College of Horticulture, Gansu Agricultural University, Lanzhou, 730070, People's Republic of China.
Planta. 2020 Jun 29;252(1):9. doi: 10.1007/s00425-020-03416-z.
NO was involved in H-induced adventitious rooting by regulating the protein and gene expressions of PM H-ATPase and 14-3-3. Simultaneously, the interaction of PM H-ATPase and 14-3-3 protein was also involved in this process. Hydrogen gas (H) and nitric oxide (NO) have been shown to be involved in plant growth and development. The results in this study revealed that NO was involved in H-induced adventitious root formation. Western blot (WB) analysis showed that the protein abundances of plasma membrane H-ATPase (PM H-ATPase) and 14-3-3 protein were increased after H, NO, H plus NO treatments, whereas their protein abundances were down regulated when NO scavenger carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTI O) was added. Moreover, the mRNA abundances of the HA3 and 14-3-3(7) gene as well as the activities of PM H-ATPase (EC 3.6.1.35) and H pump were in full agreement with the changes of protein abundance. Phosphorylation of PM H-ATPase and the interaction of PM H-ATPase and 14-3-3 protein were detected by co-immunoprecipitation analysis. H and NO significantly up regulated the phosphorylation of PM H-ATPase and the interaction of PM H-ATPase and 14-3-3 protein. Conversely, the stimulation of PM H-ATPase phosphorylation and protein interaction were significantly diminished by cPTIO. Protein interaction activator fusicoccin (FC) and inhibitor adenosine monophosphate (AMP) of PM H-ATPase and 14-3-3 were used in this study, and the results showed that FC significantly increased the abundances of PM H-ATPase and 14-3-3, while AMP showed opposite trends. We further proved the critical roles of PM H-ATPase and 14-3-3 protein interaction in NO-H-induced adventitious root formation. Taken together, our results suggested that NO might be involved in H-induced adventitious rooting by regulating the expression and the interaction of PM H-ATPase and 14-3-3 protein.
一氧化氮（NO）通过调节质膜 H+-ATP 酶和 14-3-3 蛋白的表达参与 H 诱导的不定根形成。同时，质膜 H+-ATP 酶（PM H-ATPase）和 14-3-3 蛋白的相互作用也参与了这个过程。氢气（H）和一氧化氮（NO）已被证明参与植物的生长和发育。本研究结果表明，NO 参与了 H 诱导的不定根形成。Western blot（WB）分析表明，H、NO、H+NO 处理后，质膜 H+-ATP 酶（PM H-ATPase）和 14-3-3 蛋白的含量增加，而当添加 NO 清除剂羧基-2-（4-羧基苯基）-4,4,5,5-四甲基咪唑啉-1-氧-3-氧化物（cPTIO）时，其蛋白含量下调。此外，HA3 和 14-3-3（7）基因的 mRNA 丰度以及 PM H-ATPase（EC 3.6.1.35）和 H 泵的活性与蛋白丰度的变化完全一致。通过共免疫沉淀分析检测到 PM H-ATPase 的磷酸化和 PM H-ATPase 与 14-3-3 蛋白的相互作用。H 和 NO 显著上调 PM H-ATPase 的磷酸化和 PM H-ATPase 与 14-3-3 蛋白的相互作用。相反，cPTIO 显著抑制 PM H-ATPase 磷酸化和蛋白相互作用的刺激。在这项研究中使用了 PM H-ATPase 和 14-3-3 的蛋白相互作用激活剂 Fusicoccin（FC）和抑制剂腺苷一磷酸（AMP），结果表明 FC 显著增加了 PM H-ATPase 和 14-3-3 的含量，而 AMP 则呈现相反的趋势。我们进一步证明了 PM H-ATPase 和 14-3-3 蛋白相互作用在 NO-H 诱导不定根形成中的关键作用。综上所述，我们的研究结果表明，NO 可能通过调节 PM H-ATPase 和 14-3-3 蛋白的表达和相互作用参与 H 诱导的不定根形成。
