TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

CYCLOIDEA (CYC) and DICHOTOMA (DICH) of the CYC2 clade of the TCP gene family have been shown to play a significant role in regulating the identity of the dorsal petals and abortion of the single dorsal stamen in Antirrhinum majus. It is believed that CYC2-like genes are responsible for the convergent evolution of floral zygomorphy, but their role in the development of actinomorphic flowers is still unknown. In Solanaceae, previous analysis has identified two paralogs of CYC2-like genes, CYC2A and CYC2B, resulting from a gene duplication that predates the origin the family. Virus-induced gene silencing (VIGS) is a technique to study the gene function by silencing specific target genes of interest, which is shown to be useful in diverse plant species. Here, we report on the role of CYC2-like genes during floral development in Solanaceae based on the results of VIGS using tobacco rattle virus (TRV)-based vector in Solanum lycopersicum having completely actinomorphic flowers and Nicotiana obtusifolia having slightly zygomorphic flowers. Our VIGS experiments in So. lycopersicum show that downregulation of both CYC2A and CYC2B leads to misshaped petals, the unequal growth of the petals, and most frequently increased number of petals, stamens and sepals, while the carpel and ovule morphology remain the same as the wild type. On the contrary, downregulation of CYC2A and CYC2B in N. obtusifolia results in reduced number of flower organs in sepals, stamens, and petals, however carpels remained the same. For both solanaceous species, silencing CYC2A and CYC2B changes the property of cytoplasm and retards the rate of pollen germination. Our findings suggest that the CYC2-like genes are likely involved in the floral development, mainly regulating the number of floral organs and pollen development in Solanaceae.

TCP4 as an integrator of key developmental events A striking aspect of plant life is their sedentary life-style. Though it relieves them of the obligation of forming a complex body organization, it exposes them to environmental challenges. Plants have evolved a flexible pattern of post-embryonic growth. The major phases in their life cycle are photomorphogenesis, vegetative growth with phase transitions, reproductive growth and senescence. The phase transitions are coordinated temporally to ensure proper maturation of organism. Flexibility is built in the re-iterated programs of organogenesis, which provides a plant with an option to adopt an architecture best suited to prevailing environmental conditions. Organogenesis occurs by processes of cell division and maturation (expansion). Cell division determines the growth potential by generating the requisite number of cells and cell maturation fulfils the potential by elaborating the organ form. Organ growth requires spatially- and temporally-controlled cellular maturation. The TCP class of plant-specific transcription factors, conserved from bryophytes to angiosperms, control diverse developmental and morphological traits, such as plant architecture, floral asymmetry, seed germination, male and female gametophyte development and photomorphogenesis (Martín-Trillo and Cubas, 2010). Class II TCPs, which are targets of miR319, are best known for their role in leaf morphogenesis. They are believed to function by redundantly regulating the onset of cellular maturation ( Efroni et al., 2008; Koyama et al., 2007; Nath et al., 2003; Ori et al., 2007; Palatnik et al., 2003; Schommer et al., 2008). To establish the link between level of TCP activity and organ growth, we undertook the approach of hyper-activating the function of TCP4, a representative class II TCP, by fusing it with a strong transactivation domain. Enhanced level of TCP4 activity reduced organ growth by causing precocious cellular maturation. It also accelerated the process of organ initiation, maturation and its progression into the final stage of senescence. Hyper-active TCP4-expressing plants underwent faster maturation of shoot apex into reproductive phase. In general, hyper-activation of TCP4 advanced cellular, organ and organism maturation programs in Arabidopsis life cycle (Fig. 1). Traits such as organ initiation rate, organ size, flowering time and seed yield contribute to the fitness of the plant. Faster rate of organ initiation, bigger organ size, early onset of flowering and higher seed yield are obvious desirable traits. However, they rarely occur simultaneously in a mutant or a natural variant, suggesting that there is a trade-off among different traits. Studies have shown that such traits are linked and are controlled by multiple loci that contribute quantitatively to the phenotype. A change that benefits one trait may adversely affect another (Colautti et al., 2011; Kozlowski, 1992; Mendez-Vigo et al., 2010) . Our study shows that TCP4 activity can potentially coordinate these inter-connected traits. Though hyper-active TCP4-expressing plants have faster rate of organ initiation, the final organ size is reduced and senescence is advanced. These plants reach reproductive phase faster, but produce fewer seeds, hence limiting their propagation and lowering their fitness in comparison to the wild type. Such a genetic constraint on the traits limits the phenotypic variation that can be produced in plants and, hence, their adaptation to the environment. Our study suggests that TCP4 can link organ growth with that of the whole organism. It acts as a heterochronic regulator which possibly affects timing of multiple maturation programs. Any perturbation in the TCP activity may have far-reaching effects on plant growth and thus, optimal level of TCP activity is crucial for plant homeostasis. One possible explanation for the developmental pleitropy in TCP4 hyper-activation line is an alteration in hormone biosynthesis or sensitivity. A combination of microarray and hormone application studies on hyper-active TCP4-expressing line has indicated a reduction in the levels of GA and auxin and an increase in cytokinin and MeJA levels. There may also be an inhibition of auxin signaling and upregulation of MeJA and ethylene signaling. In addition, TCP4 appeared to regulate both GA biosynthesis and response in opposing manner. The molecular mechanisms involved in TCP4-mediated integration of hormonal pathways are still unclear. Answering these questions would require identification of its direct downstream targets.

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.