Exogenous MeJA And Its Combination With Melatonin Regulate The Physiological And Phenolic Acid Metabolism Of Barley Seedings

Barley is rich in phenolic acids,β-glucan,flavonoids,and other bioactive substances,among which phenolic acids have antioxidant,hypoglycemic and hypolipidemic effects on human body.However,the content of phenolic acids in barley is lower,and it is found that the biosynthesis of phenolic acids in higher plants is regulated by the growth environment.Previous studies have shown that treatment with Methyl jasmonate（MeJA）,SA（Salicylic acid）,and ZnSO₄ during germination can effectively promote phenolic acid enrichment in barley seedings,but the action mechanism of these three treatments on physiological metabolism of barley seedlings and promote phenolic acid enrichment is still unclear.At the same time,melatonin,as a new plant regulatory substance,not only regulates the growth and development of plants,but also has been confirmed to be involved in the biosynthesis of secondary metabolites in plants in recent years.The new cultivation method combined with melatonin and MeJA treatment promoted the accumulation of secondary metabolites,and provided a new idea for the development of new types of seedings health food.In this study,the effects of MeJA/ZnSO₄/SA treatment and MeJA combined with melatonin treatment on that physiology and biochemistry as well as phenolic acid metabolism of barley seedings were investigated from the aspects of physiological metabolism,enzyme and gene level.The main results were as follows:1.The effects of MeJA,ZnSO₄,and SA treatments on physiological metabolism and phenolic acid enrichment of barley seedings were studied.After 4 days of treatment with three exogenous conditions,the growth of barley seedings was inhibited to different degrees,and the sprout length and fresh weight were significantly decreased（p<0.05）.The MeJA,S A,and ZnSO₄ treatments caused cell damage,which showed that the contents of H₂O₂,O₂^·-,and malondialdehyde（MDA）were significantly increased（p<0.05）.At the same time,the total phenolic content increased,soluble protein and free amino acid contents changed significantly after the three exogenous treatments.The antioxidant capacity of the seedings was increased,the activities of CAT,POD,SOD,and APX were enhanced,and the scavenging ability of ABTS and DPPH was significantly increased.The fluorescence intensity of free Ca²⁺,H₂O₂,and O₂^·-increased in root tip of barley.The MeJA,SA,and ZnSO₄ treatments can increase PAL and C4H activities to achieve high efficiency enrichment of phenolic acid.2.The changes in the expression of key enzyme genes for phenolic acid synthesis,antioxidant enzyme genes and calcium target protein genes in barley seedings under MeJA,SA,and ZnSO₄ treatment were studied.On 6th day of germination,the expression levels of PAL,C4H,4CL,C3H,and F5H were significantly increased under the treatment of MeJA.The expression levels of C4H,4CL,and COMT in the seedings were significantly increased under ZnSO₄ treatment,while SA treatment promoted the accumulation of phenolic acid by up-regulating COMT expression.At the same time,it was found that MeJA treatment improved the antioxidant enzyme system by up-regulating CAT,APX SOD,and GST,the treatment with ZnSO₄ treatment improved the antioxidant enzyme system by up-regulating the expressions of CAT and APX,while SA treatment improved the antioxidant enzyme system by up-regulating the expression of APX.The MeJA and ZnSO₄ treatments promoted the accumulation of calcium ions in cells by up-regulating the expressions of CDPK,CAMK1 and Ca²⁺-ATP in response to environmental stress.3.The effects of exogenous melatonin on physiological metabolism and phenolic acid enrichment of barley seedings under MeJA stress were studied.Under the treatment of MeJA alone,the phenolic acid content of barley seedings was significantly increased but the growth was inhibited,and the sprout length and fresh weight were significantly decreased（p<0.05）.The MDA content,H₂O₂ content,and O₂^·-content in the sprout were significantly increased（p<0.05）.After treatment with MeJA and exogenous melatonin,the antioxidant enzyme activities of CAT,POD,and SOD were significantly increased（p<0.05）.To alleviate the inhibitory effect of MeJA treatment on the growth of barley seedings,the sprout length and fresh weight were significantly increased（p<0.05）.The MDA,H₂O₂,and O₂^·-content were decreased significantly（p<0.05）.The activities of PAL and C4H in barley seedings treated with MeJA combined with exogenous melatonin were significantly increased（p<0.05）.The total phenolic acid content in barley on 4th day and 6th day of germination was 1.10 times and 1.19 times that of the barley treated with MeJA alone.4.The changes of phenolic acid synthase gene and antioxidant enzyme gene expression in barley seedings under exogenous melatonin combined with MeJA treatment were studied.The MeJA combined with melatonin treatment promoted the accumulation of phenolic acid by significantly up-regulating the expressions of PAL,C4H,4CL,C3H,F5H,and COMT.The MeJA combined with melatonin treatment enhanced the antioxidant enzyme system of barley by significantly up-regulating the expressions of POD,APX,and SOD.The MeJA combined with melatonin treatment promoted the accumulation of melatonin and increased the tolerance of plants to the environment by up-regulating the expression of TDC.