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Comprendre comment la coordination des cellules entre elles permet l’émergence d’une forme est une des questions les plus fascinantes en biologie du développement. Au cours de cette thèse, nous avons utilisé les premiers stades de développement des feuilles dentelées d'Arabidopsis thaliana comme modèle pour étudier la relation entre les évènements cellulaires et la morphogenèse. Pendant le développement des feuilles d'Arabidopsis thaliana, le contrôle fin de la prolifération et de l'expansion cellulaire permet la croissance différentielle au niveau de la marge foliaire, nécessaire à la formation des indentations. Dans ce modèle, la croissance différentielle est le résultat de l'interaction entre la signalisation de l’auxine et l’activité des facteurs de transcription CUP SHAPED COTYLEDONS impliqués dans le maintien de l'identité des domaines frontières. Pour affiner la compréhension des relations complexes entre les facteurs de transcriptions CUC, les réponses auxiniques et les événements cellulaires à l'origine des indentations foliaires, nous avons utilisé des expériences d’imagerie en temps réel sur des primordia foliaires de lignées exprimant des rapporteurs de développement et/ou de réponse auxinique. Nos résultats ont révélé un contrôle dynamique de la croissance différentielle à la marge des feuilles et l'implication critique de CUC3 dans la répression locale de la croissance cellulaire. / How a shape arises from the coordinated behavior of cells is one of the most fascinating questions in developmental biology. Here we used the early stages of development of serrated leaves in Arabidopsis thaliana as a model to study the tight relation between cellular behaviour and morphogenesis. During Arabidopsis thaliana leaf development the fine control of cell proliferation and cell expansion sustains differential growth at the margin required for the formation of leaf outgrowth named teeth. In this model, differential growth is the result of interplay between auxin signaling and CUC transcription factors that are involved in the maintenance of boundary domain identity. To clarify the interconnected relations between patterns of CUC TFs and auxin responses as well as the cellular events behind serrations we used time-lapse experiments on vegetative primordia of lines expressing developmental and/or auxin response reporters. Our results revealed a tight and dynamic control of differential growth at the leaf margin and the critical involvement of CUC3 in the local repression of cell growth in combination with low auxin responses.

L’auxine est une hormone essentielle au développement des plantes, tant pour la division quel’expansion cellulaire. La transcription des gènes de réponses à l’auxine est permise par un mécanisme designalisation faisant intervenir des complexes ubiquitine ligase E3 impliquant des protéines à F-boxTIR1/AFBs, qui en présence d’auxine peuvent interagir avec les répresseurs transcriptionnels AUX/IAA pourinduire leur dégradation et activer la transcription de gènes de réponses à l’auxine. Cette transcriptionimplique des facteurs de transcription ARFs (Auxin Response Factors). Dans la feuille, un maximum d’auxinerésultant de l’activité des transporteurs d’efflux d’auxine PIN1 participe à l’initiation des dents à la marge. Lefacteur de transcription CUC2 qui permet la formation des domaines frontières intervient dans le contrôle del’orientation des transporteurs PIN1. L’auxine réprime également CUC2 limitant ainsi son expression au sinus,ce qui semble nécessaire pour la formation des dents. Les acteurs de la signalisation auxinique impliquésdans la formation des dents ne sont néanmoins pas connus chez Arabidopsis.Dans cette thèse, les profils d’expression des ARFs ont été mis en évidence dans les jeunes feuillesd’Arabidopsis au moment de la formation des dents et une cartographie fine a été établie. Trois ARFspotentiellement répresseurs ARF1, ARF3, ARF18 et trois activateurs ARF5, ARF6, ARF8 ont été identifiéscomme étant exprimés dans la zone dent/sinus. La modification de leurs profils d’expression dans des formesde feuilles modifiées par des variations d’expression de CUC2 a également été étudiée. Afin de déterminer sices ARFs sont impliqués dans la morphogénèse foliaire et plus particulièrement dans l’initiation des dents, lesimple mutant arf5-2/mpS319, et les doubles mutants arf6-2 arf8-3, arf1-5 arf3-1, arf1-5 arf18-2 et arf3-1arf18-2 ont été générés et analysés. Les feuilles matures du mutant nul arf5-2/ mpS319 présentent une tailleplus importante que celles de Col-0, suggérant que ARF5 serait impliqué dans l’expansion cellulaire. / Auxin is essential for plant development, more particularly by participating in cellular division andexpansion. Transcription of auxin response genes is allowed by the auxin signaling pathway involving the F-boxTIR1/AFBs proteins associated to ubiquitin ligase E3, which can interact with the AUX/IAA repressors in thepresence of auxin to trigger their degradation and activate transcription of early auxin response genes involvingthe Auxin Response Factors (ARF). At the leaf margin, an auxin maximum resulting from the transport of auxinmediated by the auxin efflux carrier PIN1 is required for teeth formation. The transcription factor CUC2 thatdefines boundary domains, somehow redirects PIN1 to create a convergent flux to the apex of the tooth. Auxinrepresses CUC2 thus limiting its expression to the sinus. In Arabidopsis, the actors of the auxin signalingpathway involved in leaf serration are unknown.In this thesis, the expression profiles of ARFs genes have been investigated in young leaves of Arabidopsis, morespecifically during teeth formation and a detailed map was built. Three ARFs acting as putative repressors ARF1,ARF3 and ARF18 and three activators ARF5, ARF6 and ARF8 were identified as been expressed in thesinus/teeth area. Their expression profiles were also studied in leaves exhibiting modified shapes as a result ofvariations in CUC2 expression. To determine if these ARFs are involved in leaf morphogenesis and moreparticularly in tooth initiation, the arf5-2 null mutant and the double null mutants arf6-2 arf8-3, arf1-5 arf3-1,arf1-5 arf18-2 and arf3-1 arf18-2 were generated and serration was analyzed. Length of arf5-2 mature leaves islonger than the wild type, suggesting that ARF5 could be involved in cell expansion.

Much is known about how organs acquire their identity, yet we are only beginning to learn how their shape is regulated. Recent work has elucidated the role of coordinated cell division & expansion in determining plant organ shape. For instance, in Antirrhinum, leaf shape is affected in the cincinnata (cin) mutant because of an alteration in the cell division pattern. CIN codes for a TCP transcription factor and controls cell proliferation. It is unclear how exactly CIN-like genes regulate leaf morphogenesis. We have taken biochemical and genetic approach to understand the TCP function in general and the role of CIN-like genes in leaf morphogenesis in Antirrhinum and Arabidopsis. Targets of CINCINNATA To understand how CIN controls Antirrhinum leaf shape, we first determined the consensus target site of CIN as GTGGTCCC by carrying out RBSS assay. Mutating each of this target sequence, we determined the core binding sequence as TGGNCC. Hence, all potential direct targets of CIN are expected to contain a TGGNCC sequence. Earlier studies suggested that CIN activates certain target genes that in turn repress cell proliferation. To identify these targets, we compared global transcripts of WT and cin leaves by differential display PCR and have identified 18 unique, differentially expressed transcripts. To screen the entire repertoire of differentially expressed transcripts, we have carried out extensive micro-array analysis using 44K Arabidopsis chips as well as 13K custom-made Antirrhinum chips. Combining the RBSS data with the results obtained from the micro-array experiments, we identified several targets of CIN. In short, CIN controls expression of the differentiation-specific genes from tip to base in a gradient manner. In cin, such gradient is delayed, thereby delaying differentiation. We also find that gibberellic acid, cytokinin and auxin play important role in controlling leaf growth. Genetic characterization of CIN-homologues in Arabidopsis Arabidopsis has 24 TCP genes. Our work and reports from other groups have shown that TCP2, 4 and 10 are likely to be involved in leaf morphogenesis. These genes are controlled by a micro RNA miR319. To study the role of TCP4, the likely orthologue of CIN, we generated both stable and inducible RNAi lines. Down-regulation of TCP4 transcript resulted in crinkly leaves, establishing the role of TCP4 in leaf shape. To study the function of TCP2, 4 & 10 in more detail, we isolated insertion mutants in these loci. The strongest allele of TCP4 showed embryonic lethal phenotype, indicating a role for TCP4 in embryo growth. All other mutants showed mild effect on leaf shape, suggesting their redundant role. Therefore, we generated and studied various combinations of double and triple mutants to learn the concerted role of these genes on leaf morphogenesis. To further study the role of TCP4 in leaf development, we generated inducible RNAi and miRNA-resistant TCP4 transgenic lines and carried out studies with transient down-regulation and up-regulation of TCP4 function. Upon induction, leaf size increased in RNAi transgenic plants whereas reduced drastically in miR319 resistant lines, suggesting that both temporal & spatial regulation of TCP4 is required for leaf development. Biochemical characterization of TCP domain To study the DNA-binding properties of TCP4, random binding site selection assay (RBSS) was carried out and it was found that TCP4 binds to a consensus sequence of GTGGTCCC. By patmatch search and RT-PCR analysis, we have shown that one among 74 putative targets, EEL (a gene involved in embryo development), was down regulated in the RNAi lines of TCP4. This suggests that EEL could be the direct target of TCP4. We have tested this possibility in planta by generating transgenic lines in which GUS reporter gene is driven by EEL upstream region with either wild type or mutated TCP4 binding site. GUS analysis of embryos shows that transgenic with mutated upstream region had significantly reduced reporter activity in comparison to wild type, suggesting that EEL is a direct target of TCP4. We have further shown that TCP4 also binds to the upstream region of LOX2, a gene involved in Jasmonic acid (JA) biosynthesis (in collaboration with D. Weigel, MPI, Tubingen, Germany). TCP domain has a stretch of basic residues followed by a predicted helix-loop-helix region (bHLH), although it has little sequence homology with canonical bHLH proteins. This suggests that TCP is a novel and uncharacterized bHLH domain. We have characterized DNA-binding specificities of TCP4 domain. We show that TCP domain binds to the major groove of DNA with binding specificity comparable to that of bHLH proteins. We also show that helical structure is induced in the basic region upon DNA binding. To determine the amino acid residues important for DNA binding, we have generated point mutants of TCP domain that bind to the DNA with varied strength. Our analysis shows that the basic region is important for DNA binding whereas the helix-loop-helix region is involved in dimerization. Based on these results, we have generated a molecular model for TCP domain bound to DNA (in Collaboration with Prof. N. Srinivasan, IISc, Bangalore). This model was validated by further site-directed mutagenesis of key residues and in vitro assay. Functional analysis of TCP4 in budding yeast To assess TCP4 function in regulation of eukaryotic cell division, we have introduced TCP4 in S. cerevisiae under the GAL inducible promoter. TCP4 induction in yeast cells always slowed down its growth, indicative of its detrimental effect on yeast cell division. Flow cytometry analysis of synchronized cells revealed that TCP4 arrests yeast cell division specifically at G1→S boundary. Moreover, induced cells showed distorted cell morphology resembling shmoo phenotype. Shmooing is a developmental process which usually happened when the haploid cells get exposed to the cells of opposite mating type and get arrested at late G1 phase due to the inhibition of cdc28-cln2 complex. This suggested that TCP4-induced yeast cells are arrested at late G1 phase probably by the inhibition of cdc28-cln2 complex. To further investigate how TCP4 induce G1→S arrest, we carried out microarray analysis and found expression of several cell cycle markers significantly altered in TCP4-induced yeast cells. Studies on crinkly1, a novel leaf mutant in Arabidopsis To identify new genes involved in leaf morphogenesis, we have identified crinkly1 (crk1), a mutant where leaf shape and size are altered. We observed that crk1 also makes more number of leaves compared to wild type. Phenotypic analysis showed that crk1 leaf size is ~5 times smaller than that of wild type. Scanning electron microscopy (SEM) showed that both cell size and number are reduced in the mutant leaf, which explains its smaller size. We have mapped CRK1 within 3 cM on IV chromosome.