The R2R3-type MYB transcription factor MdMYB90-like is responsible for the enhanced skin color of an apple bud sport mutant

Abstract

The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (-1183 to -988 and -2018 to -1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

Fig. 1. Differentially expressed genes (DEGs) between the Fuji apple and the mutant.

A Fruits of Fuji and M_Fuji were exposed to different durations of light treatment after bag removal. B Anthocyanin contents in skins of Fuji and M_Fuji fruits. Samples were assayed on light-treated days after bag removal. Error bars are SEs for three replicates. C Venn diagram of unique and common DEGs at three different stages. D Number of DEGs upregulated (blue) or downregulated (orange) at three different stages (O1-VS-M1, O2-VS-M2, O3-VS-M3)

Fig. 2. RNA-seq and qRT-PCR results of 20 selected DEGs in Fuji and mutant apples.

The left y axis indicates the corresponding expression data from RNA-seq (blue histogram). The right y axis shows the relative gene expression level measured by qRT-PCR (red lines). Bars represent the standard error (SE; n = 3)

Fig. 3. Anthocyanin biosynthesis and gene expression in apple transgenic lines.

A, B Anthocyanin accumulation in agroinfiltrated Fuji (A) and Golden Delicious (B) apple skin after 3 and 5 days of treatment, respectively. Agrobacterium harboring the MdMYB90-like overexpression vector and the 62SK empty vector were infiltrated into apple skins and exposed to light and dark, respectively. C Accumulation of anthocyanin in MdMYB90-like overexpression calli (MdMYB90-like-OE) and wild-type calli (WT) after 5 days of light and dark treatment. WT calli were used as the control. D Anthocyanin contents of transgenic (MdMYB90-like-OE) and wild-type calli (WT). E Expression levels of MdMYB90-like, MdSIMYB1, MdMYB1, MdCHS, MdCHI, MdANS, and MdUFGT in transgenic and wild-type apple calli. Asterisks (*) and (**) denote significant differences between samples at P < 0.05 and P < 0.01 significance levels, respectively

Fig. 4. Transcriptional activity of MdMYB90-like.

A Yeast one-hybrid (Y1H) analysis of interactions of MdMYB90-like (left panels) and MdMYB1 (middle panels) with the promoters of anthocyanin biosynthesis-related genes. The pB42AD vector was used as the negative control (right panels). B Dual-luciferase detection experiments showed that MdMYB90-like promoted the expression of the MdCHS, MdBHLH3, MdUFGT, and MdMYB1 genes. C LUC/REN activities of constructs: 35Spro:MdMYB90-like/MdMYB1pro:LUC and 35Spro:62-SK/MdMYB1pro:LUC; 35Spro:MdMYB90-like/MdbHLH3pro:LUC and 35Spro:62-SK-MdbHLH3pro:LUC; 35Spro:MdMYB90-like/MdUFGTpro:LUC and 35Spro:62-SK-MdUFGTpro:LUC; 35Spro:MdMYB90-like/MdCHSpro:LUC and 35Spro:62-SK-MdCHSpro:LUC

Fig. 5. DNA methylation analysis by McrBC-PCR and bisulfite sequencing.

A, B DNA methylation analysis by McrBC-PCR. Genomic DNAs from both Fuji and mutant (M_Fuji) apple skins were treated with McrBC digestion reactions with (+) or without (−) GTP. The promoters and CDSs of MdMYB1 (A) and MdMYB90-like (B) of both Fuji and the mutant (M_Fuji) were divided into fourteen regions, and each fragment was PCR-amplified. The numbers denote the start and end positions of each fragment relative to the “A” nucleotide (+1) of the translation initiation codon. C–H DNA methylation analysis by bisulfite sequencing. Types of cytosine methylation sites (CG, CHG, and CHH) in the −1997 to −1800 (C) and −1162 to −1009 (D) regions of the MdMYB90-like promoter; methylation levels in the −1997 to −1800 (E) and −1162 to −1009 (F) regions in MdMYB90-like promoters of both Fuji and mutant (M_Fuji) apple skins; methylation levels of individual cytosine across the two regions: −1997 to −1800 (G) and −1162 to −1009 (H) in both Fuji and the mutant (M_Fuji). The methylation level at each cytosine position represented the average of nine sequenced bisulfite-PCR clones. Three biological replications were performed, and the means and SEs of methylation levels (percentage of methylated nucleotides) were calculated. Asterisks (*) denote significant differences at the P < 0.05 level

Fig. 6. Regulatory network of anthocyanin biosynthesis in the Fuji apple mutant.

MdMYB90-like plays a key role in the regulation of anthocyanin biosynthesis. Arrows denote direct activation of downstream genes. The MYB-bHLH3-WD40 regulatory complex was grouped into a cycle. The arrow with a dotted line denotes possible gene activation