氢气调控苜蓿干旱胁迫耐性的机理研究

Our recent findings demonstrated that hydrogen gas (H2) acts as a novel signaling gas in the improvement of plant tolerance against salt and oxidative stresses (Plant Cell Environ). Further results showed that drought stress induces an increase of endogenous H2 release in Medicago sativa seedlings. Exogenous application of the hydrogen rich water (HRW) not only mimics the induction of endogenous H2 release upon drought stress, but also effectively decreases stress-induced injury. However, the mechanisms of the inducible H2 synthesis and cytoprotective roles are still unknown. In this project, to explore the potential enzyme responsible for H2 synthesis and its subcellular localization, candidate H2-synthesis genes were cloned from Medicago sativa firstly, and then prokaryotic and transient expressed. To further assess the enhancement of drought resistance by H2, hydrogenase from chlamydomonas reinhardtii is also heterologous expressed in Medicago sativa; vital components of H2-signalling transduction were screened and verified by transcriptomics and protemics methods. By using ABA and its metabolism inhibitor, NO-releasing compounds, its scavenger and synthesis inhibitor, the spatial and temporal variation of NO signal and corresponding relaying processes regulated by H2 were analyzed. Combined with the differences of corresponding drought tolerance phenotypes, we emphatically identified the key components involved in the H2-signalling, in particularly focus on the stomatal movement regulated by ABA and redox homeostasis. Overall, above results will not only illustrate the molecular mechanism of H2 in the regulation of drought tolerance in Medicago sativa, but also provide new ideas for the practices of related agricultural production.

我们最近的研究表明，氢气（H2）是一种新的植物耐盐和抵御氧化伤害的信号分子（Plant Cell Environ）。进一步研究发现，干旱胁迫能诱导紫花苜蓿幼苗H2的释放，外源富氢水不仅能模拟干旱胁迫下的H2释放，而且能明显缓解胁迫伤害，但是不清楚H2的诱导合成机制和保护作用机理。本课题将首先克隆苜蓿H2合成候选编码基因，通过原核表达和瞬时表达探索H2的酶学来源和定位；把莱茵衣藻的氢酶基因导入苜蓿，验证H2对耐旱性的调节作用；通过转录组和蛋白组学方法筛选并验证H2信号转导的关键组分；采用ABA及其合成抑制剂、一氧化氮（NO）供体、清除剂和合成抑制剂，分析受H2调节的NO时空变化及其信号转导，结合耐旱表型的改变，重点分析H2信号转导的关键因子尤其是气孔关闭过程中的ABA敏感性和氧化还原稳态的改变。上述研究结果将阐明H2调控苜蓿干旱胁迫耐性的分子机理，同时也为相关的农业生产实践提供新的思路。

我们先前的研究表明，干旱胁迫能诱导紫花苜蓿幼苗氢气（H2）的释放，外源富氢水不仅模拟干旱胁迫诱导的H2释放，而且能明显缓解胁迫伤害。为了进一步探查H2的诱导合成机制及其作用机理，本课题首先采用拟南芥突变体来进行相关研究，实验发现ABA能提高拟南芥H2的产生，从而诱导叶片气孔关闭以及增强抗旱性；ABA信号下游存在与H2相关的信号转导级联过程，包括依赖于NADPH氧化酶F（RbohF）的活性氧（ROS）以及硝酸还原酶（NR）酶促产生的一氧化氮（NO）。进一步研究发现，外源ABA处理诱导苜蓿幼苗H2的产生，H2也可以增强紫花苜蓿对外源ABA的敏感性；富氢水预处理可以增强ABA诱导的过氧化氢（H2O2）产生，富氢水对苜蓿气孔张度和相对含水量的调控作用可以被H2O2代谢的抑制剂以及清除剂抑制，提示NADPH氧化酶产生的H2O2参与了H2诱导的苜蓿干旱胁迫耐性；H2O2可能作为血红素加氧酶/一氧化碳（HO-1/CO）的上游信号参与H2介导的苜蓿对渗透胁迫的耐性；外源H2、NO供体硝普钠（SNP）和脯氨酸均能增强紫花苜蓿对干旱胁迫的耐性，外源H2还可以诱导苜蓿内源NO的产生，外源添加NO专一性清除剂后，H2缓解紫花苜蓿渗透胁迫的效应（包括诱导脯氨酸的积累和维持氧化还原稳态）均被逆转，提示NO可能参与了H2对苜蓿渗透胁迫的缓解，其机理与诱导脯氨酸积累和重建氧化还原稳态有关；克隆了莱茵衣藻氢酶基因(HYD1), 构建并获得了拟南芥转基因株系；在干旱条件下，发现转基因株系的萌发速率明显高于野生型，其植株失水速度也相对较慢，因此从遗传学角度也初步证明了H2可以提高耐旱性。上述研究结果不仅初步阐明了H2调控干旱胁迫耐性的分子机理，同时也为相关的农业生产实践提供新的思路。此外，本课题在SCI源刊物（Plant Physiol等）上发表相关论文4篇，获得国家发明专利和实用新型专利各一项。