Characterization of cold shock domain proteins and SUMOylation system from Oryza sativa.

摘要

In the first part of this study, two novel c&barbelow;old s&barbelow;hock domain p&barbelow;roteins from rice (OsCSP) were cloned and subsequently characterized their roles during stress conditions and development. OsCSP1 and OsCSP2 ( Oryza sativa CSD protein) encode putative proteins consisting of an N-terminal CSD and glycine-rich regions that are interspersed by 4 and 2 CX2CX4HX4C (CCHC) retroviral-like zinc fingers, respectively. Using an in vitro DNA binding assay, I demonstrate that OsCSPs exhibit conserved ssDNA binding activity. In vivo functional complementation in a cold-sensitive bacterial strain, that lacks four cold inducible cold shock domain proteins revealed that OsCSPs function as RNA chaperones, similar to their bacterial and winter wheat counterparts. To understand the functions of these genes in rice, I studied the transcriptional regulation in response to abiotic stress conditions. Under cold stress, OsCSP transcript levels are only transiently and marginally increased and the encoded proteins did not accumulate. These transcript and protein data are in sharp contrast with the observed data for winter wheat and Arabidopsis cold shock domain proteins under cold stress. In these species, both transcripts and protein levels of CSPs are increased upon cold stress. Based on these data, it can be hypothesized that the accumulation of cold shock domain proteins may play an important role in determining the cold acclimation capability of the plants. Expression analysis at the protein and RNA levels during development revealed that OsCSPs are highly expressed in the reproductive and meristematic tissues. These results indicate a potential role for rice cold shock domain proteins in plant growth and reproductive development.;In this study, I also characterized the post-translational modification of plant cold shock domain proteins by SUMOylation. Post-translational modifications can impart rapid changes in protein function. SUMOylation involves the reversible attachment of a small protein called SUMO (small ubiquitin-like modifier) to target proteins. The SUMO protein has a similar three dimensional structure as that of ubiquitin and the process of SUMOylation is very similar to that of ubiquitination. However, unlike ubiquitination, SUMOylation is not implicated in protein degradation. SUMO modification can affect the target protein stability, sub-cellular localization protein-protein interactions. Using a computational approach on rice and Arabidopsis cold shock domain proteins, I identified canonical SUMOylation motifs in both rice CSPs and one of the Arabidopsis CSPs. Using in vitro assays, I demonstrated that both OsCSPs can undergo SUMOylation. Using mutational approaches, I identified an important lysine residue for SUMOylation in Arabidopsis AtCSP1. By employing GFP-tagged wild-type and variant AtCSP1 proteins, I demonstrate that SUMOylation appears to affect AtCSP1 protein localization.;In another study, I characterized the entire SUMO conjugation system in rice. The process of SUMOylation involves a cascade of enzymatic reactions involving activation (E1) enzymes, conjugation (E2) enzymes and ligation (E3) enzymes. I compared the protein sequences of all these genes from rice with those from Arabidopsis, yeast and human. This revealed a high amino acid sequence conservation of individual SUMOylation components from yeast to plants and animals. In Arabidopsis, the SUMOylation system has been implicated in plant development and in mediating abiotic stress responses. To understand the role of the rice SUMOylation system during development, I studied the SUMO conjugate profiles and the expression of individual SUMO component genes in various tissues at different stages of plant development. The highest levels of SUMOylated proteins were observed in panicles and meristematic tissues. Expression studies revealed that SUMO component genes are highly expressed in reproductive tissues like developing seeds and panicles. Together, these data implicate an important role for the rice SUMOylation system in plant growth and reproductive development. To understand the role of SUMOylation system in rice, I studied SUMO conjugate profiles and the transcriptional regulation of individual SUMO components during cold, salt and ABA stress conditions. Rice responds to these stresses by accumulating SUMO conjugated proteins, suggesting that protein SUMOylation helps to mediate plant stress responses. Studies on the transcriptional regulation of individual SUMO pathway genes during these stress conditions revealed that most are transcriptionally down-regulated. However, a particular SUMO E3 ligase gene (OsSIZ2) is transiently up-regulated upon exposure to all three stress conditions. Considering the importance of E3 ligases in improving the efficiency and specificity of the SUMO conjugation reactions, OsSIZ2 may mediate accumulation of SUMO conjugates during these stress conditions. Taken together, these data suggest a role for SUMOylation in rice development and stress responses. (Abstract shortened by UMI.)