Study On The Regulation Mechanism Of Nitric Oxide On Salt Tolerance Of Hibiscus Hamabo Sieb.et Zucc.

Nitric oxide?NO?,as an important signaling molecule in plants,has obvious effects on alleviating biotic and abiotic stress.In this study,sodium nitroprusside?SNP?was used as an exogenous NO donor,methylene blue?MB?as the NO transduction pathway inhibitors,to explore the influence of SNP on the seed germination and seedling salt-tolerant ability of Hibiscus hamabo Sieb.et Zucc.under salt stress.In addition,2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl?PTIO?were used as the NO scavenger to explore NO participation in the salt-tolerant response mechanism and molecular regulation of Hibiscus hamabo Sieb.et Zucc..1.The influence of SNP on the seed germination and physiological and biochemical indexes of Hibiscus hamabo Sieb.et Zucc.?1?The germination of Hibiscus hamabo Sieb.et Zucc.seeds under salt stress was inhibited.The seed germination under salt stress was promoted by the application of low concentration of SNP,inhabited by the application of high concentration of sodium nitroprusside.When SNP and MB were applied at the same time,SNP promoting the seed germination under salt stress was restraint.This suggests that SNP promotes the seed germination under salt stress via by NO pathway.The SNP has a certain concentration effect,ex.the low concentration promotes the seed germination,and the high concentration inhibits the seed germination.?2?The concentration of Na⁺and K⁺of the seed embryo of Hibiscus hamabo Sieb.et Zucc.was increased by salt stress.Suitable concentration of exogenous SNP could inhibit the seed embryo absorption of Na⁺,but it had no obvious effect on K⁺.This indicates that SNP inhibits the seed embryo absorption of Na⁺,so that the seeds maintain a lower Na⁺/K⁺in the germination process,which is beneficial to the maintenance of intracellular ion homeostasis.?3?The ascorbic acid?AsA?content,superoxide dismutase?SOD?activity and APX?ascorbate peroxidase?activity increased of the seed embryo of Hibiscus hamabo Sieb.et Zucc.under salt stress,but applicating suitable content of SNP inhibited AsA content,SOD activity and APX activity of the seed embryo under salt stress.The hydrogen peroxide?H₂O₂?,malondialdehyde?MDA?content and relative electrical conductivity of the seed embryo were also increased by salt stress,and applicating suitable content of SNP reduced the H₂O₂,MDA content and relative electrical conductivity of the seed embryo under salt stress.This indicates that SNP can remove the reactive oxygen produced by salt stress,reduce the secondary stress such as oxidative damage,and inhibit the enhancement of antioxidant system capacity.2.The effects of SNP on the growth and physiological and biochemical indexes of Hibiscus hamabo Sieb.et Zucc.seedling under salt stress?1?Salt stress significantly inhibited Hibiscus hamabo Sieb.et Zucc.seedling biomass,leaf number and leaf area,applying suitable concentration of SNP can improve the seedling NO content,and promote the seedling growth,biomass leaf number and leaf area.This indicates that SNP increases salt stress tolerance and promotes the seedling growth under salt stress via by increasing NO content.?2?Under salt stress,the relative water content of Hibiscus hamabo Sieb.et Zucc.seedling was reduced,but proline,soluble protein and soluble sugar content was increased.If applying the appropriate concentration of SNP,the relative water content,Proline,soluble protein and soluble sugar content of seedling under salt stress was further increased,but the content of soluble sugar decreased.This indicates that SNP mainly reduces water potential by increasing the content of proline and soluble protein,ensuring that the cells absorb water and maintain relatively high relative water content.?3?The content of Na⁺,K⁺and Ca²⁺was increased if Hibiscus hamabo Sieb.et Zucc.seedling under salt stress,and applying the appropriate concentration of SNP inhibited the seedling absorption Na⁺under salt stress,which further increased the content of K⁺and Ca²⁺.This indicates that SNP mainly by inhibiting Na⁺absorption,promoting the absorption of Ca²⁺and K⁺,reducing Na⁺/K⁺,and maintaining the ion homeostasis.?4?The activity of SOD,APX and glutathione S-transferase?GST?of Hibiscus hamabo Sieb.et Zucc.seedling was increased under salt stress,and the H₂O₂,MDA content and relative conductivity of the seedlings were also increased under salt stress.The activity of SOD,APX and GST of seedlings were further increased by applying the appropriate concentration of SNP under severe salt stress.And applying the appropriate concentration of SNP can reduce the H₂O₂,MDA content and relative electrical conductivity of the seedlings under salt stress.This indicates that SNP mainly increases the activity of plant antioxidant enzymes in late stage of salt stress and reduces the damage of oxidative.3.NO participated in response of Hibiscus hamabo Sieb.et Zucc.under salt stress and regulation of salt-tolerant genes?1?The content of NO in the root and leaf under salt stress was significantly higher than that in the control group.This indicates that the increase of NO content is a response to salt stress in the Hibiscus hamabo Sieb.et Zucc..The activity of nitrate reductase?NR?in the root was increased and the activity of NR in the leaves decreased under salt stress.The activity of nitric oxide synthase?NOS?in the root and neutral leaves all decreased under salt stress.If NR activity was inhibited by sodium azide?NaN₃?,NO content of Hibiscus hamabo Sieb.et Zucc.under salt stress declined and decreased compared with the control group.If the NOS activity was inhibited by N'-nitro-L-arginine-methylesterhydrochloride?L-NAME?,NO content of Hibiscus hamabo Sieb.et Zucc.under salt stress declined,but increased compared with the control group.If NR and NOS activity were inhibited at the same time,NO content of Hibiscus hamabo Sieb.et Zucc.under salt stress declined and decreased compared with the control group.This indicates that NR pathway is the main way to increase NO content of Hibiscus hamabo Sieb.et Zucc.under salt stress.?2?As is reveald by qPCR,SNP can promote relative expression of HhAKT1,HhAPX and HhGST of Hibiscus hamabo Sieb.et Zucc.,but inhibit the relative expression of HhSOD.However,combination of SNP and PTIO processing or PTIO single processing inhibited the relative expression of HhAKT1,HhSOD,HhAPX,and HhGST of Hibiscus hamabo Sieb.et Zucc..This reveals that ion balance and antioxidant enzymes are regulated by NO at the transcriptional level.