Functional Identification Of Wheat Phenolamine Acyltransferase

Phenamine is a secondary metabolite in plants.Phenolamines are mostly formed by enzymes of the BAHD family,widely present in plants such as Poaceae and Solanaceae,and can resist biotic stress.At present,there are few studies on BAHD enzymes in wheat,and only three genes(TaACT1-1/1-2/2)have been confirmed to be involved in the synthesis of phenolamines.We used a population of 368 natural wheat varieties to detect the contents of 9phenolamines in leaves at the two-leaf and one-heart stage by liquid chromatography-mass spectrometry(LC-MS).Eight candidate genes encoding BAHD acyltransferases were screened out by metabolic genome-wide association analysis(mGWAS),gene annotation and homology alignment.Among them,TaACT1-1/1-2/2on chromosome 2D has been shown to be able to catalyze agmatine acylation.The other 5 genes were located on chromosomes 2A and 2B,which were named TaACT3-1/3-2 and TaACT4-1/4-2/4-3 by evolutionary analysis,respectively.Through haplotype analysis and specific amplification,we found that multiple non-synonymous variant sites in the CDS regions of TaACT3-2 and TaACT4-1 were significantly correlated with changes in the content of sinapoyl agmatine(Sin-Agm).In vitro enzymatic assay verified that 5 genes in addition to the ability to acylate agmatine,some genes also have weak activity on putrescine or tyramine.In order to further enrich the synthesis pathway of wheat phenolamines,we identified genes that may catalyze the acylation of putrescine,tryptamine,tyramine and serotonin in wheat through genome annotation and homology alignment,and named them TaPHT1 and TaTHT1.The vitro enzymatic assay proved that TaPHT1 has relatively strong activity on putrescine.TaTHT1 has the strongest relative activity on serotonin.To verify that the candidate genes can resist biotic stress by producing these phenolamines in wheat,such as resistance to Fusarium graminearum,we added0.5m M acylated agmatine and serotonin to the medium,and observed that the growth of Fusarium was significantly inhibited.This study enriched the functional study of wheat BAHD acyltransferase,revealing the process of wheat phenolamine accumulation,and the inhibitory effect of phenamine on pathogenic bacteria,which is for the analysis of phenotypic traits and metabolites.The genetic basis provides reference and theoretical support for the research on wheat stress resistance breeding.