NaCl胁迫下一氧化氮调控灌木柳液泡膜H+-ATPase的分子机制

Shrub willow is an ideal tree species that can be used for afforestation on coastal beach. There are still many scientific issues need to be clarified in the study of the response of shrub willow to salt stress. It has drawn wide attention on the mechanisms of the regulation of vacuolar H+-ATPase (V-H+-ATPase) activity under salt stress. However, the exact mode of regulation is not well defined. In order to explore whether the enhancement of V-H +-ATPase activity in shrub willow was induced by nitric oxide (NO), the NO scavenger was used, and the activity and protein expression and distribution of Nitrate reductase (NR) and Nitric oxide synthase (NOS) were further investigated. To explore the molecular mechanisms of the regulation of V-H +-ATPase, exogenous NO was supplied using SNP. Subsequently, the expression of V-H +-ATPase gene was investigated by RT-PCR, and protein expression and distribution of V-H +-ATPase were examined by Western blot and laser confocal microscopy. Furthermore, the probable involvement of MAPK signaling pathway in the regulation process was also studied. This study may provide new insight into understanding of the signaling cascade involved in the response of V-H +-ATPase to salt stress.

灌木柳是沿海滩涂造林的理想选择，其在响应盐逆境的过程中还有很多需要探明的科学问题。液泡膜H+-ATPase的活性调节作为耐盐机理研究的一个重要方向已受到普遍关注，但其具体的调控方式迄今未获得清晰的认识。本项目利用药物阻断技术，探究灌木柳液泡膜H+-ATPase活性增强是否由NO诱导，并进一步研究硝酸还原酶（NR）和一氧化氮合酶（NOS）的变化，阐明NO含量与液泡膜H+-ATPase活性的相关性；通过外源供给NO，利用RT-PCR、Western blot和激光共聚焦等技术手段，从液泡膜H+-ATPase基因和蛋白表达水平变化、MAPK信号传导通路三个方面揭示NO调控液泡膜H+-ATPase的分子机制。项目的实施有望为揭示液泡膜H+-ATPase响应盐胁迫的信号传导机制提供新的思路。

液泡膜H+-ATPase在植物耐受盐胁迫过程中扮演重要角色。有报道一氧化氮能够提高植物的耐盐性，两者间是否存在协同性关系目前尚不清楚。本研究中，我们选择两种耐盐能力不同的灌木柳，盐耐受型JW2345和盐敏感型JW2367作为研究对象。利用药物阻断技术，探究灌木柳液泡膜H+-ATPase活性增强是否由NO诱导。并通过外源供给NO揭示NO调控液泡膜H+-ATPase的分子机制。研究结果表明，使用NO清除剂可以降低灌木柳体内的NO含量，同时液泡膜H+-ATPase活性下降；而外源供给NO则可以提高液泡膜H+-ATPase活性。说明NO确实参与液泡膜H+-ATPase的活性调节。进一步研究发现，NO可能是通过调节液泡膜H+-ATPase的蛋白水平实现其作用的。此外，我们的研究发现NaCl对灌木柳液泡膜H+-ATPase活性的调控不仅是通过影响液泡膜H+-ATPase的蛋白表达，还可能是通过影响液泡膜H+-ATPase所处的膜微环境实现的。这一结果表明盐耐受型灌木柳可能一方面通过提高液泡膜H+-ATPase活性将更多Na+泵入液泡中进行区隔化，另一方面通过降低液泡膜的流动性减少离子外漏实现其对NaCl耐受性的。我们的研究结果可能为木本植物耐盐机理的研究提供新的思路。