The Regulation Mechanism Of UV-B Stress And Ca²⁺ Treatment On Phenolic Acid Accumulation In Wheat Seedlings

Wheat is an important cereal crop.In addition to being rich in nutrients such as starch,protein and minerals,wheat is also rich in dietary fiber and bioactives substances.As an important class of secondary metabolites,phenolic acids contain phenolic hydroxyl group and carboxyl group,and they are mainly present in bound form in wheat.Phenolic acids have anti-oxidation,anti-cancer,anti-microbial and other physiological effects.After germination,wheat grains can effectively promote the accumulation of phenolic compounds and increase the antioxidant activity.UV-B radiation is an electromagnetic radiation with a wavelength between 280 and 320 nm.As a kind of stress,UV-B radiation can effectively induce the formation of phenolic compounds in plants and cause morphological changes and a series of physiological reactions.As an essential nutrient and the second messenger for plant growth and development,Ca2+can couple extracellular signals and intracellular physiological signals.It has very important physiological functions in maintaining cell morphology,regulating ion balance and osmotic pressure,and it can regulate plant growth and respond to various abiotic stresses.In the present study,the regulation mechanism of UV-B stress and Ca2+treatment on phenolic acid accumulation in wheat seedlings was explored from physiological and biochemical level,enzyme level and gene level.The main results are as follows:1.The changes of phenolic content,phenolic acid composition and content,antioxidant capacity,and relevant enzyme activity during wheat germination were studied.Results showed that free and bound phenolic content significantly increased in germinated wheat and contributed to enhanced antioxidant capacity.Sumai188 showed the highest total phenolic content,total DPPH·scavenging activity and total ABTS+·scavenging activity after 4-day germination,followed by Luyuan502 and Jimai22.Ferulic acid and p-coumaric acid were most abundant in wheat seedlings and mainly existed in the bound form.In the three wheat seeds of Jimai22,Sumai188 and Luyuan502,the ratios of bound to free ferulic acid content were 218:1,423:1 and 268:1,respectively.Compared to the bound ferulic acid content in wheat seeds,bound ferulic acid increased by 1.8,1.3 and 1.4 times for Jimai22,Sumai188 and Luyuan502 after germination for four days,respectively.Meanwhile,the activities of phenylalanine ammonia-lyase,peroxidase,catalase and ascorbate peroxidase were also significantly activated during wheat germination.Correlation analysis suggested that PAL activity had high significant positive correlation with TPC,with correlation coefficients of 0.999,0.884 and 0.949 for Jimai22,Sumai188 and Luyuan502,respectively.In addition,Multiple linear regression analysis(MLRA)and principal component analysis(PCA)indicated that there were highly positive correlation among total phenolic content,phenolic acid content and antioxidant activity,and ferulic acid contributed the most to the bound phenolic content,bound DPPH·and ABTS+·scavenging activities in wheat seedling.2.The effects of UV-B stress on the phenolic content,phenolic acid content,antioxidant activity,and physiological changes in wheat seedlings were investigated.Results showed that the free,bound and total phenolic content and antioxidant activities significantly increased in wheat seedlings after UV-B exposure from the third and fourth day.UV-B radiation of 20 ?W/cm2 led to the highest accumulation of phenolics.Total phenolic content,DPPH value and ABTS value significantly increased by 26.3%,25.1%and 12.0%,respectively,on day 4 as compared to unirradiated wheat seedlings.UV-B radiation significantly increased the content of bound p-coumaric acid and ferulic acid in wheat seedlings.The highest respiratory rate and phenylalanine ammonia-lyase activity were also observed with the radiation.The increased UV-B radiation intensified the lipid peroxidation in wheat seedlings.At a radiation intensity of 30 ?W/cm2,the MDA content in wheat seedlings increased by 40.2%as compared to the control.The activities of superoxide dismutase,peroxidase and catalase decreased,while the ascorbate peroxidase activity increased with the enhanced UV-B radiation intensity.This study showed that UV-B stress could increase the phenolic content,phenolic acid content and antioxidant activity in wheat seedlings.3.The effects of exogenous Ca2+ and Ca2+ channel inhibitor LaCl3 on Ca2+transmembrane behavior and physiological metabolism,phenolic acid formation,phenylpropanoid metabolic pathway-related enzyme activities,gene expression and endogenous hormones were studied.Results showed that exogenous Ca2+ promoted the growth and respiration of wheat seedlings,while LaCl3 adversely affected the growth and respiration of wheat seedlings.After 3-day and 4-day germination,exogenous Ca2+significantly increased the free phenolic content in wheat seedlings,which were 13.6%and 16.0%higher than that of the control,respectively.After 4-day germination,bound phenolic content in wheat seedlings were 1.25 and 1.56 times as compared to the control and LaCl3 treatment,respectively.Exogenous Ca2+ had an effect on the bound p-hydroxybenzoic acid,vanillic acid,syringic acid,p-coumaric acid,ferulic acid and sinapic acid in wheat seedlings.After 4-day germination,the p-coumaric acid content in wheat seedlings treated with exogenous Ca2+ increased by 31.1%compared with the control,and exogenous Ca2+ treatment had the highest ferulic acid content,which were 1.17 and 1.48 times as compared to the control and LaCl3 treatment,respectively.Ca2+ significantly reversed the inhibitory effect of ferulic acid synthesis caused by LaCl3.Exogenous Ca2+activated the PAL,C4H and 4CL activities and upregulated PAL1,C3H and COMT1 expression.LaCl3 significantly inhibited the key enzyme activities and gene expression of phenylpropanoid metabolic pathway,leading to the inhibition of the synthesis of phenolic compounds and phenolic acids.After 4-day germination,exogenous Ca2+ significantly increased the CaM content in wheat seedlings,which were 1.44 and 1.79 times as compared to the control and LaCl3 treatment,respectively,and there was a high positive correlation with the total phenolic content(r=0.929).Exogenous Ca2+increased the content of endogenous GA3,IAA and SA in wheat seedlings,and the content was highly positive correlation with total phenolic content and ferulic acid content.4.Under the UV-B stress,the effects of exogenous Ca2+on the physiological indexes and phenolic acid metabolism of wheat seedlings were studied.Results showed that exogenous Ca2+significantly alleviated membrane lipid peroxidation of wheat seedlings induced by UV-B stress and restored MDA content to control level.The increase of proline content in wheat seedlings under exogenous Ca2+treatment mainly occurred after 3-day germination,while UV-B radiation treatment occurred after 4-day germination.After 4-day germination,the total phenolic content of wheat seedlings under UV-B stress combined with Ca2+treatment increased by 30.9%and 10.3%,respectively,compared with the control and UV-B radiation treatment.Exogenous Ca2+effectively increased the bound ferulic acid,p-coumaric acid and vanillic acid content with ultraviolet absorption properties in wheat seedlings under UV-B stress,while LaCl3 significantly decreased the bound ferulic acid,p-coumaric acid,p-hydroxybenzoic acid,vanillic acid and syringic acid content in wheat seedlings.Under UV-B stress,exogenous Ca2+significantly upregulated the relative expression of PAL1,C4H,C3H and COMT2 in the early stage of wheat germination,while LaCl3 significantly inhibited the expression of key genes in phenolic acid synthesis in phenylpropanoid metabolic pathway.Under UV-B stress,exogenous Ca2+increased reactive oxygen scavenging efficiency by activating the antioxidant enzyme system in advance,and alleviated the damage of reactive oxygen species in wheat seedlings.Exogenous Ca2+ significantly increased the CaM content in wheat seedlings and promoted the activation of Ca2+/CaM signaling system in wheat seedlings under UV-B stress.Exogenous Ca2+ was also involved in the regulation of different types of hormone levels in wheat seedlings under UV-B stress.5.Exogenous Ca2+ activated the key enzyme activities of phenylpropanoid metabolic pathway and upregulated the expression of PAL,C4H,C3H and COMT,induced the increase of CaM content,and activated Ca2+/CaM signaling system,which significantly increased the phenolic content and phenolic acid content in wheat seedlings under UV-B stress and non-stress.However,Ca2+ channel inhibitor LaCl3 inhibited this effect.In addition,UV-B stress and Ca2+ have synergistic effects on phenolic acid accumulation in wheat seedlings,and Ca2+ could alleviate the adverse damage caused by UV-B stress on wheat seedlings by regulating a series of physiological and biochemical changes.Ca2+improved the resistance of wheat seedlings to UV-B radiation.