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Zinc finger proteins are among the most conserved proteins in eukaryotes. Despite numerous lines of evidence have indicated that Tandem CCCH Zinc Finger (TZF) proteins are important regulators for plant growth and stress responses, the functions of some of the tissue-specific TZFs remain elusive. The objective of this research is to characterize three seed-specific Arabidopsis thaliana TZFs in seed germination responses and to dissect the molecular and cellular mechanisms underlying their functions. A comprehensive literature review and genomic analysis of plant TZF genes are presented in Chapter 1. Plants usually have higher number of TZF genes compared to animals. Notably, plant TZF proteins are over-represented by members with a TZF motif preceded by an arginine (R)-rich (RR) region called RR-TZF domain. While the majority of plant RR-TZF genes express ubiquitously, some show strict expression patterns in time and space. Whereas animal TZFs regulate different types of inflammation, homeostasis and developmental responses, plant RR-TZFs apparently play critical roles in hormone-mediated growth and stress responses. They are likely nucleocytoplasmic shuttling proteins because almost all contain both Nuclear Localization Signal (NLS) and Nuclear Exit Signal (NES) sequences. Plant RR-TZFs are localized in cytoplasmic foci characteristic of processing bodies (P-bodies, PBs) and stress granules (SGs). Given plant RR-TZFs can bind specific RNA elements and trigger RNA degradation, they may control growth and stress responses via modulation of mRNA metabolism taking place in PBs and SGs. The first goal of the research was to functionally characterize three seed-specific AtTZF genes, AtTZF4, AtTZF5 and AtTZF6, using reverse genetic, molecular and cellular analyses (chapter 2). Results showed that AtTZF4, AtTZF5 and AtTZF6 are up-regulated by ABA and down-regulated by GA. As such, transcripts of these three genes are rapidly diminished during seed imbibition. Mutant analyses indicate that AtTZF4, 5 and 6 act as positive regulators for ABA- and negative regulators for light- and GA-mediated seed germination responses. Additional analyses reveal that they affect genes critical for ABA and GA accumulation and response during seed germination. AtTZF4, 5 and 6 are localized in cytoplasmic foci and can co-localize with both PB and SG markers. To further understand how AtTZF4, 5, and 6 work in concert with other molecules, protein-protein interactions were analyzed (chapter 3). Yeast two-hybrid (Y-2-H) library screen by using AtTZF5 as bait revealed 35 unique interacting partners. Among them, Mediator of ABA-Regulated Dormancy 1 (MARD1) and Responsive to Dehydration 21A (RD21A) were selected to study in detail. Y-2-H and bimolecular fluorescence complementation (BiFC) assays were used to confirm these protein-protein interactions in yeast and plant cells, respectively. Results of co-immunoprecipitation assay revealed that TZF domain is sufficient for the interaction between AtTZF5 and MARD1. As AtTZF5 interacted with MARD1 and RD21A in cytoplasmic foci and they could all co-localize with both PB and SG markers, it is likely that these protein-protein interactions are taken place in PBs and SGs.In summary, my research has revealed both general knowledge and novel insights into the roles of RR-TZF proteins in hormone-mediated plant growth, stress, and gene expression responses. |
