Effect Of Exogenous Plant Hormones On Phenolic Content In The Leaves Of Rhus Chinensis

Objective:This study aimed to explore the influence of exogenous plant hormones on the accumulation of R.chinensis secondary metabolites and to seek a way for taking the replace of the traditional way for extracting the phenolic compounds from Chinese galls.If so,the production of phenolic compounds such as the gallic acid,epigallocatechin gallate,epicatechin gallate will not depend on aphid and gallnut,and this will be beneficial to the development of industry related to the production of phenolic compounds in China.This study will provide a reference for research related to plant phenolic metabolism.Moreover,this study researched the antioxidant activity of R.chinensis phenolic content in order to deriving more practical value from R.chinensis.Methods:First of all,the endogenous hormones of R.chinensis leaves and Chinese galls were detected by liquid/mass spectrometry,and the differences in the types and contents of plant hormones between the two samples were compared.Meanwhile,the extraction conditions of polyphenols in leaves of R.chinensis were optimized.The exogenous plant hormones at different concentrations were sprayed on R.chinensis leaves at an appropriate time,and the polyphenol content of these leaves treated by plant hormones was detected.Compared with the control group at the same period,the optimal combination and dose of exogenous hormones were screened with the highest yield of polyphenols as the screening target,and antioxidant comparison was made between the group with the best spraying effect of exogenous plant hormones and the control group.the components were measured by ultra high performance liquid chromatography（HPLC）for analyzing the role of the secondary metabolites in the increase of oxidation resistance of these leaves.Finally,unreferenced transcriptomics was used to analyze the molecular mechanism of exogenous plant hormones increasing the content of secondary metabolites.Results:1.Comparison of Endogenous Plant Hormones in Rhus chinensis Leaves and Chinese gallsThe contents of IAA,TZR and GA4 in the Chinese galls were significantly different from those in the leaves of R.chinensis.The contents of IAA and TZR in the Chinese galls were 12.87±11.51 ng/g,13.66±7.01 ng/g,respectively,which were significantly higher than those in the leaves of R.chinensis,1.72±0.29 ng/g,0.35±0.05 ng/g.However,the content of GA4 in Chinese galls was 0.48±0.6 ng/g and was significantly lower than that in the leaves of R.chinensis（102.31±31.26 ng/g）.2.Optimization of the process for extracting polyphenols from the leaves of R.chinensisThe results showed that the polyphenol yield could reach 88.97 mg/g when the water content of the deep eutectic solvent was 48%,the ultrasonic temperature was 41℃,and the ultrasonic time was 37 min.3.Effects of Exogenous Plant Hormones on Secondary Metabolites and Antioxidant Activity of Leaves of R.chinensisBy detecting the change of polyphenols in the leaves of R.chinensis in a year,it was found that the change of polyphenols in R.chinensis was relatively rapid from June to July,and the content reached a peak of 114.5±0.78 mg/g from July to August.Therefore,exogenous hormones were sprayed on the leaves of R.chinensis in June to stimulate the growth of leaves.The contents of polyphenols and their components in R.chinensis leaves were 62.42±18.07 mg/g,63.71±15.99 mg/g and 54.78±10.23 mg/g,respectively.However,further study found that epigallocatechin gallate,epigallocatechin gallate and phloperidin were significantly different,and the contents of these three medicinal components in R.chinensis leaves were more prominent.A study of 1-year and 3-year salinoides showed that 1-year R.chinensis had higher ethyl gallate and lower rhizopetin content than that of 3-year R.chinensis.IAA,6-BA and GA3 increased significantly the total phenol content in leaves of R.chinensis at concentrations of 50,30 and 30 mg/L,respectively.The highest accumulation of total phenols of R.chinensis leaves separately sprayed IAA,6-BA,and GA3 appeared on 15th,10th and 15th day,respectively,which increased by46.54%,48.48%,and 70.58%,respectively,compared with the control group.Moreover,when leaves were sprayed by IAA+GA3,IAA+6-BA,6-BA+GA3,and IAA+6-BA+GA3,their highest total phenol content occurred on 10th,10th,10th and15th day and increased by 42.27%,52.73%,33.07%and 74.37%,respectively,compared with the control group.The IC₅₀of DPPH and ABTS in the hormone stimulated group were 49.70 mg/L and2.15 mg/L,respectively.The IC₅₀of DPPH and ABTS free radical scavenging were79.90 mg/L and 4.77 mg/L,respectively.In addition,the antioxidant capacity of the experimental group was significantly higher than that of the control group.The results showed that the contents of gallic acid,epigallocatechin gallate,epigallocatechin gallate and phloperidin in experimental group were significantly higher than those in control group.4 Transcriptomics analysis of experimental group and control groupA total of 52359 Unigene samples from the trial group（G）and control group（H）were functionally classified by GO database,including 20 functional classifications of cell components.Molecular function has 10 functional categories;There are 26 functional categories of biological processes.A total of 4130 DEGs were annotated to 128pathways using KEGG database,among which carbohydrate metabolism accounted for 15 pathways,followed by amino acid and lipid metabolism with 14 pathways,and other secondary biosynthesis with 12 pathways.Comparison of high-throughput transcriptome sequencing data between group G and group H revealed 12545differentially expressed genes,among which 7379 differentially expressed genes were up-regulated（the expression of differentially expressed genes in the experimental group was higher than that in the control group）and 5166 differentially expressed genes were down-regulated（the expression of differentially expressed genes in the experimental group was lower than that in the control group）.The analysis of secondary metabolic pathways that may be related to antioxidant components showed that the key genes of phenylalanine metabolic pathway,phenylpropane,tyrosine and tryptophan synthesis pathway were significantly different between the two groups.Conclusion:In this study,the significant difference of the hormones between the leaves of R.chinensis and Chinese galls was found by liquid/mass spectrometry.the optimal extraction process was obtained by response surface test for polyphenols extraction.Through single factor experiment,the influencing factors of secondary metabolites in the leaves of R.chinensis were explored.The results showed that the contents of gallic acid,phlorizin and gallocatechin in mixed hormone group were significantly higher than those in the control group,which indicated that appropriate exogenous plant hormones could help to improve the content of secondary metabolites in leaves of R.chinensis and thus improve its antioxidant activity.This may make the idea of"spraying exogenous plant hormones to increase the content of secondary metabolites in leaves of R.chinensis,and directly extract gallic acid from leaves,so as to get rid of the dependence of polyaphid and galls for the acquisition of phenols"come true.This is conducive to promoting the development of the industry related to phenolic compounds in China galls,and will also provide a new idea for the study of plant phenolic metabolism and the improvement of antioxidant activity.Finally,we preliminarily explored the molecular mechanism of exogenous plant hormones affecting plant secondary metabolites,and exogenous mixed plant hormones may regulate phenylpropane synthesis to regulate secondary metabolites synthesis.