| Isoflavone,mainly found in legumes,are a class of secondary metabolites formed in higher plants through the metabolic pathways of phenylpropanoids and flavonoids.Isoflavone has a variety of physiological effects such as anti-cancer,antioxidant and anti-ageing.Increasing isoflavone content in plants is a hot topic in functional food research.Soybean（Glycine max L.）germination under NaCl or UV-B stress increases isoflavone content several fold,but the growth and development of soybean sprouts are severely inhibited.How to maintain the biomass of soybean sprouts and promote the biosynthesis of isoflavone simultaneously is an urgent and essential problem to be solved.Melatonin（MT）is not only regulate plant growth and protects against various abiotic stresses,but is also involved in the synthesis of secondary metabolites in higher plants.In the present study,the mechanism of isoflavone accumulation in germinating soybeans under NaCl or UV-B combining MT treatment were explored.The main results were as follows:1.The effect of exogenous MT on the physiological metabolism and isoflavone enrichment of soybean sprouts under NaCl/UV-B treatment was investigated.NaCl（60 mM）or UV-B（9 h）was optimized to obtain.Soybean sprouts were inhibited to varying degrees,sprout length and fresh weight were significantly decreased,MDA,H2O2 and were increased,and the content of isoflavone was significantly increased after stress.The enrichment of isoflavone in soybean sprouts were achieved,but the growth of soybean sprouts were inhibited.After adding exogenous MT on the base of NaCl or UV-B treatment,the sprouts length and fresh weight were significantly increased compared with NaCl or UV-B treatment,the contents of MDA and H2O2 were decreased,the activities of SOD and CAT and the content of isoflavone was significantly increase.The activities of PAL and 4CL were significantly increased compared with NaCl treatment,The activity of PAL was significantly increased compared with UV-B treatment.The results showed that exogenous MT calleviated the damage of sprouts under NaCl/UV-B stress,and further increased the isoflavone content in sprouts under NaCl/UV-B stress.2.The effect of exogenous MT plus NaCl/UV-B treatment on the isoflavone synthesis related enzyme genes expression in germinating soybeans was investigated.The expression levels of PAL1,C4H,4CL,IFS1 and RH3 in soybean sprouts after NaCl stress were significantly up-regulated.Compared with NaCl treatment,the expression levels of C4H,CHI1A and IFS1 were significantly increased in NaCl Plus MT treatment.Indicating that,under NaCl stress,isoflavone was regulated mainly by promoting the activities of PAL and 4CL,and by increasing the expression levels of phenylpropanoid and flavonoid metabolic synthesis related genes.The expression levels of PAL1,C4H,4CL,CHS and IFSA in soybean sprouts after UV-B stress were significantly up-regulated.Compared with UV-B treatment,the expression level of PAL1 was significantly increased in UV-B plus MT treatment.Indicating that,under UV-B stress,isoflavone was regulated mainly by promoting the activity of PAL,and by increasing the expression levels of phenylpropanoid and flavonoid metabolic synthesis related genes.The above indicates that exogenous MT regulates the mechanism of action of enrichment of isoflavone under NaCl or UV-B stress differently.3.The effect of exogenous NO-mediated MT-regulated NaCl/UV-B stress on the physiology and isoflavone accumulation of soybean sprouts was investigated.The NO content and NR activity in soybean sprouts were significantly increased and root tip Ca2+ fluorescence intensity was significantly enhanced by exogenous MT in combination with NaCl/UV-B treatment.The results showed that MT combined NaCl or UV-B treatment promoted the production of NO by activating the nitrate reduction pathway and increasing root tip Ca2+content.Under MT combined with NaCl treatment,activities of POD,SOD and CAT were increased by SNP,and inhibited by cPTIO.The results suggested that MT further regulated the antioxidant system of soybean through NO signalling in order to enhance resistance to NaCl stress.Compared with UV-B plus MT treatment,POD activity was significantly increased by SNP treatment,while POD activity was significantly reduced by cPTIO treatment.The results showed that NO promoted the antioxidant capacity of MT plus UV-B treatment by increasing the activity of POD.Compared with NaCl Plus MT treatment,C4H activity and isoflavone contents were significantly increased by SNP treatment,while PAL activity and isoflavone content were significantly reduced by cPTIO treatment.The results showed that NO is a downstream molecule of MT,and NO promoted isoflavone content of NaCl stress by increasing the activities of PAL and C4H.Compared with UV-B plus MT treatment,PAL activity and isoflavone content were significantly increased by SNP treatment,while PAL activity and isoflavone content were significantly reduced by cPTIO treatment.The results showed that NO is a downstream molecule of MT,and NO promoted isoflavone content of NaCl stress by increasing the activities of PAL.4.The effect of exogenous NO-mediated MT regulation of NaCl/UV-B stress on the expression of NO synthase genes and isoflavone synthesis key enzyme genes in soybean sprouts was investigated.After adding MT on the base of NaCl treatment,the expression levels of NR1,NR2 and NOS1 were significantly up-graded,and after adding MT on the base of UVB treatment,the expression level of NR1 and NOS1 were significantly up-graded.It was shown that MT activates the NR and NOS pathway by regulating the expression of different genes to increase NO levels under NaCl or UV-B stress.The expression levels of C4H,CHR,CHI1A,IFS1 and RH3 in soybean sprouts after adding MT on the base of NaCl treatment were significantly up-regulated.Compared with NaCl Plus MT treatment,the expression level of PALI was increased in SNP treatment.Compared with NaCl Plus MT treatment,the expression levels of PAL1,C4H,4CL,CHR and CHI1A were significantly decreased in cPTIO treatment.Indicating that,NO as a signalling molecule downstream of MT,and under NaCl stress,isoflavone was regulated mainly by increasing the expression levels of phenylpropanoid and flavonoid metabolic synthesis related genes.The expression levels of C4H and CHS in soybean sprouts after adding MT on the base of UV-B treatment were significantly upregulated.Compared with UV-B plus MT treatment,the expression levels of PAL1 and 4CL were increased in SNP treatment.Compared with NaCl Plus MT treatment,the expression levels of PAL1,C4H,4CL,CHS,IFS and MAPK were significantly decreased in cPTIO treatment.Indicating that,NO as a signalling molecule downstream of MT,and under UV-B stress,isoflavone was regulated mainly by increasing the expression levels of phenylpropanoid and flavonoid metabolic synthesis related genes. |
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