Effect Of UV-B Radiation On The Isoflavones Accumulation During Germination Of Soybeans And The Mechanisms Mediated By H₂O₂-H₂ Pathway

Isoflavones are the main phenolic substances in soybeans,which have many physiological effects such as anti-cancer,reduction of cardiovascular disease incidence and immune response.Therefore,it is of great significance to develop soy-flavored foods rich in isoflavones.UV-B radiation inhibits plant growth,but at the same time regulates the synthesis of plant secondary metabolites.H2O2 and H2,as two novel signal molecules discovered in recent years,act as signal transduction in oxidative stress response in plants.This paper investigated the signal transduction mechanism of H2O2-H2 pathway in UV-B promoting the accumulation of germinated soybean isoflavones,the main research results are as follows:Firstly,the changes in nutrient composition,functional components and antioxidant activities of soybean during UV-B radiation were systematically studied.It was found that UV-B radiation inhibited the decrease of total sugar content and the increase of free amino acid content during soybean germination;during UV-B radiation,the content of aspartic acid(Asp)in soybean increased significantly,the content of glutamic acid(Glu)decreased,and the proportion of linoleic acid decreased,but the ratio of linolenic acid and erucic acid increased significantly;the activity of anti-nutritional factors in germinated soybean was further reduced under UV-B,the content of antioxidant functional components such as isoflavones,phenolic acids(both bound and free),vitamin C,folic acid and chlorophyll were significantly increased,DPPH free radical scavenging ability and reducing ability were enhanced.At the same time,UV-B radiation significantly increased the soluble protein content,especially in the protein-peptide region of 1-75 kDa.Correlation analysis and principal component analysis of main index changes showed that the content of isoflavones,phenolic acids and chlorophyll were most affected by UV-B radiation,and the changes of isoflavone content in germinated soybean under UV-B radiation were significantly positively correlated with the changes of antioxidant functional components such as vitamin C,phenolic acid and folic acid,as well as DPPH free radical scavenging ability and reducing ability.It has a significant negative correlation with the change of unsaturated fatty acids.It indicates that the significant accumulation of isoflavones in germinated soybean under UV-B radiation is directly correlated with oxidative stress which was induced by UV-B.Secondly,the relationship between UV-B radiation and isoflavones synthesis were investigated,including the oxidative stress induced by UV-B.It was found that the isoflavones content of different varieties of soybeans increased significantly under UV-B radiation with a certain dose during germination.However,the isoflavones content did not increase further and even showed a significant decrease in Yunhe Soybean under a high dose of radiation.The variation of aglycon monomer content with UV-B radiation dose accorded with the cubic polynomial model,and the variation of glucoside and malonyl glycoside monomer content with UV-B radiation dose accorded with the quadratic polynomial model.At the same time,the activities of isoflavone synthesis related enzymes(including PAL,CHS,C4H,4CL,CHI,CHR,and IFS)under UV-B radiation were significantly improved.H2O2 content,electrolyte permeability,malonaldehyde(MDA)content and intercellular water ratio increased under UV-B stress,and cell volume was reduced.It was demonstrated that UV-B induced H2O2 formation through oxidative stress,leading to lipid peroxidation and cell damage,which in turn activates enzymes include activity of SOD,CAT,and POD and isoflavones accumulation,enhanced ability of defending oxidative damage.Thirdly,the synthesis process of isoflavones in germinated soybean under UV-B stress was studied from the aspects of enzymology level,gene and protein expression level,system redox state and energy state.The results showed that UV-B stress resulted in the up-regulation of the activity,gene and protein expression of NADPH oxidase in germinated soybean,which induced a significant increase of endogenous H2O2 content.NADPH oxidase inhibitor-DPI inhibited the H2O2 produced under UV-B stress,while exogenous H2O2 significantly reversed this inhibition.Both UV-B irradiation and exogenous H2O2 treatment significantly up-regulated the activities of PAL,CHS and IFS,and the expression of their genes and proteins,which led to the enrichment of isoflavones.The addition of DPI inhibited the increase of UV-B-induced isoflavone accumulation,which was only 50%of the UV-B group,and DPI weakened the up-regulation effect of UV-B on the activity and protein expression of isoflavone synthase,and these inhibitions can be reversed by exogenous H2O2,indicating that H2O2 is a necessary signal molecule in the process of UV-B stress-inducing isoflavone accumulation in soybean during germination.In addition,UV-B radiation and exogenous H2O2 treatment can change the redox state and energy state of germinated soybean,promote it to synthesize more reduced ASC and GSH,and improve the energy state of the system to resist external stress;after the addition of DPI,the system changes gradually to the oxidation state,and the energy state is significantly reduced,while the exogenous H2O2 can reverse imbalance of the redox state and the energy state.This indicates that H2O2 is a necessary signal factor for maintaining normal physiological metabolism of the body under UV-B radiation.It also indicates that the redox state as a receptor of H2O2,induces corresponding transcripts and promotes isoflavone accumulation.Finally,the role of H2 on the isoflavones accumulation induced by H2O2 under UV-B stress was investigated.H2 treatment can alleviate the inhibition of germinated soybean growth by UV-B radiation,and significantly increase the content of isoflavones in germinated soybeans;exogenous H2 and UV-B combined treatment has superimposed effect on the accumulation of isoflavones,and the content of isoflavones decreased significantly under EDTA treatment.Exogenous H2 could reverse this inhibition under EDTA treatment;H2 treatment significantly induced the enhancement of activity,gene and protein expression of PAL,CHS,and IFS;these enhancements could be inhibited by EDTA,while the inhibition can be reversed by the treatment of exogenous H2.It indicated that H2 played a role as a signal molecule in the process of UV-B-induced isoflavone accumulation.H2O2 treatment significantly increased the H2 content in germinated soybean,and the H2 content in DPI treatment decreased significantly,and this inhibition was alleviated by exogenous H2O2.DPI treatment significantly reduced isoflavone content,exogenous H2 treatment could alleviate the inhibition of DPI.EDTA treatment significantly reduced isoflavone content.H2O2 treatment could not reverse the inhibition of EDTA.These indicate that H2 plays a role in the downstream of H2O2.