The Mechanism Of Phenolic Acid Accumulation Mediated By GABA In Barley Seedlings Under NaCl Stress

Phenolic acid is a kind of plant secondary metabolites with phenolic hydroxyl structure,which has antioxidant,anti-cancer,bacteriostasis,anti-infection,hypoglycemic and cholesterol-lowering effects on human body.Phenolic acids in human body are derived from the diet.Therefore,it is of great significance to develop food rich in phenolic acid.When grain germinated under adverse conditions,a series of physiological and biochemical processes were regulated and the metabolic flow could be changed,so phenolic acid could be greatly enriched.Gamma-aminobutyric acid(GABA),a non-protein amino acid,is a signaling molecule that plays a regulatory role in plant growth and development.Nitric oxide(NO)is an important bioactive molecule,which is involved in plant growth and development.As a second messenger coupled extracellular signal and intracellular physiological response,Ca2+ is a major regulator of plant metabolism and growth.In this study,the signal transduction mechanism of GABA/NO/Ca2+ in the synthesis of phenolic acid in barley seedlings in NaCl stress was discussed from the level of physiological,biochemical metabolism,enzyme,gene and metabolomics.The main results were as follows:1.The effects of NaCl stress on physiological metabolism,phenolic acid synthesis and antioxidant capacity of barley seedlings were investigated.NaCl stress caused oxidative damage.Increased ROS content caused stress response in barley seedlings.Therefore,phenolic acid content and antioxidant enzyme activity increased,and the antioxidant capacity of barley seedlings enhanced.Among them,the seedlings treated with 60 mM NaCl had the best effect of phenolic acid enrichment,and the contents of p-hydroxybenzoic acid,vanillic acid,caffeic acid,syringic acid,p-coumaric acid,ferulic acid and sinapic acid significantly increased.The antioxidant capacity of barley seedlings was strongest under 60 mM NaCl treatment.2.The effect of GABA treatment on physiological metabolism of barley seedlings under NaCl stress was investigated.GABA treatment alleviated NaCl stress,which exhibited concentration-dependent effects.The 0.5 mM GABA showed the best effect.On 4 d and 6 d of germination,GABA increased sprout length by 18%and 15%,and decreased malondialdehyde content by 10%and 34%.GABA decreased O2-· production by 23%and 30%,and decreased H2O2 content by 27%and 7%,respectively,compared with that of NaCl stress.GABA treatment increased cross-sectional area of roots and diameter of endothelial cells,and effectively alleviated the folds of epithelial cells caused by NaCl stress.GABA treatment significantly increased GAD activity and induced GABA accumulation in barley seedlings.On 4 d and 6 d of germination,the content of endogenous GABA in barley seedlings increased by 28%and 18%,respectively,compared with that under NaCl stress.3.The effects of GABA treatment on phenolic acid synthesis and antioxidant capacity of barley seedlings under NaCl stress were investigated.GABA significantly improved activities of PAL,C4H,4CL and COMT,and induced protein expression of PAL,C4H,4CL and F5H by 38%,55%,38%and 5%compared with NaCl stress alone.3-MP treatment reduced activities of PAL,C4H,4CL,C3H and COMT,and this inhibitory effect could be effectively alleviated by GABA.GABA significantly improved contents of total phenolic,protocatechuic acid,p-hydroxybenzoic acid,vanillic acid,p-coumaric acid,ferulic acid and sinapic acid.3-MP treatment inhibited the synthesis of total phenolic and phenolic acids mentioned above,and this inhibitory effect could be reversed by GABA.On 4 d of germination,ABTS and DPPH values increased by 22%and 66%,respectively,under GABA treatment,and by 5%and 11%on 6 d of germination,compared with that of NaCl-treated seedlings.3-MP significantly reduced antioxidant capacity.GABA reversed this inhibitory effect.4.The effect of NO on the synthesis of phenolic acid in barley seedlings under NaCl stress was investigated.SNP significantly induced gene and protein expressions of PAL,C4H and 4CL,and increased activities of PAL,C4H and 4CL by 15%,9%,and 52%,respectively,on 4 d of germination,and by 28%,69%,and 10%,respectively,on 6 d of germination,compared with that of NaCl-treated seedlings.COMT activity increased only on 4 d of germination.cPTIO significantly inhibited activities of PAL,C4H,4CL,C3H and COMT,which was consistent with the gene expression of the corresponding enzymes.The addition of SNP effectively alleviated the effect of cPTIO on the expression of these genes and corresponding enzymes activities.SNP treatment significantly increased contents of total phenolic,protocatechuic acid,p-hydroxybenzoic acid,vanillic acid,caffeic acid,syringic acid and ferulic acid.cPTIO significantly reduced contents of total phenolic,protocatechuic acid,p-hydroxybenzoic acid,vanillic acid,syringic acid,p-coumaric acid and ferulic acid.GABA effectively alleviated this inhibitory effect.The results indicated that NO played an important role in phenolic acid synthesis of barley seedlings.5.The effect of GABA-NO on synthesis of phenolic acid in barley seedlings under NaCl stress was investigated.3-MP significantly reduced content of phenolic acid,and SNP treatment alleviated the inhibitory effect of 3-MP.The content of phenolic acid was also significantly decreased under cPTIO treatment,and the inhibitory effect of cPTIO was alleviated by GABA treatment.Moreover,SNP reversed the inhibitory effect of 3-MP on PAL,C4H and 4CL activities,and GABA also reversed the inhibitory effect of cPTIO on PAL,C4H,4CL and F5H activities.Therefore,GABA and NO interacted with each other in the synthesis of phenolic acid in barley seedlings under NaCl stress.6.The effect of Ca2+ on synthesis of phenolic acid in barley seedlings under NaCl stress was investigated.CaCl2 treatment induced protein expressions of PAL,C4H,4CL and F5H,but inhibited the induction effect of NaCl stress on expression of C3H and COMT proteins,which was consistent with their activities changes.On 6 d of germination,except for the abnormal up-regulation of gene expression of EGTA-treated barley seedlings,LaCl2,EGTA and 2-APB significantly inhibited the gene expression of PAL,C4H,4CL,C3H,COMT and F5H,which was consistent with the change trend of enzymes activities.CaCl2 treatment significantly increased contents of total phenolic and phenolic acids.On 4 d of germination,p-hydroxybenzoic acid,vanillic acid,caffeic acid,syringic acid,p-coumaric acid,ferulic acid and sinapic acid increased by 246%,214%,21%,92%,47%,76%and 155%,respectively,under CaCl2 treatment compared with NaCl stress.On 6 d of germination,protocatechuic acid,p-hydroxybenzoic acid,vanillic acid,p-coumaric acid,ferulic acid and sinapic acid increased by 2189%,80%,34%,67%,6%and 36%respectively.LaCl3,EGTA and 2-APB treatment significantly reduced the contents of p-hydroxybenzoic acid by 76%,27%and 81%,vanillic acid by 49%,29%and 56%,syringic acid by 86%,83%and 84%,p-coumaric acid by 53%,36%,63%,ferulic acid by 24%,19%and 39%,and sinapic acid by 66%,51%and 60%respectively on 6 d of germination.CaCl2 treatment effectively alleviated this inhibitory effect.This indicated that Ca2+ was involved in the synthesis of phenolic acid in barley seedlings under NaCl stress..7.The effect of GABA-Ca2+ on the synthesis of phenolic acid in barley seedlings under NaCl stress was investigated.GABA induced Ca2+ influx into root tip cells of barley seedlings.The amount of calcium precipitation increased significantly.The content of total calcium and water-soluble calcium also increased significantly.The contents of total phenolic,total phenolic acid and major phenolic acid in barley seedlings were significantly reduced by 3-MP treatment,and the inhibition effect of 3-MP was alleviated by 3-MP+CaCl2 treatment.The phenolic acid content in LaCl3,EGTA and 2-APB treatments was significantly reduced,and the inhibition was alleviated by GABA treatment.In addition,CaCl2 reversed the inhibitory effect of 3-MP on protein expression of PAL,C4H and F5H,and GABA also effectively reversed the inhibitory effect of LaCl3,EGTA and 2-APB on the protein expression of 4CL,C3H and COMT.Therefore,GABA and Ca2+interacted with each other in the synthesis of phenolic acid in barley seedlings under NaCl stress.8.The effect of NO-Ca2+on the synthesis of phenolic acid in barley seedlings under NaCl stress was investigated.SNP reversed the inhibitory effect of LaCl3,EGTA and 2-APB on the synthesis of phenolic acid.Exogenous CaCl2 could not reverse the inhibitory effect of cPTIO.During the whole germination period,CaCl2 only reversed the inhibitory effect of cPTIO on the protein expression of PAL and F5H,while exogenous SNP effectively reversed the inhibitory effect of LaCl3,EGTA and 2-APB on the protein expression of PAL,C4H,4CL,C3H and COMT.Therefore,Ca2+acted as a upstream signal of NO in the synthesis of phenolic acid in barley seedlings under NaCl stress.9.In the metabolomic analysis of barley seedlings treated with NaCl alone and in combination with 3-MP,cPTIO and LaCl3,various significant different metabolites were identified in pos and neg mode,respectively.Among them,NaCl stress promoted phenolic acid synthesis resulting from the enhancement of phenylpropanoid biosynthesis.glycolysis pathway,pentose phosphate pathway,amino acids metabolism and phytohormone biosynthesis.NaCl-3-MP,NaCl-cPTIO and NaCl-LaCl3 inhibited phenolic acid synthesis resulting from the weakening of phenylpropanoid biosynthesis,glycolysis pathway,pentose phosphate pathway,amino acids metabolism and phytohormone biosynthesis.