| Walnut is rich in rutin,especially in the kernel pellicle,and its content directly affects the accumulation of walnut kernel polyphenols,and plays a key role in the quality of the kernel.Rutin is a functional and active substance to protect human health with its good antioxidant,anti-aging and immune function.Studying the biosynthesis of walnut rutin is of great significance for regulating the metabolism of rutin and related phenols.Previously,the research group cloned the genes of rutin biosynthetic pathway and measured the transcriptome information of the fruit development process in J.sigillata,and explored the metabolism pathway of rutin and other related phenolic substances.Relevant studies have shown that rutin synthesis is regulated by a variety of metabolic enzymes and genes,and is related to development process and organ specificity.However,its main metabolic pathways and key enzymes are not yet clear,so it still needs to be explored from multiple perspectives and levels.In this study,the fruit of walnut（J.sigillata Dode.cv.‘Qianhe 7’）was used as the material,using the existing transcriptome data,through a widely targeted metabolomics techniques for the young fruit periode（20 DAF）,fruit enlargement period（50 DAF）,hard kernel period（80 DAF）,seed filling period（110 DAF）,and mature period（140 DAF）were analyzed by LC-MS/MS.Through the association analysis between transcriptome and metabolome,we can understand the changing rules of compounds from the gene transcription level and the metabolite phenotype.Besides,determine the content of rutin and related metabolites,analyze the expression of related genes and enzyme activity,and further analyze the biosynthetic pathway of rutin during the development of walnut fruit in Juglans sigillata.The main results of this study are as follows:1.A total of 719 metabolites of 23 types were identified from the metabolite analysis of fruit in’Qianhe 7’,among which organic acids and derivatives were the most,109 in total,and procyanidins and steroids were the least,with only 3.The highest content in the fruit is lipids,accounting for 20.62%of the total metabolite content,followed by organic acids and derivatives（11.33%）,amino acids and derivatives（10.92%）,flavonols（8.40%）,Sugar（6.05%）.Have 167polyphenols and flavonoids were detected in the walnut fruit,which the procyanidin B2 content was the highest,accounting for 11.39%of the total metabolite content,followed by myricetin3-O-galactoside（6.45%）,and luteolin O-eudesmic acid-O-hexoside（0.05?）is the lowest.2.During fruit development,the metabolites of green husk were most significantly reduced from the young fruit period（20 DAF）to the fruit enlargement period（50 DAF）,72.35%of the358 different metabolites decreased significantly,followed by the fruit enlargement period（50DAF）to the hard kernel period（80 DAF）,62.41%of the 133 different species metabolites were down-regulated.And the content of metabolites increased overall in the kernel filling period（110DAF）and mature period（140 DAF）,among the 117 and 178 different components in the two stages,the content of metabolites increased by 58.12%and 52.25%,respectively.The contents of226 different metabolites were significantly decreased from hard kernel period（80 DAF）to kernel filling period（110 DAF）,while the contents of 240 different metabolites were significantly increased（65.00%）from kernel filling period（110 DAF）to mature period（140 DAF）.In the mature period（140 DAF）,there were the largest differences in metabolite types and contents between green husk and pellicle,with a total of 500 different components,among which 221 were up-regulated（44.20%）and 279 were down-regulated（55.80%）.3.Phenolic acid and flavonoids in coumarin-CoA and caffeoyl-CoA pathways were detected in the metabolome analysis of walnut fruit,indicating that both pathways can synthesize secondary metabolites such as polyphenols and flavonoids.KEGG pathway analysis found that caffeoyl-CoA focuses on synthesizing coniferyl alcohol and sinapyl alcohol into lignin metabolism,while flavonol substances such as rutin are mainly biosynthesized by coumarin-CoA pathway.Correlation analysis showed that,rutin was significant or extremely significant positive correlation with L-phenylalanine,trans-cinnamate,naringenin chalcone,naringenin,dihydrokaempferol,kaempferol,quercetin,and isotrifoliin in green husk.In addition,rutin was significant and extremely significantl negative correlate with p-coumaric acid and kaempferol in the kernel pellicle,while it is significant or extremely significant positive correlation with trans-cinnamic acid,naringenin chalcone,naringenin,dihydrokaempferol,quercetin and isotrifoliin.It shows that these metabolites are closely related to the synthesis of rutin,and the level of precursor metabolites may determine the final accumulation of rutin.4.Transcriptome and metabolome association analysis screened 19 kinds of rutin-related synthase,including phenylpropanoid biosynthesis（Ko00940）:PAL,C4H,4CL,CAD,C3’H,COMT,CCoAOMT,β-Glucosidase,POD;Flavonoid biosynthesis（Ko00941）:CHS,CHI,FLS,F3H,DFR,LAR;Flavonol biosynthesis（Ko00944）:FOMT,F3`H,F3`5`H,UGT.The related gene expression and enzyme activity in the three pathways are closely related to the content of polyphenols and flavonoids,especially the content of phenols in the kernel pellicle increases significantly with the up-regulate of related gene expression and enzyme activity.5.In green husk,the rutin content in the young fruit period（20 DAF）to the hard kernel period（80 DAF）decreased significantly,the kernel filling period（110 DAF）increased slightly,and the the content decreases again in mature period（140 DAF）;In the kernel pellicle,rutin gradually accumulates from the hard kernel period（80 DAF）to the maturity period（140 DAF）,and its content in the maturity period is significantly higher than that in other stages.At this time,the content of rutin in the kernel peillicle is much higher than that in green husk.Correlation analysis of gene expression and enzyme activity found that rutin was mostly negatively correlated with PAL,C4H,4CL,COMT,CCoAOMT,CAD and other upstream gene expression levels,but was related to FLS,FOMT,F3`H,F3`5`H,UGT gene expression and enzyme activity showed significant or extremely significant positive correlation.The expression of genes in green husk was weakly correlated with enzyme activity,while the expression of genes in kernel pellicle was strongly correlated with enzyme activity.Especially the flavonol biosynthesis pathway related synthases in latter such as FLS,FOMT,F3’H,F3’5’H,UGT were significantly positively correlated with rutin,indicating that the enrichment of rutin in kernel pellicle was the result of specific expression of genes related to flavonol synthesis. |
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