Transcriptome Profiling Reveals Transcriptional Regulation by DNA Methyltransferase Inhibitor 5-Aza-2'-Deoxycytidine Enhancing Red Pigmentation in Bagged "Granny Smith" Apples (Malus domestica)

Abstract

The red color of apples (Malus domestica) is an attractive trait for consumers. The green skinned "Granny Smith" cultivar develops red pigmentation after bagging treatment. DNA methylation plays an important role in various developmental processes in plants. To explore the possible functions of DNA methylation in the pigmentation of bagged "Granny Smith" apples, we first analyzed the anthocyanin content of fruit skin following treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The results revealed an increase in anthocyanin content in bagged fruits following 5-aza-dC treatment, while no anthocyanins were detected in unbagged fruits. In addition, 8482 differentially expressed genes between 5-aza-dC-treated and control groups were identified in bagged fruits by RNA sequencing, including genes encoding transcription factors, enzymes related to anthocyanin accumulation, and methylases. Changes in the expression of these genes may be responsible for 5-aza-dC-induced red pigmentation in bagged fruits of "Granny Smith". The findings provide novel evidence for the involvement of DNA methylation in the red pigmentation of non-red-skinned apples.

Keywords: 5-aza-dC; DNA methylation; Granny Smith; MYB; anthocyanin; transcriptome.

Figure 1

Effect of 5-aza-2′-deoxycytidine (5-aza-dC) treatment on skin color and pigmentation in “Granny Smith” apples. (A) Changes in skin color. (B) Dynamic characteristics of cyanidin3-galactoside and (C) differences in chlorophyll content. Error bars indicate standard deviation (SD) obtained from four biological replicates. Different lowercase letters indicate significant differences between the treated and control groups of bagged or unbagged fruits by Tukey’s multiple range test (p < 0.05).

Figure 2

Venn diagrams of all genes and differentially expressed genes (DEGs) in skin samples between 5-aza-dC-treated and control groups of bagged “Granny Smith” apples. (A) Intersection of the Venn diagram showing all DEGs identified in pair analyses (CK1 vs. CK2 and T1 vs. T2). (B,C) Intersection of the Venn diagram showing up- and down-regulated DEGs between pairs, respectively.

Figure 3

Clusters of annotated Gene Ontology (GO) terms in the “biological process” category enriched in 3281 DEGs in “Granny Smith” apple skins. DEGs were classified into specific “biological process” categories using DAVID (available online: https://david.ncifcrf.gov) with high classification stringency (p < 0.05). The horizontal ordinate represents the number of genes in the category.

Figure 4

Simplified scheme and heat map of the expression of genes related to anthocyanin biosynthesis in “Granny Smith” apple skins. (A) Structural gene expression in “Granny Smith” apple skins. Straight-line and dashed-line arrows indicated continuous and discontinuous steps, respectively. (B) Regulatory gene expression in “Granny Smith” apple skins. Arrows with different colors indicated different regulatory genes. PAL, phenylalanine ammonia lyase; 4CL, 4-coumarate coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol-4-reductase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP-glucose: flavonoid-3-O-glucosyltransferase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase. Enzyme names, gene IDs, and expression patterns are indicated at the side of each step. The color scale on the right represents the log-transformed Fragments Per Kilobase of transcript per Million (FPKM) value.

Figure 5

Heat map representation of the expression patterns of genes encoding transcription factors in “Granny Smith” apple skins. bZIP, basic region/leucine zipper motif; GATA, GATA zinc finger domain; NAC, NAM/ATAF/CUC; WRKY, protein domain containing a tryptophan-arginine-lysine-tyrosine amino acid motif; SPL, SQUAMOSA promoter-binding protein-like; ATHB, homeodomain leucine zipper proteins. Columns and rows in the heat map represent samples collected at different time points for which bags were removed. The color scale on the right represents the log-transformed FPKM value.

Figure 6

Heat map representation of expression patterns of methylase genes in “Granny Smith” apple skins. DME, demethylase; ROS, repressor of gene silencing; DRM, DNA (cytosine-5)-methyltransferase; MET, methyltransferase. Columns and rows in the heat map represent samples collected at different time points for which bags were removed. The color scale on the right represents the log-transformed FPKM value.

Figure 7

Quantitative real-time PCR (qRT-PCR) analysis of DEGs in “Granny Smith” apple skins. (A) Transcript levels and qRT-PCR results of 13 selected genes identified in RNA-sequencing. The left y-axis indicates the corresponding expression data from RNA-seq (gray histogram). The right y-axis shows the relative gene expression level measured by qRT-PCR (black lines). The x-axis represents the time (days) after 5-aza-dC treatment. Bars represent standard error (SE; n = 4). (B) Comparison between the log₂ values of gene expression ratios obtained from RNA-seq and qRT-PCR methods.