Mechanism Of MdHY5-MdWRKY41-MdMYBs Transcription Factor Cascade Regulates The Biosynthesis Of Anthocyanins And Procyanidins In Red-fleshed Apple

Apple is a worldwide fruit,and its related industries are also an important part of the development of the global agricultural economy.The bright color and rich flavonoid content of apple fruit are of positive significance for improving its commodity value and human health.In recent years,red-fleshed apple has been widely sought after and welcomed due to their attractive color and rich anthocyanin content.The biosynthesis of flavonoids such as anthocyanins is regulated by many factors,such as light,temperature,nutrition,Osmotic pressure,etc.;internal factors such as plant hormones,structural genes and transcriptional regulators.Although MdMYB10,MdMYB11 and their homologues have been identified as key regulators of the fruit coloring process,the mechanisms by which other transcription factors（TFs）regulate the color formation of the fruit need to be further studied.In this study,we used the red callus induced by the fruits of the F2 generation of the Xinjiang red-fleshed apple hybrid and the leaves of the high flavonoid strain"Zihong 3"as experimental materials,Studied the effect of MdWRKY41 on the synthesis of anthocyanins and proanthocyanidins in red-fleshed apple.The overexpression of MdWRKY41 in red apple callus inhibits the accumulation of anthocyanins and PA by down-regulating the expression of a MYB gene（MdMYB12）and specific structural genes（MdLAR,MdUFGT and MdANR）.In addition,MdWRKY41 also interacts with the anthocyanin synthesis inhibitor MdMYB16 to form a protein complex,which further inhibits the expression of MdANR and MdUFGT.What is interesting is that the transcription of MdWRKY41 is targeted to be inhibited by the light-responsive factor MdHY5.In summary,this study provides a new MdHY5-MdWRKY41-MdMYBs regulatory module that affects the synthesis of anthocyanins and PA in red-fleshed apple.The specific research results are as follows:1.Fluorescence quantitative polymerase chain reaction analysis was performed using c DNA isolated from red-fleshed apple fruits（R1-R5）with different flesh colors.It was found that the transcription level of MdWRKY41 gradually decreased with the increase of anthocyanin and PA content.It indicates that MdWRKY41 may have a negative regulatory effect on apple fruit coloring.Protein sequence analysis showed that MdWRKY41 contains a conserved WRKY domain（137-143aa）and a C-X₇-C-X₂₃-H-X-C zinc finger-like motif（157-191aa）at the nitrogen end.2.Introduce 35S::MdWRKY41 into the callus of red apple to produce transgenic callus overexpressing MdWRKY41.It was found that compared with the wild-type control,the transgenic callus overexpressing MdWRKY41 accumulated less anthocyanins and PA,and the transcription levels of the two biosynthesis-related genes（MdUFGT,MdANR,MdLAR and MdMYB12）were also reduced accordingly.These results indicate that MdWRKY41 inhibits the accumulation of anthocyanins and PA in red apple callus.3.The yeast monohybrid（Y1H）and EMSA gel blocking experiments verified that MdWRKY41 specifically binds to the target gene promoter.Y1H analysis showed that MdWRKY41 can bind to the promoters of anthocyanin and PA synthesis related genes（MdUFGT,MdANR and MdMYB12）in the cell.In addition,we detected different numbers of W-boxes in the promoter sequences of MdANR,MdUFGT and MdMYB12.EMSA experiments verified that MdWRKY41 binds to the promoters of MdANR,MdUFGT and MdMYB12 in vitro through W-box.4.Using Yeast two-hybrid（Y2H）,protein pull-down and Bimolecular Fluorescence Complementation（Bi FC）experiments to verify the interaction of MdWRKY41 and anthocyanin synthesis inhibitor MdMYB16 in cells and in vitro,further LUC reporter gene experiments proved that MdMYB16 enhances the inhibitory effect of MdWRKY41 on the transcription of MdUFGT and MdANR.5.The transcriptional expression of MdWRKY41 is inhibited by the light signal mediated by MdHY5.The light signal cis-acting element G-box was detected in the promoter of MdWRKY41,which was subsequently proved by yeast monohybrid,EMSA gel block and LUC reporter gene experiments.MdHY5 binds to the promoter of MdWRKY41 via G-box and inhibits its activity.This study proved that MdWRKY41 negatively regulates the accumulation of anthocyanins and PA by inhibiting the transcription of MdUFGT and MdANR,and MdMYB16 interacts with it to enhance this inhibition;MdWRKY41 also indirectly inhibits the transcription of MdLAR by directly inhibiting the expression of MdMYB12,thereby Reduce the synthesis and accumulation of procyanidins;And the expression of MdWRKY41 is also inhibited by the light signal mediated by MdHY5.Our research provides new insights into the role of WRKY transcription factors in regulating the accumulation of red-fleshed apple anthocyanins and proanthocyanidins.