Mechanism Of Brassinosteroid On Regulation Flavonoid Biosynthesis In Red-fleshed Apple

Flavonoid content is an important indicator of nutritional value in fruits and vegetables,which directly determines the marketability of many horticultural crops.Therefore,the breeding and cultivation of horticultural crops with high flavonoid content has gradually become an important field of plant research.As a precious wild germplasm resource,Xinjiang red-fleshed apple(Malus sieversii f.niedzwetzkyana(Dieck)Langenf)is rich in antioxidants such as flavonoids and polyphenols,which have high ornamental value and rich medical and health care functions.In 2016,our research group took the lead in constructing a hybrid group of cultivated varieties such as Xinjiang red-fleshed apples and Fuji,focusing on the research on the red development mechanism of high-flavonoid apples.Flavonoids biosynthesis is affected by many external factors including light,temperature,sugar and hormones.In agricultural production,brassinolide(BR),as a new type of plant hormone,plays an important role in regulating fruit ripening.However,the molecular mechanism of BR in regulating fruit coloring and flavonoid synthesis is still unclear.The BRASSINAZOLE RESISTANT family protein(BZR)is a class of b HLH transcription factors,which play a key role in the regulation of plant response by BR signals.In Arabidopsis,BZR proteins include BZR1,BZR2/BES1 and four homologous family proteins(BEH1,BEH2,BEH3 and BEH4).Among them,BZR1 and BZR2/BES1 are hotspot transcription factors,and their functions in light morphogenesis,adversity stress,and secondary metabolism have been discussed in depth.However,the functions of BEH transcription factors,especially in the regulation of flavonoid biosynthesis,remain unclear.In order to further explore the potential regulation mechanism of BR signaling and BZR transcription factors in apple fruit development,this study used the hybrid progeny of Xinjiang red-fleshed apple as test materials,screened by differential gene expression,and analyzed the BZR transcription factors in apples through systematic evolution.In-depth discussions on its function and regulation mechanism are carried out,and the main results are as follows:1.Using the c DNA of the red-fleshed apples with different degrees of coloring as templates,fluorescence quantitative expression analysis was performed on 9members of the BZR transcription factor family in apples.RT-q PCR results proved that the expression level of MdBEH2.2 gradually decreased with the increase of flavonoid content.Therefore,we targeted the candidate gene MdBEH2.2 and further verified its function.2.MdBEH2.2 was overexpressed in red-fleshed apple seedlings and then cultured under conditions containing BR synthesis inhibitor Brz.The results showed that overexpression of MdBEH2.2 caused the redness of apple seedling leaves to fade.Compared with the wild type,the content of flavonoids,anthocyanins and procyanidins in the transgenic MdBEH2.2 seedlings was reduced.Correspondingly,q RT-PCR detection revealed that the expression levels of flavonoid synthesis structural genes(MdANR,MdFLS,MdLAR and MdANS)and transcription factor genes(MdMYB9 and MdMYB11)were significantly down-regulated.In addition,the phosphorylated form of MdBEH2.2 protein decreased after Brz treatment,and the inhibitory effect of overexpression of MdBEH2.2 on the synthesis of flavonoids in apple seedlings was weakened.Therefore,MdBEH2.2 responds to BR signals and negatively regulates the synthesis of flavonoids.3.Yeast one-hybrid and Ch IP experiments verified that MdBEH2.2 can specifically recognize and bind the promoters of downstream structural genes MdFLS,MdLAR,MdMYB9 and MdMYB11 in vivo.It was further verified by EMSA test that MdBEH2.2 can bind BRRE elements and E-box elements on their promoters respectively.4.The yeast two-hybrid experiment,pull-down and bimolecular fluorescence complementation experiments proved that MdBEH2.2 can interact with MdMYB60 in vivo and in vitro.According to the analysis of the protein structure of MdBEH2.2,we found that the conserved domain of MdBEH2.2 protein containing b HLH,Phospho and PEST structures is an essential part of the interaction with MdMYB60.5.MdMYB60 was overexpressed in red-fleshed apple callus,and then the transgenic line was cultured under Brz conditions.The results showed that the phenotype of the callus overexpressing MdMYB60 changed from red to yellow.Compared with the wild type control,the content of flavonoids,anthocyanins and procyanidins in the transgenic lines were reduced.Correspondingly,the q RT-PCR test indicated that the expression levels of flavonoid synthesis structural genes(MdANR,MdFLS,MdLAR,MdUFGT and MdANS)were significantly down-regulated.In addition,the protein abundance of MdMYB60 increased after Brz treatment,and the inhibitory effect of overexpression of MdMYB60 on the synthesis of flavonoids in apple seedlings was weakened.Therefore,MdMYB60 participates in BR's inhibition of the biosynthesis of flavonoids in red-fleshed apple.6.Yeast two-hybrid experiment and Pull down experiment proved that MdMYB60 cannot interact with Mdb HLH3 and Mdb HLH33 to form a complex in vivo and in vitro.The yeast one-hybrid and Ch IP test verified that MdMYB60 can specifically recognize and bind the promoters of the downstream structural genes MdANS and MdANR in vivo.The EMSA test further verified that MdMYB60 can bind to MYB recognition elements on their promoters respectively.7.Because both MdBEH2.2 and MdMYB60 can independently act as regulators of the upstream flavonoid synthesis pathway,we further examined the effect of the interaction between the two on downstream gene transcription.The EMSA binding test proved that the MdBEH2.2-MdMYB60 complex enhanced the binding of MdBEH2.2 and MdMYB60 to the downstream target gene promoter.In addition,the LUC fluorescence report experiment further verified this result.In addition,the Co-IP test proved that enhancing the signal transmission of BR enhances the stability of the MdBEH2.2-MdMYB60 complex.This study shows that BR signaling inhibits the biosynthesis of apple flavonoids through the MdBEH2.2-MdMYB60 regulatory module.Our findings provide a theoretical basis for elucidating the influence of BR signaling on the secondary metabolism of horticultural plants.