Identification And Functional Analysis Of XHS Gene Family In Rice

Rice (Oryza Sativa L.) as a main crop in China and worldwide, its productivity and quality are seriously constrained by abiotic stress such as drought, salinity and low temperature, and the reduced production and quality has a far-reaching influence on agricultural production and even social life. Therefore, identifying new stress-related genes and understanding their functions will have an important practical significance in improving the tolerance to adverse growing conditions and variety upgrading.In this study, a novel function-unknown gene family was discovered through expression profiling and bioimformatics analysis. The protein sequences of the family contain conserved domains including XH, XS, Zf-XS (thus named XHS family). Through genetic transformation and stress resistance screening, several stress- and/or development-related genes in this family were identified. Moreover, the stress resistance mechanisms of a few genes were explored. The main results are as follows:1. Through the analysis of a panicle development-related gene 01A19, a novel plant specific family (XHS family) was discovered. Protein sequences of XHS family in rice contained 2-4 of four domains including Zf-XS, XS, Coiled-Coil, and XH, and their functions are unknown.2. Expression analyses of the XHS genes in rice (OXHS family) suggest that most of them are induced by stress treatments and all the XHS genes are predominantly expressed in floral organs. Based on phenotypic identification of mutants or overexpression plants, three genes (OXHS6,OXHS2, and OXHS4) were found playing important roles in rice development and stress tolerance.3. OXHS6 is a typical XHS protein, and was identified as a nuclear protein. Growth performance of OXHS6RNAi plant showed stronger sensitivity to salt stress and abscisic acid treatment. Measurement of Na+ and K+ and microscopic assay under salt stress revealed that accumulation of Na+ in shoot leaded to severe damage to vascular. XHS6RNAi transgenic plants showed reduced sensitivity to salt stress as pH increased. This result suggested that OXHS6 may be involved in the regulation of Na+/H+ transporter. 4. Phenotype of mutant oxhs6 was extremely abnormal during growth and development, particularly the stigma-absent phenotype. The expression of C class gene DL controlling pistil development was obviously down-regulated in the mutant oxsh6. Interestingly, some OXHS genes were also changed remarkably in the oxsh6 mutant.5. OXHS2 is not a typical XHS protein, it lacks Zf-XS domain and localizes in cytoplasm,. Over-expression of OXHS2 in rice resulted in reduced tolerance to salt and drought stress, and these results were verified by various physiological parameters (Proline and chlorophyl) related to stress.6. OXHS4 is also a typical XHS protein. The result of subcellular localization showed it was a nuclear protein like OXHS6. The plant height and fertility of oxhs4 mutant were significantly decreased. Over-expression of OXHS4 in rice resulted in significantly increased tolerance to drought while dramatically reduced drought resistance capacity was observed in oxhs4. These results were validated by detecting proline content which is a commomly used physiological indicator of drought tolerance.7. We compared the root length and root volume between OXHS4 over-expression plants and oxhs4 mutant at reproductive stage under normal and drought stress conditions. The results showed that, under normal condition, over-expression plants had longer root and larger root volume than wild-type control plants, and these differences became more evident under drought stress. When the mutant was compared to the corresponding control, the results of root length and root volume were exactly opposite to that of overexpression plants. These results indicated that the OXHS4 may mediate rice drought tolerance by controlling the root growth.8. Over-expression of OXHS4 enhanced root elongation and numbers of lateral root, while loss of OXHS4 leaded to opposite result. Furthermore,OXHS4 was induced by some hormones (auxin, abscisic acid and brassinosteriod), indicating that OXHS4 may be involved in some phytohormone-mediated pathways to affect the growth of root.9. OsNMCP1 is an OXHS4-interacting protein, obtained by yeast two-hybrid screening with OXHS4 as bait, and is located in nucleus. Mutant nmcpl showed similar phenotypes to oxhs4, such as decreased plant height, vestigial growth of root and reduced drought tolerance. So we predict that OXHS4 may mediate drought resistance by regulating elongation of root and numbers of lateral root through interaction with OsNMCP1.In conclusion, OXHS genes were sysmeticaly identified in this work. Functional analysis of three OXHS genes suggests that XHS family play important roles in development and stress tolerance in rice.