Protein S-Nitrosylation Was Involved In Ca²⁺-Induced Adventitious Rooting Of Cucumber Under Cd Stress

In heavy metal pollution,cadmium（Cd）might be as one of the most toxic pollutants in plants,which causes a series of reactions from gene expression to cell metabolism in plants,thus affecting plant growth and development.Nitric oxide（NO）which as a free radical,is involved in the regulation of various biological and physiological processes in plants.Moreover,the process of S-nitrosylation modification is the most important mechanism of NO bioactivity transduction in plants.In addition,calcium ion(Ca²⁺),as an important signaling molecule,participates in the regulation of plant growth and development,and plays an essential role in plant response to abiotic stress.However,it is not clear that whether the process of protein S-nitrosylation is involved in Ca²⁺-induced the process of adventitious rooting in cucumber explants under Cd stress.Therefore,in this experiment,cucumber explants were used as experimental materials to study the role of S-nitrosylation during adventitious root development and the effect of Ca²⁺on the level of nitrosation during adventitious root formation under Cd stress,in order to reveal the functional role of S-nitrosylation as a post-translational modification in regulating adventitious rooting,meanwhile,provide sufficient theoretical basis for the mechanism of root growth and development in plants.The main results are as follows:1.Proteomic investigation of S-nitrosylated proteins during NO-induced adventitious rooting of cucumber.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.In addition,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.The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting.2.During adventitious rooting,the root number and root length of cucumber explants were significantly inhibited by different concentrations of CdCl₂ treatment.In our experiment,the adventitious root number and root length of cucumber which treated with 1μM CdCl₂ were reduced to half of that of the control.Therefore,1μM CdCl₂ was selected in the following experiment.In addition,we also verified the effects of different Ca²⁺concentrations on adventitious root development of cucumber under Cd stress.In our experiment,200μM CaCl₂ had the greatest biological effect on promoting adventitious rooting under Cd stress.Furthermore,removing endogenous Ca²⁺significantly inhibited the development of adventitious root under Cd stress.The results suggested that Ca²⁺is indispensable for promoting the adventitious root development of cucumber under Cd stress.3.The effect of Ca²⁺on the level of endogenous nitrosylation during adventitious rooting of cucumber under Cd stress was investigated in our study.The results showed that Ca²⁺played an important role in regulating the changes of endogenous NO content during adventitious root development under Cd stress.In addition,exogenous Ca²⁺significantly increased the level of endogenous SNO under Cd stress.However,we found that exogenous application of Ca²⁺significantly decreased the activity of GSNOR in cucumber explants.These results suggested that Ca²⁺may affect the development of cucumber adventitious roots under Cd stress by regulating the process of protein S-nitrosylation modification in cucumber explants.4.The effect of Ca²⁺on cell cycle during adventitious rooting of cucumber under Cd stress was studied.These results showed that CdCl₂ treatment significantly decreased the gene expression levels of key enzymes in the process of cell cycle.On the contrary,exogenous application of CaCl₂ significantly up-regulated the expression of cell cycle-related genes.These results indicated that Ca²⁺played a critical role in regulating the cell cycle during adventitious rooting under Cd stress.In addition,the S-nitrosylation level of TUA was significantly up-regulated by exogenous CaCl₂under Cd stress.However,removing Ca²⁺significantly decreased the level of TUA S-nitrosyaltion,which further implying that Ca²⁺had a positive effect on the S-nitrosylation of TUA during the process of adventitious root development under Cd stress.Furthermore,we tested a total of 13 proteins which could interact with S-nitrosylated TUA proteins under the treatment of CdCl₂ and CdCl₂+CaCl₂.These results indicated that S-nitrosylated TUA not only played an important role in cell division and differentiation during adventitious rooting under Cd stress,but also further interacts with related proteins which might be involved in these processes of carbon and energy metabolism,protein metabolism,transcription and translation,and photosynthesis to affect the adventitious rooting of cucumber under Cd stress.5.We investigated the effect of Ca²⁺on AsA-GSH cycle during adventitious root development in cucumber under Cd stress.These results indicated that Ca²⁺may be a positive regulator of AsA-GSH cycle during adventitious root formation to alleviate the oxidative stress damage caused by Cd stress,and finally induce the development of adventitious roots in cucumber.Furthermore,Ca²⁺may activate the production of antioxidants AsA or GSH by increasing the activity of endogenous GR,and significantly increase the level of GR S-nitrosylation in order to improve the level of antioxidation during adventitious root development.Additionally,we further detected the proteins that interact with S-nitrosylated GR.These identified proteins are mainly involved in the process of cytoskeleton development,transcription and translation,response to DNA damage,photosynthesis and cell wall formation during Ca²⁺-induced adventitious rooting under Cd stress.6.In our study,the effect of Ca²⁺on glycolysis pathway during adventitious rooting of cucumber under Cd stress was studied.Our results revealed that Cd stress significantly down-regulated the gene expression levels of glycolysis key enzymes during adventitious root formation.However,exogenous application of Ca²⁺could significantly up-regulate these gene expression levels under Cd stress.Therefore,in our experiment,Ca²⁺may enhance the glycolysis pathway during adventitious rooting under Cd stress by increasing the gene expression of key enzymes in glycolysis pathway.In addition,Ca²⁺treatment significantly enhanced the level of S-nitrosylation of GAPDH,indicating that Ca²⁺may enhance the S-nitrosylation modification of GAPDH,a key enzyme of glycolysis,through increasing the level of endogenous NO during adventitious rooting under Cd stress.On the contrary,the activity of GAPDH was significantly inhibited by exogenous Ca²⁺,thus responding to the negative effect of Cd stress during the development of adventitious root in cucumber.Similarly,in order to further study whether S-nitrosylated GAPDH interacts with downstream proteins during adventitious rooting to transmit oxidation signals,we identified these proteins which might interact with S-nitrosylated GAPDH.The identified proteins are mainly involved in these processes including energy metabolism,transcription and translation,balance of cellular redox and hormone response during adventitious rooting.