Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 μM S-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 S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-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 S-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 S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-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.

Calcium and ethylene are essential in plant growth and development. In this study, we investigated the effects of calcium and ethylene on adventitious root formation in cucumber explants under salt stress. The results revealed that 10 μM calcium chloride (CaCl₂) or 0.1 μM ethrel (ethylene donor) treatment have a maximum biological effect on promoting the adventitious rooting in cucumber under salt stress. Meanwhile, we investigated that removal of ethylene suppressed calcium ion (Ca²⁺)-induced the formation of adventitious root under salt stress indicated that ethylene participates in this process. Moreover, the application of Ca²⁺ promoted the activities of 1-aminocyclopropane-l-carboxylic acid synthase (ACS) and ACC Oxidase (ACO), as well as the production of 1-aminocyclopropane-l-carboxylic acid (ACC) and ethylene under salt stress. Furthermore, we discovered that Ca²⁺ greatly up-regulated the expression level of CsACS3, CsACO1 and CsACO2 under salt stress. Meanwhile, Ca²⁺ significantly down-regulated CsETR1, CsETR2, CsERS, and CsCTR1, but positively up-regulated the expression of CsEIN2 and CsEIN3 under salt stress; however, the application of Ca²⁺ chelators or channel inhibitors could obviously reverse the effects of Ca²⁺ on the expression of the above genes. These results indicated that Ca²⁺ played a vital role in promoting the adventitious root development in cucumber under salt stress through regulating endogenous ethylene synthesis and activating the ethylene signal transduction pathway.

Cadmium (Cd), as one of the most toxic nonessential elements, severely prohibits plant growth and development. Hydrogen sulfide (H₂S) and methyl jasmonate (MeJA) play essential roles in plant response to abiotic stress. However, the potential mechanism of H₂S and MeJA in alleviating Cd stress in plants remains unclear. In the current study, the importance and crosstalk of H₂S and MeJA in the Cd tolerance of cucumber seedlings have been investigated. Our results revealed that Cd stress obviously prohibited the growth of cucumber seedlings. Optimal concentrations of H₂S donor sodium hydrosulfide (NaHS) or MeJA treatment, respectively, or in combination, significantly enhanced seedling growth under Cd stress. However, the positive effects of H₂S during seedling growth were obviously reversed by the application of MeJA biosynthesis inhibitors, which implied that MeJA might be involved in the H₂S-improved growth of cucumber seedlings under Cd stress. Moreover, Cd stress resulted in the increase in hydrogen peroxide (H₂O₂), superoxide radical (O₂^·−) accumulation, and impaired the functioning of the ascorbate–glutathione cycle. Both H₂S and MeJA decreased the reactive oxygen species (ROS) level and ameliorated the negative effects of Cd stress through significantly increasing the ratio of ascorbate (AsA)/dehydroascorbic acid (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG). Besides that, the expression level of ROS scavenge genes was significantly upregulated by the application of exogenous H₂S or MeJA treatment. Moreover, H₂S and MeJA significantly enhanced the chlorophyll concentration and inhibited chlorophyll degradation through decreasing the expression levels of chlorophyll catabolic enzymes. Additionally, exogenous H₂S and MeJA obviously enhanced the chlorophyll fluorescence. However, MeJA biosynthesis inhibitors significantly suppressed the positive role of H₂S. The above results suggested MeJA is involved in H₂S-induced Cd stress alleviation in cucumber seedlings through enhancing ROS-scavenge capacity and improving the photosynthesis system.