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Parmi les signaux floraux, les odeurs florales sont remarquables pour leur complexité en composés odorants et leur variation entre, et au sein des taxa. Elles interviennent dans de nombreuses interactions que les plantes entretiennent avec les organismes de leur environnement. Cette diversité chimique gouverne de multiples fonctions, telles que l’attraction de pollinisateurs, l’encouragement à la constance florale et la défense contre des antagonistes. Bien que les fonctions écologiques des odeurs florales soient relativement bien étudiées, les facteurs évolutifs qui gouvernent la composition et les variations de ce signal complexe sont très mal connus. C’est dans ce contexte que ma thèse s’inscrit. J’ai étudié les variations de ce trait floral particulier : les odeurs florales. Ma thèse se focalise sur une espèce de plante, la gueule-de-loup, Antirrhinum majus, utilisée comme espèce modèle en biologie depuis des décennies. Cette espèce, native des Pyrénées, elle présente deux sous-espèces, l’une à fleurs magenta, A. m. pseudomajus, et l’autre à fleurs jaunes, A. m. striatum. Alors que ces deux sous-espèces peuvent s’inter-féconder, elles ne coexistent jamais dans la nature et leurs hybrides, reconnaissables par une grande diversité de colorations florales, sont peu fréquents. Le mécanisme de cet isolement reproducteur n’est pas connu, mais le comportement des pollinisateurs a été envisagé dans de précédentes études. Les principaux résultats de ma thèse montrent que les deux sous-espèces d’A. majus se distinguent par leurs odeurs florales. Certains composés volatils, en particulier trois benzénoïdes, ne sont émis que par A. m. pseudomajus, et ceci de manière constante entre les populations et pour différents environnements. Quant aux hybrides, les ratios de composés volatils floraux sont très variables par rapport aux signaux reproductibles parentaux, avec un patron de ségrégation chez les hybrides F2. En utilisant des bourdons commercialisés (Bombus terrestris), donc naïfs de toutes odeurs florales, j’ai montré que ces bourdons sont capables de détecter les principaux composés d’odeurs d’A. majus et qu’ils préfèrent de manière innée un mélange de composés volatils d’A. m. striatum. Finalement, en conditions naturelles, c’est-à-dire avec des odeurs florales naturelles et des pollinisateurs sauvages, ces derniers sont attirés préférentiellement par les odeurs florales de leur sous espèce d’origine. J’ai finalement montré que le patron associatif odeur-nectar qu’apprennent les pollinisateurs fait intervenir uniquement les composés odorants floraux et la quantité de nectar, puisque les différences d’odeurs florales entre les deux sous-espèces sont associées à une plus grande quantité de nectar par fleur chez A. m. pseudomajus mais à une plus faible concentration en sucres. En d’autres termes, les plantes contiennent autant de sucre total dans leurs fleurs dans une sous-espèce ou dans une autre. Ces résultats, pris dans leur ensemble, semblent montrer que les composés volatils floraux sont bien impliqués dans l’isolement reproducteur de ces deux sous-espèces. Même si les odeurs florales ne peuvent pas expliquer à elles seules la distribution spatiale des deux sous-espèces d’A. majus, elles peuvent jouer un rôle supplémentaire de barrière aux flux de gènes. En effet, les pollinisateurs sont susceptibles de montrer un phénomène de constance envers l’un des phénotypes floraux, limitant ainsi les flux de gènes entre les deux sous-espèces. Dans cette thèse, je propose différentes perspectives possibles à mes résultats de thèse / Manquant

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.

During photosynthesis Plantaginaceae species can produce glucose derivatives such as iridoid glycosides and alcohol sugars that in addition to sucrose can be exported from leaves. Plantago lanceolata transported sorbitol in addition to sucrose especially at warmer leaf temperatures. However, two iridoids, catalpol and aucubin, found in P. lanceolata were not readily labelled from 14CO2 under any conditions examined. In contrast, in two greenhouse, cut-flower cultivars of Antirrhinum majus the iridoids, antirrhinoside and antirrhide, were readily 14C-labelled along with sucrose but little 14C was recovered in alcohol sugars (e.g., mannitol). The amount of 14C-partitioned into antirrhinoside increased at higher temperatures. Exposing leaves of P. lanceolata and A. majus to reduced-photorespiratory conditions (e.g. short-term CO2 enrichment and/or low O2) increased fixation and export. Under low O2 in P. lanceolata sorbitol 14C-labelling increased relative to sucrose and in A. majus 14C-labelling of sucrose increased relative to antirrhinoside. Also 14C-labelling of antirrhide increased more than antirrhinoside. During both short-term and long-term acclimation to high CO2, whole plant NCER, leaf photosynthesis and export increased in A. majus. Taken together the temperature and CO2 enrichment studies show plasticity in Plantaginaceae species to synthesize and transport sucrose and auxiliary glucose esters and alcohol sugars in a species-specific manner (depending on the rate of carboxylation).

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.