Mechanism Of DNA Methylation And MicroRNA Regulating Peel Coloration In Red Pear

Peel color is one of the most important fruit qualities,and the red coloration of pear skin is caused by deposition of anthocyanin.The mechanism of regulation of anthocyanin biosynthesis has been well studied by using classical genetic methods,but little is known about the mechanism of DNA methylation and miRNA in regulating peel coloration in red pear.In this study,green pear cultivar 'Zaosu',a hybrid of 'Pingguoli'(Pyrus pyrifolia Nakai)× 'Mishirazu'(Pyrus communis L.),its red bud sport 'Zaosu Red',red Chinese sand pear(P.pyrifolia Nakai)cultivar 'Meirensu' and 'Mantianhong',and red European pear(P.communis L.)cultivar 'Cascade' were used as materials.The light sensitive red pear materials were selected,and the mechanisms of DNA methylation and miRNA in regulating red pear coloration were investigated.The main results are as follows:Tissues of 'Zaosu Red' contained much higher concentration of anthocyanin,than tissues of 'Zaosu' and 'Green Zaosu Red'.For the mature fruit of 'Zaosu Red',peels of the fully red phenotype showed the highest anthocyanin concentration.The anthocyanin concentration in red-stripe enriched peel was much higher than that in green-stripe enriched peel.The transcript levels of most genes related to anthocyanin biosynthesis(PpPAL1,PpPAL2,PpCHS1,PpCHS2,PpCHIl,PpCHI2,PpCHI3,PpF3H,PpDFRl,and PpANS)were significantly higher in young tissues than in mature tissues,regardless of the anthocyanin concentration in these tissues.The transcript levels of the genes PpUFGT2 and PpMYB10 were highly coordinated with this anthocyanin accumulation.The sequences of the open reading frames(ORF)and promoter regions of these two key genes were cloned and compared among 'Zaosu'and its bud sports,but no sequence mutations were found.A yeast one-hybrid assay showed that PpMYB10 associated with the-658 to-172 bp fragment of the PpUFGT2 promoter,probably via a MYB binding site(MBS)located at-466 bp.The PpMYBI 0 promoter had lower methylation levels in anthocyanin-rich tissues,indicating that the red bud sport of 'Zaosu' pear and the striped pigmentation pattern of 'Zaosu Red' pear are associated with demethylation of the PpMYB10 promoter.The pigmentation patterns of 'Mantianhong'(Pyrus pyrifolia Nakai)and 'Cascade'(Pyrus communis L.)that developed under bagging and postharvest UV-B/visible irradiation conditions were evaluated in this study.The results showed that,unlike'Cascade,' in which almost no anthocyanin was detected,'Mantianhong' developed good red coloration under bagging treatment and postharvest irradiation.The activity of PAL and DFR increased during anthocyanin accumulation.The expression of PpCHS3,PpCHI3,PpUFGT1,and PpMYB10 was up-egulated 'Mantianhong' but not in 'Cascade' during both treatments.This suggests that these four genes may be limiting factors for peel coloration in 'Cascade.'The upregulated expression of anthocyanin biosynthetic and regulatory genes and downregulated expression of PpSPL2,PpSPL5,PpSPL7,PpSPL9,PpSPL10,PpSPL13,PpSPL16,PpSPL17,and PpSPL18 were observed in pear fruits after bags were removed from plants during the anthocyanin accumulation period.Based on high-throughput miRNA and degradome sequencing data,we determined that two miR156 members(ie.,miR1 56a and miR156ba)were expressed in pear fruit peels,and targeted four SPL genes.Light-responsive elements were detected in the promoter regions of the miR156a and miR156ba precursors.We identified 19 miR156-targeted SPL genes using the 'Suli' pear(Pyrus pyrifolia Chinese White Pear Group)genome database,of which seven members were targets of miR156.Additionally,miR156a/ba abundance increased after bags were removed.Yeast two-hybrid results revealed that the MYB10/bHLH/WD40 protein complex exists in pear,likely to regulate anthocyanin biosynthesis.Additionally,PpSPL10 and PpSPL13 interacted with PpMYB10.We proposed the folowing model for miRNA-mediated light-induced anthocyanin accumulation in pear fruits:Because of the low abundance of miR156 in darkness,high SPL levels repress anthocyanin accumulation by destabilizing the MYB/bHLH/WD40 complex and directly inhibiting the expression of anthocyanin biosynthetic genes such as CHS,ANS,and UFGT.Exposure to light increases the abundance of miR156,which degrades SPL genes,resulting in the formation of the MYB/bHLH/WD40 complex and activation of anthocyanin biosynthetic genes.This ultimately induces anthocyanin accumulation and the coloration of red pear fiuits.