Novel and conserved miRNAs in the halophyte Suaeda maritima identified by deep sequencing and computational predictions using the ESTs of two mangrove plants

Abstract

Background: Although miRNAs are reportedly involved in the salt stress tolerance of plants, miRNA profiling in plants has largely remained restricted to glycophytes, including certain crop species that do not exhibit any tolerance to salinity. Hence, this manuscript describes the results from the miRNA profiling of the halophyte Suaeda maritima, which is used worldwide to study salt tolerance in plants.

Results: A total of 134 conserved miRNAs were identified from unique sRNA reads, with 126 identified using miRBase 21.0 and an additional eight identified using the Plant Non-coding RNA Database. The presence of the precursors of seven conserved miRNAs was validated in S. maritima. In addition, 13 novel miRNAs were predicted using the ESTs of two mangrove plants, Rhizophora mangle and Heritiera littoralis, and the precursors of seven miRNAs were found in S. maritima. Most of the miRNAs considered for characterization were responsive to NaCl application, indicating their importance in the regulation of metabolic activities in plants exposed to salinity. An expression study of the novel miRNAs in plants of diverse ecological and taxonomic groups revealed that two of the miRNAs, sma-miR6 and sma-miR7, were also expressed in Oryza sativa, whereas another two, sma-miR2 and sma-miR5, were only expressed in plants growing under the influence of seawater, similar to S. maritima.

Conclusion: The distribution of conserved miRNAs among only 25 families indicated the possibility of identifying a greater number of miRNAs with increase in knowledge of the genomes of more halophytes. The expression of two novel miRNAs, sma-miR2 and sma-miR5, only in plants growing under the influence of seawater suggested their metabolic regulatory roles specific to saline environments, and such behavior might be mediated by alterations in the expression of certain genes, modifications of proteins leading to changes in their activity and production of secondary metabolites as revealed by the miRNA target predictions. Moreover, the auxin responsive factor targeted by sma-miR7 could also be involved in salt tolerance because the target is conserved between species. This study also indicated that the transcriptome of one species can be successfully used to computationally predict the miRNAs in other species, especially those that have similar metabolism, even if they are taxonomically separated.

Fig. 1

Abundance of the conserved miRNAs and their distribution in the miRNA families. Abundance is expressed in terms of the number of reads in the individual miRNA families in S. maritima in the controls and after exposure to 340 mM NaCl for 9 h. The numerical figs. at the top of the bars for the individual miRNA families are the numbers of miRNAs that were found to belong to these families out of 134 known miRNAs identified

Fig. 2

Changes in the abundance of select conserved miRNAs in S. maritima in response to exposure to 340 mM NaCl, which is represented as a fold change relative to the control level. a The conserved miRNAs that showed twofold or more change. b The conserved miRNAs reported to be salt stress responsive. Negative and positive values represent decrease and increase, respectively, in abundance of a miRNA in response to NaCl treatment. RPM- Reads per million; the values are of the control or treated reads, whichever the maximum. The homologous miRNAs of sma-miR165a*, sma-miR165a**, sma-miR166e⁺, sma-miR166e⁺⁺, sma-miR159a^# and sma-miR159a^## are ath-miR165a, aly-miR165a, bdi-miR166e, osa-miR166e, pta-miR159a and ath-miR159a respectively

Fig. 3

Changes in the expression of select conserved miRNAs in S. maritima in response to exposure to 340 mM NaCl, as determined by Northern blot analysis. The upper blot of each panel represents a hybridization signal of the anti-sense probe with a specific miRNA. U6 served as the loading control and is shown in the lower panel for the individual miRNA blot analyses. The signal intensities of U6 and miRNAs were analyzed densitometrically and plotted as a histogram representing relative changes in the hybridization intensities in the NaCl (340 mM) treated sample (filled bar) relative to the control sample (empty bar)

Fig. 4

Abundance of novel miRNAs in S. maritima. Abundance is expressed in terms of the numbers of reads in the controls and after exposure to 340 mM NaCl for 9 h

Fig. 5

Changes in the expression of select miRNAs in S. maritima in response to exposure to 340 mM NaCl as determined by stem-loop PCR. The bars for the individual miRNAs represent fold changes in their expression in response to 340 mM NaCl treatment relative to the mean expression level of the control plants. The data are the mean ± SD of six independent estimations. Asterisks in the individual columns indicate that NaCl-responsive changes in the expression of the respective miRNAs differed significantly from the control expression level at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***). ns = not significant

Fig. 6

Changes in the expression of sma-miR6 and sma-miR7 in the test plants in response to their exposure to 255 mM NaCl (O. sativa cv. Badami/Pokkali) or 340 mM NaCl (S. maritima) for 9 h. The bars for the individual miRNAs represent fold changes in their expression in the three test plants in response to NaCl treatment relative to the mean expression level in the control plants. The data are the mean ± SD of six independent estimations for S. maritima and 3 independent estimations for the other plants. Variations in the expression of the individual miRNAs in the four plants resulting from NaCl treatment were statistically analyzed with an ‘F’ test and Duncan’s multiple range test. The mean fold change in the expression of an miRNA (bars) of individual species marked with at least one common letter do not differ significantly at p ≤ 0.05

Fig. 7

Fold changes in the expression of the S. maritima transcripts targeted by the miRNAs identified in the plant after exposure to 340 mM NaCl for 9 h. The data are the mean ± SD of six independent estimations. The values less than one represent decreases, and values more than one represent increases in the expression/abundance of the target mRNAs in the NaCl-treated plants relative to the non-treated (control) plants. The details regarding the statistical significance are as in Fig. 5