Effect Of Hydrogen Sulfide On Programmed Cell Death Of Cucumber(Cucumis Sativus L.) Root Tip Under Cadmium Stress

Hydrogen sulfide?H₂S?,as the third gas signal molecule after nitric oxide?NO?and carbon monoxide?CO?,has been widely studied and concerned in recent years.It has been reported that H₂S can regulate many physiological functions,including seed germination,root organogenesis,stomatal movement,photosynthesis,senescence and yield.At the same time,H₂S is involved in the response of plants to stress,including temperature stress,salt stress,hypoxia,heavy metal stress and drought stress.In this study,the effects of H₂S on programmed cell death induced by cadmium?Cd?in cucumber root tip cells were studied.The negative regulation of H₂S on Cd induced cell death was explained by phenotype,cell physiology and molecular level,which provided a theoretical reference for H₂S to resist heavy metal stress.The main results are as follows:1.The root length and fresh weight of cucumber seedlings decreased significantly with the increase of Cd?50,100,200 and 300?M?concentration.When the concentration was 200?M,the root length was half of the control.At the same time,the relative conductivity and the content of MDA in the roots also increased with the increase of Cd concentration.When treated with 200?M CdCl₂ for 48 h,the contents of hydrogen peroxide?H₂O₂?and superoxide anion(O₂^·-)in roots increased significantly with treatment time.With the accumulation of ROS,the cell death of root tip increased significantly with the extension of stress time.It is suggested that the inhibition of root elongation by Cd was influenced by the cell death of root tip.2.Through different concentrations of NaHS pretreatment,we found that the effect of H₂S on relieving Cd stress was concentration dependent.With the increase of H₂S concentration,the root length and fresh weight first increased and then decreased,and the best effect was when the concentration was 100?M.Cd stress induced a significant increase in the content of endogenous H₂S,while the content of endogenous H₂S in NaHS pretreatment was significantly higher than that in other treatments.H₂S could significantly improve root activity and reduce ROS accumulation and membrane lipid peroxidations by activating antioxidant system to improve antioxidant enzyme activity.The results of Evans blue staining also showed that H₂S reduced the cell death induced by Cd stress.qRT-PCR showed that H₂S up regulated HSP70 expression to enhance Cd resistance,down regulated ATG8f expression to reduce Cd-induced autophagy.These results indicated that H₂S could inhibit the Cd toxicity on cucumber seedlings by reducing oxidative damage and cell death.3.Cd stress reduced the content of ascorbate?AsA?,significantly increased the content of dehydroascorbate?DHA?and oxidized glutathione?GSSG?,and destroyed the redox balance.H₂S significantly increased the content of AsA and glutathione?GSH?,and decreased the accumulation of DHA and GSSG under Cd stress.The activity of glutathione reduucttase?GR?,DHAR and MDHAR was significantly increased induced by Cd.The activity of GR,dehydroascorbate reductase?DHAR?and monodehydroascorbate reductase?MDHAR?after NaHS pretreatment was higher than that of Cd alone.Moreover,H₂S promoted the expression of GR,DHAR and MDHAR genes,enhanced the reduction ability of cells and reduced oxidative damage.4.By DAPI staining and caspase-3-like activity measurement,we found that Cd could cause programmed cell death?PCD?,while H₂S could reduce DAPI fluorescence and caspase-3-like activity,and inhibit the occurrence of this process.At the same time,Cd stress leaded to the release of mitochondrial cytochrome c?Cyt c?and the opening of mitochondrial membrane transition pore?MPTP?.H₂S increased the ratio of Cyt c/a,reduced the absorption value of MPTP,and inhibited the downstream reaction of PCD caused by the release of Cyt c into the cytoplasm.5.Under Cd stress,the Ca²⁺-ATPase?H⁺-ATPase and Mg²⁺-ATPase activity of mitochondria decreased significantly,MPTP opened,and the expression of related MPTP genes CsVDAC and CsANT increased,which led to the release of Cyt c and Ca²⁺into the cytoplasm,the accumulation of mitochondrial H₂O₂ and the activation of caspase-3-like activity,and finally PCD.H₂S negatively regulated this process,reduced the release of Ca²⁺and the increase of mitochondrial H₂O₂,down-regulated the expression of CsVDAC and CsANT genes,and maintained the activity and physiological function of mitochondrial ATPase.However,endogenous H₂S scavenger HT could reverse this process and aggravate the occurrence of PCD.