Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing

Abstract

Alfalfa (Medicago sativa L.) is a high quality leguminous forage. Drought stress is one of the main factors that restrict the development of the alfalfa industry. High-throughput sequencing was used to analyze the microRNA (miRNA) profiles of alfalfa plants treated with CK (normal water), PEG (polyethylene glycol-6000; drought stress), and PEG + SNP (sodium nitroprusside; nitric oxide (NO) sprayed externally under drought stress). We identified 90 known miRNAs belonging to 46 families and predicted 177 new miRNAs. Real-time quantitative fluorescent PCR (qRT-PCR) was used to validate high-throughput expression analysis data. A total of 32 (14 known miRNAs and 18 new miRNAs) and 55 (24 known miRNAs and 31 new miRNAs) differentially expressed miRNAs were identified in PEG and PEG + SNP samples. This suggested that exogenous NO can induce more new miRNAs. The differentially expressed miRNA maturation sequences in the two treatment groups were targeted by 86 and 157 potential target genes, separately. The function of target genes was annotated by gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) analysis. The expression profiles of nine selected miRNAs and their target genes verified that their expression patterns were opposite. This study has documented that analysis of miRNA under PEG and PEG + SNP conditions provides important insights into the improvement of drought resistance of alfalfa by exogenous NO at the molecular level. This has important scientific value and practical significance for the improvement of plant drought resistance by exogenous NO.

Keywords: alfalfa; differential expression; drought stress; exogenous nitric oxide; miRNAs.

Figure 1

The length distribution of sRNAs in the libraries of CK, PEG, and PEG + SNP in alfalfa.

Figure 2

Abundance of the top 10 highly-expressed conserved miRNAs in the seven alfalfa libraries.

Figure 3

Differentially-expressed conserved miRNAs upon different treatment in alfalfa. (a) Conserved miRNAs from CK and PEG samples. (b) Conserved miRNAs from CK and PEG + SNP samples. The upregulated miRNAs are showed in red, whereas downregulated miRNAs are shown in green.

Figure 3

Differentially-expressed conserved miRNAs upon different treatment in alfalfa. (a) Conserved miRNAs from CK and PEG samples. (b) Conserved miRNAs from CK and PEG + SNP samples. The upregulated miRNAs are showed in red, whereas downregulated miRNAs are shown in green.

Figure 4

Diagrams of alfalfa miRNAs identified in the three samples: (a) conserved miRNAs, (b) novel miRNAs.

Figure 5

The number of gene ontology (GO) terms in different comparisons.

Figure 6

qRT-PCR validation of nine miRNAs and target genes in alfalfa. (a in the x-coordinate) miRNA expression patterns from deep sequencing, (b in the x-coordinate) miRNA expression patterns from qRT-PCR, (c in the x-coordinate) target genes expression patterns.

Figure 6

qRT-PCR validation of nine miRNAs and target genes in alfalfa. (a in the x-coordinate) miRNA expression patterns from deep sequencing, (b in the x-coordinate) miRNA expression patterns from qRT-PCR, (c in the x-coordinate) target genes expression patterns.

Figure 6

qRT-PCR validation of nine miRNAs and target genes in alfalfa. (a in the x-coordinate) miRNA expression patterns from deep sequencing, (b in the x-coordinate) miRNA expression patterns from qRT-PCR, (c in the x-coordinate) target genes expression patterns.

Figure 6

qRT-PCR validation of nine miRNAs and target genes in alfalfa. (a in the x-coordinate) miRNA expression patterns from deep sequencing, (b in the x-coordinate) miRNA expression patterns from qRT-PCR, (c in the x-coordinate) target genes expression patterns.