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Thiourea priming enhances salt tolerance through co-ordinated regulation of microRNAs and hormones in Brassica juncea

View Article: PubMed Central - PubMed

ABSTRACT

Activation of stress tolerance mechanisms demands transcriptional reprogramming. Salt stress, a major threat to plant growth, enhances ROS production and affects transcription through modulation of miRNAs and hormones. The present study delineates salt stress ameliorating action of thiourea (TU, a ROS scavenger) in Brassica juncea and provides mechanistic link between redox, microRNA and hormones. The ameliorative potential of TU towards NaCl stress was related with its ability to decrease ROS accumulation in roots and increase Na+ accumulation in shoots. Small RNA sequencing revealed enrichment of down-regulated miRNAs in NaCl + TU treated roots, indicating transcriptional activation. Ranking analysis identified three key genes including BRX4, CBL10 and PHO1, showing inverse relationship with corresponding miRNA expression, which were responsible for TU mediated stress mitigation. Additionally, ABA level was consistently higher till 24 h in NaCl, while NaCl + TU treated roots showed only transient increase at 4 h suggesting an effective stress management. Jasmonate and auxin levels were also increased, which prioritized defence and facilitated root growth, respectively. Thus, the study highlights redox as one of the “core” components regulating miRNA and hormone levels, and also strengthens the use of TU as a redox priming agent for imparting crop resilience to salt stress.

No MeSH data available.


Distribution and differential expression of conserved/novel miRNAs and corresponding targets in B. juncea seedlings.The seedlings were grown hydroponically for 15 d and then subjected to different treatments such as control (1/2 MS), NaCl (125 mM), NaCl (125 mM) + TU (75 μM) and TU (75 μM). For NaCl + TU and TU alone treatments, 24 h pre-treatment with same concentration of TU was also given. At 4 h after treatment, root and shoot were harvested independently and subjected to small RNA sequencing using SOLiD platform. The differentially expressed miRNAs and target genes under different treatments were analyzed using venn-diagram, independently for root (A) and shoot (B). The heat-map represents the average change in miRNA and corresponding target gene expression from two biological replicates. The data was clustered into two distinct sets on the basis of up- (Cluster-A) and down-regulated (Cluster-B) miRNAs. The ranking analysis was performed using expression difference of target genes between NaCl + TU and TU and top-ranked genes along with corresponding miRNAs are highlighted as “bold red”. The absolute expression change can be found as Supplementary Table 10 (root) and 11 (shoot).
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f3: Distribution and differential expression of conserved/novel miRNAs and corresponding targets in B. juncea seedlings.The seedlings were grown hydroponically for 15 d and then subjected to different treatments such as control (1/2 MS), NaCl (125 mM), NaCl (125 mM) + TU (75 μM) and TU (75 μM). For NaCl + TU and TU alone treatments, 24 h pre-treatment with same concentration of TU was also given. At 4 h after treatment, root and shoot were harvested independently and subjected to small RNA sequencing using SOLiD platform. The differentially expressed miRNAs and target genes under different treatments were analyzed using venn-diagram, independently for root (A) and shoot (B). The heat-map represents the average change in miRNA and corresponding target gene expression from two biological replicates. The data was clustered into two distinct sets on the basis of up- (Cluster-A) and down-regulated (Cluster-B) miRNAs. The ranking analysis was performed using expression difference of target genes between NaCl + TU and TU and top-ranked genes along with corresponding miRNAs are highlighted as “bold red”. The absolute expression change can be found as Supplementary Table 10 (root) and 11 (shoot).

Mentions: The expression data of conserved (on the basis of RNAseq) and novel miRNAs (on the basis of stem-loop) were pooled together and their expression pattern was analysed using venn-diagram, independently for root (Fig. 3A and Supplementary Table 6) and shoot (Fig. 3B and Supplementary Table 7). The miRNA pattern in different treatments was found distinct as early as 4 h after treatment, which suggested that miRNA expression is very versatile and can be seen as the direct reflection of external treatment condition. For the entire set of differentially expressed miRNAs, target gene expressions were quantified through quantitative real-time PCR and analyzed using venn-diagram, independently for root (Fig. 3A and Supplementary Table 8) and shoot (Fig. 3B and Supplementary Table 9). In both the organs, proportion of elements shared between NaCl and NaCl + TU treatment were found more than NaCl + TU specific elements which clearly indicated that TU effect on restoration of gene expression was only partial. This also explained why we did not observe complete amelioration of growth phenotype under NaCl + TU treatment (Fig. 1). The expression data of miRNAs along with corresponding target genes were clustered together which yielded two distinct clusters: cluster-A and B representing up- and down-regulated miRNAs, respectively. The overview heat-map for these clusters reveals an inverse relationship between miRNA and target gene expression in root (Fig. 3C) and shoot (Fig. 3D). This validated the accuracy and robustness of target gene prediction pipeline used in the present study.


Thiourea priming enhances salt tolerance through co-ordinated regulation of microRNAs and hormones in Brassica juncea
Distribution and differential expression of conserved/novel miRNAs and corresponding targets in B. juncea seedlings.The seedlings were grown hydroponically for 15 d and then subjected to different treatments such as control (1/2 MS), NaCl (125 mM), NaCl (125 mM) + TU (75 μM) and TU (75 μM). For NaCl + TU and TU alone treatments, 24 h pre-treatment with same concentration of TU was also given. At 4 h after treatment, root and shoot were harvested independently and subjected to small RNA sequencing using SOLiD platform. The differentially expressed miRNAs and target genes under different treatments were analyzed using venn-diagram, independently for root (A) and shoot (B). The heat-map represents the average change in miRNA and corresponding target gene expression from two biological replicates. The data was clustered into two distinct sets on the basis of up- (Cluster-A) and down-regulated (Cluster-B) miRNAs. The ranking analysis was performed using expression difference of target genes between NaCl + TU and TU and top-ranked genes along with corresponding miRNAs are highlighted as “bold red”. The absolute expression change can be found as Supplementary Table 10 (root) and 11 (shoot).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382540&req=5

f3: Distribution and differential expression of conserved/novel miRNAs and corresponding targets in B. juncea seedlings.The seedlings were grown hydroponically for 15 d and then subjected to different treatments such as control (1/2 MS), NaCl (125 mM), NaCl (125 mM) + TU (75 μM) and TU (75 μM). For NaCl + TU and TU alone treatments, 24 h pre-treatment with same concentration of TU was also given. At 4 h after treatment, root and shoot were harvested independently and subjected to small RNA sequencing using SOLiD platform. The differentially expressed miRNAs and target genes under different treatments were analyzed using venn-diagram, independently for root (A) and shoot (B). The heat-map represents the average change in miRNA and corresponding target gene expression from two biological replicates. The data was clustered into two distinct sets on the basis of up- (Cluster-A) and down-regulated (Cluster-B) miRNAs. The ranking analysis was performed using expression difference of target genes between NaCl + TU and TU and top-ranked genes along with corresponding miRNAs are highlighted as “bold red”. The absolute expression change can be found as Supplementary Table 10 (root) and 11 (shoot).
Mentions: The expression data of conserved (on the basis of RNAseq) and novel miRNAs (on the basis of stem-loop) were pooled together and their expression pattern was analysed using venn-diagram, independently for root (Fig. 3A and Supplementary Table 6) and shoot (Fig. 3B and Supplementary Table 7). The miRNA pattern in different treatments was found distinct as early as 4 h after treatment, which suggested that miRNA expression is very versatile and can be seen as the direct reflection of external treatment condition. For the entire set of differentially expressed miRNAs, target gene expressions were quantified through quantitative real-time PCR and analyzed using venn-diagram, independently for root (Fig. 3A and Supplementary Table 8) and shoot (Fig. 3B and Supplementary Table 9). In both the organs, proportion of elements shared between NaCl and NaCl + TU treatment were found more than NaCl + TU specific elements which clearly indicated that TU effect on restoration of gene expression was only partial. This also explained why we did not observe complete amelioration of growth phenotype under NaCl + TU treatment (Fig. 1). The expression data of miRNAs along with corresponding target genes were clustered together which yielded two distinct clusters: cluster-A and B representing up- and down-regulated miRNAs, respectively. The overview heat-map for these clusters reveals an inverse relationship between miRNA and target gene expression in root (Fig. 3C) and shoot (Fig. 3D). This validated the accuracy and robustness of target gene prediction pipeline used in the present study.

View Article: PubMed Central - PubMed

ABSTRACT

Activation of stress tolerance mechanisms demands transcriptional reprogramming. Salt stress, a major threat to plant growth, enhances ROS production and affects transcription through modulation of miRNAs and hormones. The present study delineates salt stress ameliorating action of thiourea (TU, a ROS scavenger) in Brassica juncea and provides mechanistic link between redox, microRNA and hormones. The ameliorative potential of TU towards NaCl stress was related with its ability to decrease ROS accumulation in roots and increase Na+ accumulation in shoots. Small RNA sequencing revealed enrichment of down-regulated miRNAs in NaCl + TU treated roots, indicating transcriptional activation. Ranking analysis identified three key genes including BRX4, CBL10 and PHO1, showing inverse relationship with corresponding miRNA expression, which were responsible for TU mediated stress mitigation. Additionally, ABA level was consistently higher till 24 h in NaCl, while NaCl + TU treated roots showed only transient increase at 4 h suggesting an effective stress management. Jasmonate and auxin levels were also increased, which prioritized defence and facilitated root growth, respectively. Thus, the study highlights redox as one of the “core” components regulating miRNA and hormone levels, and also strengthens the use of TU as a redox priming agent for imparting crop resilience to salt stress.

No MeSH data available.