Identification of Rapeseed MicroRNAs Involved in Early Stage Seed Germination under Salt and Drought Stresses

Abstract

Drought and salinity are severe and wide-ranging abiotic stresses that substantially affect crop germination, development and productivity, and seed germination is the first critical step in plant growth and development. To comprehensively investigate small-RNA targets and improve our understanding of miRNA-mediated post-transcriptional regulation networks during Brassica napus seed imbibition under drought and salt stresses, we constructed three small-RNA libraries from B. napus variety ZS11 embryos exposed to salt (200 mM NaCl, denoted "S"), drought (200 g L(-1) PEG-6000, denoted "D"), and distilled water (denoted "CK") during imbibition and sequenced them using an Illumina Genome Analyzer. A total of 11,528,557, 12,080,081, and 12,315,608 raw reads were obtained from the CK, D, and S libraries, respectively. Further analysis identified 85 known miRNAs belonging to 31 miRNA families and 882 novel miRNAs among the three libraries. Comparison of the D and CK libraries revealed significant down-regulation of six miRNA families, miR156, miR169, miR860, miR399, miR171, and miR395, whereas only miR172 was significantly up-regulated. In contrast, comparison of the S library with the CK library showed significant down-regulation of only two miRNA families: miRNA393 and miRNA399. Putative targets for 336, 376, and 340 novel miRNAs were successfully predicted in the CK, D, and S libraries, respectively, and 271 miRNA families and 20 target gene families [including disease resistance protein (DIRP), drought-responsive family protein (DRRP), early responsive to dehydration stress protein (ERD), stress-responsive alpha-beta barrel domain protein (SRAP), and salt tolerance homolog2 (STH2)] were confirmed as being core miRNAs and genes involved in the seed imbibition response to salt and drought stresses. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs as well as the predicted target genes. Our data suggest that diverse and complex miRNAs are involved in seed imbibition, indicating that miRNAs are involved in plant hormone regulation, and may play important roles during seed germination under salt- or drought-stress conditions.

Keywords: Brassica napus; drought stress; microRNA; salt stress; seed germination.

Figure 1

Size distribution of small RNAs in CK, D, and S libraries from rapeseed. CK, control; D, drought stress; S, salt stress.

Figure 2

Number of family members per miRNA family detected in the three libraries. Candidate miRNA families were considered together and grouped by their miRBase numerical identifiers. CK, control; D, drought stress; S, salt stress.

Figure 3

Abundance of conserved miRNAs in the CK, D, and S libraries in rapeseed. CK, control; D, drought stress; S, salt stress.

Figure 4

Diagram of novel miRNAs identified in three libraries. CK, control; D, drought stress; S, salt stress.

Figure 5

Diagram of significantly different expression novel miRNAs between CK&D, CK&S, and S&D. CK, control; D, drought stress; S, salt stress.

Figure 6

Quantitative RT-PCR validation of mature miRNAs. The expression profiles of mature miRNAs were consistent with the results obtained by small-RNA deep sequencing. The expression values presented are the means of three technical replicates. U6 was used for each sample as an endogenous control. (A,B) Represent the expression pattern of mature conserved and novel miRNAs, respectively.

Figure 7

Quantitative RT-PCR analyses of relative expression levels of various predicted target genes. The rapeseed housekeeping gene actin7 was used as the internal control. The values presented are the means of three technical replicates.