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.

A filosfera foi considerada um ambiente extremo durante muitos anos para os microrganismos. Estudos recentes envolvendo comunidades bacterianas na superfície das plantas têm demonstrado que esta é composta por uma enorme e variável diversidade, principalmente em plantas de ambientes naturais como as da Mata Atlântica. Comparar as comunidades de bactérias de plantas de ambientes naturais com as de sistemas agrícola é de extrema importância, visto que as matas abrigam uma enorme diversidade vegetal e esta vem sendo diariamente substituída pela agricultura. Fatores ambientais como radiação UV, compostos orgânicos liberados pelas plantas em resposta à proteção, estresse osmótico e disponibilidade de nutrientes também podem induzir ou reprimir a colonização dessas bactérias na filosfera das plantas. Portanto, este trabalho traçou dois objetivos, o primeiro foi avaliar a variabilidade na comunidade bacteriana da filosfera de plantas cultivadas (soja, cana-de-açúcar, e eucalipto), localizadas em regiões geograficamente distintas, utilizando sequenciamento de bibliotecas de clones de DNA do gene rRNA 16S e comparar com as comunidades da filosfera de plantas nativas (Trichilia clausenii, Trichilia catigua e Campomanesia xanthocarpa), e o segundo, foi verificar a influência dos compostos orgânicos voláteis (COVs) emitidos pela planta, os diferentes níveis de radiação solar e a sazonalidade na seleção das comunidades bacterianas na filosfera de Trichilia clausenii, usando sequenciamento de bibliotecas de clones de cDNA do gene rRNA 16S e cromatografia gasosa acoplada à espectrometria de massas (CGEM). A análise comparativa das bibliotecas de clones do gene rRNA 16S de plantas de ambientes naturais com cultivadas, confirmou que cada espécie de planta possui sua própria comunidade de bactérias, sendo a diversidade de UTOs na filosfera de C. xanthocarpa e T. clusenii maior que a de plantas cultivadas. O filo Proteobacteria. foi predominante em ambas as filosferas de plantas cultivadas e nativas, sendo as Alphaproteobacteria dominantes na soja e na cana-de-açúcar e as Gamaproteobacteria nas filosferas das três árvores da Mata Atlântica. Este resultado sugere que a filosfera de plantas cultivadas pode selecionar grupos específicos de bactérias que não são encontradas na filosfera de espécies arbóreas da Mata Atlântica, e vice-versa, e que a substituição de florestas por culturas agrícolas pode levar a uma redução da beta-diversidade de bactérias. Já os resultados da afiliação filogenética das amostras da filosfera de Tichilia clausenii submetida a diferentes intensidades de radiações UV, também mostraram uma predominância de clones associados à Proteobacteria em todas as bibliotecas avaliadas. O fator luminosidade e o da planta, quando avaliados na mesma época, não mostraram efeito sob a comunidade bacteriana, entretanto, o efeito da sazonalidade foi bastante evidente, apresentando uma maior diversidade e riqueza de espécies durante a estação chuvosa. Os dados sugerem que as bactérias epifíticas possuem um elevado grau de adaptação a diferentes condições ambientais que são expostas. / The phyllosphere has been considered an extreme environment to microorganisms for many years. Recent studies involving bacterial communities on plant surfaces has shown that they comprise an abundant and dynamic variety, mainly in natural environments, such as the Atlantic Forest. The comparison of bacterial communities from plants in natural environments with those of agricultural systems is extremely important, since forests contain an enormous plant diversity which is being replaced daily by agricultural cultivation. Environmental factors such as UV radiation, organic compounds released by plants in response to protection, osmotic pressure, and nutrient availability can either induce or suppress the colonization of bacteria in the phyllosphere of plants. Therefore, this study aimed to evaluate the variability in phyllospheric bacterial communities of native plants (Trichilia clausenii, Trichilia catigua and Campomanesia xanthocarpa) and crops (soybean, sugar cane and eucalyptus) located in geographically distinct regions, using 16S rRNA gene sequences from clone libraries, the influence of volatile organic compounds (VOCs) emitted by each plant, the different levels of solar radiation, and seasonality in the selection of bacterial communities in the phyllosphere of Trichilia clausenii using 16S rRNA gene sequences from cDNA clone libraries and gas chromatography-mass spectrometry (GCMS). The comparative analysis of the 16S rRNA gene sequences from clone libraries of plants from natural environments confirmed that each plant species has its own community of bacteria, where the diversity of OTUs in the phyllosphere of C. xanthocarpa and T. clusenii was greater than that of cultivated plants. The phylum Proteobacteria was predominant in the phyllosphere of both cultivated and native plants, where Alphaproteobacteria was dominant in phyllospheres of soybean and sugar cane and Gamaproteobacteria was harbored on the three trees of the Atlantic Forest. These results suggest that the phyllosphere of cultivated plants can select for specific groups of bacteria that are not found in the phyllosphere of tree species in the Atlantic Forest, and vice versa, where the conversion of forests to agriculture may lead to a reduction of bacterial beta diversity. The results of the phylogenetic affiliation between the phyllospheres of Tichilia clausenii, subjected to different intensities of UV radiation, also showed a predominance of clones associated with Proteobacteria in the evaluated clone libraries. The light factor, which was evaluated during the same period, showed no effect on the bacterial community, however, seasonal effects were quite evident, showing a higher diversity and species richness during the rainy season. This data suggests that epiphytic bacteria are equipped with a high degree of adaptation to different environmental conditions that they are exposed to.

A filosfera foi considerada um ambiente extremo durante muitos anos para os microrganismos. Estudos recentes envolvendo comunidades bacterianas na superfície das plantas têm demonstrado que esta é composta por uma enorme e variável diversidade, principalmente em plantas de ambientes naturais como as da Mata Atlântica. Comparar as comunidades de bactérias de plantas de ambientes naturais com as de sistemas agrícola é de extrema importância, visto que as matas abrigam uma enorme diversidade vegetal e esta vem sendo diariamente substituída pela agricultura. Fatores ambientais como radiação UV, compostos orgânicos liberados pelas plantas em resposta à proteção, estresse osmótico e disponibilidade de nutrientes também podem induzir ou reprimir a colonização dessas bactérias na filosfera das plantas. Portanto, este trabalho traçou dois objetivos, o primeiro foi avaliar a variabilidade na comunidade bacteriana da filosfera de plantas cultivadas (soja, cana-de-açúcar, e eucalipto), localizadas em regiões geograficamente distintas, utilizando sequenciamento de bibliotecas de clones de DNA do gene rRNA 16S e comparar com as comunidades da filosfera de plantas nativas (Trichilia clausenii, Trichilia catigua e Campomanesia xanthocarpa), e o segundo, foi verificar a influência dos compostos orgânicos voláteis (COVs) emitidos pela planta, os diferentes níveis de radiação solar e a sazonalidade na seleção das comunidades bacterianas na filosfera de Trichilia clausenii, usando sequenciamento de bibliotecas de clones de cDNA do gene rRNA 16S e cromatografia gasosa acoplada à espectrometria de massas (CGEM). A análise comparativa das bibliotecas de clones do gene rRNA 16S de plantas de ambientes naturais com cultivadas, confirmou que cada espécie de planta possui sua própria comunidade de bactérias, sendo a diversidade de UTOs na filosfera de C. xanthocarpa e T. clusenii maior que a de plantas cultivadas. O filo Proteobacteria. foi predominante em ambas as filosferas de plantas cultivadas e nativas, sendo as Alphaproteobacteria dominantes na soja e na cana-de-açúcar e as Gamaproteobacteria nas filosferas das três árvores da Mata Atlântica. Este resultado sugere que a filosfera de plantas cultivadas pode selecionar grupos específicos de bactérias que não são encontradas na filosfera de espécies arbóreas da Mata Atlântica, e vice-versa, e que a substituição de florestas por culturas agrícolas pode levar a uma redução da beta-diversidade de bactérias. Já os resultados da afiliação filogenética das amostras da filosfera de Tichilia clausenii submetida a diferentes intensidades de radiações UV, também mostraram uma predominância de clones associados à Proteobacteria em todas as bibliotecas avaliadas. O fator luminosidade e o da planta, quando avaliados na mesma época, não mostraram efeito sob a comunidade bacteriana, entretanto, o efeito da sazonalidade foi bastante evidente, apresentando uma maior diversidade e riqueza de espécies durante a estação chuvosa. Os dados sugerem que as bactérias epifíticas possuem um elevado grau de adaptação a diferentes condições ambientais que são expostas. / The phyllosphere has been considered an extreme environment to microorganisms for many years. Recent studies involving bacterial communities on plant surfaces has shown that they comprise an abundant and dynamic variety, mainly in natural environments, such as the Atlantic Forest. The comparison of bacterial communities from plants in natural environments with those of agricultural systems is extremely important, since forests contain an enormous plant diversity which is being replaced daily by agricultural cultivation. Environmental factors such as UV radiation, organic compounds released by plants in response to protection, osmotic pressure, and nutrient availability can either induce or suppress the colonization of bacteria in the phyllosphere of plants. Therefore, this study aimed to evaluate the variability in phyllospheric bacterial communities of native plants (Trichilia clausenii, Trichilia catigua and Campomanesia xanthocarpa) and crops (soybean, sugar cane and eucalyptus) located in geographically distinct regions, using 16S rRNA gene sequences from clone libraries, the influence of volatile organic compounds (VOCs) emitted by each plant, the different levels of solar radiation, and seasonality in the selection of bacterial communities in the phyllosphere of Trichilia clausenii using 16S rRNA gene sequences from cDNA clone libraries and gas chromatography-mass spectrometry (GCMS). The comparative analysis of the 16S rRNA gene sequences from clone libraries of plants from natural environments confirmed that each plant species has its own community of bacteria, where the diversity of OTUs in the phyllosphere of C. xanthocarpa and T. clusenii was greater than that of cultivated plants. The phylum Proteobacteria was predominant in the phyllosphere of both cultivated and native plants, where Alphaproteobacteria was dominant in phyllospheres of soybean and sugar cane and Gamaproteobacteria was harbored on the three trees of the Atlantic Forest. These results suggest that the phyllosphere of cultivated plants can select for specific groups of bacteria that are not found in the phyllosphere of tree species in the Atlantic Forest, and vice versa, where the conversion of forests to agriculture may lead to a reduction of bacterial beta diversity. The results of the phylogenetic affiliation between the phyllospheres of Tichilia clausenii, subjected to different intensities of UV radiation, also showed a predominance of clones associated with Proteobacteria in the evaluated clone libraries. The light factor, which was evaluated during the same period, showed no effect on the bacterial community, however, seasonal effects were quite evident, showing a higher diversity and species richness during the rainy season. This data suggests that epiphytic bacteria are equipped with a high degree of adaptation to different environmental conditions that they are exposed to.