we compared the expression clusters from the different timepoints, 6, 9 and 12DPA. To do so, the overlap between two clusters was estimated by determining the number of common genes and by calculating the significance of the overlap (Figure 31).

For each cluster, except 6-1, we found at least one significant overlapping cluster. The lack of significant overlap for cluster 6-1 might be explained by the fact that it contains genes with high expression in 2C nuclei, not studied at 9 and 12DPA. : In general, when comparing clusters with similar ploidy levels, significant overlaps were found between clusters presenting similar expression trends, such as when comparing 9-1 and 12-1 (Figure 31). Significant overlap for P <0.05 is highlighted by light blue and for P < 0.001 by dark blue.

To identify the biological processes in which genes with common expression profiles over time take part, we compared GO terms from clusters showing common expression trend and significant overlap in the pairwise comparison. Of note, the dataset from 6DPA includes 2C nuclei not studied in 9 and 12DPA, but not 32C nuclei, so attention has to be pay when analyzing the expression trends. We thus compared the GO terms for clusters showing decreasing expression with ploidy levels (6-2, 9-1, 12-1) and the opposite trending clusters with increase expression in higher ploidy levels (6-4,9-4,12-4 and 6-5,9-5,12-5).

In the clusters 6-2, 9-1 and 12-1 in which high expression is found in 4C nuclei, we observed common enrichment in GO-terms related to cuticle development such as "fatty acid derivative biosynthetic process", "cutin biosynthetic process" and "wax metabolic process". We also found additional genes associated to cuticle synthesis in other "low ploidy" clusters (clusters 6-1, 6-2, 6-3, 9-1, 9-2 or 12-1 and 12-2). Due to the high representation of cuticle related genes, we searched in literature for key genes implicated in cuticle synthesis, transport and degradation in Tomato fruits (Figure 32, [START_REF] Lashbrooke | The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly[END_REF][START_REF] Petit | Breeding for cuticle-associated traits in crop species: traits, targets, and strategies[END_REF][START_REF] Trivedi | Developmental and Environmental Regulation of Cuticular Wax Biosynthesis in Fleshy Fruits[END_REF]. We found 50 genes involved in these different steps among which 40 were DE in our dataset. In 6-9-12DPA fruits, 4C (2C at 6DPA as well) cells expressed all known genes necessary for the synthesis of both cutin and wax (Figure 32) such as SlGPAT4 (in cluster 6-2/9-1 = 2C and 4C) and three tomato WSD1 orthologs (in clusters 6-2/6-3/9-1/9-2/12-1/12-3 = 2C/4C/8C, [START_REF] Li-Beisson | Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester[END_REF][START_REF] Petit | The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis[END_REF] and some of their transcriptional regulators such as for example SlSHINE1 and SlSHINE3 (Clusters 6-2/9-1/9-3/12-1/12-2 = 2C to 8C, Figure 32, Shi et al., 2013). SlSHINE 1/3 are master activators of most of the cutin and wax synthesis genes and their expression has been associated to epidermal cells [START_REF] Shi | The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning[END_REF][START_REF] Al-Abdallat | Over-Expression of SlSHN1 Gene Improves Drought Tolerance by Increasing Cuticular Wax Accumulation in Tomato[END_REF]. Transporters ABCG's and nsLTP's necessary to export bricks of cuticle to the extracellular space [START_REF] Fabre | The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis[END_REF][START_REF] Lashbrooke | The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly[END_REF] were found in the clusters 6-2, 9-1, 9-2, 9-3, 12-1, 12-2 and 12-3 indicating expression in 2C to 8C cell. Interestingly, 8C cells (Cluster 3) of 9 and 12DPA fruits also expressed genes encoding enzymes necessary for the synthesis and exportation of cutin (Figure 32). Cuticle is synthesized in the epidermal layers of organs [START_REF] Fernández | Cuticle Structure in Relation to Chemical Composition: Re-assessing the Prevailing Model[END_REF], and Tomato pericarp is no exception since the three outer-epidermal layers are the main location of cuticle production (Dominguez et al. 2008[START_REF] Segado | Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development[END_REF]. Thus, this could indicate that the epidermal layers contain mainly 2C and 4C cells at 6DPA and up to 8C at 9DPA and 12DPA, with 8C cells specialized in cutin production.

Clusters with higher expression in 4C nuclei at 6DPA and 8C cells in 9DPA and 12DPA fruits shared a significant number of genes (6-3x9-3 = 41, 9-3x12-3 = 61 and 6-3x12-3 = 23) however, none of these overlaps was enriched in any GO terms. Among the genes from these overlaps, we found genes important for Tomato fruit development such as TOMATO AGAMOUS LIKE (TAGL1 and MBP3) genes, OVATE LIKE (annotated LONGIFOLIA PROTEIN), and SUN-likes (SUN-like 16 and SUN-like 30). [START_REF] Zhao | Role of the tomato TAGL1 gene in regulating fruit metabolites elucidated using RNA sequence and metabolomics analyses[END_REF], Zhang et al., 2018), and OVATE and SUN are necessary for the determination of the round fruit shape (Rodriguez et al., 2011[START_REF] Wu | A common genetic mechanism underlies morphological diversity in fruits and other plant organs[END_REF]. Moreover, the Tomato ortholog of Arabidopsis PERIANTHA was found in the overlap and in Arabidopsis PERIANTHA is a direct activator of AGAMOUS expression [START_REF] Maier | Dual roles of the bZIP transcription factor PERIANTHIA in the control of floral architecture and homeotic gene expression[END_REF]. OVATE, SUN and PERIANTHA indicate that 4C and 8C cells express to signal the regulation of fruit shape and organ identity [START_REF] Monniaux | How to Evolve a Perianth: A Review of Cadastral Mechanisms for Perianth Identity[END_REF]. The multiple processes found in 8C cells could be explained by the fact that several cell types are composing the 8C cell population or that 8C cells are having roles of interface between non-endoreduplicated cells and highly endoreduplicated cells.

For clusters of genes highly expressed in endoreduplicated nuclei (6-4, 6-5, 9-4, 9-5 and 12-4 12-5), few GO-enrichments were detected. Similar enrichment was found between 6-4x9-4 and 9-4x12-4 and corresponds to "Sorbitol response". Two additional enriched GO terms were found between 6-5x9-4 and 6-5x12-4 and correspond to "Carbohydrate metabolic process" and "response to reactive oxygen species", respectively. Among the 38 and 48 genes in the overlap between 6-5x9-4 and 6-5x12-4, we found representation (not enrichment) of terms associated to Actin filament, terpenoid biosynthesis (6-4x9-4,6-4x12-4 and 9-4x12-4). The term "Terpenoid biosynthesis" was represented by the TERPENE SYNTHASE (Beta-phellandrene synthase, PHS1) and the PHYSTOENE SYNTHASE (PSY1) involved in carotenoids pathway precursor of the synthesis of several hormones, Brassinosteroids, ABA, or Gibberellins [START_REF] Gutensohn | Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate[END_REF][START_REF] Fraser | Manipulation of Phytoene Levels in Tomato Fruit: Effects on Isoprenoids, Plastids, and Intermediary Metabolism[END_REF]. The comparison of the clusters with high expression in high ploidy levels (6-5,9-5 and 12-5) contained terms related to "ion transports", "cytosqueletal organization", "energy reserve metabolism" and "cell wall modifications" (only 9-5x12-5). The term "cytoskeletal organization" contains the ACTIN RELATED PROTEIN3 (ARP3) gene, important for normal cell expansion in highly endoreduplicated trichomes [START_REF] Yanagisawa | Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis[END_REF], indicating a potential role in cell expansion in endoreduplicated pericarp cells. The term "energy reserve metabolism" contains an encoding gene for a starch degrading enzyme ALPHA-GLUCAN PHOSPHORYLASE 2 (Emes et al., 2018) and cell wall modification genes (PECTINESTERASE, [START_REF] Phan | Silencing of the Major Salt-Dependent Isoform of Pectinesterase in Tomato Alters Fruit Softening[END_REF]. Finally, most of the genes involved in cuticle and cell division in low ploidy levels are shared during early fruit development. However central metabolism shifted from sucrose degradation to glycolysis when passing from 6DPA to 12DPA and secondary metabolism is enriched in 9DPA and 12DPA fruits.

Comparing ploidy specific transcriptome with laser microdissected pericarp transcriptome and ploidy transcriptome of Arabidopsis root.

After nuclei sorting, we lost the spatial information of cells within the pericarp tissue. We have seen that specific biological processes are expressed in a ploidy-specific manner and associated to low or high ploidy levels. Thus, to retrieve spatial information and try to localize the cells within the pericarp based on the ploidy specific gene expression, we compared our transcriptome data to genome wide pericarp cell type/tissue specific gene expression obtained by Laser Microdissection (LM) or handmicrodissection of five pericarp cell types/tissues from the M82 Tomato cultivar [START_REF] Shinozaki | High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening[END_REF]. To identify specific expression patterns among the Tomato tissues and the pericarp cell/tissue types, the authors clustered the expressed genes in 43 modules. For the comparison, we used the 1977 so-called "Hub genes" from the LM dataset that correspond to the genes having the strongest correlation with their modules [START_REF] Shinozaki | High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening[END_REF]. We used expression data from 5 and 10DPA samples that approximately correspond to the developmental stages studied here, 6-9 and 12DPA.

We found a total of 337 unique genes in common between the ploidy transcriptome (3330 genes) and the selected "Hub genes" (2645 genes) after the comparison. More specifically, we calculated the significance of the overlap between ploidy specific clusters and modules from the LM transcriptome dataset and found overlap enrichment for 13 of the ploidy clusters and 9 of the LM modules (Figure 33A). Five of the nine modules correspond to genes related to only one zone of the pericarp: the vascular tissue for M14, M17 and M19, and the outer epidermis for M20 and M21. DPA. The rest of the modules contain genes expressed in two zones of the pericarp: genes from M2 are mainly expressed in the outer epidermis and the vascular tissues; genes from M7 and M8 are expressed in the collenchyma and the parenchyma, corresponding to the upper cell layers and inner cell layers surrounding vascular tissue of mesocarp, respectively. Genes from M26 are lowly expressed in the vascular tissues and the most in collenchyma. We used this spatial information and the ploidy specific expression to infer/propose a ploidy identity to the different zones of the pericarp tissue and thus to locate the nuclei with specific ploidy level (Figure 33B).

Clusters with genes expressed in low ploidy levels nuclei showed significant overlap with modules containing genes mainly expressed in the outer epidermis (M2, M21), in the vascular tissues (M14, M17, M19) and the inner epidermis (M20, Figure 33A). High ploidy level clusters were significantly overlapping with gene modules mainly expressed in collenchyma and parenchyma (M7 and M8) and cluster 12-5 was also overlapping with a module expressed in every tissue except vascular tissues (M26, Figure 33A). No overlap was significant for the comparisons with clusters 9-3 or 12-3. This comparison between ploidy-specific transcriptome and LM transcriptome is indicating that cells with lower ploidy levels (2C and 4C at 6DPA and 4C at 9-12DPA) are found in the epidermis (outer and inner, Figure 33B) and the vasculature. This is in accordance with the GO enrichment analysis highlighting epidermal related processes (cuticle synthesis and cell division) and vasculature related processes (transport of sugar from vascular cells). We found enrichment between high ploidy clusters and collenchyma and parenchyma related clusters for 6 and 9DPA, but, at 12DPA we could also associate some high ploidy related gene expression with an expression in all the pericarp except the vascular tissue. This finding might indicate that endoreduplication is not restricted to collenchyma and parenchyma after 12DPA. Shared ploidy marker genes across different tissues of Arabidopsis thaliana and Solanum lycopersicum could be universal ploidy markers.

To search for potential universal ploidy markers, we compared our ploidy specific gene expression data from tomato pericarp to the ploidy specific gene expression data from the Arabidopsis root cortex [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. This later transcriptome data was obtained from sorted nuclei based on their ploidy level ranging from 2C to 16C and the authors found 3737 genes that were differentially expressed between the studied ploidy levels.

For the comparison, we used a set of 954 Arabidopsis genes associated to a specific ploidy level: 407 genes have a peak of expression at 2C, 162 at 4C, 302 at 8C, and 83 at 16C [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. To compare the datasets, we identified Tomato orthologous genes of Arabidopsis markers by using integrative orthology method provided by Plaza4. 5 (Van Bel et al., 2018). 1329 orthologous Tomato genes were found to correspond to the 954 input Arabidopsis ploidy markers. We overlapped these 1329 orthologous genes with the genes from the tomato ploidy clusters and found 237 of the Arabidopsis marker orthologs have a peak of expression at one ploidy level.

Even if not all Arabidopsis ploidy markers showed coherent expression profiles in Tomato, for each Tomato cluster, we found markers that match the ploidy expression pattern. For example, in cluster 6-1, in which genes are peaking in 2C nuclei, most of the common genes with the Arabidopsis dataset are 2C markers (35/50, Figure 34A). For cluster 6-5, 10 over 17 genes were found to be 8C or 16C markers, the rest of the genes corresponding to 2C (6/17) and 4C (1/17) markers.

To test the universality of a ploidy marker in the two species, we focused on Arabidopsis ploidy markers expressed in tomato clusters with a matching expression pattern, for example 4C markers present in clusters 9-1, 9-2 or 12-1, 6-1 and searched for their presence in in a least two developmental stages. Among the clusters with high expression in low ploidy levels (6-1,6-2,6-3,9-1,9-2,12-1 and 12-2), we first studied cluster 6-1 since expression in this cluster is high in 2C nuclei, not studied at 9 and 12DPA. In this cluster, we found 35 genes that are 2C marker genes and correspond to DNA replication related genes. In clusters 9-3 and 12-3, with expression peaking in 8C, only two Arabidopsis 8C marker genes were found one coding for a gene of unknown function and the other GPAT1 that is involved in glycerolipid metabolism in Arabidopsis flowers, siliques, and seeds.

In clusters with high expression in high ploidy levels nuclei (6-4, 6-5, 9-4, 9-5, 12-4 and 12-5) we searched for 8C markers in clusters We found in these clusters 10 common markers for 8C (4 genes) and 16C (6 genes). The genes are associated to cell wall synthesis or modification (4 genes), ion transport (2 genes), transport of molecules (a kinesin of unknown function), carotenoid biosynthesis (PSY1) and transcription regulation with a bHLH of unknown function in Tomato. In conclusion, by comparing our dataset to the ploidy specific transcriptome from Arabidopsis thaliana root cortex we have been able to identify genes that could be ploidy markers from 2C to 16C in different species, Tomato and Arabidopsis, and tissues, fruit pericarp and root cortex. Quantification of ploidy on sorted nuclei by FisH: Assessing the efficiency of a 5s rDNA oligo probe.

To complete and precise the spatial information on ploidy distribution predicted from the ploidy-specific transcriptome data (Figure 33), we aimed at determining the dynamic of ploidy changes in situ at 6DPA, 9DPA and 12DPA. To quantify ploidy in situ, we decided to use Oligo-FisH which is based on the use of synthetic oligonucleotides coupled to fluorescent dyes aready used successfully to image different DNA loci in plant [START_REF] Simon | Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana[END_REF][START_REF] Easterling | 3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation[END_REF]. Oligo-FisH is particularly powerful when used for targeting repeated sequences. Therefore, we chose to target the 5s rDNA locus of Tomato because this sequence is organized in tandem repeats and localized on a peri-centromeric locus of only one chromosome, chromosome 1 [START_REF] Chang | FISH mapping and molecular organization of the major repetitive sequences of tomato[END_REF][START_REF] Lapitan | Organization of the 5S ribosomal RNA genes in the genome of tomato[END_REF].To test if the 5s rDNA oligo-FisH probe was suitable for ploidy quantification, we determined ploidy on nuclei with known ploidy level. To do so, Fluorescence Activated Cell Sorting (FACS) was used to sort nuclei based on their ploidy levels. Nuclei ranging from 2C to 128C were collected on microscope slides and hybridized with the 5s rDNA oligo FisH probe.

By using this probe, we observed clear dot signals whose number increased with ploidy levels (Figure 35A). The number of dots was then counted for the different sorted nuclei. First, we found that the number of dots was significantly different between two consecutive ploidy levels (Figure 35B). Second, the number of dots counted was always close to the expected ploidy level studied, for example 3.36±0.49 dots and 26±4.21 for 4C and 32C nuclei, respectively. Third, the number of counted dots was more variable in higher ploidy levels but still significantly different between ploidy levels (Figure 35B). These results thus show that the use of the 5s rDNA oligo-FisH probe allows correct quantification of ploidy in situ since the number of dots allows classifying nuclei into specific ploidy level classes.

We also observed that nuclear volume was increasing with ploidy levels allowing to distinguish each ploidy class except for the 16C and 32C nuclei that did not differ in sizes (Figure 35C). Nuclear volume was small and relatively similar for low ploidy levels, 2C and 4C, 62 and 103 µm 3 , respectively, and then strongly increased in 8C, around 6x. For higher ploidy levels, the change in size between ploidy level was around 1.46x between 16C and 8C, 2.05x between 32C and 16C, 3x between 64C and 32C and 2x between 128C and 64C. The coefficient of determination R² between ploidy level and nuclear volume was 0.87 indicating a good correlation between these two traits. [START_REF] Baloban | Complementary and dose-dependent action of AtCCS52A isoforms in endoreduplication and plant size control[END_REF][START_REF] Berdasco | Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells[END_REF][START_REF] Ballicora | ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis[END_REF][START_REF] Atmodjo | Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex[END_REF][START_REF] Atmodjo | Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex[END_REF][START_REF] Ballicora | ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis[END_REF][START_REF] Al-Abdallat | Over-Expression of SlSHN1 Gene Improves Drought Tolerance by Increasing Cuticular Wax Accumulation in Tomato[END_REF]. The associated significance codes are: 0 '****' 0.001 '***' 0.01 '**' 0.05 '* Although nuclear volume increased from 2C to 16C, nuclear morphology stayed quite similar with a round shape. However, we noticed, as previously described [START_REF] Bourdon | Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth[END_REF] a major difference in the 32C nuclei shape compared to 16C nuclei: nuclear shape changed from round in 16C to invaginated nuclei in 32C (Figure 36A and 36B, Bourdon et al., 2012). Invagination were also visible in some 16C nuclei but less often. Compared to 32C nuclei, invaginations were more complex in 64C nuclei and 128C (Figure 36A and36B). In conclusion, Oligo-FisH using a synthetic probe targeting the 5s rDNA locus allowed ploidy quantification on sorted nuclei, thus the method could be transferred to analyze ploidy distribution in the pericarp of young developing tomato. To understand the dynamic of endoreduplication in young Tomato pericarp, we quantified in situ ploidy levels and generated ploidy maps of 6, 9 and 12DPA fruit pericarps. We also measured nuclei volumes and cell area in these samples.

To generate ploidy maps, we needed to acquire nuclei on the whole pericarp image and locate them into the tissue. By measuring the distance between the cells and the border of the pericarp, we were able to attribute ploidy levels of the cells to cell layers (see Chapter 2.I.2.B) c.: Semi automated imaging).

These cell layers were defined using the nomenclature from Renaudin et al, 2017. Cell layers were distributed in 7 classes based on pericarp structure at anthesis. At this stage, the pericarp is composed of nine layers including, from outside to inside the fruit, the outer epidermal layer E1, two-sub outer epidermal layers, E2 and E3, four rows of mesocarp cells grouped under the name of M layer and lastly two inner layers, the sub inner epidermal layer I2 and the inner epidermal cell layer I1. The latest class corresponds to the new layers formed after anthesis by periclinal cell division and forming M-like cell layers named M'. In our experiment, we found between 14 and 15 layers at 6, 9 and 12DPA, thus the M' layer contained five to six mesocarp layers localized below the E3 layer.

At 6DPA, ploidy levels ranged from 2C to 32C (figure 37A). 2C and 4C cells were found in the epidermal layers, E1, E2, I2 and I1. In E1 layer, there were 50% of 2C and 4C nuclei, while in the E2 layer there were 33% of 2C and 48% of 4C nuclei (Figure 37A). 8C cells were found in nearly all layers except E1 and represented nearly half of the pericarp cells at 6DPA. The proportion of 8C nuclei was nearly 50% in E3, M', I2 and I1, 34% in M layers and 19% in E2 (Figure 37A). 16C cells were absent from the outer and inner epidermal layers and were found from E3 layer to M layers and represented 36% of E3, 44% in M' cells and most of the M cells with >50%. In the M region, the most internal layers of the pericarp, layers 8, 9 and 10, had the highest proportion of 16C with 62%, 70%, 54%, respectively. Finally, 32 C cells were also found in 6DPA pericarp fruits, but only in very low proportions <10% in M' and most of the M layers. Only layer 11 contained more than 10% of 32C, with 17% (Figure 37A).

In the 9DPA fruit pericarps, ploidy levels were also ranging from 2C to 32C (Figure 37B). The low ploidy levels, 2C and 4C were found in the outer and inner epidermal layers. The proportion of 2C was of 47% in E1, ~26% in E2 and E3 and ~20% in I2 and I1 while the proportion of 4C was of 55% in E1, E2 and E3 and 60% in I2 and I1 (Figure 37B). The decrease of 2C cells compared to 6DPA was accompanied by an increase of 8C proportion in the outer and inner epidermal layers with 15% in E2 and E3 and 20% in I2 and I1. In the mesocarp layers, the proportion of 8C decreased compared to 6DPA with 28% in M' and ~24% in the layers 10, 11 and 12 (Figure 37B). (G) to (I) Cell area of each observed nucleus per layer colored by its associated ploidy level (color scale on the right) at 6 (G), 9 (H) and 12DPA (I).

The absence of 8C cells in layers 8 and 9 was accompanied by an increase in 16C and 32C nuclei. 16 C represented almost the majority of M' layers cells with a percentage of 47 and in M layers with 51% of the cells. Finally, 32C cells were found in all mesocarp layers with proportion of 12% in M' and 33% in M layers with a maximum of 50% in the layer 9 (Figure 37B).

In the latest developmental stage studied, 12DPA, ploidy levels in the pericarp increased compared to the previous time points and ranged from 2C to 64C (Figure 37C). The outer epidermal cell layers, as in 9DPA fruit, were composed of 2C and 4C in majority with ~20% of 2C and 55% of 4C cells. 4C cells were also found in I1 with a proportion of 11% and 2C cells were absent from the I2 and I1 layers (Figure 37C). Finally, cells in the inner epidermal layers started to endoreduplicate since 8C and 16C nuclei represented nearly all cells with a percentage of ~90-100%. The ploidy levels observed in M' and M layers were differentially distributed compared to 6-or 9DPA pericarps. 8C cells were more present in the outer epidermal layers (21%), in I2 layer with 75% of the cells and in I1 with 33% of the cells. In the other layers, M' and M, the proportion of 8C cells was strongly decreased compared to 6 and 9DPA with only ~10% of the cells (Figure 37C). In the M' layers, ~40% of the cells were 16C and the other half were 32C (~37%) with the exception of the inner M' layer (layer 8) that had more 32C (61%) and even only one 64C nucleus was found (Figure 37C). In the M layers, cells were mostly 32C (~57%) and proportions of 32C and 64C together could even reach 70. 64C cells represented < 10% of the cells in the M layers but the higher proportion was recorded for the inner M layer, layer 10, with 33% of 64C.

Often, an increase in ploidy levels has been associated with an increase in nuclear size and cell size (Cheniclet et al., 2005, Kawade and[START_REF] Kawade | Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue[END_REF]. To test if this correlation exists in our samples, we measured nuclei volumes and cell area.

If we compare same ploidy levels at different time-points, average nuclear volumes and cell areas were smaller in 6DPA fruit pericarps compared to 9DPA and 12DPA (Figure 37 D/E/F and G/H/I). 2C and 4C nuclear volumes and cell areas were similar between the different stages and were smaller than those of the other ploidy levels. 2C median nuclear volume were comprised between 120 and 130 µm 3 (Figure 38) and the median cell surface ranged between 142 and 252 µm² (Figure 37 G/H/I) whereas for 4C median nuclear volumes was between 115 and 163 µm 3 (Figure 38) and cell surface between 151 and 288µm² (Figure 37 G/H/I). From 8C to 32C, nuclear volumes and cell surfaces of 6DPA pericarp cells were smaller than for 9 and 12DPA, but at 9DPA, sizes were similar to those of 12DPA cells. For example, 16C nuclei volumes at 6DPA were comprised between 485 and 812 µm 3 (Figure 37 D) whereas 9DPA and 12DPA nuclear volumes were comprised between 877 and 1443 µm 3 at 9DPA (Figure 37 E) and 748 and 1533µm 3 at 12DPA (Figure 37 F). Similar trends were found for cells surfaces. Moreover, nuclear volume as well as cellular area was increasing in relation with ploidy levels. However, this increase distributed inside a gaussian-like distribution whose tails are overlapping between ploidy levels meaning that for example the population of 16C is smaller than the 32C population even if a given 16C nuclei can be larger than a 32C. Most of the increase in nuclear size and cell size was observed in the mesocarp where endoreduplication is taking place. The construction of ploidy maps using oligo-FisH thus allowed studying the dynamic of endoreduplication establishment during early Tomato fruit development the construction of ploidy maps using oligo-FisH thus allowed studying the dynamic of endoreduplication establishment during early Tomato fruit development showing that ploidy is increasing importantly in the center of the pericarp.

The integration of ploidy distribution with ploidy specific transcriptome reveals that ploidy specific gene expression is following specific spatiotemporal patterns.

We combined the ploidy specific transcriptome data and the ploidy mapping of the pericarp to obtain spatial information on the ploidy specific gene expression in the pericarp in situ. To represent gene expression based on the ploidy distribution across the pericarp tissue, for each layer, we weighted gene expression values for each ploidy level by the proportion of cells of a specific ploidy level, and then we summed up the expressions in each layer and normalized this expression to all layers for a given cluster.

In clusters 6-1, 6-2 and 6-3, genes had high expression in 2C and 4C and would be mainly expressed in the outer epidermis (Figure 39A), in the layers 1,2 and 3. Genes from clusters 6-2 and 6-3 would also be expressed in the inner epidermis, layers 13 and 14, while genes from clusters 6-4 and 6-5 mainly would localize to the mesocarp (Figure 39A), layers 4 to 12.

Genes from clusters 9-1 and 9-2 can be found in the outer and inner epidermis (Figure 39B). In cluster 9-3, genes would be mainly expressed in the outer and inner epidermis but also some M' mesocarp layers, the 5 th and 6 th layers, and one M mesocarp layer, layer 10 (Figure 39B). Lastly for 9DPA, cluster 9-4 contains genes expressed in cells from M' and M layers (layers 4 to 12), while cluster 9-5 contains genes mainly expressed in M' layer 5 and M layers (Figure 39B). At 12DPA, the clusters 12-1 and 12-2 contains genes that would be expressed in the outer epidermis and less in the mesocarp (Figure 39C). Genes from cluster 12-3, as for 9DPA the cluster 9-3, would localise in the outer and inner epidermal cells but also in some mesocarp cells (Figure 39C). For clusters 12-4 and 12-5, genes would be expressed in the mesocarp cells (Figure 39C and Figure 39A). At 12DPA, it is important to remind that the highest ploidy level assayed in the transcriptome was 32C while 64C are present in 5 of the 11 mesocarp layers and represent between 3% to 25% so for these layers interpretation has to be done with care.

Next, we studied in more detail the expression of selected genes having a role in pericarp development by inferring their spatial expression in the pericarp from the integration of our datasets. CYCLIN DEPENDENT KINASE proteins (CDK) are implicated in the regulation of several cellular events in the tomato pericarp such as regulation of cell number, cell size and indirectly cuticle development because of fewer cells [START_REF] Czerednik | Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes[END_REF]. Fruit specific overexpression of SlCDKB1 and SlCDKB2 resulted in increased expression of SlCDKA1 and fruit specific down regulation of SlCDKA1 resulted in the same phenotype: smaller fruits than WT, alteration of fruit shape and reduction of pericarp thickness due to less cell layers and smaller cells. Spatiotemporal gene expression is obtained from ploidy dependent gene expression weighed by the proportion of each ploidy level in the cell layer for each cluster of genes and each stage. This phenotype was also observed in plants overexpressing SlCDKB indicating an antagonistic mechanism of regulation between SlCDKA and SlCDKB in the pericarp [START_REF] Czerednik | Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes[END_REF]. In the ploidy transcriptome SlCDKB1 was found in cluster 6-3 and SlCDKB2 in clusters 6-3 and 12-1 while SlCDKA was not differentially expressed. The prediction of the spatial expression in the pericarp based on the ploidy distribution revealed that SlCDKB2 would be expressed in the outer and inner epidermis from 6DPA to 12DPA, SlCDKB1 in the outer and inner epidermis at 6DPA, in the mesocarp at 9DPA and again in epidermis at 12DPA while SlCDKA1 would be expressed in the mesocarp cells at 6DPA, 9DPA and in M' and inner epidermal layers at 12DPA (Figure 40). This analysis thus provides new insights on the potential expression localization of genes acting during pericarp development and shows expression profile that seem to be coherent with the antagonist regulation between SlCDKB and SlCDKA. We also predicted the spatial expression of Tomato orthologous of positive or negative regulators of the cell cycle or endoreduplication in Arabidopsis in function of the observed ploidy levels in the cell layers (Figure 41). Some of these regulators of the cell cycle or endoreduplication were found differentially expressed with ploidy levels such as SIM, HPY2 and SlKRP1 and were generally associated with low ploidy levels except for CCS52A1 associated to high ploidy levels (Figure 41). Among the 23 genes analyzed, only SlCC52A1, SlKRP1 and SlWEE1 have been shown to be able to regulate endoreduplication in Tomato fruit [START_REF] Mathieu-Rivet | Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato[END_REF][START_REF] Bourdon | Endoreduplication and Growth of Fleshy Fruits[END_REF][START_REF] Gonzalez | The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit[END_REF]. I will here first describe the prediction of expression for these genes. SlCCS52A1 was differentially expressed in cluster 6-5 while KRP was found DE in cluster 6-1. CCS52A1 was thus predicted to be preferentially expressed in layers containing endoreduplicated cells at all developmental stages (Figure 41A). KRP1 would be preferentially expressed in layers 1-2 and 13-14 at 6DPA, then in mesocarp layers at 9DPA and more evenly at 12DPA since most cell layers contain endoreduplicated cells (Figure 41A). Lastly, WEE1 would be first expressed preferentially the outer and inner epidermal layers, then at 9DPA and 12DPA in the mesocarp (Figure 41A). These expression patterns of CCS52A1 and WEE1 [START_REF] Mathieu-Rivet | Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato[END_REF][START_REF] Gonzalez | The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit[END_REF] are consistent with their roles in promoting endoreduplication. For KRP1, the interpretation is more complicated because the overexpression of KRP1 is inducing a reduction of ploidy levels in the pericarp [START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF] but this does not necessarily mean that the role of KRP1 is to repress endoreduplication. Moreover, KRP proteins have been hypothesized to regulate the rate of cell cycles and endoreduplication cycles [START_REF] Sizani | Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana[END_REF] and KRP1 does not necessarily mean that protein is accumulated since KRPs are actively regulated at protein level (Li et al., 2016). In addition, in general, the positive cell cycle regulators, such as CDKB1, SlE2Fa and CYCD3;1 had a stronger expression in 6DPA fruits and would locate in the epidermal cell layers while their expression decreases later during fruit development. The transcripts of negative regulators of the cell cycle also had a stronger expression in 6DPA fruits that would locate in the epidermal layers, but they also stayed expressed at 9DPA and 12DPA at high levels and their expression would be more evenly distributed inside the pericarp. The positive regulators of endoreduplication were expressed at a similar level at the three developmental stages and their transcripts would mostly accumulate in the mesocarp layers while the expression of the negative regulators of endoreduplication would be more dispersed (Figure 41A). Moreover, some exceptions are found such as ILP1 and CYCD5;1. ILP1 normally positively regulate, in Arabidopsis, the expression a of a mitotic cyclin [START_REF] Yoshizumi | INCREASED LEVEL OF POLYPLOIDY1, a Conserved Repressor of CYCLINA2 Transcription, Controls Endoreduplication in Arabidopsis[END_REF] and here is found expressed in endoreduplicated cells while CYCD5;1 expression favorize increase of ploidy level in cells (Sterken et al., 2011) but here CYCD5;1 is expressed in diving cells at 6DPA and evenly in the pericarp later.

Discussion

Endoreduplication in the pericarp induces differential gene expression.

From the ploidy dependent transcriptome, we found 1938, 1921 and 1878 genes differentially expressed at 6DPA, 9DPA and 12DPA when comparing gene expression between ploidy levels. The PCA analysis of gene expression in the samples showed that most of the variability was due to the change in ploidy levels suggesting that endoreduplication is probably an important regulatory component of pericarp development. Interestingly, the PCA analysis showed that 2C nuclei and 4C nuclei at 6DPA were very close to each other in the graph probably meaning that 4c nuclei are closer to be dividing nuclei than early endoreduplicated nuclei.

Endoreduplication during early fruit development is associated with cell specialization.

Our transcriptome data showed that cells with different ploidy levels express specific biological pathways suggesting that endoreduplication permits the establishment of specific developmental programs important for fruit growth. For example, in endoreduplicated nuclei, we found genes involved in sugar metabolism such as PK, G6PDH and GAPDH and these three genes encode enzymes responsible for the major metabolite fluxes in Tomato fruit during the cell proliferation phase thus favoring fruit biomass increase [START_REF] Beauvoit | Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[END_REF][START_REF] Colombié | Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit[END_REF] suggesting an important role of endoreduplicated cells in the metabolic activities measured in the pericarp during early fruit development. We also found genes involved in cell wall synthesis in endoreduplicated nuclei such as the UDP-GLUCOSE 4 EPIMERASE encoding an enzyme converting UDP-glucose into UDP-galactose and the GALACTOSYLGALACTOSYLXYLOSYLPROTEIN 3-BETA-GLUCUROSYLTRANSFERASE (IRX9-L) involved in hemicellulose synthesis [START_REF] Rösti | UDP-Glucose 4-Epimerase Isoforms UGE2 and UGE4 Cooperate in Providing UDP-Galactose for Cell Wall Biosynthesis and Growth of Arabidopsis thaliana[END_REF][START_REF] Meents | The cell biology of secondary cell wall biosynthesis[END_REF]. The potential relation between endoreduplication and cell wall synthesis and modification has already been described in Arabidopsis root cells or in response to stresses (Bhosale et al., 2018, Scholes andPaige 2018). Interestingly, in clusters 9-4 and 9-5, we found a MUTS homolog and Tomato orthologous of BARD1 and BRCA1, genes generally related to DNA repair. SlBRCA1 and SlBARD contain a plant homeodomain, the PHD domain, not found in animals suggesting a potential new role in plants during development [START_REF] Trapp | Homologs of Breast Cancer Genes in Plants[END_REF]. MUTS, BARD1 and BRCA1 participate in DNA damage response [START_REF] Yoshiyama | DNA Damage Response in Plants: Conserved and Variable Response Compared to Animals[END_REF] but BARD1 and BRCA1 are also expressed in plant tissues without stress [START_REF] Trapp | Homologs of Breast Cancer Genes in Plants[END_REF] and BRCA1 is overexpressed in high ploidy mutant of Arabidopsis e2fa and flb17 without DNA damage stress [START_REF] Ramirez-Parra | E2F-DP Transcription Factors[END_REF], Noir et al., 2015). These findings might be suggesting a potential role of these genes in developmentally controlled endoreduplication without DNA damage or in the control of DNA damage following replication of chromatids ensuring DNA integrity during endoreduplication. Thus, during fruit development, endoreduplication seems to be important for cell specialization.

Localization of ploidy levels in the pericarp can be inferred using ploidy transcriptome data and publicly available transcriptome.

The ploidy specific transcriptome is obtained from sorted nuclei that came from a population of pericarp cells and while tissue is disrupted and nuclei sorted, we lost the spatial information of cells. Thus, using data from literature and other transcriptome data we are able to determine a localization for ploidy sorted sequenced nuclei. We found in our dataset genes involved in cuticle synthesis in 2C nuclei at 6DPA and 4C cells at all developmental stages. Since it is known that in general cuticle is formed in the first layers of the fruit (Dominguez et al., 2008), these 2C and 4C cells might form the outer epidermis. We were interested in the spatial distribution of ploidy levels in the pericarp and we have seen that comparing our ploidy transcriptome with transcriptome data obtained after microdissection of the pericarp allowed to draw a preliminary map of ploidy level distribution in the pericarp. We found that a gradient distribution of ploidy levels toward the center of the pericarp is taking place already early during fruit development, in accordance with previously published ploidy map at 30DPA [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. The obtained map was not precise nor absolute at the cell layer scale but was indicating that all ploidy levels are not found everywhere and that the pull of sorted nuclei at a specific ploidy level might belong to multiple tissues inside the pericarp. The mapping of ploidy in the pericarp at 6-,9-and 12DPA using FisH allowed to more precisely attribute ploidy levels to the pericarp cell layers and confirmed the inferred map based on transcriptome data.

Ploidy level distribution is dynamic during early fruit development.

The ploidy maps obtained by FisH on young fruits reinforce the data obtained fully grown fruits [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF] that showed a gradual increase of ploidy levels towards the central cell layers. Overtime, we found that the ploidy levels increased in each cell layer but with a different dynamic (Figure 42). The outer epidermal layers were composed of equal proportion of 2C and 4C cells at 6DPA and at 12DPA, the layers contained 20% of 2C and 55% of 4C and almost a fifth of cells were 8C. From 6DPA to 12DPA, the decrease of 2C cells thus indicates that less cells are likely to divide, consistent with the nearly absent cell division activities recorded after 8DPA in the pericarp [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. The inner epidermal layers were already engaged in endoreduplication at 6DPA and were composed of 50% of 2C+4C cells and 50% of 8C cells while at 12DPA the inner epidermal layers were totally endoreduplicated with few 4C cells in I2 layer and >90% of 8C and 16C. The mesocarp layers M, formed before anthesis, were also the more endoreduplicated layers with already some 32C at 6DPA that became the majority of cells later. Concerning the other mesocarp layers, M', formed after anthesis, the proportion of 16C was relatively stable between 6DPA and 12DPA while 8C were largely decreasing from and 32C increased. Thus, we found that ploidy levels are increasing globally but older mesocarp layers become more endoreduplicated than M' formed later. This evolution suggests that endoreduplication might be a continuous process occurring at the same speed in each cell layer with older cells becoming more endoreduplicated. This phenomenon could be similar to what happen in epidermal pavement cells of Arabidopsis, where endoreduplication is stochastic and older cells became more endoreduplicated [START_REF] Kawade | Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue[END_REF]. Finally, we also observed a heterogeneity of ploidy levels within the same cell layer suggesting that endoreduplication is an asynchronous process inside a layer.

A positive correlation has been shown between ploidy and cell size [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF][START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Here we confirmed this correlation at early stages of fruit development. But we also observed that that the mean area of cells with similar ploidy levels is different at different time-points and increase over time. This was also the case between cells at 12 DPA and 30 DPA [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]), with, for example, 8600µm² and 15000µm² for 16C, 32C and 64C cells, respectively at 12DPA ², respectively compared to of 15000 µm² and 25000µm in 32C and 64C cells at 30DPA. The larger mean cell area at 30DPA might be indicating that endoreduplication could be giving a potential to grow bigger as already described for trichome, hypocotyl or sepal epidermal cells [START_REF] Breuer | BIN4, a Novel Component of the Plant DNA Topoisomerase VI Complex, Is Required for Endoreduplication in Arabidopsis[END_REF][START_REF] Barkla | Making Epidermal Bladder Cells Bigger: Developmental-and Salinity-Induced Endopolyploidy in a Model Halophyte[END_REF][START_REF] Gendreau | Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl[END_REF][START_REF] Roeder | Cell cycle regulates cell type in the Arabidopsis sepal[END_REF].

By integrating the transcriptome data as a function of ploidy together with the ploidy distribution information in the pericarp, we can now propose a spatial distribution of the expression of all genes thus obtaining a cartography of gene expression within the pericarp overtime. Using this information, we assay expression of known regulators of cell cycle or endoreduplication from Arabidopsis and Tomato and found for example that SlCC52A1, a positive regulator of endoreduplication in Tomato is expressed in mesocarp cells favoring their endoreduplication. This cartography could be confirmed by performing in situ hybridization of set of genes or by spatial transcriptomic experiments. liquid nitrogen before being stored at -80°C while for FisH, slides were stored in -20°C freezer after sorting 2000 nuclei.

Molecular biology

Methods of nucleic acid manipulation and analysis

Extraction of nucleus RNA

Quality controls of RNA

The quality control of nuclei RNA has been performed on the transcriptomics platform of Neurocentre Magendie (University of Bordeaux, Bordeaux, Nouvelle Aquitaine) on an Bioanalyzer 2100 (AGILENT) using an RNA 6000 Nano chip (AGILENT).

Synthesis of libraries and RNA sequencing

The synthesis of the RNA libraries and the sequencing have been performed by the GeT-Genotoul platform (INRAE Occitanie-Toulouse). After the synthesis of the libraries, we performed a Duplex-specific nuclease (DSN) treatment of the libraries to remove the maximum amount of rRNA that are present in nuclei RNA that are not yet maturated. The DNS treatment has been performed using the commercial enzyme from EVROGEN following manufacturer's instructions. Following DNS treatment, the Nuclei RNA have been paired-end sequenced with a HiSeq3000 (ILLUMINA).

Cytogenetic FisH

The FisH protocol is adapted from Bey et al., 2018. Fruit is sectioned transversally around equator of the fruit and pericarp section are then 100 to 150 µm sections were obtained using a vibrating blade microtome Microm HM 650V (Microm MicroTech France, http://mm-france.fr/). Buffer that are used to wash or handle sample in these protocol are classical cytological solution for biological sample such as Phosphate buffer saline (PBS, NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, KH2PO4: 1.8 mM and pH7 ) and saline-sodium citrate (SSC, NaCl: 3M, trisodium citrate : 300mM, pH7). Two to three slices are placed in a well in a 24 well plate containing a fixative fixation (Formaldehyde 4%, DMSO 10% in PBS1X) and samples are place 5min under vacuum infiltration at 20kDA and let stand in the solution at room pressure and room temperature (RT). After, samples are put in successive solvent bath for 5min each (2x methanol 100%, 2x ethanol 100%). In the last ethanol bath samples rest for at least 45mn at 4°C and samples are then transferred in a solution of 50%ethanol/50%xylene for 30mn at RT. Before proceeding to next steps, extra care is taken to clean the samples of xylene solution before successive solvent wash bath for 5min at RT each (2x ethanol). Then samples will be rehydrated with successive wash bath in water-based buffer for 5min at RT in each bath (2x PBS1X+0.1% v/v Tween20 and 2x SSC2X) and a last SSC2X bath is performed in presence of 100µg/mL of RNAseA for 1h at 37°C, then an equal volume of Hybridation buffer (HB, 50 mM Sodium phosphate pH 7.0, 2x SSC, 50% deionized formamide) is added and samples rests for 30mn at RT. Following step is critical because it required to physically manipulate the samples to transfer it in a PCR 0.2mL microtube containing 100µL of HB and rest for 30min before changing the solution with the probe solution (HB + 0.5 to 3µL of labelled oligonucleotide, here the dye was a TexasRed). Tubes are then incubated in a thermal cycle for 1H at 37°C, the 4mn at 88°C followed by a ramp down to 37°C in 3min and samples rest at 37°C for 12-15h. The next day, the samples are washed in a stringent bath of HB for 30mn at 42°C followed by 5 water-based baths for 10min in PBS1X, at RT 15min in PBS1X+10µg DAPI at RT and 3x 5min at RT in PBS1X. Finally, the samples are transferred to superfrost slides in VECTASHIELD containing DAPI (VECTOR LABORATORIES, catalog number: H-2000) under stereomicroscope to assure the flatness of the samples, then samples are recovered and covered with a coverslip (20x20, #1.5) and sealed with nail hardener. Samples can be imaged right after or stored several weeks at 4°C.

CRISPR-imaging

All the constructs needed for the development of the CRISPR imaging were obtained using the Goldengate cloning method (Engler et al., 2008) that allowed the assembly of the multiple transcriptional units that were necessary for our work.

First, the dCAS9 coding sequence was amplified and mutated from the pDe-CAS9-D10A plasmid (Fauser et al., 2014) by PCR and transferred into an entry GoldenGate vector. Then, this coding sequence was assembled with the 35S promoter, t the GFP at the N terminal or C terminal end and the Octopine synthase (OCS) terminator, constituting a transcription unit, into a plasmid named. The dCAS9 was tagged at its N-terminal or Cterminal part to verify if the position of the fluorescent protein influences the fluorescent signal.

In parallel, the sgRNA was cloned by PCR and assembled into a level1 plasmid under the control of the AtU6 promoter (AtU6) of shRNA (small hairpin RNA). Finally, a final plasmid was generated by assembling the transcriptional units containing the dCAS9 plasmids for multigenic expression. This final plasmid contained the dCAS9 tagged with N-GFP or C-GFP, the sgRNA targeting telomeres or 5SrDNA (sgRNA telo or 5S ) and the selective marker nptII for Kanamycin resistance in plants.

Each assembled plasmid has been used to transform E.coli. From the bacteria resistant to the selective antibiotic, PCR on single colony has been done to verify the correct construction of the plasmids by checking the size of the PCR product. If the size was corresponding to the one expected, then purified plasmids from E.coli were sent for sequencing to verify the sequence of the extremities of the inserted cassette.

These plasmids were used for the transformation of tomato protoplasts, tobacco leaves, and to perform Tomato stable.

were clustered according to hierarchical clustering of standardized expression values ((expression value) -(mean expression of gene) / (standard deviation of expression of gene X). Distances between genes were calculated with the Euclidean distance measure, and Ward method applied to square values was used as agglomeration method.

Gene ontology enrichement

Gene ontology enrichment were done using Plaza 4.5 (Van Bel et al., 2018) using pvalue corrected using Bonferroni correction <0.05 and default parameters. Then gene ontology has been cleaned from redundancy using REVIGO [START_REF] Supek | REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms[END_REF]. REVIGO files were then filtered on R following protocol from [START_REF] Bonnot | A Simple Protocol for Informative Visualization of Enriched Gene Ontology Terms[END_REF].

Ploidy mapping and analysis

Data were obtained from segmented nuclei from images, after filtering step detailed in chapter X nuclei are segmented and the voxel contained in the segmented nucleus are quantified to measure the volume. Cell areas have been determined manually by surrounding cells on their largest diameter. Ploidy counting has also been done manually checking in 3D in successions of image if close spot is a unique or a duplicated spot.

In Tomato fruit, endoreduplication is an important process taking place during development and ploidy levels can attain up to 256C and even 512C in some varieties [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. A strong correlation has been found between endoreduplication level and the mean cell size in the pericarp and also fruit weight [START_REF] Cheniclet | Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth[END_REF]. However, this correlation between mean cell size and mean ploidy level is weaker when cell size and ploidy level are measured at the cellular level instead of the tissue level [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Nevertheless, the largest cells are always highly endoreduplicated in the pericarp [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. In the pericarp of fully grown fruit, endoreduplication is not evenly distributed but organized as a gradient of increasing ploidy levels toward the center of the tissue [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Endoreduplication might thus act as a growth factor for pericarp cells. In addition, in a fully grown fruit, an increase of ploidy levels is associated with a global gene expression shift but also to specific regulation of the expression of sets of genes [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF].

Based on these observations, mainly obtained in fully grown fruits, we wanted to know i) if endoreduplication is already distributed as a gradient in young tomato fruit pericarps as observed in fully grown fruit ii) if the endoreduplication is correlated with cell growth in young fruits and iii) if a specific regulation of gene expression occurs in these young fruits. Thus, my PhD work aimed at determining the ploidy distribution in the pericarp during early fruit development while endoreduplication is taking place. The objective was also to determine the genes that have a ploidy specific expression and to identify potential molecular markers of ploidy levels. To do so, we studied during early fruit development how endoreduplication distribution progresses in the different cell layers of the pericarp and if cell specialization is already established in these endoreduplicated cells. We mapped endoreduplication in the pericarp of tomato fruit using FisH and analyzed ploidy specific transcriptome of ploidy sorted nuclei at three developmental stages, 6DPA, 9DPA and 12DPA. These stages correspond to an important step in endoreduplication progression since they correspond to the transition between a stage where numerous cells are 2C and 4C, thus dividing, at 6DPA, to a stage where most cells that are endoreduplicating and are 8C or more. The data obtained could help to better understand how endoreduplication is taking place in the pericarp and how it is regulating genetic programs in cells during early development. The results obtained pave the way for a wider project questioning the functional role of endoreduplication in the pericarp and how entering endoreduplication is genetically controlled in cells of the pericarp.

Technological development of tools for the determination of in situ ploidy levels and ploidy specific transcriptome Transcriptome related technologies

The transcriptome of sorted nuclei was easily obtained and only few adjustement of the protocol was needed to correctly extract RNA to obtaining RNA-seq data, most of the more time spent was due to logistical problem. The use of spike-ins allowed to quantify the global shift in the case of ploidy level variation and was a good quality control of the sample after sequencing because we can evaluate the sensibility of the experiment, here we were theoretically able to detect a single RNA per nucleus. However, I encounter more difficulties with the differential analysis because of the poor annotation of the tomato genome in the public databases, for example it was difficult to clearly identified in datasets genes related to central metabolism, that has been extensively studied.

FisH related technologies

The use of Oligo Fish allowed to map ploidy levels in the pericarp and optimization of sample handling and imaging allowed to map 6DPA 9DPA and 12DPA fruits. Oligo FisH is working only when multiple dyed oligonucleotides are bound on the same area, so we wanted a repeated sequence and the choice of 5SrDNA was motivated by the low number of known repeated sequence on a single chromosome in Tomato genome. We also did use a probe targeting telomere repeats, but the number of dots and lack of time did not allow ploidy quantification in these samples for now. The automatization of ploidy quantification by the help of the Image J plugin could help with the used of telomere probe to see if it can be used in ploidy quantification. However, we have seen that the plugin is not yet functional, but it should be rapidly able to be functional and will way facilitate ploidy quantification in a lot of sample.

CRISPR-imaging

CRISPR-imaging is a promising tool that need to be adapted in the use of living organisms such as plants and not cellular model as in animals because in complex organisms CAS9 is interfering with cellular activities and inhibit normal development of plants when targeting repeated sequences. However, we only tested a single approach while there are at least three other popular approaches, unfortunately transient transformation is useless to assay the "toxicity" of the method. On the point of CRIPSR imaging, it could be interesting to communicate with other team working on this specific theme to coordinate a project in order to tackle the methods and find the one that would allow to assay chromatin organization in living cells.

Global shift in expression upon endoreduplication.

A global shift in expression has been reported in the case of allopolyploidy and autopolyploidy and resulted 1.6 to 2.1 more gene expression while comparing polyploid and diploid plants (Coate andDoyle 2010, Robinson et al., 2018). Endoreduplication has also been shown to induce a global transcription increase in fully grown tomato pericarp by measuring and comparing gene expression in the different ploidy levels and this shift corresponds to a doubling of gene expression between successive ploidy levels [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF].

In this PhD work, we used spike-ins, which is a mix of RNAs from a reference library for those the sequences and concentrations are known allowing to scale gene expression. The spike-ins allowed showing that there is a global shift in gene expression corresponding to a doubling with endoreduplication at early developmental stages. This change in global expression is similar to the global shift observed in the later mature green stage [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF] suggesting that in the pericarp the doubling of gene expression is due to endoreduplication and not dependent on the developmental stage. The spike-ins have helped to determine that gene expression is increasing with ploidy levels but did not allow to determine an absolute value of increase, doubling is an approximate value. To determine the exact value more sampling will be necessary to increase statistical force of our analysis.

A subset of genes is specifically regulated by ploidy levels in the pericarp.

The global shift in expression related to the increase of ploidy levels in the pericarp is accompanied with ploidy specific regulation of subsets of genes (Chapter 2.II.2.B) Gene expression is changing during early fruit development and based on ploidy levels). We found that five trends of expression were sufficient to represent the ploidy specific expressions with genes mostly expressed in non endoreduplicated nuclei, genes expressed in newly or low endoreduplicated nuclei, and genes expressed in endoreduplicated nuclei or higher ploidy levels. Ploidy specific gene expression related to ploidy levels has also been found in Arabidopsis ploidy specific transcriptome [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. To better understand which types of genes are associated with expression trends, we performed a GO enrichment analysis of the genes showing these different trends of expression. We have found different enriched functional categories such as those related to cell cycle and cuticle metabolism mostly in non endoreduplicated cells and genes related to sugar metabolism in endoreduplicated cells. This finding is suggesting that endoreduplication could be specifying cell differentiation in the pericarp and that sugar metabolism and storage is occurring specifically in endoreduplicated cells [START_REF] Beauvoit | Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[END_REF]. While non endoreduplicated cells are responsible of the establishment of the protection of the fruit via the accumulation of cuticle [START_REF] Hen-Avivi | Scratching the surface: genetic regulation of cuticle assembly in fleshy fruit[END_REF] and the production of new cells to assure growth flexibility to the pericarp [START_REF] Liu | Generation and Analysis of an Artificial Gene Dosage Series in Tomato to Study the Mechanisms by Which the Cloned Quantitative Trait Locus fw2.2 Controls Fruit Size[END_REF].

Interestingly, in the transcriptome data, we have found that this cell type specific expression is also influenced by the developmental stage since, for cuticle synthesis, we found that as early as 6DPA cuticle synthesis is important in non endoreduplicated cells, with the expression of genes related to Cutin and Wax synthesis pathways and only wax synthesis pathway is more importantly represented later in the development (Chapter 2.II.2.D) Dynamics of ploidy specific gene expression in the course of early fruit development).

The few enriched GO terms found in endoreduplicated cells are suggesting that cells are engaged in very specific pathways related to carbohydrate metabolism, cell wall synthesis and modification or hormone synthesis and response. The enrichment of GO terms in clusters with high expression in higher ploidy levels were generally associated with central metabolism, more specifically to glycolysis and pentose phosphate pathway and this is consistent with the expression of enzymes necessary to produce the measured metabolites accumulating in the fruit at these stages of fruit development [START_REF] Beauvoit | Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[END_REF][START_REF] Colombié | Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit[END_REF]. In the genes associated with carbohydrate metabolism we also found some related with cell wall at all stages studied. We found genes involved in both synthesis and relaxation such as hemicellulose synthesis genes [START_REF] Rösti | UDP-Glucose 4-Epimerase Isoforms UGE2 and UGE4 Cooperate in Providing UDP-Galactose for Cell Wall Biosynthesis and Growth of Arabidopsis thaliana[END_REF][START_REF] Meents | The cell biology of secondary cell wall biosynthesis[END_REF] and cell wall modifying genes (Terao et al., 2013, Amos andMohmen 2019). The expression of these genes in endoreduplicated cells would be suggesting that both new cell wall production and modification of cell wall are supporting cell growth. Similar observations have been done for cell wall regulation in endoreduplicated cell of Arabidopsis roots [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF] and it has been proposed that endocycle could be an important factor in regulating cell wall production and modification to sustain rapidly expansion of cells (Bhosale et al., 2019). Interestingly, we found in the same clusters biological categories related to hormonal regulation with auxin, brassinosteroid or abscisic acid related genes, suggesting that ploidy level can modulate the response to these hormones. Similarly, in Arabidopsis endoreduplicated root cells, a ploidy specific expression of genes implicated in hormonal responses was found [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]. Further exploration of hormone accumulation using reporting lines or fruit specific hormone synthesis mutant analyses would be helpful to understand how endoreduplication is regulated in the pericarp. Indeed, it could be interesting to use fluorescent reporter lines for auxin or cytokinin [START_REF] Liao | Reporters for sensitive and quantitative measurement of auxin response[END_REF][START_REF] Steiner | Characterization of the cytokinin sensor TCSv2 in arabidopsis and tomato[END_REF] in order to study the distribution of hormones in relation to the ploidy levels in the pericarp. While fruit specific hormone synthesis mutants might be interesting to study the modification of distribution of ploidy levels in the pericarp.

We found specific set of genes expressed either in non-endoreduplicated or in endoreduplicated cells, but we also identified a singular behavior for a group of genes expressed specifically in 8C cells at 9-and 12DPA and not at 6DPA. We looked at gene ontology enrichment, however, we did not find any enriched functional categories. Specific expression of genes in 8C cells can also be noticed in data of fully grown fruit and contrary to our results, gene ontology enrichment was found for this category [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. The lack of enrichment might be due to an "undifferentiated" nature of the 8C cells that are just entering endoreduplication or because the pool of 8C sorted nuclei correspond to a heterogeneous population assuring different functions. If each cell type expresses a specific functional category, in a pool of these different cells, these categories might be hidden in our analysis. Lastly, by comparing our transcriptome data to public dataset obtained from microdissected tissues of the pericarp [START_REF] Shinozaki | High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening[END_REF], we showed that ploidy levels are not randomly distributed within the pericarp but correspond more or less to specific types of cells. We found, for example that epidermal cells express genes found highly expressed in low endoreduplicated nuclei and that mesocarp cells express genes found in highly endoreduplicated nuclei (Chapter 2.II.2.E) Comparing ploidy specific transcriptome and laser microdissected pericarp transcriptome). The use of a pool of cells sorted based on ploidy level for transcriptomic has allowed identifying ploidy specific expression patterns. However, if cells with similar ploidy level have different functions, our analysis might not be able to properly distinguish between genes expressed in each cell type. Thus, in the future, it would be interesting to perform single cell RNA-seq (scRNA-seq) or single nuclei RNA-seq (snRNA-seq) that will allow better understanding the spatial resolution of gene expression, identifying potential difference in ploidy populations, and determining cell lineage trajectories (Figure 43A).

The ploidy distribution in tomato pericarp during early development stages is already organized as a gradient.

In fully grown fruit, ploidy levels are following a gradient from the epidermis (outer and inner) to the internal part of the pericarp with low levels on the outside and high levels in the inside [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Here we studied ploidy distribution and cell size in the pericarp at early stages of fruit development. We found that between 6 and 12DPA, the ploidy levels distribution within the pericarp is also following a similar gradual increase of ploidy levels towards the central part of the and that the distribution is highly dynamic at these early stages. The epidermal layers are composed of dividing cells at 6DPA and cells are not all engaged in endoreduplication in the outer epidermis at 12DPA contrary to the inner epidermis where almost all cells became endoreduplicated. The drastic reduction of 2C cells in the epidermal layers is consistent with the measure of cell division activities that are nearly absent in the pericarp after 8DPA [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF]. The M Mesocarp layers, formed before anthesis, were also the more endoreduplicated layers with only few non-endoreduplicated cells at 6DPA. These layers contained the largest proportion of higher endoreduplication levels, 32C and 64C at 12DPA. Concerning the other mesocarp layers, M', formed after anthesis, we observed a progressive increase of endoreduplicated cells toward higher ploidy levels but only few 64C were found at 12DPA. Ploidy levels were increasing globally but older mesocarp layers, M layers, were more endoreduplicated than M' probably indicating that endoreduplication is a continuous process and that older cells become more and more endoreduplicated over time. [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF] hypothesized that 8C cells already present before anthesis would be localized in the center of the mesocarp, corresponding to M cells and our data support the hypothesis because at 12DPA, M cells, that are older than M' cells are also more endoreduplicated. Thus, this difference in ploidy levels in the mesocarp cell layers would be suggesting that the age of the cell is related to its final size. However, in fully grown fruit, endoreduplication levels are almost similar between M' and M layers [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF] potentially indicating that the difference observed between 6DPA and 12DPA tends to reduce with the age of the fruit. This observed reduction of difference in ploidy levels might be due to a reduction of endoreduplication speed or exit from endoreduplication in M layers or more rapid endoreduplication in M' layers. Moreover, a heterogeneity of ploidy levels was observed within a layer suggesting that endoreduplication is occurring in an asynchronous manner inside the same layer.

The ploidy distribution in the pericarp was determined in a wild-type plant and in optimal growth conditions. It would be interesting to study how distribution could be affected after impairment of the endoreduplication process or cell growth. For example, in the case of the overexpression of SlKRP1, it might be interesting to determine ploidy distribution using FisH because fruit have a strong reduction of ploidy levels in the pericarp without any visible growth phenotype (Figure 43D, [START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF].

We also observed that mean cell areas in 30dpa fruit pericarps were larger than in our study when comparing the same ploidy level. The larger mean cell area at 30DPA might be indicating that endoreduplication appears before the cell expand completely and could thus be giving potential to the cell to grow bigger and in SlKRP1-OE plant, even with reduce endoreudplciation in the pericarp, the normal cell size has been maintained in fully grown fruit suggesting the possibility to uncouple endoreduplication and cell growth [START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF]. Moreover, modelling approaches using Arabidopsis as a model have shown that endoreduplication process and cells growth could be stochastic in an organ (Kawate and Tsukaya 2017) and in Tomato pericarp cell age and ploidy are able to influence cell size [START_REF] Baldazzi | Organ-wide and ploidydependent regulation both contribute to cell-size determination: evidence from a computational model of tomato fruit[END_REF], it would be then possible that a minimal ploidy level is sufficient for cells to grow normally and assure their functions. We also observed that the size of endoreduplicated cells was increased with ploidy levels at all stages and that cell size of the same ploidy level in an older fruit was larger. This size difference could be due to cell expansion that can be more important or because the population of cells with the sameploidy level at 12DPA could be composed of newly formed cells but also older cells that have enlarged more.

We demonstrated that Oligo-FisH combined with a semi-automated image acquisition process can be efficiently used to determine ploidy levels in three developmental stages and plugin development would help to make this method a powerful cytogenetic approach to evaluate change of ploidy. This would be valuable to enrich the data for other time points such as earlier developmental stages for example to spot cell newly endoreduplicated in the forming M' layers in order to compare cell size and determine if cell size is increasing before or after endoreduplication. Obtaining ploidy distribution in the ovary would help confirming the hypothesis that the most central cells of the pericarp are 8C. Ploidy mapping in the developmental stages between 12DPA and 30DPA could be also interesting data to gather in order for example to see if indeed cell size in an older fruit for a given ploidy levels is higher. Lastly, the study of other zones of the fruit such as above or under the equatorial sections could help to see if endoreduplication is distributed and progressing in the same way compared to the equatorial region. This information could then be used to model the spatial and temporal organization of endoreduplication in the pericarp.

Ploidy mapping using FisH corresponds to static images of ploidy levels at different stages. Ploidy determination in living cells by CRIPSR imaging method (Chapter 2.I.C) Visualization of genomic loci in living plant cells using CRISPR/Cas9 technology) that uses the capability of a deficient Cas9 to specifically target a locus with the help of the sgRNA could give new information on dynamic of endoreduplicated loci. For example, this method could help to measure distance and map localization of several loci whose expression is differentially regulation in function of endoreduplication level or in response to abiotic stresses for example (osmotic stress, heat stress or cold stress etc.) at different ploidy levels.

Perspective How is the ploidy-specific expression regulated?

Our ploidy-dependent transcriptome data are underlying the existence of ploidy specific regulation of gene expression that is different than the global shift of expression. In the introduction ("Endoreduplication effect on genome regulation of transcription"), we have seen that chromosome positioning, chromatin condensation, DNA methylation and transcription factor binding are the different mechanisms that might allow a differential gene expression. In the case of Tomato pericarp, transcriptomic data and ploidy mapping are now available for several early developmental stages, 6DPA, 9DPA and 12DPA and at the end of the fruit growth before ripening, at 30DPA [START_REF] Bourdon | In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)[END_REF]. Using these data, we can now understand how the transcriptional dynamics during endoreduplication are regulated, particularly by searching regulating transcription factors. It is known that co-expressed genes can have common elements in their promoter allowing their co-regulation [START_REF] Babu | Structure and evolution of transcriptional regulatory networks[END_REF]). To identify the potential regulators of ploidy-specific expression, we could fisrt identify the putative Transcription Factor Binding Sites (TFBS) that would be overrepresented in the promoters of co-expressed genes during endoreduplication. Based on the enriched TFBS identified, the next step of the analysis would be to identify TF that are binding these sites for different sets of co-expressed genes. We would mainly focus on TF implicated in favoring expression of genes in higher ploidy levels to know how endoreduplication controls expression. Such approach has allowed to uncover conservation of regulatory elements implicated in leaf senescence in Wheat [START_REF] Borrill | Identification of Transcription Factors Regulating Senescence in Wheat through Gene Regulatory Network Modelling[END_REF]. The last step in the identification of potential TF implicated in these differential expressions would be to perform electrophoretic mobility shift assays and transient expression assay to confirm the ability of the TFs to bind the promoters of targets and ability to induce activation or repression of gene expression.

It would also be also interesting to study if, in the pericarp, epigenetic changes, including chromatin conformation, chromosome organization or DNA methylation, occur as a function of endoreduplication, and influence the ploidy specific gene expression. It has been shown that endoreduplication could be associated with more accessible chromatin [START_REF] Nagymihály | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF][START_REF] Dorrity | The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution[END_REF]. To study chromatin conformation, new datasets need to be produced, ideally obtained from sorted nuclei of the same ploidy levels at the same developmental stages of fruit development. These data would correspond to ChIP-seq analysis targeting active or repressive histone marks in ploidy sorted nuclei allowing to identity genes associated with changes in chromatin condensation in each ploidy levels. Hi-C would be employed to assay the 3-D chromatin conformation according to ploidy levels (Figure 43C). Overall, these data could help to better understand the effect of endoreduplication in modifying genome organization potentially related to changes in gene expression in tomato pericarp. To gain insight into genome organization as a function of endoreduplication imaging approaches could also be used. Genes found as DE in the ploidy transcriptome could be visualized and localized within the nucleus using oligo FisH at different developmental stages. By measuring their distances to nucleus elements such as nucleoli, nucleus membrane and other loci, the organization of the loci in the nucleus can be determined. Oligo FisH could be used as a mapping tool to localized structure that could be detected with Hi-C, as for example TAD-like structures (Figure 43B). This will bring new information about localization of DE genes in the nucleus in function of ploidy levels potentially revealing localization pattern for genes in relation to endoreduplication level or a co-localization of the co-expressed genes.

Lastly, concerning the ploidy specific gene expression regulation, it would be interesting to study the activity of the newly formed alleles by endoreduplication, for example to know if all copies of the genes are expressed or to assay distribution of mRNA in the cell in function of ploidy levels. To do so, gene expression could be assayed in cells using single molecule FisH (smFISH). smFISH is a variation of FisH similar to Oligo FisH using similar DNA oligonucleotides to target RNA molecules and thus allows tracking of single molecules of RNA. By a combination of two colors for the fluorochrome, one for exons and introns, the transcription loci can be visualized (Figure 43D). SmFisH has been successfully used to study single cell regulation of transcription and mRNA dynamic of flowering genes under cold influence in meristem of Arabidopsis [START_REF] Rosa | Mutually exclusive sense-antisense transcription at FLC facilitates environmentally induced gene repression[END_REF], the authors shows that a cold responsive gene is able to inhibit FLOWERING LOCUS C, a gene responsible for the positive regulation of flowering in plants. The data obtained with smFisH would allow to determine if all copies of endoreduplicated genes are transcribed for DE genes and non-DE genes for example.

Spatiotemporal gene expression in the pericarp is correlated to ploidy levels.

By combining ploidy distribution data and transcriptomic data, we have shown that by weighting gene expression as a function of ploidy level proportion in a cell layer, we were able to estimate the spatiotemporal expression of genes in the pericarp. We observed in the representation of expression clusters in function of cell layers that genes highly expressed in high ploidy levels in the mesocarp have an oscillating expression pattern from one layer to another. This oscillation is creating local minima of gene expression that could be important because it means that whole set of genes are differentially expressed between neighbor cells. These variations could be artefactual because we know that 32C in not the maximal ploidy level at 12DPA and we know that there is also 4-5% of 64C (Figure 25A) that we have identified in the pericarp of 12DPA fruit in M layers (Figure 37C). These drops of expression can probably be due to missed expression data from 64C that we can expect to be higher than 32C for genes in clusters 12-4 and 12-5. However, as the ploidy mapping is done from the outer epidermis to the inner epidermis in the pericarp, "vertically" by cell layer, the "horizontal" information, mapping within a layer, is not accessible. It could be interesting to gather absolute spatial gene expression information in the pericarp to measure the spatial effect of endoreduplication on gene expression inside a cell layer where two neighbor cells can have different ploidy levels. To obtain this absolute spatial information in the pericarp the recently developed transcriptomic method named "spatial transcriptomic" (Figure 43A) could be used. Spatial transcriptomic allows to measure the gene expression in situ within a tissue sample at around 2µm of resolution [START_REF] Vickovic | High-definition spatial transcriptomics for in situ tissue profiling[END_REF] thank to a chip that have areas with multiple wells containing unique barcoded probe allowing to map each sequencing read.

Towards the identification of ploidy marker genes

We used the tomato ploidy transcriptome in association with the Arabidopsis root ploidy transcriptome [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF] to identify genes that could be specific markers of ploidy levels. We have been able to identify common markers for 2C to 16C ploidy levels. Further experiments would be required to determine if markers are indeed specific to ploidy levels. To do so, the marker genes could be tested by qPCR in a large set of nuclei, sorted based on ploidy level, from different organs and developmental times. In addition, the promoters of these potential marker could be used to produce fluorescent transgenic reporter plants and verify their expression specifically in the expected ploidy level. The marker genes can be useful in several cases:

-to determine the ploidy levels of cells for which we would have the transcriptome. This type of analysis could be applied in the case of single cell RNA seq or spatial transcriptome analysis. Marker genes found in Arabidopsis ploidy root transcriptome have been used to predict ploidy map of the root [START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF].

-to use promoters to express reporter genes in cells of specific ploidy levels.

Using several fluorescent colors, we could have plants expressing different fluorescent colors to visualize different ploidy levels simultaneously in the tissues. These reporter plants could be used to visualize ploidy distribution in mutant plants for example after crossing reporter lines with mutant lines.

-Using ploidy specific marker gene promoters can be useful to express transgenes having a known or potential role in endoreduplication regulation to increase or limit endoreduplication such as SlCCS52A [START_REF] Mathieu-Rivet | Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato[END_REF] or SlKRP [START_REF] Nafati | The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth[END_REF], respectively. This approach will limit pleiotropic effect of overexpressing a gene in the plant or overexpressing a knock down construct. SlCCS52A for example was expressed using a 35S promoter and resulted in pleiotropic effects giving dwarf plants limiting interpretation of phenotype on fruits.

Ploidy specific promoters could be used in a cell specific genome editing approach (Decaestecker et al., 2019, Bollier et al., 2020), to knock-out a gene or more to induce precise changes in endoreduplication. Those new plants could be inspected in a ploidy specific manner using Oligo-FisH methods and/or ploidy specific transcriptome to see the influence of ploidy distribution in the pericarp.

Establishment progress and role of endoreduplication in the pericarp and Tomato fruit growth

The ploidy mapping at 6DPA revealed that endoreduplication is already well established in the pericarp at this stage and most of mesocarp cells are already 16C. Moreover, the expression profile of ploidy DE genes at 6DPA showed that 4C nuclei are identified as nuclei from cells in division but not endoreduplication, thus do not correspond to 4C endoreduplicated nuclei. Both ploidy map and transcriptome data suggest that at 6DPA we already missed the genetic switch to endoreduplication in the cell of pericarp or at least with our analysis. It could be valuable to have information about earlier developmental stage such as the day after anthesis and/or around 3DPA where normally cell expansion is highly active and maybe endoreduplication as well in the pericarp [START_REF] Renaudin | Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp[END_REF], this information could give new insight about signaling of endoreduplication in M layers and newly formed M' cells. Also, it will be highly important for future project to dissect and study the role of the ploidy specifically expressed genes in endoreduplication progress and control in the pericarp. Moreover, ploidy specific transcriptome could be supported by equivalent work in stress condition or different genetic background (other tomato varieties or mutant background) in order to assay the effect of variation of endoreduplication in the pericarp and further increase knowledge about its exact role in the Tomato fruit development.

able to use bioinformatic tool and understand the results. The student has a personnal training on their project in order to be able to do alone the first step of the analysis.

Transversal training "Anglais scientifique 2° session

PAPIRUS (Parcours d'accompagnement pour publier et communiquer en science) EDEN (Ecole des doctorants et encadrants de l'INRA

Together with Nathalie, we participated to EDEN14 (école des doctorants et encadrants de l'INRA). This training helped us to identify the difficulties presented before and how to improve team work together with Nathalie but also other coworkers.

MOOC-INTRODUCTION A LA STATISTIQUE AVEC R -SESSION 11

Find a post doc abroad

This was a presentation of the different type of post-doc and options that exists in order to find a post-doc.

Complementary activity

Teaching with a pedagogic team in practical course for first year students and animated a course about genome editing both theoretical and practical for Master2 students.

  specific transcriptome during early Tomato fruit development: experimental setup and data-preprocessing B) Gene expression is changing during early fruit development and based on ploidy levels. C) Biological processes expressed as a function of ploidy. a. Biological processes expressed in 6DPA fruits b. Biological processes expressed in 9DPA fruits c. Biological processes expressed in 12DPA fruits D) Dynamics of ploidy specific gene expression in the course of early fruit development E) Comparing ploidy specific transcriptome and laser microdissected pericarp transcriptome. F) Quantification of ploidy on sorted nuclei by FisH: Assessing the efficiency of a 5s rDNA oligo probe. G) Endoreduplication occurs early during Tomato pericarp development and ploidy levels rapidly increase in the central layers of the pericarp.

Figure 1 .

 1 Figure 1. Cell cycle and endoreduplication progression and polytene chromosome illustration.

Figure 2 .

 2 Figure 2. Endoreduplication during Arabidopsis hypocotyl.

Figure 3 .

 3 Figure 3. Endoreduplication during Arabidopsis leaves development

Figure 4 .

 4 Figure 4. Endoreduplication and trichome development.

Figure 5 .

 5 Figure 5. Schematic representation of root morphology and organization.

Figure 6 .

 6 Figure 6. Schematic representation of nodule tissue ontology.

Figure 7 .

 7 Figure 7. Illustration of Arabidopsis thaliana sepals and epidermal giant cells.

Figure 8 .

 8 Figure 8. Examples of endoreduplication occurrences in fruits.

Figure 9 .

 9 Figure 9. Endoreduplication during Tomato fruit pericarp development Figure 10. Endoreduplication occurrence through Tomato fruit pericarp development.

Figure 11 .Figure 12 .

 1112 Figure 11. Changes of gene expression in endoreduplicated root hair cells Figure 12. Endoreduplication can induce high expression of specialized genes in Maize, Arabidopsis and Tomato.

Figure 13 .

 13 Figure 13. Chromosome position within the nucleus can define its transcriptional program.

Figure 14 .Figure 15 .

 1415 Figure 14. Organization of chromosome territories in Animals and plants Figure 15. Chromatin structure is dependent on the nucleosome composition and histone post-translational modifications.

Figure 16 .

 16 Figure 16. CRISPR/Cas9 based methods for loci visualisation in living cells.

Figure 17 .

 17 Figure 17. Use of spike-ins in the context of global shift of expression in tomato pericarp cells.

Figure 18 .

 18 Figure 18. Venn diagram of non-differentially expressed genes found in 6-, 9-and 12DPA ploidy transcriptomes and their associated biological processes.

Figure 19 .

 19 Figure 19. Repeated sequences used as targets for FisH and CRISPR imaging to quantify ploidy. Figure20. Flowchart representation of the semi-automated procedure of ploidy quantification in the tomato pericarp cells.

Figure20bis.

  Figure20bis. Illustration example of automatic nuclei assignment to cell layer for ploidy mapping Figure 21. Flowchart representation of the steps performed by the plugin to identify and count FisH spots.

Figure 22 .

 22 Figure 22. Example of application of the Histogram segmentation tool before and after the filtering steps.

Figure 23 .

 23 Figure 23. Example of plugin assisted FisH spot counting and issues.

Figure 24 .

 24 Figure 24. Illustration of CRISPR-imaging observations after transient and stable transformation.

Figure 25 .

 25 Figure 25. Ploidy sorted nuclei from 6, 9 and 12 DPA pericarp fruits show differential gene expression depending on ploidy and developmental stage.

Figure 26 .Figure 27 .Figure 30 .Figure 31 .

 26273031 Figure 26. Clustered normalized expression profiles of differentially expressed genes in pericarp of 6-9-12DPA fruits Figure 27. Clustered normalized expression profiles of differentially expressed genes in pericarp of 6-9-12DPA fruits Figure 28. Overrepresented biological processes in the different expression clusters from 6 DPA pericarp Figure 29. Overrepresented biological processes in the different expression clusters from 9 DPA pericarp Figure 30. Overrepresented biological processes in the different expression clusters from 12 DPA pericarp Figure 31. Overlaps between DE genes from the different clusters of 6DPA, 9DPA and 12DPA fruits.

Figure 32 .Figure 33 .

 3233 Figure 32. Representation of cuticle synthesis genes and their expression in relation to ploidy levels in the pericarp Figure 33. Significant overlaps between ploidy specific transcriptome and LM pericarp transcriptome of Shirozaki et al., 2018.

Figure 35 .

 35 Figure 35. Morphology of Tomato pericarp sorted nuclei based on their ploidy and evaluation of the 5sr DNA oligo FisH probe for ploidy quantification.

Figure 36 .

 36 Figure 36. Nuclear shape is modified with increasing ploidy levels.

Figure 37 .

 37 Figure 37. Ploidy distribution, nuclear volumes, and cell sizes in 6, 9 and 12DPA Tomato pericarps.

Figure 38 .

 38 Figure 38. Dynamic of nuclear volumes during early fruit development Figure 39. Data integration of ploidy transcriptome and FisH ploidy quantification reveals a spatiotemporal ploidy specific gene expression.

Figure 40 .Figure 41 .Figure 42 .Figure 43 .

 40414243 Figure 40. CDKA1 and CDKB1/2 expression in relation with ploidy levels in pericarp cell layers

Figure 1 .

 1 Figure 1. Cell cycle and endoreduplication progression and polytene chromosome illustration. (A) Illustration of the different phases of cell cycle (left panel) and endoreduplication progression (right panel). The evolution of cell size and chromosome morphology during the different phase is represented. G1: Gap phase 1; S :DNA Synthesis phase; G2: Gap2 phase; M: mitosis phase, G: Gap phase. (B) Polytene chromosome visualization. Left panel: polytene chromosome within an intact larval salivary gland nucleus of Drosophila melanogaster, as revealed by green fluorescent protein (GFP) on left panel. Right panel: giant chromosomes with different degrees of polyteny visualized in light microscopy. Adapted and modified from Spradling, 2017 and Dyka et al., 2016.

Figure 2 .

 2 Figure 2. Endoreduplication during Arabidopsis hypocotyl. (A) Endoreduplication patterns in the hypocotyl of light-grown Arabidopsis seedlings. (B) Endoreduplication patterns in the hypocotyl of dark-grown Arabidopsis seedlings A and B, adapted and modified from Gendreau et al., 1999.

Figure 4 .

 4 Figure 4. Endoreduplication and trichome development.

Figure 5 .

 5 Figure 5. Schematic representation of root morphology and organization. The dash lines represent in yellow the area considered as root meristem where cells divide, in rose the transition zone where cells enter endocycle and in green the elongation and differentiation zone where cells continue to endoreduplicate and start to grow and differentiates. The black dash lines represent an artificial frontier where cells in each side have different ploidy levels, for example the 2C-4C boundary represent areas where on the left cells are mostly 2C and on the right 4C. The root colorized areas represent in yellow the auxin gradient that is diminishing when progressing toward the transition zone (local minimum of auxin concentration) and in blue the area where cytokinin is found (transition zone).Figure modified and adapted from[START_REF] Breuer | Endocycling in the path of plant development[END_REF][START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]

  Figure 5. Schematic representation of root morphology and organization. The dash lines represent in yellow the area considered as root meristem where cells divide, in rose the transition zone where cells enter endocycle and in green the elongation and differentiation zone where cells continue to endoreduplicate and start to grow and differentiates. The black dash lines represent an artificial frontier where cells in each side have different ploidy levels, for example the 2C-4C boundary represent areas where on the left cells are mostly 2C and on the right 4C. The root colorized areas represent in yellow the auxin gradient that is diminishing when progressing toward the transition zone (local minimum of auxin concentration) and in blue the area where cytokinin is found (transition zone).Figure modified and adapted from[START_REF] Breuer | Endocycling in the path of plant development[END_REF][START_REF] Bhosale | A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation[END_REF]

Figure 6 .

 6 Figure 6. Schematic representation of nodule tissue ontology. (A) Indeterminate nodule primordium with cell ontology that is indicated by the colored layers that will be involved in nodule formation. C3, C4 and C5 layers are the third, fourth and fifth layers away from epidermis. (B) Differentiated nodule with primordium cell ontology that is indicated with same color than color used for undifferentiated primordium. Between dashed lines are areas where indicated ploidy levels are observed. Double arrows indicate areas of nodules implicated in the nodule formation and differentiation.Adapted and modified fromXiao et al., 2016.

Figure 7 .

 7 Figure 7. Illustration of Arabidopsis thaliana sepals and epidermal giant cells. (A) Wild-type Arabidopsis flower. Sepals are indicated (s) (B, C) Scanning electron micrograph of a mature Arabidopsis sepal. The typical giant cells of the sepals are colored in pink. Adapted from Roeder et al., 2010.

Figure 8 .

 8 Figure 8. Examples of endoreduplication occurrences in fruits. (A) Drawing of the cells composing the pericarp of Ememurus robustus fruit showing the cellular organization and cell size diversity. (B) Drawing of nuclei observed in Ememurus robustus at different ploidy stages. The top left nuclei correspond to a polyploidy nucleus (big nucleus) and a diploid nucleus (small nucleus). On the right and at the bottom are represented chromosomes structures observed in polyploid and tetraploid nuclei, on the right and at the bottom, respectively. (C) Occurrence of endoreduplication in fleshy fruits. The maximal number of endocycles determined in fully ripened fleshy fruits was plotted against the duration of fruit growth until ripening. (D)Photographs of teosinte (maize ancestor, left) and cultivated maize (right) caryopses.(E) Ploidy map of teosinte caryopsis at 11 days after pollination (F) Ploidy map of maize caryopsis at 16 days after pollination A and B adapted from[START_REF] Lauber | Untersuchungen über das Wachstum der Früchte einiger Angiospermen unter endomitotischer Polyploidisierung[END_REF]. C from Chevalier et al., 2011. D and E from Dermastia et al., 2009. F adapted from Vilhar et al., 2002.

  flowers in Solanum lycopersicum cv. WVA106) of the flower development according toBrukhin et al., 2003.

Figure 9 .

 9 Figure 9. Endoreduplication during Tomato fruit pericarp development (A) Anatomy of a Tomato (Solanum lycopersicum cv. WVA106) fruit. Equatorial section. Scale bar = 1cm. (B) Flow cytometry histograms of DAPI stained nuclei extracted from different tissues of Tomato fruit at breaker stage. (C and D) Structure of the pericarp of the cherry tomato WVA106 at anthesis (C) and 4DPA (D). Blue circles indicate vascular bundles, yellow arrows new cell walls and red indicates extracellular spaces delimitating the M cell layers. (E) Illustration of different developmental stage of tomato fruit growth and the associated Phase according to Gillaspy et al., 1993 Figures modified and adapted from Joubès et al., 1999 (B) and Renaudin et al., 2017 (C and D). (E) from Jean-Philippe Mauxion (personal communication).

Figure 10 .

 10 Figure 10. Endoreduplication occurrence through Tomato fruit pericarp development. (A to F) Evolution of cell number and cell volume in the different pericarp cell layers during fruit development (WVA106). Variables are expressed as relative values with reference to the value of 1 at anthesis (dot line). (G) Illustration of the morphological differences between three fruits and pericarp sections of different Tomato cultivars at breaker stage. From left to right is a fruit of Grosse de Gros, a fruit of Ferum 26 and a fruit of WVA106. Scale bars: left = 1cm, right = 1mm (H) Developmental dynamic of ploidy levels in Wva106 pericarp. Top panel: evolution of 2C, 4C, 8C, and 16C ploidy levels. Bottom panel: evolution of 32C, 64C, 128C and 256C ploidy levels. (I) Ploidy mapping in a mature green tomato pericarp. The top picture is showing the pericarp structure of WVA106 at mature green stage. Scale bar = 500 μm. At the bottom is represented the ploidy map showing the frequency of nuclei by cell layer in the pericarp. Figures modified and adapted from Cheniclet et al., 2005 (A and H),Bourdon et al., 2011 (I) andRenaudin et al., 2017 (B to G)

Figure 11 .

 11 Figure 11. Changes of gene expression in endoreduplicated root hair cells (A) Top panel: Hair cells median total mRNA is decreasing across pseudotime (predicted developmental time, for hair cell from low endoreduplicated/differentiated to highly endoreduplicated/differentiated). n is the number of genes used to get Median total mRNA. Low panel: Comparison of median total mRNA for hair-cell-specific genes (in red) to a comparable random set of genes (in blue). (B) Compared mRNA production rate across ploidy levels. (A) and (B) are adapted and modified from Jean Baptiste et al., 2019.

Figure 12 .

 12 Figure 12. Endoreduplication can induce high expression of specialized genes in Maize, Arabidopsis and Tomato. (A) Analysis of ZEIN expressed genes in the Maize endosperm. The expression is represented as a percentage of the total detected gene transcript. (B) Functional enrichment of transcripts peaking at a given endoploidy level for hormone and stress response go categories in Arabidopsis root cortex cells. (C) Expression profiles of differentially expressed genes clusters found in Tomato pericarp sorted nuclei. (A) is adapted and modified from Leiva-Neto et al., 2004. (B) is adapted and modified from Bhosale et al., 2018. (C) is adapted and modified from Pirrello et al., 2018

Figure 13 .

 13 Figure 13. Chromosome position within the nucleus can define its transcriptional program. (A) Example of human genome organization. Chromosomal function is maintained by individual chromosomes positions into chromosome territories (colored patches).Gene rich portions of chromosomes are generally positioned more internally in the nucleus. Genes are less expressed when located in the middle of the chromosome territory and are delocalizes/repositioned to its periphery to be placed in interphase areas between chromosomes territories near transcriptional factories (nuclear bodies, NB) to be expressed. (B) From left to right: fluorescence microscopy observation of a ABA inducible CRISPR based tool (named CRISPR GO) allowing to delocalize genomic loci to the nuclear periphery, here a locus name Chr3q29. Next, barplots represent the number of loci delocalized to the nuclear periphery and number of cells with delocalized loci, on the left, and gene expression of genes associated to delocalized locus Chr3q29 on the right, both after ABA induction (A) is adapted and modified fromSawyer et al., 2016. (B) is adapted and modified from[START_REF] Wang | CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization[END_REF]

Figure 14 .

 14 Figure 14. Organization of chromosome territories in Animals and plants (A) Schematic representation of DNA packing in the nucleus in Animal cells. (B) Schematic representation of chromosome organization in cells of Arabidopsis with different ploidy levels. A 4C cell is compared to a 16C cell showing chromatin relaxation and conservation of chromosome territories. (C) Representation of two chromosome territories in Arabidopsis in nuclei of different ploidy levels (2C to 64C). In red is represented one arm of the chromosome 1 and in green one arm of the chromosome 3 and in pink are indicated the chromocenters. (A) is adapted and modified from Szabo et al., 2019. (B) and (C) are adapted and modified from Schubert et al., 2012.

Figure 15 .

 15 Figure 15. Chromatin structure is dependent on the nucleosome composition and histone post-translational modifications. (A) Schematic representation of nucleosome octamer structure and formation with Histone3 (H3), Histone4 (H4) and both Histone2 A and B (H2A and H2B). (B) Histone post-translational modifications effect on gene expression related to seed dormancy or germination. In red are indicated modifications with a repressive effect and in green active modifications. ATXR = ARABIDOPSIS TRITHORAX-LIKE PROTEIN, HDA = HISTONE DEACETYLASE, HUB = HISTONE MONO-UBIQUITINATION, JMJ = JUMONJI DOMAIN-CONTAINING PROTEIN, KYP = KRYPTONITE, LDL = LYSINE-SPECIFIC HISTONE DEMETHYLASE, PRC = POLYCOMB REPRESSIVE CORE COMPLEX, SNL = SIN3-LIKE (histone deacetylase), ULT = ULTRAPETALA. (A) is adapted and modified from Chen et al., 2014. (B) is adapted and modified from Zhao et al., 2019.

Figure 16 .

 16 Figure 16. CRISPR/Cas9 based methods for loci visualization in living cells. (A) Direct labelling approach using a deactivated Cas9 (dCas9). Cas9 is deactivated by two mutation in each nuclease domains (red stars) and fused to a fluorescent protein to visualized binding localization. (B) Indirect methods using dCas9 and modified sgRNA with virus aptamers or PBS. dCas9 binds the locus of interest with help of the sgRNA and sgRNA is bound by aptamer binding proteins (purple, upper representation) or PUF protein (, lower representation).(C) Indirect methods using modified dCas9 with peptides epitopes. A tail of peptide epitopes is added to the dCas9 that recruits simplified antibody called scFV fused to fluorescent protein that allows localization of the binding.

Figure 17 .

 17 Figure 17. Use of spike-ins in the context of global shift of expression in tomato pericarp cells. (A) Fold change of spike-ins expression compared to global RNA expression in all possible comparisons of ploidy levels at 6DPA, 9DPA and 12DPA. The dotted lines are indicating the expected fold change between ploidy levels in the case of doubling of expression between two successive ploidy levels. (B) MA plots representing the mean number of normalized read counts in function of the log2 of fold change of reads counts between the compared ploidy levels. The red line is indicating the absence of differential expression (Fold change=0). Most genes are expected to have a fold change value around this line if there is no shift of expression between ploidy levels. The green dotted line is indicating the expected fold change of expression if there is a shift of expression following ploidy levels.

Figure 18 .

 18 Figure 18. Venn diagram of non-differentially expressed genes found in 6-, 9-and 12DPA ploidy transcriptomes and their associated biological processes. (A) Overlap of 14843 non-DE genes found in 6-, 9-and 12DPA ploidy transcriptome. (B) Overrepresented biological processes in the non-DE genes found in common between stages. Dot plot showing the most representative and significant biological processes among non-DE genes. Dot size represents the number of genes in the concerned biological process and color scale is representing the fold enrichment of gene number compared to the expected number in the genome.

Figure 19 .

 19 Figure 19. Repeated sequences used as targets for FisH and CRISPR imaging to quantify ploidy. (A) Location of the targeted sequences used for FisH and CRISPR imaging on the chromosomes of Tomato. Three repeated sequences of interest for our study are represented, as green boxes for telomeres, a pink star for 5S rDNA locus and black circle for centromeres. (B) Sequence of a repeat unit of the 5S rDNA locus in Tomato. In red is indicated the sequence targeted by the FisH probe. In pale transparent colors are indicated the different sgRNA that have been designed and tested in transient and/or stable transformation.

Figure20.

  Figure20. Flowchart representation of the semi-automated procedure of ploidy quantification in the tomato pericarp cells.

Figure 20bis .

 20bis Figure 20bis. Illustration example of automatic nuclei assignment to cell layer for ploidy mapping (A) Example attribution of range of distance to cell layers for control image of semi thin section of pericarp (Renaudin et al., 2017) where cell layers are easily identified. (B) Example of distance calculation for sample image of FisH experiments in pericarp. Crosses and numbers indicate nuclei.

Figure 21 .

 21 Figure 21. Flowchart representation of the steps performed by the plugin to identify and count FisH spots. (A) Steps and tools used for nucleus segmentation that allow measuring nuclear volume and setting the boundaries where FisH spots can be found (B) Steps and tools used for FisH spots segmentation. The tools are applied only in the area identified by nucleus segmentation to limit segmentation of artefacts outside the nucleus.

Figure 22 .

 22 Figure 22. Example of application of the Histogram segmentation tool before and after the filtering steps. (A) Effect of the filtering steps on an image but also on the grey values that are covered by the yellow line on the image. (B) Histograms of the area represented in (A) with resulting segmentation of histograms in five classes. Most of the pixels are on the left of the histogram because these have low grey values while pixels of interests are localized on the right of the histogram.

Figure 25 .

 25 Figure 25. Ploidy sorted nuclei from 6, 9 and 12 DPA pericarp fruits show differential gene expression depending on ploidy and developmental stage. (A) Flow cytometry histograms of DNA quantities in nuclei of Tomato pericarps at 6DPA, 9DPA and 12DPA. The table represents the distribution of ploidy classes at 6, 9 and 12 DPA (2C to 64C) and n is the number of nuclei counted in a stage to determine ploidy proportions. (B) Principal component analysis (PCA) of ploidy specific transcriptome data. The two first components explain 80% of data variability. Samples are indicated as the corresponding developmental stage (e.g. the three D09.04C correspond to 4C nuclei of 9DPA pericarp). Font colors indicate the ploidy levels: yellow for 2C, green for 4C, orange for 8C, red for 16C and pink for 32C. Green contours indicate similar developmental stages and pink overlay similar ploidy levels. (C) Number of genes up-(yellow) and downregulated (blue) in pairwise comparisons of the expression values (false discovery rate ≤0.01, FC >2).

Figure 26 .

 26 Figure 26. Clustered normalized expression profiles of differentially expressed genes in pericarp of 6-9-12DPA fruits Nine clustered expression profiles of DE genes obtained with an ascendent hierarchical clustering for 6DPA (A), 9DPA (B) and 12DPA (C). On a cluster plot, the blue line represents the median profile and the top right number is the total number of genes.

Figure 28 .

 28 Figure 28. Overrepresented biological processes in the different expression clusters from 6 DPA pericarp (A) Dot plot showing the most representative and significant biological processes in each cluster. Dot size represents the number of genes in the concerned biological process and color scale the fold enrichment of gene number compared to the expected number in the genome. Below the dotblot are represented views of 6DPA ploidy specific clusters. The grey square indicates the ploidy levels analyzed at all developmental stages (4C to 16C).

Figure 29 .

 29 Figure 29. Overrepresented biological processes in the different expression clusters from 9 DPA pericarp (A) Dot plot showing the most representative and significant biological processes in each cluster. Dot size represents the number of genes in the concerned biological process and color scale is representing the fold enrichment of gene number compared to the expected number in the genome. Below the dotblot are represented views of 9DPA ploidy specific clusters. The grey square indicates the ploidy levels analyzed at all developmental stages (4C to 16C).

Figure 30 .

 30 Figure 30. Overrepresented biological processes in the different expression clusters from 12 DPA pericarp (A) Dot plot showing the most representative and significant biological processes in each cluster. Dot size represents the number of genes in the concerned biological process and color scale is representing the fold enrichment of gene number compared to the expected number in the genome. Below the dotblot are represented views of 12DPA ploidy specific clusters. The grey square indicates the ploidy levels analyzed at all developmental stages (4C to 16C).

Figure 31 .

 31 Figure 31. Overlaps between DE genes from the different clusters of 6DPA, 9DPA and 12DPA fruits. (A) Overlap between genes present in the different clusters at 6DPA, 9DPA and 12DPA. The number of genes in each overlap is shown for each comparison between two clusters. Empty boxes correspond to comparisons with no overlap.

Figure 32 .

 32 Figure 32. Representation of cuticle synthesis genes and their expression in relation to ploidy levels in the pericarp (A) Cutin and wax synthesis genes pathways are represented with only transcriptional regulators found differentially expressed in the clusters. Expression peaking at a specific ploidy level is symbolized by a colored circle (or twocolored half circles for expression peaking in two ploidy levels) in a box corresponding to a specific stage (6DPA, 9DPA and 12DPA). For example, cluster 6-2 would be represented by a purple/green circle for expression peak in 2C/4C levels whereas clusters 9-1, 9-2,12-1 and 12-2 are represented by a green circle for an expression peak in 4C and clusters 9-3 and 12-3 are represented by a red circle for 8C peak of expression. Abbreviations: KCS = KETOACYL-CoA SYNTHASE, CYP = CYTOCHROME P450, nsLTP = nonspecific LIPID TRANSPORT PROTEIN, CD2 = CUTIN DEFICIENT 2, MAH = MIDCHAIN ALKANE HYDROXYLASE, KCR = β-ketoacyl reductase, GPAT = GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE, ABCG = ATP Binding Cassette G, CER = ECERIFERUM, WSD + WAX ESTER SYNTHASE, LACS = LONG-CHAIN ACYL-CoA SYNTHETASE, CFL1 = CURLY FLAG LEAF 1, PAS = PASTICCINO, CUS = CUTIN SYNTHASE

Figure 33 .

 33 Figure 33. Significant overlaps between ploidy specific transcriptome and LM pericarp transcriptome of Shirozaki et al., 2018. (A) Overlap between genes from in the different clusters at 6DPA, 9DPA and 12DPA and Laser Microdissection Pericarp (LM) transcriptome. The number of genes in each overlap is shown for each comparison between two clusters. Empty boxes correspond to comparisons with no overlap. Significant overlap for P <0.05 is highlighted by light blue and for P < 0.001 by dark blue. (B) Ploidy map inferred from the comparison between the ploidy-specific transcriptome and the laser microdissected pericarp transcriptome. Colors are indicating LM parts of the pericarps corresponding to outer-epidermis in red, collenchyma in blue, parenchyma in yellow, vascular bundles in orange and innerepidermis in green. Adapted and modified from Shirozaki et al., 2018.

Figure 34 .

 34 Figure 34. Ploidy markers from a ploidy specific transcriptome of Arabidopsis thaliana root cortex (Bhosale et al., 2018) compared to the pericarp ploidy transcriptome. (A) Dot plot representing each overlapping gene between the ploidy dependent transcriptome of Arabidopsis root cortex and the ploidy dependent transcriptome of Tomato pericarp. A stronger opacity of dots is associated to the ploidy level a gene is supposed to be marker of, according to Bhosale et al., 2018. 2C markers are selected for 6-1 and 6-2. 4C markers are selected for 6-2,6-3,9-1,9-2,12-1 and 12-2. 8C markers are selected for 9-3 and 12-3 and 16C markers are selected for 9-4,9-5,12-4 and 12-5. (B) Venn diagram representing the overlap between selected markers in clusters of each stages for low ploidy clusters (2C and 4C). (C) Venn diagram representing the overlap between selected markers in clusters of each stages for high ploidy clusters (8C to 16C). (D) Venn diagram representing the overlap between kept markers in clusters of 9DPA and 12DPA for 8C clusters.

Figure 35 .

 35 Figure 35. Morphology of Tomato pericarp sorted nuclei based on their ploidy and evaluation of the 5SrDNA oligo FisH probe for ploidy quantification. (A) Representative illustration of nuclear morphology of different ploidy levels. The green dots correspond to the hybridization signal of the 5sr DNA oligo FisH probe. Scale bar = 5µm. (B) Boxplot representing the counted dots in nuclei sorted based on their ploidy level. (C) Boxplot representing nuclear volumes as a function of ploidy level (B) and (C) A Wilcoxon test was used to compare foci counts and nuclear size in relation to ploidy; number of nuclei studied per ploidy level =[START_REF] Baloban | Complementary and dose-dependent action of AtCCS52A isoforms in endoreduplication and plant size control[END_REF][START_REF] Berdasco | Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells[END_REF][START_REF] Ballicora | ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis[END_REF][START_REF] Atmodjo | Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex[END_REF][START_REF] Atmodjo | Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex[END_REF][START_REF] Ballicora | ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis[END_REF][START_REF] Al-Abdallat | Over-Expression of SlSHN1 Gene Improves Drought Tolerance by Increasing Cuticular Wax Accumulation in Tomato[END_REF]. The associated significance codes are: 0 '****' 0.001 '***' 0.01 '**' 0.05 '*

Figure 36 .

 36 Figure 36. Nuclear shape is modified with increasing ploidy levels. (A) Successive images following Z axis representative of nuclear morphology at different ploidy levels. Z step = 0.14µm. Scale bar = 10µm (B) Illustration of nuclear morphology of three nuclei per ploidy levels illustrating shape complexification with increasing ploidy levels. Scale bar = 5µm

Figure 37 .

 37 Figure 37. Ploidy distribution, nuclear volumes, and cell sizes in 6, 9 and 12DPA Tomato pericarps.(A) to (C) Ploidy maps associated with a representative pericarp section of 6DPA (A), 9DPA (B) and 12DPA (C) fruit. The proportion of each ploidy level per layer is associated with a color scale. n is the number of observed nuclei per layer. E1, E2 and E3 correspond to the outer epidermis (layers 1 to 3). M and M' layer correspond to distinct mesocarp layers (layers 4 to 12 or 13). I2 and I1 correspond the inner epidermis (layers 13 and 14 or 14 and 15).

(

  D) to (F) Nuclear volume of each observed nucleus per layer colored by its associated ploidy level (color scale on the right) at 6 (D), 9 (E) and 12DPA (F).

Figure 38 .

 38 Figure 38. Dynamic of nuclear volumes during early fruit development Boxplot representing nuclear volumes as a function of ploidy level. A Wilcoxon test was used to compare nuclei volumes between consecutive ploidy levels. Number of nuclei studied per ploidy level = -6DPA: 2C = 45, 4C = 65, 8C = 72, 16C = 56, 32C = 5; -9DPA: 2C = 29, 4C = 73, 8C = 60, 16C = 83, 32C = 36; -12DPA: 2C = 13, 4C = 34, 8C = 34, 16C = 58, 32C =70, 64C = 12. The associated significance codes are: 0 '****' 0.001 '***' 0.01 '**' 0.05 '*

Figure 39 .

 39 Figure 39. Data integration of ploidy transcriptome and FisH ploidy quantification reveals a spatiotemporal ploidy specific gene expression. Spatiotemporal gene expression in relation with the ploidy levels in each layer for the five clusters at 6DPA (A), 9DPA (B) and 12DPA (C).

Figure 40 .

 40 Figure 40. CDKA1 and CDKB1/2 expression in relation with ploidy levels in pericarp cell layers

Figure 41 .

 41 Figure 41. Cell cycle and endoreduplication related genes expression in relation with ploidy levels in pericarp cell layers (A) Prediction of spatial expression of. genes encoding positive (green area) or negative (red area) regulators of cell cycle, endoreduplication or both. Red stars indicate genes that are found DE. (B) Table of genes used for this figure with the associated references showing their function to regulate cell cycle, endoreduplication or both.

Figure 42 .

 42 Figure 42. Dynamic changes of ploidy levels in the different pericarp cell layers during early fruit development from 6DPA to 12DPA (A) Percentage of nuclei for each ploidy level in each cell layer for 6DPA, 9DPA and 12DPA pericarps. Color scale indicates ploidy levels.

  The ploidy specific transcriptome was obtained from 90 tomato plants that were cultivated in three cultures between April and June 2017 for a final harvest of 135 fruits pericarp for each culture (total = 405 fruits) at 3 different developmental stages, 6DPA, 9DPA and 12DPA. Pericarps from 20 fruits were used for ploidy sorting of 200k nuclei of 2C or 4C or 100k nuclei for other ploidy levels A 2mL screw cap tube contains 100k nuclei sorted in 900µL of TRIzol™ Reagent resulting in a final volume of around 1.2mL. Before extraction 1µL of a dilution 1:1000 of ERCC RNA Spike-In Mix (THERMOFISHER SCIENTIFIC) was added as a control of RNA quantification (more details in Chapter 2.I.A): Use of spike-in to normalize gene expression in a global shift in expression context). The mix of nuclei and spike-ins were shortly vortexed and rested for 5mn at room temperature before adding 200µL of Chloroform then tubes are agitated by hand for 15sec and incubated for 3min at room temperature. After the incubation, tubes are centrifuged for 15mn at 12000g and 4°C, this centrifugation allow the creation of phases and the supernatant (aqueous phase) is pipetted (around 840µL) and transferred and mixed in a new tube that contains 420µL of 100% ethanol. The next steps follow the protocol of the commercial solution from RNeasy Micro Kit (QIAGEN) from step 3. A final volume of 14µL of RNA is obtained from the extraction and due to low input of material at first, low concentrations of RNA are obtained.

Figure 43 .

 43 Figure 43. Summary diagram of PhD and its perspectives. Going from the center to the outside: PhD project in pink, information that can be obtained from the PhD data in green and new information linked to the PhD that need new experiments to be obtained in blue. (A) Increasing knowledge of ploidy specific gene expression (B) Dynamic of loci behavior during endoreduplication (C) Experiments giving new information about genome organization and loci behavior during endoreduplication (D) Experiments giving new information about the role of endoreduplication in Tomato fruit Plain arrows represent direct links between information while dotted arrow represent inferred information.

Module 1 - 5 -

 15 From Research to Publication", "Anglais scientifique 2ème session Module 4 Conference Talk" and, "Anglais scientifique 2ème session Module From text to talk". I am concentrating my transversal training on that point by doing three trainings named "Anglais scientifique 2ème session Module 4 Conference Talk", "Anglais scientifique 2° session Module 1 -From Research to Publication" and "Anglais scientifique 2ème session Module 5 -From text to talk". These three trainings should help me to learn about rhetorical methods, vocabulary, grammar and also about participating to debates, knowing how to ask a question without hesitations and for networking sessions.

I

  refreshed my statistical basis from an INRA training in March 2018. This was coupled with another training by following a MOOC (end of March 2019) done by two professors from "Université Paris Sud" (Introduction à la statistique avec R). Taken together, these two trainings helped me understanding and learning on how to use R to perform statistical analysis in order to analyze my data with more reliability.

Remerciements

Chapter 2: Results

Chapter 3: Materials and methods

Biological materials Plant materials and Cultures

Solanum lycopersicum

Tomato plants Solanum lycopersicum Mill. Cv. West Virginia 106 (WVA106) were cultivated in greenhouse in 5L pots with fertilization. Plants were cultivated with a photoperiod of 16H light condition at 25°C and 8h night at 20°C and a relative humidity between 70 and 75%. For RNA-seq and FisH, individual flowers were tagged on the day of anthesis, fully opened flowers that is wilt the next day. Fruits were harvested at specific stages 6DPA, 9DPA and 12DPA.

Nicotiana benthamiana

Tobacco plants Nicotiana benthamiana were cultivated in greenhouse in Ø13cm pots with subirrigation. Plants were cultivated with a photoperiod of 16H day at 25°C and 8h night at 20°C and a relative humidity between 70 and 75%.

Bacterial stains and culture media

Escherichia coli (E.coli) TOP10 strain from INVITROGEN (catalog number: C404052) have been used for the cloning of the plasmid used in CRISPR-imaging experiments. Agrobacterium tumefacien GV3101 and C58C1 stains have been used for stable and transient plant transformations of Tomato and Tobacco. TOP10 bacteria are cultivated at 37°C and Agrobacteria are cultivated at 28°C. Bacterial culture medium for both E.coli and Agrobacteria are commercial LB media from DUCHEFA BIOCHEMIE (catalog number: L1704) containing Bactotryptone 1% w/v, yeast extract 0.5% w/v and NaCl 1% w/v with appropriate antibiotics when necessary.

Cytological analysis

Isolation of tomato nuclei and flow cytometry

Nuclei were prepared 20 sections of tomato pericarp fruits at 6DPA and 10 sections for 9DPA and 12DPA. Nuclei isolation is performed as described in [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF] and resumed hereafter. Pericarp sections are chopped using a razor blade in 2mL of Gif Nuclear Buffer (GNB) and the suspension was filtered twice through a 48 µm nylon mesh before adding DAPI (4',6-diamidino-2-phenylindole).

GNB is composed of 45 mM MgCl2, 30 mM Sodium-Citrate and 60 mM MOPS pH 7.0, 1% PVP 10.000, 0.1% Triton X-100 and 10 mM sodium metabisulfite (S2O5Na2).

Nuclei were sorted according to their ploidy levels with a MoFlo ASTRIOS EQ (BECKMAN COULTER) and harvested either in a screw cap 2mL tube containing 900µL of TRIzol™ Reagent (THERMOFISHER SCIENTIFIC) or on a microscope glass slides (Superfrost) prepared with a 10 µl of a cushion made of 2M sucrose, 50% GNB and 1% paraformaldehyde. 100k nuclei were sorted in each tube for RNA-seq, mixed by inversions and frozen in

Imaging

All microscopy experiment has been realized on a confocal microscope Zeiss LSM880 with airyscan piloted with Zen Black 2.1. FisH acquisition were performed using x63 objective with oil immersion in airyscan mode.

Image analysis

Airyscan raw images were saved on the computer used to pilot the microscope and processed using ZenBlue

Mapping and normalization of RNA-Seq data

Sequence quality checking, read trimming and mapping was performed as in [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. The Tomato reference genome used is the Sly2.40 tomato reference genome with STAR. ERCC spike-ins has been used to normalize gene expression.

Identification of differentially expressed genes and clustering

The differential expression analyses were performed with the DESeq2 package as performed and described in [START_REF] Pirrello | Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation[END_REF]. Normalized expression values were then used to identify DEG. The data sets for all stages were processed the same way. First, log2 fold changes (Log2FC) and false discovery rates (FDRs) for each gene were filtered. We used a filter for Log2FC to be > 2 and <2 and FDR <0,01. Then DE genes Chapter 4: Discussion and perspectives

Conclusion

During the PhD we optimized in situ methods for ploidy determination in the pericarp using Oligo-FisH and the use of spike-ins for absolute gene expression determination in ploidy sorted nuclei having a global shift of gene expression. The ploidy map obtained at early stage of development allowed to determine that the gradient distribution of ploidy levels in the pericarp was already established in 6DPA fruits. We also found that cell size is increasing with endoreduplication and the developmental stage suggesting that endoreduplication is not the sole responsible of cell growth in the pericarp. Lastly, the ploidy transcriptome revealed that a global shift of expression, doubling, exists in early stage of development and is due to endoreduplication. The integration of the ploidy map data and the transcriptome data shows that gene expression controlled in a ploidy specific manner in early stage of development inducing spatialization of genetic programs in the pericarp. The data obtained during the PhD prepares for future projects that could aim at determining the potential rules of establishment and progress of endoreduplication in the pericarp, the identification of transcriptional regulators responsible for the ploidy specific expression and the study of the role of ploidy specifically expressed genes in endoreduplication control.

66.

Decaestecker W, Buono RA, Pfeiffer ML, Vangheluwe N, Jourquin J, Karimi M, Isterdael GV, Beeckman T, Nowack MK, Jacobs TB. 2019. CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis. The Plant Cell 31, 2868-2887.

67.

Del Prete S, Molitor

69.

Dermastia M, Kladnik

Workshop From Pictures to Numbers, September 2019

The aim of this course is to lower the threshold for researchers who use microscopy on a routine basis and/or demand image analysis for their own research by: a) providing fundamental knowledge on the most important image properties, basic processing algorithms and typical analyses, and b) by giving direct hands-on training on how to perform very common tasks in open source software ranging from simple image annotation to more advanced phenotypic analyses.

An overview of different image analysis platforms and an introduction to basic scripting capabilities should further enable the participants to select or devise apt solutions for their image analysis needs. The primary goal is to familiarize PhD students, post-docs and lab technicians, who work with microscopy images of biological samples and have limited or no experience with the fundamentals of biological image analysis.

After this course participants should be able to:

-Know important image properties and processing algorithms -Annotate and present images in a publication-ready format -Know which analytical routine to select and apply -Compose basic image analysis workflows -Gain proficiency in basic scripting language The organizing committee is composed of the following members:

Bioinformatic school AVIESAN-INSERM: Initiation to processing of NGS data.

This training is for Biologist implicated in project that imply NGS. The school focused on new technology and three practical courses parallelly (RNA-seq, ChIP-seq/ATACseq, Variant DNA-seq). Introduction to data integration, single cell genomic and long reads technology will be dispensed. The aim of the school is to give the bases to be Titre : Distribution spatio-temporelle des niveaux de ploïdie et du transcriptome associés durant le développement du fruit de Tomate Résumé :

L'endoréduplication, qui est la source majeure d'endopolyploïdie chez les Plantes, est un processus cellulaire durant lequel la quantité d'ADN nucléaire (ploïdie) est augmentée par une succession de réplications du génome sans division cellulaire. L'endoréduplication est le résultat de la capacité des cellules à transformer le classique cycle cellulaire en un cycle cellulaire partiel, l'endocycle, où la synthèse de l'ADN intervient indépendamment de la mitose. Chez les plants, l'endoréduplication est présente dans de nombreux organes et types cellulaires et notamment ceux ayant un fort intérêt agronomique, comme la partie charnue (péricarpe) du fruit de Tomate dans lequel les niveaux de ploïdie sont important. A la fin du développement du fruit de tomate, le péricarpe est composé d'une population hétérogène de cellules dont les niveaux de ploïdie et la tailles varient. De plus, à ce stade, des gènes présentent des profils d'expression spécifiquement lié aux niveaux de ploïdie. Cependant, peu de données sont disponible à propos de l'engagement et la progression de l'endoreduplication dans les cellules durant le développement du fruit de tomate ou son effet sur l'expression des gènes. Afin d'expliquer pourquoi une population hétérogène de cellule co-existent dans le péricarpe et comme base de description des régulations transcriptionnelles par l'endoréduplication dans le péricarpe, la thèse a eu d'étudier cette population hétérogène de cellule afin d'établir les bases de détermination des programmes génétiques mis en place par l'endoreduplication. Donc, j'ai cartographié le dynamisme des niveaux de ploïdie et de l'expression des gènes en fonction des niveaux de ploïdie dans le péricarpe au cours du développement précoce du fruit, à 6-, 9-et 12 jours après anthèse. Les données ont montré qu'un doublement de l'expression était présent en relation avec le doublement de ploïdie et l'endoreduplication est déjà bien en place, organisée en gradient dans le péricarpe à 6DPA. En utilisant les données produites, une carte virtuelle des niveaux de ploïdie et de l'expression des gènes sera établie au cours de du développement du péricarpe du fruit de Tomate. Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication results from the capacity of cells to modify their classical cell cycle into a partial cell cycle, or endocycle, where DNA synthesis occurs independently from mitosis. In plants, endoreduplication is found in numerous organs and cell types and particularly in organs or tissues of agronomical high value, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. At the end of tomato fruit development, the pericarp is composed of a heterogeneous population of cells in term of ploidy levels and cell sizes. In addition, at this stage, ploidy-specific gene expression profiles have been found. However, little is known about the onset and progression of endoreduplication during tomato fruit growth and its consequences on the regulation of cell size and gene expression. the aim of my PhD project was to study the heterogeneous population of cells that co-exists within the pericarp tissue and as a basis to dissect the genetic regulation of pericarp endoreduplication. Thus, I mapped the ploidy and transcriptome dynamics of the tomato fruit pericarp during early growth at three stages, 6-, 9-and 12 days post-anthesis (dpa) during fruit growth. The data showed a global shift of expression in relation to ploidy level as well as ploidy specific gene expression and that ploidy levels are already gradiently distributed in the pericarp. Using this data, a virtual map of ploidy distribution and gene expression will be done for the early development of Tomato fruit pericarp.

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