Study On Technology And Mechanism Of Enriching Melatonin In Mustard Sprouts With Heat Shock,MeJA And ABA

Mustard(Brassica juncea L.)is an important vegetable crop in China,with a characteristic of rich in nutrients.During the germination of mustard,the endogenenous enzymes of seeds are activated or produced,functional substances such as melatonin,which have various physiological effects are enriched.In plants,melatonin synthesis from tryptophan involves the usage of a stepwise sequence of four enzymes,namely tryptophan decarboxylase(TDC),tryptamine 5-hydroxylase(T5H),serotonin N-acetyltransferase(SNAT),and N-acetylserotonin methyltransferase(ASMT).Under stress conditions,the synthetases of melatonin were activated which endogenous melatonin levels are significantly increased.Heat,MeJA and ABA treatments could significantly increase the melatonin-related enzymes activity resulting in the accumulation of melatonin.In the present study,the mechanism of melatonin accumulation in mustard sprouts under Heat,MeJA and ABA treatments were explored.The main results were as follows:1.The effects of Heat,MeJA and ABA treatments on physiological metabolism and melatonin accumulation in mustard sprouts were investigated.Under different treatments,the growth of mustard sprouts was suppressed,the sprout length,respiratory rate,dry weight and fresh weight of each mustard sprouts were decreased significantly,and resulted in cell membrane damage in mustard sprouts,which showed significant increase in electrolyte permeability and H2O2 content;Meanwhile,the content of Ca2+and soluble sugar increased,the total phenol,anthocyanin and isothiocyanate(ITC)content changed significantly after treatments,and the antioxidant capacity of sprouts were increased;After germinating for 4 days under Heat,MeJA and ABA treatments,melatonin content was 1.88-,10.04-and 3.67 fold of the control in mustard sprouts,respectively,and the melatonin enrichment in sprouts was achieved.2.The effect of Heat,MeJA and ABA treatments on the melatonin synthesis related enzymes gene expression in mustard sprouts were investigated.In this study,eleven key genes homologous for melatonin biosynthesis enzyme were identified in mustard by using molecular biological methods.These include two BjTDCs,two BjT5Hs,four BjSNATs,and three BjASMTs.The gene expression levels of BjTDC 1 and BjTDC 2 in mustard sprouts by heat stress treatment were significantly increased compared with the control,and BjTDC 2 was coincided with the trend of melatonin content.The expression levels of BjT5H1 and BjT5H2 at 4 d were significantly higher than control group,while at 7 d were decreased(p<0.05).Under heat stress,the transcriptional levels of BjSNAT1 and BjSNAT4 at 4 d sprouts increased,while the expressions of BjSNAT2 and BjSNAT 3 were decreased.Furthermore,BjASMT 1 expression was opposite to that of BjASMT 2 and BjASMT 3 at 4 d and 7 d.The expression of BjASMT 1 was increased at 4 d,while BjASMT 2 and BjASMT 3 were decreased.After MeJA treatment,the transcriptional of BjTDC 1 and BjTDC 2 at 4 d were increased compared with the control(p<0.05),and there was no significant difference in expression at 7 d(p>0.05).The expressions of BjT5H 1 and BjT5H 2 were at 4 d and 7 d were increased significantly(p<0.05).The transcriptional of BjSNAT 1,BjSNAT 2 and BjSNAT 4 increased significantly at 4 d(p<0.05).The expression of BjSNAT 3 was decreased at 4 d while increased at 7 d.The expression of BjASMT 1 was increased significantly at 4 d,while BjASMT 2 and BjASMT 3 were decreased significantly(p<0.05).After ABA treatment,only BjSNAT 1 and BjASMT 3 were significantly down-regulated at 4 days(p<0.05),and the rest of melatonin synthase genes were significantly up-regulated(p<0.05).The results showed that the three treatments all up-regulated BjTDC,BjT5H and BjSNAT 3,and regulated the expression of other key enzyme genes of melatonin synthesis to influenced the accumulation of melatonin.3.The effect of Heat,MeJA and ABA treatments on proteome expression in mustard sprouts were investigated.A total of 172,156 and 237 differentially expressed proteins were identified in mustard sprouts under Heat,MeJA and ABA treatments,respectively.The remaining proteins involved in carbohydrate metabolism,energy,response to stress,nucleotide metabolism,amino acid metabolism,secondary metabolism,protein biosynthesis,signal transmission,etc.These differentially expressed proteins were localized in nucleus,ribosome,vacuole,mitochondria,chloroplast,endoplasmic reticulum and cytoplasmic skeleton.Each treatment had regulatory effect on the tryptophan metabolism pathway in mustard sprouts,while the regulatory protein site and protein expression abundance are different between treatment groups,resulted the melatonin accumulated between each treatment group.In addition,Heat treatment mainly enriched the pathways of protein processing at the endoplasmic reticulum and photosynthesis,MeJA and ABA treatments major impacted the pathways of ribosomes and secondary metabolism.ABA treatments also enriched the pathways of glutathione metabolism.These three processes indirectly resulted melatonin synthesis by regulate signal transmission,nucleic acid transcription and translation,improved energy metabolism,promoted protein transport and synthesis,and strengthened the antioxidant system,accumulated active secondary metabolites and other physiological functions regulatory substances work together to increase the tolerance to stress.