Publications of NIPGR Scientists

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Author: search.filters.author.Ram, HasthiSubject: search.filters.subject.Rice

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Now showing 1 - 7 of 7
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    A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice
    (Springer Nature Publishing AG, 2025) Yadav, Banita; Sardar, Shaswati; Yadav, Anil; Kumari, Annapurna; Gautam, Mohini; Mandlik, Rushil; Arora, Simran; Kumar, Shailesh; Jewaria, Pawan Kumar; Sonah, Humira; Deshmukh, Rupesh; Chinnusamy, Viswanathan; Ram, Hasthi
    Advent of CRISPR-Cas9 library approach has revolutionized the field of high throughput targeted mutagenesis in plants. By identifying an sgRNA spacer that can target multiple paralogous genes in a genome, higher-order knockout plants can be developed. Using this concept, we developed ten CRISPR-Cas9 pool libraries and generated higher-order knockout plants in rice. Towards this, firstly we identified genome-wide sets of genes which are co-expressed and have high sequence similarity and can be targeted by a single sgRNA. Based on the expression pattern, these genes were divided into ten groups, and subsequently ten CRISPR-Cas9 plasmid libraries were developed. One such library designed against seed-expressed genes was transformed into rice and higher-order knockout plants were developed. Genotyping revealed that around 90% T0 plants had editing, and among the edited plants majority of them were higher-order knockouts. Phenotypic analysis in the next generation discovered functions of several seed specific genes in grain length, width, number and 100-grain weight. By analyzing single and double mutants for two Agenet domain-containing proteins, we have discovered an epistatic interaction between them for grain development. Further application of our approach will help to uncover hidden functions of the targeted genes and accelerate functional genomics research in rice. The CRISPR-Cas9 library is a useful approach to generate higher-order knockout mutants and identify functions of the targeted genes in rice.
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    Genome-wide identification, in-silico characterisation and expression analysis of multiprotein bridging factor 1 gene family members in rice
    (Springer Nature Publishing AG, 2025) Bishnoi, Alka; Ram, Hasthi; Soni, Praveen
    The multiprotein bridging factor 1 (MBF1) proteins are evolutionarily conserved transcription co-factors. However, little is known about rice MBF1 gene family and its role. A genome-wide search led to the identification of two MBF1 genes in the rice genome. Their proteins contained characteristic MBF1 and helix-turn-helix domains. Phylogenetic analysis showed that they belong to two different groups. Exploration of publicly available rice transcriptome data revealed that OsMBF1b exhibits constitutively high transcript abundance in all tissues and developmental stages of rice with a little alteration in its expression. Contrarily, OsMBF1c exhibited a prominent alteration in its expression in response to environmental perturbations. Both OsMBF1s showed the highest expression in endosperm. Analysis of publicly-available rice transcriptome data also showed that both OsMBF1s have a role in response to different stresses, especially in heat. Transcript analysis using qRT-PCR confirmed heat inducibility of OsMBF1c in contrasting genotypes i.e. IR64 (heat sensitive) and Nagina 22 (heat tolerant). qRT-PCR also confirmed the drought inducibility of both genes in the IR64 genotype which is sensitive to drought stress also as revealed by analysis of different parameters. In-silico interaction study also indicated their role in heat response as a number of proteins required to cope with high temperatures were predicated to be their interacting partners. Several heat-responsive genes were found to co-express with OsMBF1s. In-silico promoter analysis revealed the occurrence of stress-responsive elements in their putative promoters. Interestingly, both OsMBF1s showed diurnal rhythmic expressions having peaks during the daytime when the temperature rises. Altogether, this study indicates an active role of OsMBF1s in thermotolerance in rice. This is the first report regarding the characterization of rice MBF1 members.
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    Meta-analysis of transcriptomics studies identifies novel attributes and set of genes involved in iron homeostasis in rice
    (Springer Nature Publishing AG, 2023) Shekhawat, Pooja Kanwar; Sardar, Shaswati; Yadav, Banita; Salvi, Prafull; Soni, Praveen; Ram, Hasthi
    Iron (Fe) is an important micronutrient for humans as well as for plant growth and development. Rice employs multiple mechanisms to counteract the negative effects of Fe deficiency and Fe toxicity. Previously, many transcriptomics studies have identified hundreds of genes affected by Fe deficiency and/or Fe toxicity. These studies are highly valuable to identify novel genes involved in Fe homeostasis. However, in the absence of their systematic integration, they remain underutilized. A systematic meta-analysis of transcriptomics data from such ten previous studies was performed here to identify various common attributes. From this meta-analysis, it is revealed that under Fe deficiency conditions, root transcriptome is more sensitive and exhibits greater similarity across multiple studies than the shoot transcriptome. Furthermore, under Fe toxicity conditions, upregulated genes are more reliable and consistent than downregulated genes in susceptible cultivars. The integration of data from Fe deficiency and Fe toxicity conditions helped to identify key marker genes for Fe stress. As a proof-of-concept of the analysis, among the genes consistently regulated in opposite directions under Fe deficiency and toxicity conditions, two genes were selected: a proton-dependent oligopeptide transporter (POT) family protein and Vacuolar Iron Transporter (VIT)-Like (VTL) gene, and validated their expression and sub-cellular localization. Since VIT genes are known to play an important role in Fe homeostasis in plants, the entire OsVTL gene family in rice was characterized. This meta-analysis has identified many novel candidate genes that exhibit consistent expression patterns across multiple tissues, conditions, and studies. This makes them potential targets for future research aimed at developing Fe-biofortified rice varieties, as well as varieties tolerant to sub-optimal Fe levels in soil.
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    Negative regulators of grain yield and mineral contents in rice: potential targets for CRISPR-Cas9-mediated genome editing
    (Springer Nature Publishing AG, 2023) Yadav, Banita; Majhi, Ashis; Phagna, Kanika; Meena, Mukesh Kumar; Ram, Hasthi
    Rice is a major global staple food crop, and improving its grain yield and nutritional quality has been a major thrust research area since last decades. Yield and nutritional quality are complex traits which are controlled by multiple signaling pathways. Sincere efforts during past decades of research have identified several key genetic and molecular regulators that governed these complex traits. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene knockout approaches has accelerated the development of improved varieties; however, finding out target gene with negative regulatory function in particular trait without giving any pleiotropic effect remains a challenge. Here, we have reviewed past and recent literature and identified important negative regulators of grain yield and mineral contents which could be potential targets for CRISPR-Cas9-mediated gene knockout. Additionally, we have also compiled a list of microRNAs (miRNAs), which target positive regulators of grain yield, plant stress tolerance, and grain mineral contents. Knocking out these miRNAs could help to increase expression of such positive regulators and thus improve the plant trait. The knowledge presented in this review would help to further accelerate the CRISPR-Cas9-mediated trait improvement in rice.
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    Heavy metal stress in rice: uptake, transport, signaling and tolerance mechanisms
    (John Wiley & Sons, 2021) Kaur, Ravneet; Das, Susmita; Bansal, Sakshi; Singh, Gurbir; Sardar, Shaswati; Dhar, Hena; Ram, Hasthi
    Heavy metal contamination of agricultural fields has become a global concern as it causes a direct impact on human health. Rice is the major food crop for almost half of the world population and is grown under diverse environmental conditions, including heavy metal-contaminated soil. In recent years, the impact of heavy metal contamination on rice yield and grain quality has been shown through multiple approaches. In this review article, different aspects of heavy metal stress, i.e. uptake, transport, signalling and tolerance mechanisms, are comprehensively discussed with special emphasis on rice. For uptake, some of the transporters have specificity to one or two metal ions, whereas many other transporters are able to transport many different ions. After uptake, the intercellular signalling is mediated through different signaling pathways involving the regulation of various hormones, alteration of calcium levels and the activation of Mitogen-Activated Protein kinases. Heavy metal stress signals from various intermediate molecules activate various transcription factors, which triggers the expression of various antioxidant enzymes. Activated antioxidant enzymes then scavenge various reactive oxygen species, which eventually leads to stress tolerance in plants. Non-enzymatic antioxidants, such as ascorbate, metalloids and even metal-binding peptides (metallothionein and phytochelatin) can also help to reduce metal toxicity in plants. Genetic engineering has been successfully used in rice and many other crops to increase metal tolerance and reduce heavy metals accumulation. A comprehensive understanding of uptake, transport, signalling and tolerance mechanisms will help to grow rice plants in agricultural fields with less heavy metal accumulation in grains.
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    Identification and molecular characterization of rice bran-specific lipases
    (Springer Nature Publishing AG, 2021) Bansal, Sakshi; Sardar, Shaswati; Sinha, Kshitija; Bhunia, Rupam Kumar; Katoch, Megha; Sonah, Humira; Deshmukh, Rupesh; Ram, Hasthi
    Key message Among the 113 lipases present in rice genome, bran and endosperm-specifc lipases were identifed and lipase activity for one of the selected lipase gene is demonstrated in yeast. Abstract: Rice bran is nutritionally superior than endosperm as it has major reservoirs of various minerals, vitamins, essential mineral oils and other bioactive compounds, however it is often under-utilized as a food product due to bran instability after milling. Various hydrolytic enzymes, such as lipases, present in bran causes degradation of the lipids present and are responsible for the bran instability. Here, in this study, we have systematically analyzed the 113 lipase genes present in rice genome, and identifed 21 seed-specifc lipases. By analyzing the expression of these genes in diferent seed tissues during seed development, we have identifed three bran-specifc and three endosperm-specifc lipases, and one lipase which expresses in both bran and endosperm tissues. Further analysis of these genes during seed maturation and seed germination revealed that their expression increases during seed maturation and decreases during seed germination. Finally, we have shown the lipase activity for one of the selected genes, LOC_Os05g30900, in heterologous system yeast. The bran-specifc lipases identifed in this study would be very valuable for engineering designer rice varieties having increased bran stability in post-milling.
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    Reference gene identification for gene expression analysis in rice under different metal stress
    (Elsevier B.V., 2021) Soni, Praveen; Shivhare, Radha; Kaur, Amandeep; Bansal, Sakshi; Sonah, Humira; Deshmukh, Rupesh; Giri, Jitender; Lata, Charu; Ram, Hasthi
    Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common approach to quantify changes in gene expression. Appropriate internal reference genes are essential for normalization of data of RT-qPCR. In the present study, we identified suitable reference genes for gene expression analysis in rice seedlings subjected to different heavy metal stresses such as deficiencies of iron and zinc and toxicities of cobalt, cadmium and nickel. First, from publically available RNA-Seq data we identified 10 candidate genes having stable expression. We also included commonly used house-keeping gene OsUBQ5 (Ubiquitin 5) in our analysis. Expression stability of all the 11 genes was determined by two independent tools, NormFinder and geNorm. Our results show that selected candidate reference genes have higher stability in their expression compared to that of OsUBQ5. Genes with locus ID LOC_Os03g16690, encoding an oxysterol-binding protein (OsOBP) and LOC_Os01g56580, encoding Casein Kinase_1a.3 (OsCK1a.3) were identified to be the most stably expressed reference genes under most of the conditions tested. Finally, the study reveals that it is better to use a specific reference gene for a specific heavy metal stress condition rather than using a common reference gene for multiple heavy metal stress conditions. The reference genes identified here would be very useful for gene expression studies under heavy metal stresses in rice.