Potential Role of TIP3 Aquaporins in the Transport of H2O2 and Boric Acid During Seed Development and Germination in Soybean (Glycine max L.) Sreeja Sudhakaran, Vandana Thakral, Rushil Mandlik, Badal Mahakalkar, Yogesh Sharma, Virender Kumar, Rupesh Deshmukh, Tilak Raj Sharma, Humira Sonah Plant Cell and Environment, 2026 Tonoplast intrinsic proteins (TIPs) are the channel‐forming proteins predominantly found in the tonoplast of plant cells. Despite the identification of TIPs in numerous plant species, very less is known about the precise role of different TIP subgroups. In the present study, two genes belonging to the TIP3 subgroup were studied to understand tissue‐specific role and solute transport activity. The soybean GmTIP3‐1 and GmTIP3‐2 were found to be expressed exclusively in seeds. Unlike rest of the aquaporins (AQPs), the expression of GmTIP3s gradually increased during seed maturation. The GmTIP3s also show higher expression during the initiation of seed germination, suggesting their potential role in the solute transport during seed maturation and germination. Further, GmTIP3‐1 and GmTIP3‐2 were functionally characterised to understand the structure, pore morphology, pore hydrophobicity, sub‐cellular localization, and solute specificity. The solute specificity of TIPs is crucial in various physiological and developmental processes. Solute transport activity studied using yeast growth and survivability assay suggests that GmTIP3‐1 and GmTIP3‐2 can transport hydrogen peroxide (H2O2) and boric acid, both of which are known to play significant role in seed germination. The information provided here will help to understand the precise role of TIP3 genes in seed development and germination.
Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns Yanhao Cheng, Gen Li, Man Zhou, Rushil Mandlik, Doris Wang, Yiping Qi Journal of Integrative Plant Biology, 2026 Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR‐Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off‐target effects, base editors derived from the CRISPR‐Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a‐RRV variant to introduce herbicide‐resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a‐RRV. This intron‐containing Cas12a‐CBE shows substantial improvement in editing efficiency in rice, compared to the non‐intron counterparts. By contrast, the improvement of ABE with the intron‐containing dLbCas12a‐RRV is very limited, partly due to the already high baseline editing efficiency of the intron‐less dLbCas12a‐RRV ABE. Testing of these base editors in poplar shows elevated C‐to‐T base editing by dLbCas12a‐RRV–intron‐CBE. For A‐to‐G editing, ABEs built upon dLbCas12a‐RV and dLbCas12a‐RRV variants showed significant improvement over ABEs derived from wild‐type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a‐RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron‐containing Cas12a CBE and ABE barely introduce guide RNA‐dependent off‐target mutations. However, they can generate guide RNA‐independent off‐target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.
A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice Banita Yadav, Shaswati Sardar, Anil Yadav, Annapurna Kumari, Mohini Gautam, Rushil Mandlik, Simran Arora, Shailesh Kumar, Pawan Kumar Jewaria, Humira Sonah, Rupesh Deshmukh, Viswanathan Chinnusamy, Hasthi Ram BMC Plant Biology, 2025 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.
Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers Dhiôvanna Corrêia Rocha, Miracle Osazee Omoregbee, Danyel Fernandes Contiliani, Rushil Mandlik, Gen Li, Juliet Mascoveto, Gary Coleman, James N. Culver, Daniel Rodriguez Leal, Alessandra Alves de Souza, Yiping Qi Plant Cell Reports, 2025 Key message Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. Abstract CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium -mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.
Deciphering the role of monoacylglycerol lipases (MAGL) under abiotic stress and lipid metabolism in soybean (Glycine max L.) Virender Kumar, Rushil Mandlik, Surbhi Kumawat, Badal Mahakalkar, Nitika Rana, Yogesh Sharma, Nitika Rajora, Sreeja Sudhakaran, Sanskriti Vats, Rupesh Deshmukh, Henry T. Nguyen, Tilak Raj Sharma, Humira Sonah Plant Biotechnology Journal, 2025 SummaryMonoacylglycerol lipase (MAGL) is involved in the last step of triacylglycerol breakdown by hydrolysing the monoacylglycerol (MAG) to free fatty acid and glycerol. In the present study, 21 and 38 MAGL genes were identified in Glycine max (cultivated soybean) and Glycine soja (wild) genomes, respectively. Gene‐specific association performed using whole genome resequencing data by mixed linear model showed a significant association with total seed oil, linolenic, and oleic acid content. Subsequent haplotypic analysis revealed allelic variations for MAGL genes in soybean germplasm. Diversity analysis indicated a balancing selection of MAGL genes in cultivated soybean compared to wild soybean. Transient expression of three candidate MAGL proteins in Nicotiana tabacum leaves showed chloroplast‐specific localization, which is the site for fatty acid biosynthesis. An extensive transcriptomic evaluation revealed comparatively higher expression of five genes in soybean seeds, and nine genes showed higher expression under abiotic stress conditions like drought and heat. The quantitative real‐time PCR analysis of three candidate MAGL genes showed differential expression under arsenic and silicon supplementation. Gene co‐expression analysis showed the interaction of MAGL with diacylglycerol acyltransferase and triacylglycerol. In addition, confocal microscopy and fluorescence‐activated cell sorting (FACS) analysis of yeast expressing four GmMAGL genes showed altered lipid deposition, leading to smaller and more dispersed lipid droplets, suggesting its significant role in lipid metabolism. Manipulation of MAGL can be a pragmatic strategy to improve abiotic stress tolerance, likely by membrane lipid remodeling under environmental stress. Similarly, MAGL could be strategically utilized to enhance oil yield by regulating lipid metabolism.
A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants Yanhao Cheng, Gen Li, Aileen Qi, Rushil Mandlik, Changtian Pan, Doris Wang, Sophia Ge, Yiping Qi Plant Cell, 2025 Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants.
Challenges and Opportunities with CRISPR-Based Genome Editing in Legume Crops P Kumar, H Yadav, B Mahakalkar, R Mandlik, S Vats, V Thakral, V Kumar, ... Functional & Integrative Genomics 26 (1), 24 , 2026 2026
Enhancing CRISPR‐Cas12a base editing in plants with LbCas12a variants and introns Y Cheng, G Li, M Zhou, R Mandlik, D Wang, Y Qi Journal of Integrative Plant Biology , 2026 2026
Role of beneficial metalloids, silicon and selenium, in enhancing yield and quality in floricultural crops NS Gawade, MC Pagariya, PR Jadhav, MM Pk, NA Shinde, R Mandlik, ... Plant Science 364, 112971 , 2026 2026 Citations: 2
A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice B Yadav, S Sardar, A Yadav, A Kumari, M Gautam, R Mandlik, S Arora, ... BMC Plant Biology 25 (1), 1769 , 2025 2025
Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers DC Rocha, MO Omoregbee, DF Contiliani, R Mandlik, G Li, J Mascoveto, ... Plant Cell Reports 44 (11), 1-17 , 2025 2025 Citations: 2
Multigene engineering in plants: Technologies, applications, and future prospects R Rajput, BA Boone, R Mandlik, MT Islam, Y Qi, J Wang, R Barrangou, ... Biotechnology Advances, 108697 , 2025 2025 Citations: 7
Deciphering the role of monoacylglycerol lipases (MAGL) under abiotic stress and lipid metabolism in soybean (Glycine max L.) V Kumar, R Mandlik, S Kumawat, B Mahakalkar, N Rana, Y Sharma, ... Plant Biotechnology Journal , 2025 2025 Citations: 3
A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants Y Cheng, G Li, A Qi, R Mandlik, C Pan, D Wang, S Ge, Y Qi The Plant Cell 37 (4), koaf081 , 2025 2025 Citations: 8
Exploration of advanced omics tools and resources for the improvement of industrial oil crops B Mahakalkar, V Kumar, S Sudhakaran, V Thakral, S Vats, R Mandlik, ... Plant Science 352, 112367 , 2025 2025
Potential Role of TIP3 Aquaporins in the Transport of H 2 O 2 and Boric Acid During Seed Development and Germination in Soybean ( Glycine max L.) S Sudhakaran, V Thakral, R Mandlik, B Mahakalkar, Y Sharma, V Kumar, ... Plant, Cell & Environment , 2025 2025 Citations: 3
Evolutionary analysis of tonoplast intrinsic proteins (TIPs) unraveling the role of TIP3s in plant seed development S Sudhakaran, R Mandlik, S Kumawat, G Raturi, SK Gupta, SM Shivaraj, ... Plant Physiology and Biochemistry 215, 109022 , 2024 2024 Citations: 4
Transgenic approaches for accelerating breeding of ornamental crops S Sudhakaran, R Mandlik, P Singh, P Kumar, M Meghwal, B Mahakalkar, ... Ornamental horticulture: latest cultivation practices and breeding … , 2024 2024 Citations: 3
Genome-wide identification and characterisation of Aquaporin s in Rosa chinensis R Mandlik, S Sharma, P Rout, S Singh, G Raturi, N Rana, H Sonah, ... The Journal of Horticultural Science and Biotechnology 99 (3), 311-325 , 2024 2024 Citations: 2
Silicon, a quasi-essential element: Availability in soil, fertilizer regime, optimum dosage, and uptake in plants V Thakral, G Raturi, S Sudhakaran, R Mandlik, Y Sharma, SM Shivaraj, ... Plant Physiology and Biochemistry 208, 108459 , 2024 2024 Citations: 63
Genome-Wide Association Study (GWAS) for Identifying SNPs and Genes Related to Phosphate-Induced Phenotypic Traits in Tomato ( Solanum lycopersicum L.) HR Hakla, S Sharma, M Urfan, R Mandlik, S Kumawat, P Rajput, ... Plants 13 (3), 457 , 2024 2024 Citations: 13
Genome-Wide Association Study (GWAS) for Identifying SNPs and Genes Related to Phosphate-Induced Phenotypic Traits in Tomato (Solanum lycopersicum L.). Plants 2024, 13, 457 HR Hakla, S Sharma, M Urfan, R Mandlik, S Kumawat, P Rajput, ... 2024 Citations: 1
Identification of VrNIP2-1 aquaporin with novel selective filter regulating the transport of beneficial as well as hazardous metalloids in mungbean (Vigna radiata L.) V Thakral, Y Sharma, R Mandlik, S Kumawat, G Patil, H Sonah, P Isenring, ... Plant Physiology and Biochemistry 203, 108057 , 2023 2023 Citations: 12
Overexpression of peanut ( Arachis hypogaea L.) AhGRFi gene enhanced root growth inhibition under exogenous NAA treatment in Arabidopsis thaliana Z Zhang, SS Gangurde, S Chen, RR Mandlik, H Liu, R Deshmukh, J Xu, ... Frontiers in Plant Science 14, 1184058 , 2023 2023 Citations: 1
Deciphering the Role of Aquaporins Under Different Abiotic Stress Conditions in Watermelon ( Citrullus lanatus ) G Raturi, S Kumawat, R Mandlik, D Duhan, V Thakral, S Sudhakaran, ... Journal of Plant Growth Regulation 42 (5), 3137-3149 , 2023 2023 Citations: 10
Necessity and challenges for exploration of nutritional potential of staple-food grade soybean G Padalkar, R Mandlik, S Sudhakaran, S Vats, S Kumawat, V Kumar, ... Journal of food composition and analysis 117, 105093 , 2023 2023 Citations: 47
MOST CITED SCHOLAR PUBLICATIONS
Transcription factors as key molecular target to strengthen the drought stress tolerance in plants M Manna, T Thakur, O Chirom, R Mandlik, R Deshmukh, P Salvi Physiologia Plantarum 172 (2), 847-868 , 2021 2021 Citations: 391
Significance of silicon uptake, transport, and deposition in plants R Mandlik, V Thakral, G Raturi, S Shinde, M Nikolić, DK Tripathi, H Sonah, ... Journal of experimental botany 71 (21), 6703-6718 , 2020 2020 Citations: 294
Role of silicon in elevating resistance against sheath blight and blast diseases in rice (Oryza sativa L.) AP Sathe, A Kumar, R Mandlik, G Raturi, H Yadav, N Kumar, SM Shivaraj, ... Plant Physiology and Biochemistry 166, 128-139 , 2021 2021 Citations: 67
Outstanding questions on the beneficial role of silicon in crop plants SM Shivaraj, R Mandlik, JA Bhat, G Raturi, R Elbaum, L Alexander, ... Plant and Cell Physiology 63 (1), 4-18 , 2022 2022 Citations: 64
Silicon, a quasi-essential element: Availability in soil, fertilizer regime, optimum dosage, and uptake in plants V Thakral, G Raturi, S Sudhakaran, R Mandlik, Y Sharma, SM Shivaraj, ... Plant Physiology and Biochemistry 208, 108459 , 2024 2024 Citations: 63
Microbial remediation and plant-microbe interaction under arsenic pollution G Raturi, A Chaudhary, V Rana, R Mandlik, Y Sharma, V Barvkar, P Salvi, ... Science of the Total Environment 864, 160972 , 2023 2023 Citations: 62
Necessity and challenges for exploration of nutritional potential of staple-food grade soybean G Padalkar, R Mandlik, S Sudhakaran, S Vats, S Kumawat, V Kumar, ... Journal of food composition and analysis 117, 105093 , 2023 2023 Citations: 47
Versatile role of silicon in cereals: Health benefits, uptake mechanism, and evolution BN Devanna, R Mandlik, G Raturi, SS Sudhakaran, Y Sharma, S Sharma, ... Plant Physiology and Biochemistry 165, 173-186 , 2021 2021 Citations: 46
Targeting aquaporins to alleviate hazardous metal (loid) s imposed stress in plants S Vats, S Sudhakaran, A Bhardwaj, R Mandlik, Y Sharma, S Kumar, ... Journal of hazardous materials 408, 124910 , 2021 2021 Citations: 38
Silicon‐induced mitigatory effects in salt‐stressed hemp leaves R Berni, R Mandlik, JF Hausman, G Guerriero Physiologia plantarum 171 (4), 476-482 , 2021 2021 Citations: 36
Understanding aquaporin transport system, silicon and other metalloids uptake and deposition in bottle gourd (Lagenaria siceraria) S Kumawat, P Khatri, A Ahmed, S Vats, V Kumar, R Jaswal, Y Wang, P Xu, ... Journal of hazardous materials 409, 124598 , 2021 2021 Citations: 32
Understanding aquaporin regulation defining silicon uptake and role in arsenic, antimony and germanium stress in pigeonpea (Cajanus cajan) R Mandlik, P Singla, S Kumawat, P Khatri, W Ansari, A Singh, Y Sharma, ... Environmental Pollution 294, 118606 , 2022 2022 Citations: 24
Pinpointing genomic regions and candidate genes associated with seed oil and protein content in soybean through an integrative transcriptomic and QTL meta-analysis V Kumar, V Goyal, R Mandlik, S Kumawat, S Sudhakaran, G Padalkar, ... Cells 12 (1), 97 , 2022 2022 Citations: 21
Understanding the role of SWEET genes in fruit development and abiotic stress in pomegranate ( Punica granatum L.) S Kumawat, Y Sharma, S Vats, S Sudhakaran, S Sharma, R Mandlik, ... Molecular biology reports 49 (2), 1329-1339 , 2022 2022 Citations: 16
Genome-Wide Association Study (GWAS) for Identifying SNPs and Genes Related to Phosphate-Induced Phenotypic Traits in Tomato ( Solanum lycopersicum L.) HR Hakla, S Sharma, M Urfan, R Mandlik, S Kumawat, P Rajput, ... Plants 13 (3), 457 , 2024 2024 Citations: 13
Identification of VrNIP2-1 aquaporin with novel selective filter regulating the transport of beneficial as well as hazardous metalloids in mungbean (Vigna radiata L.) V Thakral, Y Sharma, R Mandlik, S Kumawat, G Patil, H Sonah, P Isenring, ... Plant Physiology and Biochemistry 203, 108057 , 2023 2023 Citations: 12
Genomic landscape highlights molecular mechanisms involved in silicate solubilization, stress tolerance, and potential growth-promoting activity of bacterium Enterobacter sp. LR6 G Raturi, Y Sharma, R Mandlik, S Kumawat, N Rana, H Dhar, DK Tripathi, ... Cells 11 (22), 3622 , 2022 2022 Citations: 11
Deciphering the Role of Aquaporins Under Different Abiotic Stress Conditions in Watermelon ( Citrullus lanatus ) G Raturi, S Kumawat, R Mandlik, D Duhan, V Thakral, S Sudhakaran, ... Journal of Plant Growth Regulation 42 (5), 3137-3149 , 2023 2023 Citations: 10
Regulation of metalloid uptake in plants by transporters and their solute specificity Y Sharma, P Soni, G Raturi, R Mandlik, VK Rachappanavar, M Kumar, ... Environmental and Experimental Botany 206, 105180 , 2023 2023 Citations: 10
A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants Y Cheng, G Li, A Qi, R Mandlik, C Pan, D Wang, S Ge, Y Qi The Plant Cell 37 (4), koaf081 , 2025 2025 Citations: 8
