Regulatory Mechanism Of Anthocyanin By Transcription Factor MdHB1 In Apple Cultivar ’granny Smith’

The red coloration of the apple(Malus × domestica)peel and flesh is mainly caused by the accumulation of anthocyanins,which directly affected its commodity value and nutritional value.Red-fleshed apple is rich in anthocyanins and is valuable in both economics and biology.Research on the regulation mechanism of anthocyanin accumulation is important for apple molecular breeding and fruit quality improvement.HD-Zip protein is a kind of transcription factor unique to the plant community.It is widely involved in biological processes,such as plant growth and stress tolerance,but its regulation on the accumulation of anthocyanin is not clear now.In this study,an HD-Zip I transcription factor MdHB1 was cloned from ‘Granny Smith’,and the regulation of MdHB1 on anthocyanin biosynthesis was studied by Agrobacterium tumefaciens-mediated transient transformation.Then,combined with physiological and biochemical experiments,we suggested a possible mechanism for the regulation of anthocyanin by MdHB1 in ‘Granny Smith’.The main results are as follows:(1)MdHB1 encodes a protein of 336 amino acids,and is a member of HD-Zip I family.Evolutionary tree analysis showed that MdHB1 was closely clustered with LeHB1 in tomato and AtHB1 in Arabidopsis thaliana.Quantitative RT-PCR results showed that MdHB1 expressed widely,and was highly expressed in flesh of ‘Granny Smith’ fruit at young and picking mature stages.Subcellular localization assays showed that MdHB1-GFP was diffuse in the cytoplasm and was concentrated in the nucleus under the driven of Cauliflower Mosaic Virus 35 S promoter;the GFP protein controlled by MdHB1 promoter was mainly distributed in the cytoplasm.(2)MdHB1 silencing led to red flesh in mature ‘Granny Smith’.High performance liquid chromatography analysis showed the red color was caused by the accumulation of anthocyanins and the major one was cyanidin 3-O-arabinoside.Quantitative RT-PCR analysis showed that the application of transient gene silencing decreased the expression of MdHB1,but increased the transcription levels of anthocyanin accumulation genes,including MdDFR,MdUFGT and MdMYB10.In contrast,transient overexpression of MdHB1 reduced the flesh content of anthocyanin and the mRNA levels of MdDFR,MdUFGT and MdMYB10 in ‘Ballerina’,whose flesh is red.Moreover,flowers of MdHB1-overexpressing tobacco(Nicotiana tabacum ‘NC89’)showed a remarkable reduction in pigmentation relative to those in the wild plant.Thus,the HD-Zip I transcription factor MdHB1 negatively regulates the synthesis of anthocyanin in specific tissues involving the expression of DFR and UFGT and MYB10.(3)Using dual-luciferase assays and GUS reporter genes,we showed that MdMYB10 activates the promoter activities of MdDFR and MdUFGT,and the addition of MdbHLH3 and MdTTG1 enhances these activities.In contrast,MdHB1 inhibits the promoter activities of MdDFR and MdUFGT,and disturbs the activation of MdMYB10,MdbHLH3 and MdTTG1 proteins on those promoters.Yeast one-hybrid showed that MdHB1 did not bind to the promoters of Md DFR and MdUFGT,indicating the negative regulation of MdDFR and MdUFGT by MdHB1 is indirect.(4)In ‘Granny Smith’ flesh,MdMYB10 silencing significantly reduced the expression levels of MdDFR and MdUFGT,but caused no color phenotype;the simultaneous silencing of MdMYB10 and MdHB1 blocked the red color caused by MdHB1 silencing.Therefore,MdMYB10 is essential for flesh coloration.(5)The transient transformation of GUS reporter genes showed that MdHB1 and MdMYB10 inhibited the promoter activities of MdMYB10 and MdHB1 in tobacco,suggesting that MdHB1 and MdMYB10 negatively regulated the transcription of each other in ‘Granny Smith’.(6)Yeast two-hybrid and bimolecular fluorescence complementation assay showed that two MdHB1 proteins can form homodimers in the cytoplasm.The interaction of MdHB1 with MdMYB10,MdbHLH3 and MdTTG1 also occurs in the cytoplasm,with the weakest binding to MdbHLH3 and the strongest binding to MdMYB10-MdbHLH3-MdTTG1 ternary complex.Taken all these results together,we suggest a working model to show the association of MdHB1 with anthocyanin biosynthesis in white-fleshed ‘Granny Smith’.In mature fruit under normal conditions,MdHB1 recruits MdMYB10,MdbHLH3,MdTTG1 transcription factors to the cytoplasm.These combinations indirectly suppress the transcription of MdDFR and MdUFGT,encoding key enzymes in anthocyanin biosynthesis.When MdHB1 mRNA levels are decreased by RNAi approaches,the MdMYB10,MdbHLH3,MdTTG1 transcription factors are released to activate the expression of MdDFR and MdUFGT in the nucleus,leading to red pigmentation in ‘Granny Smith’ flesh.This study not only enhances our functional understanding of HD-Zip I transcription factors,but also provides a deeper understanding of the control of anthocyanin biosynthesis,supplying evidences for the molecular breeding of red-fleshed apple.