Acknowledgement

I am very grateful for having the chance to work in IJPB during the past 4 years. During my Ph.D. thesis, a lot of people have helped me with either works or life. I really appreciate your kindness, without your help I could not finishing my journey of Ph.D. thesis.

First of all, I would like to thank my supervisor Enrico, thank you for helping me come to France and supervise my work for 4 years, I really love to share the discussion with you about all the hypothesis on my project, and thanks for your patience and techniquecally supports, with all the helps and inspirations from you with the project, now I am more interested in Science and selfconfident that I want to continue with Science, even if I still have a lot to learn. I also would like to thank my thesis committee members, Rozenn, Robert, Christel and Gwyneth, as well as the reporter of my first and mid thesis presentation, Jean-Christophe and Magalie who had gave me a lot of good suggestions and inspired me to think in different angles.

I also thank to my team Chromatin dynamics and signaling. Valerie, thank you so much for your kindness and all the helps that you have offered me with both works and life, I appreciated so much and also thanks for the delicious sweets and ice cream. Jeff, thank you for sharing a lot of interesting conversations with me and thanks a lot for your great home-made bread and ice cream.

Marcela, thanks for your help with experiments and sharing happy lunch time with me. I also send my thanks to my previous SEEDEV team, I thank to Loï c who is very kind to me and helped me a lot with administration works and I thank to the whole SEEDEV team for all the helps you had offered to me.

I thank to all my collaborators, I thank to Rozenn who did sugar quantification for me. It was a pleasure for me to work with you, thanks for your patience and generous advises. I learned a lot knowledge about sugar from you. I also thanks to Sophie, thanks for your help with lipid, protein, quantification during the difficult pandemic time. I thank to Christine who is from INRAE de Jouyen-Josas, she did the beautiful electronic microscopy for us. I thank to Emmanuelle and Lysiane who are from CNRS, Université de Bordeaux and they are helping me with the immune-gold experiments.

I would like to thank to Ivan who taught me how to perform qpcr, Katia and Jean-Christophe gave me good suggestions for my images. Adeline who taught me how to perform RNA in situ hybridization. Gladys and Bertrand who help me solved issues with microscopes. Also, I will warmly thank to the greenhouse staff, Amé lie, Sé bastien, Hervé , Lilian, thank your patience and kindness and helped me with taking care of the plants. I thank to the administration team, Vanessa, Maria, Nazneen, thank you for your warmly helps for dealing with procedures for me. I also thank to the Chinese Scholarship Council, thanks for funding me 4 years for my thesis. I thank to the people from doctoral school for your help. And I also thank to IJPB very much for the incredible equipments and tools that I could benefit during my thesis.

I will thank my families, thank for your endless supports and understanding. I will thank to my friends, Olivier, thank you so much for your generous help and great patience, I feel very lucky that I can meet you in IJPB (as Fadi said you are a gift for everyone). Yassir, thank you be with me all the time, and we are the team of share hardships, especially for searching appartments. Emilie, thank you for a lot of heart to heart conversations and cares, I wish you a great future. Fadi, Julien, Isabelle, Naoto, Cyril, Marion, Greta thank for your kindness and sharing the great time with me. I will also thank to my Chinese friends, Fan and Yu, thank you so much for your family-like friendship, I won't forget your superb cook skills, I will also thank Liuxiao, Yuanyuan, Chenguang, Chuande, Zeyun, Yin, Luo, Tia, Dongdong, Shenbing thank you so much for your generous help and sharing a lot of great time with me. I will especially thank my best friend Xianqiang, thank you so much for bearing me all the time. MATERIAL AND METHODS ............................................................................................

Chapter I -General introduction

The seed

The seed habit allows the dispersal of the progeny and represents a remarkable evolutionary advance in plant sexual reproduction that contributed to the successful colonization of land by gymnosperms and angiosperms (seed plants). Compared to spores, a more primitive mechanism of progeny dispersal used by ferns and mosses, seeds rely less on water, carry more nutrients, are better protected and can disperse in different ways.

Seed development

Seeds develop from ovules after fertilization. A typical ovule initiates as primordium in the ovary. The ovule primordium consists of three tissues: the proximal funiculus, the medial chalaza and the distal nucellus (Fig. 1). The funiculus is connected to the placenta, which provides all the nutrients, the chalaza forms one or two protective integuments, and the nucellus undergoes meiosis to produce the megaspore (Schneitz et al., 1995a) (Fig. 2). Compared to mosses and ferns, which release the spores, the ovule retains the megaspore in the nucellus (Fig. 3). As a consequence, the megaspore develops into the female gametophyte (embryo sac) and grows inside the ovule at the expense of the nucellus (Magnani, 2018) (Fig. 3 and4).

Seed development starts with the single (in gymnosperms) or double (in angiosperms) fertilization of the female gametophyte, which generates the embryo, the next plant generation, and, in the case of angiosperms, also the endosperm, a nourishing tissue (Figs. 5 and6) [START_REF] Skinner | Regulation of ovule development[END_REF]. Whereas the maternal tissues do not actively participate in the fertilization process, they undergo drastic changes in response to it. The integuments develop into the seed coat which surrounds embryo and endosperm and protects them from biotic and abiotic stresses [START_REF] Coen | Seed coat thickness in the evolution of angiosperms[END_REF] (Figs. 6 and 7). The funiculus transports nutrients from the placental tissue and unload them into the chalaza from where are distributed to the rest of the seed. Finally, the nucellus is either eliminated to allow the growth for the fertilization products (for example in Arabidopsis) (Figs. 6 and7) or it grows to become a nutrient storage tissue (for example in amaranth).

Seed tissue partitioning

Despite having a common function and same tissues, seeds evolved different architectures. In gymnosperms, the female gametophyte grows at the expense of the nucellus to become the main storage tissue alongside the embryo. Angiosperm seeds have been classified into three major architectures according to the relative volumes of the fertilization products, embryo and endosperm, and the nucellus (Fig. 8). In endospermic seeds (e.g., Arabidopsis and cereals), the endosperm surrounds the embryo and plays an important role in nutrient storing [START_REF] Sreenivasulu | Seed-development programs: a systems biology-based comparison between dicots and monocots[END_REF]. By contrast, the endosperm of non-endospermic seeds (e.g., most legumes) is completely consumed by the embryo, which becomes the primary storage tissue [START_REF] Weber | Molecular physiology of legume seed development[END_REF]. Finally, perispermic seeds (e.g., pseudocereals such as amaranth and quinoa) develop a large perisperm, a tissue originating from the nucellus, alongside a minute endosperm (Burrieza et al., 2014). The ancestral condition of angiosperm seeds is still debated between endospermic and perispermic as basal angiosperms display either a large nucellus or endosperm as primary seed storage compartment [START_REF] Friedman | Seed development in Trimenia (Trimeniaceae) and its bearing on the evolution of embryo-nourishing strategies in early flowering plant lineages[END_REF]. Plants shifted several times between the endospermic and perispermic seed condition highlighting the antagonistic development of endosperm and nucellus as a defining mechanism for seed evolution (Lu and Magnani, 2018) Figure 8. Angiosperm seed architectures.

In seed plants, the development of different seed architectures is achieved by the complete elimination of unwanted cells (cell elimination) of one tissue in favor of another tissue. Compared to other cell death programs that are 'structure-conserving', cell elimination results in the complete vanishing of cells, including the cell wall [START_REF] Daneva | Functions and Regulation of Programmed Cell Death in Plant Development[END_REF]Ingram, 2017;van Doorn et al., 2011). In seeds, cell elimination has been observed in different tissues during development (Ingram, 2017). For example, the elimination of three out of four megaspores after meiosis and the degeneration of female gametophyte cells during or shortly after fertilization are observed in most angiosperm species. Cell elimination takes also place in maternal sporophyte tissues, as the nucellus or the integuments. After fertilization, cell elimination can also occur in the fertilization products, endosperm and the suspensor tissue of the embryo. Despite being a recurrent theme in seed development, cell elimination is still a poorly understood mechanism both at the biochemical and molecular level.

Seed nutrient partitioning

Tissue and nutrient partitioning are two inextricably linked processes. Such a diverse panorama of seed structures, described above, correlates therefore with an equally broad spectrum of nutrient-storing strategies. Different seed tissues tend to accumulate different nutrients, thus different seed architectures may vary considerably in their nutrient content. The endosperm, which is rich in carbohydrates (mainly starch), accounts for most of the volume of the endospermic seed of the Poaceae family. As a consequence, about 80% of the nutrient composition of such grains is made of starch [START_REF] Beloshapka | Compositional Analysis of Whole Grains, Processed Grains, Grain Co-Products, and Other Carbohydrate Sources with Applicability to Pet Animal Nutrition[END_REF]. By contrast, non-endospermic seeds like soybeans, which carry a relatively large embryo, accumulate predominantly proteins and lipids [START_REF] Yazdi-Samadi | Components of developing soybean seeds: Oil, protein, sugars, starch, organic acids, and amino acids 1[END_REF].

Sugars, lipids and proteins reserves in seeds

Starch is the main polysaccharide stored in seeds and it is the main source of carbohydrates for human nutrition. The biosynthesis of starch in both photosynthetic organs like leaves and in heterotrophic sink organs like seeds has been described. In plants, the photosynthetic tissues produce sucrose (Suc) as the major form of carbohydrates to be transported to the heterotrophic sink tissues (flower, root, seed, fruit) through the phloem (Fig 9). Once Suc reaches the sink tissue, it can be stored in vacuoles, further transported, hydrolyzed by invertases (INVs) or sucrose synthases (SuSys) into hexoses (glucose, Glc, and fructose, Fru), or metabolize into fructans by fructosyl transferases (FTs) [START_REF] Ruan | Sucrose metabolism: gateway to diverse carbon use and sugar signaling[END_REF] (Fig 9). After being phosphorylated, hexoses are used for starch synthesis in the Amyloplasts or undergo glycolysis in Mitochondria [START_REF] Stein | An Overview of Sucrose Synthases in Plants[END_REF] Oils are synthesized and stored in many plant tissues, where they act as energy reserves [START_REF] Baud | Compared analysis of the regulatory systems controlling lipogenesis in hepatocytes of mice and in maturing oilseeds of Arabidopsis[END_REF]. The storage lipids of seeds consist mainly of triacylglycerols (TAGs). The fatty acid profiles of seed oil vary greatly from one species to another [START_REF] Voelker | Variations in the Biosynthesis of Seed-Storage Lipids[END_REF].

TAGs are stored in subcellular structures called oil bodies [START_REF] Murphy | Storage lipid bodies in plants and other organisms[END_REF]. The abundance and location of these oil bodies in seed tissues vary considerably among species. For example, the embryos of soybean (Glycinemax) and sunflower (Helianthus annuus) is rich in oil bodies [START_REF] Baud | Physiological and developmental regulation of seed oil production[END_REF]. However, in castor bean, oils are mostly accumulated in the endosperm [START_REF] Chen | Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in castor bean (Ricinus communis L.)[END_REF][START_REF] Marriott | The breakdown of lipid reserves in the endosperm of germinating castor beans[END_REF].

Plants provide most (around 58%) of the dietary protein for human nutrition compared to animal [START_REF] Van Vliet | The Skeletal Muscle Anabolic Response to Plant-versus Animal-Based Protein Consumption[END_REF]. In seeds, proteins account for from 10% of dry seed weight in cereals to 40% in some legumes and oilseeds. With the exception of proteins with metabolic or structural roles, seed storage proteins (SSPs) are accumulated in the cotyledon and embryo in dicotyledonous plants and of the endosperm in monocotyledonous plants [START_REF] Krishnan | Seed Storage Proteins[END_REF].

SSPs have been classified into four main groups according to their solubility: water (albumins), dilute saline (globulins), alcohol-water mixtures (prolamines) and dilute acid or alkali (glutelins) [START_REF] Osborne | The vegetable proteins[END_REF]. SSPs serve as source of nitrogen, carbon and sulfur during seed germination and seedling growth.

Nutrients accumulation in the Arabidopsis seed

Arabidopsis seed development can be divided into three stages: the early embryogenesis stage, which is from fertilization until 7 days after flowering (DAF), the maturation phase, which is from 8DAF to 16 DAF, and the late maturation stage, which is from 17 DAF until dry seeds (Baud et al., 2002b). Developing Arabidopsis seeds initially accumulate starch, at 12 DAF the starch level reaches a peak and at the same time seed have very low content of lipids and protein. Starch accumulation decreases after 12DAF and the synthesis of lipids and proteins increases rapidly during the maturation phase [START_REF] Andriotis | Starch turnover in developing oilseed embryos[END_REF]Baud et al., 2002b;[START_REF] Hills | Control of storage-product synthesis in seeds[END_REF]. In the late maturation stage, starch is hardly detectable, whereas lipids and proteins account for 45% of the seed weight [START_REF] Andriotis | Plastidial glycolysis in developing Arabidopsis embryos[END_REF][START_REF] O'neill | Natural variation for seed oil composition in Arabidopsis thaliana[END_REF].

The distribution of starch in seed coat and embryo was examined by iodine staining. In the seed coat, starch is visible at 4DAF (Fig. 10). The staining intensity increases until the cotyledon embryo stage. After the cotyledon embryo stage, starch in the seed coat decreases and is not detectable in mature seeds. At the torpedo embryo stage, starch granules are detectable in two regions, one is above the tip of the radicle and a second one is in the hypocotyl (Fig. 10). At the walking stick embryo stage, starch accumulates both in radicle and hypocotyl. At the early cotyledon embryo stage, starch is present in cotyledons as well as in radicle and hypocotyl. After 12 DAF, starch accumulation in the embryo decreases rapidly. At the expanded cotyledon embryo stage, starch is present only in the radicle tip [START_REF] Andriotis | Starch turnover in developing oilseed embryos[END_REF].

Nutrient transport in seeds

What all angiosperm seeds have in common is the allocation of resources from the placental maternal tissue, through the chalaza, to the storage tissues following a source-sink nutrient gradient [START_REF] Patrick | Compartmentation of transport and transfer events in developing seeds[END_REF]. In most angiosperms, vascularization arrests at the chalaza, and nutrients follow a combination of symplastic and apoplastic pathways to reach the sink tissues. Though, there are examples of nucellar tracheids, an ancestral character also observed in extinct gymnosperms, and vascularized seed coats [START_REF] Johri | Comparative embryology of angiosperms[END_REF].

Symplastic pathway

Seed maternal tissues have been shown to be symplastically connected in most studied species.

Therefore, nutrients supposed to be unloaded from the phloem symplastically and move along the maternal tissues through plasmodesmata [START_REF] Ellis | Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. I. The Use of Steady-State Labeling[END_REF][START_REF] Offler | Cellular Structures, Plasma Membrane Surface Areas and Plasmodesmatal Frequencies of Seed Coats of Phaseolus vulgaris L. In Relation to Photosynthate Transfer[END_REF]Stadler et al., 2005;[START_REF] Thorne | Phloem Unloading of C and N Assimilates in Developing Seeds. Annual Review of Plant Biology[END_REF]. In Arabidopsis, GFP mobility assays have shown that sucrose reaches the seed through the funicular phloem, which is symplastic connected to the seed coat (Stadler et al., 2005). However endosperm and embryo are not symplastic connected to the surrounding maternal tissues and are, therefore, supposed to receive nutrients from the apoplast (Stadler et al., 2005).

Apoplastic pathway

Apoplast transport has been broadly described in angiosperms seeds. Many studies have shown that transporters allow the release of nutrients from the maternal tissues into the apoplast and the uptake of the same nutrients by the fertilization products. The SWEET passive facilitators, have been shown to allow the export of sucrose and hexoses from seed maternal tissue cells into the apoplast in different species (Chen et al., 2012b;Sosso et al., 2015;Yang et al., 2018a). Plant SWEET transporters have seven transmembrane helixes (TMs) arranged into two triple-helixbundles (THBs) (Fig. 11). The two THBs are organised in a parallel orientation and function as a single unit. The THBs at the N and C terminus of the protein are fused together by the TM4 linker helix (Chen et al., 2010). Plant SWEET transporters originated from gene duplication of prokaryotic semiSWEETs, which contain only one THB (Chen et al., 2010;[START_REF] Xuan | Functional role of oligomerization for bacterial and plant SWEET sugar transporter family[END_REF] (Fig. 11).

There are 17 SWEET genes in Arabidopsis: AtSWEET1/2/4/5/7/8/13/17 are monosaccharide transporters [START_REF] Chardon | Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis[END_REF]Chen et al., 2010) whereas AtSWEET9/10/11/12/14/15 function as sucrose transporters (Chen et al., 2015;Chen et al., 2012b;[START_REF] Kanno | AtSWEET13 and AtSWEET14 regulate gibberellin-mediated physiological processes[END_REF][START_REF] Lin | Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9[END_REF]Rottmann et al., 2018). AtSWEET16 is able to transport sucrose, fructose, glucose [START_REF] Klemens | Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis[END_REF]. AtSWEET15, AtSWEET12, and AtSWEET11 are expressed in the seed coat and release sucrose in the apoplast (Chen et al., 2015). AtSWEET 11 is also expressed in the endosperm, thus suggesting that it might play a role in reimporting sucrose in the endosperm. Triple sweet11;12;15 mutant seeds showed retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a "wrinkled" seed phenotype, thus indicating their paramount importance in sugar allocation to the seed (Chen et al., 2015). [START_REF] Ruan | Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat[END_REF]. In maize, the export of sucrose in the endosperm apoplast by the ZmSWEET4c facilitator and its further hydroxylation into hexoses by a cell wall INV has been shown to be a key step in seed filling (Sosso et al., 2015). Loss of an endosperm specific cell wall INV results, indeed, in a miniature seed phenotype [START_REF] Miller | The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development[END_REF].

Seed tissue and nutrient partitioning a case for nucellus (Lu and Magnani, 2018) , attached below

Introduction

Tissue partitioning is the driving force that shapes the development of different seed structures. The relative contribution of each tissue to the final seed mass varies considerably among species and underlies different nutrientstoring strategies. Tissue partitioning is achieved through cell elimination programs that regulate the degeneration of one tissue in favor of another (Ingram 2017). The nucellus, the most distal maternal tissue of the ovule primordium (the seed precursor) responsible for the formation of the female gametophyte, plays a key role in defining the seed structure together with the fertilization product/s. In gymnosperms, most of the nucellus is eliminated and replaced by the female gametophyte, the main storage tissue, which will be in turn absorbed by the developing embryo, the only fertilization product (Fig. 1) (Linkies et al. 2010). Angiosperm seeds have been classified into three major architectures according to the relative volumes of the fertilization products, embryo and endosperm, and the nucellus (Fig. 1). In mature endospermic seeds (e.g., cereals), the endosperm surrounds the embryo and plays an important role in nutrient storing [START_REF] Sreenivasulu | Seed-development programs: a systems biology-based comparison between dicots and monocots[END_REF]. By contrast, the endosperm of non-endospermic seeds (e.g., most legumes) is completely consumed by the embryo, which becomes the primary storage tissue [START_REF] Weber | Molecular physiology of legume seed development[END_REF]. Finally, perispermic seeds (e.g., pseudocereals such as amaranth and quinoa) develop a large perisperm, a tissue originating from the nucellus, along with a minute endosperm (Burrieza et al. 2014). The ancestral condition of angiosperm seeds is still debated between endospermic and perispermic as basal angiosperms display either a large nucellus or endosperm as primary seed storage compartment [START_REF] Friedman | Seed development in Trimenia (Trimeniaceae) and its bearing on the evolution of embryo-nourishing strategies in early flowering plant lineages[END_REF]. Plants shifted several times between the endospermic and perispermic seed condition highlighting the antagonistic development of endosperm and nucellus as a defining mechanism for seed evolution.

Recent discoveries on the molecular regulation of nucellus elimination have given an insight into the process of seed tissues partitioning. Here, we discuss them in the context of angiosperm seed natural diversity. Finally, we review nutrient transport and accumulation in the nucellus across different seed architectures to present seed tissue and nutrient partitioning as two coherent and inextricably linked aspects of seed development.

3

Natural diversity in nucellus morphology

Angiosperm ovules have been classified according to their nucellus position and thickness (Endress 2011). A first general distinction is made between ovules that bear nucellus hypodermal cells above the megaspore mother cell (MMC) (crassinucellar) and those that display only distal epidermal nucellus cells (tenuinucellar). Crassinucellar ovules are considered ancestral to tenuinucellar, as they are present in basal angiosperms, magnoliids, most monocots, and basal and part of the core eudicots. They are further classified into (1) truly crassinucellar, if they carry two or more distal hypodermal nucellus cell layers, (2) weakly crassinucellar when they display only one hypodermal cell layer, or (3) pseudo-crassinucellar if the distal nucellus epidermal cell layer divides periclinally to form additional cell layers, in the absence of hypodermal cells. The tenuinucellar condition, observed in several monocots and part of the core eudicots, includes (4) incompletely tenuinucellar ovules, which display hypodermal nucellus cells proximal and/or lateral to the MMC, (5) truly tenuinucellar ovules, without any hypodermal nucellus cell, and (6) reduced tenuinucellar ovules, when the proximal region of the MMC is not fully enclosed by the nucellus. Further terminology has been created to describe specific nucellus regions. In pseudo-crassinucellar ovules, the dermal layers of the nucellus apex (at the micropylar region) undergoing periclinal cell divisions are called "nucellar cap." In extreme cases, the nucellus apex divides massively to form a "nucellar beak" that can extend outside the seed coat and define the micropyle. Nucellus epidermal cells can also elongate radially around the female gametophyte to form a so-called nucellar pad [START_REF] Johri | Comparative embryology of angiosperms[END_REF]. A persistent nucellus base, at the chalazal side, is instead referred to as "podium" or "postament" if only its axial part persists [START_REF] Johri | Comparative embryology of angiosperms[END_REF]. Overall, this classification highlights the great natural diversity in ovule nucellus size, which sets the premises for tissue partitioning programs later on in development.

Nucellus architecture changes during ovule and seed development. The female gametophyte grows at the expense of the nucellus which is partially eliminated, a process that is still almost completely unexplored [START_REF] Johri | Comparative embryology of angiosperms[END_REF]. After fertilization, the nucellus of endospermic and non-endospermic seeds partially degenerates to make space to endosperm and embryo growth. By contrast, perispermic seeds display a large central nucellus (perisperm) that grows to become the main storage tissue along a minute endosperm. Variations have been observed in between these extreme seed architectures. A retard in the elimination of the nucellus is a hallmark of coffee grains. In coffee, the nucellus grows to define seed size and is then replaced by the endosperm (Alves et al. 2016;Mayne 1937). Similarly, the nucellus of Austrobaileya scandens seeds drives early seed growth and is then eliminated by the endosperm, whose further development determines final seed size (Losada et al. 2017). By contrast, nucellus and endosperm coexist and display a similar volume in Acorus calamus seeds (Floyd and Friedman 2000). Furthermore, the structure of Malpighiaceae seeds appears perispermic during early seed development but the nucellus is fully eliminated by the embryo at later stages (Souto and Oliveira 2014). Finally, Podostemaceae ovules do not undergo central cell fertilization and lack an endosperm. In these species, the nucellus cell walls proximal to the female gametophyte break down to produce a multinucleate cytoplasmic structure termed "nucellar plasmodium" (Arekal andNagendran 1975, 1977).

A mechanical role for the nucellus has also been hypothesized. The anticlinal cell walls of the rice nucellus epidermis, surrounding the endosperm, are uniquely thickened with cellulosic material and have been speculated to provide mechanical support [START_REF] Krishnan | Structural and histochemical studies on grain-filling in the caryopsis of rice (Oryza sativa L.)[END_REF]. Similarly, the chalazal or micropylar nucellus cells can differentiate into the so-called hypostase and epistase, respectively. The cell walls of these nucellar structures thicken and accumulate cutin, suberin, lignin, or callose. Hypostase and epistase have not been assigned a clear function yet but are thought to play a mechanical role or work as apoplastic barriers [START_REF] Johri | Comparative embryology of angiosperms[END_REF].

Tissue partitioning

Nucellus elimination in Arabidopsis

In Arabidopsis seeds, nucellus elimination begins 2 days after flowering (DAF) and progresses in a distal-proximal fashion to achieve the loss of 50% of its cells by 8 DAF. A few layers of proximal nucellus cells persist and expand with the rest of the ovules to form a gate between chalaza and endosperm till embryo maturity (Xu et al. 2016).

Elimination of the nucellus, as well as seed coat growth, is triggered by the endosperm (Fig. 2) (Roszak and Kohler 2011; Xu et al. 2016). Single fertilization of the central cell is necessary and sufficient to initiate nucellus degeneration. The MADS box transcription factor AGAMOUS LIKE 62 (AGL62) is specifically expressed in the endosperm and essential for nucellus-endosperm communication. agl62 mutant seeds display precocious endosperm cellularization and fail to undergo nucellus degeneration and seed coat differentiation. Figueiredo and co-workers have recently proposed that AGL62 regulates auxin efflux, considered the fertilization signal that coordinates the development of endosperm and maternal tissues (Figueiredo et al. 2016). Nevertheless, this model has been tested solely on seed coat growth and not on nucellus degeneration. Two alternative scenarios have been proposed to explain nucellus elimination: The endosperm might generate mechanical signals while growing against the nucellus or act as strong nutrient sink, thus triggering death of neighboring tissues by nutrient deprivation (Ingram 2017). It has been argued that the latter two models are less favorable to explain endospermmaternal tissue developmental coordination as titan 2 mutant seeds, which undergo early endosperm arrest comparable to agl62 (Liu and Meinke 1998), show signs of seed coat growth (Roszak and Kohler 2011) and nucellus degeneration (personal observations).

Regardless of the nature of the signaling mechanism, it has been shown that endosperm growth relieves the repressive action mediated by Fertilization-Independent Seed (FIS) Polycomb Group (PcG) proteins on nucellus degeneration (Xu et al. 2016). Compared to other FIS genes that are solely expressed in the ovule central cell, Fertilization-Independent Endosperm (FIE) and Multicopy Suppressor of IRA1 (MSI1) are also expressed in the nucellus and seed coat (Kohler et al. 2003;Xu et al. 2016). Both fie/+ and msi1/+ mutants display high penetrance of autonomous seed coat growth (Roszak and Kohler 2011) and nucellus degeneration (Xu et al. 2016) in the absence of fertilization. Downstream of PcG proteins, TRANSPARENT TESTA 16 (TT16) and GORDITA (GOA) MADS BOX transcription factors promote nucellus elimination and inhibit cell division (Xu et al. 2016). TT16 regulates nucellus cell elimination in part by repressing the expression of HVA22d, which inhibits gibberellin-mediated programmed cell death (PCD) and autophagy. Furthermore, a papain-type KDEL-tailed cysteine endopeptidase (CysEP), involved in PCD of vegetative tissues, has been shown to be expressed in the distal nucellus undergoing degeneration [START_REF] Zhou | Expression analysis of KDEL-CysEPs programmed cell death markers during reproduction in Arabidopsis[END_REF]). Nevertheless, nucellus elimination has not been entirely assigned to any known cell death program. As in vacuolar PCD (van Doorn et al. 2011), nucellus cells undergo autophagy. By contrast, the nucellus displays protoplast shrinkage and largely unprocessed cell corpses, which are hallmarks of necrosis (van Doorn et al. 2011). Another example of PCD that combines signs of vacuolar and necrotic cell death is induced by the successful recognition of pathogens during hypersensitive response (HR) (van Doorn et al. 2011). Nevertheless, PCD associated with HR does not exhibit degradation of the cell wall as in the nucellus. Furthermore, mutations in the METACASPASE1 and LESION SIMULATING DISEASE1 genes, which encode components of the HR-PCD machinery (Coll et al. 2010), do not affect nucellus development (Xu et al. 2016).

As endosperm growth is necessary to initiate nucellus elimination, the persistence of the nucellus in tt16 mutant seeds negatively affects endosperm development revealing an antagonistic development of endosperm and nucellus (Xu et al. 2016). This antagonism is reflected in the evolution of the two most ancient seed structures, perispermic and endospermic, which rely on nucellus or endosperm as major storage tissue, respectively.

Nucellus elimination in cereals

In cereals, the nucellus accounts for most of the grain volume at anthesis and it is eliminated after fertilization in a centripetal fashion. At grain filling, only the outermost nucellus cell layer (nucellus epidermis) and a few nucellus cell layers overlaying the ovule vascular trace at the chalazal side are retained and undergo PCD more or less rapidly according to the species. The chalazal nucellus of maize grains appears as compact layers of dead cells with limited plasmodesmata connections (Felker and Shannon 1980;Kladnik et al. 2004). In sorghum, the chalazal nucellus consists of a few large cell layers which are reduced to one during development and whose symplastic connection with the chalaza is interrupted (Dwivedi et al. 2014;Maness and McBee 1986;Wang et al. 2012). Rice, Brachypodium, barley, and wheat grains develop instead the so-called nucellar projection, a tissue dedicated to nutrient transport simplastically connected to the placenta [START_REF] Krishnan | Structural and histochemical studies on grain-filling in the caryopsis of rice (Oryza sativa L.)[END_REF]Opanowicz et al. 2011;Oparka and Gates 1981;[START_REF] Radchuk | Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain[END_REF]Radchuk et al. , 2011;;Wang et al. 1994aWang et al. , b, 1995;;[START_REF] Zheng | Contrast observation and investigation of wheat endosperm transfer cells and nucellar projection transfer cells[END_REF]. The nucellar projection of barley and wheat grains is more developed and has been divided into different regions based on cell morphology: (starting from the integument inward) (1) actively dividing cells, (2) elongating cells, (3) transfer cells with wall ingrowth, and (4) cell debris (Radchuk et al. 2006;Thiel et al. 2008;Wang et al. 1994a;[START_REF] Zheng | Contrast observation and investigation of wheat endosperm transfer cells and nucellar projection transfer cells[END_REF]. By contrast, the nucellus epidermis of rice and Brachypodium appears larger and more persistent, compared to other cereals (Ellis and Chaffey 1987;Opanowicz et al. 2011;Oparka and Gates 1981). Finally, the chalazal nucellus physically touches the endosperm in maze, Brachypodium, and rice, while it is separated by a cavity filled with nucellar lysate (referred to as "endosperm or nucellar cavity" or "placental sac") in wheat, barley, and sorghum.

The Arabidopsis signaling pathway underlying nucellus development is partially conserved in rice grains (Fig. 2). The rice TT16 orthologous gene, MADS29, is expressed in the nucellus and nucellar projection and promotes cell elimination [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF][START_REF] Yang | Live and let die -the B(sister) MADS-box gene OsMADS29 controls the degeneration of cells in maternal tissues during seed development of rice (Oryza sativa)[END_REF][START_REF] Yin | The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development[END_REF]. Compared to Arabidopsis, MADS29 is also expressed in the embryo and the protein has been detected in nucellus epidermis, embryo, and endosperm but not in the nucellar projection [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF]. MADS29 directly activates the expression of nucleotide-binding site-leucine-rich repeat proteins and Cys proteases [START_REF] Yin | The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development[END_REF]. In line with the Arabidopsis endosperm-maternal tissue signaling model (Figueiredo et al. 2016), MADS29 expression is induced by auxin and regulates auxin-cytokinin homeostasis [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF][START_REF] Yin | The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development[END_REF]. Furthermore, antagonistic development of nucellus and endosperm has been observed also in rice as suppression of MADS29 expression impairs starch accumulation and endosperm growth [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF][START_REF] Yang | Live and let die -the B(sister) MADS-box gene OsMADS29 controls the degeneration of cells in maternal tissues during seed development of rice (Oryza sativa)[END_REF][START_REF] Yin | The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development[END_REF].

In barley grains, nucellus elimination correlates with the expression of genes encoding for Asp protease-like protein nucellin, vacuolar processing enzyme nucellain, Cys and Asp endopeptidases, subtilisin-like Ser proteinases, and JEKYLL protein, all known to play a role in PCD (Chen and Foolad 1997;Linnestad et al. 1998;Radchuk et al. 2006Radchuk et al. , 2011Radchuk et al. , 2018;;Thiel et al. 2008;Tran et al. 2014). Down-regulation of jekyll by RNA interference affects nucellus elimination and nucellar projection differentiation and, indirectly, endosperm development and starch accumulation (Radchuk et al. 2006). Furthermore, the differentiation gradient along the barley nucellar projection is also regulated by a 1 3 gibberellin-to-abscisic acid balance, with gibberellin promoting differentiation (Weier et al. 2014).

Morphological analyses of nucellus parenchymal cells in wheat revealed fragmentation of the cytoplasm, vacuolization, disruption of the nuclear envelope and plasma membrane, and mitochondrion structural alterations (Dominguez et al. 2001). Nevertheless, the authors of this study might have erroneously located nucellus cells as what it is indicated as nucellus parenchymal cell in Fig. 2b appears to be integument cells. In line with this interpretation, the cells analyzed do not undergo degeneration of the cell wall. Nucellus epidermis and nucellar projection of wheat grains have been shown to express genes encoding for carboxypeptidase III, thiol protease, nucellain, and nucellin, some of which are also implicated in aleurone death during germination (Domınguez and Cejudo 1998;Drea et al. 2005). A parallel has been drawn between wheat and Brachypodium nucellus, which also expresses nucellain during its elimination (Opanowicz et al. 2011).

Finally, the study of the maize invertase Miniature 1 (Mn1) gene revealed a mechanical interaction between nucellus and endosperm. Maize grains mutated for the Mn1 gene show a gap between the nucellus cells, which are rapidly emptied of their nuclear and cytoplasmic material, and the endosperm. Such a gap is not due to cell death but to an underdeveloped endosperm that results in over-expanded nucellus cells, thus suggesting that the endosperm exercises a mechanical force on the nucellus (Kladnik et al. 2004).

Overall, these data indicate that a protease-dependent cell death machinery is shared by cereals to achieve nucellus degeneration. These same types of proteases, even though not necessarily the same genes, appear to drive endosperm cell death. On the other hand, more data are necessary to highlight variations in the nucellus elimination pathways responsible for the slightly different nucellus fates observed in different cereals.

Nucellus elimination in other angiosperms

Similar to Arabidopsis and cereals, a number of other angiosperm seeds have been shown to undergo early nucellus elimination in a progressive fashion starting from the nucellus-endosperm border toward the chalazal region. Proteomics and genetic analyses revealed the presence of Cys endopeptidases and other peptidases associated with PCD in the nucellus of castor bean seeds (Greenwood et al. 2005;Nogueira et al. 2012). Cys endopeptidases are accumulated in ricinosomes, organelles derived from the endoplasmic reticulum that collapse upon nucellus degeneration releasing their content in the cytoplasm and contributing to the digestion of proteinaceous debris (Greenwood et al. 2005). In Sechium edule, nucellus elimination correlates with the induction of caspase-like proteases and high levels of hydrogen peroxide, nitric oxide, and ethylene, which has been proposed as the signaling molecule between endosperm and nucellus (Lombardi et al. 2007(Lombardi et al. , 2010(Lombardi et al. , 2012)). High level of indole acetic acid has also been detected in endosperm and nucellus of Sechium edule seeds but its role in nucellus development is still unclear (Lombardi et al. 2012). By contrast, the nucellus of peach seeds displays a pick of abscisic acid after anthesis, thus suggesting that different hormones might play a role in nucellus degeneration in different species (Piaggesi et al. 1991).

Nucellus retention

Perispermic seeds such as quinoa, amaranth, Peperomia, spinach, and Nymphaeales display a large nucellus, which defines seed size and becomes the major storage tissue, along a minute endosperm. The process has been well studied in quinoa. At anthesis, the nucellus reaches its final number of cells as its mitotic activity arrests. After fertilization, a relatively small endosperm grows at the expense of part of the nucellus and leads the way to embryo development which in turn consumes most of the endosperm and part of the nucellus. The central nucellus, termed perisperm, is not eliminated and undergoes cell expansion, endoreduplication, reserve accumulation, and PCD. Nucellus cell death involves nuclease and proteolytic activity but not cell wall degeneration, a process comparable to endosperm cell death in endospermic seeds (Burrieza et al. 2014;[START_REF] Lopez-Fernandez | Programmed cell death during quinoa perisperm development[END_REF].

Nutrient partitioning

Tissue and nutrient partitioning are two inextricably linked processes. Such a diverse panorama of seed structures correlates therefore with an equally broad spectrum of nutrient-storing strategies. What all angiosperm seeds have in common is the allocation of resources from the placental maternal tissue, through the chalaza, to the storage tissues following a source-sink nutrient gradient [START_REF] Patrick | Compartmentation of transport and transfer events in developing seeds[END_REF]. In most angiosperms, vascularization arrests at the chalaza, and nutrients follow a combination of symplastic and apoplastic pathways to reach the sink tissues. Nevertheless, there are examples of nucellar tracheids, an ancestral character also observed in extinct gymnosperms, and vascularized seed coats [START_REF] Johri | Comparative embryology of angiosperms[END_REF].

Sugar transport in endospermic seeds

The role of the nucellus in nutrient transport has been mostly addressed studying cereal grain filling. In cereals, nutrients are supposed to travel simplastically from the phloem through the maternal tissues of the chalazal region to then 1 3 being released into the apoplast. The endosperm, which is not simplastically connected to the maternal tissues, uploads nutrients from the apoplast and accumulates mostly starch while undergoing PCD [START_REF] Thorne | Phloem Unloading of C and N Assimilates in Developing Seeds. Annual Review of Plant Biology[END_REF]. The nucellus lies at the interface of maternal and endosperm tissues and can play a role in nutrient transfer.

In maize grains, sucrose moves simplastically from the phloem to the chalaza and is then released into the apoplast where cell wall-bound invertases convert it into hexoses, glucose and fructose (Felker and Shannon 1980;McLaughlin and Boyer 2004;Porter et al. 1985;Shannon 1972a, b;Tang and Boyer 2013). The nucellus is not simplastically connected to the chalaza and imports glucose during the first stages of grain development while being eliminated by endosperm growth (McLaughlin and Boyer 2004;Tang and Boyer 2013). Later in development, persistent nucellus cells undergo PCD (Felker and Shannon 1980;Kladnik et al. 2004), thus suggesting that nutrients cross the nucellus apoplastically. A similar path of sugar transport probably occurs in sorghum grains as they accumulate hexoses in the placental sac and display symplastic disconnection of chalaza and nucellus (Dwivedi et al. 2014;Maness and McBee 1986;Wang et al. 2012).

By contrast, the nucellus of wheat and barley is simplastically connected to the placenta and the nucellar projection develops transfer cells, thus expanding the nutrient unloading zone and facilitating transfer [START_REF] Radchuk | Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain[END_REF](Radchuk et al. , 2011;;Wang et al. 1994aWang et al. , b, 1995;;[START_REF] Zheng | Contrast observation and investigation of wheat endosperm transfer cells and nucellar projection transfer cells[END_REF]. At the beginning of barley seed development, starch accumulates mostly in the pericarp, which acts as a short-term sink, and only transiently in the nucellus. Alpha amylase 4 is expressed in degenerating nucellus tissue facilitating mobilization of starch toward the endosperm during nucellus elimination (Radchuk et al. 2006[START_REF] Radchuk | Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain[END_REF]. At barley grain filling, 13 C sucrose analyses revealed a flow of sucrose from the nucellar projection toward the endosperm (Melkus et al. 2011;Rolletschek et al. 2011). The nucellus projection of barley grains expresses a cell wall-bound invertase, indicating that hexoses are also released into the endosperm cavity (Weschke et al. 2003). Furthermore, barley nucellar projection and epidermis express members of the aquaporin family, which may play a role in nutrient efflux (Thiel et al. 2008). Interestingly, transfer cells of the nucellar projection and endosperm of barley and wheat express the same sucrose symporter (SUT) genes responsible for sucrose import in sink tissues (Bagnall et al. 2000;Weschke et al. 2000). The role of SUT proteins in the nucellus is not clear, and it might allow sucrose scavenging, work as sucrose passive port along concentration gradient or be an evolutionary relic of perispermic seeds. Impaired development of the barley nucellar projection leads to starch accumulation in maternal tissues at the expense of the endosperm, thus further proving the importance of this tissue in nutrient partitioning (Melkus et al. 2011;Radchuk et al. 2006;Rolletschek et al. 2011).

Rice and Brachypodium grains develop a smaller nucellar projection than barley and wheat, but display a thicker nucellus epidermis which has been proposed to play an active role in nutrient transport (Ellis and Chaffey 1987;Opanowicz et al. 2011;Oparka and Gates 1981). Defective starch synthesis in the endosperm has been observed in rice grains with suppressed MADS29 expression, highlighting the active role of the rice nucellus in transferring nutrients to the endosperm [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF][START_REF] Yin | The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development[END_REF]. During nutrient transfer, the nucellus might act as a shortterm sink as MADS29 has been found to promote the differentiation of proplastids in amyloplasts likely by regulating cytokinin biosynthesis [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF]. Finally, SWEET sucrose exporters have been found in all rice nucellar tissues, indicating that the nucellus engages in apoplastic seed filling (Yang et al. 2018).

Sugar transport in perispermic seeds

The role of the nucellus in perispermic seeds changes from nutrient-transport facilitator to long-term nutrient sink. The perisperm of quinoa seeds accumulates mostly starch while undergoing PCD [START_REF] Lopez-Fernandez | Programmed cell death during quinoa perisperm development[END_REF]. Starch accumulation follows an apical-basal pattern, with the chalazal side being the last to be filled. Such a pattern might be the result of sugars transport from the chalaza toward the perisperm while maximizing seed filling by following a source-sink gradient. Alternatively, nutrient transport through the seed coat might also explain such a nutrient accumulation pattern. At seed maturity, perisperm cells appear as thin walled and completely filled with starch grains, similar to cereal starchy endosperm cells [START_REF] Lopez-Fernandez | Programmed cell death during quinoa perisperm development[END_REF][START_REF] Prego | Seed structure and localization of reserves in Chenopodium quinoa[END_REF]. By contrast, the role of the few endosperm cells that persist at the micropylar region is less clear.

Conclusive remarks

The evolution of seed storage tissues in angiosperms has been a "battle" between endosperm and nucellus development. Both tissues can store starch and become the main source of energy for embryo germination. Indeed, the nucellus of perispermic seeds parallels the endosperm of endospermic seeds at both morphological and functional levels. Nevertheless, most angiosperm seeds evolved mutually exclusive growth of nucellus and endosperm. The nucellus offers an easier system of nutrient storage simplastically connected to the placenta. By contrast, the endosperm couples 1 3 nutrient storing to fertilization, thus possibly avoiding energy waste in case of unsuccessful fertilization.

Whereas we have a better understanding on seed nutrient transport and tissue elimination, the next challenge will be to address how nutrient and tissue partitioning are coordinated at the molecular level.

Author contribution statement JL reviewed the role of MADS box genes in nucellus development. EM wrote the rest of the paper in consultation with JL. The end of the introduction A change in nucellus cell fate led to the evolution of seed plants from non-seed plants and, subsequently, drove the evolution of different seed architectures. When I started my thesis, we had a general understanding of the nucellus elimination process in Arabidopsis, driven by the action of the MADS domain transcription factor TRANSPARENT TESTA 16 (TT16). The process of nucellus cell elimination was known to be conserved in cereals and, at least in rice, under the control of the TT16 orthologue gene MADS29. As we had a good understanding of the process of nucellus elimination, nothing was known on its mechanism of arrest in both Arabidopsis and cereals as well as unknown was the opposite process of nucellus growth in perispermic seeds. Furthermore, the physiological role of the persistent nucellus had been explored in cereals, endospermic seeds, and in the quinoa perispermic seed. In rice, for example, the persistent nucellus specializes in the transport of nutrients to the endosperm and expresses SWEET facilitators to facilitate the transit of sugars into the apoplast. In perispermic seeds, the nucellus was instead known to accumulate nutrients. Despite light was shed on the mechanisms of both nutrient and tissue partitioning in the nucellus, these processes had been studied independently and little was known on their coregulation.

I first asked what the role of the Arabidopsis nucellus is in nutrient partitioning and how it relates to nucellus elimination. As the nucellus is located between the nutrient sink (endosperm/embryo) and source (chalaza) tissues, I wondered if it played a function in nutrient transport, as shown in cereals, or accumulation, as shown in perispermic seeds. In other words, does the nucellus works as a sink or source tissue? I was also curious about the causal relationship, if any, between tissue and nutrient partitioning. Nutrient partitioning has been always described as following tissue partitioning and the hypothesis that the transient nucellus might be eliminated by a starving mechanism had never been addressed before. Finally, as tissue and nutrient portioning are two linked mechanisms, I was interested in testing if TT16 might co-regulate both processes.

I then asked how the process of nucellus is contained. The elimination of the transient nucellus happens cell layer by cell layer in a centripetal fashion, both in cereals and Arabidopsis. This mechanism is consistent with the spreading of a cell death signal or toxic materials responsible for the elimination of the nucellus. Nevertheless, the most proximal cell layers of the nucellus, the persistent nucellus, are not eliminated, thus indicating that the there is a mechanism for blocking the spreading of such hypothetical death signal. I therefore investigated if a symplastic block exists between persistent and transient nucellus. In addition, I wondered if the elimination and survival of the nucellus is genetically regulated.

I finally focused my attention on the opposite cell fate of the nucellus, growth. Compared to all other known seeds, which undergo nucellus elimination, perispermic seeds, such as amaranth, display nucellus growth. During evolution, plants shifted several times between an endospermic (dominated by the elimination of the nucellus) and perispermic (characterized by nucellus growth) seed architecture, suggesting that few genes might be responsible for such a change. I therefore asked if a change in TT16 expression or function might explain the growth of the nucellus in the amaranth perispermic seed.

Chapter II -The Arabidopsis nucellus, between cell elimination and sugar transport

The Arabidopsis nucellus, between cell elimination and sugar transport Funding:

The project was supported by the Labex Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS) grant.

INTRODUCTION

Nutrient accumulation in the seed helps the growth of the embryo and the establishment of the seedling in adverse environmental conditions. Seeds evolved different nutrient storing strategies alongside different seed structures. Seed nutrient and tissue partitioning are indeed deeply intertwined mechanisms. Cell elimination programs are put in place in the seed to eliminate unwanted tissues in favor of others that will accumulate and store nutrients, two processes whose sequential order is not fully understood (Ingram, 2017;Lu and Magnani, 2018). In angiosperms, the nucellus maternal tissue and the endosperm fertilization product grow antagonistically to become the main nutrient sink during early seed development (Lu and Magnani, 2018). Such a process defines different seed architectures (Lu and Magnani, 2018) and marked the evolution of angiosperm seeds (Magnani, 2018).

In early Arabidopsis seed development, the endosperm triggers the partial elimination of the nucellus through a signaling pathway that converges on the MADS Box transcription factor TRANSPARENT TESTA 16 (TT16) (Xu et al., 2016) and becomes the main sugar sink. Sucrose is transported from the placental tissue to the chalaza, the seed maternal tissue where vascularization ends, through the phloem. GFP mobility assays demonstrated that chalaza and seed coat maternal tissues are symplasmically connected, thus suggesting that sucrose diffuses from one tissue to the other through the plasmodesmata (Stadler et al., 2005). Nevertheless, maternal tissues and fertilization products are symplasmically isolated (Stadler et al., 2005). The discovery of SWEET sucrose facilitators on the plasma membrane of seed coat cells indicates that sucrose is exported to the apoplasm where it diffuses to the fertilization products (Chen et al., 2015). Finally, previous studies have shown that SWEET and SUCROSE-PROTON SYMPORTER (SUC) proteins allow the endosperm to reimport sucrose from the apoplasm [START_REF] Baud | The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis[END_REF]Chen et al., 2015).

Similar to Arabidopsis, the rice endosperm dominates early seed development and partially eliminates the nucellus through the action of the MADS29 transcription factor, TT16 orthologue in rice [START_REF] Nayar | Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis[END_REF][START_REF] Yang | Live and let die -the B(sister) MADS-box gene OsMADS29 controls the degeneration of cells in maternal tissues during seed development of rice (Oryza sativa)[END_REF]. By contrast, the rice nucellus takes the role of the Arabidopsis seed coat in sugar transport. The nucellus cells in between endosperm and chalaza develop into the so-called nucellar projection, a tissue dedicated to sugar transport symplasmically connected to the chalaza [START_REF] Krishnan | Structural and histochemical studies on grain-filling in the caryopsis of rice (Oryza sativa L.)[END_REF][START_REF] Wu | Rice caryopsis development I: Dynamic changes in different cell layers[END_REF]. OsSWEET11 and OsSWEET15 sucrose transporters are located on the plasma membrane of the nucellar projection and facilitate the export of sucrose to the apoplasm (Yang et al., 2018b). Cell wall bound invertases then hydrolyze part of the apoplasmic sucrose into hexoses (glucose and fructose) [START_REF] Wang | Control of rice grain-filling and yield by a gene with a potential signature of domestication[END_REF]. Finally, sucrose and glucose are reimported in the endosperm cells by the OsSWEET11, OsSWEET15, and OsSWEET4 sugar facilitators (Sosso et al., 2015;Yang et al., 2018b).

The Arabidopsis nucellus is conveniently positioned in between the nutrient unloading zone and the sink tissue (Fig. 1A) and suggests a role in nutrient transport or accumulation alongside the seed coat. In line with this hypothesis, in silico analyses by Hedhly and coworkers indicate that the seed chalazal area is a hot-spot for sugar metabolism and transport during early seed development [START_REF] Hedhly | Starch Turnover and Metabolism during Flower and Early Embryo Development[END_REF]. Here, we demonstrate that chalaza and nucellus are symplasmically connected, thus suggesting that sugars can diffuse toward the nucellus. The transient nucellus is indeed an early sugar sink in Arabidopsis seeds while releasing starch granules in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars to the apoplasm through the SWEET4 sugar facilitator. Finally, we investigate the link between the processes of tissue and nutrient partitioning in the nucellus by analyzing tt16 seeds, which do not undergo nucellus elimination.

RESULTS

The nucellus is an early sugar sink

In Arabidopsis, a few nucellus cell layers persist in between the chalaza, where nutrients are unloaded from the phloem, and the endosperm, a strong nutrient sink (Fig. 1A) (Xu et al., 2016). To test if the nucellus accumulates sugars, we monitored the starch (the main storage carbohydrate) content in the nucellus across fertilization. At the end of ovule development, Lugol staining (a starch indicator) revealed starch build-up at the boundary between nucellus and chalaza, in the female gametophyte and in proximal integument cells (Fig. 1B). After fertilization, the distal cells of the nucellus (transient nucellus, undergoing cell elimination, Fig. 1A), the boundary between nucellus and chalaza/proximal inner integument (ii), and the outer integument (oi) accumulated starch (Fig. 1C). Furthermore, we detected fainter Lugol staining in proximal nucellus cells (persistent nucellus, not undergoing cell elimination, Fig. 1A) and in distal ii cells (Fig. 1C). From the globular embryo stage of seed development on, we observed strong Lugol staining everywhere in the seed (Sup. Fig. 1), thus losing tissue resolution.

To better characterize the pattern of starch accumulation in different nucellus domains we adapted the modified pseudo-Schiff propidium iodide imaging (mPS-PI) technique (Truernit et al., 2008b;Xu et al., 2016) to the analysis of both cell wall and starch in seeds (starch-mPS-PI, see methods and Sup. Fig. 2). In line with Lugol staining analyses, we detected starch in the transient nucellus but not in the persistent nucellus, at early embryogenesis (Fig. 1D). Furthermore, we observed propidium iodide staining in the chalaza and ii cells surrounding the nucellus and in the oi2 (Fig. 1D). Starch-mPS-PI analyses revealed that the persistent nucellus does not accumulate starch even later in seed development, when most of the seed maternal tissues, with the exception of the ii1'cell layer, appear full of starch (Fig. 1E). These results indicate that the faint Lugol staining observed in the persistent nucellus is possibly due to diffusion of stained material during the seed clearing process (see methods).

We further confirmed our results by transmission electron microscopy, which showed starch granules in the apoplasmic space in between nucellus and endosperm (nucellar cavity, which contains cell corpses and debris resulting from the process of cell elimination) and in transient nucellus cells but not in the persistent nucellus (Fig. 1F).

Finally, we analyzed starch biosynthesis by fusing the promoter region of the GLUCOSE-1-PHOSPHATE ADENYLYLTRANSFERASE LARGE SUBUNIT 3 (APL3) gene, which encodes the enzyme that catalyzes the first limiting step reaction in starch biosynthesis [START_REF] Crevillen | The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme[END_REF][START_REF] Crevillen | Differential pattern of expression and sugar regulation of Arabidopsis thaliana ADP-glucose pyrophosphorylase-encoding genes[END_REF], to the chimeric GFP-GUS reporter gene (ProAPL3:GFP-GUS). In ovules, we detected GFP fluorescence in the oi1 and ii2 cell layers and the female gametophyte (Fig. 2A).

After fertilization, the promoter was active in the transient nucellus, chalaza, oi1, ii2, and endosperm of pre-globular embryo stage seeds (Fig. 2B). At bent cotyledon embryo stage, we detected fluorescence solely in the oi (Fig. 2C). Overall, GFP fluorescence in the nucellus recapitulated starch deposition as observed by Lugol, starch-mPS-PI and transmission electron microscopy analyses. These data indicate that the transient nucellus is an early sugar sink in Arabidopsis seeds.

The nucellus is symplasmically connected to the chalaza

The finding of starch in the nucellus prompted us to test if sugars can move symplasmically from the chalaza, the seed nutrient unloading zone, to the nucellus. To this mean, we conducted GFP mobility assays by expressing GFP or GFP-sporamin translational fusion in the companion cells of the chalazal phloem under the control of the SUCROSE-PROTON SYMPORTER 2 (SUC2) promoter region. It has been previously shown that the size exclusion limit of the chalazal plasmodesmata allows GFP (27 kD), but not GFP-sporamin (47 kD), to diffuse to the neighboring seed coat cells (Stadler et al., 2005). At 1-cell embryo stage, GFP-sporamin was confined in a few chalazal cells (Fig. 3A). By contrast, we detected free GFP fluorescence in chalaza, oi, proximal ii and nucellus cells at the same developmental stage (Fig. 3B). These data demonstrate that the nucellus is symplasmically connected to the chalaza and suggest that nutrients diffuse from the chalaza to the nucellus in a symplasmic fashion.

SWEET4 sugar facilitator is expressed in the nucellus

Nutrients are supposed to reach the fertilization products via the apoplasm as maternal and zygotic tissues are not symplasmically connected (Stadler et al., 2005). In Arabidopsis, SWEET transporters in the seed coat facilitate sucrose export to the apoplasm (Chen et al., 2015). By contrast, rice grains express SWEET genes in nucellar cells (Yang et al., 2018b), which play an important role in nutrient transfer to the endosperm. To verify if the Arabidopsis nucellus also plays a role in sugar apoplasmic transport, we tested if members of the SWEET gene family are expressed in such tissues. According to microarray data by Belmonte and coworkers (Belmonte et al., 2013), the SWEET4 glucose and fructose facilitator gene (Chen et al., 2010;[START_REF] Liu | AtSWEET4, a hexose facilitator, mediates sugar transport to axial sinks and affects plant development[END_REF] is specifically expressed in the chalazal region (including chalaza and nucellus) of Arabidopsis seeds throughout seed development (Sup. Fig. 3). To better characterize its expression pattern, we fused SWEET4

promoter region to the chimeric GFP-GUS reporter gene. In ProSWEET4:GFP-GUS ovules and seeds, we detected GFP fluorescence in the most proximal nucellus cell layer and first 1-3 cells of the ii (Fig. 4A-4C). Our results indicate that in Arabidopsis the persistent nucellus plays a role in sugar transport.

SWEET4 localizes to the plasma membrane

To test if SWEET4 facilitate transport across the plasma membrane, we studied its sub-cellular localization. The SWEET4-GFP chimeric protein has been shown to localize to the plasma membranes of protoplasts, when expressed under the control of the 35S promoter sequence (Liu et al., 2016). To test SWEET4 localization in planta, we imaged cells of Pro35S:SWEET4-GFP seeds.

We detected GFP fluorescence in the plasma membrane, in proximity to propidium iodide signal marking the cell wall, and in vesicles (Fig. 5A). In a number of cells we also detected fluorescence in the vacuole (Sup. Fig. 4). SWEET4-GFP localization in the vacuole might be due to incorrect protein folding or overcrowding of the secretory pathway in the transgenic lines. Overall, these data indicate that SWEET4 facilitates sugar export to the apoplasm.

SWEET4 affects seed sugar content

A number of sweet mutants have been shown to transiently and locally over-accumulate sugars as a consequence of impaired sugar export, while negatively affecting the sugar content of the sink tissues in the long run. For example, mutations in the SWEET11, SWEET12, and SWEET15 genes lead to excessive starch accumulation in the seed coat and low starch levels in the embryo, when compared to the wild type (Chen et al., 2015). In line with these data, we observed stronger Lugol staining in sweet4 mutant (see methods and Sup. Fig. 5) nucellus, chalaza and seed coat, when compared to the wild type (Fig. 6A-6C). Nevertheless, Lugol staining assays are not quantitative and we detected a certain degree of variability among seeds sharing the same genetic background.

We therefore quantified sugars content in wild type and mutant seeds. On average, sweet4 seeds at four and six days after flowering (DAF) accumulated higher levels of glucose, fructose, total soluble sugars and starch, when compared to the wild type (Fig. 6D and6E). By contrast, sweet4 dry seeds showed a slightly lower level of sucrose and soluble sugars than wild type seeds (Fig. 6F).

Finally, we showed that the perturbed sugar transport in sweet4 seeds does not affect oil and protein accumulation (Fig. 6G).

When sugars are not efficiently transported from the seed coat to the fertilization products, as in the sweet11;sweet12;sweet15 triple mutant, embryo development is delayed (Chen et al., 2015).

Similarly, we observed that the sweet4 mutation impaired embryo growth (Fig. 7). Overall, these data support the hypothesis that SWEET4 facilitates sugar export to the apoplasm during early seed development.

TT16 regulates cell elimination but not sugar export in the nucellus

We have previously shown that the B-sister MADS box transcription factor TT16 plays a role in seed tissue partitioning by promoting partial nucellus elimination in favor of endosperm growth (Xu et al., 2016). Since tissue and nutrient partitioning are known to be tightly linked mechanisms (Lu and Magnani, 2018), we investigated sugar transport and metabolism in tt16 mutant seeds, whose transient nucellus (thereafter referred to as transient nucellus*, Fig. 8A) is not eliminated.

Whereas starch accumulation in wild type and tt16 ovules appeared indistinguishable (Fig. 1B and 8B), Lugol staining and starch-mPS-PI analyses highlighted three domains in the tt16 nucellus:

from proximal to distal, 1) the persistent nucellus, which did not accumulate starch, 2) the proximal cells of the transient nucellus*, which accumulated starch, and 3) the distal cells of the transient nucellus*, which did not accumulate starch (Fig. 8C and8D). Nevertheless, the ProAPL3:GFP-GUS construct did not drive GFP expression in the nucellus of tt16 ovules and seeds (Fig. 8E and8F). Altogether, these data show that part of the transient nucellus maintains its sugar sink force if it is not eliminated.

Sugars quantification in tt16 seeds revealed a lower level of sucrose and soluble sugars at 4

DAF and a higher level of the same sugars in dry seeds, when compared to the wild type (Fig. 6D-6F). Furthermore, tt16 dry seeds contained less oil and more proteins than wild type seeds (Fig. 6G).

Finally, embryo development was delayed in tt16 seeds relative to the wild type (Fig. 7).

To test if TT16 affects nutrient transport by regulating SWEET4 expression, we introgressed the ProSWEET4:GFP-GUS line in a tt16 background. The tt16 mutation did not perturb SWEET4 spatial expression pattern (Fig. 9A to 9B), when compared to the wild type (Fig. 4). In line with these results, RT-qPCR analyses of SWEET4 expression in tt16 and wild type seeds at 2 and 11 DAF were statistically similar (Sup. Fig. 6). Altogether, these data suggest that TT16 regulates the elimination of the nucellus and, indirectly, the release of starch in the nucellar cavity but not the apoplasmic transport of sugar through the SWEET4 facilitator.

DISCUSSION

The nucellus path of nutrient transport

In Arabidopsis, it has been shown that sugars travel from the placental tissue to the chalaza through the phloem and then diffuse symplasmically towards the seed coat (Fig. 10) (Stadler et al., 2005). The seed coat has been regarded as the only tissue responsible for sugar export to the apoplasm (Chen et al., 2015), a needed step to allow sugar diffusion toward the fertilization products that are symplasmically disconnected from the maternal tissues (Stadler et al., 2005). Here, we identified a novel path of sugar transport through the nucellus (Fig. 10). Our data indicate that the chalaza is symplasmically connected not only to the seed coat but also to the nucellus. Therefore, we speculate that chalazal sucrose and/or hexoses, resulting from the activity of cytoplasmic sucrose synthases present in the chalaza [START_REF] Fallahi | Localization of sucrose synthase in developing seed and siliques of Arabidopsis thaliana reveals diverse roles for SUS during development[END_REF], diffuse from cell to cell to the nucellus. The most proximal cells of the persistent nucellus express the SWEET4 glucose and fructose facilitator (Chen et al., 2010;[START_REF] Liu | AtSWEET4, a hexose facilitator, mediates sugar transport to axial sinks and affects plant development[END_REF], indicating that part of the nucellar hexoses are exported to the apoplasmic space. We speculate that the apoplasmic glucose and fructose, provided by the nucellus, might diffuse toward the endosperm to be reimported by endosperm transporters [START_REF] Baud | The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis[END_REF]Chen et al., 2015). In line with our model, sweet4 mutant lines displayed over-accumulation of soluble sugars during early seed development. Part of the excess of hexoses observed in the sweet4 mutant might be converted into starch in the maternal tissues, as suggested by Lugol staining assays and starch quantification analyses. Later in seed development, the lower sugar content of sweet4 seeds, when compared to the wild type, might be due to the compromised transport of sugars from the nucellus to the fertilization products, the ultimate sink tissues. A correlation between sugar and lipid content in seeds has been observed in a number of mutations that affect sugar metabolism. Nevertheless, the relationship between sugar and lipid metabolism has not been fully understood as other mutations, such as sus2 and sus3, perturb starch but not lipid accumulation, similar to what observed in sweet4 seeds (Angeles-Nunez and Tiessen, 2010; [START_REF] Barratt | Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase[END_REF]. Compared to the seed coat path, which is based on sucrose transport (Chen et al., 2015), our data indicate that hexoses are mostly transported through the nucellus path.

We speculate that the seed-coat is involved in relatively longer distance transport of sugars and therefore favors the more energy-efficient and less reactive sucrose. By contrast, the nucellus offers a faster route toward the endosperm thus allowing the transport of readily available glucose and fructose.

Cell elimination as a way to recycle sugars.

We demonstrated that the transient nucellus accumulates starch across fertilization. Starch is then released into the nucellar cavity, the apoplasmic space in between nucellus and endosperm, which contains cell corpses and debris resulting from the process of cell elimination (Fig. 10). A similar phenomenon has been shown in cereals, which possess a larger transient nucellus that works as transient sugar sink and develops, in some species, a larger nucellar cavity in between nucellus and endosperm (Lu and Magnani, 2018). In Barley grains, alpha amylase 4 has been shown to be active in nucellus cells during cell elimination, thus indicating that the starch in the nucellus is hydrolyzed to facilitate its mobilization to the endosperm [START_REF] Radchuk | Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain[END_REF]. Similarly, microarray data suggest that the starch degradation pathway is active in the Arabidopsis chalazal area [START_REF] Hedhly | Starch Turnover and Metabolism during Flower and Early Embryo Development[END_REF]. In line with this hypothesis, starch granules in nucellus cells at an advanced stage of cell elimination appeared less contrasted than those in cells starting the process of elimination, when imaged by transmission electron microscopy (Fig. 1F). Therefore, the starch present in the Arabidopsis transient nucellus might be recycled in favor of the endosperm. The advantage, if any, of such a process in a relatively small nucellus is not clear. Alternatively, it might be an evolutionary relic. The nucellus, and not the endosperm, acts indeed as major sugar sink in perispermic seeds (Lopez-Fernandez and Maldonado, 2013), which might be the ancestral seed condition (Lu and Magnani, 2018;Magnani, 2018), a character that might have been retained by the Arabidopsis nucellus despite undergoing cell elimination. tt16 seeds partially resemble perispermic seeds as they carry a large nucellus that accumulates starch, thus indicating a possible molecular scenario for the evolution of such different seed architectures.

Coordination of tissue and nutrient partitioning.

Tissue and nutrient partitioning are thought to be two inextricably linked processes. In early Arabidopsis seed development, the nucellus is eliminated to allow endosperm growth and nutrient accumulation. Later in seed development, most of the endosperm is eliminated to allow embryo growth, the ultimate nutrient sink. It is still debated if nutrient partitioning follows or precedes tissue partitioning (Ingram, 2017). It has been speculated that preventing nutrient transport to a tissue might indeed lead to its elimination. Our finding of starch accumulation in the transient nucellus, alongside sugar export towards the endosperm in the persistent nucellus, argues against this hypothesis. Furthermore, TT16 allows endosperm growth by eliminating the transient nucellus (Xu et al., 2016) but does not promote nutrient export to the endosperm by activating SWEET4 expression in the persistent nucellus.

Nonetheless, the tt16 mutation affected sugar, oil and protein content in seeds. TT16 might regulate, directly or indirectly, the expression of other nutrient transporters in the seed coat or in the chalaza. Indeed, the pleiotropic nature of the tt16 seed phenotype, which shows defects in nucellus, seed coat, and chalaza development (Coen et al., 2017;Coen et al., 2019a;Coen et al., 2019b;Ehlers et al., 2016;Fiume et al., 2017a, b;Nesi et al., 2002a;Xu et al., 2016), does not allow us to conclusively interpret such data. Further experiments are necessary to understand the regulatory mechanisms underlying such physiological changes.

METHODS

Plant material

All Arabidopsis thaliana plants are in the Columbia accession. The tt16-1 allele was isolated in the Wassilewskija accession from the INRA Versailles collection (Bechtold and Pelletier, 1998) and then backcrossed to Columbia (Coen et al., 2017;Nesi et al., 2002a). sweet4-1 (SALK_072225) (Chong et al., 2014) allele is from the Salk collection (Alonso et al., 2003) whereas the sweet4-2 (GK_858G02) (Chong et al., 2014) allele is from the GABI-KAT collection (Kleinboelting et al., 2012). SWEET4 expression in sweet4-1 and sweet4-2 seeds, compared to the wild type, has been tested by RT-qPCR, as shown below, by using a set of primers

(5'CCTCAATGGTGTCGTTTGGG3' and 5'TAGCTTGTCCACTGTTGCCA3') downstream of both T-DNA insertions (Sup. Fig. 5).

Cloning and Construction

PCR amplifications were performed using the gene-specific primers described below carrying the attB1 (5′GGGGACAAGTTTGTACAAAAAAGCAGGCT3′) and attB2

(5′GGGGACCACTTTGTACAAGAAAGCTGGGTC3′) Gateway recombination sites at the 5′ends of the forward and reverse primers, respectively. All PCR products were amplified with the high-fidelity Phusion DNA polymerase (Thermo Fisher Scientific), recombined into the pDONR207 or pDONR201 vector (BP Gateway reaction) according to the manufacturer's instructions (Thermo Fisher Scientific), and then sequenced. The PCR products cloned into the pDONR vectors were then recombined into the appropriate destination vector (LR Gateway reaction) according to the manufacturer's instructions (Thermo Fisher Scientific). SWEET4 3.2 kb promoter region was amplified using the attB1-(5′TGGTTCGCATTTTGGATTCTTTGTTTAC3′) forward and attB2-(5′ TTCACTTCAAAAGAAAAATCCGAAC3') reverse primers. SWEET4 genomic sequence without the stop codon was PCR amplified using the attB1-(5′CGCTCGCTCTCTTCTTTGTT3') forward and attB2-(5′AGCTGAAACTCGTTTAGCTTGTC3') reverse primers. APL3 1.8 kb promoter region was amplified using the attB1-(5′GTCGACGATGTTTGGTTTCTTTATCC3') forward and attB2-(5′CCATGGCTTTTTTTAGCTGGAATGA3') reverse primers.

both cell walls and starch.

GFP expressing lines were analyzed one hour after mounting in a 100 µg/ml propidium iodide, 7% sucrose solution, as previously described (Figueiredo et al., 2016).

Samples were imaged by confocal laser scanning microscopy (Leica SP8). Three-dimensional Z-stack confocal laser scanning microscope images were analyzed through the Volume Viewer plug-in of the ImageJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. More than 30 seeds were analyzed for each genotype and time point.

Transmission electron microscopy

Transmission electron microscopy analyses were conducted as previously described (Coen et al., 2019a).

Enzymatic analyses

Sugars were extracted as previously described (Baud et al., 2002a) and quantified by using the Enzytech D-Glucose/Fructose/Sucrose kit (R-biopharm, https://r-biopharm.com/fr/). At least four biological replicates were performed for each genotype. 50 seeds were used for each replicate.

Seed oil and protein content by near-infrared spectroscopy

Seed samples were analyzed as previously described [START_REF] Jasinski | Arabidopsis Seed Content QTL Mapping Using High-Throughput Phenotyping: The Assets of Near Infrared Spectroscopy[END_REF]. Four biological replicates were performed for each genotype. 300 µl of seeds were used for each replicate.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: TT16 (AT5G23260), SWEET4 (AT3G28007), APL3 (AT4G39210).

INTRODUCTION

The chalaza has been shown to be the nutrient unloading zone, symplastically connected to the phloem, which provide nutrients to the seed, and to the seed coat and nucellus tissues, which export nutrients to the apoplastic space. In this study we challenged the hypothesis that the chalaza might also be involved in apoplastic sugar transport. We characterized the role of the SWEET10 transporter protein in facilitating sucrose export in the chalazal region (Chen et al., 2012a;Rottmann et al., 2018). The nature of the SWEET facilitator and our finding of a cell wall bound invertase in the chalazal area suggest that the chalaza provide sucrose and hexoses to the endosperm.

RESULTS

The SWEET10 gene is expressed in the chalaza

We tested if members of the SWEET gene family are expressed in the chalzal tissue. According to microarray data by Belmonte and coworkers (Belmonte et al., 2013), the SWEET10 sucrose facilitator gene (Rottmann et al., 2018) is specifically expressed in the chalazal region of Arabidopsis seeds throughout seed development. To better characterize its expression pattern, we fused SWEET10 promoter region to the chimeric GFP-GUS reporter gene. In ProSWEET10:GFP-GUS ovules and seeds, we detected GFP fluorescence in the chalazal cells surrounding the vasculatures, in both ovules and seeds (Fig. 1). Our results indicate that the chalaza play a role in sugar apoplastic transport.

SWEET10 localize to the plasma membrane

To test if SWEET10 facilitate transport across the plasma membrane, we studied its subcellular localization. Orthologues of the Arabidopsis SWEET10 protein in sweet potato and grapes have been shown to localize to the plasma membrane [START_REF] Li | The Plasma Membrane-Localized Sucrose Transporter IbSWEET10 Contributes to the Resistance of Sweet Potato to Fusarium oxysporum[END_REF][START_REF] Zhang | VvSWEET10 Mediates Sugar Accumulation in Grapes[END_REF]. Similarly, we observed GFP fluorescence in the plasma membrane and vacuoles (Fig. 2A) or only in the plasma membrane (Fig. 2B) of chalazal cells expressing SWEET10 promoter and genomic sequence translationally fused to GFP. These data indicate that SWEET10 facilitate sugar export to the apoplast. SWEET-GFP localization in the vacuole might be due to incorrect protein folding or overcrowding of the secretory pathway in the transgenic lines. The chalaza provides hexoses to the endosperm SWEET10 has been demonstrated to work as sucrose facilitator (Chen et al., 2012a;Rottmann et al., 2018). To test if the sucrose transported by SWEET10 to the apoplastic space is then converted into hexoses, we tested if cell wall bound invertases, which hydrolyze sucrose into hexoses, are expressed in the chalaza. According to microarray data by Belmonte and coworkers (Belmonte et al., 2013), the cell wall bound INVERTASE4 (INV4) [START_REF] Ruhlmann | CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis[END_REF]

TT16 regulates SWEET10 expression pattern

To test if TT16 affects nutrient transport by regulating SWEET10 expression, we introgressed ProSWEET10:GFP-GUS lines in a tt16 background. tt16 mutation did not appear to affect SWEET10 spatial expression pattern (Fig. 5A to 5B), when compared to the wild type (Fig. 1). By contrast, RT-qPCR analyses revealed a 2.2 fold increase of SWEET10 expression in tt16 seeds at 2 DAF, when compared to the wild type (Fig. 5C). TT16 is not expressed in the chalaza (Xu et al., 2016) but it has been shown to act non-autonomously on the development of the chalazal cells

INTRODUCTION How is cell elimination contained?

Our preliminary results suggest that cell elimination of the nucellus involves the degradation of cell walls and the release of cell components, including vacuole contents, into the apoplast. Such events undoubtedly involve the release of destructive molecules that might be expected to diffuse locally through the apoplast or through plasmodesmata. However, intriguingly, cell death has not been reported to spread to neighboring tissues in seeds. Furthermore, the nucellus is not completely eliminated. As a result, it seems clear that cell elimination is tightly spatially controlled both within and between the tissues of the developing seed.

Soon after fertilization the three genetic compartments of the seed (maternal tissues, endosperm and embryo), become symplastically isolated (Stadler et al., 2005), thus suggesting that the establishment of a symplastic barrier might be responsible of containing cell elimination.

The establishments of symplastic blocks

Previous studies have shown that the regulation of plasmodesmal connectivity among cells is a dynamic process that is tightly linked to developmental and physiological processes [START_REF] Holdaway-Clarke | Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata[END_REF][START_REF] Tucker | Mastoparan-induced intracellular Ca2+ fluxes may regulate cell-to-cell communication in plants[END_REF]. Furthermore, various signal pathways have been shown to relay primary cellular signals to modify plasmodesmata [START_REF] Sager | Plasmodesmata in integrated cell signalling: insights from development and environmental signals and stresses[END_REF]. Two general classes of mechanisms have been shown to modify the permeability of plasmodesmata: callose dependent and callose independent mechanisms. Callose (β-1,3-glucan) is a polysaccharide that is deposited at the cell plate during cytokinesis.

When cell division is completed, callose is almost completely degraded [START_REF] Northcote | Use of antisera to localize callose, xylan and arabinogalactan in the cell-plate, primary and secondary walls of plant cells[END_REF].

Nevertheless, a small portion persists and plays a role in regulating the permeability of plasmodesmata. Callose accumulation around the neck region of plasmodesmata effectively reduces plasmodesmal permeability (Zavaliev et al., 2011a), and even lead to plasmodesmal closure (Fig 1).

The level of callose accumulation at plasmodesmata is mediated mainly by the activity of two enzymes: β-1,3-glucan synthases and glucanases (Zavaliev et al., 2011a), which catalyze the synthesis and degradation of callose, respectively. In Arabidopsis, twelve genes are predicted to encode the catalytic subunit of the β-1,3-glucan synthase and are named CALLOSE SYNTHASE (CALS) or GLUCAN SYNTHASE-LIKE (GSL) genes [START_REF] Saxena | Cellulose synthases and related enzymes[END_REF][START_REF] Verma | Plant callose synthase complexes[END_REF]. Furthermore, fifty Β-1,3-GLUCANASE (BGL) genes were identified in Arabidopsis [START_REF] Doxey | Functional divergence in the Arabidopsis β-1, 3-glucanase gene family inferred by phylogenetic reconstruction of expression states[END_REF]Zavaliev et al., 2011b). Callose-dependent modulation of plasmodesmata permeability has been described in various developmental processes and in response to different cellular and environmental conditions. It has been shown that the loss of symplastic connectivity between megaspore mother cell and neighboring nucellus cells in the ovule [START_REF] Werner | A dual switch in phloem unloading during ovule development in Arabidopsis[END_REF] is due to callose deposition around the megaspore mother cell (Schneitz et al., 1995c). Similarly, the tapetal cells, surrounding the pollen grains, secrete callose, thus leading to their symplastic isolation (Mamun et al., 2005a, b;[START_REF] Regan | Cytochemical analysis of pollen development in wild-type Arabidopsis and a malesterile mutant[END_REF]. As a consequence of the symplastic isolation of the tapetum cell layer, nutrients and metabolites are transported apoplastically to the developing pollens grains (Clé ment and Audran, 1995;[START_REF] Roschzttardtz | The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development[END_REF]. It is speculated that the symplastic isolation of the tapetum during pollen development is necessary to protect the surrounding cells from potentially detrimental molecules generated during the programmed cell death of the tapetum [START_REF] Wu | Programmed cell death in plant reproduction, Programmed Cell Death in Higher Plants[END_REF].

After fertilization, the embryo is symplastically isolated from the surrounding tissues. Only the suspensor and embryo proper are symplastically connected during early embryogenesis (Stadler et al., 2005). However at embryo heart stage, the suspensor become cytoplasmically isolated as its plasmodesmata increase in structural complexity and are occluded by a callose like substance [START_REF] Kozieradzka-Kiszkurno | Are there symplastic connections between the endosperm and embryo in some angiosperms?-a lesson from the Crassulaceae family[END_REF]. The symplastic restriction escalates through embryo maturation, till an overall block in intercellular permeability in the late torpedo stage as the suspensor start degenerating (Stadler et al., 2005).

Besides the over mentioned developmental processes, callose modulates plasmodesmata closing in response to various cellular and environmental conditions, such as cold, wound, toxic metal stress, defense and dormancy. A number of callose modifying enzymes were shown to modulate plasmodesmata callose level in response to various environmental stimuli. The cals1 mutant fails to accumulate callose in response to pathogenic bacterial infection, whereas the cals8 mutant shows disrupted callose deposition in response to mechanical wounding [START_REF] Cui | Arabidopsis callose synthases CalS1/8 regulate plasmodesmal permeability during stress[END_REF].

Chilling-induced dormancy breakage at the shoot apical meristem is marked by an increase in symplastic connectivity and shows up-regulation of BGL gene expression, responsible for plasmodesmata callose degradation [START_REF] Rinne | The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy[END_REF].

Plasmodesmata can be blocked also through a callose-independent mechanism. Cytoskeletal elements like filamentous actin, myosin proteins and microtubules, have been found localized to or near plasmodesmata, and play a role in transporting viral movement proteins. For example, the cauliflower mosaic virus (CaMV) inclusion body protein P6 traffics intracellularly along microfilaments and actin disruption inhibits CaMV cell to cell movement [START_REF] Harries | The cauliflower mosaic virus protein P6 forms motile inclusions that traffic along actin microfilaments and stabilize microtubules[END_REF].

Plant MYOSIN VIII locates at the plasma membrane and in clusters near plasmodesmata and helps targeting viral cargoes to plasmodesmata [START_REF] Baluška | Actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices[END_REF][START_REF] Radford | Localization of a myosin-like protein to plasmodesmata[END_REF][START_REF] Reichelt | Characterization of the unconventional myosin VIII in plant cells and its localization at the post-cytokinetic cell wall[END_REF]. It has been shown that the disruption of tobacco MYOSIN VIII impairs the targeting of the viral protein Hsp70 to plasmodesmata [START_REF] Avisar | Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog[END_REF]. Furthermore, a study showed that increasing the concentration of cytosolic free calcium stimulates the rapid closure of plasmodesmata [START_REF] Tucker | Calcium-loaded 1, 2-bis (2-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid blocks cell-to-cell diffusion of carboxyfluorescein in staminal hairs of Setcreasea purpurea[END_REF]) by affecting calcium-responsive proteins, including certrin and myosin, which associate with plasmodesmata [START_REF] Geimer | Centrin scaffold in Chlamydomonas reinhardtii revealed by immunoelectron microscopy[END_REF][START_REF] Hu | The Mode of Action of Centrin BINDING OF Ca2+ AND A PEPTIDE FRAGMENT OF Kar1p TO THE C-TERMINAL DOMAIN[END_REF][START_REF] Knight | A myosin-like protein from a higher plant[END_REF][START_REF] Sanders | Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii[END_REF]. This rapid but transient contractions of the pore, which is physiologically relevant, takes only 10 seconds compared to the callose-dependent transient closure which takes hours [START_REF] Holdaway-Clarke | Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata[END_REF][START_REF] Tucker | Mastoparan-induced intracellular Ca2+ fluxes may regulate cell-to-cell communication in plants[END_REF]. Based on their localization in plasmodesmata and detrimental effect on intercellular movement, it has been speculated that filamentous actin and myosin regulate the plasmodesmal size exclusion limit [START_REF] Tilsner | Staying tight: plasmodesmal membrane contact sites and the control of cell-to-cell connectivity in plants[END_REF].

In addition to β-1,3-glucan synthases and glucanases, a number of other plasmodesmatalocalized proteins have been identified to regulate the permeability of plasmodesmata through both callose-dependent and independent modes. For example, ectopic overexpression of the Arabidopsis PLASMODESMATA CALLOSE BINDING PROTEIN1 (PDCB1) gene, which encodes an extracellular protein that contains a glycosylphosphatidylinositol anchor, increases callose deposition in the plasmodesmata [START_REF] Simpson | An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking[END_REF]. Furthermore, PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) and 5 genes encode receptor-like integral membrane proteins in Arabidopsis whose overexpression results in reduced plasmodesmata connectivity [START_REF] Cui | Arabidopsis callose synthases CalS1/8 regulate plasmodesmal permeability during stress[END_REF][START_REF] Thomas | Specific targeting of a plasmodesmal protein affecting cell-to-cell communication[END_REF]. When cells are infected by pathogens, PDLP5 allows callose accumulation in plasmodesmata, eventually leading to the symplastic isolation of the infected cells. This response helps affected cells to isolate themselves from adjacent cells and safely execute programmed cell death [START_REF] Lee | A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis[END_REF].

RESULTS

Persistent and transient nucellus are symplastically isolated.

We have previously shown that the nucellus is symplastically connected to the chalaza by GFP mobility analyses. Free GFP, which is small enough to diffuse through the plasmodesmata, was expressed in a few chalazal cell under the control of the pSUC2 promoter and fluorescence was observed in chalaza, seed coat and persistent nucellus at 1-cell globular embryo stage (Fig. 2B). As a control, GFP-sporamin, which exceeds the plasmodesmata size exclusion limit, was confined in chalaza cells at the same development stage (Fig. 2A). Interestingly, we did not detect free GFP in the transient nucellus cells that have not undergone elimination yet (Fig. 2B). These data suggest that the transient nucellus is not symplastically connected to the persistent nucellus.

We than asked when such a symplastic barrier is established during seed development. In particular, if it is fertilization dependent, as many other seed developmental pathways. To address these questions we analyzed symplastic connections in mature ovules using the same pSUC2:GFP and pSUC2:GFP-sporamin lines described above. Free GFP was detected in chalaza, oi, proximal part of ii and persistent nucellus of mature ovules, but not in the transient nucellus (Fig. 2D). As control, GFP-sporamin was detected only in a few cells of the chalaza where the phloem ends, at the same develop stage (Fig. 2C). These results indicate that the symplastic barrier is already created between transient and persistent nucellus before fertilization.

To characterize the precise time point at which the symplastic block is established we looked earlier in ovule development. We detected GFP fluorescence in the all nucellus of ovule primordia (Supplementary Fig. 1). Interestingly, at stage 3-IV of ovule development (Schneitz et al., 1995b) the entire nucellus, including its proximal region, appeared symplastically disconnected from the chalaza (Supplementary Fig. 1). At stage 3-V we detected GFP in the persistent but not transient nucellus, thus indicating that the symplastic block is established at this stage of ovule development (Supplementary Fig. 1).

TT16 does not regulate the nucellus symplastic block

Considering that TT16 promotes the elimination of the transient nucellus, we tested if the nucellus symplastic block is altered by the tt16 mutation. We therefore introgressed ProSUC2:GFP-sporamin and ProSUC2:GFP lines in a tt16 background. We found that the symplastic connection pattern in the tt16 mutant is comparable to that of the wild type. Free GFP was not observed in the large transient nucellus, which does not undergo cell elimination, of tt16 seeds (Fig. 3B). As a control, GFP-sporamin was confined only in a few cells of the chalaza at the accumulation (Fig. 5C).

Expression of callose synthases in the nucellus

To determine if callose syntheses are expressed in the nucellus and potentially contribute to the formation of the nucellus symplastic block, we looked at the microarray data by Belmonte and coworkers (Belmonte et al., 2013) obtained from laser micro-dissected domains of the seed. The GSL4 callose synthase gene was annotated as specifically expressed in the chalaza/nucellus area of the seed. To study its expression pattern, we generated a marker line carrying the GSL4 -3kb promoter region fused to the uidA reporter gene, which encodes the GUS enzyme. The GSL4 promoter drove GUS expression in the chalaza, but not the nucellus, throughout ovule and seed development (Fig. 6).

of the plasmodesmata of the persistent nucellus might be necessary to isolate the symplastic and apoplastic space of such cells once the transient nucellus is fully eliminated. Our data are based on GFP mobility assays and do not allow us to conclude if a full symplastic block or a reduction in the size exclusion limit of the nucellus plasmodesmata is put in place. Molecules smaller than 27 KD, the GFP size, might still be able to move through. We detected starch accumulation in the distal nucellus of tt16 seeds, thus indicating the sucrose or hexoses might still move through the proximaldistal axis of the nucellus.

Callose deposition created the symplastic block in nucellus

Our aniline blue staining analyses suggest that the accumulation of callose in the plasmodesmata of the transient nucellus might lead to a reduction in their size exclusion limit.

Nevertheless, the anline blue staining technique is limiting because does not allow the resolution needed to observe plasmodesmata and the fluorescence is easily quenched, thus requiring rapid imaging. TEM analysis support the hypothesis of a callose-dependent symplastic block as the distal nucellus plasmodesmata appeared swollen as observed in plasmodesmata clogged with callose.

Further experiments are required to fully address this question.

Future perspective

To confirm if callose is deposited in the plasmodesmata of the transient nucellus, we are planning to preform immunogold labeling assays using callose antibodies. Immunogold labeling could help us locate callose accurately and provide solid evidence of callose deposition in plasmodesmata. We will also keep testing the expression of members of the GSL gene family predicted to be expressed in the nucellus according to the microarray data by Belmonte and coworkers (Belmonte et al., 2013). The analysis of their mutant and over-expression lines will help us test hypotheses on the role of callose deposition in nucellus plasmodesmata and of the establishment of a symplastic block. Finally, we plan on testing if there is a decrease of plasmodesmata frequency in the transient nucellus. To address these questions we will visualize plasmodesmata during nucellus development by using fluorescent markers, such as endogenous plasmodesmal proteins fused to fluorescent proteins (e.g. members of the PDLP family [START_REF] Lee | A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis[END_REF][START_REF] Thomas | Specific targeting of a plasmodesmal protein affecting cell-to-cell communication[END_REF], PDCB1/2 [START_REF] Simpson | An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking[END_REF] and PdBG1/2(Benitez-Alfonso et al., 2013)). Furthermore, MP17, which is a movement protein of the potato leaf roll virus that binds to branched plasmodesmata, can be used as additional marker for plasmodesmata branching.

INTRODUCTION

MADS-box transcription factors are key regulators of several plant development processes.

The MADS-box gene family is characterized by the presence of the MADS box sequence motif, which encodes the DNA-binding MADS domain. The analysis of several MADS-domain proteins revealed that the 58 amino acids long MADS domain recognizes a DNA consensus sequence known as the CArG box [CC(A/T)6GG] [START_REF] Hayes | The human c-fos serum response factor and the yeast factors GRM/PRTF have related DNA-binding specificities[END_REF][START_REF] Riechmann | DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS[END_REF]. MADS-box genes have been identified in plants, fungi and metazoans. The phylogenetic analysis performed by [START_REF] Alvarez-Buylla | An ancestral MADS-box gene duplication occurred before the divergence of plants and animals[END_REF], which included 44 Arabidopsis MADS domain sequences and several MYOCYTE ENHANCER FACTOR2-like (MEF2) and SERUM RESPONSE FACTOR-like (SRF) MADS box domains from fungi and animals, suggests that a gene duplication occurred before the divergence of plants and animals, giving rise to two main lineages of MADS-box genes: type I and type II (Fig. 1). The plant type-I MADS-box transcription factors, together with the animal SRF-like domain proteins, forms the type-I lineage. The plant MIKC-type MADS-domain proteins, together with the animal MEF2-like proteins, form instead the type-II lineage [START_REF] Parenicová | Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world[END_REF]. 2). The I domain influences protein dimerization, which is driven by the K coiled-coil domain (Davies et al., 1996;Fan et al., 1997). The C domain is the least conserved domain; in some cases, it has been shown to contain a transactivation domain or to contribute to the formation of multimeric MADS-domain protein complexes [START_REF] Egea-Cortines | Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus[END_REF][START_REF] Honma | Complexes of MADS-box proteins are sufficient to convert leaves into floral organs[END_REF]. Furthermore, based on domain structure differences, MIKC-type MADS-box transcription factors have been classified into MIKC C and MIKC * types [START_REF] Henschel | Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens[END_REF]. MIKC * type proteins appear to have an altered K domain structure [START_REF] Kwantes | How MIKC* MADS-box genes originated and evidence for their conserved function throughout the evolution of vascular plant gametophytes[END_REF]. Analyses of the Arabidopsis genome revealed 107 genes encoding MADS-domain proteins.

Phylogenetic analyses indicated that the type I Arabidopsis MADS-box genes can be further subdivided into three subfamilies, named Mα, Mβ, Mγ (Fig. 3) [START_REF] Smaczniak | Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies[END_REF]. Based on the distinct sequence motifs in their C-terminal end, the Arabidopsis MIKC C type genes are divided into several distinctive subfamilies [START_REF] Vandenbussche | Structural diversification and neo-functionalization during floral MADS-box gene evolution by C-terminal frameshift mutations[END_REF]: B-sister (GGM13), AGAMOUS (AG), AGL12, SOLANUM TUBEROSUM MADS-BOX 11 (STMADS11), AGL15, AGL17, TOMATO MADS-BOX3 (TM3), SOC1, TM8, SEPALLATA (SEP), AGL6, SQUAMOSA (SQUA), FLOWERING LOCUS C (FLC), DEFICIENS (DEF) and GLOBOSA (GLO) (Fig. 4).

Several members of the MIKC C -type subfamilies have been shown to have conserved functions in different flowering plant species [START_REF] Smaczniak | Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies[END_REF]. B-sister (Bs) MADS box genes are the closest relatives of the homeotic B MADS box genes (DEF/GLO subfamily of MIKC C type genes). Divergence of B and Bs lineages happened by the duplication of an ancestor gene between 400MYA (million years ago) and 300MYA, which corresponds to the divergence of fern and seed plant lineages (400MYA) and separation of gymnosperm and angiosperm lineages (300MYA). Expression analyses revealed that Bs genes are predominately expressed in female reproductive organs whereas B genes are mainly expressed in male reproductive organs [START_REF] Becker | A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes[END_REF]. To date, Bs genes have been only isolated from seed plants and gymnosperm [START_REF] Becker | A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes[END_REF]. In Arabidopsis there are two Bs genes (Fig 5 ): TRANSPARENT TESTA 16 (TT16), also named ARABIDOPSIS B-SISTER (ABS), and its orthologue GORDITA (GOA), also named AGL63 [START_REF] Parenicová | Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world[END_REF]. TT16 is involved in cell differentiation and pigmentation of the seed coat (Coen et al., 2017;Coen et al., 2019a;Coen et al., 2019c;Fiume et al., 2017a;Nesi et al., 2002b;Xu et al., 2016). It promotes cuticle deposition and flavonoid accumulation in the endothelium, the innermost cell layer of the seed coat (Coen et al., 2019a;Nesi et al., 2002b). Mutations in the TT16 gene also affect the cell shape and polarity of the inner integument cell layers of the seed coat (Coen et al., 2017;Coen et al., 2019c;Fiume et al., 2017a;Nesi et al., 2002b). Finally, Xu et al. showed that TT16 promotes nucellus elimination (Xu et al., 2016). Before fertilization, Fertilization-Independent Seed (FIS) Polycomb Group (PcG) proteins repress nucellus degeneration. After fertilization, the endosperm initiates a signal, through the action of the AGAMOUS-LIKE 62 MADS box transcription factor that relieves the repressive action mediated by the FIS PcG proteins.

TT16 functions downstream of PcG proteins to promote nucellus elimination. TT16 is expressed in the persistent nucellus and regulates non-cell autonomously the elimination of the transient nucellus in part by repressing the expression of the HVA22d gene, which inhibits gibberellin-mediated program cell death (PCD) and autophagy. However, nucellus elimination has not been entirely assigned to any known cell death program. TT16 paralogue, GOA, has been identified as regulator of fruit growth [START_REF] Erdmann | GORDITA (AGL63) is a young paralog of the Arabidopsis thaliana B(sister) MADS box gene ABS (TT16) that has undergone neofunctionalization[END_REF]Prasad et al., 2010a). Furthermore, GOA is expressed in the transient nucellus and acts redundantly with TT16 in repressing nucellus cell division after fertilization (Xu et al., 2016) .

A handful of other Arabidopsis type-2 MADS box genes have been involved in ovule and seed development. WUSCHEL (WUS) acts non-cell-autonomously from the nucellus to induce integument growth from chalaza. wus mutant ovules fail to develop integuments and eventually degenerate [START_REF] Gross-Hardt | WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development[END_REF]. SHATTERPROOF1 (SHP1) and SHP2, first discovered for their redundant role in fruit dehiscence [START_REF] Liljegren | SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis[END_REF], have been shown to play an antagonistic role with TT16 in seed coat development. shp1 and shp2 partially rescue the defects in the development of the inner integument of tt16 seeds (Ehlers et al., 2016). SEEDSTICK (STK) acts redundantly with SHP1 and SHP2 to control ovule identity: stk;shp1;shp2 triple mutant ovules develop as leaf-like or carpel-like structures [START_REF] Favaro | MADS-box protein complexes control carpel and ovule development in Arabidopsis[END_REF][START_REF] Pinyopich | Assessing the redundancy of MADS-box genes during carpel and ovule development[END_REF]Rodrí guez-Cazorla et al., 2018). Furthermore, STK and TT16 redundantly regulate the development of the inner integument, as a total absence of the inner integument 1' cell layer is observed in the tt16;stk double mutant (Coen et al., 2017;[START_REF] Mizzotti | The MADS box genes SEEDSTICK and ARABIDOPSIS Bsister play a maternal role in fertilization and seed development[END_REF]. In the stk single mutant, ovules display a relatively larger funiculus and seeds do not detach from the silique once matured [START_REF] Pinyopich | Assessing the redundancy of MADS-box genes during carpel and ovule development[END_REF].

Finally, the SEPALLATA (SEP) MADS-domain proteins have been detected in reproductive organs and act as bridges in the formation of MADS multimeric protein complexes. SEP3 has been shown to enable the interaction of TT16 with STK, SHP1 and SHP2 [START_REF] Favaro | MADS-box protein complexes control carpel and ovule development in Arabidopsis[END_REF][START_REF] Kaufmann | Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein[END_REF]. SEP3 RNA was detected in ovule and seed maternal tissues [START_REF] Mandel | The ArabidopsisAGL9 MADS box gene is expressed in young flower primordia[END_REF]. These results suggest a possible role of SEP3 in ovule and seed development.

In this study, we have characterized SHP2 and SEP3 expression pattern in ovules and seeds and found out a spatial-temporal overlap with TT16 in ovules. We also found that shp1;shp2 double mutation can rescue the nucellus phenotype of tt16;goa seeds. We therefore, speculate that SEP3 allows TT16 and SHP physical interaction, timing and regulating nucellus elimination.

RESULTS

MADS box genes expression pattern in the nucellus

To test if additional MADS-box genes are involved in nucellus development, we further characterized the expression pattern of MADS box genes known to be expressed in ovules or seeds.

It was shown that TT16 can interact in vitro with SHP1 or SHP2 through SEP3 [START_REF] Kaufmann | Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein[END_REF]. To test if such an interaction might happen in the nucellus, we studied the expression pattern SEP3 works as bridging protein to allow the formation of multimeric complexes with other MADS box transcription factors and regulate ovule development (Ehlers et al., 2016). SEP3 has been shown to be expressed at high levels in all tissues of ovule primordia till stage 2-IV [START_REF] Mandel | The ArabidopsisAGL9 MADS box gene is expressed in young flower primordia[END_REF][START_REF] Urbanus | In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana[END_REF]. To further characterize SEP3 expression pattern at later stages, we used SEP3 1.5kb promoter and genomic sequence translationally fused to GFP [START_REF] Urbanus | In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana[END_REF] (1.5ProSEP3:g SEP3-GFP). We detected GFP florescence in the ii2 and oi1 integuments and a few cells in the distal region of ii1 integument, no GFP signal was detected in the nucellus of mature ovules and seeds 1 DAF (Fig. 7).

Expression analyses of SEP1 and SEP2 genes, which show redundant function with SEP3, are currently underway.

shp1;shp2 double mutation suppresses tt16 nucellus phenotype

It has been shown that shp1 and shp2 partially rescue the phenotype of tt16 in seed coat development (Ehlers et al., 2016). To investigate the possible role of SHP1 and 2 in nucellus development and its genetic interactions with TT16, we imaged central longitudinal sections of wt, tt16;goa, shp1;shp2, and shp1;shp2;tt16;goa mutants seeds 6 DAF using the mPS-PI technique. As Xu et al. have previously described (Xu et al., 2016) To test if the shp1;shp2 mutation might lead to the elimination of the persistent nucellus, we the high-fidelity Phusion DNA polymerase (Thermo Fisher Scientific), recombined into the pDONR207 vector (BP Gateway reaction) according to the manufacturer's instructions (Thermo Fisher Scientific), and then sequenced. The PCR products cloned into the pDONR207 vectors were then recombined into the pMDC107 binary vector (Curtis and Grossniklaus, 2003), (LR Gateway reaction) according to the manufacturer's instructions (Thermo Fisher Scientific).

Transgenic plants (see chapter2)Pseudo-Schiff propidium iodide staining

This protocol allows the staining of cell walls of fixed plant material (Truernit et al., 2008a;Xu et al., 2016). Samples were mounted onto microscope slides and observed by confocal microscope (Leica SP8).

Calcofluor staining

For staining with calcofluorM2R white (fluorescent brightener 28; Sigma Aldrich), harvested septums were first immersed in a NaOH (0.2M), SDS (1%) solution at 37°C for 2h. Samples were rinsed three times in water, and then transferred to12.5% bleach solution (1.25% active Cl -) for 10 to15 min (until samples become white and transparent). Samples were rinsed three times in water, and then transferred to calcofluor solution (10 μg mL-1) at 4°C overnight. Finally, the samples were mounted onto microscope slides with water and observed by confocal microscope (Leica SP8).

Microscopy

Stained samples were analyzed with Leica TCS-SP8 spectral confocal laser scanning microscope.

The excitation wavelength for mPS-PI stained samples was 488 nm, and emission was collected at 520 to 720 nm (Truernit et al., 2008a). The excitation wavelength for calcofluor stained samples was 365 nm and emission was collected at 440 nm. Three-dimensional Z-stack confocal laser scanning microscope images of mPS-PI stained or calcofluor stained seeds were analyzed through the VolumeViewer plug-in of the ImageJ software. Mid-plane longitudinal sections were obtained. More than 30 independent seeds were analyzed for each genotype and time point.

DISCUSSION

SHP2 and TT16 regulate nucellus development

TT16 in expressed in the persistent nucellus throughout ovule and seed development and promotes non-cell autonomously the elimination of the transient nucellus after fertilization (Xu et al., 2016).

GOA is instead expressed in the transient nucellus and represses, redundantly with TT16, nucellus growth. Here, we showed that SHP2 is expressed in both transient and persistent nucellus in ovules and seeds. The characterization of SHP1 expression is currently underway. Furthermore, our genetic analyses demonstrate that shp1;shp2 mutations suppress tt16;goa phenotype, thus highlighting a genetic interaction among such MADS-box genes. While TT16 and GOA promote nucellus elimination, SHP1 and SHP2 promote nucellus survival. Finally, it has been observed that SEP3 is expressed in the nucellus in ovule primordia [START_REF] Mandel | The ArabidopsisAGL9 MADS box gene is expressed in young flower primordia[END_REF][START_REF] Urbanus | In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana[END_REF]. Our data indicate that SEP3 is not present in the nucellus across fertilization.

Previous studies have revealed that MADS-domain proteins form dimers or higher-order protein complexes that are instrumental for function and specificity [START_REF] Favaro | MADS-box protein complexes control carpel and ovule development in Arabidopsis[END_REF][START_REF] Honma | Complexes of MADS-box proteins are sufficient to convert leaves into floral organs[END_REF][START_REF] Theißen | Development of floral organ identity: stories from the MADS house[END_REF]. For example, it has been shown that TT16-AG-SEP3 and TT16-STK-SEP3 protein complexes are involved in the determination of integument cell identity in the seed coat [START_REF] De Folter | A Bsister MADS-box gene involved in ovule and seed development in petunia and Arabidopsis[END_REF][START_REF] Kaufmann | Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein[END_REF]. Interestingly, it has also been shown that TT16, SEP3, SHP1 or SHP2 can form protein trimers but their biological function has not been elucidated [START_REF] Kaufmann | Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein[END_REF]. Our analyses identified spatial-temporal overlap of such MADS-box gene expression as well as genetic interaction between TT16 and SHP1 and SHP2. We therefore, speculate that TT16 and SHP1 and 2 might time and regulate the extent of nucellus elimination by forming protein complexes. Besides, qRT-PCR has shown that TT16 and SHP1 or SHP2 mutually repress each other expression directly or indirectly (Ehlers et al., 2016). We integrated all this information in a model that requires further testing for validation (Fig. 10).

Perspectives

To better understanding the role of SHP1/SHP2 and TT16 in regulating the nucellus elimination pathway, we will analyse the expression of known TT16 downstream target genes in different mutant backgrounds. HVA22d has been shown to be regulated by TT16 and we therefore introgressed the ProHVA22d:gHVA22d-GUS line into shp1;shp2 and shp1;shp2;tt16;goa background, experiments are underway. Furthermore, we are cloning a marker line carrying the SHP1 3kb promoter region and genomic sequence, transcriptionally fused to GFP and we will observe the expression pattern of SHP1 in the near future. We also plan to observe the expression pattern of SEP1 and SEP2 genes. To overcome the ovule-less phenotype of sep1;sep2;sep3 plants, where flowers are converted into leaf-like or carpel-like structures, we plan on specifically expressing SEP3 in flower meristems under the control of the CLV1 promoter in a SEP1/sep1;sep2;sep3 background. These line might bear wild type looking flowers that sep1;sep2;sep3 ovules. We will also look into type I MADS box genes that are expressed in the nucellus such as AGL57, AGL 59, AGL 64, AGL 100, AGL 77. Overall, we hope to shed light on the role of MADS-Box transcription factors on nucellus evolution and development.

ChapterV-nucellus development in the Amaranthus perispermic seed

INTRODUCTION 1 Perispermic seeds

A number of species of the Amaranthaceae family, such as Chenopodium quinoa and Amaranthus hypochondriacus, the nucellus (also referred to as perisperm) is the primary nutritive tissue of the seed [START_REF] Lopez-Fernandez | Programmed cell death during quinoa perisperm development[END_REF], alongside the embryo and a minute endosperm. Opposite to what observed in endospermic seeds, such as Arabidopsis, perispermic seeds, such as quinoa and amaranth, develop a large nucellus at the expense of the endosperm.

Phylogenetic analyses showed that plants shifted several times between the endospermic and perispermic seed condition highlighting the antagonistic development of endosperm and nucellus as a defining mechanism for seed evolution. Nevertheless, little is known about the genetic bases underlying the perispermic seed architecture.

Amaranth as model perispermic seed plant

Quinoa and amaranth have raised the attention of agricultural ministries, food and health organization and agro-companies for their unique nutritional qualities and growth habit [START_REF] Coimbra | Amaranthus hypochondriacus: seed structure and localization of seed reserves[END_REF][START_REF] Dakhili | Quinoa protein: Composition, structure and functional properties[END_REF][START_REF] Das | Amaranthus: A promising crop of future[END_REF]. Both species have been domesticated in South America by pre-Columbian cultures [START_REF] Coimbra | Amaranthus hypochondriacus: seed structure and localization of seed reserves[END_REF][START_REF] Lopez-Fernandez | Programmed cell death during quinoa perisperm development[END_REF]. Quinoa and amaranth produce perispermic seeds with an almost indistinguishable architecture. Nevertheless, the pericarp of quinoa sticks to the seed coat and need to be removed before human consumption as it is rich in saponins compounds with a bitter flavor [START_REF] Ando | Food components in fractions of quinoa seed[END_REF][START_REF] Brady | Effects of processing on the nutraceutical profile of quinoa[END_REF][START_REF] Chauhan | Nutrients and antinutrients in quinoa seed[END_REF][START_REF] Comai | The content of proteic and nonproteic (free and protein-bound) tryptophan in quinoa and cereal flours[END_REF][START_REF] Dini | Studies on the constituents of Chenopodium quinoa seeds: isolation and characterization of new triterpene saponins[END_REF][START_REF] Farro | Desenvolvimento de filmes biodegradáveis a partir de derivados do grão de quinoa (Chenopodium quinoa Willdenow) da variedade "Real[END_REF][START_REF] James | Quinoa (Chenopodium quinoa Willd.): composition, chemistry, nutritional, and functional properties[END_REF][START_REF] Jancurová | Quinoa-a rewiev[END_REF][START_REF] Koziol | Programmed cell death during quinoa perisperm development[END_REF][START_REF] Mastebroek | Occurrence of sapogenins in leaves and seeds of quinoa (Chenopodium quinoa Willd)[END_REF][START_REF] Spehar | Quinoa: alternativa para a diversificação agrícola e alimentar[END_REF].

This process is costly and can reduce the nutritional value of the seed. On the other hand, quinoa produces larger seeds due to its polyploid genome, a favorable agronomical trait. Efforts to produce polyploid amaranth are currently underway [START_REF] Das | Amaranthus: A promising crop of future[END_REF][START_REF] Misra | Chemurgic studies on some diploid and tetraploid grain amaranths[END_REF][START_REF] Pal | Cytogenetics of the raw autotetraploid Amaranthus edulis[END_REF][START_REF] Sun | Research on polyploid grain amaranth-a preliminary study on selection of grain amaranth with character of bigger seed. The research and development of grain amaranth in China[END_REF]. The nutritional value of these pseudocereals is mostly connected to their protein content. Proteins are accumulated in the embryo [START_REF] Ando | Food components in fractions of quinoa seed[END_REF][START_REF] Gallardo | Morfología del fruto y semilla de Chenopodium quinoa Willd.(«quinoa») Chenopodiaceae[END_REF][START_REF] Oelke | Alternative Field Crops Manual University of Wisconsin-Extension[END_REF][START_REF] Prego | Seed structure and localization of reserves in Chenopodium quinoa[END_REF] and consist almost exclusively of globulins and albumins [START_REF] Abugoch | Stability of quinoa flour proteins (Chenopodium quinoa Willd.) during storage[END_REF][START_REF] Brinegar | Isolation and characterization of chenopodin, the 11S seed storage protein of quinoa (Chenopodium quinoa)[END_REF][START_REF] Brinegar | High-cysteine 2S seed storage proteins from quinoa (Chenopodium quinoa)[END_REF][START_REF] Marcone | Evidence confirming the existence of a 7S globulin-like storage protein in Amaranthus hypochondriacus seed[END_REF]. The absence of prolamins, which is the main storage proteins of cereals such as wheat, makes quinoa and amaranth suitable for gluten-free diets [START_REF] Das | Amaranthus: A promising crop of future[END_REF][START_REF] Fairbanks | Electrophoretic characterization of quinoa seed proteins[END_REF][START_REF] Thanapornpoonpong | Influence of various nitrogen applications on protein and amino acid profiles of amaranth and quinoa[END_REF]. Despite the protein content of these pseudocereals is comparable to that of cereals, their quality is superior. Protein nutritional quality is determined by the content of essential amino acids, which cannot be synthetized by the human body. Pseudocereals carry a complete and balanced set of amino acids rich in lysine, which is limiting in cereals [START_REF] Das | Amaranthus: A promising crop of future[END_REF]. Their amino acid profile matches the FAO/WHO suggested requirements for children [START_REF] Das | Amaranthus: A promising crop of future[END_REF][START_REF] James | Quinoa (Chenopodium quinoa Willd.): composition, chemistry, nutritional, and functional properties[END_REF][START_REF] Miranda | Impact of airdrying temperature on nutritional properties, total phenolic content and antioxidant capacity of quinoa seeds (Chenopodium quinoa Willd.)[END_REF][START_REF] Morales | Nutritional value for young children of grain amaranth and maizeamaranth mixtures: effect of processing[END_REF]Valcá rcel-Yamani and Lannes, 2012;[START_REF] Wright | Composition of Atriplex hortensis, sweet and bitter Chenopodium quinoa seeds[END_REF].

Furthermore, quinoa and amaranth proteins are highly bioavailable and their nutritional quality equals that of casein, the standard of nutritional excellence [START_REF] Das | Amaranthus: A promising crop of future[END_REF]. Amaranth and quinoa grains are considered nutraceutical food because of their carbohydrate content that has hypocholesterolemic and beneficial hypoglycemic effects and induce lowering of free fatty acids [START_REF] Filho | Quinoa: Nutritional, functional, and antinutritional aspects[END_REF][START_REF] Girija | Anti-diabetic and anti-cholesterolemic activity of methanol extracts of three species of Amaranthus[END_REF][START_REF] Sangameswaran | Anti-diabetic, anti-hyperlipidemic and spermatogenic effects of Amaranthus spinosus Linn. on streptozotocin-induced diabetic rats[END_REF]. The seed perisperm is responsible for the accumulation of carbohydrates, mainly in the form of starch [START_REF] Coimbra | Amaranthus hypochondriacus: seed structure and localization of seed reserves[END_REF][START_REF] Lopez-Fernandez | Programmed cell death during quinoa perisperm development[END_REF][START_REF] Prego | Seed structure and localization of reserves in Chenopodium quinoa[END_REF]. The lipid content of quinoa and amaranth is more than double that of other cereals such as barley and wheat [START_REF] Tang | Phytochemicals in quinoa and amaranth grains and their antioxidant, anti-inflammatory, and potential health beneficial effects: a review[END_REF]. Pseudocereals have been indeed considered alternative oilseed crops as their fatty acid profile, with a high unsaturated/saturated ratio, is similar to that of soybean oil [START_REF] Filho | Quinoa: Nutritional, functional, and antinutritional aspects[END_REF][START_REF] Singhal | Amaranths-an underutilized resource[END_REF]. Both pseudocereals are rich in vitamins, especially vitamin E that contributes to the stability and antioxidant properties of their oils, and minerals [START_REF] Kozioł | Chemical composition and nutritional evaluation of quinoa (Chenopodium quinoa Willd.)[END_REF][START_REF] Ruales | Content of fat, vitamins and minerals in quinoa (Chenopodium quinoa, Willd) seeds[END_REF][START_REF] Tang | Phytochemicals in quinoa and amaranth grains and their antioxidant, anti-inflammatory, and potential health beneficial effects: a review[END_REF]. Amaranth is also the plant with the highest content of squalene, a triterpene of high economic value for its anti-cancer properties and pharmaceutical and industrial use.

Whereas quinoa and amaranth are both promising crops, they are not equally feasible model organism for basic research. The genome of A. hypochondriacus is diploid and relatively small (466Mbp), only 3.4 times that of Arabidopsis. Its complete sequence is available at the online computational platform of the Joint Genome Institute. By contrast the genome of C. quinoa is allotetraploid and considerably larger (1.385Gbp) than A. hypochondriacus. Genome size and ploidy make A. hypochondriacus a better candidate for forward and reverse genetic analyses compared to C. quinoa. Furthermore, transgenic A. hypochondriacus plants have been obtained through Agrobacterium transformation and plant regeneration [START_REF] Jofre-Garfias | Agrobacterium-mediated transformation of Amaranthus hypochondriacus: light-and tissue-specific expression of a pea chlorophyll a/b-binding protein promoter[END_REF]. We are currently growing A. hypochondriacus both in vitro and in soil. It is a robust plant that can be easily grown in a range of conditions [START_REF] Brenner | Genetic resources and breeding of Amaranthus[END_REF]. All considered, we believe that A. hypochondriacus has the potential to become the mode plant for perispermic pseudocereals and is therefore the plant of choice for our study. Finally, the high degree of orthology between A. hypochondriacus and A. hypochondriacus genomes facilitate translational biology analyses between such species.

RESULTS

Nucellus development in Amaranthus seed

To characterize nucellus development in Amaranthus seeds, we adapted the Propidium-Pseudo-Schiff/Iodide (PS/PI) imaging technique (Xu et al., 2016). We followed nucellus patterning from ovule primordia till the torpedo embryo seed stage (Fig. 1234). Contrary to what previously believed, we detected a domain of the amaranth nucellus that undergoes cell elimination after fertilization. We therefore divided the amaranth nucellus into two domains: the persistent nucellus, which is not eliminated, after fertilization, and the transient nucellus, which is consumed after fertilization (Fig. 5). Similar to what observed in Arabidopsis, the transient nucellus of amaranth mature ovules is adjacent to the proximal side of the female gametophyte (Fig. 5).

Figure1. Longitudinal section of an

The Amaranthus TT16 gene

Our team characterized the role of the TT16 transcription factor in promoting nucellus elimination in Arabidopsis. Arabidopsis tt16 mutant seeds mimic perispermic seeds as they develop a large nucellus, at the expense of the endosperm. It has been shown that forms in nature evolve largely by altering the expression of functionally conserved proteins [START_REF] Carroll | Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution[END_REF]. To test if different TT16 expression patterns in Arabidopsis and amaranth might account for different seed architectures, we cloned TT16 orthologue gene in amaranth and studied its expression pattern by RNA in situ hybridization analyses. We detected TT16 expression in the cell layer of the persistent nucellus surrounding the female gametophyte and the transient nucellus and in the endothelium, the innermost seed coat layer of the inner integument, of amaranth seeds right after fertilization (Fig. 6A). Older amaranth seeds displayed a similar TT16 expression pattern in the nucellus, whereas we did not detect signal in the endothelium (Fig. 6B). We used TT16 sense probe as a negative control (Fig. 6D). As a positive control, we instead hybridized seed sections with a HISTONE4 (HIS4) antisense probe and observed its characteristic patchy expression pattern in actively dividing cells (Fig. 6C) [START_REF] Blein | A conserved molecular framework for compound leaf development[END_REF].

Figure6. Amaranthus TT16 gene is expressed in nucellus cell layer surrounding the female gametophyte. Amaranthus TT16 expression was detected by RNA in situ hybridization with a TT16 antisense probe on Amaranthus hypochondriacus seed longitudinal sections (A and B). Black arrows point to the expression signal in the outermost cell layer of the perisperm. HIS4 expression was detected with a HIS4 antisense probe on Amaranthus hypochondriacus seed longitudinal sections (C). No Amaranthus TT16 expression was detected with a TT16 sense probe on Amaranthus hypochondriacus seed longitudinal sections (D). Scale bars = 50 μm.

Alternatively, perispermic and endospermic seed structures might have evolved due to the functional divergence of the TT16 protein. To test this hypothesis, we cloned the whole genome sequence and seven splicing forms (two of which corresponding to the only two known Arabidopsis TT16 splicing forms) of Amaranthus hyoochondriacus TT16 gene (Fig. 7). We performed domain and motif amalyses on all the TT16 splicing forms of Amaranthus hyoochondriacus and Arabidopsis thaliana by using the SMART (http://smart.embl-heidelberg.de/) domain analysis tool.

MADS domain was found at N-terminal end of all splicing forms in both Amaranthus hyoochondriacus and Arabidopsis thaliana (Fig. 7). Beside the MADS domain, the K coiled-coil domain, known to drive protein-protein interaction (Davies et al., 1996;Fan et al., 1997), was found in three splicing forms (Ah TT16-I, Ah TT16-II, Ah TT16-III) of Amaranthus hyoochondriacus and two splicing forms of Arabidopsis thaliana TT16 gene. To study if Amaranthus and Arabidopsis TT16 proteins share the same function we performed complementation analyses in the Arabidopsis tt16 mutant background. We cloned all amaranth TT16 splicing forms and genomic sequences downstream the TT1 promoter region, which drives expression specifically in the inner integument.

We could not express them in the nucellus because, to date, we do not have a promoter specific for the nucellus and Arabidopsis TT16 promoter alone is not sufficient to drive expression in the nucellus as it requires TT16 genomic sequence. Nevertheless, the laboratory already demonstrated that TT16 works non-cell autonomously and TT16 expression under the control of the TT1 promoter complemented all known Arabidopsis tt16 phenotypes. We already obtained several complementation lines and their analysis is underway.

MATERIAL AND METHODS

Material

Amaranthus hyoochondriacus seeds were obtained by the USDA amaranth germplasm station, Nebraska, USA. at greenhouse of INRAE Versailles. All Arabidopsis thaliana plants are in the Columbia accession. The tt16-1 mutant comes from Prasad (Prasad et al., 2010). pDONR221-gAtTT16; pDONR221-At5G23260.1 and pDONR221-At5G23260.2 plasmids were obtained from Wenjia Xu, pGUCB2-ProTT1 modified binary vector were obtained from Olivier Coen.

We found out that a domain of the amaranth nucellus also undergoes cell elimination, thus showing that both Arabidopsis and amaranth have transient and persistent nucellus domains. These data indicate that the basic tissue organization of endospermic and perispermic seeds is more similar than previously thought. Our discovery suggests that Arabidopsis and amaranth seed structures might be driven by different cell fates of the persistent nucellus more than a change in the nucellus elimination program.

Angiosperms shifted several times between the endospermic and perispermic seed condition, thus suggesting that a relatively simple genetic mechanism is underlying these different seed structures. During my thesis I challenged the hypothesis that a change in TT16 expression or function might be responsible for the evolution of endospermic and perispermic seed structures.

Amaranth seeds express TT16 in the nucellus cells surrounding the female gametophyte and the transient nucellus, similar to what observed in Arabidopsis. These data suggest that TT16 might be responsible for the elimination of the transient nucellus also in amaranth seeds. Further confirmation will come from the complementation analyses. If the amaranth TT16 will complement the Arabidopsis tt16 nucellus phenotype, we will be able to speculate that TT16 function in Arabidopsis and amaranth is conserved. To fully address this question, the team has created an amaranth EMS mutant library and it is currently looking for tt16 mutant lines by TILLING.

The amaranth TT16 is not expressed in the entire persistent nucellus domain, in contrast to what observed in Arabidopsis. Therefore, TT16 exclusion from most of the persistent nucellus might explain its different cell fate, when compared to Arabidopsis. This hypothesis could be tested by ectopically expressing TT16 in the perisperm of amaranth seeds. To address this question, the group is working on a transformation protocol for amaranth.

Finally, we demonstrated that the SHP transcription factors promotes nucellus growth in Arabidopsis. Therefore, it would be interesting to study SHP expression and function in amaranth following the same steps used for TT16 analysis.

GENERAL CONCLUSION

My work has shed light on some of the genetic and physiological mechanisms underlying the development of the nucellus in Arabidopsis and amaranth seeds, which have opposite architectures. The Arabidopsis nucellus is almost completely eliminated to make space to the endosperm whereas the amaranth nucellus grows to become the main storage tissue.

In Arabidopsis the distal nucellus (transient nucellus) is eliminated by the endosperm whereas a few cell layers of the proximal domain of the nucellus (persistent nucellus) persist.

Whereas we had a general understanding of the nucellus elimination process, driven by the action of the MADS domain transcription factor TRANSPARENT TESTA 16 (TT16), we did not know 1) its physiological implications and 2) mechanisms of containment.

Here, I show that the nucellus is capable of receiving nutrients as it is symplastically connected to the chalaza, the nutrient unloading zone. The transient nucellus accumulates starch and releases it in the apoplastic space during its elimination. This result indicates that the process of nucellus elimination is not triggered by nutrient starvation and highlights a novel mechanism of sharing resources. By contrast, the transient nucellus does not accumulate starch and, together with the chalaza, contributes to the apoplastic transport of sugars to the fertilization products by expressing the SWEET4 and SWEET10 sugar facilitator genes, respectively. I bring evidence for a symplastic block between transient and persistent nucellus that might explain why the cell elimination process arrests. Furthermore, I was able to identify the SHATTERPROOF (SHP) MADS box genes as genetic suppressors of the tt16 mutation. This result indicates that the survival of the nucellus is also regulated at the genetic level.

Finally, I shifted my attention toward the amaranth seed that is characterized by a large nucellus tissue. Compared to what previously thought, I identified a domain of the amaranth nucellus tissue that is eliminated. Interestingly, this transient nucellus domain is surrounded by a layer of persistent nucellus cells expressing the amaranth gene orthologous to TT16. This results indicate that the nucellus elimination mechanism identified in Arabidopsis might be conserved in Amaranth but limited only two a small region of the nucellus.

GENERAL DISCUSSION

In the literature, the study of nucellus development is considerably behind other seed tissues.

More attention has been given to the study of the fertilization products, embryo and endosperm, which constitute a unique biological model. Furthermore, perispermic seed plant models, such as quinoa or amaranth, which display a prominent nucellus, have emerged only recently.

The study of nutrient transport in cereals has advanced the understanding of the physiological role of the nucellus. In most cereals, the nucellus plays a dominant or sole role in transferring nutrients to the endosperm and develop into specialized structures, such as the nucellus projection. By contrast, in Arabidopsis the seed coat was thought to be the only tissue dedicated to nutrient transport. The discovery of a persistent nucellus domain in Arabidopsis made us postulate the hypothesis that the nucellus might play a role too. Our results show that the Arabidopsis seed evolved three paths for sugar transport, one for sucrose through the seed coat, one for hexoses through the persistent nucellus and chalaza, and one based on the release of starch in the apoplastic space through the elimination of the distal nucellus. We speculate that the persistent nucellus offers a shorter path toward the endosperm and that is why it might move hexoses, known to be more reactive compared to sucrose. Nevertheless, other hypotheses are possible. For example, hexoses are known to work as developmental signals as well. Therefore the nucellus might also play a role in signaling, a scenario that should be investigated further. We observed a delay in embryo development in the chalaza and nucellus sweet mutants, compared to the wild type, which could be interpreted as lack of nutrient export. Alternatively, it might be interpreted as a disruption of a signaling pathway between embryo and maternal tissues, yet to be discovered. A comparable hexose transport pathway has been shown in the rice nucellar projection, which also rely on SWEETs for sucrose export and invertases for its conversion into hexoses. In rice, seed coat and endosperm are separated by one nucellus cell layer, the nucellus epidermis, which might be the functional analogue in sugar transport of the Arabidopsis seed coat. Finally, the accumulation of starch by the transient nucellus was also observed in cereals, where evidence of hydrolytic activity has been found. A similar mechanism might be put in place also in Arabidopsis to allow the recycling of such starch. Unclear is instead the advantage, if any, behind the accumulation of starch in the transient nucellus, since its elimination rapidly follows. We speculate that it might be an evolutionary relic, if the perispermic seed architecture will appear to have evolved before the endospermic, a topic still debated. Our results brought also evidence of co-regulation of the tissue and nutrient partitioning processes by TT16. On the one hand, TT16 promotes the elimination of the transient nucellus and the release of starch in the apoplastic space. On the other hand, it negatively regulates SWEET10 expression, possibly favoring the nucellus SWEET4 pathway of sugar transport. This opens a novel perspective in the field that should be further explored. In the past, nutrient and tissue partitioning have been analyzed independently but they are clearly linked processes. In the future, the focus should be put in better understanding such regulation. Furthermore, it would be interesting to address the co-regulation, if any, of different sugar pathways. Are the seed coat and nucellus pathways independent? Is TT16 regulating the seed coat pathway as well? Finally, the same approach should be used to analyze the transport of other nutrients such as amino acids.

The mechanism that leads to the elimination of the nucellus is still unknown and clearly differs from other known ones as it involves the complete degradation of the cell wall. The analysis of cell wall components as well as genes known to underlie modifications of the cells wall in different domains of the nucellus might be the key to study this process in the future. I did not directly study the physical mechanism behind cell elimination but shed more light on its genetic regulation. The role of the TT16 transcription factor in promoting nucellus elimination had been shown in Arabidopsis and rice. The discovery of the SHPs proteins as putatively responsible for the survival of the persistent nucellus domain opens to novel interpretations. SHP and TT16 were shown to physically interact through the action of the SEPALLATA3 (SEP3) MADS domain transcription factor. Nevertheless, I showed that these transcription factors are co-expressed in the nucellus only during early ovule development. The protein complex might, therefore, specify the cell fate of different domains of the nucellus early on in development. Alternatively, the absence of SEP3 in the nucellus of the Arabidopsis seed might allow TT16 and SHPs to independently specify different nucellus domains. To complicate the interpretation of the data is the non-cell autonomous effect of TT16 that promotes the elimination of the transient nucellus from the persistent nucellus.

Preliminary transcriptional data do not show a drastic effect of TT16 or SHPs on each other expression pattern, but we cannot exclude that even a lower decrease in the expression level might affect the stoichiometry of transcriptional complexes. Our discovery of a genetic control for cell survival is also important to understand how the mechanism of cell elimination is contained. We can suppose that the breakage of a cell might be a toxic event and affect neighboring tissues. By contrast, the seed coat but also the proximal domain of the nucellus survives to such an event. The elimination of the nucellus has been shown to spread centripetally, from distal to proximal, suggesting that the diffusion of a death signal or toxic material might spread the elimination process in the transient nucellus. The clear-cut transition from transient to persistent nucellus domains called instead for the presence of a physical barrier. Our finding of a symplastic block between the two domains of the nucellus appear to confirm such an hypothesis. More biochemical and genetic evidences are necessary to confirm our model. We are planning to analyze the function of callose synthases putatively expressed in the nucellus. Furthermore, we wonder if SHP regulates the formation of the above-mentioned symplastic block. The analysis of symplastic connections in the shp1;2 mutant background will further challenge our model. The nucellus is also known to be isolated from the neighboring seed coat cell by a cutin apoplastic barrier. The analyses of mutant defective in the formation of such cuticle layers will help us understand if apoplastic barriers might also have a role in the process of containing the cell elimination process.

Finally, my study of the amaranth seed is the first attempt to understand nucellus development in perispermic seeds. The finding of a transient nucellus domain and the expression of TT16 in the surrounding nucellus cells of the amaranth seed seem to indicate that there is a higher level of conservation between endospermic and perispermic seeds. Our complementation analyses using the Amaranth TT16 gene in the Arabidopsis tt16 mutant will help us understand if TT16 function is also conserved between these two species but the analysis of an amaranth tt16 mutant is necessary to finally prove this hypothesis. The absence of TT16 expression in certain domains of the amaranth nucellus might explain their different cell fate. Alternatively, the analysis of SHP genes in amaranth seeds might be a novel path of investigation to follow. The creation of an amaranth mutant library, currently underway, to be screen for mutants defective in nucellus growth will be necessary to better understand the process. Such a forward genetic approach has the potential to reveal novel genes and help us test novel hypotheses also in Arabidopsis. Finally, our approach to the analysis of nutrient partitioning in Arabidopsis should be applied also in Amaranth to understand how nutrients flow in a perispermic seed. Amaranth seeds are economically valued because of their content in protein, similar to that of animal sources. It would therefore be especially relevant to study the transport of amino acids through the nucellus in perispermic seeds and be confronted to that of endospermic ones.

  ....................................................................................................................... GENERAL CONCLUSION .................................................................................................... GENERAL DISCUSSION ...................................................................................................... Ré sumé en franç ais ..................................................................................................................

Figure 1 .

 1 Figure 1. Arabidopsis ovule primordium.

Figure 2 .

 2 Figure 2. Arabidopsis ovule at stage 2-III (Schneitz et al., 1995a).

Figure 3 .

 3 Figure 3. Megaspore retention and female gametophyte accommodation (Magnani, 2018).Female reproductive organ of a heterosporous non-seed vascular plant (A) and an ovule of a seed plant (B). The nucellus is highlighted in orange whereas megaspores and female gametophyte in purple.

Figure 4 .

 4 Figure 4. Arabidopsis mature ovule.

Figure 5 .

 5 Figure 5. Double fertilization in Arabidopsis.

Figure 6 .

 6 Figure 6. Arabidopsis globular embryo stage seed.

Figure 7 .

 7 Figure 7. Arabidopsis walking stick embryo stage seed.

Figure 9

 9 Figure 9 Sugar metabolism in sink tissue cells (Stein and Granot, 2019)

Figure 10

 10 Figure 10 Starch accumulation during seed development (Andriotis et al., 2010). (a) The embryo developmental stages. From left to right: globular embryo, heart embryo, torpedo embryo, walking stick embryo, and expanded cotyledon embryo. Bars, 50µm for the first four stages; 100 µm for the last two stages. (b) Starch accumulation in seeds, corresponding to the embryo stages above, stained with iodine solution. (c)-(f) dissected embryos stained with iodine solution to visualize starch and observed under DIC optics. (c) Embryo at the torpedo stage, 8 DAF. (d) Embryo at the walking stick stage, 10 DAF. (e) Embryo at the early cotyledon stage, 12 DAF. (f) Embryo at the expanded cotyledon stage.

Figure 11

 11 Figure 11 SWEETs domain structure (Jeena et al., 2019)

Fig. 1

 1 Fig. 1 Seed architectures. Diagrammatic representation in longitudinal sections of pine (gymnosperm) (a), Arabidopsis (angiosperm, endospermic) (b), rice (angiosperm, endospermic) (c), and quinoa (angiosperm, perispermic) (d) seeds right after fertilization and at an early embryogenesis stage. The figure is not in scale. Female gametophyte, nucellus, endosperm, and embryo are highlighted in violet, orange, blue, and yellow, respectively

Fig

  Fig. 2 Signaling pathways underlying nucellus and endosperm antagonistic development. Arrows indicate functional relationships

  Short title: Sugar transport in the nucellus One sentence summary: This study identifies a novel path of sugar transport through the nucellus maternal tissue of the Arabidopsis seed and its coordination with the process of nucellus cell elimination. Author contribution: J.L. performed the research, analyzed the data, and helped to write the article. D.G. performed the embryo analysis and part of the expression analysis, O.C. performed part of the expression analysis, R.LH performed sugar quantifications, S.J. performed oil and protein quantifications, C.P. performed transmission electron microscopy analyses. E.M. designed the research, analyzed the data and wrote the article.

Figure 1

 1 Figure 1 SWEET10 is expressed in chalaza (A) to (C) Propidium iodide(PI), GFP, and PI-GFP superimposed fluorescence images of proSWEET10:GFP-GUS ovule stage 3-VI(A), and seed at pre-globular (B), and globular (C) embryo stages. Orange, purple and white arrows indicate persistent and transient nucellus and vascular bundle, prospectively. Bars=50μm.

Figure 2

 2 Figure 2 SWEET10 localize to the plasma membrane (A) to (B) Propidium iodide (PI), GFP, and PI-GFP superimposed fluorescence images of proSWEET10: SWEET10-GFP seeds at pre-globular stage. White and yellow arrows indicate plasma membranes and vacuoles, respectively. Bars=25μm.

  is expressed in the chalazal region of Arabidopsis seeds throughout seed development. To better characterize its expression pattern, we translationally fused the INV4 promoter and genomic sequence to GFP. ProINV4:INV4-GFP ovules and seeds displayed GFP fluorescence in the central domain of the chalaza (Fig. 3A-3C), an expression pattern similar to that observed in ProSWEET10:GFP-GUS lines. These data indicate that INV4 might hydrolyze the sucrose exported to the apoplast by SWEET10.

Figure 4

 4 Figure 4 The sweet10 mutation delays embryo development Distribution of different embryo developmental stages (1: globular, 2: transition, 3: heart, 4: early torpedo, 5: late torpedo, 6: walking stick, 7: bent cotyledon, 8: mature embryo) in wild type and mutant seeds at 8 and 10 days after fertilization (DAF). N > 100. Bars=100μm.

Fig 1

 1 Fig 1 Callose dependent closure of plasmodesmata (Maule et al., 2012)

Figure 2

 2 Figure 2 The transient nucellus is symplastically isolated from the persistent nucellus (A) and (B) Propidium iodide (PI), GFP and PI-GFP superimposed fluorescence images of ProSUC2:GFP-sporamin (A) and ProSUC2:GFP (B) wild type seeds at pre-globular embryo stage. (C) and (D) Propidium iodide (PI), GFP and PI-GFP superimposed fluorescence images of ProSUC2:GFP-sporamin (C) and ProSUC2:GFP (D) wild type mature ovules. Orange and purple arrows indicate persistent and transient nucellus, respectively, Bars=50 µm.

Figure 5

 5 Figure 5 The morphology of plasmodesmata in the nucellus of mature ovule (A) TEM image of a nucellus (highlighted in yellow) longitudinal section of a mature ovule. The distal region of the nucellus (transient nucellus) is on the left of the image A. (B and C) Close-up images of plasmodesmata in the persistent and transient nucellus, respectively. Orange and red arrowheads indicate plasmodesmata. Bars=10µm (A), 500=nm(B) and (C).

Figure 1

 1 Figure 1 MADS-box genes have been identified in plants, fungi and metazoans.

Figure 2

 2 Figure 2 Domain structures of MADS-domain proteins.

Figure3

  Figure3 Phylogenetic analysis of selected representatives of type I MADS-box transcription factors from thale cress (Arabidopsis thaliana, At), grape (Vitis vinifera,Vv) and tomato (Solanum lycopersicum, Sl) (Smaczniak et al., 2012).

Figure 5

 5 Figure 5 Identified homologue B-sister genes in plants (Kaufmann et al., 2005).

Figure 6

 6 Figure 6 SHP2 expression pattern in wild type (A-C) and tt16 (D-F) ovules and seeds. (A) to (C) Propidium iodide (PI), GFP and PI-GFP superimposed fluorescence images of ProSHP2:gSHP2-GFP stage 2-V ovule (A), stage 3-V ovule (B), and pre-globular embryo seed (C). (D) to (F) Propidium iodide (PI), GFP and PI-GFP superimposed fluorescence images of ProSHP2:gSHP2-GFP;tt16 stage 2-V ovule (D), stage 3-V ovule (E), and pre-globular stage seed (F). Bars=50 µm.

Figure 7

 7 Figure 7 SEP3 expression pattern in wild type ovules and seeds. Propidium iodide (PI), GFP and PI-GFP superimposed fluorescence images of ProSEP3:gSEP3-GFP ovules at stage 4-I (A) and seeds at 1 DAF (B). Bars=50 µm.

  , a large part of the wild type distal nucellus degenerates during the first 6 DAF in wild type seeds (Fig 8A) whereas it grows bigger in tt16;goa seeds (Fig 8C). Similar to wild-type, the nucellus of shp1;shp2 mutant seeds underwent elimination of their distal region (Fig 8B). Interestingly, the nucellus of a number of shp1;shp2;tt16;goa seeds also appeared to display nucellus elimination as in wild type and shp1;shp2 seeds (Fig 8D). To quantify such a phenotype we calculated the percentage of seeds at 6DAF showing a nucellus that crossed the distal line of the gynoapical seed coat (orange lines in Fig. 8) in seed longitudinal sectionsAll shp1;shp2 seeds tested (n=30) displayed a nucellus well below the reference line, as observed in the wild type (Fig 8B). By contrast, 100% of tt16;goa seeds analyzed (n = 30) showed a nucellus above the reference line (Fig 8C). Finally, about 75% of shp1;shp2;tt16;goa seeds showed a nucellus below the reference line (Fig 8D), whereas the rest 25% of seeds above the reference line (Fig 8E) (n = 60). Overall, these data indicate a genetic interaction between SHP1 and SHP2 and TT16. Whereas TT16 promotes transient nucellus elimination, SHP1 and SHP2 promote transient nucellus survival.

Figure 8

 8 Figure 8 shp1;shp2 rescued tt16 nucellus phenotype. (A) to (E) mPS-PI images of longitudinal sections of wt (A), shp1;shp2 (B), tt16;goa (C), and shp1;shp2;tt16;goa (D and E) seeds at 6 DAF. Orange horizontal lines indicate the distal lines of the gynoapical seed coatused as reference. Bars=50 µm.

Figure 10 A

 10 Figure 10 A model for TT16, SHP1, SHP2 and SEP3 function. Full lines indicate genetic interactions whereas dotted line indicate functional relationships.

  Figure1. Longitudinal section of an Amaranthus hypochondriacus pistillate flower (PF) imaged by mPS-PI. Ovary (OV), inner integuments (II), outer integuments (OI), megaspore mother cell (MMC), nucellus (N), chalaza (C) and funiculus (F). Scale bar = 100 μm.

Figure2.

  Figure2. Longitudinal section of a mature ovule of Amaranthus hypochondriacus imaged by mPS-PI. inner integuments (II), outer integuments (OI), nucellus (N), female gametophyte (FG), chalaza (C), micropyle (M) and funicle (F). Scale bar = 100 μm.

Figure3.

  Figure3. Longitudinal section of a young Amaranthus hypochondriacus seed imaged by mPS-PI. Inner integuments (II), outer integuments (OI), perisperm (PE), endosperm (EN), chalaza (C), micropyle (M) and funicle (F). Scale bar = 100 μm.

Figure5.

  Figure5. Longitudinal section of Amaranthus hypochondriacus mature ovule imaged by mPS-PI. Transient Nucellus (TN); Persistent Nucellus (PN). Scale bar = 100 μm.

Figure7

  Figure7 Protein sequence alignment and domain analysis of Arabidopsis and Amaranthus hyoochondriacus TT16 splicing forms. At5G23260.1 and At5G23260.2 are from Arabidopsis and AhTT16-I to AhTT16-VII are from Amaranthus hyoochondriacus. AHYPO_002883RA is the only amaranth splicing form annotated in Phytozome. The alignment is made with the DNA MAN software. Red and orange squares indicate MADS domain and K domain, respectively.

  symplastiquement à la chalaze, la zone de de dé chargement des nutriments. Le nucelle fonctionne à la fois comme puits pour les sucres et source au côté du té gument de la graine. Le nucelle transitoire accumule de l'amidon très tôt après la fécondation et le restitue dans l'espace apoplastique lors de son é limination. Le nucelle persistant et la chalaze, au contraire, exportent les sucres vers l'endosperme grâce aux facilitateurs de sucres SWEET4 et SWEET10, processus partiellement ré gulé par TT16. De faç on inté ressante, nous avons dé couvert que les nucelles, persistant et transitoire, é taient dé connecté s symplastiquement. Nous avons dé tecté assez peu de plasmodesmes dans le nucelle transitoire et leurs formes rappellent celles de plasmodesmes bloqué s par l'accumulation de callose. Nous émettons l'hypothèse que le développement d'une barrière symplastique entre les nucelles persistant et transitoire pourrait empê cher la propagation du processus d'élimination cellulaire, qui en effet n'affecte que la région distale du nucelle. Enfin, nous avons caracté risé le dé veloppement du nucelle dans la graine d'Amaranthe, qui, au contraire de la graine d'Arabidopsis, est très important et lui confère le statut de tissu primaire de réserve.Pour tester l'hypothèse selon laquelle le facteur de transcription TT16 pourrait être responsable de l'évolution de l'architectures des graines, nous avons cloné le gène TT16 orthologue chez l'Amaranthe et déterminé son profil expression et sa fonction. En conclusion, ce travail présente les mé canismes de ré gulation qui coordonnent les processus de partitionnement des tissus et des nutriments dans les graines.

Titre:

  Contrôle génétique et moléculaire du développement de la graine et de l' accumulation des réserves : le rôle du nucelle Mots clés : nucelle, régulation transcriptionnelle, répartition des réserves, développement de la graine Résumé : L'architecture de la graine est dictée par des processus de partitionnement de tissus, déterminant le ratio volumique entre les tissus maternels et les tissus zygotiques, et par des processus de partitionnement nutritif régulant l'allocation des nutriments entre ces tissus. Chez les angiospermes, le développement précoce de la graine se caractérise par un développement antagoniste, pour la mise en place de tissus puits pour les sucres, entre le nucelle, un tissu maternel, et l'endosperme, issu de la fécondation. Un tel processus témoigne de l'évolution des angiospermes et contribue aux plus anciennes architectures de graines Durant ma thèse, le rôle du nucelle lors du partitionnement tissulaire et nutritif a été étudié dans la graine d'Arabidopsis. Le laboratoire avait précédemment démontré que le facteur de transcription à MADS-box TRANSPARENT TESTA 16 (TT16) promeut l'élimination de la région distale du nucelle, appelée nucelle transitoire, en réponse à un signal provenant de l'endosperme. Nous avons caractérisé deux suppresseurs du mutant tt16, les gènes à MADS-box SHATTERPROOF 1 (SHP1) et SHP2, qui promeuvent le maintien du nucelle. SHPs et TT16 répriment mutuellement leurs expressions. Cependant, nous avons pu détecter une superposition des expressions des gènes à MADSbox SHPs, TT16 et SEPALLATA3 (SEP3), dans la région proximale du nucelle, appelée nucelle persistant du fait de son absence de dégénérescence. Il a été montré que SEP3 établit un pont lors des interactions physiques entre TT16 et SHPs, suggérant ainsi que l'élimination du nucelle est coordonnée par la formation de complexes protéiques impliquant TT16 et SHPs. De plus, nous avons identifié une nouvelle voie de transport des sucres passant par le nucelle dans la graine d'Arabidopsis. Nous avons montré que le nucelle est connecté symplastiquement à la chalaze, la zone de de déchargement des nutriments. Le nucelle fonctionne à la fois comme puits pour les sucres et source au côté du tégument de la graine. Le nucelle transitoire accumule de l'amidon très tôt après la fécondation et le restitue dans l'espace apoplastique lors de son élimination. Le nucelle persistant et la chalaze, au contraire, exportent les sucres vers l'endosperme grâce aux facilitateurs de sucres SWEET4 et SWEET10, processus partiellement régulé par TT16. De façon intéressante, nous avons découvert que les nucelles, persistant et transitoire, étaient déconnectés symplastiquement. Nous avons détecté assez peu de plasmodesmes dans le nucelle transitoire et leurs formes rappellent celles de plasmodesmes bloqués par l'accumulation de callose. Nous émettons l'hypothèse que le développement d'une barrière symplastique entre les nucelles persistant et transitoire pourrait empêcher la propagation du processus d'élimination cellulaire, qui en effet n'affecte que la région distale du nucelle. Enfin, nous avons caractérisé le développement du nucelle dans la graine d'Amaranthe, qui, au contraire de la graine d'Arabidopsis, est très important et lui confère le statut de tissu primaire de réserve. Pour tester l'hypothèse selon laquelle le facteur de transcription TT16 pourrait être responsable de l'évolution de l'architectures des graines, nous avons cloné le gène TT16 orthologue chez l'Amaranthe et déterminé son profil expression et sa fonction. En conclusion, ce travail présente les mécanismes de régulation qui coordonnent les processus de partitionnement des tissus et des nutriments dans les graines.

  ... Material ............................................................................................................................ RNA extraction and Reverse transcription ...................................................................... Cloning and construction ................................................................................................. Arabidopsis transformation .............................................................................................. Pseudo-Schiff propidium iodide staining ......................................................................... RNA in situ hybridization ................................................................................................ DISCUSSION ...

  Funding This work was supported by the INRA BAP starter, Labex Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS), and China Scholarship Council Ph.D. grants. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. /10.1105/tpc.105.03405 8 Dwivedi KK, Roche DJ, Clemente TE, Ge Z, Carman JG (2014) The OCL3 promoter from Sorghum bicolor directs gene expression to abscission and nutrient-transfer zones at the bases of floral organs. Ann Bot 114:489-498. https ://doi.org/10.1093/aob/mcu14 8 Ellis JR, Chaffey NJ (1987) Structural differentiation of the nucellar epidermis in the caryopsis of rice (Oryza sativa). Ann Bot 60:671-675 Endress PK (2011) Evolutionary diversification of the flowers in angiosperms. Am J Bot 98:370-396. https ://doi.org/10.3732/ ajb.10002 99 Felker FC, Shannon JC (1980) Movement of C-labeled assimilates into kernels of Zea mays L.: III. An anatomical examination and microautoradiographic study of assimilate transfer. Plant Physiol 65:864-870 Figueiredo DD, Batista RA, Roszak PJ, Hennig L, Kohler C (2016) Auxin production in the endosperm drives seed coat development in Arabidopsis. Elife. https ://doi.org/10.7554/elife .20542 Floyd SK, Friedman WE (2000) Evolution of endosperm developmental patterns among basal flowering plants. Int J Plant Sci 161:S57-S81 Friedman WE, Bachelier JB (2013) Seed development in Trimenia Sechium edule nucellus. Physiol Plant 140:89-102. https ://doi.org/10.1111/j.1399-3054.2010.01381 .x Lombardi L, Mariotti L, Picciarelli P, Ceccarelli N, Lorenzi R (2012) Ethylene produced by the endosperm is involved in the regulation of nucellus programmed cell death in Sechium edule Sw. Plant Sci 187:31-38. https ://doi.org/10.1016/j.plant sci.2012.01.011 Lopez-Fernandez MP, Maldonado S (2013) Programmed cell death during quinoa perisperm development. J Exp Bot 64:3313-3325. Kohler C (2011) Polycomb group proteins are required to couple seed coat initiation to fertilization. Proc Natl Acad Sci USA 108:20826-20831. https ://doi.org/10.1073/pnas.11171 11108 Shannon JC (1972a) Movement of C-labeled assimilates into kernels of Zea mays L: I. Pattern and rate of sugar movement.

	programmed cell death of https ://doi.org/10.1093/jxb/ert17 0 Losada JM, Bachelier JB, Friedman WE (2017) Prolonged embryogen-esis in Austrobaileya scandens (Austrobaileyaceae): its ecological and evolutionary significance. New Phytol 215:851-864. https :// doi.org/10.1111/nph.14621 Maness NO, McBee GG (1986) Role of placental sac in endosperm cor-bohydrate import in sorghum caryopses. Crop Sci 26:1201-1207 Mayne WW (1937) Mysore Coffee. Kip Sta Bull 16:6 McLaughlin JE, Boyer JS (2004) Glucose localization in maize ovaries when kernel number decreases at low water potential and sucrose is fed to the stems. Ann Bot 94:75-86. https ://doi.org/10.1093/ aob/mch12 3 Melkus G et al (2011) Dynamic (1)(3)C/(1) H NMR imaging uncovers sugar allocation in the living seed. Plant Biotechnol J 9:1022-1037. https ://doi.org/10.1111/j.1467-7652.2011.00618 .x Nayar S, Sharma R, Tyagi AK, Kapoor S (2013) Functional delineation of rice MADS29 reveals its role in embryo and endosperm devel-opment by affecting hormone homeostasis. J Exp Bot 64:4239-4253. https ://doi.org/10.1093/jxb/ert23 1 Nogueira FC et al (2012) Proteomic profile of the nucellus of castor	endosperm. Plant Cell 23:3041-3054. https ://doi.org/10.1105/ tpc.111.08701 5 Roszak P, Plant Physiol 49:198-202 Shannon JC (1972b) Movement of C-labeled assimilates into kernels of Zea mays L: II. Invertase activity of the pedicel and placento-chalazal. Tissues Plant Physiol 49:203-206 Souto LS, Oliveira DM (2014) Seed development in Malpighiaceae species with an emphasis on the relationships between nutri-tive tissues. C R Biol 337:62-70. https ://doi.org/10.1016/j. crvi.2013.11.001 Sreenivasulu N, Wobus U (2013) Seed-development programs: a sys-tems biology-based comparison between dicots and monocots. Annu Rev Plant Biol 64:189-217. https ://doi.org/10.1146/annur ev-arpla nt-05031 2-12021 5 Tang AC, Boyer JS (2013) Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop. Ann Bot 111:551-562. https ://doi.org/10.1093/aob/mct01 2 Thiel J et al (2008) Different hormonal regulation of cellular differen-tiation and function in nucellar projection and endosperm transfer cells: a microdissection-based transcriptome study of young bar-ley grains. Plant Physiol 148:1436-1452. https ://doi.org/10.1104/ pp.108.12700 1 Thorne JH (1985) Phloem unloading of C and N assimilates in develop-ing seeds. Annu Rev Plant Physiol 36:317-343
		Tran V, Weier D, Radchuk R, Thiel J, Radchuk V (2014) Caspase-like
		activities accompany programmed cell death events in develop-
	Plant Mol Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, Dangl Biol 35:821-831 JL, Epple P (2010) Arabidopsis type I metacaspases control cell death. Science 330:1393-1397. https ://doi.org/10.1126/scien ce.11949 80 Domınguez F, Cejudo FJ (1998) Germination-related genes encoding proteolytic enzymes are expressed in the nucellus of developing wheat grains. Plant J 15:569-574 Dominguez F, Moreno J, Cejudo FJ (2001) The nucellus degenerates 1093/jxb/ erq30 9 Oparka KJ, Gates P (1981) Transport of assimilates in the developing caryopsis of rice (Oryza sativa L.): ultrastructure of the pericarp vascular bundle and its connections with the aleurone layer. Planta 151:561-573. https ://doi.org/10.1007/BF003 87436 Patrick JW, Offler CE (2001) Compartmentation of transport and trans-fer events in developing seeds. J Exp Bot 52:551-564 Piaggesi A, Perata P, Vitagliano C, Alpi A (1991) Level of abscisic acid in integuments, nucellus, endosperm, and embryo of peach seeds (Prunus persica L. cv Springcrest) during development. Plant Physiol 97:793-797 Porter GA, Knievel DP, Shannon JC (1985) Sugar efflux from maize (Zea mays L.) pedicel tissue. Plant Physiol 77:524-531 Prego I, Maldonado S, Otegui M (1998) Seed structure and localization of reserves in Chenopodium quinoa. Ann Bot 82:481-488 Radchuk V, Borisjuk L, Radchuk R, Steinbiss HH, Rolletschek H, Broeders S, Wobus U (2006) Jekyll encodes a novel protein involved in the sexual reproduction of barley. Plant Cell 18:1652-1666. https ://doi.org/10.1105/tpc.106.04133 5 Radchuk VV et al (2009) Spatiotemporal profiling of starch biosynthe-sis and degradation in the developing barley grain. Plant Physiol 150:190-204. https ://doi.org/10.1104/pp.108.13352 0 Radchuk V, Weier D, Radchuk R, Weschke W, Weber H (2011) Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth. J Exp Bot 62:1217-1227. https ://doi. org/10.1093/jxb/erq34 8 Radchuk V et al (2018) Vacuolar processing enzyme 4 contributes to maternal control of grain size in barley by executing programmed	Plant Physiol 118:1169-1180 Liu CM, Meinke DW (1998) The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed develop-ment. Plant J 16:21-31 Lombardi L, Casani S, Ceccarelli N, Galleschi L, Picciarelli P, Lor-enzi R (2007) Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activa-tion of caspase-like proteases. J Exp Bot 58:2949-2958. https :// ing barley grains. PLoS ONE 9:e109426. https ://doi.org/10.1371/ journ al.pone.01094 26 van Doorn WG et al (2011) Morphological classification of plant cell deaths. Cell Death Differ 18:1241-1246. https ://doi.org/10.1038/ cdd.2011.36 Wang HL, Offler CE, Patrick JW (1994a) Nucellar projection transfer cells in the developing wheat grain. Protoplasma 182:39-52 Wang HL, Offler CE, Patrick JW, Ugalde TD (1994b) The cellular pathway of photosynthate transfer in the developing wheat grain. I. Delineation of a potential transfer pathway using fluorescent dyes. Plant, Cell Environ 17:257-266 Wang HL, Offler CE, Patrick JW (1995) The cellular pathway of pho-tosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity. Plant, Cell Environ 18:373-388 Wang HH, Wang Z, Wang F, Gu YJ, Liu Z (2012) Development of basal endosperm transfer cells in Sorghum bicolor (L.) Moench and its relationship with caryopsis growth. Protoplasma 249:309-321. https ://doi.org/10.1007/s0070 9-011-0281-6 Weber H, Borisjuk L, Wobus U (2005) Molecular physiology of leg-ume seed development. Annu Rev Plant Biol 56:253-279. https :// doi.org/10.1146/annur ev.arpla nt.56.03260 4.14420 1 Weier D et al (2014) Gibberellin-to-abscisic acid balances govern development and differentiation of the nucellar projection of bar-ley grains. J Exp Bot 65:5291-5304. https ://doi.org/10.1093/jxb/ eru28 9 Weschke W, Panitz R, Sauer N, Wang Q, Neubohn B, Weber H, Wobus U (2000) Sucrose transport into barley seeds: molecular charac-terization of two transporters and implications for seed develop-ment and starch accumulation. Plant J 21:455-467
	cell death in the pericarp. New Phytol 218:1127-1142. https ://doi.	doi.org/10.1093/jxb/erm13 7
	org/10.1111/nph.14729	Lombardi L, Ceccarelli N, Picciarelli P, Sorce C, Lorenzi R (2010)
	Rolletschek H et al (2011) Combined noninvasive imaging and mod-	Nitric oxide and hydrogen peroxide involvement during
	eling approaches reveal metabolic compartmentation in the barley
	1 3

References

Alves

LC et al (2016)

Differentially accumulated proteins in Coffea arabica seeds during perisperm tissue development and their relationship to coffee grain size. J Agric Food Chem 64:1635-1647. https ://doi.org/10.1021/acs.jafc.5b043 76 Arekal GD, Nagendran CR (1975) Is there a Podostcmum type of embryo sac in the genus Farmeria? Caryologia 28:229-235 Arekal GD, Nagendran CR (1977) The female gametophyte in two Indian genera of Tristichoideae (Podostemaceae)-A reinvestigation. Proc Indian Acad Sci 86:287-294 Bagnall N, Wang X-D, Scofield GN, Furbank RT, Offler CE, Patrick JW (2000) Sucrose transport-related genes are expressed in both maternal and filial tissues of developing wheat seeds. Aust J Plant Physiol 27:1009-1020 Burrieza HP, Lopez-Fernandez MP, Maldonado S (2014) Analogous reserve distribution and tissue characteristics in quinoa and grass seeds suggest convergent evolution. Front Plant Sci 5:546. https ://doi.org/10.3389/fpls.2014.00546 Chen F, Foolad MR (1997) Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration. by a process of programmed cell death during the early stages of wheat grain development. Planta 213:352-360 Drea S, Leader DJ, Arnold BC, Shaw P, Dolan L, Doonan JH (2005) Systematic spatial analysis of gene expression during wheat caryopsis development. Plant Cell 17:2172-2185. https ://doi. org(Trimeniaceae) and its bearing on the evolution of embryonourishing strategies in early flowering plant lineages. Am J Bot 100:906-915. https ://doi.org/10.3732/ajb.12006 32 Greenwood JS, Helm M, Gietl C (2005) Ricinosomes and endosperm transfer cell structure in programmed cell death of the nucellus during Ricinus seed development. Proc Natl Acad Sci USA 102:2238-2243. https ://doi.org/10.1073/pnas.04094 29102 Ingram GC (2017) Dying to live: cell elimination as a developmental strategy in angiosperm seeds. J Exp Bot 68:785-796. https ://doi. org/10.1093/jxb/erw36 4 Johri BM, Ambegaokar KB, Srivastava PS (2013) Comparative embryology of angiosperms. Springer, Berlin Kladnik A, Chamusco K, Dermastia M, Chourey P (2004) Evidence of programmed cell death in post-phloem transport cells of the maternal pedicel tissue in developing caryopsis of maize. Plant Physiol 136:3572-3581. https ://doi.org/10.1104/pp.104.04519 5 Kohler C, Hennig L, Bouveret R, Gheyselinck J, Grossniklaus U, Gruissem W (2003) Arabidopsis MSI1 is a component of the MEA/FIE Polycomb group complex and required for seed development. EMBO J 22:4804-4814. https ://doi.org/10.1093/emboj /cdg44 4 Krishnan S, Dayanandan P (2003) Structural and histochemical studies on grain-filling in the caryopsis of rice (Oryza sativa L.). J Biosci 28:455-469 Linkies A, Graeber K, Knight C, Leubner-Metzger G (2010) The evolution of seeds. New Phytol 186:817-831. https ://doi.org/10.111 1/j.1469-8137.2010.03249 .x Linnestad C, Doan DN, Brown RC, Lemmon BE, Meyer DJ, Jung R, Olsen OA (1998) Nucellain, a barley homolog of the dicot vacuolar-processing protease, is localized in nucellar cell walls. bean (Ricinus communis L.) seeds during development. J Proteomics 75:1933-1939. https ://doi.org/10.1016/j.jprot .2012.01.002 Opanowicz M et al (2011) Endosperm development in Brachypodium distachyon. J Exp Bot 62:735-748. https ://doi.org/10.Weschke W, Panitz R, Gubatz S, Wang Q, Radchuk R, Weber H, Wobus U (2003) The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues of barley caryopses during early development. Plant J 33:395-411

Jing Lu 1,2 , Rozenn Le Hir 1 , Dennys-Marcela Gomez-Paez 1 , Olivier Coen 1,2 , Christine Pé choux 3 , Sophie Jasinski 1 , and Enrico Magnani 1,*

	1 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, University of Paris-Saclay, Route de
	St-Cyr (RD10), 78026 Versailles Cedex, France
	2 École Doctorale 567 Sciences du Vé gé tal, University Paris-Sud, University of Paris-Saclay, bat
	360, 91405 Orsay Cedex, France
	3 INRA, Génétique Animale et Biologie Intégrative, Domaine de Vilvert, 78352 Jouy-en-Josas
	Cedex, France

*Corresponding author: enrico.magnani@inrae.fr

Université Paris-SaclayEspace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France

ABSTRACT

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by the antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. Such a process marked the evolution of angiosperms and outline the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works both as sugar sink and source alongside the seed coat. The transient nucellus accumulates starch early on after fertilization and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator.

Finally, we analyze sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, and shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a novel path of sugar transport in the Arabidopsis seed and describes its link to the nucellus cell-elimination program.

The ProSWEET4 sequence was recombined into the pBGWFS7.0 binary vector (Karimi et al., 2002) whereas the SWEET4 genomic sequence was recombined into the pMDC83 binary vector (Curtis and Grossniklaus, 2003).

Constructs carrying GFP or GFP-sporamin under the control of the SUCROSE-PROTON SYMPORTER 2 (SUC2) promoter region were previously described (Stadler et al., 2005).

Transgenic plants

The Agrobacterium tumefaciens strain C58C1 was used to stably transform Arabidopsis plants using the floral dip method (Clough and Bent, 1998). Transformants were selected with the appropriate resistance and subsequently transferred to soil for further characterization. More than 10 independent transgenic lines were tested for each construct transformed. One transgenic line for each construct is presented as representative of the majority of lines showing consistent results.

Expression analysis

At least three independent biological samples were used for each analysis. Each replicate comprised the content in seeds of ten siliques. Total RNA was extracted using the RNeasy Mini kit (Qiagen), including RNase-Free DNase Set (Qiagen) treatment during washing, according to the manufacturer's instructions. The Superscript Reverse Transcriptase II kit (Invitrogen) was used to generate cDNA from 1μg of total RNA. Quantitative PCRs were performed with the SYBR Green kit (Bio-Rad) on a Bio-Rad CFX real-time PCR machine. SWEET4 was PCR amplified using the (5'CCTCAATGGTGTCGTTTGGG3') forward and (5'TAGCTTGTCCACTGTTGCCA3') reverse primers. SWEET4 expression levels were normalized to the expression levels of three reference genes (AT4G12590, AT4G02080, and AT3G25800) [START_REF] Dekkers | Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds[END_REF].

Lugol staining

Harvested seeds and ovules were incubated in a 1% SDS, 0.2 N NaOH solution at 37°C for 15 minutes to clear the tissue. Samples were rinsed in water and incubated in a 12.5% bleach solution (1.25% active Cl-) for 10 minutes. Samples were then rinsed in water and transferred to a 33% Lugol solution and incubated for 30 seconds. Finally, samples were analyzed by differential interference contrast microscopy with an Axioplan 2 microscope (Zeiss). More than 30 seeds were analyzed for each genotype and time point.

Confocal microscopy

Modified pseudo-schiff propidium iodide (mPS-PI) analyses were conducted as previously described (Xu et al., 2016). Starch-mPS-PI staining analyses were conducted following the mPS-PI protocol but omitting the α-amylase step. In starch-mPS-PI treated samples, propidium iodide stains

Nutrient transport in the chalazal region

The sweet10 mutation delays embryo development

Similar to sweet4, we observed that the sweet10 mutation impaired embryo growth (Fig. 4).

The sweet10 mutant embryo development was largely delayed. These data support the hypothesis that SWEET10 facilitates sugar export to the embryo. (Fiume et al., 2017a).

DISCUSSION

The chalaza-nucellus path of nutrient transport

Our data indicate that part of the sucrose transported to the seed through the phloem is immediately exported to the apoplast by the SWEET10 sucrose exporter and converted into hexoses by the INV4 cell wall bound invertase (Fig. 6). We speculate that the apoplastic glucose, provided by nucellus and chalaza, might diffuse toward the endosperm to be reimported by endosperm SWEET facilitators. Compared to the seed coat pathway, which is based on sucrose transport, the chalaza-nucellus path offers a faster route toward the endosperm thus allowing the transport of readily available glucose and fructose.

METHODS

Plant material

All Arabidopsis thaliana plants are in the Columbia accession. The tt16-1 allele was isolated in the Wassilewskija accession from the INRA Versailles collection (Bechtold and Pelletier, 1998) and then backcrossed to Columbia (Coen et al., 2017;Nesi et al., 2002a). sweet10-1 (SALK_144826) alleles are from the Salk collection (Alonso et al., 2003) whereas the sweet4-2 (GK_858G02) (Chong et al., 2014) allele is from the GABI-KAT collection (Kleinboelting et al., 2012).

Cloning and Construction

PCR amplifications were performed using the gene-specific primers described below carrying ProSWEET10 sequences were recombined into the pBGWFS7.0 binary vector (Karimi et al., 2002), ProSWEET10:SWEET10 and ProINV4:INV4 sequences were recombined into the pMDC107 binary vector (Curtis and Grossniklaus, 2003).

Transgenic plants

The Agrobacterium tumefaciens strain C58C1 was used to stably transform Arabidopsis plants using the floral dip method (Clough and Bent, 1998). Transformants were selected with the appropriate resistance and subsequently transferred to soil for further characterization. More than 10 independent transgenic lines were tested for each construct transformed. One transgenic line for each construct is presented as representative of the majority of lines showing consistent results.

Expression analysis

Four independent biological samples were used for each analysis. Each replicate comprised the content in seeds of ten siliques. Total RNA was extracted using the RNeasy Mini kit (Qiagen),

including RNase-Free DNase Set (Qiagen) treatment during washing, according to the manufacturer's instructions. The Superscript Reverse Transcriptase II kit (Invitrogen) was used to generate cDNA from 1μg of total RNA. Quantitative PCRs were performed with the SYBR Green kit (Bio-Rad) on a Bio-Rad CFX real-time PCR machine. SWEET10 was PCR amplified using the (5'CCTTATCGGCTGTCATGTGG3') forward and (5' AAGCTTAATCCCTGGTGGCT3') reverse primers. Expression levels were first normalized to the geometrical mean of the expression levels of four reference genes (GAPDH, AT4G12590, AT4G02080 and AT3G25800) and subsequently normalized to the expression level of the adequate control.

Confocal microscopy (see chapter2) Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: TT16 (AT5G23260), SWEET4 (AT3G28007), SWEET10

(AT5G50790), INV4 (AT2G36190).

Chapter III -Symplastic block in the nucellus

same development stage (Fig. 3A).

At the mature ovule stage, free GFP was not detected in the transient nucellus of tt16 lines, as observed in the wild type (Fig. 3D). GFP-sporamin was detected in a few cells of the chalaza where the phloem ends (Fig. 3C). Wild type and tt16 ovules appeared indistinguishable at earlier ovule stages (Supplementary Fig. 2). These data suggest that TT16 does not regulate the establishment of the symplastic block between persistent and transient nucellus.

Callose deposition in the nucellus

Callose accumulation around the neck region of plasmodesmata can lead to the closure of plasmodesmata. To test whether callose deposition affects permeability of nucellus plasmodesmata, we used aniline blue, which is a visual marker of callose [START_REF] Zavaliev | Imaging callose at plasmodesmata using aniline blue: quantitative confocal microscopy[END_REF]. We detected a strong aniline blue signal in the distal part of transient nucellus in both mature ovules and seeds at the pre-globular embryo stage (Fig. 4). Similar data were obtained in tt16 ovules and seeds

The morphology of plasmodesmata in the nucellus

Plasmodesmata that accumulate callose at their neck region have a characteristic "buldgy"

appearance when observed at the transmission electron microscope (TEM). We, therefore, analyzed plasmodesmata by TEM in nucellus longitudinal sections of mature ovules (Fig. 5A). We found a relatively high number of plasmodesmata connecting the cells of the persistent nucellus (Fig. 5B).

However, in the transient nucellus, we hardly found plasmodesmata, and the morphology of the relatively few ones that we detected resembled that of plasmodesmata closed by callose

MATERIAL AND METHODS

Plant materials

All Arabidopsis thaliana plants are in the Columbia accession. ProSUC2:GFP and ProSUC2:GFP-sporamin lines have been previously described (Stadler et al., 2005). The tt16-1 allele was isolated in the Wassilewskija accession from the INRA Versailles collection (Bechtold and Pelletier, 1998) and then backcrossed to Columbia (Coen et al., 2017;Nesi et al., 2002a). We crossed ProSUC2:GFP and ProSUC2:GFP-sporamin lines into the tt16-1 background.

Aniline blue staining

Harvested seeds and ovules were incubated in a 1% SDS, 0.2 N NaOH solution at 37°C for 2hrs to clear the tissue. Samples were rinsed in water and incubated in a 12.5% bleach solution

(1.25% active Cl-) for 10 minutes. Samples were then rinsed in water and transferred to the staining solution of 0.01% (w/v) aniline blue in water and incubated for 1hr at room temperature. Finally, samples were analyzed at the confocal microscope.

Confocal microscopy

GFP expressing lines were analyzed one hour after mounting in a 100 µg/ml propidium iodide diluted in a 7% sucrose solution (Figueiredo et al., 2016). Samples were imaged by confocal laser scanning microscopy (Leica SP8). More than 30 seeds were analyzed for each genotype and time point.

Transmission electron microscopy

For transmission electron microscopy (TEM), septums with seeds were immersed immediately after harvest in a fresh fixative solution which is Glutaraldehyde (Sigma, 2%), fresh formaldehyde (Sigma, 0.5%) in cacodylate buffer 0.1 M at pH 7. Fixation was performed for 4h at room temperature and 1 week at 4°C. Samples were subsequently contrasted with Oolong Tea Extract (OTE) (Delta Microscopies-France) 0.5% in cacodylate buffer 0.1M pH 7.4 for 1h, post-fixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate for 2h, gradually dehydrated in ethanol series (10% to 90%, 1h for each bath), and dehydrated twice for 1h in ethanol 100%.

Samples were then gradually treated with mixtures of ethanol-epon (ratios of 1:2, 1:1 and 2:1, for 2h time) and finally transferred to pure epon (Delta Microscopie) overnight under vacuum. For embedding, ovules/seeds were spread on silanized glass slides. One drop of epon was added before overlaying with a second glass slide. After polymerisation (48h at 56°C), the epon layer was removed from the slide. Selected ovules and seeds were cut out and stuck on the top of Beem capsules (EMS) pre-filled with epon. Semi-thin sections (500 nm) were collected and colored with azure II/methylene blue to check tissue integrity before thin sections. Thin sections (70 nm) were collected onto either 125/200 mesh copper grids or slot grids and counter-stained with lead citrate.

TEM samples were examined with Hitachi HT7700 electron microscope operated at 80 kV (Elexience-France), and images were acquired with a charge-coupled device camera (AMT).

Cloning

GSL4

-3kb promoter region was amplified using the attB1-(5′-GCGGCATGGAGATTATCAAC-3′) forward and attB2-(5′-GGGACGATTTCGTGAGACAT-3') reverse primers. AttB1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′) and attB2 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTC-3′) carrying the Gateway recombination sites at the 5′-ends of the forward and reverse primers, respectively. The PCR products were amplified with the high-fidelity Phusion DNA polymerase (Thermo Fisher Scientific), recombined into the Pdonr221 vector (BP Gateway reaction) according to the manufacturer's instructions (Thermo Fisher Scientific), and then sequenced. The PCR products cloned into the pDONR221 vectors were then recombined into the pGWBFS7 binary vector (Curtis and Grossniklaus, 2003), (LR Gateway reaction) according to the manufacturer's instructions (Thermo Fisher Scientific).

DISCUSSION

Why is the transient nucellus symplastically isolated from other tissues?

Cell elimination of the transient nucellus does not spread to the neighboring persistent nucellus, thus indicating that a mechanism to contain cell elimination is put in place. Our data indicate that the persistent nucellus is symplastically isolated from the transient nucellus already before fertilization, in mature ovules. We speculate that the symplastic block is put in place to stop the spreading of the cell elimination process that occurs in the transient nucellus.

Chapter IV -SHATTERPROOF MADSbox genes promote nucellus survival.

of SHP genes. We cloned SHP2 3kb promoter and genomic sequence and translationally fused to GFP. The transgene was transferred into both wild type and tt16 plants. We detected the expression of SHP2 in both inner (ii) and outer integuments (oi), chalaza and nucellus, with the only exception of the most distal nucellus cells above the tetrad, in wild type and tt16 ovule primordia at stage 2-V (Fig. 6A and6D). In ovules at stage 3-V, SHP2 was expressed in the proximal part of the ii and oi, in the most distal cells of the oi, chalaza and nucellus in both wild type and tt16 plants (Fig. 6B and6E). In pre-globular embryo seeds, we detected stronger GFP fluorescence in the proximal oi and everywhere in the nucellus, and a weaker signal in the proximal ii and in the chalaza in both wild type and tt16 plants (Fig. 6C and6F). Cloning of SHP1 promoter and genomic sequence is currently underway.

followed nucellus development in shp1;shp2 seeds. A fraction of shp1;shp2 appeared to have completely lost the persistent nucellus at torpedo embryo stage (Fig 9B), which suggests that SHP1 and SHP2 are important for the survival of the persistent.

Finally, we could not meaningfully analyse SEP function because of gene redundancy between SEP1, 2 and 3 and ovule-less phenotype of sep1;sep2;sep3 triple mutant [START_REF] Favaro | MADS-box protein complexes control carpel and ovule development in Arabidopsis[END_REF].

MATERIAL AND METHODS

Plant materials

All Arabidopsis thaliana plants are in the Columbia accession. The tt16;goa and shp1;shp2 mutant lines have been previously described [START_REF] Liljegren | SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis[END_REF] (Prasad et al., 2010b). Days after flowering were counted starting from the emergence of the pistil from closed flowers; 0 DAF equals stage 3-V of ovule development (Schneitz et al., 1995c). Both DAF and embryo development have been used to determine seed developmental stages. RNA extraction and reverse transcription were performed as described above (see chapter 2).

Cloning

Cloning and construction

Amaranthus hyoochondriacus TT16 splicing forms were amplified using the attB1-

Arabidopsis transformation

Arabidopsis transformation was performed as described above (see chapter2).

Pseudo-Schiff propidium iodide staining

Pseudo-Schiff propidium iodide staining was performed as described above (see chapter4).

RNA in situ hybridization

RNA in situ hybridization analyses were conducted as previously shown (Xu et al., 2016).

Primers used for probes:

Name Sequences Probes

TT16as F 5'-GAAGCAATTCACAACACTGAGC TT16as

TT16as R 5'-TGTAATACGACTCACTATAGGGCATTAGGATGAGTGGGTTGAAGG TT16s F 5'-TGTAATACGACTCACTATAGGGCGAAGCAATTCACAACACTGAGC

TT16s

TT16s R 5'-ATTAGGATGAGTGGGTTGAAGG

HIS4as F 5'-GGAAGAGGAAAGGGAGGAAA HIS4as

HIS4as R 5'-TGTAATACGACTCACTATAGGGCCCACCAAAACCATAAAGAGTACG

DISCUSSION

Arabidopsis and amaranth display opposite seed architectures. Whereas Arabidopsis seeds eliminate most of the nucellus tissue in favor of the endosperm, amaranth seeds undergo nucellus growth alongside a minute endosperm. MADS-box genes in the proximal region of the nucellus, referred to as persistent nucellus as it does not degenerate. SEP3 has been shown to bridge TT16 and SHPs physical interaction, thus suggesting that nucellus elimination is coordinated by the formation of TT16 and SHPs protein complexes. Furthermore, we identified a novel path of sugar transport through the nucellus in the Arabidopsis seed. We showed that the nucellus is symplastically connected to the chalaza, the seed nutrient unloading zone, and works both as sugar sink and source alongside the seed coat. The transient nucellus accumulates starch early on after fertilization and releases it in the apoplastic space during its elimination. By contrast, the persistent nucellus and the chalaza export sugars toward the endosperm through the SWEET4 and SWEET10 sugar facilitators, a process that is partially regulated by TT16. Interestingly, we discovered that persistent and transient nucellus are symplastically disconnected. We detected relatively few plasmodesmata in the transient nucellus and their shape was reminiscent of plasmodesmata blocked by the accumulation of callose. We hypothesize that the development of a symplastic barrier between persistent and transient nucellus might prevent the spreading of the cell elimination process, which indeed affects only the distal region of the nucellus. Finally, we characterized nucellus development in the Amaranthus seed that, opposite to Arabidopsis, grows a large nucellus as primary storage tissue. To challenge the hypothesis that the TT16 transcription factor might be responsible for the evolution of such different seed architectures, we cloned TT16 orthologous gene in Amaranthus and characterized its expression pattern and function. Overall, this work gives an insight in the regulatory mechanisms that coordinate the processes of nutrient and tissue partitioning in seeds.