Publications of NIPGR Scientists

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Author: search.filters.author.Mathur, Saloni

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    Genome-wide DNA methylation dynamics at "heading" stage of panicle and flag leaf in contrasting rice cultivars under field drought conditions
    (Frontiers Media S.A., 2025) Jajo, Ringyao; Kansal, Shivani; Mathur, Saloni; Raghuvanshi, Saurabh
    Abstract Introduction: Drought stress induces widespread genome-wide alterations in DNA methylation of rice. These changes work to alter gene expression and are relatively unexplored in reproductive tissues like flag leaf and panicle under field drought conditions. This study aims to explore the same in the panicle and flag leaf tissue of IR64 (drought-sensitive) and N22 (drought-tolerant) rice cultivars under field-drought conditions during the 'heading' stage of development. Methods: For the same, we generated whole-genome bisulfite sequencing libraries from the corresponding tissues and analysed them in detail. Results and discussion: The DNA methylation dynamics in adult tissue (flowering stage) was found to be clearly distinct from that of the seedling stage. Further, the contrasting rice genotypes also exhibited cultivar-specific and drought-induced dynamism in the methylation signatures. Notably, the two cultivars demonstrate inherent distinctions in sequence preferences of hyper- and hypo-methylation even prior to experiencing drought stress, and these preferences persist under the influence of the stress. Approximately 90% of the drought-induced differentially methylated region (DMR) are cultivar-specific, and about 70% of the cultivar differences (cultivar-DMR) under stress are unique compared to control condition. There is higher prevalence of hyper-methylated DMR that co-localized with differentially expressed genes in panicle. DMR of CHH sequence exhibit stronger negative correlation with expression compared to CpG and CHG sequence. Examination of differentially expressed genes with DMR highlights their functional relevance under drought stress, especially with DMR found in gene bodies and promoter regions. Notably, in panicle, methylation divergence of the two cultivars influences flowering regulation genes. Additionally, the findings also suggest a regulatory role for DNA methylation in drought induced response of miRNA genes, particularly in the panicle of N22 cultivars.
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    The MIR169:NF-YA module enhances biomass and yield via ARGOS in Arabidopsis and tomato
    (John Wiley & Sons, 2025) Gupta, Apoorva; Rao, Sombir; Jha, Sarita; Ghosh, Debasish; Virmani, Richa; Shrivastava, Monika; Kumar, Adesh; Sahoo, Laxmipriaya; Das, Jaishri Rubina; Crespi, Martin; Mathur, Saloni
    Molecular links between miRNA: target modules regulating downstream genes for crop maturation/yield are poorly understood. Here, we report that elevated miR169d expression and concomitant reduced NF-YA2 (Nuclear Factor-Y subunit-A) target levels positively regulate vegetative growth and yield in Arabidopsis along with a shorter life cycle. In agreement, increased NF-YA2 levels in (1) NF-YA2-OE (overexpression) lines, (2) miR169d-target-mimicry lines (in which miR169d is chelated), and (3) miR169d-non-cleavable NF-YA2 resistant target lines show the opposite phenotype. Further, we find increased auxin levels in MIR169d-OE and nf-ya2 mutant lines, supporting the enrichment of ‘auxin terms’ in MIR169-OE transcriptome data. We show that ARGOS (auxin-regulated gene involved in organ size) is upregulated in MIR169d-OE due to reduced NF-YA2 repressor levels and that NF-YA2 directly binds the ARGOS promoter. Genetic screens of this module show that neither overexpressing miR169d in an argos mutant background nor the nf-ya2:argos double mutants rescue the argos mutant phenotype, suggesting a parallel pathway of ARGOS regulation via the MIR169:NF-YA2 node, independent of auxin. To assess the translational potential of this module in a crop, we show that Sly-MIR169-OE lines in tomato, having reduced target Sly-NF-YA10 levels, also regulate Sly-ARGOS resulting in early flowering, larger sized fruits, more fruit fresh weight, higher fruit set, early fruiting, and better shelf life than wild-type plants. In contrast, Sly-STTM169 plants inhibited for Sly-miR169 action and having increased levels of Sly-NF-YA10 have a longer life cycle with reduced biomass, decreased fruit set, and an overall reduction in yield. Thus, our findings show a conserved MIR169:NF-YA:ARGOS module which can be applied to crops for addressing future food demands.
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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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    Deciphering the role of MIR169d:NF-YA2 module under individual as well as combined drought and heat stress in Arabidopsis
    (Springer Nature Publishing AG, 2024) Gupta, Apoorva; Ghosh, Debasish; Rao, Sombir; Mathur, Saloni
    Plants are often subjected to a combination of abiotic stresses under natural environmental conditions. The response of plants to combined stresses can be very diferent from that to the individual stress. Several regulatory mechanisms work in harmony to maintain plant’s homeostasis during stress conditions. Among them the roles of microRNAs (miRNAs) in combined stresses are beginning to be unravelled. In this study, we evaluated the MIR169d: NF-YA2 target module in individual as well as combined drought and heat stress (HS) in Arabidopsis. We found that MIR169d is highly HS inducible, however, contrary to the reported downregulation of MIR169a/c forms in drought stress in literature, MIR169d is upregulated in drought. Moreover, while MIR169d expression is upregulated during combined stress, the response is less than individual stresses. Further, Arabidopsis plants overexpressing MIR169d or target nf-ya2 knockout mutant plants are more tolerant to both individual as well as combined heat and drought stress as indicated by the higher expression of stress responsive genes and less Trypan blue staining, while plants overexpressing NF-YA2 or those in which miR169defg isoform is sponged up (MIM169defg) are more prone to individual as well as combined heat and drought stress. The MIR169d promoter harbours both heat and drought stress-responsive cis-elements. Assessment of GUS expression in MIR169d-promoter:GUS and NF-YA2-promoter:GUS transgenic lines shows increased and reduced reporter expression in all the three stress conditions as compared to control, respectively. This suggests a stress-induced transcriptional regulation of the MIR169d:NF-YA2 pair. Thus, the MIR169d:NF-YA2 module can be potentially exploited to engineer crops for resistance to multiple abiotic stresses.
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    HSFA1a: the quarterback of heat stress response and 3D-chromatin organization
    (Elsevier B.V., 2023) Das, Jaishri Rubina; Mathur, Saloni
    In eukaryotes, the highly complex chromatin exhibits structural dynamism to modulate cellular responses. Recently, Huang et al. have shown a novel role of the heat stress master regulator, HSFA1a, in transiently reorganizing the 3D chromatin to promote distal and proximal enhancer-promoter contacts to regulate stress-responsive genes in tomato.
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    A conserved HSF:miR169:NF-YA loop involved in tomato and Arabidopsis heat stress tolerance
    (John Wiley & Sons, 2022) Rao, Sombir; Gupta, Apoorva; Bansal, Chandni; Sorin, Celine; Crespi, Martin; Mathur, Saloni
    Heat stress transcription factors (HSFs) and microRNAs (miRNAs) regulate different stress and developmental networks in plants. Regulatory feedbacks are at the basis of these networks. Here, we report that plants improve their heat stress tolerance through HSF-mediated transcriptional regulation of MIR169 and post-transcriptional regulation of Nuclear Factor- YA (NF-YA) transcription factors. We show that HSFs recognize tomato and Arabidopsis MIR169 promoters using yeast-one-hybrid/ChIP-qPCR. Silencing tomato HSFs using virus induced gene silencing (VIGS) reduced Sly-MIR169 levels and enhanced Sly-NF- YA9/A10 target expression. Further, Sly-NF-YA9/A10-VIGS knock-down tomato plants and Arabidopsis plants overexpressing At-MIR169d or At-nf-ya2 mutants showed a link with increased heat tolerance. In contrast, Arabidopsis plants overexpressing At-NF-YA2, or those expressing a non-cleavable At-NF-YA2 form (miR169d-resistant At-NF-YA2) as well as plants inhibited for At-miRNA169d regulation (miR169d mimic plants) were more sensitive to heat stress, highlighting NF-YA as negative regulator of heat tolerance. Furthermore, post-transcriptional cleavage of NF-YA by elevated miR169 levels resulted in alleviating the repression of heat stress effectors HSFA7 in tomato and Arabidopsis revealing a retroactive control of HSFs by the miR169:NF-YA node. Hence, a regulatory feedback loop involving HSFs, miR169s and NF-YAs plays a critical role in the regulation of heat stress response in tomato and Arabidopsis plants.
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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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    Cultivar-biased regulation of HSFA7 and HSFB4a govern high-temperature tolerance in tomato
    (Springer Nature Publishing AG, 2022) Rao, Sombir; Das, Jaishri Rubina; Balyan, Sonia; Verma, Radhika; Mathur, Saloni
    Heat shock factors (HSFs) are at the core of heat stress (HS) response in plants. However, the contribution of HSFs governing the inherent thermo-tolerance mechanism in tomato from sub-tropical hot climates is poorly understood. With the above aim, comparative expression profiles of the HSF family in a HS-tolerant (CLN1621L) and -sensitive cultivars (CA4 and Pusa Ruby) of tomato under HS revealed cultivar-biased regulation of an activator (HSFA7) and a repressor (HSFB4a) class HSF. HSFA7 exhibited strong upregulation while HSFB4a showed downregulation in tolerant tomato cultivar upon HS. Functional characterization of HSFA7 and HSFB4a in a tolerant–sensitive cultivar pair by virus-induced gene silencing (VIGS)-based silencing and transient overexpression established them as a positive and a negative regulator of HS tolerance, respectively. Promoter:GUS reporter assays and promoter sequence analyses suggest heat-mediated transcriptional control of both the HSF genes in the contrasting cultivars. Moreover, degradome data highlighted HSFB4a is a probable target of microRNA Sly-miR4200. Transient in-planta Sly-MIR4200-effector:HSFB4a-reporter assays showed miRNA-dependent target down-regulation. Chelation of miRNA by short-tandem-target-mimic of Sly-miR4200 increased target abundance, highlighting a link between Sly-miR4200 and HSFB4a. This miRNA has induced several folds upon HS in the tolerant cultivar where HSFB4a levels are reduced, thus exhibiting the inverse miR:target expression. Thus, we speculate that the alleviation of HSFB4a and increased HSFA7 levels govern thermo-tolerance in the tolerant cultivar by regulating downstream heat stress-responsive genes.
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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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    Variety-specific transcript accumulation during reproductive stage in drought- stressed rice
    (John Wiley & Sons, 2022) Gour, Pratibha; Kansal, Shivani; Agarwal, Priyanka; Mishra, Bhuwaneshwar Sharan; Sharma, Deepika; Mathur, Saloni; Raghuvanshi, Saurabh
    The divergence of natural stress tolerance mechanisms between species is an intriguing phenomenon. To study it in rice, a comparative transcriptome analysis was carried out in ‘heading’ stage tissue (flag leaf, panicles and roots) of Nagina 22 (N22; drought-tolerant) and IR64 (drought-sensitive) plants subjected to field drought. Interestingly, N22 showed almost double the number of differentially expressed genes (DEGs) than IR64. Many DEGs colocalized within drought-related QTLs responsible for grain yield and drought tolerance and also associated with drought tolerance and critical drought-related plant traits such as leaf rolling, trehalose content, sucrose and cellulose content. Besides, co-expression analysis of the DEGs revealed several ‘hub’ genes known to actively regulate drought stress response. Strikingly, 1366 DEGs, including 21 ‘hub’ genes, showed a distinct opposite regulation in the two rice varieties under similar drought conditions. Annotation of these variety-specific DEGs (VS-DEGs) revealed that they are distributed in various biological pathways. Furthermore, 103 VS-DEGs were found to physically interact with over 1300 genes, including 32 that physically interact with other VS-DEGs as well. The promoter region of these genes have sequence variations among the two rice varieties, which might be in part responsible for their unique expression pattern.