SgMDH2调控根系合成苹果酸参与柱花草活化难溶性磷的分子机制

Phosphorus (P) deficiency is one of the major constrains limiting crop growth in acid soil. Stylosanthes (Stylo) is a dominant legume well adaptation to low P acid soils, and has superior ability to utilize insoluble P. However, the molecular mechanisms underlying this process are still unknown. In our previous study, we found that both malate concentration and SgMDH2 (malate dehydrogenase) transcript in roots of stylo were increased by low P stress. Furthermore, overexpression of SgMDH2 increased malate synthesis in transgenic Arabidopsis plants, which exhibited higher capability of insoluble P utilization compared to wild type, suggesting that SgMDH2 involves in P utilization in stylo. In this study, we plan to analyze the pattern of SgMDH2 in response to low P availability through identification of SgMDH2 gene and protein expression, as well as tissue specific expression of SgMDH2 promoter. Biochemical properties analysis of SgMDH2 combined with phenotypic analysis of SgMDH2 overexpressing bean hairy roots are performed to reveal the biological function of SgMDH2 involved in mobilization of insoluble P. Furthermore, we plan to dissect the regulation mechanism of SgMDH2 enzyme activity in term of malate synthesis through identification of the key amino acid within coenzyme binding domain, substrate binding domain and active center of SgMDH2. Our results could not only elucidate the molecular mechanisms underlying legume plant utilize soil P, but also provide theoretical base for developing crop cultivar with P efficient utilization.

低磷胁迫是酸性土壤中限制作物生长的主要因素。柱花草是一种能在酸性土壤上良好生长的耐低磷豆科植物，具有活化利用土壤难溶性磷能力，但其潜在机理仍不清楚。我们前期工作表明，低磷胁迫增加了柱花草根系苹果酸浓度和苹果酸脱氢酶SgMDH2基因表达；超量表达SgMDH2提高了转基因拟南芥苹果酸浓度和活化难溶性磷的能力，说明该基因可能参与了柱花草对磷活化利用。在此结果基础上，本项目通过分析SgMDH2基因和蛋白表达、启动子组织表达，研究SgMDH2对外源磷营养的响应；通过生化酶学特性和SgMDH2转基因菜豆毛根表型分析，阐明SgMDH2合成苹果酸活化难溶性磷的生物学功能；通过比较SgMDH2辅酶、底物结合结构域及活性中心的关键氨基酸位点突变对SgMDH2合成苹果酸的影响，揭示该蛋白合成苹果酸的酶活性调节机制。研究结果不仅可以剖析豆科植物活化土壤磷的分子机理，还可为磷高效利用作物品种选育提供参考依据。

土壤磷素匮乏是限制作物生长的重要因素。柱花草是一种能在缺磷土壤上良好生长的热带豆科植物，研究发现其具有活化利用土壤难溶性磷能力，但其潜在机理仍不清楚。本研究首先明确了缺磷处理能够增强SgMDH2基因和蛋白在柱花草根系中的表达。蛋白生化酶学分析表明，SgMDH2蛋白最适酶学反应温度为40℃，最适pH为7.5，其酶活性受金属离子的影响。酶动力学分析表明，相对malate和NAD+，SgMDH2蛋白对OAA和NADH具有较高的催化效率，表明SgMDH2具有催化合成苹果酸的酶学特性。进一步分析发现，超量表达SgMDH2基因显著提高了菜豆毛根苹果酸脱氢酶活性、增加根系内源苹果酸浓度和促进了根系分泌苹果酸。并且，超量表达SgMDH2基因提高了菜豆毛根利用难溶性铝磷的能力，增加了铝磷处理下的毛根干重及磷浓度，表明SgMDH2具有调控苹果酸合成与分泌，从而活化难溶性磷的生物学功能。本研究也发现了影响SgMDH2合成苹果酸功能的重要氨基酸位点，这些位点显著影响了SgMDH2催化合成苹果酸的生物学功能。另外，本研究发现柱花草利用难溶性磷的能力与SgMDH2基因表达具有相关性，表明SgMDH2可能是柱花草利用难溶性磷的重要基因。本研究结果不仅解析了热带豆科植物利用土壤磷的机理，而且为培育高效利用土壤难溶性磷的作物新品种提供了种质资源和依据。