| The formation of fruit color in red pear is a complex process.Light is important environmental factors for affecting the color of the fruit peel,but there is little about the light-responsive genes involved in the regulation of anthocyanin synthesis in pears.Consequently,it is crucial to study the mechanism of the anthocyanin synthesis and degradation regulated by light-responsive genes in red pear.In this study,six different red pear cultivars were used to analyze the differences of anthocyanin coloring patterns.Then,the light-responsive genes PbCOP1.1,PbPIF3.1 and PbZAT12 regulating anthocyanin synthesis were screened out by transcriptome of ‘Zaosu’ fruit peel reddening under high temperature and high light.Our studies have shown that PbCOP1.1 negatively regulates anthocyanin accumulation,while PbPIF3.1 and PbZAT12 positively regulate anthocyanin accumulation.The main results are as follows:1.Differences among the anthocyanin accumulation patterns in red pears.The anthocyanin contents analysis of six red pear cultivar(‘5 Hao’,‘Red Bartlett’,‘Starkrimson’,‘Red Sichou’,‘Red Zaosu’ and ‘Palacer’)showed that the peak accumulations of anthocyanin in six cultivars occurred during the middle stages of fruit development(55 and 75 day after flowering).The anthocyanin contents of all six cultivars showed a rise–drop tendency.The different expression levels of the structural genes F3 H and UFGT2 were important for anthocyanin accumulation in the six cultivars.The transcription factors MYB10 and bHLH3 were also important for anthocyanin accumulation.A principal component analysis(PCA)and hierarchical clusteranalysis were used to distinguish the types of cultivars and the genes important for each type ofanthocyanin accumulation pattern.The six cultivars were divided into three groups,with ‘Red Zaosu’(Asian pear)clustered into one group,which was determined by the genes PAL,ANS1,CHS,DFR1,bHLH33,MYB10 b and PIF3.1.‘Red Sichou’ and ‘Starkrimson’(European pears)clustered into another group,which was determined by the genes UFGT1,MYB10,COP1 and ANR.‘Palacer’(European pears),‘Red Bartlett’(European pears),and ‘5 Hao’(interspecific Pyrus hybrid)clustered into athird group,which was determined by all of these genes.2.PbCOP1.1 negatively regulates anthocyanin biosynthesis.PbCOP1 s was cloned from ‘Red Zaosu’.Tissue specific expression analysis and different development stages of leaves showed that PbCOP1 s was highly expressed in leaves,and the expression levels in green leaves was significantly higher than that in red leaves.After over-expressing PbCOP1 s,the expression levels of PbCOP1.1 in the peel around injection areas increased and the anthocyanin levels decreased significantly,when compared with the empty vector.However,there was no significant change in PbCOP1.2.After over-expressing PbCOP1.1,the expression levels of anthocyanin synthesis-related genes,except for ANS,decreased to different degrees,and CHI,DFR,UFGT2,bHLH3,HY5 and GST were significantly down-regulated.3.PbPIF3.1 and PbZAT12 positively regulate anthocyanin synthesis.PbPIF3.1 and PbZAT12 overexpression in ‘Zaosu’ peel promoted anthocyanin accumulation,while PbPIF3.1 and PbZAT12 RNAi in ‘Starkrimson’ inhibition anthocyanin accumulation.The results of subcellular localization showed that PbPIF3.1 and PbZAT12 protein were located in the nucleus.PbPIF3.1 overexpression showed that anthocyanin-related synthesis genes were significantly up-regulated,except for MYB10 b.In addition,the expression levels of PbZAT12 was also significantly up-regulated.After PbPIF3.1 RNAi,only UFGT2,MYB10 and GST were significantly down-regulated.Dual luciferase assay revealed that PbPIF3.1 can activate the activity of F3 H promoter.After over-expressing PbZAT12,the expression levels of CHS,DFR1,ANS1 and UFGT2 were up-regulated,but there was no significant difference among other genes.After PbZAT12 RNAi,the expression levels of anthocyanin-related synthesis genes were down-regulated,and PAL,UFGT2,MYB10,MYB10 b,bHLH3 and bHLH33 were significantly down-regulated. |
