Function Analysis Of Hydrogen Sulfide-Mediated Alleviation Of Cadmium Stress In Kenaf(Hibiscus Cannabinus L.)Seedlings

Cadmium（Cd）,well-known for its high toxicity and strong biological mobility,is easy to be absorbed and then accumulated in plants,causing a serious threat to food security in China.Kenaf（Hibiscus cannabinus L.）is an excellent species for phytoremediation with such advantages as rapid-growth,huge-biomass,high tolerance to heavy metals.Furthermore,kenaf,one of the most important fiber crops,can accumulate large amounts of heavy metals and thereby keep these toxic pollutants out of food chain.Hydrogen sulfide（H₂S）,a signal molecule,can alleviate the Cd toxicity in many plants,but its mechanism is still unclear.In this study,RNA-Seq,qRT-PCR and over expression techniques were utilized to excavate key Cd-tolerance-related genes in kenaf,clarify their regulatory mechanism on kenaf’s Cd-homeostasis,and analyze the role of H₂S and its regulatory mechanism in kenaf resisting Cd stress.This study will lay foundation for improving the Cd-tolerance of crops by utilizing H₂S,as well as selecting and breeding the new kenaf varieties with high Cd-tolerance.The main findings are as follows:（1）In the Cd-contaminated farmland,six kenaf varieties exhibited a strong e capacity in absorbing and transporting Cd.Especially,China Kenaf 13 and 16 are excellent species for the phytoremediation of Cd contaminated farmland,with the highest biomass and highest yield.（2）When treated with Cd of different concentrations in a hydroponic system,both kenaf roots and shoots could accumulate more than 100 mg/kg Cd and the translocation factors were beyond 1.The AsA contents in kenaf roots and leaves,as well as the CAT activity in kenaf leaves,were positively correlated with Cd concentration.Under each treatment,HcHMA2,HcGSH1,HcPCS1,HcMT2,HcLCD in kenaf roots as well as HcGSH1,HcMT2 and HcLCD in kenaf leaves were significantly up-regulated by Cd stress.（3）Exogenous H₂S-mediated alleviation of Cadmium Stress in Kenaf seedlings was found for the first time,with the best effect at 50μmol/L.H₂S can significantly increase the root length,shoot length,Cd content,as well as the dry weight and fresh weight of kenaf roots and leaves under Cd stress.H₂S induced a significant enhancement of HcGSH1,HcPCS1,HcABCC3,HcAOX4 and HcNR in both kenaf roots and leaves,as well as HcMT2 in kenaf roots under Cd stress.In addition,exogenous H₂S significantly enhanced SOD,POD and CAT in kenaf roots,as well as SOD activity in kenaf leaves under Cd stress,Exogenous H₂S significantly decreased H₂O₂ and MDA content,and increased chlorophyll content of kenaf under Cd stress.（4）By construcing the transcriptome libraries under 4 treatments,101,154 unigenes that are longer than 200 bp were obtained,involving 32 different metabolic pathways.There are 38 specifically differentially-expressed-genes between H₂S and Cd.Among them,15 genes were down-regulated and23 were up-regulated,involved in glutathione metabolism,metal ion transport,nitrogen metabolism,phenylalanine metabolism,electron transport,oxidoreductase,and carbohydrate metabolism.（5）HcGST and HcMGL in full length were cloned and their functions were further studied in Arabidopsis.According to the phenotypic identification,overexpressing HcGST could confer Arabidopsis higher Cd-sensitivity,while overexpressing of HcMGL could confer Arabidopsis higher Cd-tolerance.Overexpressing HcGST reduced the root length,fresh weight and Cd content of Arabidopsis under Cd stress.Also,overexpressing HcGST caused a significant down-regulation of AtPCS2 and SOD activity in Arabidopsis under Cd stress.Overexpressing HcMGL reduced the Cd content,as well as increased the root length and fresh weight of Arabidopsis under Cd stress.Also,overexpressing HcMGL caused a significant up-regulation of AtGSH1,AsA content and activities of SOD,CAT and POD in Arabidopsis under Cd stress.H₂S greatly increased the root length and fresh weight of three varieties of Arabidopsis thaliana under Cd stress.H₂S induced a significant up-regulation of AtGSH1,AtMT2 and AtLCD,and increased GSH content as well as SOD,POD and CAT activities in three varieties of Arabidopsis thaliana under Cd stress.H₂S could enhance the Cd-tolerance of WT,HcGST and HcMGL overexpression transgenic lines,implying that H₂S signaling pathway might play a critical role in plants’tolerance to Cd stress.