Browsing by Author "Ram, Hasthi"

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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.
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 Raj
Rice, 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.
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, Rupesh
Prolonged 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.
Genome editing for nutritional improvement of crops
(John Wiley & Sons, 2024) Shekhawat, Pooja Kanwar; Ram, Hasthi; Soni, Praveen
Genome editing techniques have offered excellent opportunities to develop new crop varieties with enhanced growth, productivity, stress tolerance, and quality. Targeted alterations in nutrition-related traits can provide a big solution for mitigating hidden hunger and ensuring nutritional security. Compared to traditional breeding methods, these novel techniques have provided scientists the ability to manipulate desirable traits precisely and quickly. The advancements and breakthroughs in these techniques are making them more efficient and cost effective. In this chapter, we have discussed the application of these approaches in the improvement of key agronomic traits attributed to nutritional quality of staple food crops, oilseed crops, and horticulture plants.
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.
Global gene regulatory network underlying miR165a in Arabidopsis shoot apical meristem
(Springer Nature Publishing AG, 2023) Sinha, Sonali; Sahadevan, Sudeep; Ohno, Carolyn; Ram, Hasthi; Heisler, Marcus G
Arabidopsis microRNA165a (miR165a) targets Class III Homeodomain Leucine-Zipper (HD-ZIPIII) transcription factors to regulate various aspects of plant development and stress response. Overexpression of miR165a mimics the loss-of-function phenotype of HD-ZIPIII genes and leading to ectopic organ formation, shoot apical meristem (SAM) termination, loss of leaf polarity, and defective vasculature development. However, the molecular mechanisms underlying these phenotypes remain unresolved. Here, we over-expressed miR165a in a dexamethasone inducible manner and identifed diferentially expressed genes in the SAM through RNA-Seq. Simultaneously, using multichannel FACS combined with RNA-Seq approach, we characterized global transcriptome patterns in miR165a expressing cell-types compared to HD-ZIPIII expressing cell-types and other cell-types in SAM. By integrating our results we identifed sets of genes which are up-regulated by miR165a as well have enriched expression in miR165a cell-types, and vice-versa. Known plant development related genes such as HD-ZIPIII and their targets LITTLE ZIPPERs, Like AUXIN RESISTANT 2, BEL1-like homeodomain 6, ROTUNDIFOLIA like 16 were found to be down-regulated. Among the up-regulated genes, GIBBERELLIN 2-OXIDASEs, various elemental transporters (YSL3, ZIFL1, SULTR), and other transporter genes were prominent. Thus, the genes identifed in this study help to unravel the molecular mechanism of miR165a and HD-ZIPIII regulated plant development and stress response.
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.
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.
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.
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.
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.
Regulation of metalloid uptake in plants by transporters and their solute specificity
(Elsevier B.V., 2023) Sharma, Yogesh; Soni, Praveen; Raturi, Gaurav; Mandlik, Rushil; Rachappanavar, Vinay Kumar; Kumar, Manish; Salvi, Prafull; Tripathi, Durgesh Kumar; Ram, Hasthi; Deshmukh, Rupesh
Metalloids are a class of elements having properties like metals and non-metals which act as beneficial as well as hazardous for plant growth. Here, the precise role and molecular mechanism involved in the uptake and transport of different metalloids to different plant tissues is discussed. We have also described the efforts made to engineer the metalloid transport i.e. influx/efflux of metalloids that improve the uptake of beneficial metalloids and reduce hazardous metalloids. Metalloids like boron (B) and silicon (Si) play a beneficial role in plant growth and development. Optimum levels of these metalloids improve plant growth and immunity by various direct and indirect effects. On the other hand, metalloids like arsenic (As) and germanium (Ge) are detrimental for plants even at lower concentrations. The presence of these toxic entities inside cells disrupts cellular homeostasis by affecting the molecular, biochemical and physiological processes. The prospect to increase the uptake of beneficial metalloids and limit the hazardous metalloids and the challenges associated with the structural analogy and common transport mechanism is also discussed. The molecular insights into the biochemical and physiological aspects of metalloid transport and detoxification mechanisms will be helpful to exploit metalloid-derived benefits for crop improvement and accomplish food safety.
Rice lipases: a conundrum in rice bran stabilization: a review on their impact and biotechnological interventions
(Springer Nature Publishing AG, 2023) Bansal, Sakshi; Sundararajan, Sathish; Shekhawat, Pooja Kanwar; Singh, Shivangi; Soni, Praveen; Tripathy, Manas K.; Ram, Hasthi
Rice is a primary food and is one of the most important constituents of diets all around the world. Rice bran is a valuable component of rice, containing many oil-soluble vitamins, minerals, and oil. It is known for its ability to improve the economic value of rice. Further, it contains substantial quantities of minerals like potassium, calcium, magnesium, iron and antioxidants like tocopherols, tocotrienols, and γ-oryzanol, indicating that rice bran can be utilized efectively against several life-threatening disorders. It is difcult to fully utilize the necessary nutrients due to the presence of lipases in rice bran. These lipases break down lipids, specifcally Triacylglycerol, into free fatty acids and glycerol. This review discusses physicochemical properties, mechanism of action, distribution, and activity of lipases in various components of rice seeds. The phylogenetic and gene expression analysis helped to understand the diferential expression pattern of lipase genes at diferent growth phases of rice plant. Further, this review discusses various genetic and biotechnological approaches to decrease lipase activity in rice and other plants, which could potentially prevent the degradation of bran oil. The goal is to establish whether lipases are a major contributor to this issue and to develop rice varieties with improved bran stability. This information sets the stage for upcoming molecular research in this area.
Role of Serendipita indica in enhancing drought tolerance in crops
(Elsevier B.V., 2021) Jangir, Pooja; Shekhawat, Pooja Kanwar; Bishnoi, Alka; Ram, Hasthi; Soni, Praveen
The root endophytic fungus Serendipita indica (S. indica) acts as a bio-fertilizer, immuno-modulator, stress-buster and enhances plant growth, and protects the host plant from various biotic and abiotic stresses. Recent high-throughput omics approaches have helped in understanding the role of S. indica in host plants under stress conditions. The present review focuses on the mechanism of S. indica mediated drought-tolerance in crops by analyzing morphological, physiological, biochemical, and molecular processes. Interactions of S. indica with other plant growth-promoting microorganisms have been discussed. The knowledge gap and future prospects have been pointed out.
Serendipita indica: Harnessing its versatile potential for food and nutritional security
(Elsevier B.V., 2021) Shekhawat, Pooja Kanwar; Jangir, Pooja; Bishnoi, Alka; Roy, Shweta; Ram, Hasthi; Soni, Praveen
Quantity as well as quality of food is a major concern for increasing human population. Serendipita indica, a root endophytic fungus, has multifarious applications in agriculture. It increases nutrient and water uptake, carbon assimilation efficiency and stress-resistance which in turn enhances the growth and yield of plants. Moreover, it improves contents of minerals, fatty acids and essential oil, composition of oil, and fruit firmness in different plants. It also enhances the production of secondary metabolites having medicinal properties. This review highlights the role of Serendipita indica in increasing crop production and improving food quality. The underlying mechanism has also been illustrated.
Silicon supplementation as a promising approach to induce thermotolerance in plants: current understanding and future perspectives
(Springer Nature Publishing AG, 2023) Bishnoi, Alka; Jangir, Pooja; Shekhawat, Pooja Kanwar; Ram, Hasthi; Soni, Praveen
In the current situation of climate change, heat is the foremost abiotic stress that is fueling food insecurity by reducing crop production, especially in arid regions around the globe. Therefore, ecofriendly and sustainable solutions are needed to address this challenge. Recent findings have established silicon (Si) as an important stress reliever element in plants which tremendously improves their health under different environmental constraints. Exogenous application of Si via fertigation, foliar spray, or seed priming acts as a booster for the already existing defense machinery of plants to cope with the drastic effects of heat. Si fertigation also improves soil properties including its water holding capacity which indirectly aids to improve plant health. Rhizospheric microorganisms also contribute by increasing the bioavailability of Si in soil. Thus, versatile interactions of Si with soil, plant, and microbes modulate the micro-environment of plants exposed to heat stress which help in mitigating the heat-induced damage to plant growth and fertility. In this review, we focus on the elucidation of the role of Si in heat tolerance at the molecular level. Silicon-derived improvements in various morpho-agronomic, physiological, biochemical, anatomical, and molecular parameters have been discussed in detail. Si-uptake and transport mechanism has been addressed. We have also discussed the knowledge gaps and scope of Si as a biostimulant for future-oriented sustainable agriculture.
Vacuolar iron transporter (Like) proteins: Regulators of cellular iron accumulation in plants
(John Wiley & Sons, 2021) Ram, Hasthi; Sardar, Shaswati; Gandass, Nishu
Iron is not only important for plant physiology, but also a very important micronutrient in human diets. The vacuole is the main site for accumulation of excess amounts of various nutrients and toxic substances in plant cells. During the past decade, many Vacuolar Iron Transporter (VIT) and VIT‐Like (VTL) genes have been identified and shown to play important roles in iron homeostasis in different plants. Furthermore, recent reports identified novel roles of these transporter genes in Symbiotic Nitrogen Fixation (SNF) in legume crops as well as in the blue coloration of petals in flowers. The literature indicates their universal role in Fe transport across different tissues (grains, nodules, flowers) to different biological processes (cellular iron homeostasis, SNF, petal coloration) in different plants. Here, we have systematically reviewed different aspects, such as structure, molecular evolution, expression and function of VIT/VTL proteins. This will help future studies aimed at functional analysis of VIT/VTL genes in other plant species, vacuolar transportation mechanisms and iron biofortification at large.