High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea

Abstract

Background: microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far.

Results: Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5'-RACE.

Conclusions: Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable lignin biosynthesis.

Figure 1

Length distribution of small RNAs in different libraries. nt, nucleotides. S-0 h, S-12 h, and S-7 d represent the shoots treated with 200 mM NaCl for 0 h, 12 h, and 7 d, respectively. R-0 h, R-12 h, and R-7 d denote the roots treated with 200 mM NaCl for 0 h, 12 h, and 7 d, respectively.

Figure 2

Number of conserved miRNAs in each family in S. europaea.

Figure 3

Differentially expressed S. europaea miRNAs after salt treatment or between the shoots and roots. A to E, Heat map of differentially expressed S. europaea miRNAs after salt treatment. These miRNAs were divided into five categories based on their expression patterns. (A) miRNAs down-regulated in salt-treated shoots. (B) miRNAs down-regulated in salt-treated roots. (C) miRNAs down-regulated in salt-treated shoots and roots. (D) miRNAs up-regulated by salt. (E) miRNAs dynamically regulated during salt treatment. Relative expression level was calculated using Log₂(RPM_Salt/RPM_{salt-0 h}). F to H, Heat map of differentially expressed S. europaea miRNAs between the shoots and roots. (F) miRNAs expressed higher in the roots than those in the shoots. (G) miRNAs expressed higher in the shoots than those in in the roots. (H) miRNAs distributed dynamically between the shoots and roots during salt treatment. Relative expression level was calculated with Log₂(RPM_root/RPM_shoot). S-0 h, S-12 h, and S-7 d represent the shoots treated with 200 mM NaCl for 0 h, 12 h, and 7 d, respectively. R-0 h, R-12 h, and R-7 d denote the roots treated with 200 mM NaCl for 0 h, 12 h, and 7 d, respectively.

Figure 4

Validation of expression profiles of miRNAs. (A) Heat map of sequencing (S) and stem-loop qRT-PCR data (P). Relative expression level was calculated using Log₂(Salt/Salt-0 h), and qRT-PCR data were averaged using the results from three technical repeats to represent three independent experiments. (B) Scatterplot of miRNA expression showing the correlation between deep sequencing (DS) and qRT-PCR (qRT) results. S-0 h, S-12 h, and S-7 d represent the shoots treated with 200 mM NaCl for 0 h, 12 h, and 7 d, respectively. R-0 h, R-12 h, and R-7 d denote the roots treated with 200 mM NaCl for 0 h, 12 h, and 7 d, respectively.

Figure 5

Functional classification of the target genes of conserved and novel miRNAs in S. europaea . Only the annotated target genes are shown. (A) Annotated target genes of conserved miRNAs. (B) Annotated target genes of novel miRNAs. The numbers of target genes are shown in the bracket.

Figure 6

Verification of miRNA-mediated target gene cleavage through 5′-RACE. For each miRNA, the partial sequence of the target unigene is shown at the top (blue) and the miRNA sequence at the bottom (red). The perfectly complementary bases are connected by solid lines, G:U wobble pairings are represented by ellipse, and the cleavage sites are shown by the arrows. Numbers indicate the fraction of cloned PCR products that were terminated at the cleavage site.

Figure 7

Potential regulatory network of salt-responsive miRNAs in S. europaea . Up- and down-regulated miRNAs are highlighted in red and green, respectively. MiR394 and miR11 were dynamically regulated, whereas the members of miR169 differentially changed. In addition, miR24 was only detected in S. europaea shoots treated with salt for 7 d.