Functional Studies On Rice High Temperature Tolerance Gene OsHTAS At Seedling Stage

With global warming, heat stress seriously affects crop yield and quality, and constrains the global agricultural production. Ubiquitin-proteasome pathway is one of the most important regulatory mechanisms for post-translational modification in plants. Studies showed that the ubiquitin-proteasome pathway was also involved in biotic and abiotic stress regulation. Reactive oxygen species (ROS) generated under stress has a dual role:If the ROS remains low, it can be used as a signal to trigger defense responses, bringing stomatal closurein plants; If ROS is more than which can be withstood in plants, it will cause damage even death.OsHTAS encodes a RING type ubiquitin ligase. We took this gene as a target to try to figure out its functions using molecular biology tools and methods in the present thesis. The main results and conclusions about this gene are shown below:(1) OsHTAS was constitutively expressed in root and shoot at seedling stage, and flag leaf blade, flag leaf sheath, clum and panicle at reproductive stage. The highest expression level of OsHTAS was detected in flag leaf blade. High temperature, high salinity, cold, hydrogen peroxide and abscisic acid could induce the expression of OsHTAS, and the strongest expression was induced by abscisic acid.(2) Bioinformatics prediction showed that OsHTAS contained four transmembrane domains at N-terminal and a RING (Really Interesting New Gene) finger domain at C-terminal.(3) OsHTAS RNA interference lines were more sensitive to high temperature than wild-type at seedling stage. In contrast, its over-expressing lines and gain-of-function mutant oshtas showed more tolerant to high temperature than wild-type.(4) Transiently expressing in tobacco epidermal cells and rice protoplasts showed that OsHTAS distributed both in nucleus and cytoplasm.(5) Yeast two-hybrid assay displayed that not only the N-terminal of OsHTAS, but also RING finger domain and the whole protein could not be self-activated. Using RING finger domain as a bait, we found four interacting proteins, which included two ubiquitin-conjugating enzymes (E2), a ubiquitin extension protein (S27a) and a ribosomal protein (40SRPS); similarly, using the N-terminal of OsHTAS as a bait, two interacting proteins were found, which included an ascorbate peroxidase 8 (APX8) and an auxin efflux carrier.(6) Transiently expressing in rice protoplasts showed OsHTAS and S27a were co-localized in nucleus. Point mutation of a conserved amino acid in RING finger domain could eliminate the interaction between RING finger and two ubiquitin-conjugating enzymes (E2), thus demonstrating that OsHTAS interacted with E2s by RING finger domain.(7) In the OsHTAS RNA interfering line, the genes belong to stress related and interaction with OsHTAS were all downregulated; but in the over-expressing line well as gain-of-founction mutant oshtas, majority of these genes were upregulated. Among them, the most up regulated gene is NECED4 that encodes the rate limiting enzyme in ABA synthesis process.(8) The APX and CAT activity in oshtas was lower than that in wild type after high temperature treatment, and correspondingly the H2O2 content in oshtas was higher than that in wild type.(9) The ABA content in oshtas was significantly higher than wild-type. Corresponding to this, the percentage of completely closed stomata in oshtas was also significantly higher than that in the wild type after heat stress although there is no obvious difference between the two materials before the heat stress. In addition, the percentage of completely opened stomata in oshtas was significantly lower than that in wild type.(10) The agronomic trait examination of the OsHTAS RNA interfering lines indicated that OsHTAS had some effects on the agronomic trait performance when both RNA interfering lines and wild type were planted under normal growth conditions. Comparing with the wild type, the OsHTAS RNA interfering lines had shorter plant height, less number oftillers, reduced number of grainsor secondary branchesper panicle, and increased number of primary branches, but there was no difference on seed setting rates.Taking together, all these results demonstrated that OsHTAS ws a positive regulatory factor of rice in the process of resisting heat stress, that improved the tolerance of rice seedlings against heat stress through up regulating the transcripts of stress related genes, increasing the plant ABA contents, H2O2 contents and inducing stomata closure.