Browsing by Author "Manna, Mrinalini"

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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
Mitogen-activated protein kinase 3/6 regulates the stability of AtIAA3 and AtIAA7 during auxin signaling in Arabidopsis
(Elsevier B.V., 2026) Noryang, Stanzin; Manna, Mrinalini; Verma, Neetu; Singh, Kirti; Tayyeba, Sumaira; Sinha, Alok Krishna
Auxin mediated Aux/IAA degradation is required to release the ARFs from the control of IAAs, and ARFs in the free forms perform their role of transcription activation or suppression in response to developmental ques. Auxin is known to tag IAAs for proteasomal degradation, but how this tagging is regulated has not been widely explored. Here we report that, in Arabidopsis, exogenous application of auxin activates MPK3/6 which in turn phosphorylate IAA3 and IAA7 at Ser-58 and Ser-26, respectively. Further, incubation of IAA3 and IAA7 with the protein extracts from auxin treated mpk3 or mpk6 single mutants increase the rate of degradation of IAAs. Consequently, the phospho-null mutants, IAA3S58A and IAA7S26A were observed to be comparatively more stable. Thus, MAP kinase-mediated phosphorylation destabilised IAA3 and IAA7 leading to their degradation. Additionally, over-expression of the phospho-dead mutant of IAA3 (35S:IAA3S58A) and complementation of iaa3 mutant with this phospho-dead mutant resulted in reduced primary root length because of increased stability and accumulation of IAA3. Interestingly, we found that a member of ARF, ARF7 regulated the expression of MPKs by binding to their respective promoters.
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.
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.
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.