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    AraNSdb: a dedicated database of stress-responsive non-coding RNAs in Arabidopsis thaliana
    (Springer Nature Publishing AG, 2026) Vivek, A.T.; Bhatia, Manika; Sahu, Namrata; Kalakoti, Garima; Kaushik, Love; Mukherjee, Kanka; Kumar, Shailesh
    Plants, as sessile organisms, are constantly exposed to biotic and abiotic stresses, making their ability to respond crucial for survival. Non-coding RNAs (ncRNAs) have emerged as key regulators in these stress responses, with several studies identifying numerous stress-responsive ncRNAs (SRNs). However, a comprehensive collection of SRNs derived from sequencing data in Arabidopsis thaliana has been lacking. To address this, we utilized high-throughput experimental data and mined published studies to construct AraNSdb (Arabidopsis ncRNA Stress Database), a systematic resource for storing and querying SRNs. AraNSdb documents over 1,000 expression profiles from diverse stress datasets, encompassing 6,616 SRNs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). The database features an intuitive web interface for exploring SRNs associated with specific stress types and provides detailed ncRNA annotations to support functional and regulatory studies. AraNSdb offers a valuable platform for advancing our understanding of ncRNA-mediated stress responses and is freely accessible at http://www.nipgr.ac.in/AraNSdb.
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    Comparative transcriptome profiling of two contrasting foxtail millet cultivars provides insights into molecular mechanisms underlying dehydration stress response
    (Springer Nature Publishing AG, 2023) Muthamilarasan, Mehanathan; Suresh, Bonthala Venkata; Singh, Roshan Kumar; Choudhary, Pooja; Aggarwal, Pooja Rani; Prasad, Manoj
    Foxtail millet (Setaria italica L.) has emerged as a model system to understand its adaptation to environmental stresses in the past decade. However, studies on understanding the molecular mechanism underlying the adaptation to dehydration stress and the regulatory network involved in the process remain elusive. In the present study, RNA-seq was performed during dehydration stress in the tolerant (IC4) and sensitive (IC41) cultivars at different time points (0, 6, and 12 h). A total of 2467 and 3318 differentially expressed genes (DEGs) were identified in IC4, and 2535 and 5572 in IC41 at 6 h and 12 h compared to control (0 h), respectively. Gene ontology (GO) analysis revealed that the DEGs were enriched in water transport, response to water deprivation, oxidative stress, amino acid and sugar transport, lipid biosynthesis, and regulation of stomatal opening. Pathway analysis suggested a significant modulation of genes involved in the metabolism of glutathione and tryptophan and biosynthesis of flavonoid, ascorbate, arginine, and proline in IC4 compared to IC41. Genes encoding for DIVARICATA, SBP family protein (teosinte glume architecture 1), and SRS family proteins (LATERAL ROOT PRIMORDIUM 1 and SHI-RELATED SEQUENCE 1) were found to be exclusively upregulated in IC4 during dehydration stress. Gene co-expression networks constructed based on the expression data showed the key modules and hubs that play critical roles during dehydration stress. Altogether, the present study has identified key genes, pathways, and regulatory modules that would serve as a base for further studies to gain insights into the dehydration-responsive molecular circuitry in foxtail millet.
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    Root hair-specific transcriptome reveals response to low phosphorus in Cicer arietinum
    (Frontiers Media S.A., 2022) Kohli, Pawandeep Singh; Pazhamala, Lekha T; Mani, Balaji; Thakur, Jitendra K.; Giri, Jitender
    Root hairs (RH) are a single-cell extension of root epidermal cells. In low phosphorus (LP) availability, RH length and density increase thus expanding the total root surface area for phosphate (Pi) acquisition. However, details on genes involved in RH development and response to LP are missing in an agronomically important leguminous crop, chickpea. To elucidate this response in chickpea, we performed tissue-specific RNA-sequencing and analyzed the transcriptome modulation for RH and root without RH (Root-RH) under LP. Root hair initiation and cellular differentiation genes like RSL TFs and ROPGEFs are upregulated in Root-RH, explaining denser, and ectopic RH in LP. In RH, genes involved in tip growth processes and phytohormonal biosynthesis like cell wall synthesis and loosening (cellulose synthase A catalytic subunit, CaEXPA2, CaGRP2, and CaXTH2), cytoskeleton/vesicle transport, and ethylene biosynthesis are upregulated. Besides RH development, genes involved in LP responses like lipid and/or pectin P remobilization and acid phosphatases are induced in these tissues summarizing a complete molecular response to LP. Further, RH displayed preferential enrichment of processes involved in symbiotic interactions, which provide an additional benefit during LP. In conclusion, RH shows a multi-faceted response that starts with molecular changes for epidermal cell differentiation and RH initiation in Root-RH and later induction of tip growth and various LP responses in elongated RH.
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    Computational methods for annotation of plant regulatory non-coding RNAs using RNA-seq
    (Oxford University Press, 2021) Vivek, A.T.; Kumar, Shailesh
    Plant transcriptome encompasses numerous endogenous, regulatory non-coding RNAs (ncRNAs) that play a major biological role in regulating key physiological mechanisms. While studies have shown that ncRNAs are extremely diverse and ubiquitous, the functions of the vast majority of ncRNAs are still unknown. With ever-increasing ncRNAs under study, it is essential to identify, categorize and annotate these ncRNAs on a genome-wide scale. The use of high-throughput RNA sequencing (RNA-seq) technologies provides a broader picture of the non-coding component of transcriptome, enabling the comprehensive identification and annotation of all major ncRNAs across samples. However, the detection of known and emerging class of ncRNAs from RNA-seq data demands complex computational methods owing to their unique as well as similar characteristics. Here, we discuss major plant endogenous, regulatory ncRNAs in an RNA sample followed by computational strategies applied to discover each class of ncRNAs using RNA-seq. We also provide a collection of relevant software packages and databases to present a comprehensive bioinformatics toolbox for plant ncRNA researchers. We assume that the discussions in this review will provide a rationale for the discovery of all major categories of plant ncRNAs.
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    Transcriptomic analysis suggests a key role for SQUAMOSA PROMOTER BINDING PROTEIN LIKE, NAC and YUCCA genes in the heteroblastic development of the temperate rainforest tree Gevuina avellana (Proteaceae)
    (John Wiley & Sons, 2016) Ostria-Gallardo, Enrique; Ranjan, Aashish; Zumstein, Kristina; Chitwood, Daniel H.; Kumar, Ravi; Townsley, Brad T.; Ichihashi, Yasunori; Corcuera, Luis J.; Sinha, Neelima R.
    Heteroblasty, the temporal development of the meristem, can produce diverse leaf shapes within a plant. Gevuina avellana, a tree from the South American temperate rainforest shows strong heteroblasty affecting leaf shape, transitioning from juvenile simple leaves to highly pinnate adult leaves. Light availability within the forest canopy also modulates its leaf size and complexity. Here we studied how the interaction between the light environment and the heteroblastic progression of leaves is coordinated in this species. We used RNA-seq on the Illumina platform to compare the range of transcriptional responses in leaf primordia of G. avellana at different heteroblastic stages and growing under different light environments. We found a steady up-regulation of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL), NAC, YUCCA and AGAMOUS-LIKE genes associated with increases in age, leaf complexity, and light availability. In contrast, expression of TCP, TPR and KNOTTED1 homeobox genes showed a sustained down-regulation. Additionally, genes involved in auxin synthesis/transport and jasmonate activity were differentially expressed, indicating an active regulation of processes controlled by these hormones. Our large-scale transcriptional analysis of the leaf primordia of G. avellana sheds light on the integration of internal and external cues during heteroblastic development in this species.
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    Genome-wide analysis and expression profiling suggest diverse roles of GH3 genes during development and abiotic stress responses in legumes
    (Frontiers Media S.A., 2015) Singh, Vikash K.; Jain, Mukesh; Garg, Rohini
    Growth hormone auxin regulates various cellular processes by altering the expression of diverse genes in plants. Among various auxin-responsive genes, GH3 genes maintain endogenous auxin homeostasis by conjugating excess of auxin with amino acids. GH3 genes have been characterized in many plant species, but not in legumes. In the present work, we identified members of GH3 gene family and analyzed their chromosomal distribution, gene structure, gene duplication and phylogenetic analysis in different legumes, including chickpea, soybean, Medicago, and Lotus. A comprehensive expression analysis in different vegetative and reproductive tissues/stages revealed that many of GH3 genes were expressed in a tissue-specific manner. Notably, chickpea CaGH3-3, soybean GmGH3-8 and -25, and Lotus LjGH3-4, -5, -9 and -18 genes were up-regulated in root, indicating their putative role in root development. In addition, chickpea CaGH3-1 and -7, and Medicago MtGH3-7, -8, and -9 were found to be highly induced under drought and/or salt stresses, suggesting their role in abiotic stress responses. We also observed the examples of differential expression pattern of duplicated GH3 genes in soybean, indicating their functional diversification. Furthermore, analyses of three-dimensional structures, active site residues and ligand preferences provided molecular insights into function of GH3 genes in legumes. The analysis presented here would help in investigation of precise function of GH3 genes in legumes during development and stress conditions.
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    Transcriptome profiling for discovery of genes involved in shoot apical meristem and flower development
    (Elsevier B.V., 2014) Singh, Vikash K.; Jain, Mukesh
    Flower development is one of the major developmental processes that governs seed setting in angiosperms. However, little is known about the molecular mechanisms underlying flower development in legumes. Employing RNA-seq for various stages of flower development and few vegetative tissues in chickpea, we identified differentially expressed genes in flower tissues/stages in comparison to vegetative tissues, which are related to various biological processes and molecular functions during flower development. Here, we provide details of experimental methods, RNA-seq data (available at Gene Expression Omnibus database under GSE42679) and analysis pipeline published by Singh and colleagues in the Plant Biotechnology Journal (Singh et al., 2013), along with additional analysis for discovery of genes involved in shoot apical meristem (SAM) development. Our data provide a resource for exploring the complex molecular mechanisms underlying SAM and flower development and identification of gene targets for functional and applied genomics in legumes.
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    Expanding frontiers in plant transcriptomics in aid of functional genomics and molecular breeding
    (John Wiley & Sons Ltd, 2014) Agarwal, Pinky; Parida, Swarup K.; Mahto, Arunima; Das, Sweta; Mathew, Iny Elizebeth; Malik, Naveen; Tyagi, Akhilesh K.
    The transcript pool of a plant part, under any given condition, is a collection of mRNAs that will pave the way for a biochemical reaction of the plant to stimuli. Over the past decades, transcriptome study has advanced from Northern blotting to RNA sequencing (RNA-seq), through other techniques, of which real-time quantitative polymerase chain reaction (PCR) and microarray are the most significant ones. The questions being addressed by such studies have also matured from a solitary process to expression atlas and marker-assisted genetic enhancement. Not only genes and their networks involved in various developmental processes of plant parts have been elucidated, but also stress tolerant genes have been highlighted. The transcriptome of a plant with altered expression of a target gene has given information about the downstream genes. Marker information has been used for breeding improved varieties. Fortunately, the data generated by transcriptome analysis has been made freely available for ample utilization and comparison. The review discusses this wide variety of transcriptome data being generated in plants, which includes developmental stages, abiotic and biotic stress, effect of altered gene expression, as well as comparative transcriptomics, with a special emphasis on microarray and RNA-seq. Such data can be used to determine the regulatory gene networks, which can subsequently be utilized for generating improved plant varieties.
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    A global view of transcriptome dynamics during flower development in chickpea by deep sequencing
    (John Wiley & Sons Ltd, 2013) Singh, Vikash K.; Garg, Rohini; Jain, Mukesh
    Measurement of gene expression can provide important clues about gene function and molecular basis of developmental processes. Here, we have analysed the chickpea transcriptome in vegetative and flower tissues by exploiting the potential of high-throughput sequencing to measure gene expression. We mapped more than 295 million reads to quantify the transcript abundance during flower development. We detected the expression of more than 90% genes in at least one tissue analysed. We found quite a large number of genes were differentially expressed during flower development as compared to vegetative tissues. Further, we identified several genes expressed in a stage-specific manner. Various transcription factor families and metabolic pathways involved in flower development were elucidated. The members of MADS-box family were most represented among the transcription factor genes up-regulated during various stages of flower development. The abundant expression of several well-known genes implicated in flower development in chickpea flower development stages confirmed our results. In addition, we detected the expression specificities of lineage-specific genes during flower development. The expression data presented in this study is the most comprehensive dataset available for chickpea as of now and will serve as resource for unraveling the functions of many specific genes involved in flower development in chickpea and other legumes.
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    RNA-seq for transcriptome analysis in non-model plants
    (Springer, 2013) Garg, Rohini; Jain, Mukesh
    Sequencing of mRNA using next-generation sequencing (NGS) technologies (RNA-seq) has the potential to reveal unprecedented complexity of the transcriptomes. The transcriptome sequencing of an organism provides quick insights into the gene space, opportunity to isolate genes of interest, development of functional markers, quantitation of gene expression, and comparative genomic studies. Although becoming cheaper, transcriptome sequencing still remains an expensive endeavor. Further, the assembly of millions and billions of RNA-seq reads to construct the complete transcriptome poses great informatics challenges. Here, first we outline various important issues from experimental design to data analysis, including various strategies of transcriptome assembly, which need substantial consideration for a successful RNA-seq experiment. Further, we describe a method for using RNA-seq to characterize the transcriptome of a plant species, taking the example of a legume crop plant chickpea. Our aim is to provide a quick start guide to the nonexpert researchers for NGS-based transcriptome analysis.