Identification of copper (Cu) stress-responsive grapevine microRNAs and their target genes by high-throughput sequencing

Abstract

MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs (sRNAs) that are 20-24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play important roles in plant growth, development and stress responses. With more copper (Cu) and copper containing compounds used as bactericides and fungicides in plants, Cu stress has become one of the serious environmental problems that affect plant growth and development. In order to uncover the hidden response mechanisms of Cu stress, two small RNA libraries were constructed from Cu-treated and water-treated (Control) leaves of 'Summer Black' grapevine. Following high-throughput sequencing and filtering, a total of 158 known and 98 putative novel miRNAs were identified in the two libraries. Among these, 100 known and 47 novel miRNAs were identified as differentially expressed under Cu stress. Subsequently, the expression patterns of nine Cu-responsive miRNAs were validated by quantitative real-time PCR (qRT-PCR). There existed some consistency in expression levels of Cu-responsive miRNAs between high throughput sequencing and qRT-PCR assays. The targets prediction of miRNAs indicates that miRNA may regulate some transcription factors, including AP2, SBP, NAC, MYB and ARF during Cu stress. The target genes for two known and two novel miRNAs showed specific cleavage sites at the 10th and/or 11th nucleotide from the 5'-end of the miRNA corresponding to their miRNA complementary sequences. The findings will lay the foundation for exploring the role of the regulation of miRNAs in response to Cu stress and provide valuable gene information for breeding some Cu-tolerant grapevine cultivars.

Keywords: Cu stress; grapevine; high-throughput sequencing; microRNAs.

Figure 1.

Cluster analysis of known miRNAs expression. The similar expression pattern in known miRNA level was grouped together. Red colours indicated that the expression of miRNA in Cu stress was higher than that of the control. Green colours indicated that the expression of miRNA in Cu stress was lower than that of the control. Grey indicated that there was at least one sample without expression of miRNA.

Figure 2.

Cluster analysis of novel miRNAs expression. The similar expression pattern in novel miRNA level was grouped together. Red colours indicated that the expression of miRNA in Cu stress was higher than that of the control. Green colours indicated that the expression of miRNA in Cu stress was lower than that of the control. Grey indicated that there was at least one sample without expression of miRNA.

Figure 3.

qRT-PCR validations of Cu-responsive known and novel vvi-miRNAs in grapevine. Reference gene was 5.8S rRNA. The known and novel miRNAs levels in control were arbitrarily set to 1. Each PCR reaction was repeated three times and standard error was shown with bars in the diagram.

Figure 4.

qRT-PCR validations of target genes for differentially expressed vvi-miRNAs in grapevine. The reference gene was Actin. The target genes with lower expression amounts were arbitrarily set to 1. Each PCR reaction was repeated three times and standard error was pointed with bars in the diagram.

Figure 5.

Verification of target genes for vvi-miRNAs by 5′-RLM-RACE. Each top strand (green) depicts a miRNA-complementary site in the target mRNA, and each bottom strand depicts the miRNA (black). Watson–Crick pairing (vertical dashes) and guanine–uracil wobble pairing (ellipses) are indicated. The arrows indicate the 5′ termini of mRNA fragments isolated from grapevine, as identified by cloned RLM-RACE products, with the frequency of clones shown. Only the cloned sequences that matched the correct gene and had 5′ ends within a 100 nt window centred on the miRNA validation are included. The partial mRNA sequences from the target genes were aligned with the miRNAs. The numbers indicate the fraction of cloned PCR products terminating at different positions.

Figure 6.

A schematic model for Cu-stress-responsive miRNAs and targets in grapevine. Brown boxes indicate downregulated miRNAs, yellow boxes indicate upregulated miRNAs and green ellipse boxes indicate the target genes.