A number of mechanisms that contribute to epigenetic regulation of gene expression have been described in plants. The concert actions of these mechanisms contribute to proper patterning of gene expression within organs and tissues, therefore insuring their morphology and functions. However, while the general effects of components of the transcriptional (TGS) and post-transcriptional gene silencing (PTGS) pathways have been reported, the impact of individual elements of these pathways on silencing of target genes at a tissue specific level remained to be investigated.
In this thesis, we addressed the question of regulation of tissue specific gene expression using a transgenic GFP reporter line containing the epigenetically controlled endogenous promoter of APUM9. Previous studies have revealed that APUM9 is under complex epigenetic control. Plants of this line exhibited GFP expression only in siliques (therefore, the line was named ÒsilexÓ), suggesting that the GFP transgene was silenced in the other tissues. To investigate the role of TGS and PTGS factors in silencing the GFP transgene and to identify novel factors that contribute to this process, forward and reverse genetic approaches were used.
To study the impact of different silencing pathways on suppression of the GFP transgene in silex, plants of the reporter line were crossed to mutants defective in components of the TGS (nrpe1) and PTGS (ago1, se, sgs3, dcl4) pathways. The study, presented in this thesis, revealed that these factors were all required to suppress of GFP expression in different tissues. This indicated that both TGS and PTGS are involved in silencing of the GFP transgene in silex.
In order to identify novel epigenetic factors, contributing to tissue specific silencing of the GFP transgene, a forward genetic mutant screen was performed on the silex reporter line. This thesis reports on mutant alleles of SPLAYED (SYD, syd-10 and syd-11), a chromatin remodeling ATPase of Arabidopsis that were recovered in this screen. syd-10 and syd-11, expressed GFP in the vascular tissues of leaves, stems and in inflorescences. SYD has previously been known to be an important regulator of flower organ identity and homeotic gene expression. The findings described in this thesis now indicate that SYD also contributes to silencing of the GFP transgene in parental silex line and suggest a possible wider role of SYD in silencing.
Further study of the syd phenotype indicated a possible connection between SYD and the highly conserved micro-RNA miR156 that plays important roles in regulation of juvenile-to-adult and vegetative-to-reproductive developmental phase transitions. The level of miR156 was reported to be affected by external factors, such as temperature and carbohydrate accumulation. However, so far, only a few molecular factors involved in direct transcriptional control of MIR156 genes have been identified. This thesis provides evidence that SYD takes part in regulation of developmental phase changes by directly modulating transcription of several MIR156 and SPL genes in Arabidopsis.
Generally, the results, presented in this thesis allow us to conclude that the tissue specific silencing of the GFP transgene in the silex line is dependent on components of PTGS as well as TGS, and that both systems may act in a complementary manner. Also, silencing of the GFP expression in the vasculature and inflorescences of silex plants is dependent on the SYD chromatin remodeler and possibly mediated by miR156. Moreover, SYD plays a role in direct transcriptional regulation of miR156 suggesting that it acts in the regulation of miR156-dependent and miR156-independent pathways during plant development
