Publications of NIPGR Scientists

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Author: search.filters.author.Mathur, SaloniSubject: search.filters.subject.Degradome

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    Functional diversification of miR172 isoforms in tomato under abiotic stress
    (Elsevier B.V., 2024) Bansal, Chandni; Kumar, Adesh; Shrivastava, Monika; Mathur, Saloni
    Plant gene families have expanded many folds as opposed to animals to compensate for being sessile as well as having unique features like ability to photosynthesise. While different protein families are well characterised in plants, similar knowledge on miRNA families is still in its infancy. The MIR172 family plays important role in various plant development processes including vegetative to reproductive phase change, floral patterning, nodulation, and fruit ripening as well as also in response to different environmental cues. However, in-depth analysis of this family in tomato (Solanum lycopersicum) is limited. In this study, we identified four new MIR172 loci (Sly-MIR172a1/a2/e/f) and two new isoforms, other than those reported at the miRBase repository. The MIR172 family has expanded by segmental duplication events and is conserved between the wild (S. pennellii and S. pimpinellifolium) and the cultivated tomato varieties. However, phylogenetic analysis showed that S. pennellii formed the most divergent member within each clade and S. pimpinellifolium is closer to the cultivated varieties. Additionally, investigations in 42 plant species highlighted that miR172a/b is the most abundant form in the plant kingdom. In addition to the classical target Apetala2 (AP2), degradome analysis identified SEC14p-like phosphatidylinositol transfer family protein (SEC14p) as a novel target of Sly-miR172 that was validated using precursor:effector and target:reporter transient assays. Further, we report dual mode of Sly-miR172-mediated silencing of targets Sly-AP2 and Sly-SEC14p by post-transcriptional transcript cleavage as well as translational repression. Different members of Sly-MIR172s:Sly-AP2s and Sly-MIR172s:Sly-SEC14p exhibit inverse expression correlation in response to different abiotic stresses, suggesting their role in stress response. Functional investigation of MIR172 showed that tomato plants performed better in different abiotic stresses (heat, drought, and salt) upon MIR172 overexpression or target knock-down by virus-induced-gene-silencing. Conversely, when miRNA is chelated using short-tandem-target-mimic, the plants exhibit sensitivity to these stresses. Thus, SlymiR172 acts as a positive regulator while its targets Sly-AP2a and Sly-SEC14p as negative regulators of different abiotic stresses.
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    An integrated bioinformatics and functional approach for miRNA validation
    (Springer Nature Publishing AG, 2022) Rao, Sombir; Balyan, Sonia; Bansal, Chandni; Mathur, Saloni
    MicroRNAs (miRNAs) are small (20–24 nucleotides) non-coding ribo-regulatory molecules with significant roles in regulating target mRNA and long non-coding RNAs at transcriptional and post-transcriptional levels. Rapid advancement in the small RNA sequencing methods with integration of degradome sequencing has accelerated the understanding of miRNA-mediated regulatory hubs in plants and yielded extensive annotation of miRNAs and corresponding targets. However, it is becoming clear that large numbers of such annotations are questionable. Therefore, it is imperative to adopt reliable and strict bioinformatics pipelines for miRNA identification. Furthermore, sensitive methods are needed for validation and functional characterization of miRNA and its target(s). In this chapter, we have provided a comprehensive and streamlined methodology for miRNA identification and its functional validation in plants. This includes a combination of various in silico and experimental methodologies. To identify miRNA compendium from large-scale Next-Generation Sequencing (NGS) small RNA datasets, the miR-PREFeR (miRNA PREdiction From small RNA-Seq data) bioinformatics tool has been described. Also, a homology-based search protocol for finding members of a specific miRNA family has been discussed. The chapter also includes techniques to ascertain miRNA:target pair specificity using in silico target prediction from degradome NGS libraries using CleaveLand pipeline, miRNA:target validation by in planta transient assays, 5′ RLM-RACE and expression analysis as well as functional techniques like miRNA overexpression, short tandem target mimic and resistant target approaches. The proposed strategy offers a reliable and sensitive way for miRNA:target identification and validation. Additionally, we strongly promulgate the use of multiple methodologies to validate a miRNA as well as its target.
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    Inferring the regulatory network of the miRNA-mediated response to individual and combined heat and drought stress in tomato
    (Springer Nature Publishing AG, 2021) Bansal, Chandni; Balyan, Sonia; Mathur, Saloni
    Under natural environmental conditions, plants are prone to be challenged simultaneously by combination of stresses like heat and drought stress together, thus affecting their overall growth, development and reproduction. Moreover, future climatic conditions are predicted to be warmer and drier, thus, warranting deep understanding of the stress-responsive regulatory networks for developing stress-management strategies. The role of microRNAs (miRNAs) that are key regulators of different stress signalling cascades in such dual stress conditions using varieties growing in warmer climatic conditions is completely lacking. In this study, we have investigated the effect of drought, heat and the two stresses together (combined stress) on a heat-tolerant tomato (Solanum lycopersicum) variety by evaluating physiological parameters as well as, some stress-responsive miRNA-target modules. Taqman-based qRT-PCR miRNA expression analysis showed enhanced expression of sly-miR482d-3p, sly-miR172d-3p, sly-miR164b-3p, sly-miR398b in individual drought and heat stress with an additive upregulation effect under combined stresses. On the other hand, the expression of sly-miR397-5p and sly-miR396b-3p was less when these two stresses co-occurred than the individual stresses and an antagonistic response was observed for sly-miR166a expression in combined versus single stresses. Several high confidence miRNA targets (101) were identified in-silico using degradome data and were functionally annotated using Gene Ontology enrichment analysis into various stress regulatory networks. The comparative analysis confirmed the inverse expression regulation of the miRNA:target pairs for sly-miR398b:Solyc07g006180, sly-miR164b-3p:Solyc08g061500, sly-miR172d:Solyc04g049800, sly-miR396b-3p:Solyc01g102810 and sly-miR396b-3p:Solyc05g017930 under all the three stress conditions. Since miRNAs are highly conserved across diverse plant species, these miRNAs can be candidates for engineering climate resilient crop plants.
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    Novel insights into expansion and functional diversification of MIR169 family in tomato
    (Springer Nature Publishing AG, 2020) Rao, Sombir; Balyan, Sonia; Jha, Sarita; Mathur, Saloni
    MIR169 family is an evolutionarily conserved miRNA family in plants. A systematic in-depth analysis of MIR169 family in tomato is lacking. We report 18 miR169 precursors, annotating new loci for MIR169a, b and d, as well as 3 novel mature isoforms (MIR169f/g/h). The family has expanded by both tandem- and segmental-duplication events during evolution. A tandem-pair MIR169b/b-1 and MIR169b-2/h is polycistronic in nature coding for three MIR169b isoforms and a new variant miR169h, that is evidently absent in the wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets including RNA-binding protein, protein-phosphatase, aminotransferase, chaperone, tetratricopeptide-repeat-protein, and transcription factors ARF-9B and SEPELLATA-3 were established by efficient target cleavage in the presence of specific precursors as well as increased target abundance upon miR169 chelation by short-tandem-target-mimic construct in transient assays. Comparative antagonistic expression profiles of MIR169:target pairs suggest MIR169 family as ubiquitous regulator of various abiotic stresses (heat, cold, dehydration and salt) and developmental pathways. This regulation is partly brought about by acquisition of new promoters as demonstrated by promoter MIR169:GUS reporter assays as well as differential processivity of different precursors and miRNA cleavage efficiencies. Thus, the current study augments the functional horizon of MIR169 family with applications for stress tolerance in crops.