Molecular Mechanism Of The Flavonoid Biosynthesis Regulated By CsMYB60 In Cucumber

Flavonoids are widely distributed in plants kingdom and play important roles in plant growth and development,disease resistance,and so on.They also can protect human from chronic diseases such as cancer,cardiovascular disease,and neurodegenerative diseases.In recent years,flavonoids have received much attention in their exploitation and utilization because of their wide biological activities and pharmacological functions.Our previous results indicate that the pigments in black-spine of cucumber mainly are proanthocyandins and flavonols,and CsMYB60 plays a key role in the regulation of flavonoid biosynthesis.However,in cucumber,the resistance of PAs to phytopathogen and the flavonoid distribution pattern,and the molecular mechanism of flavonoid biosynthesis regulated by CsMYB60 still remain unclear.Therefore,this study aims to analyze the resistance of PAs to cucumber root rot,the flavonoid distribution pattern,and the molecular mechanism of flavonoid biosynthesis regulated by CsMYB60 in cucumber,which can provide theoretical basis,target genes and technical support for the molecular designed breeding to improve cucumber quality and disease resistance.In order to achieve such research goals,a series of experiments have been carried out.The results are as follows:1.It was confirmed by DMACA staining and HPLC that in all tissues of black-spined cucumber,PAs and flavonols were detected and the content of PAs was the highest in spines,but the flavonol types were different in different tissues.PAs were also accumulated in the pulp,pericarp and seeds of white-spined cucumber,but their content was much lower than that of the black-spined cucumber.Flavonols were only detected in the leaves and flowers in white-spined cucumber.2.The 35S:CsMYB60 lines with black spine have more PAs and flavonols in all tissues,which confirmed the key regulation of CsMYB60.The overexpression of CsMYB60 increased the expressions of the structural genes(Cs4CL ? CsCHS ? CsFLS and CsLAR)in the biosynthetic pathway of flavonols and PAs.3.Y1 H showed that CsMYB60 could directly bind the promoters of the CsFLS and CsLAR genes in the biosynthesis pathway of flavonoids to activate their expressions,but not directly bind the promoters of the key enzyme gene Cs4 CL and other structural genes.Transient expression assay in the tobacco leaf showed that CsMYB60 significantly promoted the expressions of CsFLS and CsLAR.The cis-acting elements combined with CsMYB60 on the CsFLS promoter were screened by Y1 H,and 1 motif on the CsFLS promoter and 2 motifs on the CsLAR promoter were obtained.EMSA experiment showed that these three cis-acting elements were indeed the binding sites of CsMYB60.4.CsMYB60 could activate the expression of the key genes,Cs4 CL and CsCHS,but it could not directly bind their promoters,indicating that the activation was indirect.CsMYB60 were used as a bait to screen a cucumber cDNA library for Y2 H and 12 proteins that interacted with CsMYB60 were obtained.Of the 12 proteins,four transcription factors,CsMYC1,CsbHLH42,CsWD40 and CsTATA-box,were screened out several times.We used BiFC and Pull-down experiments to confirm their interactions with CsMYB60.Moreover,the CsMYB60 could directly bind the CsbHLH42 and CsTATA-box promoters to activate their expressions,but indirectly activate the expressions of CsMYC1 and CsWD40.5.Y1 H assays proved that CsMYC1,CsbHLH42,CsWD40,CsTATA-box all could directly bind to the promoter of the key gene Cs4 CL,and that only CsTATA-box protein could directly bind to the promoter of CsCHS.The transient expression experiments in the tobacco leaf or cucumber cotyledons showed that these four proteins could promote the expression of the GUS gene driven by the Cs4 CL promoter to a certain extent.when CsMYB60 was co-transfected with them,respectively,the GUS expression was enhanced.And when the five proteins were co-injected at the same time,the expression of GUS gene was significantly enhanced.However,only the CsTATA-box protein could effectively enhance the expression of the GUS driven by the CsCHS promoter.So,these rusults indicated that through its interacting proteins,CsMYB60 regulate the expression of Cs4 CL and CsCHS which boost the flavonoid biosynthesis in cucumber.In addition,Y2 H,BiFC and Pull-down experiments showed that CsMYC1 could interact with CsWD40,CsbHLH42 and itself,respectively.6.In order to rapidly characterize the biological function of CsMYC1,CsbHLH42,CsWD40,CsTATA-box,the four genes were overexpressed in Arabidopsis,respectively.The accumulation of anthocyanin and PAs was increased in the 35S:CsMYC1 and 35S:CsbHLH42 lines,but not in 35S:CsWD40 and 35S:CsTATA-box lines,compared to wild type.7.PAs could improve the resistance of cucumber to root rot.Catechin could inhibit the growth of cucumber root rot pathogen.Moreover,the extract liquid from cucumber seedlings treated with catechin could inhibit the growth of root rot pathogen,and the positive correlation exists between catechin concentration treated and the inhibition to root rot.The seedlings treated with different concentrations of catechins for 3 days before inoculating root rot showed more resistance to root rot than that treated with water.After inoculating pathogens,the CsMYB60 transgenic lines showed obvious growth vigor comparing with the non-transgenic siblings isolated from the same generation.Moreover,the extract liquid from the transgenic seedlings could effectively inhibit the growth of root rot pathogen.8.To clarify the reason that anthocyanin did not accumulate in cucumber,the sequence and gene expression of ANS,a key enzyme for anthocyanin biosynthesis,were analyzed.The results indicated that CsANS1 had only a low expression in the leaves of cucumber,but not in other tissues.The expressions of CsANS2 and CsANS3 were not detected in all tissues of cucumber.Comparing with the ANS promoters of other species,the promoters of CsANS1,CsANS2 and CsANS3 did not contain G-Box motif,a light response element.Therefore,the expression loss or low expression of the ANS genes was the key factor to explain the lack of anthocyanins in cucumber.In short,this study showed that PAs could enhance the resistance of cucumber to root rot.Besides spines,PAs and flavonols also accumulated in other tissues of black-spined cucumber.CsMYB60 directly activated CsFLS and CsLAR,and directly or indirectly activated CsMYC1,CsWD40,CsbHLH42 and CsTATA-box,and interacted with these four proteins to form complexes to regulate the expression of the key gene Cs4 CL,and CsMYB60 also interacted with CsTATA-box to regulate the expression of CsCHS,thereby regulating the synthesis of PAs and flavonols in cucumber.