Identification and characterization of salt-responsive novel miRNAs and their targets in O. sativa

Salinity is an inevitable environmental constraint leading to devastated crop productivity. Rice, being a glycophyte, is highly susceptible to salt stress specifically during early vegetative and late reproductive stages. To cope up, the plant has evolved a considerable degree of developmental plasticity so that the potential stress impacts are minimized. One such mechanism is driven by a class of endogenously expressed small RNAs, miRNAs, which have emerged as ubiquitous post-transcriptional gene regulatory molecules. Sequenced genome coupled with high throughput sequencing significantly advances our ability to unravel miRNA-guided stress tolerance mechanisms. Computational analysis revealed hundreds of miRNAs and their potential targets in different plant conditions. A total of eight conserved, and nine novel miRNAs were tested for their expression profiles in non-stressed and stressed conditions. All of these were found to be differentially expressing in different tissues and varied concentrations. Their presence was also checked in a distant wild relative (O. coarctata) and a halophyte (S. maritima). Targets of respective novel miRNAs were identified and the credibility was duly checked. Target prediction revealed several proteins directly or indirectly involved in imparting salt tolerance to the plant. Our study demonstrates genotype-specific miRNA regulation under salinity stress and evidence for their role in mediating expression of target genes for abiotic stress response. It can also be contemplated in developing transgenic crop cultivar which has increased salt stress tolerance

Transcriptome Analysis of the Response to NaCl in <i>Suaeda maritima</i> Provides an Insight into Salt Tolerance Mechanisms in Halophytes

<div><p>Although salt tolerance is a feature representative of halophytes, most studies on this topic in plants have been conducted on glycophytes. Transcriptome profiles are also available for only a limited number of halophytes. Hence, the present study was conducted to understand the molecular basis of salt tolerance through the transcriptome profiling of the halophyte <i>Suaeda maritima</i>, which is an emerging plant model for research on salt tolerance. Illumina sequencing revealed 72,588 clustered transcripts, including 27,434 that were annotated using BLASTX. Salt application resulted in the 2-fold or greater upregulation of 647 genes and downregulation of 735 genes. Of these, 391 proteins were homologous to proteins in the COGs (cluster of orthologous groups) database, and the majorities were grouped into the poorly characterized category. Approximately 50% of the genes assigned to MapMan pathways showed homology to <i>S</i>. <i>maritima</i>. The majority of such genes represented transcription factors. Several genes also contributed to cell wall and carbohydrate metabolism, ion relation, redox responses and G protein, phosphoinositide and hormone signaling. Real-time PCR was used to validate the results of the deep sequencing for the most of the genes. This study demonstrates the expression of protein kinase C, the target of diacylglycerol in phosphoinositide signaling, for the first time in plants. This study further reveals that the biochemical and molecular responses occurring at several levels are associated with salt tolerance in <i>S</i>. <i>maritima</i>. At the structural level, adaptations to high salinity levels include the remodeling of cell walls and the modification of membrane lipids. At the cellular level, the accumulation of glycinebetaine and the sequestration and exclusion of Na⁺ appear to be important. Moreover, this study also shows that the processes related to salt tolerance might be highly complex, as reflected by the salt-induced enhancement of transcription factor expression, including hormone-responsive factors, and that this process might be initially triggered by G protein and phosphoinositide signaling.</p></div

Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity.

Soil salinization is a serious problem for cultivation of rice, as among cereals rice is the most salt sensitive crop, and more than 40% of the total agricultural land amounting to approximately 80 million ha the world over is salt affected. Salinity affects a plant in a varieties of ways, including ion toxicity, osmotic stress and oxidative damage. Since miRNAs occupy the top place in biochemical events determining a trait, understanding their role in salt tolerance is highly desirable, which may allow introduction of the trait in the rice cultivars of choice through biotechnological interventions. High throughput sequencing of sRNAs in the root and shoot tissues of the seedlings of the control and NaCl treated Pokkali, a salt-tolerant rice variety, identified 75 conserved miRNAs and mapped 200 sRNAs to the rice genome as novel miRNAs. Expression of nine novel miRNAs and two conserved miRNAs were confirmed by Northern blotting. Several of both conserved and novel miRNAs that expressed differentially in root and/or shoot tissues targeted transcription factors like AP2/EREBP domain protein, ARF, NAC, MYB, NF-YA, HD-Zip III, TCP and SBP reported to be involved in salt tolerance or in abiotic stress tolerance in general. Most of the novel miRNAs expressed in the salt tolerant wild rice Oryza coarctata, suggesting conservation of miRNAs in taxonomically related species. One of the novel miRNAs, osa-miR12477, also targeted L-ascorbate oxidase (LAO), indicating build-up of oxidative stress in the plant upon salt treatment, which was confirmed by DAB staining. Thus, salt tolerance might involve miRNA-mediated regulation of 1) cellular abundance of the hormone signaling components like EREBP and ARF, 2) synthesis of abiotic stress related transcription factors, and 3) antioxidative component like LAO for mitigation of oxidative damage. The study clearly indicated importance of osa-miR12477 regulated expression of LAO in salt tolerance in the plant

Details of the raw data and the quality control used for the assembly of the control and 2.0% NaCl-treated (9 h) <i>S</i>. <i>maritima</i> libraries.

<p>Details of the raw data and the quality control used for the assembly of the control and 2.0% NaCl-treated (9 h) <i>S</i>. <i>maritima</i> libraries.</p

Relative expression of the proteins/enzymes involved in G protein and phosphoinositide signaling.

<p>The grey bars represent the sequencing results, while the corresponding black bars represent the results that were obtained by qPCR. The individual black bars, representing the qPCR data, are the means ± SD of six determinations (three technical replicates each for two biological samples). * and ** next to a column indicate significant changes in expression at <i>p</i> ≤ 0.05 and <i>p</i> ≤ 0.01, respectively. Ns = not significant.</p

Pie chart showing the influence of NaCl on the distribution of reads among the three GO components, i.e., biological process, molecular function and cellular component.

<p>Pie chart showing the influence of NaCl on the distribution of reads among the three GO components, i.e., biological process, molecular function and cellular component.</p

Top 20 Cellular component categories as revealed by the GO enrichment of the transcripts.

<p>A) The individual bars represent the percent contribution of the reads to the individual GO terms in decreasing order in the control and NaCl-treated plants. The individual bars in B) and C) represent increases and decreases, respectively, in the expression of the individual genes in the cellular components categories presented as changes in the read values (fold changes) in decreasing order in the plants following NaCl application.</p

Primary assembly statistics of the control and 2.0% NaCl-treated (9 h) <i>S</i>. <i>maritima</i> libraries generated using Trinity assembler.

<p>Primary assembly statistics of the control and 2.0% NaCl-treated (9 h) <i>S</i>. <i>maritima</i> libraries generated using Trinity assembler.</p

Secondary assembly statistics of the control and 2.0% NaCl-treated (9 h) <i>S</i>. <i>maritima</i> libraries following CD-HIT EST analysis.

<p>Secondary assembly statistics of the control and 2.0% NaCl-treated (9 h) <i>S</i>. <i>maritima</i> libraries following CD-HIT EST analysis.</p