Bak1 Bri1-Associated

Receptor Kinase

Kinase

Bsu1 Bri1-Suppressor

Brassinazole Resistant1

Cal Cauliflower

Cupshaped Cotyledon

Dreb2a Dre-Binding Protein

AUXIN/INDOLE ACETIC ACID BAS1

Summary

The process of morphogenesis is driven by cell division and expansion, which are controlled in a differential manner among cell types and tissues. In plants, the above ground organs are continuously produced by the shoot apical meristem (SAM), where the initiation of new primordia is triggered by the local accumulation of the plant hormone auxin. We study the process of morphogenesis in the inflorescence of Arabidopsis thaliana, where flowers are formed in a regular pattern from the SAM.

The DNA-binding auxin response factor ARF5/MP plays a central role in the initiation of flowers. After its activation, it induces the expression of LEAFY, AINTEGUMENTA and AINTEGUMENTA-LIKE6 transcription factors necessary for the specification of floral identity and proliferative growth. However, at the cellular level, the initiation of lateral outgrowths depends on regional differences in growth. In plant cells, these processes are regulated via modifications of the cell wall. Auxin and its downstream targets are also involved in these processes, by activating changes in the dynamics of the cortical microtubules, which result in changes in growth direction. Auxin also slightly reduces wall rigidity prior to organ outgrowth in the SAM, which results in changes in growth rate. This is correlated with the transcriptional activation of a number of cell wall modifying genes.

Thus, auxin signaling regulates primordium initiation by integrating the activation of a transcriptional regulatory network and both the stiffness and anisotropy of the cell wall, which directly influence the rate and direction of growth.

The findings of this thesis provide evidence indicating that the mechanisms of organ initiation at the SAM involve feedbacks where changes in the local properties of the cell wall influence the molecular regulation of the transcriptional regulatory network. Our results suggest that this might require the influence from other hormones, different from auxin, that funnel the initiation of lateral outgrowths.

Résumé

Le phénomène de morphogenèse est le fruit de la division des cellules et de leur expansion, qui sont controllées de façon différentielle selon les types cellulaires et les tissus. Dans le cas des plantes, le méristème apical caulinaire (MAC) produit de façon continue les organes aériens à partir de primordia qui sont initiés suite à l'accumulation locale d'une hormone végétale, l'auxine. Pour étudier le processus de formation des organes aériens, nous utilisons l'inflorescence d'Arabidopsis thaliana, dont les fleurs sont mises en place selon un patron régulier à partir de cellules dérivées de cellules souches. Au cours de ce processus, ARF5/MP -un facteur de réponse à l'auxine se liant à l'ADN -joue un rôle central. Une fois activé, il induit l'expression des facteurs de transcription LEAFY, AINTEGUMENTA et AINTEGUMENTA-LIKE6, qui sont nécessaires pour la spécification de l'identité florale et pour la croissance proliférative.

A l'échelle cellulaire, des excroissances latérales sont initiées suite à des hétérogénéités locales de croissance. Dans les cellules végétales, ces différences sont dues à des modifications de la paroi cellulaire impliquant l'auxine et ses cibles, qui induisent des variations dans la dynamique des microtubules corticaux résultant en des changements de direction de croissance. Dans une moindre mesure, l'auxine diminue la rigidité des parois cellulaires préalablement à la formation d'un nouvel organe, conduisant à des changements de taux de croissance. Ceci est corrélé à l'activation transcriptionnelle de nombreux gènes qui sont impliqués dans les modifications de la paroi. Ainsi, la voie de signalisation de l'auxine régule l'initiation des primordia en intégrant d'une part l'activation d'un réseau de régulation transcriptionnelle et, d'autre part, la rigidité et l'anisotropie de la paroi cellulaire, impactant directement le taux et la direction de croissance.

Cette thèse soutient l'idée selon laquelle l'initiation des organes dans le MAC repose sur des boucles de rétroaction là où des changements locaux de propriétés de la paroi cellulaire influent sur le réseau moléculaire. Il est probable que d'autres hormones soient nécessaires afin de canaliser l'initiation des organes.

To all the members of the Meristem and Mechanodevo teams, I extend my gratitude to those with whom I worked in the past and that are no longer in the lab and those who are still in the RDP. A special thanks to Amélie who gave me a lot of support in this last part of the thesis.

Thanks to Léa who helped me a lot with the summary. To all my lab mates Laia, Mar, Roberta, Massi, Carlos, Jazmin, Ana, Chie, Kateryna, Antoine et tous les copains du foyer, who offered me their friendship and their support both academic and personal. I thank to all the members of the RDP, who through their work and motivation provide the best conditions to work in this laboratory.

I want to express my gratitude to my family, Pilli, Papo, Bin, Naye y familia (Emmanuel, Andres y choquinho(a)), quienes siempre me han apoyado en todos los aspectos de mi vida.

Gracias por estar conmigo y ofrecerme su amor incondicional. Gracias tambien a mi familia extendida, Gise, Juan y Jimena, quienes tambien me ofrecen su carino y consejo. A mis amigos, que aun a la distancia siguen cercanos y me reciben siempre con una sonrisa.

Gracias a mi Uke, que es el que mas me aguanta y del que mas aprendo en muchos aspectos.

Gracias port tu compania, tu apoyo y tu carino.

Finally, I want to thank anyone who I could have forgotten to mention, to all those who have somehow shared this project with me.

Glossary of Terms

activator a short-ranging substance that promotes its own production and the synthesis of its antagonist anisotropy the existence of directions with distinctive properties anisotropic growth growth with a maximal and minimal direction canalization a valley in Waddington's epigenetic landscape that represents a cluster of similar trajectories complex system a system made of many elements that exhibits emerging global properties not directly predictable from the properties of the individual components crosstalk specific interactions between components of more than one pathway emergent property a feature that is characteristic of system-level dynamics that cannot be attributed to any of its components epigenetic landscape visual depiction of a set of developmental choices that is faced by a cell in the embryo elastic deformation reversible extension of the cell wall feedback regulation control mechanism that uses the consequence of a process to regulate the rate at which the process occurs feed-forward loop a biochemical pattern in a transcription network , a three-gene pattern, composed of two input transcription factors, one of which regulates the other, both jointly regulating a target gene hydrogel network of polymer chains that are hydrophilic, they are highly flexible due to their significant water content inhibitor rapidly difussing antagonist of an activator, it slows down the production of the activator or catalyzes its decay lateral inhibition strategy for emphasizing differences between inputs, a chemical inhibitor diffusing faster through neighboring cells prevents the accumulation of the activator creating a zone of lateral inhibition microtubule anisotropy indicates a dominant microtubule orientation over a population of microtubules morphogen a diffusible signal that acts at a distance to regulate pattern formation in a dose-dependent manner phyllotaxis the pattern at which new leaf and flower primordia emerges plastic deformation irreversible extension of the cell wall plastochron interval between the initiation of two consecutive primordia self-organization evolution of a system into an organized form in the absence of external pressures strain deformation of an object induced by stress, corresponds to growth rate in living organisms stiffness the extent to which an object resists deformation stress force applied on a surface normalized by the surface area upon which it is exerted stress anisotropy stress with maximal and minimal directions tensile strength the resistance of a material to breaking under tension yield threshold level of stress that needs to be applied to a structure to induce an irreversible deformation wall creep cell wall extension that involves the breaking of hydrogen bonds between cell wall polymers Introduction

Generalities of pattern formation and morphogenesis in multicellular organisms

One of the most fundamental questions in biology is that of biological pattern formation: how do the individual cells of a multicellular organism differentiate and how is this related to the overall structures and shapes that arise during development? In this first part of the introduction I will present the main theories that have addressed the problem of biological pattern formation and that have largely influenced modern developmental biology.

On Growth and Form

In 1917 D'Arcy Thompson published an extensive study on growth and form during development. He hereby underlined the fact that biological form is the consequence of physical processes and mechanical forces [START_REF] Thompson | On Growth and Form[END_REF]. Thompson eloquently described form as a direct product of growth, and emphasized that growth and form are inseparably associated. According to his view the form of an organism is determined by its rate of growth in various directions. "Every growing organism; and every part of such a growing organism, has its own specific rate of growth, referred to this or that particular direction; and it is by the ratio between these rates in different directions that we must account for the external forms of all save certain very minute organisms".

On Growth and Form is Thompson's most famous work containing its most influential ideas.

In the book, he offers a descriptive explanation of the shapes of various parts of multicellular organisms. These descriptions were more than anything else mathematical descriptions. Due to his preference for mathematical and biophysical concepts, but most likely also because ideas about molecular regulation were not known at that time, Thompson did not invoke biochemical explanations for his thesis. In this way, he excluded any explanation for the function of such shapes. Since then, the elaboration of biological knowledge has made it possible to address the question of pattern formation not only as a consequence of biophysical forces, but also in terms that consider biochemical signaling and downstream molecular regulation.

Regardless of the limitations [START_REF] Mcclung | Growth and Form[END_REF]), Thompson's ideas provided an important basis for shaping modern developmental biology, and have gained renewed interest. I will now present a number of concepts that have focused more on biochemical regulation.

Reaction-diffusion model

The assembly of basic physical laws served Alan Turing to propose a key explanation for the formation of biological patterns [START_REF] Turing | The chemical basis of morphogenesis[END_REF]). Turing's hypothesis was that patterns in biological tissues can arise from simple chemical processes that can be described precisely and mathematically. He suggested "a system of chemical substances, which he termed morphogens, reacting together and diffusing through a tissue, is adequate to account for the main phenomena of morphogenesis".

The essential feature of the reaction-diffusion model proposed by Turing is that a small perturbation in the concentration of two substances, initially distributed homogeneously can become spatially distributed heterogeneously given the differences in their diffusion properties and cross-regulation. Over time, the theory of Turing of biological pattern formation was further developed by Gierer and Meinhardt, who introduced to the reactiondiffusion model, the role of autocatalysis in conjunction with lateral inhibition [START_REF] Gierer | A theory of biological pattern formation[END_REF][START_REF] Meinhardt | Pattern formation by local self-activation and lateral inhibition[END_REF]. They proposed that one of the two substances is a short-range "activator", a chemical that can make more of itself; the other one a longrange "inhibitor", slows the production of the "activator". Each of these substances acts on itself as well as the other. This dynamical interaction between the morphogens allows the mechanism to become self regulated and endows the ability to produce spontaneously a pattern when starting from a uniform field of cells. The system could be illustrated as follows (Figure 1a). (i) Molecular fluctuations of the morphogens, will cause some cells to accumulate slightly higher levels of activator. (ii) The activator self-regulation will increase its concentration enhancing also the production of the inhibitor. (iii) The activator positive feedback stabilises its own levels. (iv) However, since the inhibitor diffuses faster, it will increase its level in neighbouring cells, preventing the accumulation of the activator, creating a zone of "lateral inhibition" where no new peaks of activator can form (v) The whole system dynamically changes until a regular array of peaks and valleys is formed across the whole field of cells [START_REF] Green | Positional information and reaction-diffusion: two big ideas in developmental biology combine[END_REF].

In principle such a reaction-diffusion system can account for many patterning events. This could be the case, for example, for the patterning of digits during limb development in mouse embryos. This mechanism depends on the feedback loop between Wnt and Bmp signaling and the transcription factor Sox9 (Figure 1b) [START_REF] Newman | Dynamics of skeletal pattern formation in developing chick limb[END_REF][START_REF] Raspopovic | Modeling digits. Digit patterning is controlled by a Bmp-Sox9-Wnt Turing network modulated by morphogen gradients[END_REF].

Another example of a potential reaction-diffusion based mechanism is the left-right patterning of the early vertebrate embryo (Figure 1c). The distinction between the left and right side of the body is driven by the interaction between the protein Nodal, the activator, and Lefty 2, the repressor. Their interaction creates a broad gradient that allows cells to distinguish in which side of the embryo they are. Nodal-Lefty network forms spontaneously from an initial maternal bias through local auto-activation and long range inhibition [START_REF] Green | Positional information and reaction-diffusion: two big ideas in developmental biology combine[END_REF]. In plant systems, probably the best example of a mechanism potentially based on reaction-difussion is phyllotaxis, which describes the pattern at which new leaf and flower primordia emerge [START_REF] Meinhardt | Models of pattern formation and their application to plant development In Positional Controls in Plant Development[END_REF]. Various patterns can be created depending on the range of activation and inhibition, either an alternating (distichous), 90° rotated (decussate) and even spiral [START_REF] Meinhardt | Models of biological pattern formation: common mechanism in plant and animal development[END_REF]. Later on Kuhlmeier and collegues discovered that actively transported auxin is the instructive signal determining the induction and positioning of lateral organs. Although in this example, patterning is not through an inhibitor but through a redistribution of an activator by transport (see also below). Good evidence for an activatorinhibitor system also exists for the initiation of leaf hairs [START_REF] Hulskamp | Plant trichomes: a model for cell differentiation[END_REF].

Positional-information model

Another notable contribution to the concept of pattern formation was the notion of morphogen concentration and gradients proposed by Wolpert. In his aim to understand how more complex patterns were constructed from earlier tissue heterogenities, he proposed that differences in morphogen concentration across space could be enough to define different positions. In the gradient model there is a fixed source of morphogens. The morphogens leave this site by diffusing within the tissue. Since they are also degraded, they will form a concentration gradient [START_REF] Wolpert | Positional information and the spatial pattern of cellular differentiation[END_REF][START_REF] Wolpert | Positional information and pattern formation[END_REF]. Cells that are responsive to the morphogen interpret the local concentration, whereby different threshold concentrations would hereby give different reponses [START_REF] Ashe | The interpretation of morphogen gradients[END_REF][START_REF] Kondo | How animals get their skin patterns: fish pigment pattern as a live Turing wave[END_REF][START_REF] Kondo | Reaction-diffusion model as a framework for understanding biological pattern formation[END_REF].

The positional information concept explains how a prior asymmetry results in a graded distribution of a morphogen, and how cells use this distribution to acquire different identities.

This concept is commonly illustrated with a French Flag pattern (Figure 2a), in which the field of cells are divided into three different regions of cell fates (red, white and blue). After the interpretation of the morphogen threshold levels (T1, T2), cells react differently to these concentrations and adopt diverse fates. It has been proposed that the development of the Drosophila embryonic segments is based on a positional information system (Figure 2b andc). Each stripe is defined independently by its unique anterior-posterior position in a succession of local concentration gradients of the gap genes. Differences in morphogen concentration at each position of the field provide distinct inputs to the gap gene network, which convert the smooth spatial differences into more discrete molecular patterns. This more complex molecular pattern of gap genes then provides the positional information for the next level of gene regulation, the segment polarity genes, which are each expressed as a series of stripes (Figure 2b andc) [START_REF] Green | Positional information and reaction-diffusion: two big ideas in developmental biology combine[END_REF].

Both, Turing's reaction-difussion and Wolpert's positional information models are able to explain biological patterns. The key feature that differentiates a reaction-difussion system from a positional information system is that the gradient is self-organized through the dynamics of the activator-inhibitor pair, unlike the positional information concept, which explains how a prior asymmetry is converted into a specific pattern. Therefore, the two processes rather than mutually exclusive may be complementary and could function together as regionalizing mechanisms [START_REF] Green | Positional information and reaction-diffusion: two big ideas in developmental biology combine[END_REF].

A number of morphogens have been well described in animals [START_REF] Wolpert | Principles of Development[END_REF]. The first one discovered was the concentration gradient of Bicoid (Bcd) protein in Drosophila, which patterns embryo segmentation [START_REF] Driever | A gradient of bicoid protein in Drosophila embryos[END_REF][START_REF] Frigerio | Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network[END_REF]. On the other hand, in plants the morphogen concept has remained a subject of debate. The signaling molecules closest to this concept are hormones. Particularly, auxin fulfils the characteristics of a morphogen, since it functions in diverse patterning events in a concentration-dependent manner and directly regulates target cells [START_REF] Benkova | A morphogenetic trigger: is there an emerging concept in plant developmental biology?[END_REF][START_REF] Bhalerao | The case for morphogens in plants[END_REF][START_REF] Sabatini | An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root[END_REF]. (b) Initial asymmetries in the anterior-posterior axis of the early Drosophila embryo result in the graded distribution of morphogens that in turn regulate the expression of the gap genes providing the positional information for the segment polarity genes, expressed as a series of stripes.

(c) Differences in the concentration of the morphogens Bicoid and Caudal across the embryo inputs the gap genes network (giant, hunchback, Krüppel, and knirps). In turn, the complex interactions among gap genes create molecular patterns of expression of the segment polarity genes (even-skipped, runt, hairy).

Epigenetic landscape

A third theory worth to mention, is the epigenetic landscape concept proposed by Conrad Waddington. At the time he started to develop his ideas, the Mendelian laws of heredity were well accepted. Waddington agreed with the distinction between the individual's physical appearance, or the phenotype and the hereditary information contained in the germ cells and passed to the next generation, better known as the genotype. He considered the phenotype as the result of the interrelations among genetic processes, their potentialities and constraints, and the external environment.

Waddington studied the development of embryonic cells triggered by given stimuli, through a process he called induction. He proposed a hypothesis in which he emphasized the importance of the reacting tissue, in the sense that change occurs not only because cells receive a particular signal, but also because they have the 'potency' to react. In other words, cells react to different stimuli, biochemical or environmental, in a way allowed by their state at that time. With each new reaction, the cell might differentiate further into a state with more constraints or more possibilities and potentialities.

He argued that the various developmental pathways a cell might take follow an epigenetic path. Each step is defined by instructions in the genotype that interact to produce a system that moves along a trajectory. The diverse paths in development are protected or canalized by threshold reactions, providing stability and direction. Waddington illustrated this canalization concept, as a landscape, an epigenetic landscape formed by a series of ridges and valleys a cell can traverse on its way to a final tissue type [START_REF] Waddington | Principles of embryology[END_REF][START_REF] Waddington | The strategy of the genes: a discussion of some aspects of theoretical biology[END_REF] (Figure 3a). The landscape thus represents the tendency of cells to pass from an immature stage to an adult and specified condition. The path of a cell would start from a totipotent state, passing via a pluripotent state to a lineage -committed state that leads it into one of many possible fates.

The steepness of the walls, represents the stability of the path. If the walls are very high, it is hard for the cells to escape from their developmental faith and even big mutational or environmental perturbations will not be able to bring the cell out of its path. The control of the steepness of the walls in turn depends on the underlying genetic landscape (Figure 3b).

Importantly, not only genes and their products, but also gene-gene interactions and geneenvironment interactions are in control of development. According to Waddington's ideas, genes not only regulate, but they are also regulated by non-genetic factors [START_REF] Van Speybroeck | From epigenesis to epigenetics: the case of C. H. Waddington[END_REF].

General concepts: some concluding remarks

According to these theories there are multiple factors directing pattern formation. The physical properties and form diversity in D'Arcy Thompson's theory, the self-organization and biochemical patterning in Turing patterns and the importance of the interaction of these factors at multiple scales. However, in order to understand pattern formation it is necessary to analyze how are these elements acting in concert. These interactions lead to the emergence of collective properties that cannot be deduced from adding up local behaviour. These are properties common to all multicellular organisms comprised under the concept of complex systems.

Plants as complex systems are hierarchically organized and composed by interactive elements: molecules assemble into cells, cells into tissues and organs. The interactions between the individual components and the multiple feedbacks between the diverse levels of organization are fundamental for pattern formation. Therefore, these systems can only be understood by analysing them at multiple scales, leading to the use of more interdisciplinary approaches. In addition, certain constraints imposed by the developing system might limit the possible final shapes. In plant development some specific emergent properties should be considered when studying pattern formation and morphogenesis. Of them I will speak in the following section.

Generalities of pattern formation and morphogenesis in plants

The generation of form in plants is distinguished by a number of specific features [START_REF] Hernandez-Hernandez | Dynamical patterning modules in plant development and evolution[END_REF][START_REF] Niklas | The Evolution of Plant Body Plans-A Biomechanical Perspective[END_REF][START_REF] Niklas | The evolutionary development of plant body plans[END_REF]. First, plant architecture is characterized by an open and indeterminate ontogeny, with multiple growing points (or meristems) where cell proliferation persist continuously producing new tissues and organs [START_REF] Esau | Plant anatomy[END_REF]. This characteristic relates to the sessile nature of plants. It provides them the opportunity to adjust to their external environment, adapting their shape and architecture in relation to it.

A notable characteristic of plant cells that highly influences development is the presence of a relatively rigid extracellular matrix, the cell wall. The presence of this cell wall provides plants with specific mechanical properties. As a result, morphogenesis must occur in the absence of cell migration. Therefore, cell expansion, cell division and, to a lesser extent, programmed cell death, are of major importance in plant morphogenesis [START_REF] De Smet | Asymmetric cell division in land plants and algae: the driving force for differentiation[END_REF][START_REF] Van Hautegem | Only in dying, life: programmed cell death during plant development[END_REF]. Given that plant cells are immobilized they In what follows I will review the basic characteristics of plant development that define pattern formation in plants. I will firstly focus on the role of signaling molecules, hormones and their effect on establishing molecular expression patterns. Secondly, I will describe the role of the extracellular matrix in controlling growth patterns. A good amount of our knowledge in these topics has been obtained from the plant experimental system Arabidopsis thaliana (hereafter called Arabidopsis); therefore I will mainly focus on this species.

Molecular regulation: the role of hormones in plant development

As mentioned previously, positional information is perceived and transmitted via communication between different parts of the organism, locally and over long distances. This communication is based on the perception and production of mobile signals. In plants, the distribution and perception of hormones as instructive mobile signals of growth and development has been well established, involving in particular cytokinins, auxins, gibberellins, brassinosteroids and strigolactones (Santner et al., 2009;Santner and Estelle, 2009;[START_REF] Wolters | Survival of the flexible: hormonal growth control and adaptation in plant development[END_REF].

Major aspects of plant hormone synthesis, degradation, transport and signaling have been extensively studied. In what follows I will briefly summarize the role of hormones in controling gene regulation, in particular at the transcriptional level, where they play a central role [START_REF] Nemhauser | Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses[END_REF]. I will hereby focus on auxins, cytokinins and brassinosteroids, which have major roles in development of the meristems and plant architecture and are a central focus of this thesis.

Auxin

Auxin is certainly one of the most important signals, affecting plant developmental processes at cellular, tissue and organ levels. It has been considered as the closest equivalent to morphogens in plants [START_REF] Bhalerao | The case for morphogens in plants[END_REF][START_REF] Sabatini | An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root[END_REF]. During development, auxin differentially accumulates in different parts of the plant. Auxin gradients are fundamental in the regulation of many developmental processes. From the very early stages of development onwards, auxin accumulation and its graded distribution play fundamental roles in defining plant shape. Already after fertilization, the apical cell of the divided zygote is the site of auxin accumulation and activity. This is maintained this way until the 32-cell-embryo stage. Later on auxin distribution changes to establish the root pole and cotyledons [START_REF] Friml | Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis[END_REF]. Another example of the importance of differential auxin distribution is the accumulation of auxin at the location of organ initiation, either at the root (Figure 4b) [START_REF] Dubrovsky | Auxin acts as a local morphogenetic trigger to specify lateral root founder cells[END_REF] or at the shoot (Figure 4c) [START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF][START_REF] Meinhardt | Complex pattern formation by a self-destabilization of established patterns: chemotactic orientation and phyllotaxis as examples[END_REF]. There are numerous examples about the role that auxin distributions and gradients play in the regulation of plant growth and development. But how does these specific distributions arise?

One mechanism for the differential distribution of auxin is attributed to its site of biosynthesis. The most common auxin in vascular plants is indole-3-acetic acid (IAA). IAA is synthesized by one tryptophan (Trp)-independent and four Trp-dependent pathways. Two of them, the tryptamine (TAM) pathway, and the indole-3-piruvic acid (IPA) pathway are most relevant for plant development. Rate-limiting enzymes for these pathways include the flavin monooxygenase-like enzymes of the YUCCA family and the Trp aminotransferase of Arabidopsis (TAA) [START_REF] Teale | Auxin in action: signalling, transport and the control of plant growth and development[END_REF][START_REF] Woodward | Auxin: regulation, action, and interaction[END_REF] (Figure 5a). Mutations of multiple YUCCA and TAA genes impair local auxin accumulation and result in severe developmental defects, in embryogenesis, leaf venation, and floral organ patterning, among others [START_REF] Cheng | Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis[END_REF][START_REF] Cheng | Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis[END_REF][START_REF] Stepanova | TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development[END_REF].

Another major process controling auxin distribution is auxin transport. It has been well

established that auxin moves directionally through plant tissues. From the sites of its synthesis it is transported to the whole plant (reviewed in [START_REF] Peer | Seven things we think we know about auxin transport[END_REF]. A long distance source-tosink transport occurs by the loading of auxin into the phloem, from young biosynthetically active shoot tissues towards sink tissues (Figure 4a). Cell-to cell transport can also achieve auxin movement over both short and long distances. Cell-to-cell transport was predicted by the chemiosmotic model, based in the physicochemical properties of the auxin molecules [START_REF] Goldsmith | The Polar Transport of Auxin[END_REF][START_REF] Raven | TRANSPORT OF INDOLEACETIC ACID IN PLANT CELLS IN RELATION TO pH AND ELECTRICAL POTENTIAL GRADIENTS, AND ITS SIGNIFICANCE FOR POLAR IAA[END_REF][START_REF] Rubery | Carrier-mediated auxin transport[END_REF]. Auxins are weak acids, and their ability to penetrate through the membrane, depends on the pH. The plant's apoplastic pH is approximately 5.5. Under this conditions it is predicted that only a small but signficant fraction (17%) of auxin molecules are proton-associated (HA). Although protonated auxin freely diffuses from the apoplast into the cytoplasm, 83% of the auxin pool remains unavailable for diffusion in its dissociated form. Once in the cytoplasm where pH is approximately 7, the equlibrium of the auxin shifts to the anionic, dissociated form. In this circumstances auxins cannot diffuse across the cell membrane, hence the active transport of auxin is required. Indeed, three main families of transmembrane proteins provide means of active auxin transport in and out of the cell, across the plasma membrane: i) the AUX1/LIKE AUX1 (AUX1/LAX) auxin influx permeases, ii) the P-glycoproteins of the ATP-Binding Cassette family B (ABCB/PGP) efflux transporters, and iii) the PIN-FORMED (PIN) auxin efflux carriers reviewed in [START_REF] Zazimalova | Auxin transporters--why so many?[END_REF] (Figure 5b). Among them, mainly PINmediated transport seems to contribute to polar auxin transport (PAT), which is essential for defining differential auxin distribution [START_REF] Weijers | Maintenance of embryonic auxin distribution for apical-basal patterning by PIN-FORMED-dependent auxin transport in Arabidopsis[END_REF].

PINs are plant specific proteins with a predicted secondary structure of five transmembrane helices at the N and C terminus [START_REF] Galweiler | Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue[END_REF][START_REF] Krecek | The PIN-FORMED (PIN) protein family of auxin transporters[END_REF][START_REF] Paponov | The PIN auxin efflux facilitators: evolutionary and functional perspectives[END_REF] linked by an intracellular hydrophilic loop that influences protein localization and activity [START_REF] Bennett | Paralogous radiations of PIN proteins with multiple origins of noncanonical PIN structure[END_REF][START_REF] Dhonukshe | Plasma membrane-bound AGC3 kinases phosphorylate PIN auxin carriers at TPRXS(N/S) motifs to direct apical PIN recycling[END_REF][START_REF] Huang | Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport[END_REF]. Arabidopsis PIN family consists of eight members, PIN1-8. PIN1, 2, 3, 4 and 7 localize preferentially in the plasma membrane [START_REF] Adamowski | PIN-dependent auxin transport: action, regulation, and evolution[END_REF][START_REF] Petrasek | PIN proteins perform a rate-limiting function in cellular auxin efflux[END_REF][START_REF] Wisniewska | Polar PIN localization directs auxin flow in plants[END_REF]; PIN5 and 8 localization has been reported at the endoplasmic reticulum [START_REF] Dal Bosco | The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis[END_REF][START_REF] Ding | ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis[END_REF][START_REF] Mravec | Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter[END_REF]; whereas there are still doubts regarding the localization and function of PIN6 [START_REF] Nisar | The promoter of the Arabidopsis PIN6 auxin transporter enabled strong expression in the vasculature of roots, leaves, floral stems and reproductive organs[END_REF]. Plasma membrane localized PINs often display polar cellular localization, which notably correlates with the directional flow of auxin and therefore have also been used to deduce such fluxes [START_REF] Benkova | Local, efflux-dependent auxin gradients as a common module for plant organ formation[END_REF][START_REF] De Reuille | Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis[END_REF]Friml et al., 2002a;[START_REF] Galweiler | Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue[END_REF][START_REF] Wisniewska | Polar PIN localization directs auxin flow in plants[END_REF]. The auxin transport ability of PINs has been shown in Arabidopsis and heterologous systems [START_REF] Petrasek | PIN proteins perform a rate-limiting function in cellular auxin efflux[END_REF][START_REF] Yang | Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe[END_REF][START_REF] Zourelidou | Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID[END_REF]. The positioning of PINs is highly dynamic. It is crucial to the production of organs during development [START_REF] Blilou | The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots[END_REF][START_REF] Friml | Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis[END_REF][START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF] and to the modulation of patterns of growth and development in response to the environment, for example in gravitropism [START_REF] Friml | Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis[END_REF]. After their transcription, PINs are either retained in the endoplasmic reticulum (ER) or translocated through the Golgi Apparatus (GA) to the plasma membrane [START_REF] Matheson | Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond[END_REF]. PINs undergo continuous shuttling between the plasma membrane and the intracellular compartments by rounds of internalization (endocytosis) and polar recycling (exocytosis). These processes together are known as consitutive endocytic cycling [START_REF] Dhonukshe | Plasma membrane-bound AGC3 kinases phosphorylate PIN auxin carriers at TPRXS(N/S) motifs to direct apical PIN recycling[END_REF][START_REF] Dhonukshe | Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions[END_REF][START_REF] Geldner | Auxin transport inhibitors block PIN1 cycling and vesicle trafficking[END_REF][START_REF] Kleine-Vehn | Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane[END_REF]. According to current models, the regulation of PIN trafficking is based on their phosphorylation status, determined by the action of the PINOID (PID) and other AGC3

kinases and the antagonistic action of PP2A/PP6 phosphatases (Figure 5b) [START_REF] Benjamins | The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport[END_REF][START_REF] Michniewicz | Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux[END_REF]. Unphosphorylated PINs are recycled to the plasma membrane by the ADP-ribosylation factor-guanine nucleotide exchange factor (ARF-GEF)

GNOM. Phosphorylated PINs result in GNOM-independent recycling to the opposite plasma membrane [START_REF] Dhonukshe | Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis[END_REF][START_REF] Geldner | Auxin transport inhibitors block PIN1 cycling and vesicle trafficking[END_REF][START_REF] Kleine-Vehn | ARF GEF-dependent transcytosis and polar delivery of PIN auxin carriers in Arabidopsis[END_REF].

Monoubiquitination of PINs induces their endocytosis, which occurs via clathrin-coated vesicles and requires the actin cytoskeleton. Subsequently, polyubiquitination labels PIN proteins for degradation [START_REF] Leitner | Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots[END_REF]. The post-translational modifications of PINs, ubiquitination and phosphorylation, provide an entry point for various external signals, for example gravity [START_REF] Abas | Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism[END_REF][START_REF] Friml | Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis[END_REF] or light [START_REF] Ding | Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis[END_REF][START_REF] Michniewicz | Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux[END_REF][START_REF] Willige | D6PK AGCVIII kinases are required for auxin transport and phototropic hypocotyl bending in Arabidopsis[END_REF]. Thereby the abundance of PINs in the plasma membrane or their polarity can be modified in reponse to external signals. Several hormones (auxin included) may influence directly or indirectly the transcription of PINs (Bishopp et al., 2011a;[START_REF] Dello Ioio | A genetic framework for the control of cell division and differentiation in the root meristem[END_REF][START_REF] Hacham | BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers[END_REF][START_REF] Liu | Interaction of PLS and PIN and hormonal crosstalk in Arabidopsis root development[END_REF][START_REF] Zhang | Type-A response regulators are required for proper root apical meristem function through post-transcriptional regulation of PIN auxin efflux carriers[END_REF] or influence their abundance [START_REF] Crawford | Strigolactones enhance competition between shoot branches by dampening auxin transport[END_REF][START_REF] Hacham | BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers[END_REF][START_REF] Willige | Gibberellin regulates PIN-FORMED abundance and is required for auxin transport-dependent growth and development in Arabidopsis thaliana[END_REF].

Experimental evidence partially combined with modelling approaches suggests that auxin itself provides feedback regulation on its own distribution influencing transcription, turnover, and plasma membrane localization of PIN proteins [START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF][START_REF] Stoma | Flux-based transport enhancement as a plausible unifying mechanism for auxin transport in meristem development[END_REF].

Besides production and transport, auxin perception and downstream signaling have also been extensively studied in a range of developmental processes. Auxin is first perceived via one or more receptors that initiate a signaling cascade that translates the auxin concentration into diverse cellular behaviors. Mainly two auxin receptor systems have been described, involving respectively the TRANSPORT INHIBITOR RESISTANT1/AUXIN SIGNALING F-BOX (TIR1/AFB) complexes (Dharmasiri et al., 2005a;Dharmasiri et al., 2005b;[START_REF] Kepinski | The Arabidopsis F-box protein TIR1 is an auxin receptor[END_REF] and AUXIN BINDING PROTEIN 1 (ABP1) [START_REF] Woo | Crystal structure of auxin-binding protein 1 in complex with auxin[END_REF] (Figure 5c).

From these, the best characterized is the TIR1/AFB pathway that regulates auxin responses within the nucleus [START_REF] Villalobos | A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin[END_REF][START_REF] Chapman | Mechanism of auxin-regulated gene expression in plants[END_REF]Dharmasiri et al., 2005a;[START_REF] Kepinski | The Arabidopsis F-box protein TIR1 is an auxin receptor[END_REF][START_REF] Parry | Complex regulation of the TIR1/AFB family of auxin receptors[END_REF] (Figure 6a). At low auxin concentrations the transcriptional repressors AUXIN/INDOLE ACETIC ACID (Aux/IAAs) negatively regulate auxin signalling. Aux/IAAs carry out their repressor activity by binding to the DNA-binding AUXIN RESPONSE FACTOR (ARF) proteins [START_REF] Guilfoyle | Auxin response factors[END_REF][START_REF] Kim | Involvement of soluble proteinous factors in auxininduced modulation of P-type ATPase in rice (Oryza sativa L.) seedlings[END_REF], and recruiting the transcriptional co-repressors of the TOPLESS (TPL) family to ARF-bound promoters [START_REF] Ke | Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors[END_REF][START_REF] Szemenyei | TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis[END_REF]. This prevents the ARF mediated transcription of auxin responsive-genes (Figure 6a). At high auxin concentrations, auxin interacts with TIR1 or other AFBs (Dharmasiri et al., 2005a). The binding of auxin occurs within an internal pocket formed from the binding between the F-box protein and the Aux/IAA, forming a complex TIR1/AFB-auxin-Aux/IAA that targets Aux/IAA for ubiquitination and degradation via the 26S proteasome (Figure 6a) (Calderon [START_REF] Villalobos | A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin[END_REF]. Once Aux/IAA are degraded, ARF proteins can either activate or repress auxin responsive genes [START_REF] Chandler | Auxin response factors[END_REF]. More than 50 genes encoding ARF and Aux/IAA have been identified in the Arabidopis genome [START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF]. Differential expression of each of these players provides combinatorial possibilities for auxin-dependent gene regulation [START_REF] Chapman | Mechanism of auxin-regulated gene expression in plants[END_REF]. Large-scale analyses of the AUX/IAA-ARF network have been performed in order to try to understand the distribution and perception of auxin signaling; for instance, in the shoot apex of Arabidopsis [START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF].

Through a combination of expression data, a set of molecular interactions, mathematical modelling and auxin signaling sensors, [START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF] have described a key role for local auxin signaling in the regulation of the shoot apex patterning.

Notably auxin functional specificity can be generated at different levels. For instance, at the level of protein-DNA interactions, by the presence of cis regulatory elements in auxinresponsive genes. At the level of chromatin-level it has been shown that ARF5/MP transcriptional regulation requires chromatin state changes target loci. Aux/IAA repressors of auxin signalling together with co-repressors of the TPLs family and the repressive chromatin regulator histone deacetylase HDA19, prevent the expression of ARF5/MP regulated genes [START_REF] Long | TOPLESS regulates apical embryonic fate in Arabidopsis[END_REF][START_REF] Szemenyei | TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis[END_REF]. Local accumulation of auxin, drives Aux/IAA degradation, as well as the dissociation of TPL and HDA19. This in turns leaves ARF5/MP free to recruit the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatinremodeling complexes, SPLAYED (SYD) or BRAHMA (BRM). SWI/SNF complex unlock the repressed chromatin state at ARF5/MP target loci, which also increases chromatin accessibility for additional transcription factors. In contrast, in the absence of auxin, Aux/IAAs promote chromatin closure by recruiting TPL transcriptional co-repressors to ARFbound promoters [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF].

ABP1 was the first auxin-binding protein described in the literature. Its binding capacity was demonstrated by physiological and structural studies [START_REF] Hesse | Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin[END_REF][START_REF] Woo | Crystal structure of auxin-binding protein 1 in complex with auxin[END_REF].

ABP1 localizes mainly to the ER, but a small portion is likely secreted to the cell wall where it is assumed to be active [START_REF] Sauer | AUXIN BINDING PROTEIN1: the outsider[END_REF]. Described physiological responses linked to ABP1 are initiated on the outside of the plasma membrane, this requires that the signal is passed into the cell. This role is supposedly accomplished by plasma membranelocalized proteins TRANSMEMBRANE KINASE (TMK) or SPIKE (SPK1) [START_REF] Lin | A ROP GTPase-dependent auxin signaling pathway regulates the subcellular distribution of PIN2 in Arabidopsis roots[END_REF][START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF], which were reported to transfer auxin signal inside the cell via the RHO OF PLANTS (ROP)-GTPases and the ROP INTERACTIVE CRIB motif-containing (RIC)

proteins. ROP-RIC systems regulate endocytosis/exocytosis of PIN proteins on the plasma membrane, thus controlling auxin fluxes. In leaf pavement cells, for example, two ROP-RIC downstream pathways have been described [START_REF] Fu | Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF]. The ROP2-RIC4 pathway acts in the lobe outgrowth through the stabilization of cortical actin microfilaments and further inhibition of PIN1 endocytosis [START_REF] Nagawa | ROP GTPase-dependent actin microfilaments promote PIN1 polarization by localized inhibition of clathrin-dependent endocytosis[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF]. The ROP6-RIC1 pathway inhibits the indentation outgrowth by the activation of the microtubule severing protein KATANIN (KTN1), which promotes the bundling of cortical microtubules in the necks, and further inhibits PIN1 and PIN2 endocytosis [START_REF] Chen | ABP1 and ROP6 GTPase signaling regulate clathrin-mediated endocytosis in Arabidopsis roots[END_REF][START_REF] Fu | A ROP GTPase signaling pathway controls cortical microtubule ordering and cell expansion in Arabidopsis[END_REF][START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF]. In the root, ABP1 was reported to control cell cycle entry by regulating the D-type CYCLIN/RETINOBLASTOMA pathway and the PLETHORA (PLT) gradients [START_REF] Tromas | The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth[END_REF]. Although a number of non-transcriptional responses mediated by ABP1 have been reported (Chen et al., 2012a;[START_REF] Chen | Pavement cells: a model system for non-transcriptional auxin signalling and crosstalks[END_REF][START_REF] Chen | ABP1 and ROP6 GTPase signaling regulate clathrin-mediated endocytosis in Arabidopsis roots[END_REF][START_REF] Lin | A ROP GTPase-dependent auxin signaling pathway regulates the subcellular distribution of PIN2 in Arabidopsis roots[END_REF][START_REF] Nagawa | ROP GTPase-dependent actin microfilaments promote PIN1 polarization by localized inhibition of clathrin-dependent endocytosis[END_REF][START_REF] Wu | RAC/ROP GTPases and auxin signaling[END_REF][START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF] the function of ABP1 as an auxin receptor has remained unclear. The recent isolation of two Arabidopsis abp1 mutants with no obvious phenotypes raised strong questions [START_REF] Gao | Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development[END_REF]. Although ABP1 inactivation by inducible antibody-and antisense-based lines present strong phenotypes not caused by ABP1 down-regulation, which might suggest redundancy [START_REF] Michalko | Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein[END_REF].

Cytokinins

Cytokinins are known for their ability to promote cytokinesis, hence their name. Cytokinins are adenine derivatives carrying either an isoprene-derived or an aromatic side chain [START_REF] Mok | Cytokinin Metabolism and Action[END_REF]. Great diversity exists among the predominant CKs between plant species [START_REF] Sakakibara | Interactions between nitrogen and cytokinin in the regulation of metabolism and development[END_REF]. Major derivatives present in Arabidopsis are trans-zeatin (tZ), and isopentenyladenine (iP) types. The initial step of CK synthesis is catalyzed by isopentenyltransferases (IPTs), which use ADP, ATP and tRNA as isoprenoid acceptors [START_REF] Kakimoto | Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases[END_REF][START_REF] Takei | Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana[END_REF]. In higher plants two pathways for the synthesis of tZ coexist. The iP nucleotide-dependent pathway catalyzed by the cytochrome P450 monooxygenases CYP735A and the nucleotide-independent pathway, catalysed by the CK riboside 5'-monophosphate phosphoribohydrolase, LONELY GUY (LOG) [START_REF] Kurakawa | Direct control of shoot meristem activity by a cytokininactivating enzyme[END_REF][START_REF] Kuroha | Functional analyses of LONELY GUY cytokininactivating enzymes reveal the importance of the direct activation pathway in Arabidopsis[END_REF][START_REF] Tokunaga | Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOGdependent pathway in cytokinin activation[END_REF][START_REF] Zurcher | Cytokinin Synthesis, Signaling, and Function--Advances and New Insights[END_REF] Additionally steady-state levels of active CKs are determined by the rate of conjugation and degradation.

Active CKs, the free bases, are modified into ribosides and ribotides by O-glycosylation.

Modified cytokinins can be activated when needed and seem to be the major long-range transport forms in plants [START_REF] Zurcher | Cytokinin Synthesis, Signaling, and Function--Advances and New Insights[END_REF]. CYTOKININ DEHYDROGENASE/OXIDASE (CKX) proteins catalyse the degradation of CKs; they act by cleaving the CKs side chains. Genes encoding cytokinin degradation and synthesis proteins are widely expressed and active both in the shoot and the root [START_REF] Nordstrom | Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin-cytokinin-regulated development[END_REF]. Regulation over the synthesis of CKs depends on the differential expression of the basic elements of its metabolism IPTs, CKX and CYP735A. CKs homeostasis is fine-tuned by other hormones and external factors, such as nitrogen availability [START_REF] Sakakibara | Interactions between nitrogen and cytokinin in the regulation of metabolism and development[END_REF].

The signaling of CKs initiates by its perception by the membrane-bound Arabidopsis HISTIDINE KINASE (AHK) proteins, which serve as CKs receptors [START_REF] Heyl | Properties, functions and evolution of cytokinin receptors[END_REF].

Binding of cytokinin to AHK proteins triggers a phosphorelay, in which a phosphoryl group is transferred from a His residue into an Asp residue within the kinase domain of the receptor.

Afterwards, the phosphoryl is transmitted to a His residue of an Arabidopsis HISTIDINE PHOSPHOTRANSFERASE (AHP) protein. The previous is true for AHP1-5. AHP6 differs, since it cannot accept an activated phosphoryl group, which makes it unable to perform the phosphorelay [START_REF] Mahonen | Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development[END_REF]. The role of AHP6 is, however, important, since it performs an inhibiting role over cytokinin signaling by competing with the "true" AHPs and contributes to confine the CKs signaling domains [START_REF] Besnard | The AHP6 cytokinin signaling inhibitor mediates an auxin-cytokinin crosstalk that regulates the timing of organ initiation at the shoot apical meristem[END_REF][START_REF] Bishopp | Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem[END_REF]. AHP1-5 proteins continuously translocate to the nucleus enabling the phosphorylation of Arabidopsis RESPONSE REGULATOR (ARR) proteins. According to Cterminal differences, the family of ARRs has been classified into type -A -B and C [START_REF] D'agostino | Characterization of the response of the Arabidopsis response regulator gene family to cytokinin[END_REF]. Type-B ARRs have a transcription factor domain for DNA binding, once phosphorylated type-B ARRs as DNA-binding transcription factors activate transcription of cytokinin-regulated genes [START_REF] Kiba | Differential expression of genes for response regulators in response to cytokinins and nitrate in Arabidopsis thaliana[END_REF][START_REF] Mason | Type-B response regulators display overlapping expression patterns in Arabidopsis[END_REF]. Type-A ARRs lack the transcription factor domain and instead act as negative regulators of cytokinin by attenuating the signal [START_REF] Brandstatter | Two genes with similarity to bacterial response regulators are rapidly and specifically induced by cytokinin in Arabidopsis[END_REF][START_REF] D'agostino | Characterization of the response of the Arabidopsis response regulator gene family to cytokinin[END_REF][START_REF] Rashotte | Expression profiling of cytokinin action in Arabidopsis[END_REF]. Transcription of type-A ARRs is under the direct regulation of type-B ARRs (Figure 6b). Type-C ARRs are less characterized, although they might have roles as modulators, since their ectopic expression affects cytokinin signaling [START_REF] Kiba | Arabidopsis response regulator, ARR22, ectopic expression of which results in phenotypes similar to the wol cytokininreceptor mutant[END_REF].

Sites of cytokinin synthesis do not necessarily coincide with the sites of perception, suggesting the transport of cytokinins [START_REF] Zurcher | A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta[END_REF]. Long distance transport of tZtype cytokinins occurs from the root to the shoot via the xylem, whereas iP-type cytokinins move through the phloem from the shoot to the root [START_REF] Bishopp | Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem[END_REF]. Cell-to-cell transports of cytokinins seem to be mediated by the PURINE PERMEASE (PUP) proteins [START_REF] Burkle | Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis[END_REF].

The components involved in cytokinin biosynthesis, degradation and phosporelay signaling, are encoded by multigene families [START_REF] Muller | Advances in cytokinin signaling[END_REF]. The diverse but specific expression patterns of these components, suggest a broad range of cytokinin functions.

Physiological functions of cytokinins include male and female gametophyte development, root and shoot apical meristem maintenance and development, as well as vasculature development and nodule organogenesis [START_REF] Zurcher | Cytokinin Synthesis, Signaling, and Function--Advances and New Insights[END_REF].

Brassinosteroids

Brassinosteroids (BRs) are steroidal hormones that play a major role in promoting cell expansion and proliferation [START_REF] Hardtke | Phytohormone collaboration: zooming in on auxin-brassinosteroid interactions[END_REF][START_REF] Nakaya | Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana[END_REF]. Brassinolide (BL) is the most biologically active BR among more than 50 natural BRs [START_REF] Fujioka | Biosynthesis and metabolism of brassinosteroids[END_REF].

The biosynthesis of BRs involves parallel and highly branched pathways [START_REF] Fujioka | Biosynthesis and metabolism of brassinosteroids[END_REF]. The initial step is the formation of campestanol (CN) from campesterol (CR).

For this step, two branches have been proposed, the early C-22 oxidation, and the late C-22 oxidation. In Arabidopsis the early C-22 oxidation appears to be the major BR biosynthetic pathway [START_REF] Fujita | Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols[END_REF]. Two main pathways have been identified for the biosynthesis of BL from campestanol, the early and late C-6 oxidation pathways [START_REF] Ohnishi | Insights into the function and evolution of P450s in plant steroid metabolism[END_REF]. In the early C-6 oxidation pathway, C-6 oxidation occurs ahead of C-22 hydroxylation. While in the late C-6 oxidation pathway, C-22 hydroxylation takes places before C-6 oxidation [START_REF] Chung | The Regulation of Brassinosteroid Biosynthesis in Arabisopsis[END_REF]. A number of the genes relevant for brassinosteroids biosynthesis or signaling have been cloned taking advantage of BR-deficient mutants. Features such as dwarfism, dark-green and curled leaves, reduced fertility and delayed senescence, are characteristic of these mutants. When grown in the dark, such mutants are de-etiolated with

short hypocotyls and open cotyledons [START_REF] Clouse | A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development[END_REF][START_REF] Li | A role for brassinosteroids in light-dependent development of Arabidopsis[END_REF][START_REF] Szekeres | Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis[END_REF].

Two different enzymes can perfom the initial modification of CR. The cytochrome P450 monooxygenase (CYP90B1) DWARF4 (DWF4) [START_REF] Choe | The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alphahydroxylation steps in brassinosteroid biosynthesis[END_REF]; and the cytochrome P450 monooxygenase (CYP90A1) CONSTITUTIVE PHOTOMORPHOGENIC AND DWARF (CPD) [START_REF] Szekeres | Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis[END_REF]. DWF4 acts as a C-22-hydroxylase, whereas CPD functions as a C-3 dehydrogenase [START_REF] Ohnishi | CYP90A1/CPD, a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis, catalyzes C-3 oxidation[END_REF]. Depending on the availability of substrates and enzymes, the biosynthesis of BL progresses via either DWF4-or CPDmediated pathways. Interestingly, both DWF4 and CPD can act on multiple substrates, constituting multiple biosynthetic parallel pathways. Nevertheless, the overall flux of the BR biosynthetic pathway seems to be determined by the activity of DWF4 [START_REF] Chung | The Regulation of Brassinosteroid Biosynthesis in Arabisopsis[END_REF]. The expression of BR-biosynthetic genes is primarily regulated at the transcriptional level. Interestingly the expression of several biosynthetic genes is subject to feedback regulation from the BRs signaling pathway [START_REF] Bancos | Regulation of transcript levels of the Arabidopsis cytochrome p450 genes involved in brassinosteroid biosynthesis[END_REF][START_REF] He | BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses[END_REF][START_REF] Mathur | Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids[END_REF].

BRs are widely distributed throughout plant tissues, although the active forms seem to accumulate mostly in young growing regions undergoing active cell division and elongation.

BRs do not seem to undergo long-distance transport; in contrast they appear to be synthesized and function in the same tissue or even the same cell [START_REF] Bishop | The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family[END_REF][START_REF] Shimada | Organ-specific expression of brassinosteroid-biosynthetic genes and distribution of endogenous brassinosteroids in Arabidopsis[END_REF][START_REF] Symons | Brassinosteroids, de-etiolation and the re-emerging art of plant hormone quantification[END_REF]. BRs synthesis seems to take place in the endoplasmic reticulum, while its perception is located at the exterior cell surface. Thus movement of BRs is more likely to occur within and between neighbouring cells [START_REF] Symons | Brassinosteroids, de-etiolation and the re-emerging art of plant hormone quantification[END_REF].

BRs receptors have been described in Arabidopsis as plasma membrane localized leucine-rich repeat receptor kinases (LRR), BRASSINOSTEROID INSENSITIVE1 (BRI1) and its two homologues BRL1 and BRL3 [START_REF] Li | A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction[END_REF]. The kinase activity of BRI1 is activated following the binding of BR [START_REF] Kinoshita | Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1[END_REF][START_REF] Wang | BRI1 is a critical component of a plasma-membrane receptor for plant steroids[END_REF]. Upon perception of BRs by BRI1, the inhibitory protein BRI1 KINASE INHIBITOR (BKI1) is phosphorylated and dissociated from BRI1 [START_REF] Jaillais | Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor[END_REF][START_REF] Wang | Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane[END_REF]. BRI1 is then free to interact with BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [START_REF] Nam | BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling[END_REF], leading to the autophosphorylation and transphosphorylation between the kinase domains of BRI1 and BAK1 [START_REF] Wang | Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling[END_REF]. Activated BRI1 is able to phosphorylate BRASSINOSTEROID-SIGNALING KINASE 1(BSK1) and CONSTITUTIVE DIFFERENTIAL GROWTH1 (CDG1) kinases [START_REF] Kim | The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2[END_REF][START_REF] Tang | BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis[END_REF].

Subsequently, phosphorylated BSK1 and CDG1 bind and phosphorylate BRI1-SUPPRESSORS1 (BSU1) phosphatase [START_REF] Kim | The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2[END_REF][START_REF] Kim | Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors[END_REF]. Phosphorylated BSU1 inactivates by dephosphorylation the GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) [START_REF] Kim | Brassinosteroid signal transduction from receptor kinases to transcription factors[END_REF]. Activated BIN2 phosphorylates transcription factors BRASSINAZOLE RESISTANT1 (BZR1) and BZR2 (hereafter called BRI1-EMS-SUPRESSOR1 (BES1)) [START_REF] Wang | Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis[END_REF][START_REF] Yin | BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation[END_REF]. Phosphorylated BZR1 and BES1 are retained in the cytoplasm via the activity of 14-3-3 proteins [START_REF] Gampala | An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis[END_REF][START_REF] Vert | Downstream nuclear events in brassinosteroid signalling[END_REF]. Thus, under high BR levels, inactivation of BIN2 by BSU1 leads to the dephosphorylation and activation of BZR1 and BES1 [START_REF] Tang | PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1[END_REF].

Unphosphorylated BZR1 and BES1 are free to move into the nucleus and bind the promoter of their target genes [START_REF] He | BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses[END_REF][START_REF] Sun | Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis[END_REF][START_REF] Yin | A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis[END_REF][START_REF] Yu | A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana[END_REF]. The activation of BR signaling requires histone modifications and additional interacting transcription factors, among them, BES1 INTERACTING MYC-LIKE1 (BIM1) and

INTERACT WITH SPT6 1 (IWS1) (Figure 7) [START_REF] Li | Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression[END_REF][START_REF] Yin | A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis[END_REF][START_REF] Yu | Modulation of brassinosteroidregulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis[END_REF].

A number of target genes of BZR1 and BES1 have been identified, revealing diverse molecular links. Noteworthy, the activity of BZR1 is responsible for the transcriptional regulation of BR biosynthetic genes, such as DWF4 and CPD [START_REF] Kim | The regulation of DWARF4 expression is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis[END_REF][START_REF] Wang | Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis[END_REF]. When activated, BZR1 binds to the BR-responsive elements of the promoter sequence of DWF4 and CPD and repress their transcription [START_REF] He | BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses[END_REF]. BES1 might also repress DWF4 and CPD transcription to attenuate BR responses in a feedback loop, but this mechanism is primarily dependent on the repression and de-repression of transcription by BZR1 [START_REF] Yu | A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana[END_REF]. Additional targets of BRs signaling are related with cell wall modification [START_REF] Xie | Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis[END_REF] and cellular transport, in agreement with BRs effects on cell expansion and growth. Not surprisingly, BR signaling converges substantially with other hormonal and environmental signals, such as light and GA signaling [START_REF] Guo | Mechanisms and networks for brassinosteroid regulated gene expression[END_REF]Zhu et al., 2013). Of special relevance for this thesis are the interactions with auxin signaling, which I will examine next.

Hormonal crosstalk. The case of auxin and brassinosteroids

Multiple hormones are at play during plant growth and development. Their specific functions, however, are sometimes difficult to define, in particular because extensive crosstalk and signaling integration among growth regulating hormones has been demonstrated [START_REF] Nemhauser | Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses[END_REF]. For instance, cell proliferation is regulated by cytokinins and auxin, while, cell expansion is under the control of auxin, BRs and gibberellins. More recently, a role of BRs in cell proliferation has been identified [START_REF] Hardtke | Phytohormone collaboration: zooming in on auxin-brassinosteroid interactions[END_REF][START_REF] Nakaya | Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana[END_REF]. Antagonistic relationships between cytokinin and auxin have also been described in much detail. This relationship seems to keep a balance between cell proliferation and differentiation, especially during embryogenesis and during shoot and root meristem development [START_REF] Barkoulas | From genes to shape: regulatory interactions in leaf development[END_REF][START_REF] Dinneny | Plant stem cell niches: standing the test of time[END_REF][START_REF] Muller | Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis[END_REF]. Here I will focus on auxin and BRs, which modulate cell expansion and proliferation and because of their relevance for this thesis. In the following paragraphs I will describe some of the evidence that suggests molecular interactions between auxin and BRs signaling pathways.

Physiological assays of cell elongation have provided the first evidence of the interaction between auxin and BRs. In these assays segments of hypocotyls were promoted to elongate by the application of auxin or BRs. The asymmetrical application of auxin or BRs both triggered bending of the root or hypocotyl, mimicking the tropic response normally achieved by the local accumulation of auxin. This similarity in the responses triggered by both hormones, i.e. elongation by directional expansion as well as certain tropic responses, suggested interaction between the two hormone pathways. [START_REF] Clouse | Physiological and molecular effects of brassinosteroids on Arabidopsis thaliana[END_REF][START_REF] Clouse | BRASSINOSTEROIDS: Essential Regulators of Plant Growth and Development[END_REF][START_REF] Zurek | Investigation of Gene Expression, Growth Kinetics, and Wall Extensibility during Brassinosteroid-Regulated Stem Elongation[END_REF].

The close relationship between auxin and BRs very likely reflects several levels of crossregulation. One possibility is that auxin and BRs control cell elongation through different cellular mechanisms. In this case the signaling pathways and mechanisms used by each hormone can be independent from each other and the interaction is at the level of the physical properties of the system. This can happen without any interaction between the hormone signaling pathways. In addition, interference between the two hormone signaling pathways [START_REF] Mundy | Crosstalk[END_REF]) can be at the biosynthesis level, the components of the signaling pathways might interact, or the signaling pathways might share components [START_REF] Hardtke | Transcriptional auxin-brassinosteroid crosstalk: who's talking?[END_REF].

In the first case, it has been found that among the targets of BZR1 and BES1 there are genes involved in auxin biosynthesis, transport and signaling [START_REF] Bao | Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis[END_REF][START_REF] Kim | Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA[END_REF][START_REF] Nakamura | Brassinosteroid selectively regulates PIN gene expression in Arabidopsis[END_REF][START_REF] Sun | Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis[END_REF][START_REF] Yu | A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana[END_REF]. In turn, there is evidence indicating that auxin regulates BR biosynthesis [START_REF] Chung | Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis[END_REF][START_REF] Scacchi | Dynamic, auxin-responsive plasma membrane-tonucleus movement of Arabidopsis BRX[END_REF] and signaling [START_REF] Sakamoto | Auxin signal transcription factor regulates expression of the brassinosteroid receptor gene in rice[END_REF].

Nevertheless, the hypothesis which has more support is that auxin and BR pathways converge at the level of common target genes [START_REF] Hardtke | Phytohormone collaboration: zooming in on auxin-brassinosteroid interactions[END_REF][START_REF] Nemhauser | Interdependency of brassinosteroid and auxin signaling in Arabidopsis[END_REF].

Transcriptomic studies identified significant overlap of genes that respond to external application of auxin with genes that respond to external application of BR [START_REF] Goda | Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis[END_REF][START_REF] Goda | Microarray analysis of brassinosteroid-regulated genes in Arabidopsis[END_REF][START_REF] Mussig | Brassinosteroid-regulated gene expression[END_REF][START_REF] Nemhauser | Interdependency of brassinosteroid and auxin signaling in Arabidopsis[END_REF][START_REF] Yin | BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation[END_REF]. Notably, genes repressed by auxin are usually repressed by BRs, while auxin-induced genes are also BR-induced, indicating that the two hormone pathways affect gene expression in a coordinated manner [START_REF] Hardtke | Transcriptional auxin-brassinosteroid crosstalk: who's talking?[END_REF]. Many of these genes are synergistically induced by the simultaneous application of these hormones [START_REF] Chung | Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis[END_REF][START_REF] Goda | Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis[END_REF][START_REF] Mouchel | BRX mediates feedback between brassinosteroid levels and auxin signalling in root growth[END_REF][START_REF] Nemhauser | Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses[END_REF][START_REF] Vert | Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2[END_REF]. Moreover, expression changes in response to auxin require intact BR biosynthetic and signaling pathway and vice versa [START_REF] Hardtke | Transcriptional auxin-brassinosteroid crosstalk: who's talking?[END_REF][START_REF] Nakamura | Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type[END_REF]. Although, no direct interactions between ARFs and BES1/BZR1 have been demonstrated, it has been suggested that auxin and BR responses are mediated by a combination of specific cis-regulatory elements [START_REF] Walcher | Bipartite promoter element required for auxin response[END_REF]. For instance, a Hormone Up at Dawn (HUD)-type E-box combined with a nearby auxin-responsive element variant has been identified as a target for BES1 and MP.

Interestingly, their binding can be enhanced by treatment with either hormone [START_REF] Chandler | BIM1, a bHLH protein involved in brassinosteroid signalling, controls Arabidopsis embryonic patterning via interaction with DORNROSCHEN and DORNROSCHEN-LIKE[END_REF]. BES1 has been shown to interact with IWS1 protein which promotes transcriptional elongation [START_REF] Li | Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression[END_REF]. This could suggest a model where BES1 boosts the response to auxin [START_REF] Walcher | Bipartite promoter element required for auxin response[END_REF].

Both auxin and BR modulate cell expansion and proliferation, therefore ultimately their regulation must feed into cellular effectors of growth. In plant cells, growth results from the irreversible plastic yielding of the cell wall to the internal turgor pressure. Thus, the structural elements of the cell wall as well as the underlying cytoskeleton might be targets of auxin-BR regulation. Nevertheless, evidence in this regard is scarce. At least partially, BR signaling seems to affect the rearrangement of the cortical microtubules, just as auxin signaling does [START_REF] Catterou | Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant[END_REF][START_REF] Takahashi | The DIMINUTO gene of Arabidopsis is involved in regulating cell elongation[END_REF]. In addition, both auxin and BR also induce the expression of genes encoding for cell wall associated proteins [START_REF] Yin | BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation[END_REF].

In order to have a clearer idea of how growth could be regulated, we need to take a closer look at the cell wall. In the next section I will cover this aspect and present the structural components of the cell wall that make growth possible.

The cell wall in plant development and morphogenesis

In most prokaryotes, algae, fungi and plants, cells are enclosed by a stiff extracellular matrix or cell wall. Plant cell walls fulfil a wide range of biological roles; they provide support, act as defensive barrier, as conduits for information and as source of signaling molecules and developmental cues. Cell walls can be quite diverse in their composition, depending on the developmental stage, the cell type, and the plant species. Most cell walls are viscoelastic fibre composites based on a load-bearing network, infiltrated with matrix polymers.

Primary cell walls, the type of wall surrounding cells that are dividing and/or expanding, are laid down during cytokinesis. They are mainly constituted by cellulose microfibrils embedded and cross-linked into a viscous matrix of pectin and hemicellulose chains (Figure 8a).

Although growing cells have relatively thin but flexible walls (less than 1µm), they are able to resist the extremely high turgor pressure that pushes on the plasma membrane. Turgor pressure can reach up to 1 MPa [START_REF] Beauzamy | Mechanically, the Shoot Apical Meristem of Arabidopsis Behaves like a Shell Inflated by a Pressure of About 1 MPa[END_REF], but as long as it does not exceed a certain threshold, coined yielding threshold, it only leads to a reversible elastic deformation of the cell wall. [START_REF] Cosgrove | Growth of the plant cell wall[END_REF]Wolf et al., 2012a). Growth occurs when the turgor pressure exceeds the yielding threshold causing the matrix elements to break and the wall to expand in a non reversible manner (plastic deformation) [START_REF] Ali | Physical models of plant development[END_REF]. This plastic deformation is in principle accompanied by cell wall synthesis.

Since plant cells usually do not move relative to one another growth patterns are entirely defined by local cell expansion. The rate of cell wall expansion can be equivalent in all directions of the cell, in which case expansion is considered isotropic. On the contrary, when the rate of cell wall expansion in one direction differs from the rate in other directions, expansion is anisotropic. It is the integration of the local expansions which will allow the plant to reach its particular size and shape [START_REF] Baskin | Anisotropic expansion of the plant cell wall[END_REF].

Although, plant cell growth depends on both turgor pressure and cell wall mechanics, I will mostly consider the role of cell wall througout this work. Cell wall expansion implies a constant modification of the cell wall properties, for instance, through wall loosening followed by synthesis and insertion of new wall materials. To better understand plant cell growth, we will need to take a look at the composition of the primary cell wall and the roles that these elements play during cell expansion.

Cellulose microfibrils as cell wall load-bearing elements

Cellulose is a paracrystalline polysaccharide, whose primary structures are unbranched β1, 4linked glucan chains, synthesized in parallel at the cell surface by CELLULOSE SYNTHASE A (CESA) COMPLEXES (CSC) [START_REF] Schneider | Cellulose and callose synthesis and organization in focus, what's new?[END_REF]. In Arabidopsis, the CESA gene family has 10 members [START_REF] Richmond | The cellulose synthase superfamily[END_REF]. CESA1, CESA3, and CESA6 are preferentially expressed in expanding tissues [START_REF] Desprez | Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana[END_REF][START_REF] Doblin | Cellulose biosynthesis in plants: from genes to rosettes[END_REF], while CESA4, CESA7, and CESA8 have proven roles in the secondary cell wall thickening in xylem [START_REF] Scheible | Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants[END_REF][START_REF] Taylor | Interactions among three distinct CesA proteins essential for cellulose synthesis[END_REF][START_REF] Taylor | Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis[END_REF]. The remaining CESA2, CESA5, CESA9, and CESA10 genes are poorly understood but might have certain redundancy with other CESAs [START_REF] Desprez | Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana[END_REF][START_REF] Persson | Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis[END_REF]. According to the current model each CESA protein can synthesize one β1, 4-linked glucan chain. In Arabidopsis, the association of three CESA proteins into heterotrimeric CSC facilitates the interaction of dozens of these glucan chains in such a way that they associate to form fibrils of undefined length and shape (Figure 8b) [START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF][START_REF] Desprez | Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana[END_REF][START_REF] Persson | Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis[END_REF]. The regulation of CSC activity remains ill defined. However, phosphorylation might play a role in their motility and might also regulate their activation [START_REF] Sanchez-Rodriguez | BRASSINOSTEROID INSENSITIVE2 negatively regulates cellulose synthesis in Arabidopsis by phosphorylating cellulose synthase 1[END_REF].

Cellulose microfibrils provide tensile strength to the wall and regulate the degree of expansion. They are usually oriented transversely to the growth axis of elongation, which has been considered to underlie the anisotropy of expansion [START_REF] Bashline | Cell wall, cytoskeleton, and cell expansion in higher plants[END_REF][START_REF] Baskin | On the role of stress anisotropy in the growth of stems[END_REF][START_REF] Green | Mechanism for Plant Cellular Morphogenesis[END_REF][START_REF] Ivakov | Plant cell shape: modulators and measurements[END_REF]. Remarkably, a good amount of evidence indicates that cellulose microfibrils align with cortical microtubules (CMT). It has been noticed that upon transport of the CSC to the cell surface, they are inserted in the plasma membrane close to the CMT [START_REF] Gutierrez | Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments[END_REF]. There, the CMT direct the track of CSC affecting the deposition of cellulose microfibrils (Figure 8a) [START_REF] Baskin | Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis[END_REF][START_REF] Chan | The rotation of cellulose synthase trajectories is microtubule dependent and influences the texture of epidermal cell walls in Arabidopsis hypocotyls[END_REF][START_REF] Emons | Microtubules and cellulose microfibrils: how intimate is their relationship?[END_REF][START_REF] Paredez | Visualization of cellulose synthase demonstrates functional association with microtubules[END_REF][START_REF] Sanchez-Rodriguez | Chitinaselike1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis[END_REF]. The most direct link between the synthesis machinery of cellulose microfibrils and microtubules is via the protein CELLULOSE SYNTHASE INTERACTING (CSI) 1/POM2 [START_REF] Bringmann | POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis[END_REF][START_REF] Gu | Identification of a cellulose synthaseassociated protein required for cellulose biosynthesis[END_REF][START_REF] Li | Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes[END_REF], although a number of additional proteins have been described (see [START_REF] Landrein | How mechanical stress controls microtubule behavior and morphogenesis in plants: history, experiments and revisited theories[END_REF]. Disruption of the CMT via microtubuledepolymerizing drugs, such as oryzalin alters the alignment of cellulose microfibrils [START_REF] Baskin | Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis[END_REF][START_REF] Corson | Turning a plant tissue into a living cell froth through isotropic growth[END_REF]. Notably, disruption of the synthesis of cellulose using the cellulose synthesis inhibitor isoxaben, results in disorganized CMTs in tobacco culture cells [START_REF] Fisher | Extending the Microtubule/Microfibril paradigm. Cellulose synthesis is required for normal cortical microtubule alignment in elongating cells[END_REF] and pollen tubes [START_REF] Lazzaro | Disruption of cellulose synthesis by isoxaben causes tip swelling and disorganizes cortical microtubules in elongating conifer pollen tubes[END_REF], suggesting the presence of a mutual feedback relationship between CMTs and cellulose microfibril deposition (Figure 8b). Thus, CMTs are crucial for growth anisotropy, since they orient the cellulose microfibrils. But how are CMT orientations controlled? A recent hypothesis proposes that CMT respond and align in the direction of maximal stress in the cell wall [START_REF] Landrein | How mechanical stress controls microtubule behavior and morphogenesis in plants: history, experiments and revisited theories[END_REF][START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. The force patterns, ultimately generated by the turgor pressure, depend on different factors, including the geometry of the cells and tissues as well as local differences in growth rate. Mechanical stress is of great relevance since it allows the cell to sense and adapt its mechanical status. This involves a feedback loop in which microtubules affect morphogenesis through oriented cellulose deposition and anisotropic growth, which in turn defines global mechanical stress patterns that influence microtubule orientation. I will come back to this topic later, after introducing the other elements of the cell wall, which also play a role in the expansion of the cell wall. The complexity of the cell wall, involving many different polysaccharides and hundreds of proteins exceeds the scope of this work. Thus the description of the cell wall will only be partial.

Hemicelluloses and their role in cell wall architecture

Hemicelluloses are a heterogeneous group of polysaccharides, initially defined as those cell wall polysaccharides that do not solubilize in water or chelating agents but in the presence of aqueous alkali. Under this definition, the term hemicelluloses would include xyloglucan, glucomannan, mannan, xylan, arabinoxylan and arabinogalactan. Alternatively hemicelluloses are defined chemically as cell wall polysaccharides structurally homologous to cellulose characterized by β-1-(1-4)linked backbones of sugars in equatorial configuration. This definition includes xyloglucans, glucomannans, mannans, xylans, arabinoxylans, but not arabinogalactans [START_REF] Scheller | Hemicelluloses[END_REF]. A number of enzymes involved in hemicelluloses biosynthesis have been identified [START_REF] Cantarel | The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics[END_REF].

Xyloglucans (XyG) are the most abundant hemicelluloses of primary cell walls of eudicots.

They are composed of β-(1-4)-glucan backbone substituted with α-(1-6)-xylosyl residues in a regular pattern, and occasional galactosyl or fucosyl residues (Figure 9a) [START_REF] Park | Xyloglucan and its interactions with other components of the growing cell wall[END_REF]. The β-(1-4)-glucan backbone of XyG is synthesized at the Golgi by glucan synthases members of the cellulose synthase-like (CSLC) family of proteins [START_REF] Cocuron | A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase[END_REF].

Most XyG have backbone substitutions, that can reach very complex branching patterns [START_REF] Fry | An unambiguous nomenclature for xyloglucan-derived oligosaccharides[END_REF]. Their biosynthesis involves the activity of a number of glycosyltransferases, including β-(1-4)-glucan synthase [START_REF] Cocuron | A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase[END_REF], α-fucoyltransferse [START_REF] Perrin | Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis[END_REF], β-galactosyltransferase [START_REF] Levy | Simulations of the static and dynamic molecular conformations of xyloglucan. The role of the fucosylated sidechain in surface-specific sidechain folding[END_REF][START_REF] Madson | The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins[END_REF], and αxylosyltransferses [START_REF] Faik | An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis[END_REF](Figure 9b). Arabidopsis contains seven genes encoding

XyG xylosyltransferases (XXT). Of these, xylosyltransferase activity has been demonstrated

for XXT1, XXT2 and XXT5 [START_REF] Cavalier | Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexaose[END_REF][START_REF] Faik | An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis[END_REF][START_REF] Zabotina | Xyloglucan and its biosynthesis[END_REF][START_REF] Zabotina | Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis[END_REF]). Yet, although the double Arabidopsis mutant xxt1 xxt2 has no detectable levels of XyG [START_REF] Cavalier | Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component[END_REF]Park and Cosgrove, 2012a), it has only a minor phenotype [START_REF] Cavalier | Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component[END_REF]Park and Cosgrove, 2012a;[START_REF] Xiao | Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis[END_REF]. Two possibilities were discussed by the authors to explain the absence of strong phenotypes in the complete lack of XyG. They suggest that an aberrant form of XyG might be present in the xxt1 xxt2 that could not be detected by their methods. They also suggest that the lack of XyG It has been suggested that the most important role of XyG was to coat cellulose microfibrils and tether them together, thus reinforcing the load-bearing properties of the cell wall.

(Scheller and Ulvskov 2010). However, our understanding about the nature of the interaction between cellulose and XyG is evolving and other models have been proposed. In contrast to the "tethered network" model, which suggests that most of the microfibrilar surface is coated with XyG (Figure 10a), nuclear magnetic resonance (NMR) analyses of Arabidopsis cell walls indicated that the interactions between XyG and cellulose are limited [START_REF] Dick-Perez | Structure and interactions of plant cell-wall polysaccharides by two-and three-dimensional magic-angle-spinning solid-state NMR[END_REF][START_REF] Wang | Pectin-cellulose interactions in the Arabidopsis primary cell wall from two-dimensional magic-angle-spinning solid-state nuclear magnetic resonance[END_REF]. Additional tests of the tethered network model based on biochemical changes induced by substrate-specific endoglucanases (Park and Cosgrove, 2012b) led to the hypothesis that the sites where cell walls are loosened are probably digested by an enzyme with both xyloglucanase and cellulase activity. This model, coined the "biomechanical hotspot model", proposes the existence of a limited number of cellulosecellulose junctions, which are attached together by XyG. These XyGs would keep the microfibrils together, prevent their aggregation and limit enzymatic accessibility to these biomechanical hotspots (Figure 10b) (Park and Cosgrove, 2012b). These sites which would be the location of cell wall loosening during growth could also be the targets of another set of wall loosening proteins named expansins, which will be discussed in the next paragraph. Expansins are pH-dependent wall-loosening proteins [START_REF] Mcqueen-Mason | Two endogenous proteins that induce cell wall extension in plants[END_REF].

Although, no clear enzymatic activity has been reported yet for expansins, a number of studies support their ability to induce selective wall loosening that enables irreversible extension, or "wall creep" and wall relaxation [START_REF] Cosgrove | The growing world of expansins[END_REF][START_REF] Mcqueen-Mason | Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding[END_REF]. Plant expansins are divided into four families [START_REF] Kende | Nomenclature for members of the expansin superfamily of genes and proteins[END_REF] of which α-expansins (EXPAs), and β-expansins (EXPBs) have different biological roles [START_REF] Cosgrove | Plant expansins: diversity and interactions with plant cell walls[END_REF][START_REF] Sampedro | The expansin superfamily[END_REF].

In addition to expansins, other enzymes (e.g. glycoside hydrolases (GH)) have been considered as wall loosening. Such is the case of the GH16 or xyloglucan endotransglucosylase/hydrolases (XTH), encoded by a large multigene family. After XTHs were discovered they were considered as wall-loosening enzymes, but with time, experiments showed they have limited ability to induce cell wall creep. This entailed to hypothesize that the effect of XTHs in cell loosening is minimal, but that they are likely involved in the remodelling an turnover of XyG during the formation of the primary wall and after cell elongation (Cosgrove, 2016a). Among the XTHs, the xyloglucan endotransglucosylases (XETs) seem to perform a wall-strengthening action by cutting XyG backbones and bind them to chains that are already part of the wall network [START_REF] Thompson | Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells[END_REF].

Another group of enzymes that might cause wall expansion are the GH9s, also called endoglucanases or cellulases. In plants, 11 distinct clades of GH9s have been reported, which seem to have roles in cell wall modification during diverse processes. One of the best studied GH9 is a membrane-associated endoglucanase named KORRIGAN, which is part of the synthesis machinery complex of cellulose microfibrils and therefore influences the organization of cellulose in the wall [START_REF] Vain | The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex[END_REF]. Despite the efforts it is not conclusive whether GH9 enzymes are able to directly loosen the cell wall.

Pectins and their role in cell wall expansion

Pectins, like hemicelluloses, are complex and heterogeneous polysaccharides. They are characterized by chains of galacturonic acid molecules linked at their 1 and 4 positions. There are three major types: homogalacturonans (HG), rhamnogalacturonans I (RG-I), and rhamnogalacturonans II (RG-II). Distinct combinations of these polysaccharide residues covalently bind and produce different pectin molecules [START_REF] Atmodjo | Evolving views of pectin biosynthesis[END_REF].

Pectin polysaccharides are produced in the Golgi and later on deposited in the cell wall. Given that specific enzymes are needed to catalyse the formation of each glycosidic linkage and modification, at least 67 transferases are required for the synthesis of pectins. These include, glycosyltransferases (GTs), methyltransferases (MTs) and acetyltransferases (ATs) [START_REF] Mohnen | Pectin structure and biosynthesis[END_REF]. Pectins possess the ability to bind to other pectins, as well as to cellulose and hemicellulose.

This allows the formation of structures with hydrogel characteristics. HGs have the ability to form multiple intermolecular bonds in the presence of calcium cations. Prior to crosslinking they should be 'activated' via the removal of the methyl group that protects them from the action of lyases. Demethyl-esterified pectin then acquires the capacity to create big aggregates known as 'egg boxes' (Figure 11). Specialized enzymes, pectin methyl-esterases (PMEs), are in charge of this process. In counteraction, PME-inhibitors (PMEIs) modulate the activity of PMEs. 66 PMEs and 69 PMEIs have been identified so far in the Arabidopsis genome [START_REF] Levesque-Tremblay | Tuning of pectin methylesterification: consequences for cell wall biomechanics and development[END_REF]. The PME/PMEI system is potentially very important, since the degree and pattern of demethyl-esterification might strongly influence biomechanical properties of the cell wall [START_REF] Ali | Force-Driven Polymerization and Turgor-Induced Wall Expansion[END_REF]Peaucelle et al., 2011a). Indeed, modifications in the level of PME or PMEI can strongly affect plant development (see also below).

Cellulose/xyloglucan and pectin act together during morphogenesis

Pectins, like hemicelluloses, interact with cellulose microfibrils to assemble the cell wall. This complex network allows for many potential sites where loosening and expansion can be initiated. But how are the assembly and rearrangements of these elements coordinated in order to produce cell expansion? As earlier mentioned, the expansion of plant cells results from the interaction between the turgor pressure inside the cells and the irreversible expansion of the viscoelastic wall that surround plant cells. Turgor pressure is the product of the osmotic force, which is equal in all directions or isotropic, whereas morphogenetic processes require anisotropic growth, which consequently relies on the cell wall properties. It is widely accepted, that the orientation of the cellulose microfibrils, controlled by the microtubules underlies anisotropic growth [START_REF] Baskin | On the role of stress anisotropy in the growth of stems[END_REF]. Recently it has been proposed that changes in pectin configurations can trigger anisotropic organogenesis even prior to changes in cellulose orientation. This has been reported for the epidermal cells of Arabidopsis hypocotyl, where AFM analyses indicated that the walls that preferentially expand during elongation (the longitudinal anticlinal walls) experience softening previous to the initiation of anisotropic elongation [START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF]. This led to suggest a two-step mechanism for anisotropic plant cell growth (Figure 12) [START_REF] Bidhendi | Relating the mechanics of the primary plant cell wall to morphogenesis[END_REF]. This might also be true for other tissues, such as the shoot apex, where a reduction in the elastic modulus detected by AFM precedes organogenesis [START_REF] Milani | In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy[END_REF]Peaucelle et al., 2011a).

Either way, cellulose microfibrils as well as hemicelluloses and pectins, interact and contribute altogether to control cell expansion [START_REF] Cosgrove | Re-constructing our models of cellulose and primary cell wall assembly[END_REF]. The integration of the local expansions of all the growing regions gives an organ its characteristic shape. How this orchestrated expansion is regulated during morphogenesis remains poorly understood. In the previous sections we have seen that a molecular network involving a set of hormones governs morphogenesis in plants. This network interferes with cell wall composition and structure which in turn defines local growth rates and directions, driven by turgor pressure.

In this thesis I have studied the link between molecular regulation and cell wall remodelling at the shoot apical meristem. In section 3 of this introduction I will therefore review a number of aspects concerning this structure.

Pattern formation and morphogenesis at the shoot apical meristem

The shoot apical meristem (SAM) located at the tip of the growing shoot contains a group of non-differentiated stem cells. These cells continuously generate organs at the meristem flanks while maintainig themselves via asymetrical cell divisions. The lateral organs emerge following a continuous and precise pattern, called phyllotaxis (Figure 13c). The type of organ generated from the SAM depends on the developmental phase. I will focus here on the SAM in Arabidopsis which, as a typical dicotylenous plant produces leaves and side branches during the vegetative phase and flower primordia during the reproductive phase.

Functional domains in the SAM of Arabidopsis are defined by specific histological features and gene expression profiles. In what follows, I will review the SAM functional domains and the molecular pathways that underlie them.

Shoot Apical Meristem set-up during the embryonic stage

During embryogenesis, a number of stereotypic cell divisions set up the basic polarity and patterning of the plant body plan. Through this process only a very rudimentary plant is formed, that displays an apical-basal axis of polarity and perpendicular to it, a radial pattern of concentric tissues. After the first two rounds of cell divisions, the determination of shoot and root domains is separated [START_REF] Haecker | Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana[END_REF]. At the globular stage the SAM is specified at the top of the axis of polarity [START_REF] Sarkar | Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers[END_REF]. Although the molecular organization characteristic of the vegetative SAM is not yet established at this stage, it develops gradually during embryogenesis [START_REF] Lau | Early embryogenesis in flowering plants: setting up the basic body pattern[END_REF][START_REF] Ten Hove | Building a plant: cell fate specification in the early Arabidopsis embryo[END_REF].

Shoot Apical Meristem functional domains

The SAM is a dome-shaped structure (Figure 13). At its summit, in the central zone (CZ), resides the pool of stem cells. Surrounding the CZ lays the peripheral zone (PZ), consisting of small cells that frequently divide but are still undifferentiated. Underneath the PZ, the rib zone (RZ) can be distinguished, characterized by a group of cells that mainly contribute to the formation of the central tissues of the shoot axis (Figure 13a). Superimposed to this zonation, a layered organisation can be distinguished. From outside to inside, the L1 and L2 layers are composed cells that generally divide perpendicular to the surface by this means forming two different sheets of clonally distinct tissue. The L1 gives preferentially rise to the epidermis, while the L2 largely contributes to the sub-epidermal tissues. The underlying L3 cells divide in apparently random directions and generate the internal tissues of lateral organs (Figure 13b).

The Central Zone and the maintenance of a group of undifferentiated stem cells

Optimal functioning of the SAM requires a balance between the indeterminate growth at its centre and the production of lateral organs at the flanks. Local maintenance of stem cells is provided by a well-described feedback regulation, orchestrated by the homeodomain transcription factor WUSCHEL (WUS). WUS expression is restricted to a small region of the SAM better known as the organizing centre (OC) located underneath the stem cells [START_REF] Baurle | Regulation of WUSCHEL transcription in the stem cell niche of the Arabidopsis shoot meristem[END_REF][START_REF] Laux | The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis[END_REF][START_REF] Mayer | Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem[END_REF]. The WUS protein migrates into the overlying cells, where it binds to the promoter of CLAVATA3 (CLV3) to positively regulate its expression [START_REF] Brand | Regulation of CLV3 expression by two homeobox genes in Arabidopsis[END_REF][START_REF] Daum | A mechanistic framework for noncell autonomous stem cell induction in Arabidopsis[END_REF][START_REF] Yadav | WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex[END_REF][START_REF] Yadav | WUSCHEL protein movement and stem cell homeostasis[END_REF]. CLV3 encodes a peptide that diffuses from the stem cells to the underlying cell layer, where it binds to the leucine-rich repeat-like kinase (RLK) CLV1 [START_REF] Clark | The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis[END_REF][START_REF] Ogawa | Arabidopsis CLV3 peptide directly binds CLV1 ectodomain[END_REF] or to a receptor complex formed by the receptor-like protein CLV2, and CORYNE, which is a serine/threonine kinase localized at the plasma membrane [START_REF] Jeong | The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase[END_REF]Muller et al., 2008). Through this interaction the CLV signalling components negatively regulate WUS expression, by this means restricting the size of the OC [START_REF] Mayer | Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem[END_REF][START_REF] Yadav | WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex[END_REF]. In turn, CLV3 signaling is also regulated by WUS, since it directly represses CLV1 [START_REF] Busch | Transcriptional control of a plant stem cell niche[END_REF]. This interaction establishes a negative feedback loop with the potential to dynamically adjust the size of the stem-cell population and the OC, thus contributing to meristem homeostasis (Figure 14a) [START_REF] Soyars | Ready, aim, shoot: stem cell regulation of the shoot apical meristem[END_REF]. Transcriptomic analysis of the Arabidopsis shoot meristem indicated that genes activated by WUS are specific to the stem cells, whereas those repressed are mostly expressed in differentiating cells of the PZ [START_REF] Busch | Transcriptional control of a plant stem cell niche[END_REF][START_REF] Yadav | Gene expression map of the Arabidopsis shoot apical meristem stem cell niche[END_REF][START_REF] Yadav | Plant stem cell maintenance involves direct transcriptional repression of differentiation program[END_REF]. More recently the transcription factor HECATE 1 (HEC1), which promotes stem cell proliferation, was described as negatively regulated by WUS. Absence of HEC1 in the OC is necessary for stem cell maintenance, since it can uncouple the WUS-CLV feedback turning stem cells into organizer cells [START_REF] Schuster | A regulatory framework for shoot stem cell control integrating metabolic, transcriptional, and phytohormone signals[END_REF].

Parallel to the WUS-CLV pathway, the class I KNOTTED1-like homeobox (KNOX) protein family performs key roles in the maintenance of stem cell homeostasis in Arabidopsis [START_REF] Endrizzi | The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE[END_REF][START_REF] Long | A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis[END_REF]. In Arabidopsis, this family includes SHOOT MERISTEM LESS (STM), Kn1-like in Arabidopsis thaliana 1(KNAT1)/BREVIPEDICELLUS (BP), KNAT2 and KNAT6, with partially overlapping expression patterns in the SAM [START_REF] Hay | KNOX genes: versatile regulators of plant development and diversity[END_REF]. STM expression can be distinguished all throughout the shoot meristem except for the organ primordia [START_REF] Long | A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis[END_REF]. STM prevents stem cell differentiation by suppressing the expression of organ-specific regulators in the CZ such as ASYMMETRIC LEAVES 1 (AS1) and AS2. In turn, the MYB transcription factor AS1 and the LBD transcription factor AS2 seem to create an AS1/AS2 complex that represses KNAT1 and KNAT2 in the lateral organ primordia [START_REF] Byrne | Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis[END_REF][START_REF] Lodha | The ASYMMETRIC LEAVES complex maintains repression of KNOX homeobox genes via direct recruitment of Polycombrepressive complex2[END_REF]. Additionally, BLADE ON PETIOLE (BOP1) and BOP2, which encode BTB/POZ domain proteins, negatively regulate KNAT1, KNAT2 and KNAT6. KNOX genes. Both WUS and STM regulate meristem formation independently from each other but in a complementary manner. WUS contributes non-cell-autonomously with stem cell specification at the meristem summit, at the CZ. STM prevents differentiation through the meristem dome including the CZ and the PZ and at the same time is required to promote cell division in the PZ [START_REF] Lenhard | The WUSCHEL and SHOOTMERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation[END_REF].

Hormonal balance contributes to the establishment and maintenance of the meristem domains (Figure 14a andb) [START_REF] Shani | The role of hormones in shoot apical meristem function[END_REF]. The synthesis and action of cytokinin is necessary for the maintenance of an undifferentiated pool of stem cells in the centre of the meristem. In

Arabidopsis the biosynthetic enzyme LOG4 is expressed in the epidermal layer of the shoot meristem, while the expression of the cytokinin receptor AHK2 and AHK4 is localized to the organizing centre [START_REF] Chickarmane | Cytokinin signaling as a positional cue for patterning the apical-basal axis of the growing Arabidopsis shoot meristem[END_REF]. WUS positively regulates cytokinin signalling by directly repressing transcription of the negative regulators, ARR7 and ARR15 [START_REF] Jasinski | KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities[END_REF][START_REF] Leibfried | WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators[END_REF]. In turn, cytokinin signaling directly activates WUS expression [START_REF] Zhang | Type-B ARABIDOPSIS RESPONSE REGULATORs Directly Activate WUSCHEL[END_REF], generating a positive feedback loop, which contributes to maintain and even re-establish the stem cell pool [START_REF] Adibi | Centering the Organizing Center in the Arabidopsis thaliana Shoot Apical Meristem by a Combination of Cytokinin Signaling and Self-Organization[END_REF][START_REF] Chickarmane | Cytokinin signaling as a positional cue for patterning the apical-basal axis of the growing Arabidopsis shoot meristem[END_REF][START_REF] Gordon | Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem[END_REF]. Moreover, this mechanism seems to be buffered by members of the ERECTA (ER) receptor kinase family that regulate cell homeostasis by preventing an excessive increase of cytokinin in the SAM [START_REF] Uchida | ERECTA-family receptor kinases regulate stem cell homeostasis via buffering its cytokinin responsiveness in the shoot apical meristem[END_REF]. In addition, STM activates cytokinin biosynthesis, via the positive regulation of IPT7 and represses gibberellin activities in the meristem [START_REF] Jasinski | KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities[END_REF][START_REF] Leibfried | WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators[END_REF]. I will discuss auxin and its crucial role in lateral organ initiation [START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF] in section 4.1.

In addition, two pathways of miRNAs have been implicated in maintaining the position of stem cells at the shoot apical meritem. First, miR394, produced at the L1, difusses downwardly and acts as a positive stem cell cue by maintaining the expression of CLV3 [START_REF] Knauer | A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem[END_REF]. The second miRNA pathway involves miR165/miR166, which targets mRNAs of CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III). miR165/166 are sequestered in the provasculature surrounding the meristem by ARGONAUTE 10 (AGO10) also known as ZWILLE (ZLL) [START_REF] Zhu | Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development[END_REF]. This allows the expression of HD-ZIP III genes in the meristem and promotes its maintenance [START_REF] Liu | The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis[END_REF]. Interestingly, this pathway seems to be relevant for shoot meristem maintenance only in the Arabidopsis accession Landsberg erecta (Ler), in which zll mutants display stem cell exhaustion [START_REF] Tucker | Accession-specific modifiers act with ZWILLE/ARGONAUTE10 to maintain shoot meristem stem cells during embryogenesis in Arabidopsis[END_REF].

The Peripheral Zone and the generation of organ primordia : the inflorescence meristem

The initiation of organ primordia occurs at the flanks of the SAM at a certain distance from the pool of stem cells in the PZ. In Arabidopsis one of the first requirements for organ initiation, either leaf or flower, is the down-regulation of STM gene expression in the organ founder cells [START_REF] Jackson | Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot[END_REF][START_REF] Long | Initiation of axillary and floral meristems in Arabidopsis[END_REF][START_REF] Long | A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis[END_REF]. This occurs before the primordium is recognizable as a bulge in the meristem, in the incipient primordium also known as I1/P0 ("P" refers to the plastochron stage, time interval between the initiation of two consecutive primordia [START_REF] Green | ON THE PHYSICAL BASIS OF CELL MORPHOGENESIS*[END_REF]). There are a number of processes in common with the vegetative meristem and the inflorescence meristem. Since the main focus of this thesis is on the morphogenesis in the inflorescence meristem, I will hereby focus on the generation of organ primordia in this meristem.

At the inflorescence meristem of Arabidopsis, the sites of incipient primordia and high auxin levels are coincident with the expression of ARF5/MONOPTEROS (MP) [START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF]. Downstream of MP in the incipient primordia, the transcriptional regulation of a group of targets has been identified (Figure 15a) [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF]. These include the LEAFY (LFY) transcription factor, which is necessary and sufficient for specification of floral identity (Liu et al., 2009a;[START_REF] Weigel | LEAFY controls floral meristem identity in Arabidopsis[END_REF] and two members of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, i.e. AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE 6/PLETHORA3 (AIL6/PLT3), which are expressed in all organ primordia throughout the plant [START_REF] Elliott | AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth[END_REF] and have critical roles in proliferative growth of the flower [START_REF] Krizek | Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs[END_REF][START_REF] Mizukami | Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis[END_REF]. ANT and AIL6 also contribute to the auxin dependent activation of LFY in parallel with MP [START_REF] Yamaguchi | AINTEGUMENTA and AINTEGUMENTA-LIKE6/PLETHORA3 Induce LEAFY Expression in Response to Auxin to Promote the Onset of Flower Formation in Arabidopsis[END_REF]. Additional targets of MP in the flower primordia include: AHP6, TARGET OF MONOPTEROS 3 (TMO3) and FILAMENTOUS FLOWER (FIL) [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF]. AHP6 encodes a negative regulator of cytokinin signaling [START_REF] Besnard | The AHP6 cytokinin signaling inhibitor mediates an auxin-cytokinin crosstalk that regulates the timing of organ initiation at the shoot apical meristem[END_REF], TMO3 was firstly described as a target of MP during embryo development [START_REF] Schlereth | MONOPTEROS controls embryonic root initiation by regulating a mobile transcription factor[END_REF] and encodes a cytokinin response factor (CRF) [START_REF] Rashotte | A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway[END_REF]; whereas FIL encodes a member of the YABBY family of transcription factors.

After the apical extension of the flower primordium (P1), once a cleft is form between the SAM and the primordium (P2), the polarity genes acquire differential adaxial/abaxial expression [START_REF] Husbands | Signals and prepatterns: new insights into organ polarity in plants[END_REF]. Although most of our knowledge on the acquisition of organ polarity is based on Arabidopsis leaf, it is clear that the mutually exclusive mechanisms dictating adaxial/abaxial polarity in floral primordia echoes those established for leaves. The acquisition of organ polarity is a gradual process and initially relies on positional information supplied from the meristem [START_REF] Kuhlemeier | The Sussex signal: insights into leaf dorsiventrality[END_REF]. Prior to organ initiation the HD-ZIP III genes, REVOLUTA (REV), PHABULOSA (PHB) and PHAVOLUTA (PHV), are preferentially expressed at the tip of the meristem. Upon primordium initiation, the expression of these genes extends into the incipient primordium [START_REF] Mcconnell | Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots[END_REF],

where they become restricted to the adaxial side by the post transcriptional regulation of mir166 [START_REF] Yao | Two types of cis-acting elements control the abaxial epidermis-specific transcription of the MIR165a and MIR166a genes[END_REF]. Upon organ initiation KANADI (KAN) proteins relocalize to the abaxial side of the lateral organ, where they restrict the expression AS2. Thus AS1-AS2 activity becomes adaxialized and in turn polarizes the expression of additional components [START_REF] Husbands | The ASYMMETRIC LEAVES Complex Employs Multiple Modes of Regulation to Affect Adaxial-Abaxial Patterning and Leaf Complexity[END_REF][START_REF] Lin | The Arabidopsis LATERAL ORGAN BOUNDARIES-domain gene ASYMMETRIC LEAVES2 functions in the repression of KNOX gene expression and in adaxial-abaxial patterning[END_REF]. In addition to the KAN genes, YABBY (YAB) genes including FIL, YAB2, YAB3 and YAB5 in Arabidopsis [START_REF] Husbands | Signals and prepatterns: new insights into organ polarity in plants[END_REF], promote abaxial polarity, along with ARF3 and ARF4 [START_REF] Eshed | Establishment of polarity in lateral organs of plants[END_REF][START_REF] Siegfried | Members of the YABBY gene family specify abaxial cell fate in Arabidopsis[END_REF]. The spatial restriction of some adaxial / abaxial determinants is regulated via small RNA pathways [START_REF] Husbands | Signals and prepatterns: new insights into organ polarity in plants[END_REF]. For instance, miR166, expressed at the abaxial side, promotes the cleavage of HD-ZIPIII mRNA, restricting the expression of these genes in the adaxial side;

while in opposition, tasiARFs expressed at the adaxial developing primordia moves abaxially to target ARF3 and ARF4 restricting the expression of these genes in the abaxial side (Figure 15b) [START_REF] Chitwood | Small RNAs are on the move[END_REF].

In Arabidopsis the demarcation of boundaries between cells expressing STM and those lacking its expression is a requirement for the initiation of organ primordia. These boundaries, which separate lateral organs from the adjacent meristem, are characterized by low cell division rates and specific patterns of gene expressions [START_REF] Aida | Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant[END_REF][START_REF] Breuil-Broyer | High-resolution boundary analysis during Arabidopsis thaliana flower development[END_REF][START_REF] Reddy | Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana[END_REF][START_REF] Takada | The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation[END_REF]. In addition to its regulating role in the meristem, STM functions in the specification of this boundary domain. In Arabidopsis the NAM-ATAF1/2-CUC2 (NAC) family of transcription factors, CUPSHAPED COTYLEDON 1 (CUC1), CUC2 and CUC3 control the formation of organ boundaries in the shoot [START_REF] Aida | Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant[END_REF][START_REF] Aida | Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes[END_REF][START_REF] Vroemen | The CUP-SHAPED COTYLEDON3 Gene Is Required for Boundary and Shoot Meristem Formation in Arabidopsis[END_REF]. STM and CUC regulate each other's expression all throughout development [START_REF] Hibara | Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation[END_REF][START_REF] Spinelli | A mechanistic link between STM and CUC1 during Arabidopsis development[END_REF][START_REF] Vroemen | The CUP-SHAPED COTYLEDON3 Gene Is Required for Boundary and Shoot Meristem Formation in Arabidopsis[END_REF]. Since the expression of CUC genes overlaps with the slowly dividing cells of the boundaries, it has been thought that CUC proteins might repress growth and inhibit differentiation in the boundary regions [START_REF] Aida | Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant[END_REF][START_REF] Breuil-Broyer | High-resolution boundary analysis during Arabidopsis thaliana flower development[END_REF][START_REF] Souer | The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries[END_REF][START_REF] Takeda | CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells[END_REF]. Downstream targets of CUC proteins include LIGHT-DEPENDENT SHORT HYPOCOTYL 4 (LSH4) and LSH3, which are directly activated by CUCs and supress organ inititation in the boundary [START_REF] Takeda | CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells[END_REF]. Additional described regulators of boundary formation include certain members of the family of transcription factors LATERAL ORGAN BOUNDARIES DOMAIN (LBD) [START_REF] Shuai | The lateral organ boundaries gene defines a novel, plant-specific gene family[END_REF], for instance, JAGGED LATERAL ORGANS (JLO). Its expression is firstly detected at the sites of organ initiation, where JLO seems to act as positive regulator of the expression of STM and BP [START_REF] Borghi | Arabidopsis JAGGED LATERAL ORGANS is expressed in boundaries and coordinates KNOX and PIN activity[END_REF], thereby promoting the exit of these cells from a meristematic state. Once organs are established, the expression of JLO gets restricted to the boundary [START_REF] Rast | Arabidopsis JAGGED LATERAL ORGANS acts with ASYMMETRIC LEAVES2 to coordinate KNOX and PIN expression in shoot and root meristems[END_REF] [START_REF] Bell | Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries[END_REF]. As already discussed, brassinosteroids have proven functions in cell expansion and proliferation, hence low brassinosteroid activity in the boundary likely reduces cell expansion and division. In addition, the brassinosteroid transcription factor BZR1 directly represses the expression of CUC genes [START_REF] Gendron | Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis[END_REF], thus low brassinosteroid activity in the boundary zone also allows the induction of CUC genes (Figure 14b).

In addition to the regulatory gene network involved in the establishment of the boundary, a characteristic feature of boundary zones is low auxin concentrations and signaling. In contrast, organ initiation is closely correlated with the accumulation of auxin. Contrary to the meristem, the incipient primordium has a high auxin to cytokinin ratio, high levels of giberellic acid and likely high brassinosteroid activity [START_REF] Tsuda | Genome-wide study of KNOX regulatory network reveals brassinosteroid catabolic genes important for shoot meristem function in rice[END_REF]. Auxin depletion from the boundary and accumulation in the incipient primordia induces organ initiation by restricting the expression of CUC and KNOX genes [START_REF] Furutani | PIN-FORMED1 and PINOID regulate boundary formation and cotyledon development in Arabidopsis embryogenesis[END_REF][START_REF] Vernoux | PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem[END_REF]. I will further discuss the role of auxin as positive regulator of organ primordia in the inflorescence meristem in the next section.

Flower initiation as model system to understand plant morphogenesis

The SAM initially gives rise to vegetative organs, but at some point the SAM makes the transition to reproductive development. The transition from the vegetative phase to the reproductive phase, called the floral transition, is marked by the conversion of the shoot apical meristem into an inflorescence meristem. This event is characterized by several physiological changes; the elongation of the stem, the acceleration of cell division, and the formation of flowers in the flanks of the shoot apex [START_REF] Kwiatkowska | Flower primordium formation at the Arabidopsis shoot apex: quantitative analysis of surface geometry and growth[END_REF][START_REF] Steeves | Patterns in Plant Development[END_REF].

The floral transition is the culmination of a complex interaction of multiple environmental and endogenous inputs. Ultimately, these inputs promote the activation of a group of genes termed floral pathway integrators. In Arabidopsis the main integrator is LEAFY (LFY), which not only confers flower mersitem identity, but also plays a central role in controlling the identity of individual floral organs [START_REF] Parcy | A genetic framework for floral patterning[END_REF]. This gene is already weakly expressed during the vegetative phase in leaf primordia and a dramatic increase in its expression is necessary for the floral transition [START_REF] Blazquez | LEAFY expression and flower initiation in Arabidopsis[END_REF][START_REF] Hempel | Floral determination and expression of floral regulatory genes in Arabidopsis[END_REF][START_REF] Li | LEAFY controls auxin response pathways in floral primordium formation[END_REF].

Two genes termed flowering time genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) also known as AGAMOUS-LIKE 20 (AGL20) are also strongly implicated in the transition from producing leaves to the production of flowers.

Flower primordia start as new meristems, which in turn will form the successive floral organs, often arranged in concentric whorls. The characteristic features that underlie the initiation of flower primordia at the flanks of the inflorescence meristem, will be described in this section paying special attention to the role of auxin and the cell wall.

The molecular regulation of flower initiation: a central role for auxin

There is strong evidence coming from work on several plant species, that auxin is concentrated at specific locations at the meristem periphery, where it induces organ initiation [START_REF] Bayer | Integration of transport-based models for phyllotaxis and midvein formation[END_REF][START_REF] De Reuille | Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis[END_REF][START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF][START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF][START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF]. The accumulation of auxin is possible through the activity of PIN membrane transporters, which often show polar localization. Since neighbouring cells frequently show a coherent distribution of these proteins, they can generate fluxes of auxin through the tissues, causing the formation of auxin maxima at certain places and auxin depletion at others [START_REF] Murray | Systems analysis of shoot apical meristem growth and development: integrating hormonal and mechanical signaling[END_REF][START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF][START_REF] Sassi | Auxin and self-organization at the shoot apical meristem[END_REF]. Via immunological studies the localization of PIN1, the founding member of the family of PIN efflux carriers, has been determined in the surface layers of the SAM [START_REF] Benkova | Local, efflux-dependent auxin gradients as a common module for plant organ formation[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF]. Cell membranes carrying PIN1 in the epidermis seem to be preferentially oriented toward the incipient primordia, coinciding with the sites of high intracellular auxin [START_REF] Benkova | Local, efflux-dependent auxin gradients as a common module for plant organ formation[END_REF][START_REF] Brunoud | A novel sensor to map auxin response and distribution at high spatio-temporal resolution[END_REF][START_REF] De Reuille | Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis[END_REF][START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF][START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF]. Subsequently, upon bulging of the organ primordium, the polarity of PIN1 reverses at the boundary and is then directed towards the center of the SAM. Since both the organ and meristem itself continue to act as auxin sinks, a low auxin domain is created that corresponds to the boundary region [START_REF] Wang | Auxin Depletion from the Leaf Axil Conditions Competence for Axillary Meristem Formation in Arabidopsis and Tomato[END_REF]. As discussed above, it was proposed that these low auxin levels permit the activation of the CUC genes.

The precise cellular mechanisms by which the coordination of auxin transport between cells occurs is not entirely understood, but two general classes of theoretical models have been proposed to explain the patterns at the meristem surface: flux-based and concentration-based models [START_REF] Van Berkel | Polar auxin transport: models and mechanisms[END_REF]. Flux based-models were the first to be proposed and are based on the hypothesis that cells experiencing the flux of auxin in a certain direction will increase their capacity to transport the hormone in that direction [START_REF] Rolland-Lagan | Reviewing models of auxin canalization in the context of leaf vein pattern formation in Arabidopsis[END_REF][START_REF] Sachs | The Control of the Patterned Differentiation of Vascular Tissues[END_REF]. In this type of models small auxin fluxes through the membrane may be amplified by the polar localization of PINs, which point in the direction of the original flux [START_REF] Alim | Quantitative predictions on auxin-induced polar distribution of PIN proteins during vein formation in leaves[END_REF][START_REF] Feugier | Self-organization of the vascular system in plant leaves: inter-dependent dynamics of auxin flux and carrier proteins[END_REF]. Concentration-based models suggest that cells are able to sense the concentration of auxin and direct its transport towards the neighbouring cell with the highest auxin level [START_REF] Jonsson | An auxin-driven polarized transport model for phyllotaxis[END_REF][START_REF] Smith | A plausible model of phyllotaxis[END_REF].

Both models can explain the patterns of PIN polarity at the meristem surface, but only the flux based model is capable of reproducing also the behaviour of PIN induced fluxes in the internal tissues [START_REF] Bayer | Integration of transport-based models for phyllotaxis and midvein formation[END_REF][START_REF] Merks | Canalization without flux sensors: a traveling-wave hypothesis[END_REF][START_REF] Stoma | Flux-based transport enhancement as a plausible unifying mechanism for auxin transport in meristem development[END_REF]. Nevertheless, it remains unclear which precise cellular mechanism directs PIN allocation in the SAM.

The presence of PIN1 is essential for the transport of auxin in the meristem. Reduction of auxin transport has been reported in the loss-of-function mutant pin-formed 1 (pin1) [START_REF] Bennett | Morphogenesis in pinoid mutants of Arabidopsis thaliana[END_REF][START_REF] Okada | Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation[END_REF]. The pin1 phenotype is striking, with most of the effects observed at the level of flower initiation. Impaired auxin transport in the inflorescence results in the formation of naked inflorescence stems, which are unable to initiate flower primordia [START_REF] Okada | Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation[END_REF][START_REF] Vernoux | PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem[END_REF]. In a similar manner, impairment of auxin transport by polar auxin transport inhibitors, such as N-1-naphthylphthalamic acid (NPA) and 2, 3, 5triiodobenzoic acid (TIBA), results in defects in the formation of lateral primordia in the shoot apex [START_REF] Grandjean | In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis[END_REF][START_REF] Okada | Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation[END_REF]. Notably, the local application of auxin on the naked inflorescence apex of pin1 mutants and NPA treated plants restores the initiation of lateral primordia [START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF].

The transient accumulation of auxin feeds into a complex network of molecular regulators, which has been relatively well characterized. As already mentioned, the core machinery of auxin-dependent gene regulation comprises the ARFs and their binding partners the Aux/IAA.

At the inflorescence meristem of Arabidopsis, the sites of incipient primordia and high auxin levels are coincident with the expression of ARF5/ MP and a range of other ARFs [START_REF] Vernoux | The auxin signalling network translates dynamic input into robust patterning at the shoot apex[END_REF]. MP is very strongly expressed, and like pin mutants, monopteros (mp) mutants, are unable to initiate flower primordia [START_REF] Hardtke | The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development[END_REF][START_REF] Odat | Characterization of an allelic series in the MONOPTEROS gene of Arabidopsis[END_REF][START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF]. The naked meristem of mp are unresponsive to externally applied auxin [START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF], which indicates that auxin signaling largely triggers flower primordium initiation via ARF5/MP [START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF].

In the incipient primordia auxin activated MP acts at several levels. As mentioned in the previous section, MP induces the expression of a number of direct targets ultimately leading to the initiation of flower primordium (Figure 15a). Cell division and growth is promoted via the activation of expression of ANT and AIL6. The peripheral fate and the abaxial polarity is promoted via the activation of FIL. Finally, floral identity is promoted via the activation of LFY [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF]. In addition, MP induces the expression of AHP6, which encodes a negative regulator of cytokinin signaling that diffuses to neighbouring initium to prevent the initiation of new floral primordia [START_REF] Besnard | The AHP6 cytokinin signaling inhibitor mediates an auxin-cytokinin crosstalk that regulates the timing of organ initiation at the shoot apical meristem[END_REF].

MP also represses in the central region of the shoot apex two negative regulators of cytokinins, ARR7 and ARR15 [START_REF] Zhao | Hormonal control of the shoot stem-cell niche[END_REF], and in the incipient primordia MP induces the expression of the cytokinin response factor (CRF) TMO3/CRF2 [START_REF] Rashotte | A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway[END_REF], thus promoting cytokinin responses in incipient flower primordia. Additional feedbacks and feed-forward loops come into action during flower primordium initiation. For instance, ANT and AIL6 both induce LFY, and LFY in turn reinforces auxin transport [START_REF] Li | LEAFY controls auxin response pathways in floral primordium formation[END_REF][START_REF] Yamaguchi | AINTEGUMENTA and AINTEGUMENTA-LIKE6/PLETHORA3 Induce LEAFY Expression in Response to Auxin to Promote the Onset of Flower Formation in Arabidopsis[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF][START_REF] Yamaguchi | LEAFY and Polar Auxin Transport Coordinately Regulate Arabidopsis Flower Development[END_REF]. AIL/PLT genes contribute to control the transport and synthesis of auxin [START_REF] Pinon | Local auxin biosynthesis regulation by PLETHORA transcription factors controls phyllotaxis in Arabidopsis[END_REF][START_REF] Prasad | Arabidopsis PLETHORA transcription factors control phyllotaxis[END_REF]. While MP orients non-cell autonomously the polarity of PIN1, thereby facilitating a positive feedback loop [START_REF] Bhatia | Auxin Acts through MONOPTEROS to Regulate Plant Cell Polarity and Pattern Phyllotaxis[END_REF].

Once established and after an initial growth phase, the flower primordium acquires meristematic features. This includes the expression of WUS and CLV3 and the establishment of an organizing center and a stem cell niche (Figure 16a) [START_REF] Gruel | An epidermis-driven mechanism positions and scales stem cell niches in plants[END_REF]. The identity of the floral primordia relies on the activity of LFY, which later on also plays a role in controlling the identity of individual floral organs [START_REF] Parcy | A genetic framework for floral patterning[END_REF]. In the flower meristem, LFY positively regulates the expression of the MADS-domain transcription factors APETALA1 (AP1) and its homolog CAULIFLOWER (CAL) [START_REF] Kempin | Molecular basis of the cauliflower phenotype in Arabidopsis[END_REF]. AP1

together with SHORT VEGETATIVE PHASE (SVP) and AGL24 MADS-domain transcription factor specify floral identity and maintain the flower meristem in a meristematic state during the early stages of the flower [START_REF] De Folter | Comprehensive interaction map of the Arabidopsis MADS Box transcription factors[END_REF]. After establishment of the floral meristem, the patterning of the flower is activated with the upregulation of the floral homeotic genes (reviewed in [START_REF] O'maoileidigh | Gene networks controlling Arabidopsis thaliana flower development[END_REF][START_REF] Wils | Gene-regulatory networks controlling inflorescence and flower development in Arabidopsis thaliana[END_REF].

Typically, angiosperm flowers are characterized by sterile perianth organs in the outer whorls, followed by reproductive organs in the inner whorls. According to the classical ABC model, floral organ identities are specified by functional gene classes [START_REF] Causier | Floral organ identity: 20 years of ABCs[END_REF][START_REF] Haughn | Genetic control of morphogenesis in Arabidopsis[END_REF]. In Arabidopsis, sepal identity specification is mediated by A-class protein AP1 combined with E-class proteins SEPALLATA1-4. Petal identity is defined by AP1 together with the B-class proteins AP3, PISTILLATA (PI), and SEP proteins. Stamens are specified by the C-class protein AGAMOUS (AG) with AP3 and PI, while carpels are specified by AG and SEP complexes [START_REF] Honma | Complexes of MADS-box proteins are sufficient to convert leaves into floral organs[END_REF]) (Figure 16a).

As we have already seen, MP signalling activates diverse developmental processes during flower primordium inititation. It directs the specification of floral fate, cell proliferation and growth, the establishment of organ polarity, and cytokinin responses. Nevertheless, is not clear how the activation of these transcriptional regulators influence the physical events behind the shape changes that lead to the emergence of the flower primordia. In the following section I will address this issue including some putative targets of these transcription factors.

The physical regulation of flower initiation: a central role for the cell wall

As we have seen, the mechanochemical state of the cell wall is a fundamental link between molecular growth regulation and the effective shape evolution of the tissue (Figure 16b).

Therefore to control morphogenesis, the above mentioned genetic network controlling flower primordium initiation should somehow modulate the synthesis or remodelling of the cell wall.

In the two following subsections I will give an overview of the state of the art on this topic at the onset of my thesis.

Control of growth anisotropy the dialog between mechanical forces and the cytoskeleton

Several studies have focused on the regulation and role of wall anisotropy during organ initiation [START_REF] Baskin | Anisotropic expansion of the plant cell wall[END_REF][START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. As the microfibrils themselves cannot be visualized directly in vivo, these studies looked at microtubule dynamics, which are guiding cellulose deposition [START_REF] Bringmann | POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis[END_REF][START_REF] Gutierrez | Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments[END_REF][START_REF] Li | Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes[END_REF][START_REF] Paredez | Visualization of cellulose synthase demonstrates functional association with microtubules[END_REF]. An important feature of the growing meristem is that the outer (surface) wall seems to be loadbearing [START_REF] Beauzamy | Mechanically, the Shoot Apical Meristem of Arabidopsis Behaves like a Shell Inflated by a Pressure of About 1 MPa[END_REF][START_REF] Kierzkowski | Elastic domains regulate growth and organogenesis in the plant shoot apical meristem[END_REF][START_REF] Milani | In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy[END_REF][START_REF] Savaldi-Goldstein | The epidermis both drives and restricts plant shoot growth[END_REF]. This hypothesis is supported by the fact that this wall is much thicker than the inner walls (typically 200-250 nm versus 80-120 nm in the Arabidopsis inflorescence according to our own unpublished results). This feature also represents an important advantage for experimentation, as the outer cells are easy to visualize in vivo using standard confocal microscopy.

Careful analysis of microtubule dynamics at the meristem, have revealed stereotypic behaviour and supracellular arrangements of the CMT arrays (Figure 17a) [START_REF] Burian | A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem[END_REF][START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF][START_REF] Sampathkumar | Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells[END_REF][START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. At the tip of the SAM microtubules are highly dynamic, showing isotropic arrangements. Towards the flanks of the meristematic dome and along the stem, microtubules show more anisotropic arrangements, perpendicular to the apical-basal axis, in particular along organ boundaries [START_REF] Burian | A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem[END_REF][START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. How microtubule orientation is coordinated at the supra-cellular level is not understood. However, as the microtubules seem to align along the predicted force fields at the meristem surface, it was proposed that the cells are somehow able to sense these forces and to reorganize their cytoskeleton and reinforce their walls accordingly (Figure 17b) [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF][START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. Would such a mechanism be sufficient to generate the morphodynamics seen in vivo? To address this and other questions, several authors have turned to mechanical models in the form of virtual tissues [START_REF] Ali | Force-Driven Polymerization and Turgor-Induced Wall Expansion[END_REF]. Mechanical models where a feedback between mechanical forces and the cytoskeleton were simulated were able to reproduce typical morphological structures, such as cylindrical stems or an outgrowing primordium [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. It is important to note that this is a typical example of self-organization: cells locally react to a mechanical signal, which more globally leads to particular shapes of the entire cell population. Later studies have further supported the hypothesis, that a feedback between mechanics and microtubules might play an important role in morphogenesis [START_REF] Hervieux | A Mechanical Feedback Restricts Sepal Growth and Shape in Arabidopsis[END_REF][START_REF] Sampathkumar | Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells[END_REF].

Control of growth rate: cell wall remodeling proteins

As indicated above, the transcriptional regulators act in principle not only on wall anisotropy but also on wall synthesis rate and stiffness. Many of the known cell wall-modifying proteins target the matrix molecules in which the cellulose microfibrils are embedded. As discussed earlier, these can be roughly divided in three subcategories, (i) pectin modifying enzymes such as pectin methyl esterases (PMEs) or pectin methyl estherase inhibitors (PMEIs), (ii) enzymes targeting the hemicellulose chains such as xyloglucan endotransglycosylase/hydrolases (XTHs) and (iii) expansins which possibly target hydrogen bond between hemicelluloses (Wolf et al., 2012a). Little is known about the precise molecular regulation of these cell-wall modifying proteins, but many of them can be found among the published potential targets of many transcription factors [START_REF] Krizek | RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways[END_REF]Peaucelle et al., 2011a;[START_REF] Yadav | Gene expression map of the Arabidopsis shoot apical meristem stem cell niche[END_REF].

What is the precise function of these wall modifying proteins? Whereas many mutants perturbed in wall synthesis have been described, very few of them have been characterized at the level of the SAM. Nevertheless, experiments where the activities of some of these modifying enzymes are manipulated show their potential importance in organ formation at the shoot apex. For instance, local applications of expansins on SAMs of tomato induce the formation of ectopic leaf-like organs [START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF]. More recently, the possible roles of PMEs and PMEIs in organ formation at the inflorescence meristem of Arabidopsis have been explored (Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]Peaucelle et al., 2011b). These enzymes control the stiffness of the pectin matrix. Antibodies recognizing specific modifications in pectins chains indicate that specific meristematic zones, in particular organ boundaries, are likely to have a stiffer pectin matrix. Interestingly, overproduction of PMEI completely inhibits organ formation at the shoot apex, whereas local applications of PME induces extra flowers.

These experiments point to a scenario where matrix molecules are stiffened or loosened at sites where growth has to be respectively inhibited as in organ boundaries or increased as in organ primordia. Several attempts have been made to test this hypothesis using atomic force microscopy (AFM) to measure the local stiffness of the walls. Depending on the thickness of the AFM probes used, different results were obtained. Thicker probes (1-5 µm) indicated that the inner walls became more elastic at the moment of organ initiation. However, when very small probes (10 nm) were used to measure only the supposedly limiting outer wall, relatively minor differences were observed, not exceeding a reduction of 20-50 % in stiffness in the very young initium (Peaucelle et al., 2011a;[START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

Concluding remarks and Objectives of the thesis

As we have seen the production of patterns, relies on multiple factors: the physical properties and mechanical forces of the system [START_REF] Thompson | On Growth and Form[END_REF], its intrinsic system of chemical substances [START_REF] Turing | The chemical basis of morphogenesis[END_REF] with its particular properties (e.g. diffusion, reaction, autocatalysis, lateral inhibition [START_REF] Gierer | A theory of biological pattern formation[END_REF][START_REF] Meinhardt | Pattern formation by local self-activation and lateral inhibition[END_REF]), the interpretation of this chemical system by the responsive tissue [START_REF] Wolpert | Positional information and the spatial pattern of cellular differentiation[END_REF][START_REF] Wolpert | Positional information and pattern formation[END_REF] as well as the properties of the tissue which makes it responsive but resilient at same time [START_REF] Waddington | Principles of embryology[END_REF][START_REF] Waddington | The strategy of the genes: a discussion of some aspects of theoretical biology[END_REF]. In principle, these factors and their interactions are able to coordinate the production of multiple diverse and reproducible shapes.

Pattern formation in plants relies on certain aspects of their cell biology. Plant cells remain glued together through their cell walls. As a result, morphogenesis is basically a matter of localized cell growth largely through cell wall modifications. As in animals, cell-cell communication is essential to establish the local differences that guide tissue morphogenesis.

Cell communication (chemical and physical) feeds into molecular regulation, which then controls cell wall modifications. Although this general scenario is well accepted, it is not clear at all how this functions. whose activities ultimately lead to the initiation of flower primordium [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF]. However how this molecular network interferes with cell wall structure to direct the intiation of flower primordia remains unclear.

To gain insight into the patterning event of flower primordium initiation in Arabidopsis, I have studied three different aspects:

Summary

To control morphogenesis, spatiotemporal patterns of gene expression somehow must interfere with the mechanical properties of the individual cells of developing organs and tissues, ultimately resulting in different growth patterns. Nevertheless, the characterization of the mechanisms that link gene activities to cell behaviors remain to be defined. We study the process of morphogenesis in the shoot apical meristem, a group of undifferentiated cells where complex changes in growth rates and directions drive the formation of new organs in a periodic and continuous manner [START_REF] Murray | Systems analysis of shoot apical meristem growth and development: integrating hormonal and mechanical signaling[END_REF][START_REF] Sassi | Auxin and self-organization at the shoot apical meristem[END_REF]. Here, we

show that the plant hormone auxin plays an important role in this process by interfering with wall anisotropy via the regulation of cortical microtubule dynamics. We propose that to induce growth isotropy and organ outgrowth, auxin interferes with the cortical microtubuleordering activity of a network of proteins, including ROP6/RIC1 and KATANIN. Moreover

Introduction

A specific requirement for the initiation and positioning of organs in the SAM of Arabidopsis is the local accumulation of the hormone auxin (IAA). This is achieved at the meristem surface, via the PIN-FORMED1 (PIN1) auxin efflux carrier [START_REF] Benkova | Local, efflux-dependent auxin gradients as a common module for plant organ formation[END_REF][START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF][START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF]. The importance of auxin transport is clearly illustrated by the phenotype of pin1 loss-of-function mutants, which form naked inflorescence meristems, unable to initiate flower primordia [START_REF] Okada | Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation[END_REF]. This phenotype is also obtained by chemical treatments using inhibitors of auxin transport such as N-1-naphthylphthalamic acid (NPA). This and additional evidence indicate that auxin accumulation is indispensable for the initiation of lateral organs (Kuhlemeier, 2017;[START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF]. However, how auxin precisely functions in this process remains unclear.

To induce organ outgrowth, auxin has to modify directly or indirectly growth rates and growth directions which in plants largely rely on the mechanical properties of the cell wall.

Previous studies have pointed to the importance of wall remodeling and elasticity at the SAM, via wall-loosening proteins [START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF][START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]. Auxin is thought to promote primordia formation, at least in part through this process [START_REF] Murray | Systems analysis of shoot apical meristem growth and development: integrating hormonal and mechanical signaling[END_REF][START_REF] Sassi | Auxin and self-organization at the shoot apical meristem[END_REF]. However, this represents a rather one-sided view of the contribution of wall mechanics to organogenesis.

Here, we will focus on mechanical anisotropy, another essential property of the wall which mainly depends on the orientation of cellulose microfibrils and plays a central role in defining growth directions [START_REF] Bashline | Cell wall, cytoskeleton, and cell expansion in higher plants[END_REF][START_REF] Baskin | On the role of stress anisotropy in the growth of stems[END_REF][START_REF] Ivakov | Plant cell shape: modulators and measurements[END_REF].

The deposition of the cellulose microfibrils is controlled by the cortical microtubules (CMT), which guide the cellulose synthesis complexes, controlling the orientation of the cellulose microfibrils [START_REF] Bringmann | POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis[END_REF][START_REF] Gutierrez | Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments[END_REF][START_REF] Paredez | Visualization of cellulose synthase demonstrates functional association with microtubules[END_REF].

Consequently, the organization of the CMT has been used as proxy to predict the direction of the cellulose microfibrils and the anisotropy of the cell wall. We show here that auxin interferes with CMT organisation during organ initiation at the SAM. We present evidence that this involves a pathway depending on ROP6 as well as its partners RIC1 and KATANIN.

Results

Transgenic plants, expressing the green fluorescent protein (GFP) fused to a microtubulebinding domain (MBD) of the microtubule-associated protein 4 (MAP4), both under the expression of the Cauliflower mosaic virus (CaMV) promoter 35S (35S::GFP-MBD), permit the analysis of microtubules dynamics by live imaging. Since it has been considered that the epidermal L1 layer is rate limiting for growth, analyses of the cytoskeleton have been mainly

focusing on this layer [START_REF] Kierzkowski | Elastic domains regulate growth and organogenesis in the plant shoot apical meristem[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF][START_REF] Savaldi-Goldstein | The epidermis both drives and restricts plant shoot growth[END_REF]. Notably, the naked meristems of plants grown on NPA [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF], exhibited supracellular, circumferential alignments of CMT at the periphery of the meristem and the stem. This orientation is thought to impose cell expansion in one main direction, along the vertical axis. In other words, in the absence of auxin transport plants are forced to grow in one main direction because of the anisotropic arrangement of the CMT of the individual cells [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. This implies that to induce a lateral organ, auxin has to break this radial symmetry. How this occurs is not known.

To address this issue we set up an experimental system in which we could follow the dynamics of CMT from an anisotropic pin-shaped SAM. We grew Arabidopsis plants bearing the 35S::GFP-MBD transgene on NPA-containing medium, which prevents the formation of flowers. Subsequently, we treated the naked SAM with IAA, which induced changes in the organization of CMT across the SAM of 35S::GFP-MBD plants (Figure 1d ande). Already within the first 24hr following the treatment, the anisotropy of the CMT at the peripheral zone of the SAM was lost (Figure 1e). Notably, these changes in the organization of CMT preceded the production of lateral outgrowths, clearly visible after 72hr from the initial treatment with IAA (Figure 1d). After 96hr of treatment, a characteristic ring-like outgrowth emerging from the peripheral zone could be distinguished.

Discrete regions displaying disorganized CMTs were also observed in the SAM of soil-grown plants, i.e. in the presence of an active auxin transport and preexisting lateral organs. These regions displayed substantially different GFP-MBD patterns, compared to highly anisotropic organ boundaries [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF], and produced visible organ primordia 24 hr later (Figure 1a andb) [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. The appearance of these regions correlates in time and space with peaks of high auxin activity indicated by the auxin-signalling synthetic reporter DR5::VENUS-N7 (Figure 1c). These results suggest that in normal untreated meristems changes in CMT anisotropy coincide with auxin maxima and precede organ outgrowth at the SAM. It has been shown for other tissues, that auxin (IAA) induces rapid changes in the orientation of microtubules in growing cells [START_REF] Bergfeld | Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth[END_REF][START_REF] Nick | Unilateral reorientation of microtubules at the outer epidermal wall during photo-and gravitropic curvature of maize coleoptiles and sunflower hypocotyls[END_REF].

The previous findings suggested a correlation between three events, all taking place in the periphery of the SAM: the creation of local auxin maxima, the reorganization of CMT, and the formation of lateral outgrowths. The relationship between these three events was further explored by addressing whether the reorganization of CMT could be instrumental in the formation of lateral outgrowths. To do so, plants with impaired auxin transport were first exposed to the microtubule depolymerizing drug oryzalin. Since high global oryzalin treatments lead to complete loss of CMT polymerization [START_REF] Corson | Turning a plant tissue into a living cell froth through isotropic growth[END_REF][START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF], local treatments of this drug were applied. These treatments are performed by local application in the periphery of the SAM of lanolin paste emulsified with oryzalin. pin1 mutants or NPA grown plants treated locally with oryzalin initiated the production of lateral outgrowths already after 72hrs from the local treatment with oryzalin.

We next altered CMT dynamics by using the bot1-7 mutant allele of KATANIN1 (KTN1).

KTN1 encodes a microtubule-severing protein that promotes bundling and ordered alignment of CMT patterns [START_REF] Bichet | BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis[END_REF][START_REF] Burk | Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein[END_REF][START_REF] Stoppin-Mellet | Katanin's severing activity favors bundling of cortical microtubules in plants[END_REF].

Importantly, the modulation of CMT orientation via KTN1 has been linked to an auxin signaling pathway [START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF][START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF].

bot1-7 plants grown on NPA, are more prone to initiate lateral outgrowths and to produce flowers than wild type (WT) plants grown in the same conditions (Figure 1f). Notably, the double mutant pin1-6 bot1-7 reestablished the formation of lateral outgrowths (Figure 1g).

Thus, in auxin transport-depleted backgrounds, the bot1-7 mutation replicates the effect of auxin treatments on CMT organization and organ initiation. Relevantly, auxin treatments were not able to further affect CMT in bot1-7 SAMs. This suggests that (1) at low auxin concentrations, KTN1 leads to the formation of a pin, and (2) KTN1 is no longer able to maintain a pin in the presence of high auxin levels. Together, these results confirm the connection between local auxin maxima in the SAM, the reorganization of CMT, and the formation of lateral outgrowths. They suggest a causal relationship of these three events. Local auxin accumulation at the periphery of the SAM leads to the loss of CMT anisotropy, which results in a shift toward an isotropic state. As a result, the formation of lateral outgrowths is promoted. This points to a causal link between loss of CMT anisotropy and organogenesis. Importantly, bot1-7 replicates auxin treatments, suggesting that somehow the microtubule-severing activity of KTN1 might control the link between auxin and the loss of CMT anisotropy. [START_REF] Fu | Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis[END_REF][START_REF] Fu | A ROP GTPase signaling pathway controls cortical microtubule ordering and cell expansion in Arabidopsis[END_REF][START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF].

A ROP6-RIC1 signaling pathway is involved in the initiation of primordia in the SAM

To assess further the role of KTN1 and the ROP6-RIC1 pathway in the reorientation of CMT in the SAM we ascertained the expression of ROP6 and RIC1 in the shoot apex by qRT PCR.

Both ROP6 and RIC1 were expressed at the shoot apex, although at different levels. If both genes are involved in the activation of KTN, we would expect similarities between the phenotypes of bot1 and rop6 or ric1 mutants. We therefore next determined the capacity of rop6-1 and ric1-1 mutant alleles in the production of organs in absence of auxin transport.

rop6-1 and ric1-1 mutants grown on NPA medium displayed increased organ formation

The ABP1-KTN1 pathway is involved in the initiation of primordia in the SAM

The ROP6-RIC1 signalling pathway that promotes the MT-severing activity of KTN1 is activated by auxin in pavement cells [START_REF] Fu | Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis[END_REF][START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF]. The extracellular receptor AUXIN BINDING PROTEIN 1 (ABP1) and its functional partner transmembrane receptor-like kinases are reported to act upstream of this activation [START_REF] Fu | Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis[END_REF][START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF], although this is still a matter of debate (see discussion below). Thus, we investigated the role of ABP1 in the reorientation of CMTs at the SAM.

Described abp1 knockout mutants are embryo lethal [START_REF] Chen | ABP1 is required for organized cell elongation and division in Arabidopsis embryogenesis[END_REF]. We therefore used the viable abp1-5 allele, which contains a point mutation (H94Y) in the auxin-binding pocket of the protein that reduces the affinity of ABP1 to auxin [START_REF] Robert | ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF].

We firstly determined the capacity of abp1-5 mutants to produce organs in absence of auxin transport. abp1-5 mutants grown on NPA displayed significantly enhanced organogenesis compared to WT plants (Figure 3a andb). Next, in order to assess the organization of CMT we used the abp1-5 mutants bearing the microtubule marker 35S::GFP-MBD (a gift from J.

Friml). The naked meristem of abp1-5 mutants displayed minor alterations in the supracellular circumferential CMT organization at the SAM periphery compared to the WT (Figure 3c ande). In addition, abp1-5 mutants were still able to respond weakly to auxininduced CMT reorganization and lateral outgrowth formation, although to a lesser extent than the WT (Figure 3c-f).

To gain additional understanding into ABP1 function, we generated a transheterozygous mutant by crossing homozygous abp1-5 mutant plants with heterozygous abp1-1s/ABP1

plants, which bears one copy of the T-DNA insertional knock-out allele abp1-1s [START_REF] Tzafrir | Identification of genes required for embryo development in Arabidopsis[END_REF]. We maintained only one allele abp1-1s given the lethality of its phenotype. The heteroallelic combination abp1-1s/abp1-5 was not lethal for the mutant plants. Notably, when grown on NPA abp1-1s/abp1-5 mutant plants displayed enhanced formation of lateral outgrowths compared with the parental abp1-5 (Figure 3a andb). More random orientations of CMTs were also observed in abp1-1s/abp1-5 35S::GFP-MBD in comparison with abp1-5 35S::GFP-MBD (Figure 3e andg). Importantly, auxin-induced CMT reorganization and lateral outgrowth formation was completely abolished in the abp1-1s/abp1-5 mutant background, comparable to bot1-7 mutants (Figure 3e-h). The above is in line with the hypothesis that ABP1 and KTN1 belong to the same signalling pathway in charge of reorganizing the CMT in the SAM and regulating the formation of lateral outgrowths.

Finally, we produced bot1-7 abp1-5 double mutant plants and searched for additive phenotypes. Coherently with our hypothesis, no additional phenotypes could be distinguished as bot1-7 abp1-5 grown on NPA display organ initiation phenotypes similar to those observed in the parental bot1-7.

Together, our results suggest that in the absence of local auxin accumulation, a network of interacting proteins, including KTN1, ROP6/RIC1 and ABP1, keep CMT arrays at the SAM periphery in an anisotropic state. We suggest that this network inhibits the spontaneous formation of lateral outgrowths, and when active it leads to the formation of a pin-shaped stem. Conversely, in the presence of high auxin levels, these molecules are no longer able to maintain CMT anisotropy, which results in a shift toward an isotropic state, in principle this could be sufficient for the formation of lateral outgrowths.

The auxin response factor ARF5/MONOPTEROS is required for microtubule reorganization at the SAM

The ROP-based signaling pathway does in principle not require transcription. We note, however, that the reorganization of CMT partially overlaps with the activation of the auxin transcriptional reporter DR5::VENUS-N7 (Figure1c). Auxin induced transcriptional responses are regulated by transcription factors of the AUXIN RESPONSE FACTOR (ARF)

family [START_REF] Chapman | Mechanism of auxin-regulated gene expression in plants[END_REF]. In the SAM, ARF5/MONOPTEROS (MP) is the main ARF required for flower formation [START_REF] Guilfoyle | Auxin response factors[END_REF][START_REF] Hardtke | The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development[END_REF][START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF]. Auxin-activated MP directly induces the expression of a number of transcription factors that contribute to the formation of the flower primordium [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF]. These targets activate diverse developmental processes, floral fate specification, cell proliferation, cell growth as well as tissue and cell polarity. To gain insight into the connection between the reorganization of CMT and auxin induced transcriptional responses we analyzed the mutant allele arf5-1, which has a T-DNA insertion in the eighth exon of ARF5/MP, the sequence that encodes part of the DNA-binding domain [START_REF] Odat | Characterization of an allelic series in the MONOPTEROS gene of Arabidopsis[END_REF]. arf5-1 homozygous plants display a rootless phenotype due to their inability to establish an embryonic root (Figure 4a). Seeds of mp/arf5-1 mutants do germinate, however, and can be induced to form adventitious roots allowing the production of shoots. These shoots fail to produce flowers, resulting in the formation of naked pin-like stems (Figure 4b, c and f). We wondered, whether this pin-like phenotype was also correlated with transversely organized CMTs. In order to understand this, we introgressed a genetic construct of a fluorescent microtubule marker expressed only in the epidermal layer under the control of the promoter of the PROTODERMAL FACTOR 1 (PDF1::mCitrine-MBD) into fertile heterozygous arf5-1 mutants. By analyzing the CMT organization of arf5- 1PDF1::mCitrine-MBD plants, we detected circumferential supracellular CMT alignments at the periphery of the meristem and the stem, as observed in pin meristems after auxin transport has been impaired (Figure 4c). Since ARF5/MP is insensitive to auxin, these results suggest that the effect of auxin on CMT organization during organ initiation requires auxin dependent transcriptional input.

If MP regulates the reorganization of CMT that triggers the initiation of lateral outgrowths, it would suffice to change the dynamics of the CMT in the shoot apex to initiate lateral outgrowths even in the mp/arf5 mutant. This hypothesis was tested inducing the disorganization of CMT in homozygous arf5 mutants. As in our previous experiments, we firstly used oryzalin to induce the local disorganization of CMTs in the shoot apices. We initially tested the arf5-2 allele of MP/ARF5, which carries a T-DNA insertion at the 3' end of the coding region [START_REF] Odat | Characterization of an allelic series in the MONOPTEROS gene of Arabidopsis[END_REF]. Homozygous arf5-2 displayed incomplete penetrance of the rootless phenotype, allowing seedlings to form an embryonic root and grow easily on soil, however all plants are sterile [START_REF] Donner | Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves[END_REF]. The lack of lateral organs in the shoot apex of homozygous arf5-2 was observed in 12 out of 17 apices. Naked SAMs of homozygous arf5-2 treated locally with oryzalin developed lateral outgrowths in 6 out of 7 apices, which were already visible 96 hr after the initial treatment (Figure 4d ande). Note that, as previously reported [START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF], local applications of auxin on naked pinlike meristems of arf5-2 were not able to initiate lateral outgrowths.

In order to confirm these observations, we used the strong mutant allele arf5-1 to generated a mp/arf5-1 bot1-7 double mutant where CMT organisation was genetically destabilised. arf5-1 bot1-7 double homozygous mutants displayed a high number of lateral outgrowths around the SAM (80%, n=15) when compared to the parental arf5-1 (0%, n=30) (Figure 4d andf).

Discussion

Here we provide evidence for a scenario where auxin interferes with the cortical cytoskeleton to induce organ initiation. We propose that in the absence of auxin accumulation, a network of interacting proteins, possibly including KTN1, ROP6 and RIC1 keeps microtubular arrays at the SAM in an anisotropic state. This is sufficient to inhibit lateral outgrowth, leading by default to the formation of a pin-shaped stem. In the presence of high auxin levels, these molecules are no longer able to maintain CMT anisotropy, which induces a shift toward isotropic cell walls. As a result, organ outgrowth is promoted (Figure 5).

A signaling pathway known to control CMT downstream of auxin was first described for epidermal pavement cells. In this system, plasma membrane localized ROP6 directs the parallel alignment of cortical microtubules. This is done through the activation of the ROP6 effector protein RIC1. ROP6 and RIC1 in turn activate the KTN1-mediated detachment of branched microtubules, thus promoting the organization of CMT into parallel arrays (Chen et al., 2015;[START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF]. Thus, the ROP6/RIC1 signaling pathway plays an important role in controling microtubule organisation and lateral cell expansion [START_REF] Fu | Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis[END_REF]. It should be noted, however, that mutations in ROP6 and RIC1 have little phenotypic effects on plants grown on soil. This might suggest functional redundancy with other ROP and/or RIC proteins expressed in the meristem (e.g. ROP4 and ROP10).

We also obtained indications, that the activation of this ROP pathway might involve ABP1.

There is evidence, that the binding of auxin to ABP1 directly activates ROP6 [START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF][START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF] and thus induces microtubule rearrangements. In addition the model of a ROP-based cytoplasmic auxin signaling pathway able to regulate the orientation of CMT, has been demonstrated by other groups using multiple mutant alleles encoding members of the TRANSMEMBRANE KINASE (TMK) subfamily of receptor-like kinases (tmk1, 2, 3, 4), which could work as a link between ABP1 and ROP proteins [START_REF] Xu | Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling[END_REF]. Although a number of studies suggest that auxin might directly act on a cellular ABP1 signaling pathway, recent work describing Arabidopsis abp1 null mutant alleles with no obvious growth or developmental phenotypes have shed doubt on such a scenario [START_REF] Gao | Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development[END_REF]. It was shown for example that the embryo lethal phenotype of abp1-1 and abp1-1s is caused by disruption of the tightly-linked neighboring gene, BELAYA SMERT/RUGOSA2 (BSM/RUG2), suggesting that the role of ABP1 during early embryogenesis might not be essential (Dai et al., 2015;[START_REF] Michalko | Embryo-lethal phenotypes in early abp1 mutants are due to disruption of the neighboring BSM gene[END_REF][START_REF] Michalko | Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein[END_REF]. Michalko et al; 2015b however, report strong effects of partial knock-downs of ABP1 activity. In addition, other proteins with overlapping functions might be involved. The precise function of ABP1, therefore, remains an open question.

Although our results point at a direct cellular signaling response to auxin, we also have obtained evidence that the reorganization of CMTs depends on transcriptional regulation. As we have seen, arf5 mutants display phenotypes reminiscent of pin1 phenotype. We show, that these apices are also characterized by ordered circumferential CMT patterns at the periphery of the SAM that force the tissue to grow in one single direction. Although auxin is no longer effective, if is sufficient to disorganize CMTs at the periphery of the mp/arf5 SAM, to induce lateral outgrowths (Figure 5).

Several genes encoding for IQ67-domain (IQD) proteins have been identified as ARF5/MP targets [START_REF] Moller | Auxin response cell-autonomously controls ground tissue initiation in the early Arabidopsis embryo[END_REF]. In plants, IQD proteins are the largest class of targets of the calcium (Ca 2+ ) ion sensor calmodulin (CaM). 33 members form Arabidopsis thaliana IQD family. Most Arabidopsis IQD members are aligned along microtubules, and additionally often localize to the cell nucleus or to membranes to recruit CaM or other calcium sensors (Burstenbinder et al., 2017b). IQD functions are mostly unknown, links with microtubule arrangements are strongly supported by the altered MT organization and plant growth phenotypes in IQD gain-of-function lines. Bürstenbinder and colleagues suggested that auxindependent regulation of IQDs might restrict their local abundance in specific tissues in order to control growth via control of the MT (Burstenbinder et al., 2017a).

Although the reorientation of CMT at the SAM is instrumental for the initiation of lateral organ outgrowths, cell wall remodeling and changes in cell wall stiffness also play a role [START_REF] Braybrook | Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin[END_REF]Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF][START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF]. AFM measurements of the outer wall of auxin-treated SAMs grown on NPA indicated a reduction of rigidity in the induced lateral outgrowths, which become slightly softer than the apex. According to the analyses the changes in stiffness did not exceed 30% and indicated that a major reduction in the outer wall rigidity does not occur during organ initiation. Modelling approaches showed that such relatively modest reductions in stiffness would on their own be insufficient to induce organ initiation. [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Relevantly, simulations also showed that the combination of limited loosening with a shift to wall isotropy would lead to increased growth rates. This theoretical analysis therefore suggests that the anisotropy-to-isotropy shift could promote organ formation by amplifying the effect of relatively minor reductions in the outer wall rigidity at the SAM.

Conclusion and Perspectives

In summary, we have obtained evidence that auxin plays a role in the formation of new organs, by locally interfering with the properties of the cell wall. On one side the hormone interferes with cortical microtubule (CMT) dynamics, thereby inducing changes in growth direction. This shift to isotropic growth would signficantly enhance the slight reductions in cell wall stiffness induced during organ formation and thus allow the initiation of new growth axes. Our current working hypothesis proposes that the molecular mechanism by which auxin triggers the initiation of new growth axes includes on one side an auxin signaling via KTN1mediated pathway and a MP-dependent pathway. According to our results the MP-dependent pathway facilitates the reorientation of CMT towards an isotropic expansion through a yet unknown mechanism.

Experimental procedures

Plant material and Genetic Analysis

Arabidopsis thaliana ecotypes Columbia-0 (Col-0) and Wassilewskija-2 (Ws-2) were the wild types seeds used in this study. Transgenic lines and mutants used in this work were for the most part previously described. pin1-1 (introgressed in Col-0) [START_REF] Sassi | COP1 mediates the coordination of root and shoot growth by light through modulation of PIN1-and PIN2-dependent auxin transport in Arabidopsis[END_REF], pin1-6 [START_REF] Vernoux | PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem[END_REF], 35S::GFP-MBD [START_REF] Marc | A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells[END_REF]; bot1-7 35S::GFP-MBD [START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF], ric1-1 and RIC1 OX [START_REF] Fu | Arabidopsis interdigitating cell growth requires two antagonistic pathways with opposing action on cell morphogenesis[END_REF], rop6-1, and ROP6 OX [START_REF] Fu | A ROP GTPase signaling pathway controls cortical microtubule ordering and cell expansion in Arabidopsis[END_REF], abp1-5 [START_REF] Xu | Cell surface-and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis[END_REF], abp1-1s (NASC accession N16148), arf5-1 [START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF], arf5-2 [START_REF] Donner | Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves[END_REF], DR5::VENUS-N7 [START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF]. The

Growth Conditions and Treatments

For napthylphtalamic acid (NPA) treatments, plants were sown in petri dishes with a medium adapted for Arabidopsis (Duchefa) containing NPA to a final concentration of 10µM. To induceorgan formation plants were treated in petri dishes with 1mM IAA liquid solution for 3 h after imaging the t=0 time point and again the next day after imaging the t=24h time point.

No other IAA was added after the 24h time point until the completion of the experiment.

Lanolin paste for local SAM treatments was prepared as follows: [START_REF] Berleth | The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo[END_REF], then transferred to MS medium supplemented with the synthetic auxin 1-naphthaleneacetic acid (NAA) to a final concentration of 10µM and kept on these plates for 7 days, when they were transferred to free MS medium free of NAA to allow the elongation of the root. Plants were either kept in plates or transplanted to soil after rooting.

Microscopy

Confocal microscopy was carried out with a Zeiss LSM700 upright microscope equipped with 40x water immersion lens. Live imaging of NPA pins was carried out directly in petri dishes, after submerging plantlets in water. SAMs of plant grown on soil were imaged as previously described [START_REF] Fernandez | Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution[END_REF] or in whole mount preparations. Briefly, for whole mount preparations plants were grown on soil in flat vessels until bolting. After the removal of older flower buds to expose the SAM surface, plants were imaged with a droplet of water between the objective and the SAM surface. Plants were kept in humid transparent boxes for all the duration of the experiments to prevent SAM desiccation. Scanning electron microscopy was carried out with a Hirox SH-3000 table-top SEM on fresh plant material at -40°C, with an accelerating voltage of 5kV. Images of SAMs treated with lanolin paste were taken with a Leica MZ12 stereomicroscope equipped with a Leica DFC320 camera.

Image analyses

Image processing for PDF1::mCitrine-MBD was carried out with the ZEN software (Zeiss) using the 3D transparent projection. For MT visualization in 35S::GFP-MBD, confocal stacks were further processed with MerryProj (de Reuille et al., 2006) to obtain the projection of MTs on the L1 layer. Measurements of MT organization were carried out with the MT plug-in for ImageJ [START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. Briefly, for each meristem cell contours were inferred from the original confocal stack (loaded onto the FIJI release of ImageJ; http://fiji.sc/Fiji) and further saved in a single Region Of Interest (ROI) file. The ROI file was then overlaid on the corresponding MerryProj-derived image and used as template to measure MT organization with the MT plug-in as previously described [START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. Supracellular organization of MT in the SAM was assessed with FIJI by calculating the angle between the radius of the SAM and the average MT orientation from each cell as obtained by MT plug-in.

Graphs and statistics were obtained with Microsoft Excel software.

Chapter II

A transcriptional coupling between microtubule-driven growth isotropy and cell wall remodeling promotes organogenesis at the shoot apex in Arabidopsis Alessia Armezzani, Ursula Abad, Olivier Ali, Laetitia Vachez, Amélie Robin, Ann'Evodie Sallee, Antoine Larrieu, Ewa Mellerowicz, Nobu Nishikubo, Ludivine Taconnat, Virginie Battu, Teva Vernoux, Jan Traas, Massimiliano Sassi.

Summary

The shoot apical meristem (SAM) of higher plants plays a central role in the establishment of architecture, as it continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Plant cell growth is driven by the internal turgor pressure and largely depends on the cell walls, which provide external support and allow cell expansion through synthesis and structural changes. A previous study revealed the importance of growth isotropy in organ outgrowth, which involves disorganization of the cortical microtubules. Nevertheless, our results and those of others indicate that cell wall loosening is required in order to have proper organ outgrowth [START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF]Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]. We show here that this disorganization is tightly coupled to the transcriptional control of genes involved in wall loosening via pectin and hemicellulose modifications. Transcriptomic analysis combined with in situ hybridization identified a set of cell wall-remodeling genes, which are active in the SAM during organ formation.

Interestingly, some of these wall-loosening genes are induced when microtubules are

Introduction

The control of shape during growth of multicellular organisms is a fundamental, yet poorly understood process. We address this issue here in plants where in most species morphogenesis entirely depends on the local rates and directions of cell expansion [START_REF] Coen | The genetics of geometry[END_REF][START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. This is because plant cells usually do not migrate or rapidly change shape during development, as in animals [START_REF] Coen | The genetics of geometry[END_REF][START_REF] Kicheva | Limbs made to measure[END_REF] while cell death in principle does not play an important role. For this reason, plants represent an excellent model to investigate the mechanisms that determine the architecture and shape of multicellular organisms.

Plant growth depends largely on the control of the cell wall, which surrounds most cells and counteracts the high internal turgor pressure. The wall in higher plants is largely composed of relatively stiff cellulose microfibrils that are cross-linked by matrix of polysaccharides such as hemicelluloses and pectins. In order to grow, cells have to expand their cell walls irreversibly, making them yield to the internal pressure. It is therefore thought that molecular regulation controls morphogenesis for a large part by affecting the local biochemical composition and arrangements of the cell wall polysaccharides [START_REF] Braybrook | Shifting foundations: the mechanical cell wall and development[END_REF].

A plethora of cell wall remodeling and synthesizing proteins have been identified. The precise number of synthesizing proteins is not known, but it is thought that hundreds of enzymes may be involved in this process [START_REF] Yang | Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis[END_REF]. In addition, several multigene families encode proteins likely involved in modifying the existing bonds, thus affecting the mechanical properties of the wall. The best known of these so-called remodeling proteins include (i) the pectin methyl esterase (PME) family and their inhibitors (PMEIs), which control the assembly and mechanical properties of the pectin matrix, (ii) the xyloglucan endotransglucosylase/hydrolase (XTH) and (iii) the A-type expansin (EXPA) families both supposed to act on the mechanical properties of the hemicellulose matrix [START_REF] Braybrook | Shifting foundations: the mechanical cell wall and development[END_REF]Cosgrove, 2016a, b). In addition, some XTH enzymes might also interfere with the interactions between hemicellulose and cellulose.

The remodeling of pectin and hemicellulose polysaccharides in principle modifies cell wall stiffness and has thereby the potential to modulate growth rates [START_REF] Braybrook | Shifting foundations: the mechanical cell wall and development[END_REF]Cosgrove, 2016a, b). Although pectins and hemicelluloses have also been associated

with the control of growth anisotropy [START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF], growth directions are largely determined by the presence of cellulose microfibril arrays in the cell wall. The polymers can be deposited in highly ordered arrays, restricting cell expansion along their axis [START_REF] Baskin | Anisotropic expansion of the plant cell wall[END_REF][START_REF] Paredez | Visualization of cellulose synthase demonstrates functional association with microtubules[END_REF]. The orientation of the cellulose microfibrils themselves is controlled by cortical microtubules (CMTs), which guide the movement of the cellulose synthase (CesA) complexes across the plasma membranes [START_REF] Bringmann | POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis[END_REF][START_REF] Paredez | Visualization of cellulose synthase demonstrates functional association with microtubules[END_REF]. Although the exact mechanisms underlying microtubule orientation are not known, several components of the Rho Proteins of Plants (ROP) signaling pathway seem to be involved. Indeed, mutations in ROP6 and ROP INTERACTING PROTEIN AND CRIB MOTIF-CONTAINING PROTEIN 1 (RIC1) affect microtubule organization at the shoot apex, and influence organ formation [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. RIC1 itself directly interacts with KATANIN1 (KTN1) a microtubule associated protein required for microtubule severing and bundling [START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF]. Accordingly, mutations in KTN1 also affect organ initiation [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF][START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF].

In summary, to control growth directions and growth rates at the level of the cell wall, the regulatory molecular networks can act on two sets of parameters: (i) the composition and the structure of the wall and (ii) the orientation of the cellulose microfibrils, mainly via microtubule dynamics. However, the coordination of these processes as well as their relative contributions to morphogenesis, are not understood.

The shoot apical meristem (SAM) of Arabidopsis thaliana represents an ideal system to address these questions. As it harbors the stem cells, the SAM continuously generates new tissues and organs at the shoot tip, which involves complex changes in cell growth directions and in cell growth rates [START_REF] Braybrook | Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin[END_REF]Peaucelle et al., 2011a;[START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Organ formation at the SAM is initiated by the accumulation of the hormone auxin in discrete foci at the periphery of the meristem [START_REF] Benkova | Local, efflux-dependent auxin gradients as a common module for plant organ formation[END_REF][START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF]. Auxin is concentrated at the site of organ initiation by an active transport mechanism relying on the action of the PIN-FORMED 1 (PIN1) efflux carrier [START_REF] Okada | Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation[END_REF][START_REF] Reinhardt | Regulation of phyllotaxis by polar auxin transport[END_REF][START_REF] Vernoux | PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem[END_REF].

Several studies have pointed at the importance of wall remodeling downstream of auxin during morphogenesis at the meristem. The local application of expansins can induce organ formation, whereas induced changes in the degree of pectin methylation can induce or inhibit organogenesis [START_REF] Braybrook | Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin[END_REF][START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF]Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]. Mutations directly or indirectly affecting pectin composition also affect morphogenesis. Likewise, the xxt1/xxt2 mutant, that has greatly reduced xyloglucan (a hemicellulose) levels, shows abnormal plant architecture. It remains nevertheless unclear how wall composition is regulated during organ formation.

In a recent study we showed that high auxin concentrations at organ initia cause the disorganization of microtubules and we proposed that a shift to isotropic growth, through the isotropic deposition of microfibrils, plays a major role in organ formation. This shift could act in synergy to relatively modest changes in wall stiffness observed during organ formation [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Although both wall loosening and wall isotropy seem to be involved, their relative importance and their coordination during organogenesis remain to be established.

To address this question, we investigate the roles of microtubule dynamics and wall remodeling during morphogenesis at the SAM. For this purpose, we identified genes encoding XTHs, EXPAs, PMEs and PMEIs strongly expressed at the shoot meristem. We show that the transcription of the genes can be activated through changes in microtubule dynamics.

Conversely, interfering with wall loosening promotes changes in microtubule organization.

We propose that this tight coupling between cytoskeleton organization and cell wall remodeling plays a central role in coordinating growth during organ initiation at the SAM.

Results

Disorganizing the cortical microtubules increases growth rates in vivo but not in silico

Previous work suggested a central role for the organization of cortical microtubules in organ outgrowth [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. To bulge out, cells in a primordium must initially grow more rapidly than the surrounding cells, in particular the boundary cells [START_REF] Kwiatkowska | Surface growth at the reproductive shoot apex of Arabidopsis thaliana pin-formed 1 and wild type[END_REF][START_REF] Kwiatkowska | Growth and morphogenesis at the vegetative shoot apex of Anagallis arvensis L[END_REF]. The partial restoration of organ formation observed in the pin1-6 bot1-7 double mutant [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF] (Figure 1), suggests that disorganized CMTs are sufficient to cause this increase in growth rates. This is in line with the observation that the local disorganization of microtubules using the drug Oryzalin (ORY) also causes the induction of local outgrowths [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. If we accept that the disorganization of microtubules primarily affects cellulose deposition, a shift to the isotropic distribution of microfibrils must also lead to outgrowth and an overall increase in growth rates. This leads to the somewhat counterintuitive hypothesis that not only microfibril density but also their orientation has the potential to affect global growth rates. In other words, the experimental results indicate that, at identical microfibril density, cells with an isotropic wall would grow faster than cells with anisotropic walls.

To explore this hypothesis, we performed mechanical simulations of a growing meristematic dome (Figure 2). These simulations were conducted with a dedicated numerical framework, where specific values of the mechanical wall parameters (stiffness, wall synthesis rates and yielding thresholds) and turgor pressure can be set in each cell [START_REF] Boudon | A computational framework for 3D mechanical modeling of plant morphogenesis with cellular resolution[END_REF][START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Growth is implemented through a strain-based law, stating that directional (plastic) expansion of the cell wall is proportional to its elastic stretching minus a given threshold [START_REF] Boudon | A computational framework for 3D mechanical modeling of plant morphogenesis with cellular resolution[END_REF]. We assumed the reversible mechanical behavior of the cell wall to be linear elastic. Since elastic strain results from the combination of the elastic properties and turgor-induced mechanical stress, we reasoned that a change from anisotropic to isotropic microfibril deposition would reduce stiffness in certain directions and increase it in others, leading to directional changes in growth. Considering a growing tissue where microfibril deposition is highly anisotropic, we indirectly changed the growth dynamics of an embedded, small group of cells by making their microfibril deposition isotropic (Supplemental Model Description, see annex 2). Based on the experimental observations, we expected that these isotropic cells would grow faster than the otherwise anisotropic environment. However, simulations following this scenario showed a reduction in growth rate of about 20-25% in the isotropic domain (Figure 2a, see annex 2 for quantified output). Similarly, even when all the cells at the periphery of the virtual dome are made isotropic, growth rate is reduced by roughly 30% (Figure 2b, see annex 2 for quantified output). In fact, increased growth rates in primordium cells could only be obtained in simulations where this directional shift was combined with a reduction in the amplitude of the stiffness tensor, mimicking a decrease in the cellulose deposition rate or a loosening of the cell wall matrix. Accordingly, we observed that at least a 30% decrease of rigidity amplitude is needed for the initium zone to expand faster than its surroundings (Figure 2a, annex 2). In this context, it is important to note that we did observe relatively minor and variable reductions in stiffness at the wild type shoot apex in outgrowing organ primordia, not exceeding 50% [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Thus, in contrast to experimentation, simulations predict that isotropy alone is not able to increase cell growth rates. In fact, a shift to isotropic microfibril orientation would rather slow down growth unless it would be coupled to reductions in wall rigidity. This suggests that also in vivo the shift to isotropic growth must somehow lead to changes in wall stiffness. We therefore looked more in detail at regulators involved in cell wall remodeling, in particular those with the potential to modify cell wall stiffness.

A previous study investigating the role of wall synthesis in SAM morphogenesis did not reveal particular changes in the amount of cellulose correlating with organ initiation [START_REF] Yang | Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis[END_REF], Wightman personal communication). In addition, no strong overall transcriptional upregulation of genes encoding CesAs was found in outgrowing organs [START_REF] Yang | Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis[END_REF] Wightman personal communication, our own unpublished data). We therefore focused our attention on modifications of the cell wall matrix, more in particular we looked at four gene families, which have been associated with modifications in the wall matrix: pectin modifying enzymes (PMEs and PMEIs) and enzymes potentially targeting the hemicellulose matrix (XTHs and EXPAs). (d) When the cells in the primordium are made isotropic, but stiffness remains as in the periphery, the primordium grows more slowly than the surrounding periphery.

(e) When both anisotropy and stiffness are reduced, the primordium cells grow quicker than the surrounding cells.

In (b) the dome has only a peripheral zone with variable mechanical properties (f) The peripheral cells are anisotropic, (g) when cells are made isotropic, growth rate is reduced

Identification of candidate cell wall-remodeling genes involved in organ initiation

In a first step, the expression of these cell wall related genes was investigated by exploiting an RNAseq analysis conducted on dissected inflorescences on which only a minimal segment of stem and the flowers up to stage 2/3 were left. The results allowed us to identify a set of abundantly expressed candidate genes (Figure 3 A and B; Table S1). Transcripts belonging to all four families were identified in the samples. In particular genes encoding XTHs were abundantly expressed. Genes encoding PMEIs, PMEs, and EXPAs were less expressed, but nevertheless more abundantly than the cellulose synthases for example. We next investigated the expression patterns of these genes through in situ hybridization. Of the 31 most abundantly expressed genes tested (Table S1 for list of tested genes), 16 were detectable in our hands (see Figure S1 for a complete set of results).

The most abundantly expressed PMEI's (PMEI AT5G62350, PMEI3; Figure 3C; Table S1)

were detected in the outgrowing primordia, mainly in the outer cell layers of the fast expanding floral organs (Figure 3C). These zones of high PMEI activity correlated with high labeling of the JIM5 antibody, interacting with partially or entirely de-methyl esterified pectins (Figure S1, see also [START_REF] Krizek | AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development[END_REF]. The third PMEI, AT1G14890, was homogenously expressed at the meristem.

PME5 (33% of the PME transcripts) had a spotted expression pattern reminiscent of cell cycle-related genes as was reported previously (Peaucelle et al., 2011b) (Figure 3C). PME3

(14 % of the PME transcripts) was weakly and homogeneously expressed throughout the SAM. The other PMEs could not be detected.

We next investigated the putative xyloglucan remodeling genes (XTHs and EXPAs). The RNA-seq analysis identified a number of such genes expressed at the shoot apex (Table S1).

In situ hybridization confirmed that 8 XTH and 4 EXPA genes were expressed at the detectable level in apical tissues (Figure 3C, Figure S2). Interestingly, three XTHs (XTH9, XTH4 and XTH22) and three EXPAs (EXPA6, EXPA15, and EXPA4) represented the large majority of the transcripts in each family (Figure 3A, Table S1). Importantly XTH9, XTH4

and EXPA15 were strongly expressed at the shoot apex and clearly detected in the outgrowing primordia. EXPA15 showed the most restricted pattern, mainly limited to what is the future floral meristem, excluded from the cryptic bract zone. XTH9 and in particular XTH4 were expressed in the meristem, the expression decreased in the very young flower meristem, then it increased very strongly in the flower primordia as soon as they started to grow out. Two other highly active genes, EXPA4 and EXPA6, were homogenously expressed, while XTH22 was mainly expressed in the vascular tissues and in the differentiating epidermis. Taken together, these data suggest that three genes associated with hemicellulose remodeling (XTH4, XTH9 and EXPA15) are highly expressed during the early stages of primordium formation.

The organ-specific expression of these three remodeling genes is somewhere downstream of the auxin accumulation that triggers organ initiation. Indeed, RNA in situ hybridizations showed that their expression was substantially reduced when compared to the WT in pin1-6 apices, where auxin doesn't accumulate (Figure S2). Moreover, exogenous application of auxin at the flank of the SAM could restore the organ-specific expression of these genes in a pin1-6 background (Figure S2). Importantly, these effects of auxin on gene expression can take up to several days, indicating that they are largely indirect.

Perturbing cortical microtubule dynamics promotes the expression of wall remodeling genes

We next assessed whether the strongly expressed remodeling genes targeting pectins and hemicelluloses could be related to the increased growth rates observed in isotropic outgrowths, induced when microtubules become disorganized. To this end, we analyzed the expression of representatives of the PME, XTH and EXPA families in the pin/bot double mutant. In order to obtain a more global view of the expression in the irregularly shaped dome of this mutant, we used whole mount RNA in situ hybridizations. This showed that PME3, XTH9 and EXPA15 were specifically expressed in the outgrowths compared to the surrounding tissues (Figure 4). We confirmed this information for XTH9 after ORY treatment and observed that, 4 days after application, XTH9 was clearly expressed in ORY-induced outgrowths (not shown). In conclusion, the results suggest that the disruption of microtubule alignments also cause the transcriptional activation of three types of wall remodeling genes.

The existence of this link between wall isotropy and wall loosening provides a plausible explanation for the increased growth rates induced by the disruption of microtubule organization, not predicted by the simulations.

An Enhanced Yeast-One-Hybrid suggests multiple regulation of wall remodeling genes

The specific expression patterns of some of the cell wall genes as well as their transcriptional activation after perturbations of the cytoskeleton raised questions regarding their upstream regulation. In this context, in particular XTH9, EXPA4 and EXPA15 represented interesting candidates for further analysis. In order to gain some insight into the identity of upstream regulators, we carried out an Enhanced yeast-one-hybrid (eYIH) assay. eYHI assays have been used in several systems to identify transcription factor-DNA interactions at a large-scale.

They allow the production of transcriptional network models and the generation of hypotheses about gene regulation [START_REF] Deplancke | A gateway-compatible yeast one-hybrid system[END_REF][START_REF] Gaudinier | Enhanced Y1H assays for Arabidopsis[END_REF]Reece-Hoyes et al., 2011a;[START_REF] Reece-Hoyes | Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping[END_REF]. In plants, eYHI assays have been useful tools to generate large-scale models of interaction networks involved in developmental processes such as the synthesis of the secondary cell wall [START_REF] Taylor-Teeples | An Arabidopsis gene regulatory network for secondary cell wall synthesis[END_REF], or the establishment of the root ground tissue [START_REF] Sparks | Establishment of Expression in the SHORTROOT-SCARECROW Transcriptional Cascade through Opposing Activities of Both Activators and Repressors[END_REF].

For the eYIH assay, we prepared baits for XTH9, and the two EXPA genes, which show contrasting expression, EXPA4 and EXPA15. All the baits used for the eYIH assay contained portions of the 2000bp sequences immediately upstream of the translational start site, except for XTH9 bait, for which the intergenic region of 1386bp was used instead. The three promoters were Gateway cloned to the reporter genes lacZ and HIS3. In this system the expression of the reporter genes is activated when a TF interacts with the promoter bait. For the screen, a complete collection of 2000 Arabidopsis TFs was fused to a transcription activation domain and used as prey [START_REF] Pruneda-Paz | A genome-scale resource for the functional characterization of Arabidopsis transcription factors[END_REF].

A total of 31 TFs (1.55% from the complete collection) were found to potentially regulate with the XTH9 promoter fragment used, 97 TF (4.85%) were found to bind to the EXPA4 promoter fragment, and 23 TF (1.15%) to the EXPA15 promoter fragment (Table S4). The high numbers of EXPA4 interactors might reflect the homogeneous expression of this gene when compared to the other two. One TF characterized as a B3 domain protein encoded by At5g38490 displayed potential binding to the three cell wall loosening genes. XTH9 and EXPA15, both up regulated in the organ outgrowth, share only one putative upstream regulator, the MYB-related transcription TRFL10 (At5g03780). EXPA4 and EXPA15 showed potential co-regulation by the HISTONE H2A 2 (HTA2); whereas EXPA4 and XTH9 share 11 potential regulators, which represent more than 10% of the TF that might be regulating EXPA4 expression (Table S5). These included one additional B3 domain TF, the HISTONE 2A 13 (HTA13), the LOB DOMAIN-CONTAINIG PROTEIN 3 (LBD3) also known as ASYMMETRIC LEAVES 2-LIKE 9 (ASL9), as well as two C2H2-type zinc finger TFs. Four TFs of the plant specific BASIC PENTACYSTEINE (BPC) family were putative regulators of EXPA4 and XTH9, thus ATBPC1, ATBPC4, ATBPC5 and ATBPC7. These results indicate complex regulation of XTH9, EXPA15 and EXPA4 expression.

Increasing cell wall extensibility promotes the disruption of CMT organization

The results obtained thus far led to a scenario, where auxin would perturb microtubule alignments, which then would (indirectly) feed back on the transcriptional activation of wall loosening enzymes. This explains why interfering with microtubule alignments does also lead to increased growth rates and the bulging out of the cells, even in the absence of auxin transport.

Previous studies, however, have shown that interfering with wall properties through the ectopic expression of pectin modifying enzymes or expansins can also cause outgrowths [START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF][START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF] This led us to ask whether wall loosening induced by pectin or hemicellulose modifiers can also cause changes in microtubule arrangements.

We therefore investigated the effects of perturbing the pectin polymers within the wall using external PME treatment. PMEs in principle reduce wall stiffness by affecting the pectin matrix, and thereby cause the ectopic formation of outgrowths at the SAM (Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF]. In our hands, PME treatments induced organ formation in 7

(31,8%) of the tested LTI6b expressing SAMs (n=22) (Figure 5A, Figure S3). A substantial radial enlargement of the SAM was observed in the majority of the population (14 or 63.6%), with 1 plant not responding to the treatment (4.5%, Figure S3). Regardless of the final effect in all responding meristems, PME treatments affected CMT organization in 35S::GFP-MBD within 48h (Figure 5B andC). In particular we observed that 24h after the beginning of the PME treatment most of the meristematic cells still displayed an anisotropic arrangement of CMTs, although in some plants the average CMT orientation shifted from circumferential to longitudinal (Figure 5B andC). At 48h after the treatment, the majority of the cells clearly displayed isotropic CMT arrangements. It must be pointed out that the disruption of CMT organization by PME is substantially slower and less abrupt compared to that induced by IAA in control experiments (Figure 5B andC) [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

We next tested whether perturbing wall remodeling genes potentially targeting the hemicellulose matrix would also affect microtubules and cause the formation of outgrowths in the absence of auxin transport. Unfortunately, single and double xth4 and xth9 mutants as well as expa15 knockouts did not show any obvious phenotype (our unpublished results). We therefore focused our attention on the xxt1/xxt2 double mutant. The XXT1 and XXT2 genes encode XyG xylosyl-transferases, which are both expressed at the shoot apex [START_REF] Yang | Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis[END_REF]. Mutants lacking both genes have very little or no xyloglucans and form stunted plants, which are nevertheless able to produce lateral organs such as branches, leaves and flowers. In addition, certain cell walls of the mutant are more elastic and more extensible, whereas microtubule alignments are perturbed in certain tissues (Park and Cosgrove, 2012a;[START_REF] Xiao | Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis[END_REF].

Under normal conditions on soil, the xxt1/xxt2 mutant had a mild phenotype [START_REF] Xiao | Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis[END_REF]. The primary inflorescence stem was not able to grow vertically; the meristems were smaller, while phyllotaxis was perturbed. On NPA a particular phenotype was observed. In our hands, 60-70% (n=100) of wild type plants grown on NPA form pin-like stems. This is at least partially because the microtubules maintain a highly anisotropic organization of cellulose deposition, thus promoting the formation of a cylindrical stem [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF][START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Interestingly, when grown on NPA, a 40% (n=100) of the xxt1/xxt2

mutants showed the formation of multiple spontaneous outgrowths (Figure 6A and J) very rarely seen in wild type plants. A double mutant line expressing PDF1::mCitrine-MBD in the L1 layer of the meristem showed that this phenotype on NPA went along with altered microtubule organization. In particular, the zone of isotropic microtubule arrays at the meristem summit was extended to a variable degree (Figure 6D-I). Outside this zone, the cells were able to align their microtubules in the circumferentially around the stem. This implies that the level of xyloglucans can influence microtubule alignment at the shoot apex. However, this is only apparent in the absence of auxin accumulation.

Discussion

Structural isotropy and lateral organ formation

Shape changes during morphogenesis in plants are achieved through the local isotropic or anisotropic yielding of the cell wall to the internal turgor pressure. In a previous study we highlighted the regulation of structural wall anisotropy during organ initiation at the shoot apical meristem [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. The local accumulation of auxin destabilizes microtubule alignment, probably via a ROP-based signaling cascade. This supposedly causes a shift to isotropic microfibril deposition and consequently to isotropic growth and the formation of a new growth axis. Intuitively it seemed plausible that a group of structurally isotropic cells in an anisotropic environment would spontaneously bulge out. Our numerical simulations, however, showed that this is not the case. This comes from the fact that structurally isotropic walls expand more slowly than anisotropic walls if all other mechanical parameters are kept identical. This is probably because cell wall rigidity in a particular direction is not a linear function of the number of microfibers in that direction. Fibrils aligned in other, similar directions also contribute, generating a cooperative effect with a counter-intuitive consequence: when the structural anisotropy of the cell wall evolves, the average rigidity changes, although the number of fibrils remains constant. This change in wall stiffness in turn also affects growth rates (see Supplemental Model Description for detailed explanations). In our simulations this property led to decreased growth rates when the walls were made more isotropic.

Coupling anisotropy to wall loosening

The simulations left us with a contradiction: in vivo, isotropy seemed to be synonymous with increased growth rates and organ formation, while simulations and theory told us that the exact opposite should happen. We resolved this apparent contradiction by showing that in contrast to the simulations, where rigidity and anisotropy can be programmed independently, we were not able to separate both parameters experimentally. This is because a shift to isotropic growth in vivo also triggers an increase in wall loosening. At this stage it remains unclear how this transcriptional coupling functions. Our enhanced yeast one-hybrid assay showed that as many as 31 TFs have the potential to activate XTH9 promoter, 23 to the EXPA15 promoter and 97 to the EXPA4 promoter (Abad and Traas unpublished), thus indicating that the regulation of cell wall loosening depends on many inputs.

Whatever the molecular basis, the coupling between wall loosening and isotropy could provide a mechanical module, which is essential for establishing the typical branched plant architecture. It remains to be seen if and how this coupling functions in other developmental contexts. Evidence exists suggesting that it could be widely activated during plant morphogenesis. A previous study identified a regulatory cross talk between microtubules and XTHs, which control petiole elongation in shaded plants downstream of auxin action [START_REF] Sasidharan | Interactions between auxin, microtubules and XTHs mediate green shade-induced petiole elongation in arabidopsis[END_REF]. Moreover, a recent cellular analysis of the xxt1/xxt2 mutant, which shows a greatly reduced xyloglucan content, exhibited a loss of microtubule alignment in hypocotyls [START_REF] Cavalier | Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component[END_REF]Park and Cosgrove, 2012a;[START_REF] Xiao | Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis[END_REF]. It was also found that the expression of several microtubule-associated genes including MAP70-5 and CLASP as well as receptor genes such as HERK1 and WAK1 were changed in xxt1 xxt2

mutants [START_REF] Xiao | Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis[END_REF]. Together, these results indicate that the coupling between xyloglucan loosening and CMT organization is not restricted to the SAM but could be involved in the regulation of different developmental processes.

Organ initiation and the wall matrix

The cell wall matrix tethers the cellulose microfibrils together and the regulation of its composition is essential during organ development. A recent comprehensive analysis showed that the enzymes involved in hemicellulose synthesis play an important role throughout the meristematic apex [START_REF] Yang | Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis[END_REF]. Our finding that several XTHs and EXPAs are very strongly upregulated in the rapidly outgrowing organs would suggest a prominent role for xyloglucan modifications, in line with the results of previous studies on organogenesis in tomato [START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF][START_REF] Reinhardt | Localized upregulation of a new expansin gene predicts the site of leaf formation in the tomato meristem[END_REF]. Although mutants where XTH4 and 9

were knocked out show reduced XET activity (Mellerowicz et al unpublished), no strong phenotypes were observed under normal conditions in double mutants. Likewise, an EXPA15 knockout did not show any obvious effect. This probably reflects the extraordinary flexibility of the cell wall assembly and remodeling network. Nevertheless, the xxt1/xxt2 mutant produced lateral outgrowths when auxin transport was inhibited, showing that the control of xyloglucan synthesis is at least partially responsible for the pin phenotype.

Several studies have also pointed at the importance of pectins in morphogenesis at the shoot apex. However, Yang and colleagues [START_REF] Yang | Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis[END_REF] did not find obvious differences in the degree of pectin methylation related to organ initiation at the SAM. We confirmed these results and found that PMEIs are much more strongly expressed in the more differentiating, expanding cells on the abaxial side of the organ primordia, whereas the expression of PMEs seems to be rather homogeneous at the meristem (see also: [START_REF] Krizek | RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways[END_REF]). We therefore would favor a hypothesis where the degree of pectin methylation is not subject to major changes during organ initiation. This does imply, however, that to remain constant, pectin properties would need to be regulated in a strict manner at the meristem. Accordingly, modifications in the degree of pectin methylation through overexpression of PMEs or PMEIs lead to extra outgrowths or to the inhibition of organ initiation (Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF].

Conclusions and Perspectives

In conclusion, we provide evidence for the existence of a mechanical module at the shoot apex, which couples the transcriptional regulation of wall expansion to cellular anisotropy and show how this module might function during organ initiation. An important challenge will be to further unravel the coupling mechanisms and what signaling components and transcription factors are involved. More generally, it will be important to investigate how this coordination between wall loosening and anisotropy is modulated throughout plant development.

Experimental procedures

Plant material, growth conditions and chemical treatments

Arabidopsis thaliana plants of the ecotypes Columbia-0 (Col-0) and Wassilewskija-2 (Ws-2)

were used as wild type. Transgenic lines and mutants used in this study have been previously described: 35S::LTi6b-GFP, 35S::GFP-MBD, pin1-6 and bot1-7/pin1-6 [START_REF] Okada | Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation[END_REF][START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Plants were grown as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

Napthylphthalamic acid (NPA), indole-3-acetic acid (IAA) and oryzalin (ORY) treatments were carried out as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Treatments with PME were [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF].

Plant cells remain glued together through their cell walls, cell migration is not possible and plant morphogenesis is basically a matter of localized cell growth. Growth results from the irreversible, plastic yielding of the cell wall to the internal turgor pressure. This process implies the constant modification of the wall components, allowing changes in the rate and direction of growth. To induce organ outgrowth, the plant hormone auxin and its downstream targets therefore must play a central role in the regulation of the cell wall. There is evidence, that the initiation of organs at the sites of auxin accumulation at the SAM involves local changes in cell wall stiffness [START_REF] Braybrook | Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin[END_REF][START_REF] Kierzkowski | Elastic domains regulate growth and organogenesis in the plant shoot apical meristem[END_REF]Peaucelle et al., 2011a;[START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF][START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Auxin accumulation also affects the dynamics of the cortical microtubules (CMT) and the anisotropic properties of the cell wall.

The accumulation of auxin promotes the disorganization of ordered CMT arrays at the periphery of the SAM, activating changes in growth direction from anisotropic-to-isotropic.

Cell wall loosening and isotropy act synergistically to promote the initiation of the lateral outgrowths [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

Although it is assumed that auxin signaling regulates primordium initiation by integrating transcriptional regulation and the control of cell wall mechanics, the connection between these two processes is still unclear. In addition, the mechanical status of the cell wall is not just a downstream target of auxin signaling. The local application of wall loosening proteins and microtubule disorganizing agents are both able to induce organ production [START_REF] Fleming | Induction of Leaf Primordia by the Cell Wall Protein Expansin[END_REF][START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]chapter 2), indicating the presence of some type of feedback.

Here we present evidence that this feedback can even shortcut upstream auxin signaling. We propose a model where an auxin induced shift to isotropic growth -promoted in part by the reorientation of CMT -feeds back on the activation of the transcriptional regulatory network controlling flower initiation. Although the feedback mechanism itself remains elusive, our results indicate that organ initiation might involve multiple inputs, some of which might act in parallel to auxin. In particular, we further explore here the possible role of the brassinosteroidsignaling pathway.

Results

CMT disorganization promotes outgrowths that express developmental patterning genes

To obtain further information on the feedback mechanisms discussed above, we first analyzed the effects of changes in CMT dynamics on transcription factors involved in organ outgrowth.

As an experimental system, we used plants impaired in auxin transport. This was achieved chemically by growing the plants on media containing N-1-naphthylphthalamic acid (NPA) or genetically by using pin1 mutants (see material and methods). Under these conditions, plants are severely impaired in organ initiation, especially during the floral stage and produce naked stems characterized by their anisotropic growth sustained by the anisotropic microtubule arrangements. Lateral outgrowths can be induced in the system via the induction of changes in CMT dynamics, either by local oryzalin applications in lanolin paste or by using the botero1-7 allele of KATANIN1 (KTN1) [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Both approaches break down microtubule anisotropy at the periphery of the SAM.

We analyzed the expression patterns of three transcription factors with central roles in flower initiation, namely, LFY, ANT, AIL6 as well as their upstream regulator MP. As previously reported, the transcripts of these three genes accumulated in the incipient flower primordia of WT plants and at the periphery of pin1-6 mutant apices [START_REF] Vernoux | PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF] (Figure 1). We next analyzed the expression pattern of these genes in pin1-6 bot1-7 double mutant plants. pin1-6 bot1-7 SAMs characteristically display lateral outgrowths which can develop into flower like structures. We detected accumulation of transcripts of LFY, ANT, AIL6 as well as MP in the lateral outgrowths of pin1-6 bot1-7 double mutant, although at variable levels (Figure 1).

The variability in expression levels might be due to arrest of the lateral outgrowths, as not all of them continue to develop. Since the dynamics of gene expression in the outgrowths cannot be easily revealed by in situ hybridization, we analyzed in more detail LFY and ANT expression using fluorescent reporter lines pLFY>GFP and pANT::erGFP grown on NPA.

We excluded AIL6 and MP from this analysis given the lack of fluorescent reporter lines that faithfully reproduced their expression pattern.

To study gene expression dynamics in lines expressing fluorescent markers, we induced lateral outgrowths by local oryzalin applications. About 60% (n=30) of the plants react to oryzalin by producing defined bumps, which become visible in the binocular around 72hrs after application [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Differently from auxin-induced outgrowths, where pLFY>GFP was consistently expressed throughout (n=8), oryzalin-induced outgrowths expressed pLFY>GFP in irregular patches (n=17) (Figure 2a). However, pANT::erGFP expression was consistently detected in both auxin (n=14) and oryzalin-induced outgrowths (n=16)(Figure 2b).

To explore this further, we followed the expression dynamics of the pANT::erGFP fluorescent reporter. We observed that pANT::erGFP expression was initiated already after 48h of the local oryzalin application (n=10). Later on, the expression of ANT expanded throughout the lateral outgrowths (Figure 3a).

Together these results suggest that microtubule disorganization in addition to promoting lateral outgrowths at the periphery of the SAM also induces the expression of the developmental patterning gene ANT and possibly of LFY, AIL6 and MP although this remains to be confirmed.

CMT disorganization promotes outgrowths and ANT expression independently of the auxin master regulator MP

Only 6 out of 20 outgrowths in 5 pin1-6 bot1-7 meristems accumulated MP transcripts; whereas 18 out of 21 outgrowths in 7 pin1-6 bot1-7 meristems accumulated transcripts of ANT. This made us wonder if ANT could be activated independently of MP (Figure 1). To enquire this further, we tested the activation of the synthetic auxin-signalling reporter DR5::VENUS-N7 after oryzalin-induced microtubule disorganization. Although local oryzalin applications induced the production of lateral outgrowths (60%, n=24), these outgrowths did not show the activation of the fluorescent reporter (93%, n=14) (Figure 3b). In contrast, auxin-induced outgrowths systematically expressed the fluorescent DR5 reporter (100%, n=10) (Figure S1). These results suggest that the initiation of lateral outgrowths by growth isotropy may be independent of auxin transcriptional regulation.

To further explore whether the formation of lateral outgrowths following the disruption of CMT anisotropy occurs independently of MP, we examined the MP strong mutant allele arf5-1. These mutants display a phenotype reminiscent of pin1: they fail to form lateral flowers, which results in naked pin-like stems [START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF]. As in pin mutants, they exhibit supracellular circumferential CMTs alignments that reinforce growth anisotropy via the parallel deposition of cellulose microfibrils (see chapter 1). Local disorganization of the CMT of arf5-1 shoot apices, achieved by oryzalin treatments or by crossing with bot1-7, was able to restore the formation of lateral outgrowths (chapter 1, Figure 4a). These results were corroborated with a second weak mutant allele arf5-2, which was also crossed with bot1-7.

Double mutants arf5-2 bot1-7 enhanced the production of lateral outgrowths in comparison with the single mutants arf5-2 (Figure 4b).

We used the double mutants arf5-1 bot1-7 and arf5-2 bot 1-7 to investigate if the expression of ANT could be induced independently of MP regulation. Indeed, lateral outgrowths produced in the double mutant arf5-1 bot1-7 and arf5-2 bot 1-7 correlated with the expression of ANT as detected by whole mount in situ hybridization (Figure 4). Together, these findings indicate that the initiation of lateral outgrowths in the SAM as well as the expression of ANT can be induced independently of MP transcriptional regulation.

Identification of putative ANT transcriptional regulators

The previous results suggested that during organogenesis, ANT expression could be regulated by transcription factors other than MP. To explore this further, we performed an Enhanced yeast one-hybrid (eYIH) assay in order to identify putative transcription factors regulating ANT during lateral outgrowth formation. Since ANT and AIL6/PLT3 are related transcription factors with partially overlapping roles in flower primordium initiation [START_REF] Han | AINTEGUMENTA-LIKE6 can functionally replace AINTEGUMENTA but alters Arabidopsis flower development when misexpressed at high levels[END_REF][START_REF] Krizek | AINTEGUMENTA and AINTEGUMENTA-LIKE6 act redundantly to regulate Arabidopsis floral growth and patterning[END_REF][START_REF] Krizek | AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development[END_REF][START_REF] Krizek | RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways[END_REF][START_REF] Yamaguchi | AINTEGUMENTA and AINTEGUMENTA-LIKE6/PLETHORA3 Induce LEAFY Expression in Response to Auxin to Promote the Onset of Flower Formation in Arabidopsis[END_REF], we included AIL6 in our analyses. We prepared DNA baits of ANT and AIL6 promoters that contained portions of 2000 bp sequences immediately upstream of the translational start site. These promoters were sufficient for expression in the meristem as confirmed by fluorescent reporters. The bait promoters were fused to the reporter genes lacZ and HIS3 and screened against a complete collection of 2000 Arabidopsis transcription factors (TF) fused to a transcription activation domain and used as prey [START_REF] Pruneda-Paz | A genome-scale resource for the functional characterization of Arabidopsis transcription factors[END_REF]. As many as 42

TFs had the potential to bind ANT promoter (2.1% from the complete TF collection), whereas 105 TFs (5.25%) came up as putative regulators of AIL6 (Table S1). Of these, five TF appeared to be potential common regulators of both genes; the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 10 (SPL10), the C2H2-like zinc finger family TF NUCLEAR CAGE (NUC), the Acyl-CoA N-acyltransferase with RING/FYVE/PHD-type zinc finger domain-containing protein encoded by AT3G53680, the DRE-binding protein 2A (DREB2A)

and the NAC DOMAIN CONTAINING PROTEIN 4 (NAC4). Of these, only DREB2A and NAC4 are expressed in the inflorescence meristem at a relatively high level according to public resources [START_REF] Winter | An "Electronic Fluorescent Pictograph" browser for exploring and analyzing large-scale biological data sets[END_REF] and a high-throughput transcriptomic analysis [START_REF] Mantegazza | Gene coexpression patterns during early development of the native Arabidopsis reproductive meristem: novel candidate developmental regulators and patterns of functional redundancy[END_REF], which place them among the 30% transcripts with highest expression in the inflorescence meristem.

The candidates with the potential to bind to ANT promoter and that are highly expressed in the meristem (following the same criteria as above) included (Figure 5): the basic leucine zipper BZR2, also known as BRI1-EMS-SUPPRESOR 1 (BES1) and IWS1, represented two promising candidates identified in the eYIH screen based on the following criteria. First, BES1 and IWS1 interact to direct BR-regulated gene expression, thus they belong to the same signaling pathway [START_REF] Li | Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression[END_REF]. Second, both BES1 is expressed in the incipient flower primordia of WT plants and in the lateral outgrowths of pin1-6 bot1-7 (Figure 6). Third, BRs signaling in the shoot epidermis has a major role in facilitating shoot growth [START_REF] Savaldi-Goldstein | The epidermis both drives and restricts plant shoot growth[END_REF]. Fourth, BRs might play important roles in cell wall anisotropy via the rearrangement of the cortical microtubules [START_REF] Catterou | Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant[END_REF][START_REF] Takahashi | The DIMINUTO gene of Arabidopsis is involved in regulating cell elongation[END_REF] or in cell wall loosening via feedback regulation of pectin (Wolf et al., 2012b;[START_REF] Wolf | A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling[END_REF], they might also induce the expression of genes encoding for cell wall associated proteins [START_REF] Xie | Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis[END_REF][START_REF] Yin | BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation[END_REF]. Finally, evidence from Arabidopsis and rice suggest that high BR activity enhances cell proliferation and cell differentiation in the SAM, and antagonize boundary formation between organs including the SAM boundary [START_REF] Arnaud | Plant development: brassinosteroids go out of bounds[END_REF][START_REF] Bell | Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries[END_REF][START_REF] Gendron | Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis[END_REF][START_REF] Tsuda | Genome-wide study of KNOX regulatory network reveals brassinosteroid catabolic genes important for shoot meristem function in rice[END_REF].

To explore whether BR signaling could be involved in the formation of lateral outgrowths in the SAM, we tested if plants grown on NPA or pin1 mutants were able to restore the initiation of lateral outgrowths following treatments with the BR 24-epi-brassinolide (EBL). Treatments with 100nM EBL induced lateral outgrowth formation in pin1 mutants (11 out of 15 meristems tested) in comparison with DMSO treatment (2 out of 15) (Figure 4a). We next tested the induction of ANT expression following BL treatments. To this end we followed the expression pattern of NPA-grown fluorescent pANT::erGFP reporter. ANT transcriptional activity was increased already after 48h after treatment with EBL (4 out of 15 meristems) or 72h (increasing to 9 out 15 meristems) (Figure 4b), in contrast with DMSO treatment (1 out of 9 meristems). Together our results suggest that in addition to auxin, BR signaling can interfere with the cell mechanical properties that lead to organ initiation. BR signaling might even represent a link between microtubule-driven growth isotropy and the transcriptional activation of ANT.

Discussion

Microtubule disorganization triggers the transcriptional activation of ANT

The molecular pathway controlling flower primordium initiation in Arabidopsis has been relatively well characterized. The most widely accepted view proposes that auxin accumulates at precise sites of the SAM and triggers a molecular network largely via the activation of ARF5/MP [START_REF] Wu | Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate[END_REF][START_REF] Yamaguchi | A molecular framework for auxinmediated initiation of flower primordia[END_REF] and its direct downstream targets. These downstream targets then directly or indirectly control cell wall modifications that lead to growth. Several observations, however, indicate that this view of a hierarchical regulatory chain is probably oversimplified. Our previous work [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF] proposed that auxin might also more directly act on the cytoskeleton organization and wall anisotropy via ROP signaling, indicating the existence of parallel signaling pathways. We show here also the existence of feedbacks from downstream elements, as the disorganization of the CMT arrays at the periphery of the pin1 and mp/arf5 SAMs, is sufficient to induce at least ANT expression, i.e. a direct target of MP high in the hierarchy of the molecular network.

Importantly, we show here that ANT activation can occur independently from MP, suggesting additional layers of regulation.

Our eY1H analysis suggests that multiple TF and signaling pathways have the potential to regulate ANT and AIL6, most likely in parallel with MP (Figure 5, Table S1). Among the TF discovered as possible regulators of ANT, some are associated to precise signaling pathways.

For example, FD that interacts with FLOWERING LOCUS T (FT) to promote flowering [START_REF] Abe | FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex[END_REF], TOC1 that is involved in the generation of circadian rhythms [START_REF] Strayer | Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog[END_REF] and ATHB4, which is involved in shade avoidance syndrome [START_REF] Carabelli | The Arabidopsis Athb-2 and -4 genes are strongly induced by far-red-rich light[END_REF][START_REF] Sorin | ATHB4, a regulator of shade avoidance, modulates hormone response in Arabidopsis seedlings[END_REF] as well as in apical embryo development and meristem functions [START_REF] Turchi | Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function[END_REF]. Other TFs are associated with broader aspects of transcriptional regulation, for example HTA13 and DREB2A. The latter, showed up as common regulator of ANT and AIL6. DREB transcription factors recognize the dehydration-responsive element sequence motif in the promoters of stress-inducible genes. DREB2A is specifically involved in responses to salinity, heat, drought and cold [START_REF] Liu | Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and lowtemperature-responsive gene expression, respectively, in Arabidopsis[END_REF][START_REF] Nakashima | Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration-and high-salinity-responsive gene expression[END_REF].

Another putative common regulator is SPL10, that in conjunction with SPL11 and SPL12 redundantly controls the development of lateral organs and shoot maturation in the reproductive phase [START_REF] Shikata | Arabidopsis SBP-box genes SPL10, SPL11 and SPL2 control morphological change in association with shoot maturation in the reproductive phase[END_REF]. More than 5% from the complete collection of transcription factors used for this study could potentially regulate AIL6. Among them we discovered the VIRE2-INTERACTING PROTEIN1 (VIP1), which is a mechano-sensitive transcription factor that localizes to the nucleus upon hypo-osmotic treatment (Tsugama et al., 2016a, b). Although the validity of the eY1H results still needs to be verified, these assays have proven to be strong tools for hypothesis generation as illustrated by Sparks et al., who in a recent report generated an eYIH-based model for the root ground tissue, of which they validated in planta many (>50%) of the interactions obtained by the assay [START_REF] Sparks | Establishment of Expression in the SHORTROOT-SCARECROW Transcriptional Cascade through Opposing Activities of Both Activators and Repressors[END_REF].

BES1 and IWS1, putative regulators of ANT, are associated to the BR signaling pathway.

Since BR signaling has been associated with meristem function but its function remains poorly characterized, we chose to examine the possible role of this signaling pathway more in detail.

In general, BR signaling has been associated with cell expansion. Mutants impaired in BR production or signaling are dwarf, which seems to result from a reduced expansion of dividing and non dividing cells (Nakamura et al., 2006a;[START_REF] Nakaya | Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana[END_REF][START_REF] Savaldi-Goldstein | The epidermis both drives and restricts plant shoot growth[END_REF]. A limited number of studies have also revealed a role in patterning.

BR signaling in the growing primordia of the SAM prevents the expression of genes associated with the boundary domain including the CUC genes [START_REF] Bell | Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries[END_REF][START_REF] Gendron | Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis[END_REF]. Conversely, the boundary specific gene LATERAL ORGAN BOUNDARIES (LOB) inhibits the accumulation of BR in the organ boundaries [START_REF] Bell | Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries[END_REF].

Interestingly, a similar regulation might be involved in the control of ovule primordia initiation [START_REF] Cucinotta | Ovule development, a new model for lateral organ formation[END_REF]. Auxin-induced ANT promotes growth of ovule primordia, whereas CUC genes play a role in the establishment of the ovule primordia boundaries [START_REF] Galbiati | An integrative model of the control of ovule primordia formation[END_REF]. In this model, ANT expression was detected to be regulated by BR signaling, as shown by qRT-PCR and ChiP-PCR experiments [START_REF] Huang | BR signal influences Arabidopsis ovule and seed number through regulating related genes expression by BZR1[END_REF].

Our results further confirm a direct role of BR signaling in organ formation via the transcriptional regulation of ANT. This is not only suggested by the eY1H experiments, but also by the induction of pANT::erGFP after BR treatment of pin meristems and the localized expression of BES1 in the lateral outgrowths of pin1-6 bot1-7. To substantiate these results, bri1-5 insensitive mutant [START_REF] Noguchi | Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids[END_REF] has been crossed with the pin1-6 bot 1-7 double mutant. If indeed CMT disorganization acts via BR regulation, we would expect that bri1-5 suppresses lateral outgrowths triggered in the pin1-6 bot1-7 background. This work is in progress.

How the disruption of CMT anisotropy acts on gene expression remains unclear. The cells could sense changes in cell wall isotropy. A good candidate to fulfill as integrator between cell wall and BR signaling is the receptor-like protein (RLP)44 (Wolf et al., 2012b;[START_REF] Wolf | A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling[END_REF]. Presumably, the interaction of RLP44 with the BR pathway is through the BR coreceptor BAK1 [START_REF] Wolf | A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling[END_REF]. Although RLP44 is not part of the BR core pathway, it is proposed that it could favor the BR receptor complex BAK1-BRI1 and in turn activate the BR signaling pathway. Since RLP44 is expressed in the shoot apex inflorescence [START_REF] Wolf | A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling[END_REF] it would be worth testing if it could be a downstream component of the disruption of CMT anisotropy in the SAM, receiving information from the cell wall and laterally activating BR signaling. Certainly, the analyses of rlp44 mutants and RLP44 overexpression lines would give insight to this question.

Conclusions and perspectives

In conclusion our results indicate that cellular, mechanical anisotropy at the shoot apex links together organ initiation and transcriptional regulation. We previously suggested that this regulation involved cell wall remodeling genes (chapter 2). Here we propose that this coupling mechanism also affects genes involved in organ patterning. The feedback mechanism itself remains elusive but our results support a model in which organ initiation receives multiple inputs, which opens to a myriad of possibilities. In particular, we show here the potential role of BR signaling in the regulation of organ initiation and transcriptional activation. Signal integration and crosstalk with other pathways has been documented for BR signaling. Thus, BRs might act in parallel or in synergy with auxin to activate growth anisotropy and/or transcription. Now the challenge is to reveal how this coordination between anisotropy and transcriptional regulation is modulated throughout plant development

Experimental procedures

Plant materials

Arabidopsis thaliana ecotypes Columbia-0 (Col-0) were the wild types seeds used in this study. Transgenic lines and mutants used in this work were for the most part previously described. The pin1-6 [START_REF] Vernoux | PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem[END_REF], bot1-7 [START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF], pin1-6 bot1-7 [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF], arf5-1 [START_REF] Przemeck | Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization[END_REF], arf5-2 [START_REF] Donner | Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves[END_REF], bri1-5 [START_REF] Noguchi | Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids[END_REF], DR5::VENUS-N7 [START_REF] Heisler | Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem[END_REF], pANT::erGFP and pLFY>GFP [START_REF] Grandjean | In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis[END_REF]. The arf5-1 bot1-7, arf5-1 PDF1::mCitrine-MBD lines were generated by crossing and further PCR selection. Growth conditions were previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

Growth conditions and Treatments

For napthylphtalamic acid (NPA) treatments, plants were sown in petri dishes with a medium adapted for Arabidopsis (Duchefa) containing NPA to a final concentration of 10µΜ.

Lanolin paste for local SAM treatments was prepared as follows: 1 volume of chemical stock solution (3x concentrated) was added to 2 volumes of melted (55°C) lanolin and thoroughly mixed until the formation of a homogenous emulsion. The concentration of the stocks was 3mMfor IAA and 666 µg/ml for oryzalin. Local applications were made with a pipette tip und er a binocular. Equal amounts of DMSO were used for untreated controls. Treated meristems were imaged prior the treatment and then 24h, 48h, 72h and 96h after. For eBL induced organ formation, NPA grown plants were treated in petri dishes with 100nM eBL liquid solution for 3h after imaging the t=0 time point and again the next day after imaging the t=24h time point.

No further treatment was done after the 24h time point until the completion of the experiment.

pin1-6 treated plants were sown in petri dishes with MS medium. After phenotype-based selection, homozygous pin1-6 plants were transferred to new fresh MS medium, leaving enough space to allow the plants to develop in vitro. eBL treatments were performed as previously described. Plants were kept in the petri dishes for all the duration of analyses. For adventitious root formation of arf5-1, seeds were surface-sterilized and germinated on MS medium. After germination, homozygous arf5-1 seedlings were identified by the rootless phenotype. Mutant plants were wounded below cotyledons as earlier described [START_REF] Berleth | The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo[END_REF], then transferred to MS medium supplemented with the synthetic auxin 1naphthaleneacetic acid (NAA) to a final concentration of 10µM and kept on these plates for 7 days, when they were transferred to free MS medium free of NAA to allow the elongation of the root. Plants were either kept in plates or transplanted to soil after rooting, where they grew under a 16-h-light/8-h-dark photoperiod.

Confocal live Imaging and image analyses.

Confocal imaging was carried out on a Zeiss LSM700 system as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. SEM imaging was carried out on a Hirox SE-3000 system as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

Histochemistry

RNA in situ hybridization assays were performed as described previously (Ferrandiz and Sessions, 2008a, b). For whole-mount RNA in situ hybridization assays, meristems from WT plants, pin1-6, pin1-6 bot1-7, arf5-1, arf5-1 bot1-7 grown on soil were processed as previously described [START_REF] Rozier | Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization[END_REF]. At least three independent experiments for both assays were performed for each probe tested. Primers used are listed in Table S2.

Enhanced Yeast-One-Hybrid

The eYIH assays were performed as previously described [START_REF] Gaudinier | Enhanced Y1H assays for Arabidopsis[END_REF][START_REF] Taylor-Teeples | An Arabidopsis gene regulatory network for secondary cell wall synthesis[END_REF]. Briefly, gene promoters (2000bp of upstream regulatory region from the translational start site, or the next gene) were cloned and recombined to reporter vectors pMW2 (Y1H HIS3 reporter vector) and PMW3 (YIH LacZ reporter vector) [START_REF] Brady | A stele-enriched gene regulatory network in the Arabidopsis root[END_REF]. Interactions were called for transcription factors that activated at least one reporter assay. In addition gene promoters were fused to the mCitrine targeted to the nucleus through an SV40 NLS using the Gateway system. Final destination vectors were obtained by using the three-fragment recombination system using the pB7m34GW destination vector. Constructs were transformed into Arabidopsis Col-0 ecotype. Primers used are listed in Table S3.

A case study: the initiation of lateral organs in the inflorescence meristem

The shoot apical meristem (SAM) has been a very important system for studying pattern formation and morphogenesis in plants. The shape of this structure results from differential growth of its regions. The central region, which contains the stem cell niche, grows at a slow pace compared to the surrounding periphery. Cells participating in the formation of a new primordium further increase their growth rate, while expansion in the organ boundary region between the meristem and the primordium slows down [START_REF] Grandjean | In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis[END_REF][START_REF] Kwiatkowska | Growth and morphogenesis at the vegetative shoot apex of Anagallis arvensis L[END_REF][START_REF] Reddy | Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana[END_REF].

When I started my thesis, the available data suggested a relatively straightforward scenario for organ initiation, where auxin accumulation would initially activate MP. MP would then directly induce the expression of LFY, ANT and AIL6. Together with at least one additional regulator, these three transcription factors would then control the plastic, turgor driven deformation of the cell wall. The work described in this thesis has revealed additional levels of regulation and poses a number of important perspectives for further research.

Auxin might control organ formation in part via a direct control of microtubule dynamics.

Previous work has suggested the existence of a mechanical feedback loop, where microtubules would align to stress patterns and reinforce the cell wall along the main force vectors. The precise mechanism by which microtubules respond to mechanical stress is still unclear. However, KTN1 severing activity is required for responding to growth-derived mechanical stress [START_REF] Sampathkumar | Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells[END_REF][START_REF] Uyttewaal | Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis[END_REF]. KTN1 promotes microtubule bundling, which allows the reorganization of CMT into parallel arrays [START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF][START_REF] Zhang | Microtubule severing at crossover sites by katanin generates ordered cortical microtubule arrays in Arabidopsis[END_REF]. Normally, this behavior of the CMT would lead to the formation of a radial symmetric axis. Our results provide evidence for a scenario where auxin induced organ initiation would involve an inactivating of this feedback. In line with this, reduced microtubule organization in the apices of bot1-7 mutants triggers isotropic cellular growth and the formation of ectopic organs. We further show that auxin might directly act on microtubule dynamics via a ROP6/KTN1 signaling pathway. It is tempting to hypothesize that this pathway modulates microtubule ordering, thus participating in the establishment of growth patterns at the meristem. This pathway has been thoroughly studied in leaf pavement cells, where it was described to be under auxin control [START_REF] Lin | Rho GTPase signaling activates microtubule severing to promote microtubule ordering in Arabidopsis[END_REF]. Several important questions remain to be answered. First, the mechanism by which auxin activates the ROP6/RIC1 pathway remains to be clarified, especially in light of the controversial role of ABP1 in this process [START_REF] Feng | Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1[END_REF]. Alternatively other ROP activators might be considered, for instance FERONIA (FER), a receptor-like kinase (RLK) of the Catharanthus roseus family (crRLKs). FER is required for transduction of mechanical signals in

Arabidopsis thaliana roots [START_REF] Shih | The receptor-like kinase FERONIA is required for mechanical signal transduction in Arabidopsis seedlings[END_REF]. ROPs can act downstream of FER during mechanical stress transduction to regulate ROS production [START_REF] Duan | FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development[END_REF]. The relatively weak phenotypes of ROP and RIC mutants might also indicate that other ROPs are involved.

In particular ROP2, ROP9 and ROP10, which are also expressed at the meristem [START_REF] Mantegazza | Gene coexpression patterns during early development of the native Arabidopsis reproductive meristem: novel candidate developmental regulators and patterns of functional redundancy[END_REF]. A thorough genetic analysis using multiple knockouts might further shed light on this.

Although auxin thus has the possibility to interfere directly with the cytoskeleton, we also found that the reorientation of CMT requires MP [START_REF] Bhatia | Auxin Acts through MONOPTEROS to Regulate Plant Cell Polarity and Pattern Phyllotaxis[END_REF]. We speculate, that MP must somehow control one of the components of the ROP signaling pathway to modulate the mechanical feedback.

Wall anisotropy acts together with cell wall remodeling to promote organ formation

Our results, confirm and further complete results obtained by others [START_REF] Braybrook | Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin[END_REF]Peaucelle et al., 2011a;[START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF][START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF], indicate that cell wall anisotropy does not act alone to initiate organ formation as it needs to be combined with a change in cell wall stiffness. The expression of a number of xyloglucan (XyG) and pectin remodeling genes, specific of organ outgrowth, confirms this. A small number of these genes are at least indirectly regulated by auxin. Importantly, we identified a cross talk between wall remodeling and cytoskeleton organization. As described in chapter 2 PME3, XTH9, and EXPA15 expression is induced after CMT-driven growth isotropy, while perturbing xyloglucan or pectin remodeling affects microtubule alignment. How cell wall properties affect the cytoskeleton remains an open question. As such this doesn't seem to involve auxin and auxin trafic. A possibility is, that the cell somehow senses wall composition. The potential cell surface signaling capacity of RLKs makes them good candidates to explore. Among them a few are binding to pectins, such as the wall-associated kinases (WAKs) [START_REF] Decreux | Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation[END_REF][START_REF] Kohorn | Pectin activation of MAP kinase and gene expression is WAK2 dependent[END_REF][START_REF] Kohorn | The cell wall-associated kinases, WAKs, as pectin receptors[END_REF][START_REF] Wagner | Wall-associated kinases are expressed throughout plant development and are required for cell expansion[END_REF]. The previously mentioned crRLKs are other candidates to consider, especially THESEUS (THE) [START_REF] Hematy | A receptor-like kinase mediates the response of Arabidopsis cells to the inhibition of cellulose synthesis[END_REF] which is likely in charge of surveying cellulose synthesis and perceiving cellulose modification. RLKs in theory could act in synergy with other mechanosensory molecules, mechanosensitive ion channels for instance [START_REF] Hamilton | United in diversity: mechanosensitive ion channels in plants[END_REF]. The cellular signaling pathways could then interact with cytoskeleton organization, for example via the ROP/KTN pathway described in chapter 1 and [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

While cell wall modifications might directly influence cytoskeleton organization, we show here that CMT arrangements affect wall remodeling via transcriptional regulation. Identifying the transcription factors involved is an important and challenging objective. The eY1H analysis has identified a range of transcription factors that potentially control XTH4, XTH9, EXPA4 and EXPA15. Some of these have been associated with auxin signaling (e.g. ARF 18

and ARF 22). However links with other hormonal pathways can also be made (e.g. LOB, involved in BR signaling and interacting with the EXPA4 promoter in the 1H assay). This would explain why the outgrowths induced by perturbing CMT organization could induce the expression of these genes in the absence of auxin transport.

Crosstalks are not only limited to the cytoskeleton and cell wall remodeling. Different components of the wall remodeling machinery itself are also interacting. Although in the past XyGs and pectins were conceived as independent mechanisms in control of plant cell growth, recent studies have shown that the two systems are mutually dependent. Several studies revisiting cell wall structure suggests that in the absence of XyGs, pectins act as load-bearing components [START_REF] Cavalier | Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component[END_REF]Park and Cosgrove, 2012a). This and other compensation mechanisms that guarantee a robust control of wall mechanics are still poorly understood.

Microtubule-driven growth isotropy promotes the expression of patterning genes

The auxin-dependent transcriptional network is also regulated by CMT-driven isotropic growth, although only at certain level. Consistently, the expression of ANT was activated downstream of CMT disorganization at the outgrowing cells. The nature of the mechanism coupling the changes in CMT dynamics and transcriptional activation remains to be determined. Since, the activation of this mechanism can be triggered in the absence of auxin transport or signaling, it might even act in parallel or in synergy with auxin.

Beyond ARF5/MP only a few regulators of ANT have been described. Here we found that ANT cis regulation can be broad and that it might include BR signaling as was also shown for ovule development [START_REF] Huang | BR signal influences Arabidopsis ovule and seed number through regulating related genes expression by BZR1[END_REF]. BRs play essential roles in growth control and regulate the expression of cell wall-related genes. Although there is no strong evidence for this yet, we are tempted to hypothesize that BR signaling couples CMT disorganization with the transcriptional activation of both patterning and cell wall related genes. Recently, a receptor-like protein (RLP)44 was described as an integrator of cell wall homeostasis and BR signaling (Wolf et al., 2012;[START_REF] Wolf | A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling[END_REF]. Therefore, we wonder about the possibility that a cell wall mediated activation of BR signaling regulates the transcriptional feed back we have described here.

Table 1: Mechanical and growth parameters used in our simulations.

Mechanical description of the cell wall

We consider the cell walls as a linearly elastic continuum with transverse isotropy. This means that they feature a direction of higher rigidity. Their elastic behavior is characterized by the corresponding Hooke's tensor (H in equation ( 1)).

Y ie ff = Y i 1-µ x µ x Y xe f f µ x = Y ef f µ x (1)
Where Voigt notation is used (i.e. σ t = f σ xx σ σ x g and ε t = f ε xx ε 2ε x g ).

Structural considerations

From a structural perspective, the cell wall is considered as a fiber reinforced linear elastic continuum. Its structural anisotropy is quantified by its constituting fibers angular distribution ρ (θ ),aπ -periodic function for the fibers are not oriented, see figure 1-E, H & J. Note that by construction we choose ρ (θ ) to be even, meaning that its maximum is along the x-axis (i.e. max (ρ) = ρ (0)).

By assuming that each fiber has the same linear elastic behavior, characterized by the stiffness coefficient k and their resting length l 0 , we can relate the elastic coefficients displayed in Hooke's matrix (H in equation ( 1)) to the fibers angular distribution, as exposed hereafter in equation [START_REF] Bozorg | Stress and Strain Provide Positional and Directional Cues in Development[END_REF].

Y xef f = π 16 Y 0 (6 ρ0 + 4 ρ1 + ρ2 ) Y ef f = π 16 Y 0 (6 ρ0 -4 ρ1 + ρ2 ) G = π 8 Y 0 (2 ρ0 -ρ2 ) µ x = 2 ρ0 -ρ2 6 ρ0 +4 ρ1 + ρ2 µ x = 2 ρ0 -ρ2 6 ρ0 -4 ρ1 + ρ2 (2)
Where Y 0 = kl 0/2 and the ρk are the k th Fourier's coefficients of ρ (θ ): For the sake of simplicity we considered in our simulations the simplest angular distribution possible, the unit step function:

ρ (θ ) = ρ 0 ⇣ 1 - α ∆ρ πρ 0 + ∆ρ ρ 0 Π α (θ -θ 0 ) ⌘ with : Π α (θ ) = ( 1 θ 0 -α /2  θ  θ 0 + α /2 0 elsewhere (4)
With this parametrization ρ 0 stands for the fibers mean angular density ( 1 π R π ρdθ = ρ 0 ), ∆ρ stands for the amplitude step between the directions of low (ρ low = ρ 0 -∆ρ α /π ) and high (ρ hi h = ρ 0 + ∆ρ (1 -α /π )) density and α stands for the angular aperture of the distribution, see figure 1-J . This specific expression for ρ (θ ) yields the following expressions for Hooke's matrix coefficients: (5)

Y xef f = 3π 16 Y 0 ρ 0 ⇣ 1 +
To visualize the corresponding rigidity tensor, we plotted the norm of its projection in every angular directions (named angular rigidity and noted Y (θ ) hereafter):

Y (θ ) = kH : P θ k (6)
where P θ = êθ ⌦ êθ is the projector in the direction given by the unit vector êθ = f cos (θ ) sin (θ ) g t and k•k depicts the second order tensorial norm defined as follow: kM k =

Numerical implementation

We implemented the mechanical model with the numerical framework described in [START_REF] Boudon | A computational framework for 3D mechanical modeling of plant morphogenesis with cellular resolution[END_REF] .To implement the mechanical behavior prescribed by expressions (5) in the simulations, we use the Sofa module HookeOrthotropicForceField with specific definitions for the various coefficients as exposed in the python code below. Yiso = 150 muiso = 1./3. Giso = Yiso * (1 + muiso ) In the previous code the parameters "a" and "d" correspond respectively to the angular aperture (α) of the microfibrils distribution ρ (θ ) and its relative directional enrichment ( ∆ρ /ρ 0 ). We changed the aperture value to to simulate a change in the anisotropy of the fibers angular distribution. The complete list of parameters used in the various simulations are given in Tabs.2 to 4. .5 .5 0 0

9 > > > > = > > > > ; 1
g 9 > > > > > > = > > > > > > ;
Table 2: Numerical values of the parameters used in the simulations of Case #1. .5 .5 0 0

Mechanical parameters

9 > > > > = > > > > ; 1 1 8 > > > > < > > > > : 1 1 1 1 9 > > > > = > > > > ; • 150 1 /3 8 > > > > > > < > > > >

Figure 1 .

 1 Figure 1. The principles of reaction-diffusion and examples of real patterning systems (adapted from Green and Sharpe, 2015). (a) Morphogen fluctuation generates higher levels of activator, lower and more diffuse inhibitor levels. Although the inhibitor fails to repress the activator, it prevents the activator region from growing and imposes a zone of "lateral inhibition". The system dynamically changes until it reaches an equilibrium. (b) Mouse limb buds are created as a Turing pattern guided by a feedback loop between the signalling of Wnt and Bmp and Sox9. (c) Mouse embryo body sides, left (L) and right (R) are dictated by a RD system, which include Nodal and Lefty.

Figure 2 .

 2 Figure 2. The principles of positional information and examples of real patterning systems (adapted from Green and Sharpe, 2015) (a) The concept of positional information arises from molecular asymmetries that result in the graded distribution of a morphogen, cells make use of this information to acquire different cell fates, represented in the scheme by the three different colours.(b) Initial asymmetries in the anterior-posterior axis of the early Drosophila embryo result in the graded distribution of morphogens that in turn regulate the expression of the gap genes providing the positional information for the segment polarity genes, expressed as a series of stripes. (c) Differences in the concentration of the morphogens Bicoid and Caudal across the embryo inputs the gap genes network (giant, hunchback, Krüppel, and knirps). In turn, the complex interactions among gap genes create molecular patterns of expression of the segment polarity genes (even-skipped, runt, hairy).

Figure 3 .

 3 Figure 3. Waddington's epigenetic landscape (adapted from Waddington 1957). (a) The classical view of an epigenetic landscape, in which the differentiation of cell in an embryo is illustrated as a pebble that begins at the top of a hill and rolls down the epigenetic landscape though a series of branching points that represent decision events. Cells evolve according to the same laws, but because of the existence of inducing signals, cells in different regions would follow different pathways and end up at different states of differentiation represented as valleys. The effect of these signals is restricted to reacting tissue and ultimately trigger the cells select one of a few possible developmental pathways. (b) The genetic landscape underlying the architecture of the epigenetic landscape. The valleys are formed by tension on ropes attached to gene complexes represented as cylindrical pins stuck in the ground.

  rely on mobile signals to trigger the local differences that guide tissue morphogenesis. A major category of these mobile signals is plant hormones, which largely control plant growth and development and represent excellent candidates for plant morphogens.

Figure 4 .

 4 Figure 4. Auxin graded distribution in root and shoot morphogenesis[START_REF] Petrasek | Auxin transport routes in plant development[END_REF]. (a) Directional flow of auxin in the shoot and root of Arabidopsis. The accumulation of auxin at the location of organ initiation (green) is maintained by flow towards root and shoot apices (solid arrows). Reverse flow towards root and shoot basis (dashed arrows). (b) Auxin transport in the developing lateral root. Auxin maxima specify the founder cells in the pericycle, subsequent coordinated divisions form the lateral root primordium. PIN1 and PIN2 facilitate the transport of auxin that enables the development of the primordia. (c) Auxin transport in the shoot apical meristem in developing primordia (P1 and P2). Auxin is transported through the epidermis layer L1 by the activity of PIN1, maintaining an auxin maxima at the organ primordium tip. From there, a basipetal transport route is established through the interior of the primordium, marking the future vasculature tissues.

Figure 5 .

 5 Figure5. Underlying processes of auxin differential distribution[START_REF] Finet | Auxology: when auxin meets plant evo-devo[END_REF]. (a) Auxin biosynthesis and storage as inactive conjugates, which involves enzymes of the GH3 family. Intracellular homeostasis of auxin provided by ER localized PINs and PIN-LIKE proteins (PILS)[START_REF] Barbez | A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants[END_REF]. (b) Polar auxin transport depends on influx (AUX/LAX) and efflux carriers (PIN and ABCB/PGP) that promote the uptake and release of auxin to the apoplast. The endocytic trafficking and polar recycling of PINs is illustrated. Hormonal regulation of these pathways include auxin feedback regulation, cytokinin control over PIN endocytosis[START_REF] Marhavy | Cytokinin modulates endocytic trafficking of PIN1 auxin efflux carrier to control plant organogenesis[END_REF], and gibberellin regulation of PIN trafficking to lytic vacuoles[START_REF] Willige | Gibberellin regulates PIN-FORMED abundance and is required for auxin transport-dependent growth and development in Arabidopsis thaliana[END_REF] (c) Auxin perception and signalling

Figure 7 .

 7 Figure 7. Brassinosteroids signal transduction pathway (adapted from Belkhadir and Jaillais, 2014) OFF stands for inactive pathway, whilst ON portrays the active pathway. In the presence of Brassinolide, BRI1 kinase phosphorylates BKI1 and in turn interacts with BAK1. A series of phosphorelay steps follows; firstly the phosphorylation and activation of BSK1 and CDG1 kinases, which then leads to the phosphorylation of BSU1 phosphatase. BSU1 dephosphorylates and inactivates BIN2, allowing the nuclear translocation of BZR1 and BES1 transcription factors. Once inside the nucleus BZR1 and BES1 are able to bind the promoter of their target genes aided in certain cases by other transcription factors.

Figure8.

  Figure8. Cellulose synthesis at the plasma membrane (adapted from[START_REF] Mutwil | Cellulose synthesis: a complex complex[END_REF])) (a) The CESA complex (red) is transported from the Golgi to the plasma membrane via exocytosis. Cellulose microfibrils are synthesized directly in the plasma membrane. During this process the synthesis machinery is guided by the cortical microtubules (blue). The inset shows a YFP-tagged CESA6 (green) and a CFP-tagged tubulin (red). (b) Studies of cellulose biosynthesis inhibition performed in the upper hypocotyl. Treatment with isoxaben targets cellulose synthesis, while treatment with oryzalin targets MT. The summary of the observations during the drug treatments is displayed on the left, while on the right the time average images of YFP-CESA6 are displayed.

  might be compensated either by an altered β-glucan backbone able to function in a similar fashion to XyG, or by modification of other components (i.e. pectin cross-linking) of the cell wall. This unexpected result opened new questions, which contribute to the reexamination of the role of XyG in primary cell wall.

Figure 9 .

 9 Figure 9. Xyloglucan structure (adapted from Park and Cosgrove, 2015) (a) Structure of XLLG oligosaccharide, showing the β-(1-4)-glucan backbone (gray) with side chains, xylose (green) and galactose (blue). (b) XLFG oligosaccharide with its pattern of linkages with the associated glycosyl transferases indicated in upper case letters.

Figure 10 .

 10 Figure 10. Two models of primary cell wall structure (adapted from Cosgrove 2016) (a) The "tethered network" model suggests that the cellulose microfibrils (red) are well separated by xyloglucans (blue), whose role is to fasten the microfibrils resulting in a load-bearing network. (b)The "biomechanical hotspot" model proposes a mixture of xyloglucan-cellulose (green) to be in charge of the limited cellulose-cellulose connections. These sites, the biomechanical hotspots, have limited enzymatic accessibility and may be subject to the loosening activity of expansins

Figure 11 .

 11 Figure 11. Formation of pectin networks (adapted from Cosgrove, 2005) (a) Pectin domains covalently crosslink each other, thay also formlinkages involving boron and calcium. (b) Homogalacturonana form stiff gels through Ca 2+ mediated crosslinking of its carboxyl groups.

Figure 12 .

 12 Figure 12. Two-step mechanism for anisotropic plant cell growth (adapted from Bidhendi and Geitmann, 2016) (a) Longitudinally oriented cell walls soften through pectin de-methyl-esterification. (b) The differential mechanical properties of the longitudinal and transverse walls triggers expansion of longitudinal walls. (c) The newly generated geometry causes stress anisotropy and further microtubule alignment in the direction of maximal stress. (d) The deposition of cellulose microfibrils following microtubule orientation causes the longitudinally walls to become anisotropically reinforced. (e) The increase in stress anisotropy feedbacks into the orientation of microtubules. (f) The further deposition of cellulose microfibrils following microtubules alignment reinforces wall anisotropy.

Figure 13 .

 13 Figure 13. Structural and functional organization of the SAM of Arabidopsis adapted from (Landrein and Vernoux, 2014) (a) Top and orthogonal views showing the structural zonation of the SAM: central zone (CZ), peripheral zone (PZ), primordia (P), and boundaries (B). (b) Orthogonal view of the SAM showing the organization into layers. (c) Primordia spaced according to a regular pattern of phyllotaxis. In Arabidopsis phyllotaxy is spiral. P5 indicates the oldest primordium and P1 the youngest. Successive organs are separated by an angle close to 137.5°. The position at which the next primordium (i1) will be initiated.

Figure 14 .

 14 Figure 14. The maintenance of the stem cell niche and the initiation of lateral organs (a) WUS expressed in the organizing centre moves to induce CLV3 in the stem cells. CLV3 represses in turn WUS. WUS inhibits cell proliferation in the organizing centre and differentiation in the peripheral zone. WUS modulates stem cell non-cell autonomously by directly repressing HEC1 and by promoting CLV3 expression. WUS also integrates CK signaling inputs, which are positively influenced by MP and negatively by HEC1 through the regulation of A-type ARRs, (b) The demarcation of the meristem-to-organ-boundary requires brassinosteroid signalling. The boundary genes CUC1 and LOB are tightly interconnected with this signaling pathway. By regulating the BR catabolic gene BAS1, LOB creates a minimum in BR levels and allows the expression of CUC1, which is alleviated from BZR1 repression, and can specify the boundary domain.

Figure 15 .

 15 Figure 15. The initiation of flower primordia and the acquisition of organ polarity. (a) The initiation of lateral organs is determined by the auxin/PIN1 feedback loop. Downstream of auxin, MP regulates the expression of LFY, ANT, AIL6, FIL and TMO3 transcription factors, which altogether promote flower primordium initiation. MP also regulates AHP6 expression, which allows the spatiotemporal establishment of cytokinin signalling, thereby stabilizing the plastochron. (b) HD-ZIP III genes are restricted to the adaxial side by the inhibition of miR166. KAN proteins become active on the abaxial side of the primordium, they directly repress AS2 confining its activity to the adaxial side.

Figure 16 .

 16 Figure 16. General concept of floral morphogenesis (adapted from (Abad et al., 2017)) (a) Auxin activates a molecular network of transcriptional regulators associated with diverse developmental processes, such as cell or organ identity, cell proliferation and growth, organ polarity. (b) The molecular network must interfere with the cell wall mechanical properties in order to influence growth. Cellulose microfibrils in a meristematic cell wall as viewed with atomic force microscopy (left) Modifications in the cell wall make it yield to the turgor pressure at different rates and directions (right). (c) The changes in shape, here illustrated by an Arabidopsis growing flower bud.

Figure 17 .

 17 Figure 17. The mechancical feedback between stress and microtubule orientation in the SAM (adapted from Hamant et al., 2008) (a) Patterns of cortical microtubules in the SAM as visualized following the expression of p35S::GFP-MBD fluorescent marker. The arrays of microtubules at the apex do not show a clear orientation, especially in comparison with the organ boundaries (B).(b) As a result of the geometry of the SAM, which results from local differences in growth, mechanical stresses are produced. At the apex of the meristem the mechanical stress is equivalent in all directions, or isotropic; whereas in the meristem-to-organ boundary, the direction of stress is anisotropic, given the differential growth of contiguous regions (red). Cortical microtubule alignment follows the direction of maximal stress (green).

  Through this thesis I have studied patterning in plants. I have used the shoot apical meristem of Arabidopsis as a system of study and the initiation of lateral organs from the inflorescence meristem as the patterning event. The structure and function of the molecular networks orchestrating flower primordium initiation are quite well understood. The initiation of new organs is determined by the local accumulation of auxin, which exerts its function by activating the expression of a group of transcription factors. Each of them related with the activation of diverse developmental processes. An oversimplified view of this process is that auxin activates ARF5/MP, which directly regulates the transcription of LFY, ANT and AIL6

  we provide evidence that the microtubule rearrangements occuring during organ initiation require the auxin regulated transcription factor MONOPTEROS/AUXIN RESPONSIVE FACTOR 5. This chapter describes the results that are directly related to my contribution to our publication in Current Biology[START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF], see annex 1. I mainly contributed to the characterization of the cortical microtubule-ordering pathway. I performed the crosses and the isolation of the transheterozygous mutant abp1-1s/abp1-5, abp1-1s/abp1-5 35S::GFP-MBD and bot1-7 abp1-5. The characterization of wild type and these mutants as well as the rop6-1, ric1-1 and pin1-6 bot1-7 were done in collaboration with Massimiliano Sassi. In addition, the chapter contains my unpublished results regarding the role of MP/ARF5. The crosses, isolation and characterization of the mutants arf5-1 bot1-7 and the arf5-1 PDF1::mCitrine-MBD were done by myself as well as the analyses of these data. The PDF1::mCitrine-MBD fluorescent line was produced by Thomas Stanislas.

Figure 1 .

 1 Figure 1. Auxin disrupts CMT organization at the SAM before organ outgrowth (a) SAM of 35S::GFP-MBD plants grown on soil, time course showing the formation of a flower primordium (arrowhead). (b) Detailed CMT organization in the regions highlighted in red in (a) at t=0 and t=24. B, boundaries. (c) Spatiotemporal overlap between CMT disorganization and peaks of auxin activity. Upper panel shows CMT as visualized with the 35S::GFP-MBD marker (green), the incipient primordium presents DR5::VENUS-N7 (red). Lower panel shows the CMT measurements of the regions with (red) and without (green) expression of DR5::VENUS-N7. Those with expression display more disorganized CMT patterns compared to highly anisotropic boundaries (B) (d) Surface projections of a 35S::GFP-MBD SAM treated with IAA to induce organ formation (e) CMT organization of the SAM shown in (d) before (t=0) and after (24=hr) the initial application of IAA. The red lines are a representation of the of the CMT anisotropy. The direction and length of the lines indicate the average orientation and the anisotropy of the CMT in each cell. Notice that orientations are more random and the length of the lines shorter at t=24 in comparison with t=0. (f) bot1-7 mutation promote the formation of organs in absence of auxin transport. Shoot apices of NPA-grown WT and bot1-7 plants. Notice the spontaneous formation of flower in bot1-7. (g) Quantifications of the phenotypes displayed in (f). (h) bot1-7 mutation promotes the formation of outgrowths (right) in an otherwise naked pin1-6 background (left)

(rop6- 1 :

 1 86%, n = 146; ric1-1: 68%, n = 210) in comparison with the WT (36% n = 174)(Figure2a and b). Next, in order to determine the organization of CMT we generated rop6-1 mutants bearing the microtubule marker 35S::GFP-MBD. This allowed us to determine the organization of CMTs following auxin treatments in rop6-1 mutants. We treated NPA grown rop6-1 35S::GFP-MBD and ric1-1 35S::GFP-MBD with IAA as in previous experiments. This did not induce changes in CMT organization at the tissue level and led only to a minor decrease of cellular CMT anisotropy in rop6-1 mutants in comparison with the WT (Figure2c -f). This indicated that auxin is no further able to reorganize the CMTs on NPA in the absence of ROP6 and RIC1, suggesting they are part of the same pathway In contrast ROP6 and RIC1 overexpressing lines grown on NPA exhibited decreased formation of organs when compared to WT plants (Figure2b). In particular, ROP6 overexpression significantly reduced organogenesis. Instead of the 38% (n=169) of wild type plants able to produce flower primordia, only 20% (n=121) of the ROP6 overexpressing lines were able to do so. Altogether, these results suggested that microtubule organisation is an important parameter in organ initiation. They are also pointing towards a scenario where auxin would act on mechanical anisotropy via a ROP-based pathway.

Figure 2 .

 2 Figure 2. ROP6 and RIC1 regulate organ formation and auxin responsiveness of CMTs at the SAM (a) Shoot apices of NPA-grown WT, rop6-1 and ric1-1 plants. Notice the spontaneous formation of flowers in rop6-1 and ric1-1 (b) Quantification of the phenotypes of NPA-grown WT, ric1-1 and rop6-1 (left) and RIC1OX and ROP6OX (c) CMT organization in cells of the shoot apex of WT plants before (t=0) and after (t=24hr) IAA (d) Supracellular organization of CMT quantification of (c). (e) CMT organization in cells of the shoot apex of rop6-1 plants before (t=0) and after (t=24hr) IAA treatment (f) Supracellular organization of CMT quantification of (e).

Figure 3 .

 3 Figure 3. ABP1 regulates organ formation and auxin responsiveness of CMTs at the SAM (a) Shoot apices of NPA-grown WT, abp1-5 and abp1-1s/abp1-5 plants. (b) Quantification of the phenotypes in (a). Mutant vs WT: *p < 0.01; abp1-5 vs abp1-1s/abp1-5: **p < 0.01. (c) CMT organization in cells of the shoot apex of WT plants before (t=0) and after (t=24hr) IAA treatment (d) Supracellular organization of CMT quantification of WT plants in (c). (e) CMT organization in cells of the shoot apex of abp1-5 plants before (t=0) and after (t=24hr) IAA treatment (f) Supracellular organization of CMT quantification of abp1-5 plants (e). (g) CMT organization in cells of the shoot apex of abp1-1s/abp1-5 plants before (t=0) and after (t=24hr) IAA treatment (h) Supracellular organization of CMT quantification of abp1-5 plants (g).

Figure 4 .

 4 Figure 4. ARF5/MP regulates microtubule organization in the SAM (a) WT and arf5-1 mutant seedlings 10 dag, notice the lack of root in the mutant. (b) Dissected WT inflorescence and naked arf5-1 inflorescence. (c) CMT organization in cells of pin-like meristems impaired in auxin transport (PDF1::mCitrine-MBD plants grown on NPA) and arf5-1 PDF1::mCitrine-MBD homozygous plants. (d) Quantification of phenotypes shown in (e) and (f). (e) Local oryzalin applications (right) promote the formation of lateral outgrowths in arf5-2 SAMs (left) already visible after 96 hr after the treatment. (f) bot1-7 mutation promotes the formation of lateral outgrowths (right) in otherwise naked arf5-1 background (left).

Figure 5 .

 5 Figure5. Graphical summary of our working hypothesis about the mechanisms leading to lateral organ initiation in the SAM. A KTN1-mediated pathway maintains an anisotropic expansion of cells under no auxin accumulation (left). This mechanism inhibits lateral organ initiation. In contrast, auxin accumulation (right) prevents the KTN1-mediated pathway, but activates an ARF5/MP-dependent pathway, which facilitates the reorientation of CMT. This mechanism in addition with cell wall loosening (not shown) trigger a shift towards isotropic expansion resulting in lateral organ initiation.

  PDF1::mCitrine-MBD was kindly provided by Thomas Stanislas. The DR5::VENUS-N7 35S::GFP-MBD, rop6-1 35S::GFP-MBD, ric1-1 35S::GFP-MBD, abp1-5 35S::GFP-MBD, pin1-6 bot1-7, arf5-1 bot1-7 and arf5-1 PDF1::mCitrine-MBD lines were generated by crossing and further PCR selection. Growth conditions were previously described[START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]

  disorganized and cells shift to isotropic growth. Mechanical modeling shows that this coupling has the potential to compensate for reduced growth rates induced by the isotropic deposition of microfibrils in the organ primordium. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module involved in organ outgrowth, linking microtubule arrangements to cell wall remodeling. Although the transcriptional regulators involved in this module remain to be identified, an enhanced yeast 1-hybrid screen suggests that the cell wall modifying genes investigated here are the targets of multiple transcription factors and signaling pathways.The following chapter corresponds to the manuscript that was recently submitted. I participated in the scientific discussions leading to this article. Experimentally, I verified the expression of wall-loosening genes by qRT-PCR following auxin induction. In collaboration with, Laetitia Vachez, Amélie Robin, and Ann'Evodie Sallee, we set up and performed the whole mount RNA in situ hybridizations. I produced the crosses and participated in the isolation of the mutants xxt1 xxt2 PDF1:: mCitrine-MBD, and the line expressing DR5::VENUS-N7 in the pin1-6 bot1-7 background. In addition to the manuscript, I have also incorporated, the results of the eYIH assay in this chapter, which was done in the Proteomics

Figure 2 .

 2 Figure 2. Influence of mechanical properties on cell expansion. The structure used to perform the various simulations is a 2D shell tiled with polygonal cells. Each cell is composed of several triangular first order finite elements. The meshes are generated from a segmented 3D confocal stack of images from a pin meristem. See annex 2 for details. (a) and (b) show the structure at the beginning of the simulations. (c), (d), (e), (f) and (g) show the structure after 100 steps of growth simulation based on different hypotheses. The relative growth rate is color-coded. In (a) a set of initium cells is defined (red zone) which can have variable levels of anisotropy and stiffness. The cells at the summit (dark grey in (a) and (b)) are very stiff and grow very slowly. The periphery has a constant stiffness and 50% anisotropy.(c) When the primodium cells have the same stiffness and anisotropy as the peripheral cells, all cells grow at the same rate. (d) When the cells in the primordium are made isotropic, but stiffness remains as in the periphery, the primordium grows more slowly than the surrounding periphery. (e) When both anisotropy and stiffness are reduced, the primordium cells grow quicker than the surrounding cells. In (b) the dome has only a peripheral zone with variable mechanical properties (f) The peripheral cells are anisotropic, (g) when cells are made isotropic, growth rate is reduced

Figure 1 .

 1 Figure 1. Disruption of CMT anisotropy promotes the expression of developmental patterning genes. Expression pattern of LFY (a, b and c), ANT (d, e and f), AIL6 (g, h and i) and MP (j, k, and l) detected by whole mount in situ hybridization in WT plants (a, d, g and j), pin1-6 (b, e, h and k) and pin1-6 bot1-7 (c, f, i and l) mutants.

Figure 2 .

 2 Figure 2. Microtubule disorganization induces differently LFY and ANT expression. (a) pLFY>GFP expression in auxin-induced (+iaa) and oryzalin-induced outgrowths (+oryzalin) (b) pANT::erGFP expression in auxin-induced (+iaa) and oryzalin-induced outgrowths (+oryzalin)

Figure 4 .

 4 Figure 4. CMT disorganization promotes outgrowths that express the developmental patterning gene ANT even in the absence of MP regulation Scanning electron micrograph images of arf5-1 bot1-7 (a) and arf5-2 bot1-7 apices (b). The right part of each panel shows the expression pattern of ANT detected by whole mount in situ hybridization.

(

  bZIP) FLOWERING LOCUS D (FD), the histone H2A 13 (HTA13), the CCT motifcontaining response regulator TIMING OF CAB EXPRESSION1 (TOC1), the class II homeodomain-leucine zipper ATHB4, the BTB/POZ domain-containing protein encoded by AT1G21780, the LOB DOMAIN-CONTAINING PROTEIN 18 (LBD18), the GROWTH-REGULATING FACTOR 9 (ATGRF9), the SIN3-LIKE 3 (ATSIN3) and two transcription factors involved in the brassinosteroid (BR) regulated gene expression pathway BRASSINAZOLE-RESISTANT 2 (BZR2) and INTERACT WITH SPT6 1 (IWS1). Notably, the eYIH screen did not show ARF5/MP as regulator of ANT promoter nor of AIL6 promoters, probably due to the promoter fragments used.

Figure 5 .

 5 Figure 5. Putative regulators of ANT and AIL6 transcription according to the eYIH screen. Only candidates expressed in the top 30% of all transcripts of the inflorescence meristem according to Mantegazza et al., (2014) are shown.

Figure 6 .

 6 Figure 6. Disruption of CMT anisotropy promotes the expression of developmental patterning genes. Expression pattern of BES1 (a and b) and STM as control (c and d) detected by whole mount in situ hybridization in WT plants (a and c), and pin1-6 bot1-7 (b and d) mutants.

Figure 7 .

 7 Figure 7. BRs signaling in the shoot apex might link CMT disorganization, lateral outgrowths and ANT expression (a) Scanning electron micrograph images of WT apex, pin1-6 and pin1-6 one-week after treatment with EBL (b) Expression pattern of NPA-grown fluorescent reporter plants pANT::erGFP showing ANT transcriptional activity before (0h) and after (48h) and (72h) of treatment with EBL.

σ

  = H • ε with : H = 2

fx = 1

 1 + d/(6 * np . pi ) * (8 * np . s i n ( a ) + np . s i n (2 * a)) fy = 1 -d/(6 * np . pi ) * (8 * np . s i n ( a ) -np . s i n (2 * a)) fxy = 1 -d/(2 * np . pi ) * np . s i n (2

Figure 1 :

 1 Figure 1: Zoning, structural anisotropy & cell wall rigidity of the simulated structure. A&B :Zoning used to perform the various simulations. In case A we defined an initium zone with different mechanical properties than its surrounding (i.e. the peripheral zone). In case B, we modified the structural anisotropy of the whole peripheral zone. C: Parametrization of the structure, we can attribute specific mechanical properties to every single cell of the structure, we depict here two extreme cases. D, E & F: The purely isotropic case and G, H & I: The highly anisotropic one. D&G :schematic representation of fibers distribution within the cell wall in each case. E& H: polar plots of the corresponding fiber angular density function (ρ (θ ) in the text). F&I :polar plot of the corresponding angular rigidity (Y (θ ) in equation (6)). J: polar plots of all the fibers angular density functions used in the various simulations (ρ (θ )). K: polar plots of the corresponding angular rigidity functions (Y (θ ) ).

  Numerical values of the parameters used in the simulations of Case #2. Numerical values of the parameters used in the simulations of Case #3.

Figure 2 :

 2 Figure 2: Time evolution of the mean surface area of cells in each zone for different values of the structural anisotropy in the initium. Error bars represent the standard deviation.

Figure 3 :

 3 Figure3: Time evolution of the relative mean surface area of cells in each zone (hr i (t n )i Z ) for different zones when we decrease the rigidity amplitude in the initium, in the fully isotropic case. Error bars represent the standard deviation.

Table S1 :

 S1 results RNA seq, for XTHs, PMEs, PMEIs, EXPAs and CesAs. For normalized values taking into account the length of the RNAs, the TPM (Transcripts Per Kilobase Million has been calculated)

	Summary
	XTH9	59%
	XTH4	11%
	XTH22	9%
	XTH6	7%
	rest XTH	13%
	EXPA6	41%
	EXPA15	16%
	EXPA4	13%
	EXPA3	9%
	EXPA20	6%
	rest EXPAs	15%
	CesA3	33%
	CesA1	30%
	CesA6	16%
	CesA5	12%
	CesA2	8%
	rest CesA	1%
	PME5	34%
	ATPME3	14%
	ATPME1	10%
	ATPME31	6%
	AT3G10720 5%
	AT3G49220 5%
	ATPME44	5%
	rest PMEs	21%
	AT5G62350 31%
	AT5G20740 25%
	AT1G14890 14%
	ATC/VIF2	12%
	AT2G01610 4%
	rest	14%

Table S3 .

 S3 Primers used for qRT-PCR (FigureS3) and to amplify promoter regions for YIH assays

	AT5G64610 HAM1 AT2G20880 AT2G20880 AT4G39410 ATWRKY13 AT5G05550 AT5G05550 Introduction		C2H2 AP2/EREBP WRKY TRIHELIX		XTH9 EXPA4 EXPA4 EXPA4
	Primer name AT1G50410 AT1G50410 Sequence (5'>3') AT2G21230 AT2G21230 AT2G02820 AtMYB88 AT4G34590 ATB2/AtbZIP11/GBF6	SNF2 BZIP MYB bZIP	Use	XTH9 EXPA4 EXPA4 EXPA4
	TCTP-FW AT2G45420 LBD18 AT5G10140 FLC AT4G03170 AT4G03170 GCTCAGCGAAGAAGATCAAGCTGTC LOB/AS2 MADS ABI3-VP1 AT2G30130 ASL5/LBD12/PCK1 LOB/AS2	qRT-PCR	XTH9 EXPA4 EXPA4 EXPA4
	TCTP-RV AT1G26590 AT1G26590 CCCTCCCCAACAAAGAATTGGAAG C2H2 AT1G47870 ATE2F2 E2F/DP AT5G13780 AT5G13780 GNAT AT4G03250 AT4G03250 HB The generation of shape is a central question in developmental biology. Plants represent an qRT-PCR XTH9 EXPA4 EXPA4 EXPA4
	EXPA15-FW AT2G33550 AT2G33550 TAACGCTGGTGGTTGGTGTA AT3G23240 ERF1 AT4G12670 AT4G12670 AT2G37520 AT2G37520 excellent system to study this issue, due to their dynamic and continuous organogenesis. The qRT-PCR TRIHELIX XTH9 AP2/EREBP EXPA4 MYB-related EXPA4 PHD EXPA4
	EXPA15-RV AT2G41835 AT2G41835 CTGAGCAATGCGTTGAAAAA C2H2 AT4G27410 RD26 NAC AT3G12977 AT3G12977 NAC/NAM AT4G13480 AtMYB79 MYB aerial parts of the plant are produced by small groups of non-differentiated cells, the shoot qRT-PCR XTH9 EXPA4 EXPA4 EXPA4
	XTH9-FW XTH9-RV AT5G44260 AtTZF5 AT2G01930 ATBPC1/BBR GCTGGGCTATGGATCATTGT TTCAAACCCAGCTCCAGAGT AT5G48250 AT5G48250 AT1G14350 FLP AT5G48890 LATE AT5G66870 ASL1/LBD36 AT4G00390 AT4G00390 AT1G66420 AT1G66420 apical meristems (SAMs). Here we study the initiation of new organs in the SAM of qRT-PCR qRT-PCR C3H XTH9 BBR-BPC XTH9 C2C2-CO-LIKE EXPA4 MYB EXPA4 C2H2 EXPA4 LOB/AS2 EXPA4 GeBP EXPA4 GeBP EXPA4
	EXPA4 Y1H F 1st AT5G15480 AT5G15480 TGATGTCTTTGATGGTGGTGG C2H2 AT4G38960 AT4G38960 C2C2-CO-LIKE 1st round promoter cloning Y1H XTH9 EXPA4 AT5G19650 ATOFP8 OFP EXPA4 AT3G01530 ATMYB57 MYB EXPA4 Arabidopsis thaliana. New organ primordia initiate at the accumulation points of the plant
	EXPA4 Y1H R 1st AT3G46590 ATTRP2/TRFL1 TGGGACTAACCCATTGTGC AT5G08520 AT5G08520 AT4G27230 HTA2 AT4G31615 AT4G31615 hormone auxin. It is widely accepted that auxin driven transcription activates the expression 1st round promoter cloning Y1H MYB-related XTH9 MYB EXPA4 CCAAT EXPA4 ABI3-VP1 EXPA4
	EXPA15 Y1H F 1st AT4G38910 ATBPC5BBR/BPC5 GCAACACAGTCAAAGCATACTC BBR-BPC AT2G44730 AT2G44730 TRIHELIX AT5G14170 BAF60/CHC1 SWI/SNF-BAF60 1st round promoter cloning Y1H XTH9 EXPA4 EXPA4 AT3G53680 AT3G53680 PHD EXPA4 of a group of targets ultimately resulting in the initiation of organ primordia. A central role in
	EXPA15 Y1H R 1st AT1G02040 AT1G02040 GCCATGAACAGAGCACACC AT1G04880 AT1G04880 AT3G45150 TCP16 AT5G52170 HDG7 this process is given to the DNA-binding auxin response factor AUXIN RESPONSE 1st round promoter cloning Y1H C2H2 XTH9 ARID EXPA4 TCP EXPA4 HB EXPA4
	XTH9 Y1H F 1st XTH9 Y1H R 1st AT3G53680 AT3G53680 ACTGAGGAATGGAAACTATTAGA GCCTTCGTTGACACAATG PHD AT2G21240 ATBPC4/BBR BBR-BPC AT2G26940 AT2G26940 C2H2 AT5G06950 AHBP-1B BZIP AT1G65620 AS2 LOB/AS2 AT5G60142 AT5G60142 ABI3-VP1/B3 AT5G25470 AT5G25470 ABI3-VP1/B3 AT2G21240 ATBPC4/BBR BBR-BPC FACTOR/MONOPTEROS (ARF5/MP). After its auxin-dependent activation, ARF5/MP 1st round promoter cloning Y1H 1st round promoter cloning Y1H XTH9 XTH9 EXPA4 EXPA4 EXPA4 EXPA4 EXPA4 EXPA4
	EXPA4 Y1H F 2nd AT1G24190 ATSIN3/SNL3 TTTTTTTTCAGCAAGAAGAAA Orphans AT5G05410 DREB2A AP2/EREBP AT3G14740 AT3G14740 PHD AT2G47850 AT2G47850 C3H orchestrates a number of developmental processes notably by directing the expression of 2nd round promoter cloning Y1H XTH9 EXPA4 EXPA4 EXPA4
	EXPA4 Y1H R 2nd AT5G15020 SNL2 AT4G37260 MYB73 AT5G24050 AT5G24050 TTTGTGTGTGAATTACTAGAAACAG Orphans MYB REM(B3) AT1G75530 AT1G75530 FHA transcription factors involved in the specification of floral identity and proliferative growth, 2nd round promoter cloning Y1H XTH9 EXPA4 EXPA4 EXPA4
	EXPA15 Y1H F 2nd AT5G44160 NUC AT4G01120 GBF2 AT2G35550 ATBPC7/BBR CATACTCGAACCAACAGTAAAAA C2H2 BZIP BBR-BPC LEAFY (LFY), AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6); organ 2nd round promoter cloning Y1H EXPA15 EXPA4 EXPA4
	EXPA15 Y1H R 2nd AT3G21270 ADOF2 AT2G40950 BZIP17 AT4G00238 AT4G00238 TTACTGCTTTAACTGTTTTCCCTAC C2C2-DOF BZIP GeBP polarity, FILAMENTOUS FLOWER (FIL) and cytokinin responses, TARGET OF 2nd round promoter cloning Y1H EXPA15 EXPA4 EXPA4
	XTH9 Y1H F 2nd XTH9 Y1H R 2nd AT2G22430 ATHB6 AT5G57660 ATCOL5 ACTGAGGAATGGAAACTATTAGA TTTTTTTTTAACTTATCTCTCTAAATAA HB C2C2-CO-LIKE 2nd round promoter cloning Y1H 2nd round promoter cloning Y1H EXPA15 EXPA15 AT1G51600 ZML2 C2C2-GATA EXPA4 AT1G21910 DREB26 AP2/EREBP EXPA4 AT3G63030 MBD4 zf-CW EXPA4 AT5G07500 AtTZF6/PEI1 C3H TF AG1 TF Name TF family Target EXPA4 Table S5. Identified transcription factor-XTH9 promoter -EXPA4 promoter interactions MONOPTEROS (TMO3)
	AT2G34140 AT2G34140 AT3G61830 ARF18 AT5G38490 AT5G38490 AT5G38490	REM(B3)	C2C2-DOF ARF REM(B3) XTH9, EXPA4 and EXPA15	EXPA15 EXPA4 EXPA4
	AT1G10480 ZFP5 AT1G16530 LBD3 AT1G05230 HDG2 AT5G24050	REM(B3)	C2H2 HB XTH9 and EXPA4	EXPA15 EXPA4 EXPA4
	AT3G49940 LBD38 AT1G76420 CUC3 AT5G18090 AT5G18090 AT3G20670 HTA13		NAC ABI3-VP1 XTH9 and EXPA4	EXPA15 EXPA4 EXPA4
	Table S4. Identified transcription factor-promoter interactions AT1G73870 AT1G73870 C2C2-CO-LIKE AT1G56010 NAC1 NAC AT3G25790 AT3G25790 G2-like AT1G16530 LBD3 XTH9 and EXPA4	EXPA15 EXPA4 EXPA4
	TF AGI AT1G68550 CRF10 TF Name AT2G45190 YAB1 AT5G25190 ESE3 AT2G26940 AT2G26940	C2H2	TF family AP2/EREBP C2C2-YABBY AP2-EREBP XTH9 and EXPA4	Target EXPA15 EXPA4 EXPA4
	AT1G61660 AT1G61660 AT1G19860 AT1G19860 AT5G63090 LOB AT3G12730 AT3G12730 AT5G15480 AT5G15481	C2H2	BHLH C3H G2-like XTH9 and EXPA4	XTH9 EXPA15 EXPA4 EXPA4
	AT1G26610 AT1G26610 AT4G37780 ATMYB87 AT5G57390 PLT5 AT2G42430 ASL18/LBD16 AT3G53680	PHD	C2H2 MYB AP2/EREBP LOB/AS2 XTH9 and EXPA4	XTH9 EXPA15 EXPA4 EXPA4
	AT2G26940 AT2G26940 AT3G23230 AtERF98/AtTDR1 AT1G76420 CUC3 AT2G45410 LBD19 AT2G35550 ATBPC7/BBR	BBR-BPC	C2H2 AP2-EREBP NAC LOB/AS2 XTH9 and EXPA4	XTH9 EXPA15 EXPA4 EXPA4
	AT1G16530 LBD3 AT5G10120 AT5G10120 AT1G12980 DRN AT3G27650 LBD25 AT2G01930 ATBPC1/BBR	BBR-BPC	EIL AP2/EREBP LOB/AS2 XTH9 and EXPA4	XTH9 EXPA15 EXPA4 EXPA4
	AT1G34390 ARF22 AT3G03660 WOX11 AT1G77850 ARF17 AT1G75390 AtbZIP44 AT4G38910 ATBPC5BBR/BPC5 BBR-BPC	ARF HB ARF bZIP XTH9 and EXPA4	XTH9 EXPA15 EXPA4 EXPA4
	AT4G35280 DAZ2 AT4G27230 HTA2 AT2G24430 ANAC038/ANAC039 AT2G01930 ATBPC1/BBR AT2G21240 ATBPC4/BBR	BBR-BPC	C2H2 CCAAT NAC BBR-BPC XTH9 and EXPA4	XTH9 EXPA15 EXPA4 EXPA4
	AT3G20670 HTA13 AT5G66770 AT5G66770 AT3G18400 NAC058 AT4G00270 AT4G00270		CCAAT GRAS NAC GeBP		XTH9 EXPA15 EXPA4 EXPA4
	AT1G19490 AT1G19490 AT5G03780 TRFL10 AT3G20670 HTA13 AT3G16160 AT3G16160		bZIP MYB-related CCAAT CPP		XTH9 EXPA15 EXPA4 EXPA4
	AT5G59430 ATTRP1 AT5G38490 AT5G38490 AT1G51060 HTA10 AT1G72570 AT1G72570		MYB-related REM(B3) CCAAT AP2-EREBP		XTH9 EXPA15 EXPA4 EXPA4
	AT5G24050 AT5G24050 AT1G51220 AtWIP5 AT1G14685 ATBPC2 AT2G40140 ATSZF2/CZF1/ZFAR1		REM(B3) C2H2 BBR-BPC C3H		XTH9 EXPA15 EXPA4 EXPA4
	AT5G03780 TRFL10 AT3G06220 AT3G06220 AT3G01890 AT3G01890 AT4G00130 AT4G00130		MYB-related ABI3-VP1 SWI/SNF-BAF60 GeBP	XTH9 EXPA15 EXPA4 EXPA4
	AT4G00210 LBD31 AT1G05690 BT3 AT2G40450 AT2G40450 AT5G19790 RAP2.11		LOB/AS2 TRAF/TAZ TRAF AP2-EREBP		XTH9 EXPA15 EXPA4 EXPA4
	AT2G17180 DAZ1 AT1G68800 BRC2/TCP12 AT3G11100 AT3G11100 AT5G15480 AT5G15480		C2H2 TCP TRIHELIX C2H2		XTH9 EXPA15 EXPA4 EXPA4
	AT2G35550 ATBPC7/BBR AT4G22745 MBD1 AT1G55650 AT1G55650 AT4G33280 AT4G33280		BBR-BPC MBD ARID ABI3-VP1		XTH9 EXPA15 EXPA4 EXPA4
	AT5G38490 AT5G38490 AT5G06960 OBF5 AT5G01860 AT5G01860 AT4G38910 ATBPC5BBR/BPC5		REM(B3) BZIP C2H2 BBR-BPC		XTH9 EXPA4 EXPA4 EXPA4

Table S1 .

 S1 Identified transcription factor-promoter interactions

	AT5G44210 AT1G62120 AT5G05550	ERF9 AT1G62120 AT5G05550	AP2/EREBP mTERF TRIHELIX	AIL6 AIL6 AIL6
	AT3G14180 AT1G66370 AT4G03250	AT3G14180 AtMYB113 AT4G03250	TRIHELIX MYB HB	AIL6 AIL6 AIL6
	TF AGI AT1G47870 AT3G58190 AT4G00390 AT5G44160 AT5G44160 AT4G03170 AT4G00250 AT1G19210 AT1G34190 AT3G12977 AT1G66420 AT2G01650 AT2G28510 AT2G42380 AT4G31615 AT1G26610 AT3G10500 AT5G66940 AT3G53680 AT2G26940 AT3G11580 AT4G04890 AT4G01260 AT5G05410 AT5G13330 AT5G65590 AT2G18328 AT1G73870 AT1G71130 AT2G46670 AT5G52170 AT1G16530 AT4G35900 AT1G19350 AT5G61380 AT1G32130 AT1G67260 AT2G44910 AT3G20670 AT2G40450 AT5G08790 AT5G10120 AT1G74650 AT3G03660 AT1G03790 AT5G07500 AT3G01030 AT5G38490 AT1G21780 AT1G02230 AT2G45420 AT3G27650 AT2G33550 AT5G44260 AT1G27370 AT1G26590 AT3G45260 AT2G12646 AT5G15480 AT1G02030 AT5G52830 AT1G63030 AT2G45480 AT3G53680 AT1G24190 AT5G15020 AT4G02670 AT4G38910 AT2G18160 AT4G33280 AT5G67480 AT5G19790 AT5G22890 AT2G18350 AT2G02820 AT2G25620 AT1G55650 AT1G06850 AT3G11100 AT5G65410 AT4G35700 AT1G72570 AT1G02210 AT2G01370 AT3G01890 AT3G16160 AT1G14685 AT4G00270 AT1G80580 AT2G01930 AT3G09230 AT1G75390 AT3G60670 AT4G10240 AT4G38000 AT1G27370 AT3G18400 AT1G06280 AT2G24430 AT3G57920 AT5G57390 AT5G25190 AT5G63090 AT3G12730 AT1G76420 AT5G39610 AT4G00940 AT5G19310 AT1G14580 AT5G52230 AT3G61830 AT1G02230 AT3G14230 AT3G27785 AT4G01120 AT5G18090 AT5G05410 AT1G05230 AT5G15130 AT2G21400 AT2G44730 AT5G51980 AT1G76880 AT4G00238 AT1G03840 AT2G35550 AT1G54060 AT2G17180 AT1G29160 AT2G34620 AT3G53340 AT3G14740 AT2G23290 AT5G60142 AT3G03200 AT1G65620 AT1G43700 AT5G14170 AT2G21240	TF Name ATE2F2 ASL16/LBD29 AT4G00390 NUC NUC AT4G03170 AT4G00250 AT1G19210 anac017 AT3G12977 AT1G66420 PUX2 AT2G28510 ATBZIP34 AT4G31615 AT1G26610 anac053 AT5G66940 AT3G53680 AT2G26940 AT3G11580 PDF2 AT4G01260 DREB2A Rap2.6L SCAP1 ATRL4 AT1G73870 AT1G71130 AT2G46670 HDG7 LBD3 FD BZR2 TOC1 IWS1 TCP1 ATHB4 HTA13 AT2G40450 anac081/ATAF2 AT5G10120 ATMYB31/ATY13 WOX11 AtTZF4/SOM AtTZF6/PEI1 AT3G01030 AT5G38490 AT1G21780 ANAC004 LBD18 LBD25 AT2G33550 AtTZF5 SPL10 AT1G26590 AT3G45260 AT2G12646 AT5G15480 AT1G02030 ATWRKY27 ddf2 AtGRF9 AT3G53680 ATSIN3/SNL3 SNL2 AtIDD12 ATBPC5BBR/BPC5 ATBZIP2/FTM3/GBF5 AT4G33280 ATBT4 RAP2.11 STOP2 AtHB24/ZHD6 AtMYB88 AtDBP1 AT1G55650 AtbZIP52 AT3G11100 ATHB25/ZFHD2/ZHD1 DAZ3 AT1G72570 AT1G02210 AT2G01370 AT3G01890 AT3G16160 ATBPC2 AT4G00270 AT1G80580 ATBPC1/BBR AtMYB1 AtbZIP44 AT3G60670 bbx23 DOF4.7 SPL10 NAC058 LBD2 ANAC038/ANAC039 SPL15 PLT5 ESE3 LOB AT3G12730 CUC3 ANAC092/ATNAC2/ATNAC6/ORE1 AT4G00940 AtCHR23 AT1G14580 MBD13 ARF18 ANAC004 RAP2.2 ATMYB118 GBF2 AT5G18090 DREB2A HDG2 WRKY72 SRS3 AT2G44730 AT5G51980 AT1G76880 AT4G00238 MGP ATBPC7/BBR ASIL1 DAZ1 AT1G29160 AT2G34620 NF-YB10 AT3G14740 AtMYB70 AT5G60142 anac045 AS2 VIP1 BAF60/CHC1 ATBPC4/BBR	TF family E2F/DP LOB/AS2 GeBP C2H2 C2H2 ABI3-VP1 GeBP AP2/EREBP NAC NAC/NAM GeBP C2H2 C2C2-DOF bZIP ABI3-VP1 C2H2 NAC C2C2-DOF PHD C2H2 ABI3/VP1 HB GeBP AP2/EREBP AP2/EREBP C2C2-DOF MYB-related C2C2-CO-LIKE AP2/EREBP Orphans/C2C2-CO-like HB BZIP TCP HB CCAAT TRAF NAC EIL MYB HB C3H C3H C2H2 REM(B3) TRAF NAC LOB/AS2 LOB/AS2 TRIHELIX C3H SBP C2H2 C2H2 PLATZ C2H2 C2H2 WRKY AP2-EREBP GRF PHD Orphans Orphans C2H2 BBR-BPC bZIP ABI3-VP1 TAZ AP2-EREBP C2H2 zf-HD MYB DBP ARID bZIP TRIHELIX zf-HD C2H2 AP2-EREBP NAC GeBP SWI/SNF-BAF60 CPP BBR-BPC GeBP AP2-EREBP BBR-BPC MYB bZIP PLATZ Orphans C2C2-DOF SBP NAC LOB/AS2 NAC SBP AP2/EREBP AP2-EREBP G2-like NAC NAC C2C2-Dof SNF2 C2H2 ND ARF NAC AP2/EREBP MYB BZIP ABI3-VP1 AP2/EREBP HB WRKY SRS TRIHELIX C3H TRIHELIX GeBP C2H2 BBR-BPC TRIHELIX C2H2 C2C2-DOF mTERF CCAAT-HAP3 PHD MYB ABI3-VP1/B3 NAC LOB/AS2 BZIP SWI/SNF-BAF60 BBR-BPC	AIL6 AIL6 AIL6 Target AIL6 AIL6 AIL6 ANT AIL6 AIL6 AIL6 ANT AIL6 AIL6 AIL6 ANT AIL6 AIL6 AIL6 ANT AIL6 AIL6 AIL6 ANT AIL6 AIL6 AIL6 ANT AIL6 AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 ANT AIL6 AIL6 AIL6
	AT5G54680	ILR3	BHLH	AIL6

Table S2 :

 S2 Primers used for in situ hybridization

	Gene ID	Primers used to amplify total cDNA sequence from TAIR cds
	ANT	ANT_For : ATGAAGTCTTTTTGTGATAATGATGATAAT
		ANT_T7 Rev : TGTAATACGACTCACTATAGGGCTCAAGAATCAGCCCAAGCAG
	AIL6	AIL6_For : ATGATGGCTCCGATGACGAACTGGTTAACG
		AIL6_T7 Rev : TGTAATACGACTCACTATAGGGCTTAGTAAGACTGATTAGGCC
	BES1	BES1_For : CACCATGAAAAGATTCTTCTATAATTCC
		BES1_T7 Rev : TGTAATACGACTCACTATAGGGCTCAACTATGAGCTTTACCATT
	LFY	LFY_For : ATGGATCCTGAAGGTTTCACGAGTGGCTTA
		LFY_T7 Rev : TGTAATACGACTCACTATAGGGCCTAGAAACGCAAGTCGTCGC
	MP	MP_For : ATGATGGCTTCATTGTCTTGTGTTGAAGAC
		MP_T7 Rev : TGTAATACGACTCACTATAGGGCTTATGAAACAGAAGTCTTAA
	STM	STM_For : ATGGAGAGTGGTTCCAACAGCACTTCTTGT
		STM_T7 Rev : TGTAATACGACTCACTATAGGGCTCAAAGCATGGTGGAGGAGA

Table S3 .

 S3 Primers used to amplify promoter regions for YIH assays The generation of biological shapes during development remains a central and open question in biology. Plants represent excellent systems to study these issues given their open, indeterminate development. This means that new shapes are generated over the course of their life times.

	Primer name	Sequence (5'>3')	Use
	ANT Y1H F 1st	CTCTCTGCTGCATACTTGCA	1st round promoter cloning Y1H
	ANT Y1H R 1st	ACAAGTTGAGGTGGAACAGAAG	1st round promoter cloning Y1H
	AIL6 Y1H F 1st	TGCATGTACGACAAGTGGAG	1st round promoter cloning Y1H
	AIL6 Y1H R 1st	TATCGAGGAGATAAGGAGAGGAG	1st round promoter cloning Y1H
	ANT Y1H F 2nd	TTTGTTATTTATGAAAAACAAATATT	2nd round promoter cloning Y1H
	ANT Y1H R 2nd	GGTTTCTTTTTTTGGTTTCTG	2nd round promoter cloning Y1H
	AIL6 Y1H F 2nd	GTTTTTTCCCTTTATCACTAAATC	2nd round promoter cloning Y1H
	AIL6 Y1H R 2nd	AAACTTTCTTATAAAAACAATTTTACTT 2nd round promoter cloning Y1H

(i) I firstly addressed the role of cell wall anisotropy in the initiation of organ outgrowth and how this relates with the local accumulation of auxin and the patterns of CMT organization;(ii) secondly, I studied the function of cell wall remodeling and its coupling to CMT organization;(iii) finally, I explored the transcriptional control of organ initiation by analyzing the canonical ARF5/MP-regulated pathway and how it could be linked to cellular anisotropy via feedback.

Acknowledgements

I would like to thank Naomi Nakayama, Angela Hay, François Parcy and Olivier Hamant for reviewing this thesis. Also thanks to my thesis committee, who guided me through my PhD, Patrick Laufs and Olivier Hamant. I am especially grateful to Jan Traas my thesis director, who gave me academic support as well as to Massimiliano Sassi, who assisted in supervising me and answering my questions.

Acknowledgements

This work was funded by the ERC grant (MORPHODYNAMICS); we thank Olivier Hamant Françoise Moneger and Roberta Galletti for the critical reading of the manuscript.

carried out as previously described [START_REF] Peaucelle | Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins[END_REF], with some modifications: plants were grown in vitro on NPA plates until bolting, then they were transferred onto fresh NPA plates and imaged (t=0). Immediately after imaging, plants were submerged in a PME solution (1U/100μl in phosphate buffer pH=7) for 16h. Plants were imaged again at t=24h, and the treatment was repeated immediately after. No further treatments were applied until the end of the experiment.

Confocal live Imaging and image analyses.

Confocal imaging was carried out on a Zeiss LSM700 system as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. SEM imaging was carried out on a Hirox SE-3000 system as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF]. Analyses of CMT organization were carried out with FIJI software as previously described [START_REF] Sassi | An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis[END_REF].

RNA-Seq sample preparation and sequencing analysis

Ten dissected (to P5) Col-0 meristems were pooled for each biological replicate. RNA was extracted using the Arcturus PicoPure RNA extraction kit (ThermoFisher) according to the manufacturer's instructions. Libraries were prepared using the TruSeq Strand specific protocol following manufecturers instructions. [START_REF] Gagnot | CATdb: a public access to Arabidopsis transcriptome data from the URGV-CATMA platform[END_REF] and from the Gene Expression Omnibus repository [START_REF] Barrett | NCBI GEO: mining tens of millions of expression profiles--database and tools update[END_REF] at the National Center for Biotechnology Information (GSE XXXXX).

Histochemistry

RNA in situ hybridization assays were performed as described previously (Ferrandiz and Sessions, 2008a, b). For whole-mount RNA in situ hybridization assays, untreated and treated (IAA or ORY) pin1-6 meristems from soil-grown plants were processed as previously described [START_REF] Rozier | Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization[END_REF]. At least three independent experiments for both assays were performed for each probe tested. Whole mount immunolabellings were carried out as previously described [START_REF] De Reuille | Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis[END_REF]. Cell wall antibodies were obtained from the Plant Probes service at the University of Leeds, UK (http://www.plantprobes.net/index.php)

Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma). Total RNAs were digested on-column with Turbo DNA-free DNase I (Ambion) according to the manufacturer's instructions. SuperScript VILO cDNA Synthesis Kit (Invitrogen) was used to reverse transcribe RNA. The quantitative reverse transcription PCR (qRT-PCR) was performed on a

StepOne Plus Real Time PCR System (Applied Biosystems), using FastStart Universal SYBR Green Master (Rox) (Roche). Data were analyzed using the StepOne Software v2.2 (Applied Biosystems). TCTP gene has been used as reference. Expression levels of each target gene, relative to TCTP, were determined using a modification of the Pfaffl method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

Primers are listed in the Table S3.

Enhanced Yeast-One-Hybrid

The eYIH assays were performed as previously described [START_REF] Gaudinier | Enhanced Y1H assays for Arabidopsis[END_REF][START_REF] Taylor-Teeples | An Arabidopsis gene regulatory network for secondary cell wall synthesis[END_REF]. Briefly, gene promoters (2000bp of upstream regulatory region from the translational start site, or the next gene) were cloned and recombined to reporter vectors pMW2 (Y1H HIS3 reporter vector) and PMW3 (YIH LacZ reporter vector) [START_REF] Brady | A stele-enriched gene regulatory network in the Arabidopsis root[END_REF]. Interactions were called for transcription factors that activated at least one reporter assay. Primers used are listed in Table S3.

Author contributions

Supplementary Material

123

Summary

In plants, the production of new tissues and organs is continuous and reflects the activity of meristems. All the aerial parts of the plants are generated by shoot apical meristems (SAMs).

This involves the local accumulation of auxin orchestrating a series of events that ultimately The treatments and analyses of the fluorescent reporter line pLFY>GFP, the arf5-2 bot1-7

cross and the analyses of its phenotype were done in collaboration with Massimiliano Sassi.

The eYIH assay was done by the Proteomics Core Facility of UC Davis. I have performed the analyses of the eYIH with inputs from Jan Traas.

Supplementary Material

Annex 2

Supplementary information: Modelling methods

Modus operandi

We use a segmented Pin-like meristem as shown on figure [START_REF] Bozorg | Stress and Strain Provide Positional and Directional Cues in Development[END_REF] in the main text. Following the "pressurized tissue "hypothesis [START_REF] Bozorg | Stress and Strain Provide Positional and Directional Cues in Development[END_REF], only the outermost anticlinal cell walls are taken into consideration for the mechanical simulations.

Each one of them is tiled with triangular first order finite elements (FEs). All the FEs belonging to the same cell feature the same values for their mechanical and growth characteristics which correspond to seven independent parameters listed in table 1 hereafter. Depending on the case, cells are either regrouped in three or four zones, see figure 1-A, B : The central zone (blue), the periphery (green), the initium (red) and the border zone (white). All cells in a given zone feature the same mechanical and growth characteristics.

Mechanical parameters α

Angular aperture of the fiber distribution. ρ 0 fibers mean angular density. ∆ρ /ρ 0 Relative increase of fibers density within the reenforced direction. Y 0

The elastic modulus of cellulose-based fibers within the cell wall. µ iso

Poisson's ratio of the cell wall.

Growth parameters γ

Extensibility of the cell wall. ε th Strain threshold above with growth occurs.

2 Growth of the structure

The elastic response of the structure is coupled with a plastic one, depicting growth. Following a commonly accepted idea ( [START_REF] Bozorg | Stress and Strain Provide Positional and Directional Cues in Development[END_REF]) we assumed this irreversible part of the deformation to be controlled by a viscoelastic, strainbased, constitutive equation:

where L stands for the deformation velocity gradient, εfor the Green-Lagrangian strain field, γ and ε th are two parameters exposed in Tab.1.

From an initial resting configuration, the structure is put under constrains and mechanical equilibrium is computed. If the resulting strain field (ε) overshoot the threshold parameter (ε th ) the resting configuration is updated, simulating the irreversible deformation due to growth. Once this update applied, the previously computed mechanical equilibrium is no longer valid and a new equilibrium is computed, initiating a second step of the growth process. More detail on this approach and its implemanation are available in [START_REF] Boudon | A computational framework for 3D mechanical modeling of plant morphogenesis with cellular resolution[END_REF].

Details about the various simulation produced

In the case of the present study, we performed three main sets of simulations:

Case#1: We study the influence of the structural anisotropy of the cell wall on a spatially limited zone (the initium) on its growth dynamics.

Case#2: We study the influence of the stiffness of the cell walls of the initium on its growth dynamics, in the case the structure of these walls is isotropic.

Case#3: We study the influence of the structural anisotropy of the cell wall on a large zone (the whole periphery ) on the growth dynamics of the meristem.

Each of these sets consisted in a series of six simulations in which we slightly modified one mechanical parameter of the studied zone. To investigate the influence of the structural anisotropy of the cell wall, we modified the values of the angular aperture of the fibers distribution (variable α in equation (4) and/or parameter "a" in the code).

To investigate the influence of the stiffness, we modified the overall Young's modulus (variable Y 0 in equation ( 5) and/or parameter "Yiso" in the code). Numerical values of the various parameters (elastic, growth-related and solver-related) are given in TAB.BLABLA.

Quantification of cellular expansion

From the simulations, we could compute S i (t n ), the surface area of cell number i at growth step t n . By dividing this surface area by the initial surface area of the considered cell (S i (t 0 )), we defined the relative surface area increase at growth step t n : r i (t n ) = S i (t n ) /S i (t 0 ) .Finally we averaged this ratio over all cells belonging to the same zone, examples of its evolution for various zones of interest are given on figure 2 and figure 3 .