Key Lab of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Plant Cell Physiol. 2012 Feb;53(2):344-57. doi: 10.1093/pcp/pcr181. Epub 2011 Dec 19.
Nitric oxide (NO) plays essential roles in many physiological and developmental processes in plants, including biotic and abiotic stresses, which have adverse effects on agricultural production. However, due to the lack of findings regarding nitric oxide synthase (NOS), many difficulties arise in investigating the physiological roles of NO in vivo and thus its utilization for genetic engineering. Here, to explore the possibility of manipulating the endogenous NO level, rat neuronal NOS (nNOS) was expressed in Arabidopsis thaliana. The 35S::nNOS plants showed higher NOS activity and accumulation of NO using the fluorescent probe 3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate (DAF-FM DA) assay and the hemoglobin assay. Compared with the wild type, the 35S::nNOS plants displayed improved salt and drought tolerance, which was further confirmed by changes in physiological parameters including reduced water loss rate, reduced stomatal aperture, and altered proline and malondialdehyde content. Quantitative real-time PCR analyses revealed that the expression of several stress-regulated genes was up-regulated in the transgenic lines. Furthermore, the transgenic lines also showed enhanced disease resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 by activating the expression of defense-related genes. In addition, we found that the 35S::nNOS lines flowered late by regulating the expression of CO, FLC and LFY genes. Together, these results demonstrated that it is a useful strategy to exploit the roles of plant NO in various processes by the expression of rat nNOS. The approach may also be useful for genetic engineering of crops with increased environmental adaptations.
一氧化氮（NO）在植物的许多生理和发育过程中发挥着重要作用，包括生物和非生物胁迫，这些都会对农业生产产生不利影响。然而，由于缺乏关于一氧化氮合酶（NOS）的发现，在体内研究 NO 的生理作用及其在遗传工程中的应用方面存在许多困难。在这里，为了探索操纵内源性 NO 水平的可能性，我们在拟南芥中表达了大鼠神经元型一氧化氮合酶（nNOS）。35S::nNOS 植株使用荧光探针 3-氨基，4-氨基甲基-2'，7'-二氟荧光素二乙酸酯（DAF-FM DA）测定法和血红蛋白测定法显示出更高的 NOS 活性和 NO 积累。与野生型相比，35S::nNOS 植株表现出增强的耐盐和耐旱性，这进一步通过生理参数的变化得到证实，包括降低失水率、减小气孔开度以及脯氨酸和丙二醛含量的改变。定量实时 PCR 分析显示，转基植株中几种胁迫调节基因的表达上调。此外，转基植株还通过激活防御相关基因的表达表现出对丁香假单胞菌 pv.番茄（Pst）DC3000 的增强的抗病性。此外，我们发现通过调节 CO、FLC 和 LFY 基因的表达，35S::nNOS 系开花较晚。总之，这些结果表明，通过表达大鼠 nNOS 来利用植物 NO 在各种过程中的作用是一种有用的策略。该方法也可能对提高环境适应性的作物遗传工程有用。
