Effects Of Selenium And Sulfur On The Accumulation Techniques Of Isothiocyanate And Its Mechanism In Germinated Broccli Sprouts

Broccoli(Brassica oleracea L.),also known as broccoli,is a good material for the enrichment of isothiocyanates(ITCs)and selenium.ITCs are one of the degradation products of glucosinolates under the action of endogenous myrosinase,which can effectively prevent DNA damage caused by various carcinogens in the diet。Sulfate stress promotes the enrichment of ITCs in broccoli by increasing the glucosinolate content.The content of glucosinolates is closely related to the content of sulfur in plants.Since selenium and sulfur are similar in atomic size and chemical properties.In the process of metabolism,selenium can promote the absorption of sulfur element,and can also replace sulfur to combine with sulfur-containing organic compounds,and promote the synthesis of selenium compounds.The interaction of selenium and sulfur can significantly increase the content of ITCs in plants and alleviate the damage caused by sulfate stress on plants.In this study,broccoli sprout was used as a material to explore the effects of selenium-sulfur interactions on isothiocyanate metabolism in broccoli sprouts from physiological metabolic levels,gene transcription levels and protein expression levels.The main results were as follows:1、The effects of zinc sulfate(4 mM,ZnSO4)and its combined high(0.10 mM)and low(0.01 mM)concentration of sodium selenite(Na2SeO3)treatment on the physiological metabolism and ITCs enrichment of broccoli sprouts were studied.The growth of broccoli sprouts were all inhibited under ZnSO4 treatments,the main manifestations were that the length became shorter,the fresh weight decreased,and the contents of malondialdehyde(MDA)and H2O2 increased;After germinating for 6 days,the addition of high and low concentrations of Na2SeO3 on the basis of ZnSO4 treatment can all alleviate the damage caused by ZnSO4 treatment alone,reduce the MDA content,and increase the total phenol content in broccoli sprouts.The addition of exogenous selenium on the basis of ZnSO4 treatment significantly increased the content of selenium but did not affect the total sulfur content in the sprouts(p<0.05).ZnSO4 increased the contents of ITCs and sulforaphane by increasing the content of glucosinolates and the activity of MYR in broccoli sprouts after germinating for 4 and 6 days.On the basis of ZnSO4 treatment.the addition of high and low concentration of Na2SeO3 had no significant effect on the content of ITCs while significantly reduced the sulforaphane content in the sprouts compared with ZnSO4 alone.On the basis of ZnSO4 treatment,when the concentration of 0.10 mM Na2SeO3 was added.the effect of enriching ITCs and sulforaphane was better than the concentration of 0.01 mM in broccoli sprouts after germinating for 4 days,and 0.10 mM was the optimal Na2SeO3 concentration.2、The effects of ZnSO4 stress and high-concentration Na2SeO3 treatment and ZnSO4 plus high-concentration Na2SeO3 treatment on ITCs synthesis and selenium metabolism related enzymes gene expression in broccoli sprouts were investigated.Under ZnSO4 treatment,the expression level of UGT74B1,FMOGS-OX1,STSb,BoSMT,BoCOQ5-2 at 4 days,ESP,BoSultr1;1 at 4 and 6 days in broccoli sprouts were significantly up-regulated(p<0.05);The gene expressions of MYB28,UGT74B1,FMOGS-OX1,ST5b,BoCOQ5-2 were significantly down-regulated after germinating for 6 days(p<0.05);The gene expressions of MYB28 at 4 days,BoSMT at 6 days,MYR,BoSAT1;1,BoHMT1 at 4 and 6 days had no significantly change compared with the control(p>0.05).Under high-concentration Na2SeO3 treatment,the expression level of UGT74B1,FMOGS-OX1,ST5b at 4 days,MYB28,BoSMT at 6 days,BoHMT1、BoCOQ5-2、BoSultr1;1 at 4 and 6 days were significantly up-regulated(p<0.05);The gene expressions of UGT74B1,ESP were significantly down-regulated after germinating for 6 days(p<0.05);The gene expressions of MYB28,ESP,BoSMT at 4 days,ST5b,FMOGS-OX1 at 6 days,MYR,BoSAT1;1 at 4 and 6 days had no significantly change compared with the control(p>0.05).Under ZnSO4 plus high-concentration Na2SeO3 treatment,the expression level of ST5b,FAOGS-OX1,ESP at 4 days,MYR,BoSMT,BoSAT1;1 at 6 days,UGT74B1,BoHMT1,BoCOQ5-2,BoSultr1;1 at 4 and 6 days were significantly up-regulated(p<0.05);The gene expressions of MYB28,FMOGS-OX1,ESP were significantly down-regulated after germinating for 6 days(p<0.05);The gene expressions of MYB28,MYR,BoSMT,BoSAT1;1 at 4 days,ST5b at 6 days had no significantly change compared with the control(p>0.05).The results showed that under different treatments,the response of genes related to isothiocyanate synthesis and selenium metabolism in broccoli sprouts were different,ZnSO4 plus high-concentration Na2SeO3 treatment enriched ITCs in broccoli sprouts at 6 days by up-regulating the gene expression of UGT74B1,MYR involved in isothiocyanate synthesis,increased the selenium content by promoting the gene expression of BoHMT1 involved in selenium metabolism.3、After treated with ZnSO4 and high-concentration Na2SeO3,310 and 353 differentially expressed proteins were identified.After treated with ZnSO4 plus high-concentration Na2SeO3 treatment,compared with ZnSO4,there were 202 differentially-expressed proteins,compared with high-concentration Na2SeO3,there were 170 differentially expressed proteins.The functions of these differential proteins were mainly related to amino acid metabolism,carbohydrate metabolism,defense,energy and so on.The expression abundance of MAM1,IPMI2,IIL1,IMD1,BCAT3,SOT17,SOT18,MYR1 and MYR2 in glucosinolate synthesis significantly increased under ZnSO4 treatment,were favorable for the synthesis of glucosinolate and the activity of MYR to promote the enrichment of ITCs;The expression abundance of MS2,NTRB,and APS1 involved in selenium metabolism were not significantly different from the control,and the expression abundance of other enzymes involved in selenium metabolism increased.The relative expression of IIL1 was significantly reduced by 34.63%compared with the control after treatment with high-concentration of Na2SeO3,high-concentration Na2SeO3 treatment regulated the aliphatic by reducing the protein expression of IIL1,thereby regulating ITCs,MYR activity was not related to its protein expression;The abundance of 11 enzymes participated in selenium metabolism significantly increased compared with the control,thereby promoting the accumulation of selenium.After treated with ZnSO4 plus high-concentration Na2SeO3,the expression of IIL1 and IPMDS3.MYR1,ESP and NSP2 were reduced compared with ZnSO4 or high-concentration Na2SeO3,ZnSO4 plus high-concentration Na2SeO3 treatment increased the vitality of MYR to facilitate the enrichment of ITCs by regulating the abundance of MYR2 expression;Compared with ZnSO4 alone,the expression abundance of MS2,MTO1,APS3 increased,compared with high-concentration Na2SeO3 alone,decreased or was not significant,and the expression abundance of other enzymes involved in selenium metabolism was not significantly different than that of ZnSO4 or high-concentration Na2SeO3,indicating that high-concentration Na2SeO3 had effect on selenium in either single treatment or combined stress.