Studies On The Different Mechanism Of Anthocyanin Accumulation In Fruits Of ‘Nanguo Pear’ And Its Red-skinned Bud Sport

Color is one of the important factors to measure fruit quality.As the main color substance of red fruits,anthocyanin content directly affects the nutritional value and commercial value of fruits.‘Nanguo Pear’(Pyrus ussuriensis cv.Nanguo)is an excellent variety in Pyrus ussuriensis.‘Nanhong Pear’(Pyrus ussuriensis cv.Nanhong)is a bud sport variant of NG with a naturally occurring red skin phenotype.Bud sport incubates new characters of fruit trees,but the mechanism of color difference of bud soprt varieties has not been studied intensively yet.In this study,fruits of ‘Nanguo pear’(NG)and ‘Nanhong pear’(NH)during the developmental stages were used as samples.Key genes responsible for the coloring difference between NG and NH were identified: the structural genes PuUFGT and PuGSTF6,and the two regulatory genes PuMYB110 a and PuMYB111-like of anthocyanin synthesis and transport pathway.In addition,low expression of histone deacetylase PuHDAC15 and high expression of histone acetyltransferase PuHLS1-like in NH may be the key factors that cause the histone acetylation levels of PuMYB110 a to be higher than NG in NH.The main results are as follows:1.The red phenotype of NH is caused by the accumulation of anthocyanin.The contents of anthocyanin and chlorophyll in NG and NH fruits at different developmental stages were determined.The contents of anthocyanin in NH fruits were higher than that in NG fruits at 105,120 and 140 days after full bloom(DAFB),and there was no difference in the content of chlorophyll between the two varieties.2.The glycosyl transferase PuUFGT and the glutathione S-transferase PuGSTF6 are the key genes responsible for the different accumulation of anthocyanin between NG and NH.The expression of eight anthocyanin structural genes in anthocyanin synthesis pathway was analyzed by q RT-PCR method.The results showed that the expression level of PuUFGT in NH was significantly higher than that of NG at 105-140 DAFB,and the expression pattern was basically consistent with the trend of anthocyanin accumulation during the whole development period.The transcriptome sequencing(RNA-seq)was performed using NG and NH fruits at120 DAFB,and a gene PuGSTF6 encoding glutathione S-transferase was screened,on account of its high expression in NH.The expression level of PuGSTF6 in NH increased continuously and was always significantly higher than that of NG in the middle and late stages of fruit development(90-140 DAFB).3.The expression levels of PuUFGT and PuGSTF6 genes were independent of the degree of DNA methylation and histone acetylation in the promoter region.To clarify the reason for the high expression of PuUFGT and PuGSTF6 in NH,the coding regions and promoters of PuUFGT and PuGSTF6 were cloned in NG and NH,respectively,but there were no differences in sequences between the two varietes.DNA methylation levels and histone acetylation levels in promoter regions were detected to determine whether epigenetic modifications affected the expression of PuUFGT and PuGSTF6,but no differences were observed between NG and NH.4.The transcription levels of PuUFGT and PuGSTF6 are positively regulated by two MYB transcription factors.Several binding sites of MYB transcription factor were identified on the promoters of PuUFGT and PuGSTF6 by squencing analysis.Two R2R3-MYB transcription factors PuMYB110 a and PuMYB111-like were found in RNA-seq results due to their high expression in NH.PuMYB110 a and PuMYB111-like were found to bind to the promoters of PuUFGT and PuGSTF6 and up-regulated their expression by using Y1 H,EMSA,Ch IP-PCR and GUS assays.Meanwhile,functional verification assays in NG callus showed that PuMYB110 a and PuMYB111-like could promote anthocyanin biosynthesis.5.The expression level of PuMYB110 a is closely related to the level of histone acetylation in the promoter region.To clarify the reason for the different expression levels of PuMYB110 a and PuMYB111-like between NG and NH,the coding region and promoter region of PuMYB110 a and PuMYB111-like in NG and NH were cloned and compared,and no sequence differences were found.DNA methylation levels in the promoter regions of PuMYB110 a and PuMYB111-like were detected between NG and NH,and no differences were found.Meanwhile,the histone acetylation levels of the PuMYB110 a and PuMYB111-like promoter regions were detected between NG and NH.The results showed that the histone acetylation level of the PuMYB110 a promoter in NH was significantly higher than that in NG,but there were no differences in histone acetylation level of PuMYB111-like promoter between the two varietes.In addition,the results of EMSA,Ch IP-PCR and GUS assays showed that PuMYB111-like could directly bind the promoter of PuMYB110 a and up-regulate its expression.6.PuHDAC15 and PuHLS1-like are important genes that cause the difference in histone acetylation level of PuMYB110 a promoter between NG and NH.To illuminate the different histone acetylation levels of PuMYB110 a promoter between NG and NH,the RNA-seq results were analyzed and a histone deacetylase PuHDAC15 and a histone acetyltransferase gene PuHLS1-like were screened.PuHDAC15 was highly expressed in NG and PuHLS1-like was highly expressed in NH during the late stages of fruit development.The two genes were overexpressed in NG callus respectively,and the histone acetylation levels in some regions of PuMYB110 a promoter were correspondingly changed.In addition,both PuHDAC15 and PuHLS1-like could interact with PuMYB111-like.In conclusion,we revealed that the high expression of PuMYB110 a in NH was due to its high histone acetylation level and the positive regulation effect of PuMYB111-like.Meanwhile,highly expressed PuMYB110 a and PuMYB111-like promote anthocyanin accumulation in NH by up-regulating the transcriptions of PuUFGT and PuGSTF6,which is the key factor in the formation of the coloring difference between NG and NH.