College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China.
Int J Mol Sci. 2019 Oct 28;20(21):5363. doi: 10.3390/ijms20215363.
Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether -nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 μM -nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular -nitrosothiol (SNO) levels and endogenous NO production, while decreasing the -nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that -nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of -nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and -nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the -nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, -nitrosylation of TUA increased the expression level of . The results implied that -nitrosylation of key proteins seems to regulate various pathways through differential -nitrosylation during adventitious rooting. Collectively, these results suggest that -nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.
一氧化氮（NO）作为一种重要的信号分子，参与调节植物的各种生理和生化过程。然而，-亚硝基化是否是 NO 的关键分子机制在很大程度上仍不清楚。本研究发现，50μM 硝普酸钠（GSNO）处理对促进黄瓜不定根生根具有最大的生物学效应。同时，内源 NO 的去除显著抑制不定根的发育，这意味着 NO 负责促进不定根生根的过程。此外，GSNO 的应用导致细胞内 -亚硝基硫醇（SNO）水平和内源性 NO 产生增加，同时降低不定根生根过程中的硝普酸钠还原酶（GSNOR）活性，这表明 -亚硝基化可能参与了黄瓜中 NO 诱导的不定根生根。此外，利用液相色谱/质谱/质谱（LC-MS/MS）和生物素转换技术在不定根发育过程中鉴定了 -亚硝基化蛋白。在这些蛋白质中，进一步分析了甘油醛-3-磷酸脱氢酶（GAPDH）、微管蛋白α链（TUA）和谷胱甘肽还原酶（GR）的活性和 -亚硝基化水平作为 NO 的直接靶标。我们的结果表明，NO 可能增强 GAPDH 和 GR 的 -亚硝基化水平，并随后降低这些活性和转录水平。相反，TUA 的 -亚硝基化增加了 的表达水平。结果表明，-亚硝基化关键蛋白似乎通过不定根生根过程中的差异 -亚硝基化调节各种途径。总之，这些结果表明 -亚硝基化可能参与了 NO 诱导的不定根生根，为黄瓜外植体中 NO 信号转导的分子机制提供了基础证据。
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