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.

Transcriptome Analysis of the Response to NaCl in Suaeda maritima Provides an Insight into Salt Tolerance Mechanisms in Halophytes.

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 Suaeda maritima, 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 S. maritima. 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 S. maritima. 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.

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

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.

Identification and Characterization of Differentially Expressed Genes in Inferior and Superior Spikelets of Rice Cultivars with Contrasting Panicle-Compactness and Grain-Filling Properties.

Breeding programs for increasing spikelet number in rice have resulted in compactness of the panicle, accompanied by poor grain filling in inferior spikelets. Although the inefficient utilization of assimilate has been indicated as responsible for this poor grain filling, the underlying cause remains elusive. The current study utilized the suppression subtractive hybridization technique to identify 57 and 79 genes that overexpressed in the superior and inferior spikelets (with respect to each other), respectively, of the compact-panicle rice cultivar Mahalaxmi. Functional categorization of these differentially expressed genes revealed a marked metabolic difference between the spikelets according to their spatial location on the panicle. The expression of genes encoding seed storage proteins was dominant in inferior spikelets, whereas genes encoding regulatory proteins, such as serine-threonine kinase, zinc finger protein and E3 ligase, were highly expressed in superior spikelets. The expression patterns of these genes in the inferior and superior spikelets of Mahalaxmi were similar to those observed in another compact-panicle cultivar, OR-1918, but differed from those obtained in two lax-panicle cultivars, Upahar and Lalat. The results first suggest that the regulatory proteins abundantly expressed in the superior spikelets of compact-panicle cultivars and in both the superior and inferior spikelets of lax-panicle cultivars but poorly expressed in the inferior spikelets of compact-panicle cultivars promote grain filling. Second, the high expression of seed-storage proteins observed in the inferior spikelets of compact-panicle cultivars appears to inhibit the grain filling process. Third, the low expression of enzymes of the Krebs cycle in inferior spikelets compared with superior spikelets of compact-panicle cultivars is bound to lead to poor ATP generation in the former and consequently limit starch biosynthesis, an ATP-consuming process, resulting in poor grain filling.