γ-氨基丁酸代谢在水稻响应铵胁迫中的作用机制

In recent years, ammonium (NH4+) accumulated in flooded soils. High concentrations of NH4+ can be toxic to rice, leading to severe growth depression. It is clear that NH4+ toxicity is of increasing ecological importance, and deserves renewed attention. γ- aminobutyric acid(GABA)is a four-carbon non-protein amino acid that present in plants. GABA is mainly synthesized from L-glutamate owing to the activity of the cytosolic glutamate decarboxylase (GAD). GABA is then transported into the mitochondrion to be catabolized by the GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenaase (SSADH) to produce succinate for using in the tricarboxylic-acid (TCA) cycle. This is called the GABA shunt. GABA Shunt plays important roles in the response to a variety of plant stress. Our previous studies show that NH4+ toxicity of rice can be alleviated by GABA. However, no experimental data are available on the molecular function of GABA and the involvement of its metabolism in ammonium stress tolerance in rice. Here, we propose to use rice as material to analysis the mechanism. We first investigate the regulation of GABA metabolism in rice at the metabolite, enzymatic activity and gene transcription levels upon ammonium stress for making clear the role of GABA and its metabolism play in plant responses to high ammonium. Furthermore, we performed a analysis of transgenic lines (overexpression of truncated GAD, GABA-T and RNAi-mediated knockdown of GAD, GABA-T) to assess the role of GABA and its metabolism in ammonium stress tolerance.

近年来，淹水土壤中铵态氮大量积累，高浓度的铵态氮对水稻的生长产生抑制甚至毒害作用。植物中的γ-氨基丁酸（GABA）是一种四碳非蛋白质氨基酸，其在细胞质中由谷氨酸脱羧酶（GAD）催化谷氨酸脱羧形成，之后进入线粒体中在GABA转氨酶（GABA-T）和琥珀酸半醛脱氢酶（SSADH）作用下形成琥珀酸进入到柠檬酸循环中，由此构成GABA代谢途径即GABA支路。研究发现，GABA支路在植物响应各种逆境中发挥重要的作用。我们前期的研究发现GABA可以缓解水稻铵毒害症状，但是具体机制还不清楚。因此，本项目拟以水稻为实验材料，在明确外源GABA具有缓解水稻铵毒害作用的基础上，通过内源分析铵胁迫下GABA代谢物、GABA代谢关键酶活性及关键酶基因表达的变化，进一步明确GABA代谢在水稻响应高铵环境中所扮演的重要角色。

水稻主要栽培在以NH4+ 为主的灌溉田中，由于氮肥的过量使用以及不合理的施肥模式，加之大气中 NH3 / NH4+的沉积，已经导致很多农业土壤中NH4+浓度达到毒害水稻生长的程度。因此，本项目针对目前农田中铵毒害的现状，从分析外源GABA缓解水稻铵毒害的症状入手，进一步从GABA如何调控铵的吸收和同化方面揭示其缓解铵毒害的机制，并内源分析铵毒害发生时内源GABA代谢的变化揭示GABA在缓解铵毒害中扮演的重要角色。研究结果表明，在高铵环境下，外源GABA的添加显著缓解水稻的铵毒害症状。铵毒害引起水稻根际的酸化，外源添加GABA明显减少了水稻根尖部H+瞬时外排，降低了根际的酸化；铵毒害减少了水稻根系对其他阳离子的吸收，外源GABA处理则明显增加了根部对钙、镁、铁和锌等阳离子的吸收。进一步分析外源GABA添加对NH4+吸收和同化的影响，结果表明，与单独的NH4+处理相比，外源GABA与NH4+同时处理减少了水稻根尖对NH4+的瞬时内吸，并减少水稻地上和地下NH4+的积累量；外源GABA与NH4+同时处理降低了水稻根部NH4+同化关键酶谷氨酰胺合成酶和谷氨酸合酶基因表达的水平和酶活性，说明GABA可以在转录水平上通过负调控NH4+同化关键酶来抑制调控水稻体内NH4+同化水平。基于外源GABA缓解铵毒害的作用，我们分析了铵毒害下内源GABA如何变化。结果显示，与正常生长条件相比，铵毒害下，内源GABA的含量显著上升，合成GABA的关键酶GAD基因表达量显著增加，酶活性也有所增强，表明GABA在响应铵毒害时发挥关键的作用。GABA代谢可以有效的平衡植物体内碳氮平衡，内源不同种类糖和氨基酸含量分析显示，与单独铵毒害相比，外源GABA添加增加了水稻地上和地下部分糖的含量，降低了部分氨基酸的含量，表明GABA可以通过调节水稻体内碳氮平衡来缓解铵毒害。此外，我们以拟南芥为实验材料分析了铵胁迫对其根毛发育的影响。结果表明，高铵抑制拟南芥根毛的发育，诱导畸形根毛的产生，生长素和乙烯互作参与调控高铵对根毛发育的抑制；低铵则诱导根毛的伸长生长，乙烯和活性氧参与调控低铵对根毛伸长的诱导作用。