| Phenylalanine and its downstream secondary metabolites not only have important physiological functions in plants,but also play important roles in many fields such as human nutrition,medicine,ecology and industry.(Akebia trifoliata(Thunb.)Koidz.)is a traditional genuine medicinal material in China and a new fruit at present.Phenylalanine and its downstream secondary metabolites,flavonoids,are the natural active substances of A A.trifoliata with edible and medicinal value.Therefore,it is of great significance to study the molecular mechanism of phenylalanine synthesis and metabolism in A.trifoliata.In this study,we compared the content of free amino acids,especially phenylalanine,between A.trifoliata and seven bulk fruits such as banana and apple.The phenylalanine and other related quality traits of A.trifoliata were analyzed by population selection system.The phenylalanine-flavonoid anabolic pathway was analyzed by multi-omics(genome,metabolome,transcriptome),and the key gene families regulating this biosynthetic pathway were identified.The structure,evolution,physical and chemical properties of Arogenate Dehydratase(ADT)family were analyzed by biochemical and genetic methods,and their enzymatic properties and biological functions were systematically studied.Key findings were as follows:(1)Comparative analysis of the content of free amino acids in the pulp of A.trifoliata:In order to explore the improvement potential of phenylalanine in A.trifoliata,17 free amino acids in the pulp of randomly selected undomesticated mature fruits of A.trifoliata and 7 domesticated mature bulk fruits such as banana and apple were determined by liquid chromatography.The results showed that: The contents of phenylalanine and essential amino acids in the pulp of A.trifoliate were 57.62 μg/g and 345.78 μg/g,respectively,which were significantly higher than those of the other 7 tested fruits.A primary population composed of 29 lines was further used to study the changes in phenylalanine content,which ranged from 9.29 to 72.62 μg/g.The average content of phenylalanine was27.06 μg/g,which was the highest among the essential amino acids.The content of phenylalanine in A.trifoliata is high and the variation range is large,which has good research significance and improvement prospects.(2)Metabolomics analysis showed that the types and contents of flavonoid compounds in the fruits of A.trifoliata were the most abundant,and the accumulation of phenylalanine and flavonoids was tissue and period specific.Weighted gene co-expression analysis(WGCNA)combined with the transcriptome identified three gene cluster modules that were significantly associated with the accumulation of phenylalanine and flavonoids(r> 0.7);Genomic coliningence analysis showed that the proportion of annotations from genome-wide duplication event(WGD)genes(41.7%)was much higher than that observed in the whole genome(34.5%),and this proportion was higher in the genes with high correlation expression(53.06%).These genes showed obvious tissue and developmental stage expression specificity,and were related to the type and content of flavonoids.These results suggest that the phenylalanine metabolic pathway of A.trifoliata may be enhanced by WGD events and provide a good basis for further studies on the synthesis mechanism of this pathway.(3)Identification of key candidate genes in the phenylalanine synthesis pathway:through gene annotation,homology comparison,expression level and WGCNA analysis,5key regulatory candidate genes(PAT,ADT,ASP5 and 2 GOT2)were identified in the phenylalanine synthesis pathway.Five key genes(CHI,ANS and 3 BZ1)were identified in the downstream metabolic pathway of phenylalanine,which were related to the synthesis of flavonoids(mainly anthocyanins)in peel.At the same time,three key candidate genes(F3H,DFR and LAR)involved in the synthesis of flavonoids mainly flavonols in seeds were also identified.Further analysis showed that PAT,ASP5 and ADT genes were important for the regulation of phenylalanine anabolism,and were the Hub genes of the whole pathway.Since ADT is an important rate-limiting step in the arroic acid branching pathway for phenylalanine biosynthesis,it is also a prerequisite for the existence of the phenylpyruvate branching pathway.Therefore,the structure and function of candidate ADT genes are the focus of research.(4)Structural characteristics and evolutionary origin of Akt ADT gene family: Eight ADT family members(Akt ADT1-8)were identified by bioinformatics methods,with the length of coding sequences ranging from 1032 to 1962 bp.Evolutionarily,5 Akt ADTs(62.5%)were generated by WGD.The Ka/Ks ratio for all Akt ADT orthologous gene pairs was much less than 1,indicating that this gene family has undergone primarily purifying selection.All 26 plant ADT protein sequences that have been functionally characterized were combined to cluster PAC domains that are important for the function of prephenate dehydratase(PDT).The conserved characteristic residues of PAC domains were divided into two groups.Moreover,Akt ADTs has multiple mutations in important conserved characteristic residues,which provides the possibility of new(sub)functionalization of Akt ADTs.(5)Study on the transcription characteristics of Akt ADTs gene: The data of transgenic group and q RT-PCR results showed that Akt ADTs gene showed tissue-and time-specific expression.Sequence alignment revealed that Akt ADT2,Akt ADT5 and Akt ADT8 had two start codons in the open reading frame,which were located at amino acids 133,54 and 177 downstream of the first start codon,respectively.Subsequently,the transcription start sites were predicted using the plant promoter database,and the results showed that only two transcription start sites existed in Akt ADT8,which was further confirmed by q RT-PCR experiments.This lays the foundation for further studies on the function of the Akt ADT family.(6)Functional study and subcellular localization of Akt ADT protein: All of the recombinant Akt ADT proteins expressed and purified in E.coli were tested for PDT and ADT enzyme activity at the same time.The results showed that all of them had ADT enzyme activity,among which Akt ADT1,4,7,8 and s8 also had PDT activity,and Akt ADT4 had the highest PDT activity.The Michaelis constant(Km)of Akt ADT1 was0.43,and the Km of Akt ADT1 was 2.17.Feedback regulation studies showed that Akt ADTs could be inhibited by phenylalanine feedback,with Akt ADT7 being the most sensitive and Akt ADT5 the least sensitive.Yeast complementation assay showed that Akt ADT4,Akt ADT8 and Akt ADTs8 could restore the phenotype of yeast pha2.The subcellular localization results showed that all eight Ak TADTs were localized in chloroplasts,while Akt ADTs8 was located in the cytoplasm.This further confirms the existence of a compensatory synthetic pathway for phenylalanine synthesis via phenylpyruvate in A.trifoliata.(7)Phenotypic analysis of Akt ADTs gene overexpression lines: The overexpression line of Akt ADTs in Arabidopsis was constructed by genetic transformation,and the phenotype of the overexpression line OEAkt ADT5 showed abnormal rosette leaf development,while OEAkt ADT8 showed early flowering phenotype.Subsequently,free phenylalanine and total flavonoids in young leaves of the overexpressed lines were measured.The phenylalanine accumulation level of OEAkt ADT3,OEAkt ADT6 and OEAkt ADT8 was significantly higher than that of the wild type(WT),while OEAkt ADT5 was significantly lower than that of the WT.The total flavonoid content of OEAkt ADT6 and OEAkt ADT8 was significantly higher than that of WT,and OEAkt ADT5 was significantly lower than that of WT.This reconfirmed the function and contribution of Akt ADTs in the regulation of phenylalanine and flavonoid synthesis.These results could not only enrich the theoretical knowledge on the formation and accumulation of metabolites,but also provide data support for the genetic improvement of A.trifoliata. 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