Functional Analysis Of OsWTF1and OsWTF2Genes In Wax Synthesis And Abiotic Stress Tolerance

Plant growth and development, especially crop yield are severely influenced by nu-merous abiotic stresses, such as drought, cold, and high salinity. During the long evolu-tionary process, plant gradually formed a series of strategies and mechanisms to cope with stress environment. Plant epicuticular wax is the general term for a class of compounds which cover the outer surface of plant and do not dissolve in water and soluble in organic solvents, it also a natural defensive barrier of plant and plays important roles in drought tolerance. Researches on genes that control the biosynthesis, transit and secretion of epi-dermal wax have become a hot spot. Many reports suggested that plant AP2/EREBP transcription factors are involved in biotic and abiotic stresses, such as pathogens, hor-mones, salt, low temperature, drought and radiation stresses.OsWTF1and OsWTF2are rice transcription factor genes that belong to the ERF sub-family. In this study, the functions of OsWTF1and OsWTF2in rice growth, epidermal wax content and distribution, and the stress response were analyzed in OsWTF1and OsWTF2overexpression transgenic plant. The main results are as following:1. Drought treatment experiments showed those three days after the water disrup-tion, wilt symptoms of WT plants were more severe than those of OsWTF1-OE and OsWTF2-OE transgenic rice seedlings. After ten days of rewatering, leaves of most of the transgenic overexpressing rice seedlings restored the normal green color whereas those of WT plants did not. The survival rates of OsWTF1-OE and OsWTF2-OE transgenic rice seedlings were much higher than that of WT plants.2. When germinated under different concentrations of NaCl and mannitol, the seed germination percentages of all the transgenic and wild type rice were decreased with the increasing NaCl and mannitol concentrations. However, the seed germination percentage of OsWTFl-OE and OsWTF2-OE transgenic rice were significantly higher than that of WT seeds, and the growth state of transgenic rice seedling was better than WT plants. Overexpression of OsWTF1and OsWTF2increased the tolerance of rice seeds to drought and high salt stresses during germination.3. OsWTFl-OE and OsWTF2-OE transgenic plants at the booting stage exhibited less damage symptoms than WT plants under drought stress treatment. After water with-holding for three days, most leaves of the WT plants appeared wilted. Comparatively, the leaves of OsWTF1-OE and OsWTF2-OE plants were just slightly curled, all leaves of al- most WT plants and OsWTFl-OE and OsWTF2-OE rice plants were withered. Whereas OsWTFl-OE and OsWTF2-OE plant exhibited continued growth phenotype after rewa-tering for three days while WT plants did not recoverd even after rewatering for ten days. Furthermore, compared with WT plants, OsWTF1-OE and OsWTF2-OE transgenic plants have higher proline content, lower MDA and RFC content and REC were shown in OsWTF1-OE and OsWTF2-OE transgenic plants. These results indicated that the over-expression of OWTF1and OWTF2genes increased drought tolerance in transgenic rice plants at the booting stage.4. Deposition and crystalloid morphology of epicuticular wax in transgenic and wild-type rice leaves were observed by scanning electronic microscopy. The leaf thickness and density of the epidermal wax crystal and the number and height of the papillae of OsWTF1-OE and OsWTF2-OE transgenic plants were greater as compared with wild-type paints. Furthermore, GC-MS analysis revealed that OWTF1and OWTF2probably regulate biosynthesis of very long chain fatty acid, alkans and esters. While the above components in OsWTF2-RNAi transgenic plants were lower than those of wild type plants. Over-expression of OWTF1and OWTF2genes significantly reduced the rate of leaf chlo-rophyll leaching and leaf water loss. These results indicate that OsWTFl and OsWTF2may be involved in rice epidermal wax biosynthesis and the increase in epidermal wax may contribute to the inceased tolerance to drought stress by reducing the leaf water loss.5. OsWTF1-OE transgenic rice plants of indica culltivar were obtained and con-firmed at transcriptional level. These transgenic indica rice plants can be used to check the function of OsWTF1gene in indica rice.