Identification of MicroRNAs and Their Targets Associated with Fruit-Bagging and Subsequent Sunlight Re-exposure in the "Granny Smith" Apple Exocarp Using High-Throughput Sequencing

Abstract

Bagged fruits of green apple cultivar "Granny Smith" have been found to turn cardinal red after debagging during fruit-ripening in the Loess Plateau region of China. To understand this phenomenon at post-transcriptional level, we have investigated the roles of microRNAs (miRNAs) in response to debagging. Three small RNA libraries were primarily constructed from peels of "Granny Smith" apples subjected to bagging followed by sunlight re-exposure treatments (0, 6 h, 1 day) (debagging), and from peels of apples without any bagging treatments (0, 6 h, 1 day). 201 known miRNAs belonging to 43 miRNA families and 220 novel miRNAs were identified via high-throughput sequencing. Some miRNAs were found to be differentially expressed after debagging, which indicated that miRNAs affected anthocyanin accumulation through their target genes in mature apple. To further explore the effect of debagging on miRNAs regulating the expression of anthocyanin regulatory genes, four miRNAs and their target genes regulating anthocyanin accumulation, miR156, miR828, miR858, and miR5072, were compared between green cultivar "Granny Smith" and red cultivar "Starkrimson." Results showed that mdm-miR828 and mdm-miR858 regulated anthocyanin contents in both apple cultivars, while mdm-miR156 only affected anthocyanin accumulation in "Granny Smith," and miR5072 affected anthocyanin accumulation in "Starkrimson." Additional analysis of gene ontology for the differentially expressed miRNAs after debagging treatments and their predicted target genes showed that they were involved in photo-protective response after debagging from 0 h to 1 day; they might play important roles in fruit development and adaptation to high light stress.

Keywords: anthocyanin; apple; bagging treatment; high-throughput sequencing; microRNA; target gene expression.

Figure 1

Effect of debagging on fruit coloration of “Granny Smith” and “Starkrimson.” (A) Non-bagged (left) and debagged “Granny Smith” (right) at 0 h, 1, 2, 4, 6, 8 days; (B): Non-bagged (left) and debagged “Starkrimson” (right) at 0 h, 1, 2, 4, 6, 8 days. Note: In (A), the fruits in the left and right side in the first picture are debagged and non-bagged “Granny Smith,” respectively.

Figure 2

Length and size distribution of unique sequences in six libraries from apple peel.

Figure 3

Number of known miRNAs in different miRNA families in apple peel.

Figure 4

Heatmap of differentially expressed miRNAs among N1/T1, N2/T2, and N3/T3 under bagging treatment. Each column represents a stage, and color bar indicates relative expression level from high (red) to low (blue).

Figure 5

Heatmap of differentially expressed miRNAs among T1, T2, and T3 after debagging treatment. It shows expression levels of miRNAs. Each column represents a stage, and color bar indicates relative expression level from high (red) to low (blue).

Figure 6

GO analysis of differential expression miRNAs.

Figure 7

Quantitative analysis miRNAs and expression levels of their target genes in “Granny Smith.” Quantitative analysis of miRNAs and target genes expression level in “Granny Smith” apple peel by qRT-PCR at 0, 1, 2, 4, and 6 days. Error bars indicate SD obtained from three biological replicates.

Figure 8

Quantitative analysis miRNAs and expression levels of their target in “Starkrimson.” Quantitative analysis of miRNAs and target genes expression level in “Starkrimson” apple peel by qRT-PCR at 0, 1, 2, 4, and 6 days. Error bars indicate SD obtained from three biological replicates.