Identification And Functional Analysis Of MYB Transcription Factors Of Flavonoid Biosynthesis In Pear

Flavonoids are important secondary metabolites present in plants.Flavonoids are classified into several subgroups,including flavonols,flavones,flavanones,isoflavones,anthocyanins,and proanthocyanidins.Pear(Pyrus spp.),which belongs to the family Rosaceae,is a fruit crop widely cultivated in temperate regions worldwide.Generally,MBW complex is involved in transcriptional regulation of flavonoid biosynthesis in pear through regulating structural genes of the flavonoid biosynthesis pathway.Structural genes encoding the flavonoid biosynthetic enzymes in the flavonoid biosynthesis pathway have been identified and characterized.However,the transcriptional regulation of flavonoid biosynthesis in pear fruit has not been fully characterized 1)PpMYB17 positively regulates flavonoid biosynthesis in pear MYB transcription factors are considered to be the key regulators of flavonoid biosynthesis in plants.Identification of novel regulators further expands the flavonoid regulatory network of pear.The R2R3-MYB transcription factor PpMYB17 was previously identified in our transcriptome data based on its positive correlation with the pear fruit flavonoid content.In the present study,PpMYB17 was isolated from ’Hongzaosu’(Previously known as ’Red Zaosu’)pear fruit and functionally characterized.An exposure to light upregulated the PpMYB17 expression in pear fruit.Additionally,a phylogenetic analysis indicated PpMYB17 is related to the flavonol regulators.A subcellular localization assay suggested that PpMYB 17 is a nuclear protein.The overexpression of PpMYB17 increased the flavonoid content of pear calli and Arabidopsis via the upregulated expression of structural genes in the flavonoid biosynthesis pathway,especially FLS.The LC-MS/MS analysis revealed most of the differentially accumulated flavonols,flavanones,flavones,isoflavones,and anthocyanins were significantly more abundant in PpMYB17-overexpressing calli than in wildtype calli.Moreover,PpMYB17 did not interact with PpbHLH3,PpbHLH33,or PpWD40 in a yeast system.Dual-luciferase assays demonstrated that PpMYB17 strongly activated the promoters of PpCHS,PpCHI,PpF3 H,PpFLS,and Pp UFGT which are key downstream genes in the flavonoid biosynthesis pathway,independently of the PpbHLH3 cofactor.These gene expression changes may enhance flavonoid biosynthesis in pear fruit.The data presented herein may be useful for further elucidating the flavonoid biosynthesis regulatory network,potentially leading to the development of new pear cultivars that produce fruit with increased flavonoid contents.2)PpMYB140 repress anthocyanin biosynthesis in pear In addition to MYB activators,MYB repressors also participate in the regulation of flavonoid biosynthesis.The negative regulation of flavonoid biosynthesis by MYB repressors remains largely unstudied in pear.The R2R3 MYB transcription factor,Pp MYB140 was identified as a potential repressor of flavonoid biosynthesis through analysis of our previously published transcriptome data.Pp MYB140 was isolated from ‘Hongzaosu’ pear fruit and functionally characterized.The Pp MYB140 was phylogenetically clustered in the subgroup 4 with transcriptional repressors.Furthermore,C-terminal region contained the conserved C1 repressor motif and predicted C2 repressor motif.The expression of Pp MYB140 was significantly downregulated by light treatment of ‘Hongzaosu’ pear fruit.The trans-acting activity assay further indicated the repressing activity of Pp MYB10 in yeast system.In subcellular localization assay,Pp MYB140 was detected in nuclei.These results suggest Pp MYB140 is a transcriptional repressor.Over expression of Pp MYB140 suppressed methyl jasmonate(Me JA)induced anthocyanin biosynthesis in pear calli via downregulating the expression of flavonoid biosynthetic genes(Pp CHS,Pp CHI,Pp F3 H,Pp DFR,Pp UFGT and,Pp ANS)and components of MBW complex(Pp MYB10,Ppb HLH3 and Pp WD40).Results indicated Pp MYB140 negatively regulated anthocyanin biosynthesis in pear.Pp MYB140 physically interacted with both b HLH3 and b HLH33 in the nucleus,enabling the formation of the MYB140-b HLHWDR [M(140)BW] complex.Pp MYB140 combined with Ppb HLHs to repress the promoter activity of anthocyanin biosynthetic genes.Additionally,Pp MYB140 hinders the formation of the MBW complexes by disrupting the binding of Pp MYB114 to b HLHs,thereby inhibiting anthocyanin biosynthesis in pear.The data presented herein may be important for understanding of negative regulation of anthocyanin biosynthesis in pear.3)MYB family is prominently involved in the transcriptional regulation of flavonoid biosynthesis in pear calli Flavonoid biosynthesis is influenced by diverse factors,of which phytohormones are substantially affect flavonoid biosynthesis in fruit.For example,Me JA enhances the flavonoid accumulation in pear.However,the molecular mechanism underlying the Me JA-induced flavonoid biosynthesis in pear is largely uncharacterized.Therefore,the transcriptome of pear calli treated with Me JA was analyzed to elucidate the mechanism regulating Me JA-mediated flavonoid biosynthesis.The application of exogenous Me JA significantly enhanced flavonoid accumulation,especially anthocyanin,in pear calli.A weighted gene co-expression network analysis identified the differentially expressed genes associated with Me JA-induced flavonoid biosynthesis.The Me JA treatment upregulated the expression of the flavonoid biosynthesis pathway structural genes(Pc CHS,Pc CHI,Pc F3 H,Pc DFR,Pc ANS,Pc ANR2 a,and Pc LAR1).The MYB family members were the main transcription factors regulating the Me JA-induced flavonoid biosynthesis,but the b HLH,AP2-EREBP,NAC,WRKY,and TIFY families were also involved.In addition to Pc MYB10,which is a known positive regulator of anthocyanin biosynthesis in pear,several novel MYB candidates that may regulate flavonol and proanthocyanidin biosynthesis were revealed.Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that Pc MYB10 and Pc MYC2 could directly interact with each other and bind to JAZ repressors(Pc JAZ1 and Pc JAZ2).The Pc MYB10–Pc MYC2 molecular complex was likely involved in the regulation of jasmonate-mediated flavonoid biosynthesis at the transcript level.The data generated in this study may clarify the transcriptional regulatory network associated with the Me JA-induced flavonoid accumulation in pear calli and provide a solid foundation for future studies.In conclusion,present study functionally characterized two MYB transcription factors which regulate flavonoid biosynthesis in pear.Pp MYB17 positively regulates flavonoid biosynthesis in pear fruit by activating the promoters of specific downstream genes in the flavonoid biosynthesis pathway independently of b HLH or WD40 cofactors in the MBW complex.Pp MYB140 hinders the formation of the MBW complexes by disrupting the binding of Pp MYB114 to b HLHs,thereby repress anthocyanin biosynthesis in pear.Furthermore,comprehensive transcriptome analysis of Me JA treated pear calli revealed that the MYB family is prominently involved in the transcriptional regulation of flavonoid biosynthesis in pear calli.In addition,b HLH,AP2-EREBP,NAC,WRKY,and TIFY families were also involved.Present transcriptome analysis identified a set of candidate transcription factors relevant fo r future functional studies related to the transcriptional regulation of Me JA-mediated flavonoid biosynthesis in pear.