Regulation Mechanism Of Sulforaphane Formation In Broccoli Sprouts Under Heat And Hypoxia Stress As Well As JA And ABA Treatment

Glucosinolates are a group of sulphur-and nitrogen-containing secondary metabolites in broccoli.When plant tissue is damaged,glucosinolates can be hydrolyzed by the enzyme myrosinase(MYR)into various products including isothiocyanates(ITCs),thiocyanates,nitriles,epithionitriles and oxazolidines.Glucoraphanin(GRA)is the most abundant glucosinolate and accounts for more than 50%of total glucosinolates in broccoli.It is hydrolyzed by MYR to form sulforaphane,which is known to be the most promising anticancer agent.However,in the presence of the epithiospecifier protein(ESP),GRA degrades to sulforaphane nitrile,which decreases sulforaphane yield and does not provide the same health benefits.Hence,the formation of sulforaphane may result not only from difference in glucoraphanin content but also from differences in MYR and ESP activity.The level of GRA in broccoli sprouts is 10?100 times higher than that in their mature counterparts.Abiotic stress conditions can enhance glucosinolates accumulation.Jasmonic acid(JA)and abscisic acid(ABA)content can be induced by abiotic stress to alleviate stress.JA as a signal molecule has been found to regulate glucosinolate biosynthesis.However,little is available about the effect of ABA on glucosinolate regulation.In the present study,the mechanism of GRA and sulforaphane accumulation in broccoli sprouts under heat,hypoxia and heat plus hypoxia stresses as well as JA and ABA treatments was explored.The main results were listed as following:1.Changes in the contents of ascorbic acid,total phenolics,anthocyanins,GRA and sulforaphane,as well as antioxidant capacity and MYR activity in broccoli sprouts were investigated.The contents of ascorbic acid and anthocyanins were firstly increased,and then declined during sprouts growth from day 0 to day 7.Total phenolic and GRA content as well as sulforaphane formation declined with sprouts growth.MYR activity in sprouts,stable for the first 3 days of germination,slowly increased 3-fold by 7 days.The highest antioxidant capacity was obtained in 2-day-old broccoli sprouts.GRA was the most abundant glucosinolate and accounted for about 70%of total glucosinolates in broccoli sprouts.Sprouts grown at 25?had higher GRA content and sulforaphane formation than that grown at 20?and 30?.In addition,cotyledon contained the highest GRA content and sulforaphane formation and accounted for more than 93%of GRA and sulforaphane in broccoli sprouts.However,MYR activity in cotyledon was lower than that in hypocotyl and root.The most abundant hydrolysis product in seeds and sprouts,cotyledon and hypocotyl was sulforaphane,but the major hydrolysis products in root were 4-(methylthio)-butanenitrile and 1-isothiocyanato-butane.2.The effects of heat,hypoxia and heat plus hypoxia stresses on physiological metabolism and the accumulation of GRA and sulforaphane in broccoli sprouts were investigated.Heat stress resulted in the enhancement of the activities of SOD,CAT,POD and GPX in broccoli sprouts compared with the control.However,under hypoxia stress,the activities of POD and GPX in sprouts decreased,SOD and CAT activities increased.The activities of SOD,CAT,POD and GPX in broccoli sprouts under heat plus hypoxia were higher than those of control and hypoxia stress,but lower than those of heat stress.Broccoli sprouts under heat stress had the highest antioxidant capacity.ABA was the most abundant phytohormone in broccoli sprouts,followed by JA.Three stresses significantly increased JA,ABA and BR content in broccoli sprouts,but did not affect SA and GA content and decreased ETH content.Heat,hypoxia and heat plus hypoxia stresses significant increased the accumulation of total and aliphatic glucosinolates,gluconapin(GNA),GRA and glucoerucin(GER).However,heat plus hypoxia stress did not have the synergistic effect of heat and hypoxia stresses.GRA content in 5-and 7-day-old sprouts had no significant different among three stresses,but heat stress resulted in lower contents of GNA and GER than hypoxia stress.Moreover,no significant changes were observed in total and individual glucosinolate content between heat plus hypoxia stress and hypoxia stress.Under heat stress,MYR and ESP activities in 7-day-old broccoli sprouts decreased by 39.41%and 64.28%,respectively,compared with the control.However,compared with the control,MYR activity in 7-day-old broccoli sprouts under hypoxia stress decreased by 11.90%,ESP activity enhanced by 34.30%.In addition,under heat plus hypoxia stress,no significant difference was found in MYR activity compared with hypoxia stress,and ESP activity had no significant change compared with the control.Heat stress resulted in the highest sulfoaphane formation in broccoli sprouts.Sulfoaphane formation in 7-day-old broccoli sprouts under heat stress was 2.54-fold of the control and 1.30-fold of hypoxia stress, respectively.3.The effect of JA and ABA on the accumulation of GRA and sulforaphane in broccoli sprouts was investigated.Compared with the control,the contents of GRA and total glucosinolates in 7-day-old broccoli sprouts treated with 100?M JA increased by 2.01-and 2.53-fold,respectively.JA also induced MYR activity in 5-and 7-day-old broccoli sprouts,and had no significant effect in ESP activity.However,200?M and 300?M JA dramatically induced MYR and ESP activities in all tested sprouts.Sulforaphane formation in 5-and 7-day-old broccoli sprouts treated with 100?M JA was 1.95-and 3.36-fold of control,which was higher than that of 300?M JA.ABA enhanced the contents of GRA and total glucosinolates in all tested broccoli sprouts,but no significant difference was observed between 50?M and 75?M.In 3-,5-and 7-day-old sprouts,GRA content increased by 16.64%,62.52%and 97.17%,respectively,compared with the control;total glucosinolate content increased by 18.58%,70.36%and 109.61%respectively.MYR and ESP activities in 5-and 7-day-old broccoli sprouts was dramatically induced by ABA treatment compared with the control.Sulforaphane formation in broccoli sprouts significantly increased by ABA treatment,but there was no significant change between 50?M and 75?M.4.Designed degenerate primers,outer and inner primers for 3' and 5' Race.Then using RACE technology,cDNAs of CYP79F1,CYP83A1,UGT74B1,ST5b and FMOGS-OX1 in broccoli were sequenced.The 1980 bp long of broccoli CYP79F1 cDNA was obtained which containing a 1623 bp open reading frame(ORF)that encoded 540 amino-acid peptides.The CYP83A1 cDNA was 1652 bp in length containing a 1506 bp open reading frame(ORF)that encoded 501 amino-acid peptides.The UGT74B1 cDNA was 1592 bp long containing a 1401 bp open reading frame(ORF)that encoded 466 amino-acid peptides.The 1378 bp in length of ST5b was obtained which containing a 1029 bp open reading frame(ORF)that encoded 342 amino-acid peptides.The FMOGS-OX1 cDNA was 1623 bp in length,had a 1386 bp open reading frame(ORF)that encoded 461 amino-acid peptides.5.Under heat stress,transcriptional levels of Elong,CYP83A1 and FMOGS-OX1 in broccoli sprouts significantly increased compared to the control,while MYR and ESP expression decreased.A total of 146 differentially expressed proteins were successful identified in broccoli sprouts under heat stress.These differentially expressed proteins involved in defense/stress,protein biosynthesis,energy,carbohydrate metabolism,animo acid metabolism and secordary metabolism etc.,and were predicted to be localized in chloroplast,cytoplasm,mitochondrion and nucleus etc.Heat stress decreased the expression of S-adenosylmethionine synthase 1,2 and 4(SAM1,SAM2 and SAM4),while significantly increased the expressions of 3-isopropylmalate dehydrogenase(IPMDH),3-isopropylmalate dehydratase large subunit(IIL1)and 3-isopropylmalate dehydratase small subunit 3(IPMDS3).The expressions of myrosinase 1(MYR1),epithiospecifier protein(ESP)and nitrile-specifier protein 2(NSP2)in broccoli sprouts were decreased by heat stress.6.Hypoxia and heat plus hypoxia stresses significantly increased the expressions of Elong,CYP83A1,UGT74B1,ST5b and AOP2 in broccoli sprouts compared with the control,which resulted in the higher contents of GER and GNA than heat stress.MYR and ESP espression was also ebhanced by hypoxia and heat plus hypoxia stresses.A total of 91 differentially expressed proteins were successful identified in broccoli sprouts under hypoxia stress.However,when compared to heat and hypoxia stress,106 and 19 differentially expressed proteins were successful identified in broccoli sprouts under heat plus hypoxia stress,respectively.Hypoxia and heat plus hypoxia stresses decreased the expression of SAM1 and SAM2,while increased IIL1 and IPMDS3 expression.Hypoxia stress resulted in no siginificant difference in expression of MYR1 and MYR2,while enhanced ESP expression.Under heat plus hypoxia stress,no significant changes were found in the expression of MYR1,MYR2,ESP and NSP2.7.A total of 126 and 102 differentially expressed proteins were successful identified in broccoli sprouts treated with JA and ABA,respectively.These differentially expressed proteins involved in defense,energy,protein biosynthesis,carbohydrate metabolism,animo acid metabolism,protein folding and degradation,signal transduction and transcription,and secondary metabolism,etc.;and were predicted to be localized in chloroplast,cytoplasm,mitochondria,nucleus and endoplasmic reticulum,etc.JA and ABA significantly enhanced the expression of MYB28,Elong,CYP79F1,CYP83A1,UGT74B1,ST5b,FMOGS-OX1 and AOP2.The expression of IPMDS3 was induced by JA and ABA,IPMDH expression only induced by ABA.JA also increased the expression of MYR,MYR1 and MYR2.Under ABA treatment,the expressions of MYR and ESP,as well as protein of MYR2 and ESP were significantly enhanced.