Collaborative Expression Mechanism Between Shikimate Pathway And Flavonoid Metabolism

Shikimate pathway links with primary metabolism and the secondary metabolism,which presents widely in all kinds of higher plants.An important one of the brunches after chorismate acid is the flavonoid metabolism lead by Chorismate Mutase.Flavonoid metabolites mainly consist of anthocyanins,flavanols and flavonols.All these compounds deter the color and mouthfeel of grape as well as wine.Therefore,the metabolism of flavonoid metabolites has been widely researched recently.Previous studies suggest that there was a coordinate expression between the transcription levels of genes belonging to shikimate pathway and flavonoid metabolism.However,the mechanism of this coordinate hasn’t been clear yet.In this work,the HPLC-MS and Real-time PCR analysises were performed in the determinations of metabolites accumulation and genes expression,which were from the shikimate-flavonoid metabolism,under two different cultivations:open-field cultivation and rain-shelter cultivation.After screening the co-expression genes,we cloned the key enzymes of shikimate pathway-VvDAHPS and VvCM,and then analyzed their biochemical properties.Besides,the interactions between transcription factors(VvMYBPA1 and VvMYB5b,members from MYB family)and the promoters of VvDAHPS and VvCM were verified.The purpose of all the works was to clarify the mechanism of the coordinate expression between shikimate pathway and flavonoid metabolism through three levels:metabolites accumulation,protein regulation and genes transcription.The main results were as follows:First of all,in both 2012 and 2013,rain-shelter cultivation could alter the microclimate around fruits zones and canopies.However,these changes didn’t decrease the quality of wine grape fuites.In 2012,at commercial harvest,the concentration of anthocyanins under rain-shelter cultivation was signicantly higher than that under open-field.Nevertheless,the result in 2013 was just opposite.Compared with the meteorological data provided by regional weather station and microclimate monitor,it suggested that the biosynthesis and accumulation of anthocyanins were stongly affected by vintage and weather.However,the rain-shelter cultivation could efficiently drop of the effect made by climate constant changes.Secondly,rain-shelter cultivation significantly affected the composition and proportion of different phenolic compounds,while altered little on the total concentration of flavanols and flavonols.The flow of carbon was clearly affected.The rain-shelter cultivation accelerated the methylation of flavanols and flavonols,meanwhile against the methylation of anthocyanins.Moreover,there was a remarkable positive correlation between the transcription expression of genes coding the enzyme of VvDAHPS1 and VvMYB5b,and the corresponding metabolites,such as anthocyanins and flavonols.Besides,the expression of VvCMl was related to more products.On this basis,we cloned the open reading frames of VvDAHPS1,VvDAHPS2,VvCMl and VvCM2.The recombinant plasmids were constructed and fusion proteins were purified.The enzymetic analysis in vitro suggest that VvDAHPS2 was activated by Mn2+.The activity of VvDAHPS1,VvDAHPS2 and VvCM1 was not adjusted by feedback inhibition by aromatic amino acid.However,VvCM2 was promoted by tryptophan.Further more,we cloned and analysed the transcriptional activity of promoters of VvDAHPS2,VvCM1 and VvCM2.Shading could increase the activity of promoter of VvDAHPS2,while inhibited the activities of VvCM1 and VvCM2 promoters.The MYB transcription factors VvMYBPA1 and VvMYB5b,which were reported strongly regulate the flavonoid metabolism downstream,showed a significant up-regulation on the promoters of VvDAHPS2,VvCMl and VvCM2.The interaction explained the synergistic effect between shikimate pathway and flavonoid metabolism,both on metabolites accumulation and genes transcription levels.This study provided an addtional insight on the mechanism of coordinate expression between shikimate pathway and flavonoid metabolism.It also provides reliable theoretical foundations to cultivation and management during viticulture process.Moreover,it will serve as a useful reference for future investigations including the exploration of those environmental factors that regulate secondary metabolism on the levels of transcription and metabolite in grape berries.