Molecular evolution and expression assessment of DFRs in apple

Abstract

Abstract Background Anthocyanins are the secondary metabolites of flavonoids in plants. As a key enzyme in the biosynthetic pathway of anthocyanin, dihydroflavonol 4-reductase (DFR) act as an important regulatory point, but DFR family genes has not been systematically characterized in apple (Malus domestica Borkh.). Methods The members of DFR genes in apple were identified and their gene structure, chromosome distribution, evolutionary relationships, collinearity, cis-component and protein interaction relationships were predicted using bioinformatics methods. The expression patterns of MdDFRs in various organs, such as leaves, fruit flushes, fruits, ripe fruit peels, flowers and stems were analyzed using GeneChip expression array analysis. qRT-PCR was employed to analyze the expression levels of MdDFRs in different apple varieties with varying levels of fruit skin at maturity. Results The apple database revealed 96 DFR genes, which are distributed on 17 chromosomes and can be divided into 3 subfamilies. These 96 DFR genes were mostly composed of α-helix and random coil according to secondary structure prediction, and were mainly expressed in chloroplasts and cytoplasm. MYB binding site involved in flavonoid biosynthetic genes regulation element (MBSI) was identified in the promoter of MdDFR15/76/81/89/90/91/93/94. Lignin/flavonoid synthesis-related elements of MYB recognition site and MYB-binding site were identified in the promoters of MdDFR05/09/13/19/22/24/26/30/31/33/34/46/50/52/54/64/65/69/75/76/79/86. The internal collinearity analysis of the apple MdDFR genome revealed a total of 34 pairs of duplicated gene pairs. Interspecific collinearity analysis showed that there were 66 and 57 homologous gene pairs in apple/tomato and apple/grape, respectively. GeneChip expression array analysis showed that MdDFR72 and MdDFR96 were higher expressed in ripe fruit fleshes and peel, MdDFR01/06/67/49/54/91 were higher expressed in flowers, MdDFR64 was higher expressed in ripe fruit peels and flowers than those of other tissues. Besides, 75 MdDFR proteins interacted directly or indirectly with anthocyanidin synthesis related proteins MdANS, MdF3H, MdMYB1, MdMYBPA1 to form a protein interaction network. Interestingly, MdDFR69 and MdDFR87 had direct interactions with these four proteins, MdDFR64 had direct interactions with MdANS and MdF3H. qRT-PCR analysis showed that the expression levels of MdDFR01/05/31/53/64/69/73/84/87/94/96 were up-regulated with the accumulation of anthocyanins. Conclusions This study lays a foundation for further research on the function of DFR genes in apple. Graphical Abstract

Keywords

• Apple
• Anthocyanin
• Dihydroflavonol 4-reductase (DFR)
• Molecular evolution
• GeneChip expression array
• Protein interaction network