Metabolic Regulation Mechanism Of Flavonoids In Pigeon Pea And Their Biological Functions Against Salt Stress

Flavonoids,as a multi-functional plant secondary metabolite,play an important role in resisting salt stress.In this study,the response of flavonoids to salt stress and the distribution of different tissues in pigeon pea were explored.The structural genes of flavonoid biosynthesis pathway and MYB transcription factors were screened.Structural genes and MYB transcription factors were identified to be involved in flavonoid biosynthesis and resistance to salt stress.The application potential of elicitors in the biosynthesis of flavonoids and their resistance to salt stress was explored.The main conclusions are as follows:（1）The characteristics of flavonoids in response to salt stress and the distribution rules in different tissues were clarified.The contents of apigenin,genistin,and genistein were increase under salt stress,indicating that flavonoids played an important role in the response to salt stress.Flavonoids are mainly biosynthesized and stored in roots,stems,and leaves of pigeon pea.Flavanones（pinostrobin,naringenin）accumulated in roots,stems,and leaves;flavones（isovitexin,isorhamnetin,orientin,vitexin,luteolin,apigenin）are mainly distributed in leaves;isoflavones（genistin,genistein,biochanin A,formononetin）accumulated primarily on roots.（2）Identification and expression pattern analysis of structural genes and Cc MYB transcription factor family from flavonoid pathway.65 structural genes and 30 Cc MYB transcription factors were identified that may be involved in regulating flavonoid biosynthesis metabolism in pigeon peas by genome-wide analysis.Most structural genes and Cc MYB genes from the flavonoid pathway were located on chromosomes 3 and 11.The MYB binding sites were detected in the promoters of structural genes.Various cis-acting elements,including stress,development,and hormone-responsive elements,were detected in the promoter regions of Cc MYB genes.It indicated that the structural genes of the flavonoid biosynthesis pathway might be regulated by complex networks,such as MYB transcription factors and hormones.The gene expression patterns showed that the expression levels of candidate genes were higher in roots,stems and leaves,which was highly consistent with the distribution of flavonoids.（3）The biological function of Cc PAL in flavonoid metabolism and resistance to salt stress was revealed.The Cc PAL3 gene is highly conserved in species evolution,and Cc PAL5 is a specific differentiation gene in pigeon pea.Transgenic pigeon pea lines with transient overexpression of Cc PAL3 and Cc PAL5 genes were constructed.Quantitative analysis of flavonoid content in transgenic plants confirmed that Cc PAL3 and Cc PAL5 genes promote the biosynthesis of flavonoids,such as flavanones,flavones and anthocyanins.Phenotypic observations showed that the growth of transgenic pigeon pea was inhibited under salt stress,the content of MDA and H₂O₂ was increased,the content of proline and the activity of antioxidant enzymes was decreased,the accumulation of isoflavones and the transcription level of isoflavone synthase gene were down-regulated.The results indicated that isoflavones may be important substances that Cc PAL3 and Cc PAL5 mediate the sensitivity of pigeon pea to salt stress.（4）The biological function of Cc IFS in flavonoid metabolism and resistance to salt stress was revealed.Two members of the Cc IFS family were identified by bioinformatics.The accumulation of isoflavone was quantitatively analyzed in Cc IFS1 and Cc IFS2 overexpressed transgenic pigeon pea.The results showed that Cc IFS1 and Cc IFS2 promote the accumulation of isoflavones,including genistin,genistein,biochanin A,and formononetin.The growth condition of transgenic pigeon pea was better than wild-type under salt stress.The content of isoflavones in transgenic pigeon pea was increased,while the contents of MDA and H₂O₂ were decreased,and the content of proline and the activity of antioxidant enzymes were increased.It clearly showed that Cc IFS1 and Cc IFS2 enhanced the resistance to salt stress by increasing the biosynthesis of isoflavones.（5）The biological function of Cc MYB5 in flavonoid metabolism and resistance to salt stress was revealed.Evolutionary analysis showed that Cc MYB5 transcription factors may co-regulate multiple branches of the phenylpropane pathway.The flavonoid content and expression levels of key genes in Cc MYB5 overexpressed pigeon pea were accurately quantified.The results showed that the Cc MYB5 promoted the biosynthesis of flavonoid,including flavanones,flavones,anthocyanins,inhibited the accumulation of isoflavones,and enhanced the sensitivity to salt stress.（6）Application of exogenous Me JA-mediated flavonoid biosynthesis to improve salt tolerance in pigeon pea.Based on the results of transcriptome sequencing,it was shown that the structural genes of the flavonoid pathway and MYB transcription factors were induced by Me JA.The growth of overexpressed Cc MYB5 transgenic pigeon pea was inhibited under salt stress,and the growth-inhibited phenotype was alleviated by the application of Me JA.The content of MDA and H₂O₂ was reduced,the content of proline and the activity of antioxidant enzymes were increased,and the biosynthesis of isoflavones was promoted.It was confirmed that Me JA-mediated Cc MYB5 gene to promote the isoflavones biosynthesis and improve the resistance to salt stress.In addition,the effect of exogenous of Me JA on pigeon pea was comprehensively evaluated.The results showed that the application of exogenous Me JA enhanced the photosynthesis of pigeon pea,protected the integrity of leaf cell membrane,activated the defense system by enhancing the activity of antioxidant enzymes,and significantly promoted the biosynthesis of natural compounds.It shows that the application of Me JA has outstanding potential in improving the flavonoid biosynthesis pathway and againist salt stress.In summary,this study clarified the distribution of flavonoids in different tissues of pigeon pea.The biological functions of Cc PAL and Cc IFS genes in flavonoid biosynthesis and resistance to salt stress were explored.The results clarified that isoflavones play a role in improving salt stress tolerance.The application potential of the exogenous Me JA-mediated Cc MYB5 gene to promote isoflavone biosynthesis and improve salt stress resistance was comprehensively evaluated.This study provides a theoretical basis and technical support for using molecular breeding techniques to cultivate high-quality varieties with multifunctional components and strong resistance.