Publications of NIPGR Scientists
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Item 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, HasthiAdvent 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.Item Dissecting the nutrient partitioning mechanism in rice grain using spatially resolved gene expression profiling(Oxford University Press, 2021) Ram, Hasthi; Singh, Anmol; Katoch, Megha; Kaur, Ravneet; Sardar, Shaswati; Palia, Shubham; Satyam, Rohit; Sonah, Humira; Deshmukh, Rupesh; Pandey, Ajay Kumar; Gupta, Ishaan; Sharma, Tilak RajRice, a staple food worldwide, contains varying amounts of nutrients in different grain tissues. The underlying molecular mechanism of such distinct nutrient partitioning remains poorly investigated. Here, an optimized rapid laser capture microdissection (LCM) approach was used to individually collect pericarp, aleurone, embryo and endosperm from grains 10 days after fertilization. Subsequent RNA-Seq analysis in these tissues identified 7760 differentially expressed genes. Analysis of promoter sequences of tissue-specific genes identified many known and novel cis-elements important for grain filling and seed development. Using the identified differentially expressed genes, comprehensive spatial gene expression pathways were built for accumulation of starch, proteins, lipids, and iron. The extensive transcriptomic analysis provided novel insights about nutrient partitioning mechanisms; for example, it revealed a gradient in seed storage protein accumulation across the four tissue types analysed. The analysis also revealed that the partitioning of various minerals, such as iron, is most likely regulated through transcriptional control of their transporters. We present the extensive analysis from this study as an interactive online tool that provides a much-needed resource for future functional genomics studies aimed to improve grain quality and seed development.Item 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, HasthiKey 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.Item 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, HasthiReal-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.Item Dynamic role of aquaporin transport system under drought stress in plants(Elsevier B.V., 2021) Shivaraj, SM; Sharma, Yogesh; Chaudhary, Juhi; Rajora, Nitika; Sharma, Shivani; Thakral, Vandana; Ram, Hasthi; Sonah, Humira; Singla-Pareek, Sneh L.; Sharma, Tilak Raj; Deshmukh, RupeshProlonged soil moisture deficit poses major threat to plant survival. Plants have evolved to withstand such condition by maintaining water status through adoptive mechanisms. Such mechanisms include modulation of Aquaporins (AQPs) activity. The AQPs are small integral membrane proteins which facilitate water movement across the cells. This review summarizes the important regulatory mechanisms controlling the dynamics of AQP activity to fine tune the plant water status under the water deficit condition. Numerous studies have shown differential AQP expression under drought stress in plants. Among the known AQP subfamilies, members of plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) showed most significant expression under drought condition. The activity, stability, and membrane targeting of these AQPs are known to be regulated at transcriptional as well as post-translational level. Drought induced transcription factors and hormones are also involved in direct or indirect transcriptional regulation. At post-translational level modifications such as phosphorylation, glycosylation, ubiquitination, gating and tetramerization play a role in regulation of the abundance and activity of AQP proteins. Understanding such regulatory mechanisms will help in exploration of AQPs to improve crop plants for sustainable agriculture under changing environmental conditions.Item Whole genome re-sequencing of soybean accession ec241780 providing genomic landscape of candidate genes involved in rust resistance(Bentham Science Publishers, 2020) Ratnaparkhe, Milind Balkrishna; Marmat, Niharika; Kumawat, Giriraj; Shivakumar, Maranna; Kamble, Viraj Gangadhar; Nataraj, Vennampally; Ramesh, Shunmugiah Veluchamy; Deshmukh, Milind Panjabrao; Singh, Ajay Kumar; Sonah, Humira; Deshmukh, Rupesh Kailasrao; Prasad, Manoj; Chand, Suresh; Gupta, SanjayBackground: In this study, whole genome re-sequencing of rust resistant soybean genotype EC241780 was performed to understand the genomic landscape involved in the resistance mechanism. Methods: A total of 374 million raw reads were obtained with paired-end sequencing performed with Illumina HiSeq 2500 instrument, out of which 287.3 million high quality reads were mapped to Williams 82 reference genome. Comparative sequence analysis of EC241780 with rust susceptible cultivars Williams 82 and JS 335 was performed to identify sequence variation and to prioritise the candidate genes. Results: Comparative analysis indicates that genotype EC241780 has high sequence similarity with rust resistant genotype PI 200492 and the resistance in EC241780 is conferred by the Rpp1 locus. Based on the sequence variations and functional annotations, three genes Glyma18G51715, Glyma18G51741 and Glyma18G51765 encoding for NBS-LRR family protein were identified as the most prominent candidate for Rpp1 locus. Conclusion: The study provides insights of genome-wide sequence variation more particularly at Rpp1 loci which will help to develop rust resistant soybean cultivars through efficient exploration of the genomic resource.Item Nitric oxide and hydrogen sulfide crosstalk during heavy metal stress in plants(John Wiley & Sons, 2020) Shivaraj, Sheelavanta Matha; Vats, Sanskriti; Bhat, Javid Akhter; Dhakte, Priyanka; Goyal, Vinod; Khatri, Praveen; Kumawat, Surbhi; Singh, Akshay; Prasad, Manoj; Sonah, Humira; Sharma, Tilak Raj; Deshmukh, RupeshGases such as ethylene, hydrogen peroxide (H2O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have been recognized as vital signaling molecules in plants and animals. Of these gasotransmitters, NO and H2S have recently gained momentum mainly because of their involvement in numerous cellular processes. It is therefore important to study their various attributes including their biosynthetic and signaling pathways. The present review provides an insight into various routes for the biosynthesis of NO and H2S as well as their signaling role in plant cells under different conditions, more particularly under heavy metal stress. Their beneficial roles in the plant's protection against abiotic and biotic stresses as well as their adverse effects have been addressed. This review describes how H2S and NO, being very small size molecules, can quickly pass through the cell membranes and triggers a multitude of responses to various factors, notably to various stress conditions like drought, heat, osmotic, heavy metal, and multiple biotic stresses. The versatile interactions between H2S and NO involved in the different molecular pathways have been discussed. In addition to the signaling role of H2S and NO, their direct role in post‐translational modifications is also considered. The information provided here will be helpful to better understand the multifaceted roles of H2S and NO in plants, particularly under stress conditions.