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

Gharat SA, Shaw BP - BMC Plant Biol. (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biotechnology Laboratory, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India. sachingharat113@gmail.com.

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.

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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
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Fig5: 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

Mentions: To further confirm the presence of miRNAs identified in the test species and their differential expression, a stem-loop PCR analysis (TaqMan assay) was performed using three representative conserved miRNAs and all of the novel identified miRNAs. All three conserved miRNAs showed amplification Ct values < 30 and were upregulated in response to NaCl treatment of the plants (Fig. 5), confirming the results obtained by the Northern blot analysis (Fig. 3). High upregulation was observed for sma-miR166a, which is similar to the result obtained with Northern blotting. Among the novel miRNAs showing Ct values < 30 (Additional file 7), sma-miR2 and sma-miR7 were downregulated and sma-miR6 was upregulated (Fig. 5), which is similar to the results of the abundance analysis (Fig. 4). The stem-loop PCR although demonstrated inconsistent results with that of the abundance analysis for the sma-miR5, it generally validated the results of the abundance analysis for the miRNAs and the Northern analysis for the conserved miRNAs. A paired t-test revealed the significant influence of NaCl on the expression of all of the miRNAs assessed by the TaqMan assay except for sma-miR159a (Fig. 5). Three predicted novel miRNAs (sma-miR1, sma-miR3 and sma-miR4) were not amplified in the stem-loop PCR. The traces of progress for one representative PCR for each novel miRNA are provided in Additional file 7.Fig. 5


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

Gharat SA, Shaw BP - BMC Plant Biol. (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696257&req=5

Fig5: 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
Mentions: To further confirm the presence of miRNAs identified in the test species and their differential expression, a stem-loop PCR analysis (TaqMan assay) was performed using three representative conserved miRNAs and all of the novel identified miRNAs. All three conserved miRNAs showed amplification Ct values < 30 and were upregulated in response to NaCl treatment of the plants (Fig. 5), confirming the results obtained by the Northern blot analysis (Fig. 3). High upregulation was observed for sma-miR166a, which is similar to the result obtained with Northern blotting. Among the novel miRNAs showing Ct values < 30 (Additional file 7), sma-miR2 and sma-miR7 were downregulated and sma-miR6 was upregulated (Fig. 5), which is similar to the results of the abundance analysis (Fig. 4). The stem-loop PCR although demonstrated inconsistent results with that of the abundance analysis for the sma-miR5, it generally validated the results of the abundance analysis for the miRNAs and the Northern analysis for the conserved miRNAs. A paired t-test revealed the significant influence of NaCl on the expression of all of the miRNAs assessed by the TaqMan assay except for sma-miR159a (Fig. 5). Three predicted novel miRNAs (sma-miR1, sma-miR3 and sma-miR4) were not amplified in the stem-loop PCR. The traces of progress for one representative PCR for each novel miRNA are provided in Additional file 7.Fig. 5

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biotechnology Laboratory, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India. sachingharat113@gmail.com.

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.

Show MeSH
Related in: MedlinePlus