| China is an agricultural country with severe water-shortage, whose arid areas are increasing every year and shrink the crop acreage for farming. Rice(Oryza sativa L.), one of the most important food crops worldwide, also works as a model in cereal crops for functional genomic studies on the genetic network of stress responses. Frequently occurred summer droughts in the Yangtze River basin, the main rice cultivation area in China, seriously influence the growth and yield of rice. Drought, the primary factor of the reduction of rice production, can cause the loss of pollen fertility, spikelet death or abortion of newly formed seed. The previous study of rice flower development under water deficit condition shows that microspores are most sensitive to drought.To find the drought-tolerant genes and to explore the regulation mechanism of rice reproductive development under drought conditions,13drought-response genes at reproductive stage were selected for further study. Two of them were verified to be important in improving drought-tolerance at vegetative and reproductive stages and were named OsDIL(Oryza sativa Drought-Induced LTP) and OsFID (Oryza sativa Functionally Improved Drought-tolerance). To further explore their functions, analyses of transgenic rice were executed at vegetative and reproductive stages under drought, using many approaches of morphology, anatomy, physiology, biochemistry and molecular biology. And many interesting results were obtained, as follows:1Identification of drought-tolerant genes and acquisition of transgenic plants.13drought-response genes at reproductive stage were identified in rice and were transformed to rice using Agrobacterium-mediated method. Two of them, OsDIL and OsFID, raised drought-tolerance at vegetative and reproductive stages. Enough transgenic rice plants were obtained for further study.2OsFID gene plays critical roles in improving drought-tolerance at vegetative and reproductive stages.To explore the potential gene function on improving rice reproductive development under drought, a highly drought-induced gene in rice floret, OsFID, was identified from our microarray data and selected for further study. OsFID was primarily expressed in root and leaf, and mainly responsive to abiotic stresses, including drought, cold and NaCl. OsFID was significantly lower expressed in flower under normal conditions, but was highly induced by drought in rice florets. In situ hybrid results showed that OsFID gene was primarily expressed in the tapetum and vascular tissues of anther, implying that OsFID might function in the development of male organs.Compared with the wild type, the OsF/D-overexpressing transgenic rice plants were more tolerant to drought stress during vegetative development and showed less severe tapetal defects and fewer defective anther sacs when treated with drought at the reproductive stage. Moreover, overexpression of OsFID stabalized the rice pollen fertility and rice reproduction under drought, based on the increased seed set rate, but not on1000-grain weight.OsFID is a putative R-R type MYB-like transcription factor, and locates in nucleus. The analysis on the rice flower transcriptome revealed that64genes were positively regulated by OsFID under drought. These genes involve in multiple biological processes, including stress response, cellular processes and metabolic processes; The proteins they encode had catalytic activity, binding and antioxidant activity, suggesting that OsFID might regulate the expression of redox-related genes to improve the flower fertility under drought. Moreover, OsFID also activated the expression of LTP gene, aquaporin gene and transporter protein gene, which could improve the supplement of nutrients and water for anther development under drought.32candidate proteins interacting with OsFID in rice anther were identified using yeast-2-hybrid method. These proteins were encoded by many type of genes, including redox-related genes and protein-degradation-related genes, implyng that OsFID might be involved in redox and protein-degradation processes.Therefore, OsFID is important for improving drought tolerance in rice by regulating the expression of many genes. In addition, it is an excellent candidate gene for genetic improvement of crop in adaption to unfavorable environments.3The rice OsDIL gene plays a role in drought tolerance at vegetative and reproductive stages.Another drought induced gene, OsDIL, encoding a lipid transfer protein, was also selected from our microarray data for further study. OsDIL was primarily expressed in anther and mainly responsive to abiotic stresses, including drought, cold, NaCl, and stress-related plant hormone abscisic acid (ABA). Compared with the wild type, the OsD/L-overexpressing transgenic rice plants were more tolerant to drought stress during vegetative development and showed less severe tapetal defects and fewer defective anther sacs when treated with drought at the reproductive stage.The expressions of the drought-responsive genes RD22, SODA1, bZIP46and POD, as well as the ABA synthetic gene ZEP1were up-regulated in the OsDIL-overexpression lines while the ABA degradation gene ABAOX3was down-regulated. Moreover, overexpression of OsDIL lessened the down-regulation of anther-development genes (OsC4, CYP704B2and OsCP1) caused by drought, providing a mechanism to support pollen fertility under drought. Overexpression of OsDIL significantly enhanced drought resistance in transgenic rice during reproductive development, while showing no phenotypic changes or yield penalty compared to the normal plants. Therefore, OsDIL, like OsFID, also acts as an excellent candidate for the genetic improvement of rice to maintain the yield rate under unfavorable environments.The study on the regulation network of drought-related genes will theoretically enrich people’s knowledge and understanding of the stress resistance mechanisms at reproductive stage. The specific functions of OsDIL and OsFID in enhancing drought resistance might be especially useful and of great value in producing green super tolerance rice with limited water supply. |
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