Browsing by Author "Rengasamy, Balakrishnan"

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Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples
(Springer Nature Publishing AG, 2024) Rengasamy, Balakrishnan; Manna, Mrinalini; Thajuddin, Nargis Begum; Sathiyabama, Muthukrishnan; Sinha, Alok Krishna
The impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing ofers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modifcation of plant’s genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the fnal product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe.
Characterization and expression profiling of PIN auxin efflux transporters reveal their role in developmental and abiotic stress conditions in rice
(Frontiers Media S.A., 2022) Manna, Mrinalini; Rengasamy, Balakrishnan; Ambasht, Navin Kumar; Sinha, Alok Krishna
The auxin efflux transporter proteins called PINs ferry auxin from its source to sinks in particular directions depending on their polar localizations in the plasma membrane, thus facilitating the development of the entire plant architecture. The rice genome has 12 PIN genes distributed over eight chromosomes. To study their roles in plant development, abiotic stress responsiveness, and shaping an auxin-dependent root architecture, a genome-wide analysis was carried out. Based on phylogeny, cellular localization, and hydrophilic loop domain size, the PINs were categorized into canonical and noncanonical PINs. PINs were found expressed in all of the organs of plants that emphasized their indispensable role throughout the plant's life cycle. We discovered that PIN5C and PIN9 were upregulated during salt and drought stress. We also found that regardless of its cellular level, auxin functioned as a molecular switch to turn on auxin biosynthesis genes. On the contrary, although PIN expression was upregulated upon initial treatment with auxin, prolonged auxin treatment not only led to their downregulation but also led to the development of auxin-dependent altered root formation in rice. Our study paves the way for developing stress-tolerant rice and plants with a desirable root architecture by genetic engineering
KRP3 stability controls rice plant architecture and productivity via MPK3-mediated phosphorylation
(John Wiley & Sons, 2026) Banerjee, Gopal; Jonwal, Sarvesh; Rengasamy, Balakrishnan; Pal, Uttam; Singh, Dhanraj; Mohit, Mohit; Sinha, Alok Krishna
Yield is a critical agronomic trait in cereal crops, shaped by factors like tiller and seed number, and seed weight. Understanding the factors governing these traits will help in improving the yield of plants. In this study, we identified an orphan gene, KRP3, belonging to cereal crops as a key regulator of rice plant architecture. Altered KRP3 protein homeostasis affected plant height, tiller number, and seed production, highlighting its role in maintaining rice plant vigor and productivity. The stability of the KRP3 protein is positively regulated by MPK3-mediated phosphorylation, as unphosphorylated KRP3 is targeted for degradation via the ubiquitin-proteasome pathway. Our findings reveal that the identified MPK3-KRP3 module operates as an S-phase checkpoint, modulating the pace of cell division in the actively dividing zones and maintaining a balance between cell division and elongation. These findings provide valuable insights for improving plant growth and grain yield in rice.
Nutrient and water availability influence rice physiology, root architecture and ionomic balance via auxin signalling
(John Wiley & Sons, 2025) Manna, Mrinalini; Rengasamy, Balakrishnan; Sinha, Alok Krishna
Water and soil nutrients are the vital ingredients of crop production, and their efficient uptake is essentially dependent on root development, majorly regulated by auxin. For a water-loving crop like rice, how water availability regulates nutrient acquisition, additionally, how ambient nutrient level modulates water uptake, and the role of auxin therein is not well studied. While investigating the cross-talks among these components, we found water to be essential for auxin re-distribution in roots and shaping the root architecture. We also found that supplementing rice seedlings with moderate concentrations of mineral nutrients facilitated faster water uptake and greater nutrient enrichment in leaves compared to adequate nutrient supplementation. Additionally, moderate nutrient availability favoured greater stomatal density, stomatal conductance, photosynthesis, transpiration rate and water use efficiency when water was not limiting. Further, auxin supplementation enhanced root formation in rice, while affecting their water uptake ability, photosynthesis and transpiration causing differential mineral-specific uptake trends. The present study uncovers the existence of an intricate crosstalk among water, nutrients and auxin signalling the knowledge of which will enable optimizing the growth conditions for speed breeding of rice and harnessing the components of auxin signalling to improve water and nutrient use efficiency of rice.
A rapid and robust colorimetric method for measuring relative abundance of auxins in plant tissues
(John Wiley & Sons, 2024) Manna, Mrinalini; Rengasamy, Balakrishnan; Sinha, Alok Krishna
Introduction Auxin estimation in plant tissues is a crucial component of auxin signaling studies. Despite the availability of various high-throughput auxin quantification methods like LC-MS, GC-MS, HPLC, biosensors, and DR5-gus/gfp-based assays, auxin quantification remains troublesome because these techniques are very expensive and technology intensive and they mostly involve elaborate sample preparation or require the development of transgenic plants. Objectives To find a solution to these problems, we made use of an old auxin detection system to quantify microbe derived auxins and modified it to effectively measure auxin levels in rice plants. Materials and methods Auxins from different tissues of rice plants, including root samples of seedlings exposed to IAA/TIBA or subjected to different abiotic stresses, were extracted in ethanol. The total auxin level was measured by the presently described colorimetric assay and counterchecked by other auxin estimation methods like LC-MS or gus staining of DR5-gus overexpressing lines. Results The presented colorimetric method could measure (1) the auxin levels in different tissues of rice plants, thus identifying the regions of higher auxin abundance, (2) the differential accumulation of auxins in rice roots when auxin or its transport inhibitor was supplied exogenously, and (3) the levels of auxin in roots of rice seedlings subjected to various abiotic stresses. The thus obtained auxin levels correlated well with the auxin levels determined by other methods like LC-MS or gus staining and the expression pattern of auxin biosynthesis pathway genes. Conclusions The auxin estimation method described here is simple, rapid, cost-effective, and sensitive and allows for the efficient detection of relative auxin abundances in plant tissues.
Regulation of photosynthesis by mitogen-activated protein kinase in rice: antagonistic adjustment by OsMPK3 and OsMPK6
(Springer Nature Publishing AG, 2023) Jonwal, Sarvesh; Rengasamy, Balakrishnan; Sinha, Alok Krishna
Photosynthesis is the basis of almost all life on earth and is the main component of crop yield that contributes to the carbohydrate partitioning to the grains. Maintaining the photosynthetic efficiency of plants in challenging environmental conditions by regulating the associated factors is a potential research arena which will help in the improvement of crop yield. Phosphorylation is known to play a pivotal role in the regulation of photosynthesis. Mitogen Activated Protein Kinases (MAPKs) cascade although known to regulate a diverse range of processes does not have any exact reported function in the regulation of photosynthesis. To elucidate the regulatory role of MAPKs in photosynthesis we investigated the changes in net photosynthesis rate and related parameters in DEX inducible over-expressing (OE) lines of two members of MAPK gene family namely, OsMPK3 and OsMPK6 in rice. Interestingly, significant changes were found in net photosynthesis rate and related physiological parameters in OsMPK3 and OsMPK6-OE lines compared to its wild-type relatives. OsMPK3 and OsMPK6 have regulatory effects on nuclear-encoded photosynthetic genes. Untargeted metabolite profiling reveals a higher accumulation of sugars and their derivatives in MPK6 overexpressing plants and a lower accumulation of sugars and organic acids in MPK3 overexpressing plants. The accumulation of amino acids was found in abundance in both MPK3 and MPK6 overexpressing plants. Understanding the effects of MPK3 and MPK6 on the CO2 assimilation of rice plants under normal growth conditions, will help in devising strategies that can be extended for crop improvement.
Revisiting rice transformation for a fail-safe protocol and its application for various gene functional and molecular studies
(Springer Nature Publishing AG, 2025) Manna, Mrinalini; Rengasamy, Balakrishnan; Reddy, Malireddy K.; Sinha, Alok Krishna
An efcient transformation and regeneration system is prerequisite for gene functional studies in rice, the staple food crop of the Asian subcontinent. Despite the availability of a large number of rice transformation protocols, it is hard to fnd a simple and minimum input based, but fail-safe protocol that ensures zero number of escapes (or the non-transformants). The fear of obtaining an unpredictable percentage of escapes at the end of rice tissue culture prompts carrying out multiple batches of transformation which not only consumes costly resources and time but also burdens one with screening of a large number of tissue culture derived plants. In the present study, we have described a simple but fail-safe rice transformation protocol for functional validation of the genes by overexpression and CRISPR-Cas9 mediated gene knockout systems. By taking the advantage of high transformability of japonica rice tissues, we have also revealed that the present rice transformation protocol can be efectively employed to visualize protein localization in various subcellular compartments of rice root and callus tissues, an alternative to conventional tobacco/onion peel infltration or protoplast transformation which are either time consuming or tricky. Additionally, the paper also discusses the importance of Southern blotting in gene overexpression studies, utility of non-conventional antibiotic selection approaches and signifcance of sgRNA designing for gene knockout studies in rice. Various troubleshooting advice are also being presented. Overall, the present protocol might serve as an excellent guide for functional validation of several genes present in the rice genome waiting to be explored.
Revisiting the role of MAPK signalling pathway in plants and its manipulation for crop improvement
(John Wiley & Sons, 2023) Manna, Mrinalini; Rengasamy, Balakrishnan; Sinha, Alok Krishna
The mitogen-activated protein kinase (MAPK) pathway is an important signalling event associated with every aspect of plant growth, development, yield, abiotic and biotic stress adaptation. Being a central metabolic pathway, it is a vital target for manipulation for crop improvement. In this review, we have summarised recent advancements in understanding involvement of MAPK signalling in modulating abiotic and biotic stress tolerance, architecture and yield of plants. MAPK signalling cross talks with reactive oxygen species (ROS) and abscisic acid (ABA) signalling events in bringing about abiotic stress adaptation in plants. The intricate involvement of MAPK pathway with plant's pathogen defence ability has also been identified. Further, recent research findings point towards participation of MAPK signalling in shaping plant architecture and yield. These make MAPK pathway an important target for crop improvement and we discuss here various strategies to tweak MAPK signalling components for designing future crops with improved physiology and phenotypes.
Root transcriptome of two contrasting indica rice cultivars uncovers regulators of root development and physiological responses
(Nature Publishing Group, 2016) Singh, Alka; Kumar, Pramod; Gautam, Vibhav; Rengasamy, Balakrishnan; Adhikari, Bijan; Udayakumar, Makarla; Sarkar, Ananda K.
The huge variation in root system architecture (RSA) among different rice (Oryza sativa) cultivars is conferred by their genetic makeup and different growth or climatic conditions. Unlike model plant Arabidopsis, the molecular basis of such variation in RSA is very poorly understood in rice. Cultivars with stable variation are valuable resources for identification of genes involved in RSA and related physiological traits. We have screened for RSA and identified two such indica rice cultivars, IR-64 (OsAS83) and IET-16348 (OsAS84), with stable contrasting RSA. OsAS84 produces robust RSA with more crown roots, lateral roots and root hairs than OsAS83. Using comparative root transcriptome analysis of these cultivars, we identified genes related to root development and different physiological responses like abiotic stress responses, hormone signaling, and nutrient acquisition or transport. The two cultivars differ in their response to salinity/dehydration stresses, phosphate/nitrogen deficiency, and different phytohormones. Differential expression of genes involved in salinity or dehydration response, nitrogen (N) transport, phosphate (Pi) starvation signaling, hormone signaling and root development underlies more resistance of OsAS84 towards abiotic stresses, Pi or N deficiency and its robust RSA. Thus our study uncovers gene-network involved in root development and abiotic stress responses in rice.
A simplified and improved protocol of rice transformation to cater wide range of rice cultivars
(Springer Nature Publishing AG, 2024) Rengasamy, Balakrishnan; Manna, Mrinalini; Jonwal, Sarvesh; Sathiyabama, Muthukrishnan; Thajuddin, Nargis Begum; Sinha, Alok Krishna
The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efciencies which necessities screening of large number of transgenic plants to fnd the suitable edits. In the present study, we have simplifed the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76–78 days. We also obtained about 90% regeneration efciency for both Japonica and Indica cultivars. The transformation efciency was about 97% in the case of Japonica and 69–83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efciency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efciency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.
The small RNA biogenesis in rice is regulated by MAP kinase-mediated OsCDKD phosphorylation
(John Wiley & Sons, 2024) Singh, Dhanraj; Verma, Neetu; Rengasamy, Balakrishnan; Banerjee, Gopal; Sinha, Alok Krishna
CDKs are the master regulator of cell division and their activity is controlled by the regulatory subunit cyclins and phosphorylation by the CAKs. However, the role of MAP kinases in regulating plant cell cycle or CDKs have not been explored. Here, we report that the MAP kinases OsMPK3, OsMPK4, and OsMPK6 physically interact and phosphorylate OsCDKD and its regulatory subunit OsCYCH in rice. MAP kinases phosphorylate CDKD at Ser-168 and Thr-235 residues in OsCDKD. The MAP kinase-mediated phosphorylation of OsCDKD is required for its activation to control the small RNA biogenesis. The phosphodead version of OsCDKD fails to activate the C-terminal domain of RNA Polymerase II, thereby negatively impacting small RNA transcription. Further, the overexpression lines of wild-type (WT) OsCDKD and phosphomimic OsCDKD show increased root growth, plant height, tiller number, panicle number, and seed number in comparison to WT, phosphodead OsCDKD-OE, and kinase-dead OsCDKD-OE plants. In a nutshell, our study establishes a novel regulation of OsCDKD by MAPK-mediated phosphorylation in rice. The phosphorylation of OsCDKD by MAPKs imparts a positive effect on rice growth and development by regulating miRNAs transcription.