O florescimento é um processo chave no desenvolvimento vegetal. A mudança de identidade do meristema apical de vegetativo para reprodutivo desencadeia reprogramação genética com efeitos em todo o corpo vegetal. Arabidopsis thaliana é conhecida como o principal modelo de estudo para esse processo apresentando até o momento cinco principais vias genéticas regulatórias. Tais vias apresentam redundância, sendo complexa a eliminação total da transição de fase nessa espécie. A via AGE, regulada pela idade da planta, tem como principais reguladores o mir156 e seus alvos diretos, os fatores de transcrição da família SPL/SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like). Uma segunda via é controlada pelo fitohormônio giberelina (GA), o qual atua de maneira oposta em Arabidopsis thaliana (arabidopsis) e Solanum lycopersicum L. (tomateiro). Em tomateiro, diferentemente de arabidopsis, o cruzamento entre mutantes com conteúdo alterado de GA e plantas transgênicas superexpressando o miR156 (156OE; SILVA et al., 2014) demonstraram efeito sinérgico no atraso do tempo de florescimento. A aplicação de GA3 em plantas 156OE apresenta efeito similar aos cruzamentos citados sobre a transição do meristema apical. Em um dos cruzamentos entre mutantes da via GA e plantas 156OE, foi possível obter plantas apresentando completo bloqueio da transição de fase vegetativo-reprodutivo. A oferta extra do florígeno SINGLE FLOWER TRUSS (SFT) via enxertia não foi suficiente para restaurar a transição de fase nessas plantas, sugerindo que vias associadas à GA e AGE regulam alvos em comum, os quais podem ser independentes da regulação por SFT. Além disso, a regulação transcricional, e possivelmente pós-transcricional de alguns genes SBPs por diferentes vias associadas à GA, sugere uma complexa inter-relação entre as vias GA e AGE em tomateiro durante o florescimento. A ação combinada das vias GA e AGE foi capaz de inibir completamente o florescimento em tomateiro, regulação oposta ao verificado na planta modelo Arabidopsis thaliana. O efeito inibitório de GA sobre o florescimento é também visualizado em plantas lenhosas, sugerindo que as descobertas científicas realizadas em tomateiro podem ser expandidas para essas espécies, nas quais a experimentação é lenta e laboriosa / The flowering process is a major developmental event during the plant life cicle. The meristem identity switches from vegetative to reproductive, triggering substantial genetic modifications that affect the whole plant body. Arabidopsis thaliana is a major model for flowering with five different pathways controlling this process. These pathways are redundant, making complex the complete elimination of phase change in this species. One of the pathways is termed AGE since it is regulated by the time of development. The miR156 and its direct target SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like) are the main regulators of the AGE pathway. A second pathway is controlled by the phytohormone gibberellin (GA), which acts in opposite ways when comparing Arabidopsis thaliana and tomato. In tomato, unlike Arabidopsis, the cross between mutants with altered contents of GA and transgenic plants overexpressing the miR156 (156OE; SILVA et al, 2014) showed synergistic effect in delayed flowering time. Treatments of GA3 in plants 156OE lead to similar effects visualized on the crosses above related to meristem transition. Among the crosses between GA mutants and 156OE plants, one double mutant could completely abolish the phase change in tomato. An extra offer of the florigen (SINGLE FLOWER TRUSS or SFT) by grafting experiments was unable to restore the flowering process in this double mutant. It suggests, pathways associated to GA and AGE regulate common downstream targets, which could be independent of SFT regulation. Moreover, the transcriptional regulation, and possible the post-transcriptionally regulation of some SBP targets by different pathways associated to GA, suggest a complex network between GA and AGE during the flowering in tomato. The combined action of GA and AGE pathways can complete impaired the flowering in tomato, this interaction is opposed to the model Arabidopsis thaliana. The negative effect of GA over the time of flowering is presented in wood plants, suggesting the scientific discoveries in tomato could be expanded to these species, which experiments are slow and laborious

O florescimento é um processo chave no desenvolvimento vegetal. A mudança de identidade do meristema apical de vegetativo para reprodutivo desencadeia reprogramação genética com efeitos em todo o corpo vegetal. Arabidopsis thaliana é conhecida como o principal modelo de estudo para esse processo apresentando até o momento cinco principais vias genéticas regulatórias. Tais vias apresentam redundância, sendo complexa a eliminação total da transição de fase nessa espécie. A via AGE, regulada pela idade da planta, tem como principais reguladores o mir156 e seus alvos diretos, os fatores de transcrição da família SPL/SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like). Uma segunda via é controlada pelo fitohormônio giberelina (GA), o qual atua de maneira oposta em Arabidopsis thaliana (arabidopsis) e Solanum lycopersicum L. (tomateiro). Em tomateiro, diferentemente de arabidopsis, o cruzamento entre mutantes com conteúdo alterado de GA e plantas transgênicas superexpressando o miR156 (156OE; SILVA et al., 2014) demonstraram efeito sinérgico no atraso do tempo de florescimento. A aplicação de GA3 em plantas 156OE apresenta efeito similar aos cruzamentos citados sobre a transição do meristema apical. Em um dos cruzamentos entre mutantes da via GA e plantas 156OE, foi possível obter plantas apresentando completo bloqueio da transição de fase vegetativo-reprodutivo. A oferta extra do florígeno SINGLE FLOWER TRUSS (SFT) via enxertia não foi suficiente para restaurar a transição de fase nessas plantas, sugerindo que vias associadas à GA e AGE regulam alvos em comum, os quais podem ser independentes da regulação por SFT. Além disso, a regulação transcricional, e possivelmente pós-transcricional de alguns genes SBPs por diferentes vias associadas à GA, sugere uma complexa inter-relação entre as vias GA e AGE em tomateiro durante o florescimento. A ação combinada das vias GA e AGE foi capaz de inibir completamente o florescimento em tomateiro, regulação oposta ao verificado na planta modelo Arabidopsis thaliana. O efeito inibitório de GA sobre o florescimento é também visualizado em plantas lenhosas, sugerindo que as descobertas científicas realizadas em tomateiro podem ser expandidas para essas espécies, nas quais a experimentação é lenta e laboriosa / The flowering process is a major developmental event during the plant life cicle. The meristem identity switches from vegetative to reproductive, triggering substantial genetic modifications that affect the whole plant body. Arabidopsis thaliana is a major model for flowering with five different pathways controlling this process. These pathways are redundant, making complex the complete elimination of phase change in this species. One of the pathways is termed AGE since it is regulated by the time of development. The miR156 and its direct target SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like) are the main regulators of the AGE pathway. A second pathway is controlled by the phytohormone gibberellin (GA), which acts in opposite ways when comparing Arabidopsis thaliana and tomato. In tomato, unlike Arabidopsis, the cross between mutants with altered contents of GA and transgenic plants overexpressing the miR156 (156OE; SILVA et al, 2014) showed synergistic effect in delayed flowering time. Treatments of GA3 in plants 156OE lead to similar effects visualized on the crosses above related to meristem transition. Among the crosses between GA mutants and 156OE plants, one double mutant could completely abolish the phase change in tomato. An extra offer of the florigen (SINGLE FLOWER TRUSS or SFT) by grafting experiments was unable to restore the flowering process in this double mutant. It suggests, pathways associated to GA and AGE regulate common downstream targets, which could be independent of SFT regulation. Moreover, the transcriptional regulation, and possible the post-transcriptionally regulation of some SBP targets by different pathways associated to GA, suggest a complex network between GA and AGE during the flowering in tomato. The combined action of GA and AGE pathways can complete impaired the flowering in tomato, this interaction is opposed to the model Arabidopsis thaliana. The negative effect of GA over the time of flowering is presented in wood plants, suggesting the scientific discoveries in tomato could be expanded to these species, which experiments are slow and laborious

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.