Browsing by Author "Verma, Lokesh"

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A citrate efflux transporter important for manganese distribution and phosphorus uptake in rice
(John Wiley & Sons, 2023) Panchal, Poonam; Bhatia, Chitra; Chen, Yi; Sharma, Meenakshi; Bhadouria, Jyoti; Verma, Lokesh; Maurya, Kanika; Miller, Anthony J.; Giri, Jitender
The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 μm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.
CRISPR-Cas9 based plant genome editing: Significance, opportunities and recent advances
(Elsevier B.V., 2018) Soda, Neelam; Verma, Lokesh; Giri, Jitender
Precise genome editing is a quantum leap in the field of plant sciences. Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas9 protein have emerged as a powerful tool for precise genome editing. CRISPR-Cas9 system introduces small heritable mutations (indels) in the genome of an organism. This system also enables precise gene characterization in plants with complex genomes. Besides, it offers new opportunities of trait stacking, where addition of desirable traits or removal of undesirable traits can be achieved simultaneously in a single event. With CRISPR-Cas9 RNPs technology, raising transgene free genetically modified plants is within realm of possibility which would be helpful in addressing regulatory concerns of transgenic plants. Several new advancements have been made in this technology which has extended its applications in almost every aspect of plant science. For example, recently developed catalytically inactive dCas9 fused with transcriptional effector domains allows targeted activation or silencing of the gene of interest. Apart from this, dCas9 fused with fluorescent labels is a budding tool in chromatin imaging studies. In this review, we summarize these recent advancements in CRISPR/Cas system and methods for analyzing the induced mutations, and its implementations in crop improvement.
Identification of purple acid phosphatases in chickpea and potential roles of CaPAP7 in seed phytate accumulation
(Nature Publishing Group, 2017) Bhadouria, Jyoti; Singh, Ajit Pal; Mehra, Poonam; Verma, Lokesh; Srivastawa, Rishi; Parida, Swarup K.; Giri, Jitender
Purple acid phosphatases (PAPs) play important roles in phosphate (Pi) acquisition and utilization. These PAPs hydrolyze organic Phosphorus (P) containing compounds in rhizosphere as well as inside the plant cell. However, roles of PAPs in one of the most widely cultivated legumes, chickpea (Cicer arietnum L.), have not been unraveled so far. In the present study, we identified 25 putative PAPs in chickpea (CaPAPs) which possess functional PAP motifs and domains. Differential regulation of CaPAPs under different nutrient deficiencies revealed their roles under multiple nutrient stresses including Pi deficiency. Interestingly, most of the CaPAPs were prominently expressed in flowers and young pods indicating their roles in flower and seed development. Association mapping of SNPs underlying CaPAPs with seed traits revealed significant association of low Pi inducible CaPAP7 with seed weight and phytate content. Biochemical characterization of recombinant CaPAP7 established it to be a functional acid phosphatase with highest activity on most abundant organic-P substrate, phytate. Exogenous application of recombinant CaPAP7 enhanced biomass and Pi content of Arabidopsis seedlings supplemented with phytate as sole P source. Taken together, our results uncover the PAPs in chickpea and potential roles of CaPAP7 in seed phytate accumulation.
A lipid synthase maintains metabolic flux for jasmonate synthesis to regulate root growth and phosphate homeostasis
(Oxford University Press, 2025) Pandey, Mandavi; Verma, Lokesh; Kohli, Pawandeep Singh; Singh, Bhagat; Kochi, Abhijith; Giri, Jitender
Plants require phosphate (Pi) for proper growth and development but often face scarcity of this vital nutrient in the soil. Pi-starvation triggers membrane lipid remodeling to utilize the membrane phospholipid-bound Pi in plants. In this process, phospholipids are replaced by non-Pi-containing galactolipids (MGDG, DGDG) and sulfolipids. The galactolipids ratio (MGDG:DGDG) is suggested to influence jasmonic acid (JA) biosynthesis. However, how the MGDG:DGDG ratio, JA levels, and root growth are coordinated under Pi deficiency in rice (Oryza sativa) remains unknown. Here, we characterized DGDG synthase 1 (OsDGD1) for its role in regulating root development by maintaining metabolic flux for JA biosynthesis. We showed that OsDGD1 is responsive under low Pi and is under the direct control of Phosphate Starvation Response 2 (OsPHR2), the master regulator of low Pi adaptations. Further, OsDGD1 knockout (KO) lines showed marked phenotypic differences compared to the wild type (WT), including a significant reduction in root length and biomass, leading to reduced Pi uptake. Further, lipidome analyses revealed reduced DGDG levels in the KO line, leading to reduced membrane remodeling, thus affecting P utilization efficiency. We also observed an increase in the MGDG: DGDG ratio in KO lines, which enhanced the endogenous JA levels and signaling. This imbalance of JA in KO plants led to changes in auxin levels, causing drastic root growth inhibition. These findings indicate the critical role of OsDGD1 in maintaining optimum levels of JA during Pi deficiency for conducive root growth. Besides acting as signaling molecules and structural components, our study widens the role of lipids as metabolic flux controllers for phytohormone biosynthesis.
Monogalactosyl Diacylglycerol Synthase 3 affects phosphate utilization and acquisition in rice
(Oxford University Press, 2022) Verma, Lokesh; Bhadouria, Jyoti; Rupam, Bhunia Kumar; Singh, Shweta; Panchal, Poonam; Bhatia, Chitra; Eastmond, Peter J; Giri, Jitender
Galactolipids are essential for compensating for the loss of phospholipids by “membrane lipid remodeling” in plants under phosphorus (P) deficiency. Monogalactosyl diacylglycerol (MGDG) synthases catalyze the synthesis of MGDG which is further converted into digalactosyl diacylglycerol (DGDG), later replacing phospholipids in the extraplastidial membranes. However, the roles of these enzymes are not well explored in rice. In this study, the rice MGDG synthase gene, OsMGD3 was identified and functionally characterized. We showed that plant phosphate (Pi) status and transcription factor OsPHR2 are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout (KO) and overexpression (OE) lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared to WT, OsMGD3 KO lines displayed a reduction while OE lines showed an enhanced, Pi acquisition and utilization. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed the role of OsMGD3 in membrane lipid remodeling in addition to a role in regulating diacylglycerol and phosphatidic acid levels that affected the expression of Pi transporters. Our study highlights the role of OsMGD3 in affecting both internal P utilization and P acquisition in rice.
A novel glycerophosphodiester phosphodiesterase improves phosphate deficiency tolerance in rice
(John Wiley & Sons, 2019) Mehra, Poonam; Pandey, Bipin K.; Verma, Lokesh; Giri, Jitender
Soil phosphate (Pi) deficiency is major constraint for rice cultivation world‐wide. Cellular membranes account for one‐third of cellular P (Phosphorus) in the form of phospholipids. Therefore, remobilization of Pi from membrane phospholipids under Pi deficiency can be an important strategy to improve PUE (Phosphorus Use Efficiency). GDPDs (Glycerophosphodiester phosphodiesterases) hydrolyse intermediate product of phospholipid catabolism, glycerophosphodiesters to glycerol‐3‐phosphate (G3P); a precursor for P and non P‐lipid biosynthesis. Here, we show that OsGDPD2 is a Pi deficiency responsive gene which is transcriptionally regulated by OsPHR2. In silico analysis of active site residues and enzymatic assays confirmed phosphodiesterase activity of OsGDPD2. All overexpression lines showed higher GDPD activity, Pi content, root growth and biomass accumulation as compared to wild‐type. Conversely, silencing of OsGDPD2 led to decreased GDPD activity and Pi content. Notably, most of the P‐containing metabolites and fatty acids were elevated in transgenic lines. Further, quantitative analysis of polar lipids revealed higher accumulation of several classes of phospholipids and galactolipids in overexpression lines indicating a potential role of OsGDPD2 in de novo glycerolipid biosynthesis. Thus, present study provides insights into novel physiological roles of OsGDPD2 in low Pi acclimation in rice.
OsHAD1, a haloacid dehalogenase-like APase enhances phosphate accumulation
(American Society of Plant Biologists, 2017) Pandey, Bipin Kumar; Mehra, Poonam; Verma, Lokesh; Bhadouria, Jyoti; Giri, Jitender
Phosphorus (P) deficiency limits plant growth and crop yield. Since, plants can absorb only inorganic form of P (Pi), a large portion of soil P (organic and inorganic P complexes) remains largely unused. Here, we identified and characterized a PHR2 regulated; novel low Pi responsive haloacid dehalogenase (HAD)-like hydrolase, OsHAD1. While, OsHAD1 is a functional HAD protein having both acid phosphatase and phytase activity; it showed little homology with other known low Pi responsive HAD superfamily members. Recombinant OsHAD1 is highly active at acidic pH and dephosphorylates broad range of organic and inorganic P containing substrates including protein phosphates and Na-phytate. Exogenous application of recombinant OsHAD1 protein in growth media supplemented with phytate, led to marked increase in growth and total P content of Pi deficient WT rice seedlings. Further, overexpression of OsHAD1 in rice resulted in enhanced phosphatase activity, biomass, total and soluble P content in Pi deficient transgenic seedlings treated with phytate as restricted Pi source. Gene expression and metabolite profiling revealed enhanced Pi starvation responses such as upregulation of multiple genes involved in Pi uptake and solubilization, accumulation of organic acids, enhanced secretory phosphatase activity and depletion of ATP in overexpression lines as compared to WT. To elucidate the underlying regulatory mechanisms of OsHAD1, we performed in-vitro pull down assays which revealed association of OsHAD1 with protein kinases. We conclude that besides dephosphorylation of cellular organic-P, OsHAD1 in coordination with kinases may regulate phosphorylation status of downstream targets to accomplish Pi homeostasis under limited Pi supply.
OsJAZ11 regulates phosphate starvation responses in rice
(Springer Nature Publishing AG, 2021) Pandey, Bipin K.; Verma, Lokesh; Prusty, Ankita; Singh, Ajit Pal; Bennett, Malcolm J.; Tyagi, Akhilesh K.; Giri, Jitender; Mehra, Poonam
Jasmonic Acid (JA) is a key plant signaling molecule which negatively regulates growth processes including root elongation. JAZ (JASMONATE ZIM-DOMAIN) proteins function as transcriptional repressors of JA signaling. Therefore, targeting JA signaling by deploying JAZ repressors may enhance root length in crops. In this study, we overexpressed JAZ repressor OsJAZ11 in rice to alleviate the root growth inhibitory action of JA. OsJAZ11 is a low phosphate (Pi) responsive gene which is transcriptionally regulated by OsPHR2. We report that OsJAZ11 overexpression promoted primary and seminal root elongation which enhanced Pi foraging. Expression studies revealed that overexpression of OsJAZ11 also reduced Pi starvation response (PSR) under Pi limiting conditions. Moreover, OsJAZ11 overexpression also suppressed JA signaling and biosynthesis as compared to wild type (WT). We further demonstrated that the C-terminal region of OsJAZ11 was crucial for stimulating root elongation in overexpression lines. Rice transgenics overexpressing truncated OsJAZ11ΔC transgene (i.e., missing C-terminal region) exhibited reduced root length and Pi uptake. Interestingly, OsJAZ11 also regulates Pi homeostasis via physical interaction with a key Pi sensing protein, OsSPX1. Our study highlights the functional connections between JA and Pi signaling and reveals JAZ repressors as a promising candidate for improving low Pi tolerance of elite rice genotypes.
OsJAZ11 regulates spikelet and seed development in rice
(John Wiley & Sons, 2022) Mehra, Poonam; Pandey, Bipin K.; Verma, Lokesh; Prusty, Ankita; Singh, Ajit Pal; Sharma, Shivam; Malik, Naveen; Bennett, Malcolm J.; Parida, Swarup K.; Giri, Jitender; Tyagi, Akhilesh K.
Seed size is one of the major determinants of seed weight and eventually, crop yield. As the global population is increasing beyond the capacity of current food production, enhancing seed size is a key target for crop breeders. Despite the identification of several genes and QTLs, current understanding about the molecular regulation of seed size/weight remains fragmentary. In the present study, we report novel role of a jasmonic acid (JA) signaling repressor, OsJAZ11 controlling rice seed width and weight. Transgenic rice lines overexpressing OsJAZ11 exhibited up to a 14% increase in seed width and ~30% increase in seed weight compared to wild type (WT). Constitutive expression of OsJAZ11 dramatically influenced spikelet morphogenesis leading to extra glume-like structures, open hull, and abnormal numbers of floral organs. Furthermore, overexpression lines accumulated higher JA levels in spikelets and developing seeds. Expression studies uncovered altered expression of JA biosynthesis/signaling and MADS box genes in overexpression lines compared to WT. Yeast two-hybrid and pull-down assays revealed that OsJAZ11 interacts with OsMADS29 and OsMADS68. Remarkably, expression of OsGW7, a key negative regulator of grain size, was significantly reduced in overexpression lines. We propose that OsJAZ11 participates in the regulation of seed size and spikelet development by coordinating the expression of JA-related, OsGW7 and MADS genes.
Phosphate deficiency inducible OsGDPD5 affects root growth by regulating sugar-auxin crosstalk
(John Wiley & Sons, 2025) Verma, Lokesh; Pandey, Mandavi; Bhatia, Chitra; Mehra, Poonam; Singh, Bhagat; Giri, Jitender
Glycerophosphodiester phosphodiesterases (GDPDs) enzymes are known to be involved in phospholipids degradation pathways, where glycerophosphodiesters are hydrolyzed to glycerol-3-phosphate (G3P) and corresponding alcohol. In plants, GDPDs are involved in phosphate deficiency adaptive responses and have been shown to impact root length, but the precise mechanism remains unclear. This study focuses on the rice GDPD5 gene and its role in regulating primary root growth. Our research demonstrates that OsGDPD5 encodes a functional GDPD enzyme and could hydrolyze glycerophosphocholine and glycerophosphorylethanolamine. At transcriptional levels, OsGDPD5 is preferentially expressed in the root tip and regulated by transcription factor OsPHR2. We have used CRISPR/Cas9 to generate OsGDPD5 knock-out lines, allowing us to explore its role in root growth. Our findings show that osgdpd5 mutants had a shorter primary root, which could be restored to a normal level by the exogenous application of sugar or G3P. Further, knocking out OsGDPD5 alters endogenous levels of G3P and sugars, affecting auxin biosynthesis in the root and, ultimately, primary root growth. In this manner, OsGDPD5 has a crucial role in regulating physiological processes, specifically sugar and auxin signaling, which are known to be involved in root growth regulation in rice. Our research thus unraveled a link between rice phosphate deficiency-responsive lipid remodeling and root growth via sugar-hormone signaling.
Phosphate deficiency response and membrane lipid remodeling in plants
(Springer Nature Publishing AG, 2021) Verma, Lokesh; Rumi; Sinha, Alok Krishna; Giri, Jitender
Phosphorus (P) deficiency represents one of the significant constraints affecting plants growth and development. Plants have evolved with various adaptations to maximise the uptake of soil P and its distribution in the different organs. Membrane phospholipids store a significant amount of P as the cellular resource that is remobilised under the P deficiency condition. To utilise this P resource, plants initiate membrane lipid remodeling in which metabolic pathways are activated to allow Pi extraction from the phospholipids and reduce the P demand by increasing the synthesis of P-free lipids for replacing the phospholipids in the membrane. Many organisms, including non-photosynthetic/photosynthetic bacterium to plants employ this adaptation to survive under P deficiency conditions. The candidate genes and encoded enzymes seem to be broadly conserved among the different organisms; however, there are variations in a spatiotemporal manner. Here, we discuss the molecular mechanisms of membrane lipid remodelling and its regulation crucial for plants adaption to Pi deficiency.
PRpnp, a novel dual activity PNP family protein improves plant vigor and confers multiple stress tolerance in Citrus aurantifolia
(John Wiley & Sons, 2023) Singh, Sweta; Chaudhary, Chanderkant; Bharsakale, Rushikesh D.; Gazal, Snehi; Verma, Lokesh; Tarannum, Zeba; Behere, G.T.; Giri, Jitender; Germain, Hugo; Ghosh, Dilip K; Sharma, Ashwani K; Chauhan, Harsh
Under field conditions, plants are often simultaneously exposed to several abiotic and biotic stresses resulting in significant reductions in growth and yield; thus, developing a multi-stress tolerant variety is imperative. Previously, we reported the neofunctionalization of a novel PNP family protein, Putranjiva roxburghii purine nucleoside phosphorylase (PRpnp) to trypsin inhibitor to cater to the needs of plant defense. However, to date, no study has revealed the potential role and mechanism of either member of this protein group in plant defense. Here, we overexpressed PRpnp in Citrus aurantifolia which showed nuclear-cytoplasmic localization, where it functions in maintaining the intracellular purine reservoir. Overexpression of PRpnp significantly enhanced tolerance to salt, oxidative stress, alkaline pH, drought, and two pests, Papilio demoleus and Scirtothrips citri in transgenic plants. Global gene expression studies revealed that PRpnp overexpression up-regulated differentially expressed genes (DEGs) related to ABA- and JA- biosynthesis and signaling, plant defense, growth, and development. LC-MS/MS analysis validated higher endogenous ABA and JA accumulation in transgenic plants. Taken together, our results suggest that PRpnp functions by enhancing the endogenous ABA and JA, which interact synergistically and it also inhibits trypsin proteases in the insect gut. Also, like other purine salvage genes, PRpnp also regulates CK metabolism and increases the levels of CK-free bases in transgenic Mexican lime. We also suggest that PRpnp can be used as a potential candidate to develop new varieties with improved plant vigor and enhanced multiple stress resistance.
Rice phosphate transporter reduces the low phosphate response through jasmonate signaling
(Oxford University Press, 2026) Mani, Balaji; Maurya, Kanika; Verma, Lokesh; Gupta, Priya; Kohli, Pawandeep Singh; Gupta, Gagan; Jaskolowski, Aime; Poirier, Yves; Giri, Jitender
Phosphorus (P) is an essential macronutrient for plant growth, and its deficiency severely limits crop productivity. The PHOSPHATE1 (PHO1) protein family, defined by an N-terminal SPX domain, four transmembrane (4TM) domains, and a C-terminal EXS domain, mediates phosphate (Pi) loading into the xylem for root-to-shoot transport. In rice, OsPHO1;2 is critical for Pi export, and loss-of-function mutants exhibit severe growth retardation and Pi deficiency symptoms despite sufficient external Pi. To dissect the functional contributions of PHO1 domains beyond Pi transport, we generated CRISPR/Cas9 rice lines expressing either the EXS domain containing part of the SPX domain (S-EXS) or the 4TM+EXS domains (T-EXS) of OsPHO1;2. Phenotypic analyses under Pi-sufficient and Pi-deficient conditions revealed that S-EXS lines displayed improved early growth compared to ospho1;2 mutants, despite similar shoot Pi levels. These plants exhibited reduced jasmonic acid accumulation and attenuated phosphate starvation responses, resembling wild-type hormone profiles. In contrast, T-EXS lines mirrored the growth defects of ospho1;2 mutants. Transcriptome profiling confirmed that defense and phosphate starvation pathways were less activated in S-EXS lines relative to mutants. However, both S-EXS and T-EXS lines retained seed development defects and reduced seed phosphorus content, consistent with ospho1;2 phenotypes. Heterozygous plants carrying one functional OsPHO1;2 allele exhibited normal growth and seed development, confirming the recessive nature of the mutation. Collectively, these findings demonstrate that the S-EXS domain of OsPHO1;2 promotes plant growth independently of Pi transport by modulating jasmonate signaling and suppressing phosphate starvation responses. This highlights a signaling role for PHO1 domains, offering new insights into Pi homeostasis and potential strategies for breeding Pi-efficient crops.
Root-expressed rice PAP3b enhances secreted APase activity and helps utilize organic phosphate
(Oxford University Press, 2023) Bhadouria, Jyoti; Mehra, Poonam; Verma, Lokesh; Pazhamala, Lekha T; Rumi, Rumi; Panchal, Poonam; Sinha, Alok Krishna; Giri, Jitender
Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylates organic phosphorus complexes in the rhizosphere and intracellular compartments to release Pi. In this study, we demonstrate that OsPAP3b belongs to group III low molecular weight PAP, and is low Pi responsive, preferentially in roots. The expression of OsPAP3b is negatively regulated with Pi re-supply. Interestingly, OsPAP3b was found to be dual localized to the nucleus and secretome. Furthermore, OsPAP3b is transcriptionally regulated by OsPHR2 as substantiated by DNA-protein binding assay. Through in-vitro biochemical assays, we further demonstrate that OsPAP3b is a functional acid phosphatase with broad substrate specificity. Overexpression of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, reflected in better growth of transgenics compared to wild type when grown on organic P as exogenous P substrate. Under Pi deprivation, OsPAP3b knockdown and knockout lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced (PSI) genes and the levels of metabolites were found to be altered in the overexpression and knockdown lines. In addition, in-vitro pull-down assay revealed multiple putative interacting proteins of OsPAP3b. Our data collectively suggest that OsPAP3b can aid in organic P utilization in rice. The APase isoforms behavior and nuclear localization indicate its additional role, possibly in stress signaling. Considering its important roles, OsPAP3b could be a potential target for improving low Pi adaptation in rice.
Specific galactolipids species correlate with rice genotypic variability for phosphate utilization efficiency
(Elsevier B.V., 2021) Verma, Lokesh; Kohli, Pawandeep Singh; Maurya, Kanika; K B, Abhijith; Thakur, Jitendra K.; Giri, Jitender
Membrane lipid remodeling helps in the efficient utilization of phosphorus (P) by replacing phospholipids with galactolipids during P deficiency. Previous studies have shown lipid remodeling in rice under P deficiency; however, main lipid classes did not show association with superior P-use-efficiency in rice genotypes. Here, diverse rice genotypes were extensively phenotyped in normal (NP) and low P (LP) conditions. Based on the phenotypic response to P deficiency, genotypes were identified as tolerant and sensitive. Further, bulks were generated differing in their physiological P-use-efficiency (PPUE) during LP condition. Shoot lipidome profiling of genotypes was performed and used to correlate the abundance of various lipid classes and their constituent species with the PPUE of the genotypes. Lipid remodeling was observed as a P-starvation-induced response in all the genotypes. However, neither total galacto- and phospholipids nor the lipid classes correlated with PPUE during P deficiency. However, the difference in PPUE in the two bulks correlated with specific lipid species of galactolipids (DGDG, MGDG). Further, DGDG34:3 had the highest Mol% among the differentially accumulated lipid species between the two bulks. Our study reveals the importance of specific galactolipids species in rice adaptation to P deficient soils and thus opens new targets for future research.