Browsing by Subject "Asteraceae"

Inflorescence meristem development in plants generally falls into two types: one is the indeterminate type when the main axis keep growing and new flowers are initiated on its flank; while the other is the determinate type when the main axis terminates as a flower and growth continues sympodially. The Asteraceae plant family harbors a unique type of inflorescence, called the flower head. It combines up to hundreds of individual florets into a single structure, but its infloresecence meristem has a determinate fate and can only produce a certain number of florets before getting consumed. The genetic regulation of such determinacy has been recently brought into attention in Gerbera hybrida, a model system used for studying inflorescence and flower development in Asteraceae. So far, several genetic regulators have been identified regulating the determinacy of inflorescence meristem in Gerbera. This thesis aims to characterize new Gerbera transgenic lines to study the function of the Gerbera homolog of TERMINAL FLOWER 1 (GhTFL1). In Arabidopsis and other species, TFL1 is known to counteract with the flower meristem identity genes and maintain the indeterminacy of the inflorescence meristem. Previous results by overexpressing GhTFL1 under constitutive 35S promoter in Gerbera convert the determinate fate of inflorescence meristem into indeterminate. To better study the GhTFL1 function, an inducible overexpression system pOpON2-pOp6/LhGR-N was introduced, and the transgenic lines have been made for GhTFL1. In this this thesis, in total of 19 candidate lines were screened with GUS staining assay and RT-PCR. Two promising lines (TR4 and TR7) with strongest responses in GUS staining and high expression of the target gene were identified. Further treatment of dexamethasone was conducted in these two lines to the growing rosette; however, no clear phenotypes was observed in these lines. The treatment results suggested that further optimization should be made, in particular, the timing of treatment shall be the determining factor for a successful treatment.

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.

Inflorescence meristems (IMs) either keep producing new flowers, thereby being indeterminate, or terminate after initiation of a finite number of flowers, thereby being determinate. Gerbera hybrida (Asteraceae) has determinate inflorescences. It has been found that the LEAFY (LFY) homolog GhLFY and SEPALLATA (SEP)-like GRCD2/7 promote IM termination in Gerbera as LFY and SEP genes do in Arabidopsis. Downregulation of their expression in Gerbera leads to indeterminate inflorescences. Considering the roles of WUSCHEL (WUS), CLAVATA3 (CLV3) and SHOOT MERISTEMLESS (STM) in meristem maintenance and of TERMINAL FLOWER 1 (TFL1) in IM determinacy in Arabidopsis, it is possible that GhLFY and GRCD2/7 contribute to IM termination by regulating the expression of the homologs of these genes, namely GhWUSb, GhWOX2a, GhCLV3, GhSTM and GhTFL1. It is also possible that GhLFY upregulates the expression of GRCD2/7 in Gerbera, since LFY upregulates SEP gene expression in Arabidopsis. To test these hypotheses, the expression of these candidate genes in Gerbera IMs was compared between IM-expanding and terminating stages, as well as between transgenic plants with downregulated GhLFY or GRCD2/7 expression and wild-type plants. In addition, the pOpOn2 inducible expression vector was used in Gerbera for the first time, in order to induce GhWUSb overexpression in transgenic plants and to study the function of GhWUSb in IM determinacy. Expression analysis showed that during IM termination, GRCD7 expression was upregulated, while the expression of GhWUSb, GhCLV3 and GhWOX2a was downregulated. GhLFY upregulated the expression of GRCD7, and both of them downregulated the expression of GhWUSb, GhCLV3 and GhWOX2a. The expression of GhSTM and GRCD2 was neither affected by IM termination nor regulated by GhLFY or GRCD7. GhTFL1 expression was not detected in any IM samples. Induced GhWUSb overexpression delayed IM termination, confirming the role of GhWUSb in meristem maintenance. These results suggest that GRCD7 may contribute to IM termination by suppressing the expression of the meristem maintenance gene GhWUSb, and the upregulation of GRCD7 by GhLFY may be required for IM termination. GRCD2 and GhSTM may not play a significant role in inflorescence determinacy.