NO is involved in H 2 -induced adventitious rooting in cucumber by regulating the expression and interaction of plasma membrane H + -ATPase and 14-3-3.

Main Conclusion: NO was involved in H ₂ -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 ₂ ) 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 ₂ -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 ₂ , NO, H ₂ 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 ₂ 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 ₂ -induced adventitious root formation. Taken together, our results suggested that NO might be involved in H ₂ -induced adventitious rooting by regulating the expression and the interaction of PM H ⁺ -ATPase and 14-3-3 protein.