The Molecular Mechanism Underlying Anthocyanin Biosynthesis In Apple Using BHLH33 And MYB Repressors

Anthocyanin is flavonoids-compound formed by anthocyanidin and sugars with glycosidic bonds.In plant,it is an important secondary metabolite,mainly distributed in vacuoles of plant seed,fruit,flower,leave and other organ and tissue.There are high contents of anthocyanins in grape,apple,strawberrie,red pear,mangosteen and other mature fruit,which is an important factor in fruit color.Anthocyanin not only make fruit rich in color,but also play an important role in preventing cardiovascular disease,reducing obesity and improving blood sugar balance.Anthocyanin biosynthesis is regulated by various transcription factors and structural genes,as well as environmental factors.Thes ANS and UFGT of structural genes and the MYB-bHLH-WD40 complex involved in regulating structural genes play a key role in anthocyanin biosynthesis.At present,homologous genes of AtMYB75 related to anthocyanin biosynthesis in Arabidopsis thaliana have been found in pear(PyMYB10),peach(PpMYB10),grapes(VvMYBA1 and VvMYBA2),strawberry(FaMYB10)and apple(MdMYB1).In red-fleshed apple,MdMYB10 can bind its own promoter to enhance the regulation of anthocyanin,in addition,bHLH3/33 can participate in the anthocyanin accumulation involved of low temperature.However,the mechanism of MYB repressor and bHLH protein that participated in anthocyanin biosynthesis is unclear.Therefore,‘Hongcui ‘1-5' apples of Malus sieversii f.Neidzwetzkyana F1 population are selected as test materials,and the flesh of ‘Hongcui ‘1-5' apples are red to varying degrees.We conduct a series of studies including the analyses of anthocyanin content and expressions of MYB repressors and MdbHLH33 in fruit development and mature period,the interaction between MYB repressors and MdbHLH33,and identification of downstream target genes of MYB repressors.The main results are as follow:1.The anthocyanin content and the expressions of MdMYB9,MdMYB10,MdMYB11,MdMYBPA1,MdbHLH3 and MdbHLH33 are gradually decreasing of ‘Hongcui 1' apple in fruit development period(60-130d),while the expressions of MdMYB16 and MdMYB6 are gradually increasing.The anyhocyanin content in ‘Hongcui 3,4' are higher than that in ‘Hongcui 1,2,5' at fruit mature period(130d),while the expression of MdMYB16 and MdMYB6 are fewer.So,the differential expressions of MdMYB16 and MdMYB6 plays an important role in the difference in coloration of ‘Hongcui 1,2,3,4,5' apple.2.The phylogenetic tree of MYB related transcription factors show shows that MdMYB16 and AtMYB4 are on the same clade,which is different from other MYB repressor,such as FaMYB1.Overexpression of MdMYB16 in red-fleshed callus decrease the anthocyanin content and expressions of MdUFGT and MdANS.Yeast one-hybrid analysis shows MdMYB16 bind the promoter of MdANS and MdUFGT.So,MdMYB16 can directly bind the promoter of MdANS and MdUFGT associated with anthocyanin biosynthesis and inhibit their expression,thereby decreasing anthocyanin content.3.The analyses of yeast two-hybrid,Bifc and pull-down show MdMYB16 can interact with itself to form homodimers and interact with MdbHLH33 to form heterodimers.When the bHLH motif of MdMYB16 is removed,we find that LBSMdMYB16(removed bHLH motif of MdMYB16)can not interact with MdbHLH33.Overexpression of MdbHLH33 in callus that overexpressed MdMYB16 can weaken the inhibitory effect of MdMYB16 on anthocyanin.However,overexpression of MdbHLH33 in callus that overexpressed LBSMdMYB16 can not weaken the inhibitory effect of MdMYB16 on anthocyanin.So,MdbHLH33 can weaken the inhibitory effect of MdMYB16 on anthocyanin by interacting with MdMYB16 to form heterodimers.4.We find a EAR inhibitory motif in C-terminal of MdMYB16,and name it as LESMdMYB16 by removing its EAR inhibitory motif.Overexpression of LESMdMYB16 can not decrease the expressions of MdANS and MdUFGT and anthocyanin content.Yeast one-hybrid analysis indicates LESMdMYB16 can also bind the promoter of MdANS and MdUFGT.So,MdMYB16 can inhibit anthocyanin biosynthesis by its own EAR inhibitory motif.5.MdbHLH33 is a known activator involved of anthocyanin biosynthesis,overexpressing it in red-fleshed callus can increase the anthocyanin content.The analyses of yeast two-hybrid and pull-down show MdMYB15 L can interact with MdbHLH33.Overexpression of MdMYB15 L in red-fleshed callus can not influence the anthocyanin content.However,overexpression of MdMYB15 L in callus that overexpressed MdbHLH33 can weaken stimulative effect of MdbHLH33 on anthocyanin.So,we knock out the bHLH motif of MdMYB15 L and name it as LBSMdMYB15 L.Yeast two-hybrid shows LBSMdMYB15 L can not interact with MdbHLH33.Overexpression of LBSMdMYB15 L in callus that overexpressed MdbHLH33 can not weaken stimulative effect of MdbHLH33 on anthocyanin.6.Overexpressing MdMYB6 in red-fleshed callus can inhibit anthocyanin biosynthesis.There are 1086 differentially expressed proteins between red-fleshed callus and red-fleshed callus that overexpressed MdMYB6 using TMT quantitative proteomics technology.The GO functional analysis shows that they mainly perform the functions of binding,catalytic activity,transporter activity and molecular function regulators,and the KEGG pathway analysis shows that they mainly involve in cellular processes,metabolic processes,biological regulation and localization and other important biological processes.Overexpressing MdMYB6 in red-fleshed callus can down-regulate the proteins level including MdANS,MdFLS,MdGSTF12,MdCHS and MdF3 H,in which MdANS and MdGSTF12 are key proteins in anthocyanin biosynthesis.The analyses of yeast one-hybrid and EMSA show MdMYB6 can bind MRE element of MdANS promoter and MBS element of MdGSTF12 promoter.Taken together,MdMYB6 can directly inhibit the expression of MdANS and MdGSTF12,thereby inhibiting anthocyanin biosynthesis.7.Further study shows there is a up-regulated monosaccharide transporter protein named MdTMT1 in callus that overexpressed MdMB6.MdTMT1 protein has 12 transmembrane structures by amino acid sequence analysis and locate at tonoplast in apple callus by subcellular localization analysis.Overexpressing MdTMT1 in red-fleshed callus can reduce the contents of UDP-glu and UDP-gal which are indispensable substrate for anthocyanin biosynthesis and anthocyanin content,so MdTMT1 may play important role in anthocyanin biosynthesis.The analyses of GUS activity,yeast one-hybrid,Chip-PCR and EMSA show MdMYB6 can bind the promoter of MdTMT1 and improve its activity.Overexpressing MdMYB6 in callus that overexpressed MdTMT1 can increase the MdTMT1 expression and reduce the content of UDP-glu and UDP-gal.So,MdMYB6 can indirectly reduce anthocyanin content by regulating MdTMT1.