Browsing by Subject "TCP"

Asteraceae comprises of approximately 10% of all angiosperm plant species. These species are well known for their highly compressed inflorescences known as capitula which consists of morphologically different types of flowers: ray, trans and disc flowers. This immense morphological difference excels Gerbera as an ideal plant to study flower type differentiations. Even though this complex process is governed by several genes, the ray flower identity is believed to be greatly influenced by GhCYC3 promoter mediated gene regulations. In previous studies two TCP transcription factors (TF): GhCIN1and GhCIN2, and two MADS TFs: GAGA1 and RCD5 were identified as the potential upstream regulators of GhCYC3. So, the aim of this study is to test whether these potential upstream regulators physically bind to GhCYC3 promoter in in vitro conditions. In order to achieve the goal, these transcription factor proteins from Gerbera hybrida were successfully expressed in E. coli and purified as fusion proteins to maltose-binding protein (MBP). Physical binding of the purified fusion proteins to the putative target DNA sites in the promoter region of GhCYC3 gene was tested by electrophoretic mobility shift assay (EMSA). The results showed that none of the gerbera transcription factors (GhCIN1, GhCIN2, GAGA1 and RCD5) bind to their putative target sites under the condition tested in this study. However, it might not be justifiable to deduce that these TFs do not interact with GhCYC3 promoter. The absence of in vitro interaction between the tested TFs and GhCYC3 promoter might be caused by either lack of proper folding and activity of the TFs or absence of co-factors which are available in vivo.

The complex inflorescence architecture in Asteraceae is characterized by the presence of morphologically distinct flowers having specialized functions. In gerbera, the presence of three different flower types (ray, trans and disc), intricate inflorescence organization and an underlying complex molecular control makes it an excellent target for research on functional genomics, flower development and evolution. The genes governing flower development have been shown to have undergone sub- and neo-functionalization in gerbera leading to morphological features that are not present in the conventional model plant species. The genes encoding the CYC2 subclade of CYC/TB1-like TCP domain transcription factors are known to regulate the flower type identity in gerbera inflorescence. Although most genes of the CYC2 subclade have demonstrated functional redundancy, one such gene, GhCYC5, was found to have diverged function in regulating rate of initiation of flowers. This study aimed at investigating and affirming the function of GhCYC5 by studying two RNAi lines. Phenotypic analysis of the RNAi inflorescences showed that the length of ray ligules was significantly decreased along with the number of disc flowers and the number of involucral bracts. However, gene expression analysis could not confirm the downregulation of GhCYC5 in the RNAi lines mainly due to low endogenous expression of the gene. However, GhCYC3, another CYC2 clade gene was found to be downregulated due to the off-target silencing effect of the RNAi product and the observed phenotype was associated with suppression of GhCYC3. Further, the role of GhCYC5 in regulating the rate of involucral bract and flower initiation was unclear based on the SEM imaging and expression analysis of the inflorescence meristem, and still requires further studies.