Regulation Of The Methylation Of The Mdmyb1 Promoter On Fruit Coloration Of Malus After Bagging Treatment

Apple market acceptance greatly depends on their exterior qualities.Peel coloration is one of the most important exterior qualities for apple.Different apple cultivars have different pigmentations,which can result from genetic or environmental factors.The red color of apple peel is ascribed mainly to the accumulation of anthocyanin pigments,MdMYB1 are important regulators in the biosynthesis of anthocyanins in apple.Bagging is a commonly used treatment to enhance the red pigmentation in apple peel,and can also be used in analyse of fruit pigmentation.In this study,the changes in peel color in different color fruits were studied after bag removal.RNA-seq analysis revealed that numerous genes were differentially expressed in green-skinned 'Granny Smith'.Moreover,the methylation levels in the MdMYB1 promoter were investigated in 'Granny Smith' and 'Golden Delicious' after bag removal.The main results are documented as follows:1.In this study,the pigmentation in apple cultivars 'Granny Smith','Golden Delicious','Starkrimson','Qinguan','Ruiyang','Fuji','Ruixue' and 'Cripp's Pink' fruits were investigated after bag removal.The fruit peels of the eight cultivars showed differences in pigmentation after bag treatment.The red pigmentation induced in the non-red skinned cultivar 'Granny Smith' fruit was more intense than in 'Golden Delicious' and 'Ruixue' after bag removal.The expression of structural genes and regulatory genes,involved in the biosynthesis of anthocyanins,increased with different extents in eight apple cultivars after bag removal.When bagged fruits were exposed to light,transcript levels of MdMYB1 showed strong correlations with anthocyanin levels in 'Granny Smith','Golden Delicious','Starkrimson',etc.Additionally,the expression pattern of the structural genes basically paralleled MdMYB1 transcription level in 'Granny Smith','Golden Delicious' and 'Starkrimson',etc.after bag removal.Therefore,MdMYB1 is associated with anthocyanin biosynthesis in apple peel after bag removal.2.Peel samples of 'Granny Smith' from 0,4,and 10 days after bag removal(DABR)were subjected to transcriptome sequencing and expression analysis.Fruits without bagging treatment and dark-grown apple fruits were used for comparison.RNA-seq was used to identify 18,152 differentially expressed genes(DEGs)among unbagged,bagged,and bag-removed fruits at 0,4,and 10 DABR.The identified DEGs are implicated in light signal perception and transduction,plant hormone signal transduction,antioxidant systems,and anthocyanin biosynthesis.In addition,weighted gene co-expression network analysis of DEGs generated a network module('darkolivegreen 4')highly correlated with anthocyanin content,and the module harbors MdMYB1.These results further indicating the major effect of MdMYB1 on the red pigmentation in 'Granny Smith' fruits.3.In order to further explore the molecular mechanism of pigmentation differences in apple,the MdMYB1 genomic sequences were compared in 'Granny Smith','Golden Delicious' and 'Starkrimson',which showed that the green-fruited cultivar 'Granny Smith' harbors the MdMYB1-1 and MdMYB1-2 alleles,the yellow-fruited cultivar 'Golden Delicious' harbors only MdMYB1-2,the red-fruited 'Starkrimson' harbors only MdMYB1-1.A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in 'Granny Smith'.An investigation of the MdMYB1 promoter in 'Granny Smith' showed reduced methylation in the regions-2026 to-1870 bp,-1898 to-1633 bp,-1062 to-964,and-541 to-435 bp after bag removal treatments.However,the methylation levels of MdMYB1 promoter region in 'Golden Delicious' showed no significant differences between the unbagged fruits and those removed from the bags.Differences in anthocyanin levels between 'Granny Smith' and 'Golden Delicious' could be explained by differential methylation levels in the MdMYB1 promoter.4.The 'Granny Smith','Golden Delicious' and 'Starkrimson' fruits removed from the bags were treated with 5-aza-2'-deoxycytidine(5-aza-dC).Compared with control fruits,'Granny Smith' gradually and markedly turned red in color during the 5-aza-dC treatment,whereas 'Golden Delicious' and 'Starkrimson' fruits did not change color.The expression levels of MdMYB1 in 'Granny Smith' were associated with methylation levels in the promoter.The-2026 to-1870 bp,-1898 to-1633 bp and-1062 to-964 bp regions of MdMYB1 promoter element were essential for MdMYB1-mediated regulation of anthocyanin accumulation and red pigmentation in the 'Granny Smith' apple cultivar.However,there were negligible differences in methylation levels between the treatment and control groups in 'Golden Delicious'.The peels of candidate samples after 5-aza-dC treatment were subjected to transcriptome sequencing and expression analysis.Fruits without 5-aza-dC treatment were used for comparison.RNA-seq revealed a total of 8,482 DEGs between 5-aza-dC-treated and control groups of bagged fruits,including genes encoding transcription factors(bZIP,GATA,NAC,SPL,WRKY and ATHB),enzymes related to anthocyanin accumulation(CHS,CHI,F3 H,DFR,ANS,UFGT,MYB and bHLH),methylases(MET and DRM)and demethylases(DME and ROS).Changes in the expression of these genes may be responsible for 5-aza-dC-induced red pigmentation in bagged fruits of 'Granny Smith'.