Mitigation Effect And Functional Analysis Of Hydrogen Sulfide On Alfalfa Injury Under Cadmium Stress

Cadmium（Cd）is one of the heavy metals in polluted soil.Excessive Cd in soil is easily absorbed and accumulated by plants.It enters the human body through the food chain.Hydrogen sulfide（H₂S）,as a third gaseous signal molecule besides nitric oxide（NO）and carbon monoxide（CO）,has been found to have the effect of alleviating a variety of plant poisons,and is involved in plant growth and development and response to stress.In order to further study the regulation ability of plants to Cd resistance under the action of H₂S,this study used alfalfa（Medicago sativa L.）as the experimental material,and carried out seed germination experiments and pot experiments.The effects of H₂S on seed germination,plant growth,Cd accumulation ability,physiological characteristics and related gene expression under Cd stress in alfalfa were analyzed.It is used to preliminarily explore the mechanism of H₂S improving plant resistance,and to provide certain reference data for phytoremediation of heavy metal-contaminated soil.The main findings of this experiment are as follows:1.Seed Germination ExperimentAfter soaking with 0,0.05,0.1,0.2,0.4,0.6 mmol/L sodium hydrosulfide（Na HS,as H₂S donor）,the seeds were cultured in 0.5 mmol/L Cd Cl₂solution,and distilled water was set as the control for seed germination test.The results showed that Cd stress significantly reduced the germination rate of seeds and inhibited the growth of radicle and germ,while H₂S could alleviate Cd stress,and the effect of seed soaking with 0.2 mmol/L Na HS was the most significant.The contents of soluble protein,soluble sugar and free proline（Pro）in alfalfa seedlings under Cd stress increased;the contents of malondialdehyde（MDA）,H₂O₂,O₂·^-increased;the activities of SOD,POD and CAT did not change significantly,APX activity was significantly increased.Seed soaking with H₂S enhanced the activities of several antioxidant enzymes,and the optimum concentration was 0.2 mmol/L.2.Pot ExperimentCd Cl₂solution was poured into the soil of alfalfa plants planted for 40 days,so that the cadmium content in the soil was 0,20,40,and 60 mg/kg,respectively.The leaves were sprayed with 0,0.05,0.1,0.2 mmol/L Na HS solution,respectively,and after 14 days of treatment,the growth,physiological indexes and expression of related genes were determined.The results showed that the root length,plant height and biomass decreased with the increase of Cd stress.Spraying Na HS could alleviate the inhibitory effect of Cd stress on the growth of alfalfa,and in general,the effect was more obvious when the concentration of Na HS was 0.2mmol/L.With the increase of Cd stress,the Cd content in the above-ground and underground parts also increased gradually.Under medium and low Cd stress,H₂S increased the transfer coefficient,but under high Cd stress,H₂S decreased the transfer coefficient,indicating that H₂S could increase the transfer coefficient under medium and low Cd stress.The absorption of Cd increases the transport of Cd to the aerial part,while under high Cd stress,the trans-lineage coefficient decreases,which inhibits the upward transport of Cd.With the increase of Cd concentration,Fv/Fm andΦPSII decreased significantly,and H₂S increased Fv/Fm significantly.This indicates that H₂S can reduce the degree of photoinhibition of PSII reaction center in alfalfa leaves under Cd stress,and improve PSII electron transfer rate and light energy conversion efficiency..The increase of NPQ and the decrease of q P indicated that Cd stress reduced the photochemical efficiency of PSII and activated the mechanism of excess excitation energy dissipation to prevent further damage to the photosynthetic system.H₂S increased q P and decreased NPQ,indicating that H₂S alleviated the photoinhibitory damage of Cd stress on the photosynthetic system.Soluble sugar,free proline,MDA,H₂O₂,O₂·^-in leaves increased with the increase of Cd stress concentration,SOD,POD,CAT,APX showed an increasing and then decreased trend with the increase of Cd stress concentration.Several indicators showed a trend towards alleviating stress.It indicated that H₂S could resist stress injury by increasing the activity of antioxidant enzymes.The relative expression measurement of SOD,POD,and CAT showed that Cd stress can up-regulate several antioxidant enzyme expression,and Na Hs makes it more significant,indicating that H2S increases its activity by increasing the expression of antioxidant.To resist Cd stress.Cd stress will inhibit the expression of IRT1,and Na HS can play a role in medium and low Cd stress,and Na HS does not work when high Cd stress.Cd stress also raised the relative expression of Nramp1 and MT1 genes,and Na HS spraying promoted the increase of its expression,and the Na HS concentration required with the Cd concentration increased.The MT1 will significantly increase its relative expression quantity whether the Cd stress level H₂S is remarkably helped to alleviate Cd stress.The above test results show that H₂S can alleviate the damage of Cd stress to Alfalfa by increasing the activity of antioxidant enzymes,enhancing the ability of osmotic regulation and promoting the up-regulation of some related genes.Based on multiple indicators,it can be seen that 0.2 mmol/L Na HS is the optimal seed soaking concentration for seed germination;when the soil is under 20 mg/kg Cd stress,the optimal Na HS spraying concentration is 0.1 mmol/L;under 40 and 60 mg/kg Cd stress,the optimal Na HS spray concentration was 0.2 mmol/L.