The Crosstalk Between Hydrogen Sulfide And Calcium Signaling In Plants Defense Against Chromium Stress

Gasotransmitters,the gas molecules generated endogenously by organisms,involves in numerous cellular signal transductions,and their particularity and flexibility enable gasotransmitter to be the irreplaceable messenger in diverse physiological processes of living organisms.Hydrogen sulfide（H₂S）has been verified as the third multifunctional gasotransmitter after nitric oxide（NO）and carbon monoxide（CO）.The endogenous generation and physiological functions of H₂ S in mammals have been fully confirmed,while the studies pertaining to H₂ S signaling in plants is lagging far behind,and the potential mechanisms of H₂ S remains limited though they are attracting ever-growing attention.H₂ S can trigger various defense responses and help reduce heavy metals（HMs）accumulation in plants,however,little is known about the regulatory mechanism of endogenous H₂ S generation and the H₂ S signaling transduction during plants response to HMs stress.As increased industrial wastes,sewage irrigation,chemical fertilizers and pesticides have caused excessive release of toxic metals into the environment,HMs contamination of agricultural soil is becoming a serious environmental problem,which not only reduces crop yield but also causes human health concerns.Thus,it is imperative to research the mechanism of plants adaption to HMs stress.Understanding the endogenous H₂ S generation and its important roles in plants defense against HMs stress would provide novel strategies for the genetic improvement of plants tolerance to HMs stress and reducing the economic risk caused by HMs contamination in cultivated soil.In this research,the crosstalk between gasotransmitter H₂ S and calcium signaling during plant resistance to Cr⁶⁺ stress was studied.The regulatory pathway of calcium(Ca²⁺)/calmodulin2（CaM2）on the transcription of LCD,H₂S-producing enzyme encoding gene,was further analyzed to provide evidence for the long-standing question how H₂ S production is elevated in plant stress defense,and the potential interaction between the only sulfur-containing gasotransmitter H₂ S and its main endogenously generated substrate,cysteine（Cys）,in Cr⁶⁺ stress defense was also discussed.The main results are as follows:1.Cr⁶⁺ stress increased the endogenous H₂ S generation in Setaria italica through up-regulating the expression of H₂S-producing enzymes coding genes（LCD,DCD1,DCD2,DES1）,suggesting that H₂ S involved in plants response to Cr⁶⁺ stress.Moreover,the physiological concentration of exogenous H₂ S treatment reduced Cr⁶⁺ stress-caused H₂O₂ accumulation,and then alleviated the oxidative damage,while the H₂ S synthesis inhibitor HA treatment aggravated the Cr⁶⁺ toxicity.These results implied that H₂ S had positive effects on protecting Setaria italica from Cr⁶⁺ stress.2.The Cr⁶⁺ stress-increased H₂ S production rate was hugely strengthened by Ca²⁺ pretreatment,but weakened by EGTA treatment,suggesting that Cr⁶⁺ stress could activate the H₂ S emission in a Ca²⁺-dependent manner.Furthermore,Cr⁶⁺ stress increased the cytoplasm Ca²⁺ concentration,and initiated the calcium signaling,then up-regulated the expression of H₂S-producing associated genes to promote the endogenous H₂ S emission,and H₂ S further affected the expression of CaM and CBL integrating into the calcium signaling to complete the induction of Cr⁶⁺ resistance.Overall,Ca²⁺ signaling interacted with H₂ S to induce complex physiological responses,which enhanced the Cr⁶⁺ tolerance in Setaria italica.3.H₂ S and Ca²⁺ improved the Cr⁶⁺ tolerance by regulating the HMs ion transporters,restricting the uptake and transport of Cr⁶⁺,and reducing the accumulation of toxic ion in Setaria italic.Ca²⁺ enhanced the MTs and PCs accumulation by up-regulating the expressions of MT3 A and PCS1 in a H₂S-dependent manner to deal with the Cr⁶⁺ stress,and Ca²⁺-activated H₂ S was essential for this adaptive response.In the aspect of regulating antioxidant system,Ca²⁺ not only enhanced the antioxidant enzymes SOD and POD activities but also up-regulating the antioxidant molecules in both H₂ S dependent and independent manners to fight against Cr⁶⁺ stress caused oxidative damage.Overall,H₂ S and Ca²⁺ synergistically regulated the HMs ion transport,antioxidant system and HMs chelation system to improve Setaria italica tolerance to Cr⁶⁺ stress.4.To further understand the mechanism of Ca²⁺ promoting the endogenous H₂ S generation during plants response to the Cr⁶⁺ stress,the Arabidopsis thaliana WT（Col-0）and H₂ S producing defective mutant lcd were employed to detect the effect of Ca²⁺ on endogenous H₂ S production.The activation in the transcription and translation levels of LCD as well as the H₂ S content triggered by Cr⁶⁺ stress were strengthened by Ca²⁺ addition,but decreased by EGTA.In Cr⁶⁺ stressed lcd,the positive effects of Ca²⁺ on the H₂ S emission was markedly attenuated,indicating that LCD is a key mediator in Ca²⁺-dependent H₂ S emission during Cr⁶⁺ stress responses.5.During Arabidopsis’ response to Cr⁶⁺ stress,the transcription of LCD was enhanced through the calcium(Ca²⁺)/calmodulin2（CaM2）-mediated regulatory pathway.Biochemistry and molecular biology studies demonstrated that Ca²⁺/CaM2 physically interacted with the b ZIP transcription factor TGA3,a member of the “TGACG”-binding factor family,to enhance TGA3 binding to the LCD promoter and increase LCD expression,which promoted H₂ S emission to induce Cr⁶⁺ stress tolerance.6.The endogenous H₂ S contents in Cr⁶⁺-stressed cam2 and tga3 were much lower than that in WT,which made mutants more sensitive to Cr⁶⁺ stress,and the Cr⁶⁺ stress caused toxic symptoms in the double mutants tga3/lcd and cam2/lcd were more serious than that in WT,highlighting the importance of Ca²⁺/CaM2-mediated TGA3 in enhancing LCD expression and endogenous H₂ S emission during Arabidopsis defensing aginst Cr⁶⁺ stress.7.The response mode of the only sulfur-containing gasotransmitter H₂ S and its main endogenously generated substrate Cys in plant Cr⁶⁺ stress defense was also explored.Both the H₂ S and Cys contents were increased by Cr⁶⁺ stress time-dependently,and H₂ S production was activated earlier than Cys.Moreover,the qRT-PCR results revealed that H₂ S significantly up-regulated the Cys generation-related genes SAT1 and SAT5 expression,suggesting that H₂ S mainly increased the SAT-mediated intermediate OAS generation to involve in Cr⁶⁺ stress induced Cys accumulation.Morever,the Cys consumed by Cr⁶⁺ stress-induced H₂S-generation,which only accounted for 7.5% of the Cys in Cr⁶⁺ stress plants,did not affect the Cr⁶⁺ stress mediated Cys increase.8.Both H₂ S and Cys enhanced GSH generation and activated PCs synthesis by up-regulating PCS1 and PCS2 expression to fight against Cr⁶⁺ stress.Moreover,H₂ S also promoted MTs accumulation by significantly increasing the MT2 A gene expression.Overall,H₂ S and H₂S-induced Cys accumulation（H₂S-Cys system）was critical in imparting Cr⁶⁺ tolerance in Arabidopsis.To sum up,during plants response to Cr⁶⁺ stress,the Ca²⁺/CaM2 mediated calcium signal enhanced the binding affinity of transcription factor TGA3 to the LCD promoter,up-regulated the LCD expression and promoted endogenous H₂ S emission,and H₂ S further affected the downstream genes involving in the calcium signaling to complete the induction of Cr⁶⁺ resistance.For one hand,H₂ S interacted with calcium signal to limit HMs ion uptake and transport,activate the antioxidant system and HMs chelating system to enhance Cr⁶⁺ tolerance.For another,H₂ S functioned as a signal molecule to further increase the expression of Cys synthesis related genes,involving in Cr⁶⁺ stress induced Cys accumulation,and then H₂S-Cys increased the synthesis of GSH,promoted the accumulation of HMs chelator PCs and MTs to assist plants defense against Cr⁶⁺ stress.