Molecular Mechanism Of Fruit Skin Coloration In Apple(Malus Domestica Borkh.) Cv.'Red Delicious' Bud Sport Mutants

Apple(Malus domestica Borkh.)skin color is an important agronomic character,which directly affects the value of apple.The color of apple fruit skin is mainly determined by the content of anthocyanins and carotenoids.Bud sport is an important method for discovering new varieties or lines superior to the parent,and is one of the important ways to improve fruit tree varieties.Fuir skin color is one of the main basis for budding selection of apple and other varieties.For example,the selection of‘Red Delicious'apple varieties is mainly related to fruit skin color.At present,although there is a deep understanding of the molecular mechanism of apple and other fruit coloring,the mechanism of skin coloring difference among different bud varieties is still unclear.Therefore,‘Red Delicious'(G0)and its four generations of continuous natural mutants(‘Starking Red'(G1),‘Starkrimson'(G2),‘Campbell Redchief'(G3)and‘Vallee spur'(G4))were selected as research materials in this paper.Through the determination of physiological and biochemical indexes related to fruit coloring,combined with whole genome DNA methylation sequencing and transcriptome sequencing technologies,the genome-wide methylation levels and transcriptional levels between parents and each generation of natural mutants were compared,and the candidate factors related to fruit skin coloring were found out,in order to explore the mechanism of fruit skin coloring in‘Red Delicious'and its mutants,and provide a theoretical basis for genetic improvement of apple fruit color.The main results are as follows:1.Pigmentation in the skin gradually proliferated from G0 to G4.The anthocyanin content was higher in the mutants than in‘Red Delicious'.The expression trends of the early phenylpropanoid biosynthesis genes,including ASP3,PAL,4CL,PER,CHS,CYP98A and F3'H,are consistent with the change trend of anthocyanin content in‘Red Delicious'and its fourth-generation bud sport mutants.2.At the color break stage,the change trend of anthocyanin content was the same as that of endogenous IAA and ABA,but opposite to that of GA.Tryptophan metabolism of IAA,diterpenoid biosynthesis of GA and carotenoid biosynthesis of ABA are all branches of terpene skeleton synthesis pathway.The terpene biosynthesis genes,including AACT1,HMGS,HMGR,MVK,MVD2,IDI1 and FPPS2,were all downregulated.However,AUX1,SAUR,GID2,PP2C and SnRK2 were upregulated.3.The number of differentially methylated regions(DMRs)and differentially methylated genes(DMGs)was considerably increased from G1 to G4 versus the number observed in G0.The ~mCHH context was dominant in apple,but the levels of ~mCG and ~m CHG of DMGs were significantly higher than that of the ~mCHH.4.Methylation levels of ~mCG and ~mCHG contexts in flavonoid biosynthesis pathway genes(PAL,4CL,CYP98A,PER,CCoAOMT,CHS,and F3'H),as well as ~mCHG context in MYB10 at upstream were negatively correlated with anthocyanin accumulation.5.Bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF]at the C-terminal end of the motif and were located in the nucleus of onion epidermal cells.Overexpression of MdLUX and MdPCL-like in‘Gala'calli and Arabidopsis thaliana promoted the accumulation of anthocyanin.Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H.Dual-luciferase assays indicated that MdLUX and MdPCL-like activated the expression of MdF3H in vivo.The whole-genome DNA methylation evidence revealed that the methylation levels of the ~mCG context at the upstream(i.e.,promoter region)of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation.In summary,anthocyanin accumulation in the fruit skin of bud sport mutants is related to the upregulated expression of early phenylpropane biosynthesis genes,and the down-regulated expression of terpene biosynthesis genes positively induced the expression of hormone signal transduction related genes,thus promoting the synthesis of IAA and ABA,reducing the synthesis of GA,and regulating anthocyanin accumulation.In addition,the evolution of fruit skin color in‘Red Delicious'apple bud sport mutants is related to the difference of DNA methylation.The ~mCG hypomethylation of some structural genes related to anthocyanin synthesis,as well as transcription factors MdLUX and MdPCL-like in promoter regions,leads to transcriptional activation,which is conducive to anthocyanin accumulation.