Browsing by Author "Singh, Pooja"

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Alternative oxidase plays a role in minimizing ROS and RNS produced under salinity stress in Arabidopsis thaliana
(John Wiley & Sons, 2022) Manbir; Singh, Pooja; Kumari, Aprajita; Gupta, Kapuganti Jagadis
Under stress conditions, the overproduction of different reactive oxygen species (ROS) and reactive nitrogen species (RNS) causes imbalance in the redox homeostasis of the cell leading to nitro-oxidative stress in plants. Alternative oxidase (AOX) is a conserving terminal oxidase of the mitochondrial electron transport chain, which can minimize the ROS. Still, the role of AOX in the regulation of RNS during nitro-oxidative stress imposed by salinity stress is not known. Here, we investigated the role of AOX in minimizing ROS and RNS induced by 150 mM NaCl in Arabidopsis using transgenic plants overexpressing (AOX OE) and antisense lines (AOX AS) of AOX. Imposing NaCl treatment leads to a 4-fold enhanced expression of AOX accompanied by enhanced AOX capacity in WT Col-0. Further AOX-OE seedlings displayed enhanced growth compared with the AOX-AS line under stress. Examination of NO levels by DAF-FM fluorescence and chemiluminescence revealed that AOX overexpression leads to reduced levels of NO. The total NR activity was elevated under NaCl, but no significant change was observed in wild-type (WT), AOX OE, and AS lines. The total ROS, superoxide, H2O2 levels, and lipid peroxidation were higher in the AOX-AS line than in WT and AOX-OE lines. The peroxynitrite levels were also higher in the AOX-AS line than in WT and AOX-OE lines; further, the expression of antioxidant genes was elevated in AOX-AS. Taken together, our results suggest that AOX plays an important role in the mitigation of ROS and RNS levels and enhances plant growth, thus providing tolerance against nitro-oxidative stress exerted by NaCl.
Chickpea chitinases responsive to Helicoverpa herbivory and phytohormone signaling: genome-wide identification, field expression profiling, and structure-guided prioritization
(BioMed Central Ltd, 2026) Konda, Aravind Kumar; Annapragada, Harika; G K, Sujayanand; Singh, Pooja; Bhuvanachandra, Bhoopal; Chinnasamy, Hariharan V; Dixit, Girish Prasad; Gupta, Kapuganti Jagadis; Matheshwaran, Saravanan
Background: Chitinases can contribute to plant defence against fungal pathogens and insect herbivores, but their family organization, inducible deployment, and putative ligand-recognition behaviour remain poorly resolved in chickpea. We combined genome-wide identification, field expression profiling under controlled Helicoverpa armigera infestation, hormone treatments, and structure-guided comparison of representative proteins to prioritize defence-associated chickpea chitinases. Results: We identified 28 chickpea chitinase loci (Car_Chits), comprising 22 glycosyl hydrolase family 18 (GH18) genes and 6 GH19 genes. Local duplication, especially tandem duplication within GH18, was the main contributor to family expansion, and interpretable duplicate pairs were retained mainly under purifying selection. Promoter scans indicated broad enrichment of defence- and hormone-associated cis-elements. Field quantitative real-time PCR (qRT-PCR) profiling of 11 candidate genes in field-grown plants subjected to controlled H. armigera infestation and hormone treatments showed treatment-specific temporal regulation. Car_Chit-4 (GH19) was strongly induced by salicylic acid (7.81-fold at 0.5 h; q < 0.05) but transiently repressed shortly after H. armigera feeding (0.15-fold at 0.5 h; q = 0.030). Car_Chit-19 (GH18) was the clearest herbivory-responsive gene, with late induction at 8 h (1.62-fold; q = 0.050) and 48 h (1.85-fold; q = 0.050). Jasmonic acid caused broad early repression across several genes, followed by delayed induction of Car_Chit-4 at 24 h. Seven Car_Chit-(GlcNAc)₄ complexes were modelled, docked, and simulated for 100 ns. GH18 proteins generally showed more favourable predicted MM-PBSA binding energies than GH19 proteins, but the structural metrics were interpreted as relative ligand-recognition indicators rather than direct evidence of anti-herbivore function. Car_Chit-17 had the most favourable predicted binding energy (ΔG_bind = - 18.51 ± 6.75 kcal/mol), whereas Car_Chit-14 and Car_Chit-27 retained the most stable ligand poses and Car_Chit-19 displayed the most stable protein scaffold. Conclusions: Chickpea chitinases show differentiated temporal responses to herbivory and hormone signalling. The study supports a working model in which GH19 Car_Chit-4 marks a rapid salicylic-acid-responsive arm, whereas GH18 Car_Chit-19 marks a delayed herbivory-responsive arm. A tiered prioritization framework separates expression-deployed candidates from structure-guided biochemical candidates, explaining why different genes emerge from qRT-PCR and molecular modelling analyses. The structural analyses provide complementary prioritization of Car_Chit-17, Car_Chit-14, and Car_Chit-27 for biochemical characterization. Together, these results provide a resource for dissecting chitinase-mediated defence in chickpea and for selecting candidates for functional validation.
Detection of nitric oxide from chickpea using DAF fluorescence and chemiluminescence methods
(John Wiley & Sons, 2022) Kumari, Aprajita; Bhatoee, Manbir; Singh, Pooja; Kaladhar, Vemula Chandra; Yadav, Nidhi; Paul, Debarati; Loake, Gary J.; Gupta, Kapuganti Jagadis
The free radical nitric oxide (NO) has emerged as an important signal molecule in plants, due to its involvement in various plant growth, development, and stress responses. For elucidating the role of NO, it is very important to precisely determine, localize, and quantify NO levels. Due to a relatively short half-life and its rapid, complex reactivity with other radicals, together with its capacity to diffuse from the source of production, the quantification of NO in whole plants, tissues, organelles, and extracts is notoriously difficult. Hence, it is essential to employ sensitive procedures for precise detection of NO. Currently available methods can fulfill many requirements to precisely determine NO, but each method has several advantages and pitfalls. In this article, we describe a detailed procedure for the measurement of NO by diaminofluorescein (DAF) in cell-permeable forms (DAF-FM-DA). In this method, the tissues are immersed in DAF-FM DA, leading to their diffusion from the plasma membrane to the inside of the cell, where intracellular esterases cleave the ester bonds, leading to DAF-FM release. The resulting DAF-FM reacts with intracellularly generated NO and forms highly fluorescent triazolofluorescein (DAF-FMT), which can be localized and monitored by fluorescence or confocal microscopy, and can also be detected via fluorimetry and flow cytometry. DAF dyes are very popular as they are non-invasive, relatively easy to handle, and commercially available. Another precise and very sensitive method is chemiluminescence detection of NO, where NO reacts with ozone (O3), leading to emission of a quantum of light from which NO can be calculated. Using chickpea seedlings, we describe in detail the measurement of NO using DAF-FM-DA and chemiluminescence methods. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Measurement of nitric oxide from chickpea seedlings using DAF-FM DA fluorescence with fluorescence and confocal microscopy Basic Protocol 2: Chemiluminescence detection of nitric oxide from chickpea seedlings
An efficient method of mitochondrial DNA isolation from Vigna radiata for genomic studies
(Springer Nature Publishing AG, 2020) Singh, Pooja; Sahoo, Ranjan Kumar; Bulle, Mallesham; Gupta, Kapuganti Jagadis
Isolation of mitochondrial DNA from root tissues of mung bean (Vigna radiata) is quite tedious, complex, and often results in low yield. Hence here we show a simple, rapid, and improved protocol for isolation of mitochondrial DNA from root tissues of hydroponically grown mung bean plants. This method involves purification of mitochondria and subsequent isolation of DNA from obtained purified mitochondria. For this purpose, mitochondria were isolated using a discontinuous Percoll gradient centrifugation followed by RNase I treatment to the isolated DNA to remove any traces of RNA contamination. The mitochondrial DNA was isolated from mitochondrial samples by commonly used CTAB method. The specificity of isolated mitochondrial DNA was confirmed using mtDNA-specific genes (NAD1 and COX3). β-Actin primer was used to check the nuclear DNA contamination. PCR amplification was observed in mtDNA specific genes NAD1 and COX3 except nuclear encoded β-actin gene suggesting that mitochondrial DNA was not contaminated by nuclear DNA.
Greenhouse and field experiments revealed that clove oil can effectively reduce bacterial blight and increase yield in pomegranate
(John Wiley & Sons, 2021) Kumar, Pavan; Lokesh, Veeresh; Doddaraju, Pushpa; Kumari, Aprajita; Singh, Pooja; Meti, Bharati S.; Sharma, Jyotsana; Gupta, Kapuganti Jagadis; Manjunatha, Girigowda
Bacterial blight in pomegranate is a devastating disease caused by bacterial pathogen Xanthomonas axonopodis pv. punicae (XAP), recording huge damage to pomegranate crop worldwide. Antibiotics and copper-based chemicals are being used for the management of this blight, while in this present work, we investigated the effect of eugenol and clove oil either singly or in combination with copper oxychloride (COC) on the induction of plant defense responses and concomitant prevention of bacterial blight. Our results provided evidence that clove oil (0.2%–1%) and eugenol (0.1% and 0.2%) successfully inhibit the growth of XAP in paper disk diffusion assay. Strikingly under the greenhouse condition, clove oil (0.2%) as foliar application 24 h before XAP inoculation recorded the lowest disease severity of 7.34%, whereas eugenol (0.2%) recorded maximum disease severity of 14.56%. However, the combination of clove oil (0.2%) and copper oxychloride (0.3%) recorded the least disease severity of 2.38%. A similar trend was observed in field conditions. Prophylactic application of clove oil leads to enhanced nitrate reductase activity and nitric oxide production which was further enhanced in clove oil pre-treated plants challenged with XAP. Strikingly, the total ROS and H2O2 levels were reduced in response to clove oil application. Clove oil also induced the systemic response by inducing expression levels of defense genes. The reduction of disease severity by clove oil and COC combination also reflected on total yield recording via large-scale field experiments where maximum yield of 14.04 tonnes/acre was observed, whereas streptocycline application recorded 11.12 tonnes/acre. Application of COC and clove oil resulted in a high remunerative value of ₹ 1:5.6, compared to streptocycline (1:4.85) and control (1:1.85). The present study revealed that clove oil as a plant derivative and eugenol as a synthetic option can be effectively used for the successful management of bacterial blight in pomegranate.
Isolation and measurement of respiration and structural studies of purified mitochondria from heterotrophic plant tissues
(John Wiley & Sons, 2021) Pandey, Sonika; Kumari, Aprajita; Singh, Pooja; Gupta, Kapuganti Jagadis
Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy.
Measurement of reactive oxygen species and nitric oxide from tomato plants in response to abiotic and biotic stresses
(Springer Nature Publishing AG, 2024) Samant, Sanjib Bal; Manbir; Rekha; Swain, Jagannath; Singh, Pooja; Kumari, Aprajita; Gupta, Kapuganti Jagadis
Nitric oxide (NO) is a free radical molecule that has been known to influence several cellular processes such as plant growth, development, and stress responses. NO together with reactive oxygen species (ROS) play a role in signaling process. Due to extremely low half-life of these radicals in cellular environment, it is often difficult to precisely monitor them. Each method has some advantages and disadvantages; hence, it is important to measure using multiple methods. To interpret the role of each signaling molecule in numerous biological processes, sensitive and focused methods must be used. In addition to this complexity, these Reactive Oxygen Species (ROS) and NO react with each other leads to nitro-oxidative stress in plants. Using tomato as a model system here, we demonstrate stepwise protocols for measurement of NO by chemiluminescence, DAF fluorescence, nitrosative stress by western blot, and ROS measurement by NBT and DAB under stress conditions such as osmotic stress and Botrytis infection. While describing methods, we also emphasized on benefits, drawbacks, and broader applications of these methods.
Metabolism and signalling in pea (Pisum sativum) leaves exposed to drought and subsequent recovery
(John Wiley & Sons, 2026) Pandey, Jayendra; Mantena, Chakradhar; Kumari, Aprajita; Singh, Pooja; Foyer, Christine H.; Gupta, Kapuganti Jagadis; Subramanyam, Rajagopal
Uncovering the metabolic and molecular mechanisms involved in plant responses to drought and subsequent recovery, is essential to identify drought tolerance mechanisms that can be used to improve crop plants. Here we combine plant physiology and biochemistry, with gene expression, quantitative proteomics and metabolite profiling to identify the genetic and metabolic networks that operate in plants experiencing and recovering from drought. Network analysis of transcripts, proteins and metabolites revealed that certain biological processes such as the tricarboxylic acid cycle and lipid metabolism had a strong impact on the overall control of leaf responses to drought and recovery. The stimulation of carbohydrate oxidation pathways is demonstrated to be a key node in the generation of energy and precursors required to support diverse survival pathways of defence.
Metabolomic response to high light from pgrl1 and pgr5 mutants of Chlamydomonas reinhardtii
(Springer Nature Publishing AG, 2023) Chouhan, Nisha; Marriboina, Sureshbabu; Kumari, Aprajita; Singh, Pooja; Yadav, Ranay Mohan; Gupta, Kapuganti Jagadis; Subramanyam, Rajagopal
Chlamydomonas (C.) reinhardtii metabolomic changes in cyclic electron flow-dependent mutants are still unknown. Here, we used mass spectrometric analysis to monitor the changes in metabolite levels in wild-type, cyclic electron-deficient mutants pgrl1 and pgr5 grown under high-light stress. A total of 55 metabolites were detected using GC-MS analysis. High-light stress-induced selective anaplerotic amino acids in pgr5. In addition, pgr5 showed enhancement in carbohydrate, polyamine, and polyol metabolism by 2.5-fold under high light. In response to high light, pgr5 triggers an increase in several metabolites involved in regulating osmotic pressure. Among these metabolites are glycerol pathway compounds such as glycerol-3-phosphate and glyceryl-glycoside, which increase significantly by 1.55 and 3.07 times, respectively. In addition, pgr5 also enhanced proline and putrescine levels by 2.6- and 1.36-fold under high light. On the other hand, pgrl1-induced metabolites, such as alanine and serine, are crucial for photorespiration when subjected to high-light stress. We also observed a significant increase in levels of polyols and glycerol by 1.37- and 2.97-fold in pgrl1 under high-light stress. Both correlation network studies and KEGG pathway enrichment analysis revealed that metabolites related to several biological pathways, such as amino acid, carbohydrate, TCA cycle, and fatty acid metabolism, were positively correlated in pgrl1 and pgr5 under high-light stress conditions. The relative mRNA expression levels of genes related to the TCA cycle, including PDC3, ACH1, OGD2, OGD3, IDH3, and MDH4, were significantly upregulated in pgrl1 and pgr5 under HL. In pgr5, the MDH1 level was significantly increased, while ACS1, ACS3, IDH2, and IDH3 levels were reduced considerably in pgrl1 under high-light stress. The current study demonstrates both pgr5 and prgl1 showed a differential defense response to high-light stress at the primary metabolites and mRNA expression level, which can be added to the existing knowledge to explore molecular regulatory responses of prg5 and pgrl1 to high-light stress.
Nitric oxide accelerates germination via the regulation of respiration in chickpea
(Oxford University Press, 2019) Pandey, Sonika; Kumari, Aprajita; Shree, Manu; Kumar, Vinod; Singh, Pooja; Bharadwaj, Chellapilla; Loake, Gary J.; Parida, Swarup K.; Masakapalli, Shyam Kumar; Gupta, Kapuganti Jagadis
Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1 H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.
Nitric oxide regulates mitochondrial biogenesis in plants
(John Wiley & Sons, 2023) Kumari, Aprajita; Kaladhar, Vemula Chandra; Yadav, Nidhi; Singh, Pooja; Reddy, Kishorekumar; Gupta, Kapuganti Jagadis
The site of nitric oxide (NO) production in mitochondrial cytochrome c oxidase and the role of NO in mitochondrial biogenesis are not known in plants. By imposing osmotic stress and recovery on Arabidopsis seedlings we investigated the site of NO production and its role in mitochondrial biogenesis. Osmotic stress reduced growth and mitochondrial number while increasing NO production. During the recovery phase the mitochondrial number increased and this increase was higher in wild type and the high NO-producing Pgb1 silencing line in comparison to the NO-deficient nitrate reductase double mutant (nia1/nia2). Application of nitrite stimulated NO production and mitochondrial number in the nia1/nia2 mutant. Osmotic stress induced COX6b−3 and COA6-L genes encoding subunits of COX. The mutants cox6b-3 and coa6-l were impaired both in NO production and mitochondrial number during stress to recovery suggesting the involvement of these subunits in nitrite-dependent NO production. Transcripts encoding the mitochondrial protein import machinery showed reduced expression in cox6b-3 and coa6-l mutants. Finally, COX6b-3 and COA6-L interacted with the VQ27 motif-containing protein in the presence of NO. The vq27 mutant was impaired in mitochondrial biogenesis. Our results suggest the involvement of COX derived NO in mitochondrial biogenesis.
Overexpression of phytoglobin1 in rice leads to enhanced nitrogen use efficiency via modulation of nitric oxide
(John Wiley & Sons, 2025) Samant, Sanjib Bal; Swain, Jagannath; Yadav, Nidhi; Yadav, Reena; Singh, Pooja; Rai, Preeti; Sheri, Vijay; Sreeman, Sheshshayee; Subramanyam, Rajagopal; Pareek, Ashwani; Gupta, Kapuganti Jagadis
Nitric oxide (NO) is one of the byproducts of nitrogen metabolism. Excess amount of NO is scavenged by phytoglobins. The role of phytoglobin mediated NO homoeostasis in modulation of nitrate transporters was investigated using NO scavenger cPTIO, phytoglobin overexpressing rice and Arabidopsis. Growing plants under low nitrate leads to generation of reduced levels of NO accompanied by elevated expression of high affinity transporters (HATs) such as NRT2.1, NRT2.3 and NRT2.4. Scavenging of NO by cPTIO under optimal nitrate caused enhanced HATs expression. Phytoglobin overexpressing Arabidopsis showed improved growth and enhanced expression of HATs under low nitrogen in comparison to WT. Pretreatment of optimal nitrate grown plants with NO scavenger cPTIO enhanced HATs expression and shifting of these primed plants from optimal to low nitrate leads to further elevation of HATs expression accompanied by enhanced nitrogen uptake and its accumulation with positive effect on growth. Phytoglobin overexpression in rice leads to enhanced HATs expression, improved growth, nitrogen accumulation under low nitrate. Pgb OE lines showed enhanced accumulation of amino acids. Taken together our results suggest an important role of phytoglobins in nitrogen uptake and assimilation.
Phytoglobin-NO cycle and AOX pathway play a role in anaerobic germination and growth of deepwater rice
(John Wiley & Sons, 2021) Kumari, Aprajita; Singh, Pooja; Kaladhar, Vemula Chandra; Manbir; Paul, Debarati; Pathak, Pradeep Kumar; Gupta, Kapuganti Jagadis
An important and interesting feature of rice is that it can germinate under anoxic conditions. Though several biochemical adaptive mechanisms play an important role in the anaerobic germination of rice but the role of phytoglobin-nitric oxide cycle and alternative oxidase pathway is not known, therefore in this study we investigated the role of these pathways in anaerobic germination. Under anoxic conditions, deepwater rice germinated much higher and rapidly than aerobic condition and the anaerobic germination and growth were much higher in the presence of nitrite. The addition of nitrite stimulated NR activity and NO production. Important components of phytoglobin-NO cycle such as methaemoglobin reductase activity, expression of Phytoglobin1, NIA1 were elevated under anaerobic conditions in the presence of nitrite. The operation of phytoglobin-NO cycle also enhanced anaerobic ATP generation, LDH, ADH activities and in parallel ethylene levels were also enhanced. Interestingly nitrite suppressed the ROS production and lipid peroxidation. The reduction of ROS was accompanied by enhanced expression of mitochondrial alternative oxidase protein and its capacity. Application of AOX inhibitor SHAM inhibited the anoxic growth mediated by nitrite. In addition, nitrite improved the submergence tolerance of seedlings. Our study revealed that nitrite driven phytoglobin-NO cycle and AOX are crucial players in anaerobic germination and growth of deepwater rice.
The power of the phytoglobin-NO cycle in the regulation of nodulation and symbiotic nitrogen fixation
(John Wiley & Sons, Inc., 2020) Singh, Pooja; Kumari, Aprajita; Foyer, Christine H.; Gupta, Kapuganti Jagadis
Recent years have seen a large increase in our understanding of the multifaceted roles of nitric oxide (NO) in plant biology, particularly in the regulation of symbiotic interactions with soil microorganisms. NO serves key functions in the initiation and maintenance of legume–rhizobium symbiosis. Extensive molecular crosstalk between the host legume and the soil rhizobium results in the initiation of symbiosis and the establishment of a highly specialized organ called the nodule, which provides the conditions required for bacterial nitrogen fixation. Within the nodules, the rhizobium bacteria are housed in bacteroids, in which the oxygen is maintained at very low levels allowing the bacterial nitrogenase to function. This enzyme is oxygen sensitive (half maximal inhibitory concentration (IC50) oxygen for nitrogenase is 2.9 µM; Goldberg et al., 1987). Hence, a key feature of the nodule is the maintenance of a strong oxygen diffusion barrier, in which leghaemoglobin is used to transfer oxygen to the plant mitochondria to sustain respiration.
Serine hydroxymethyltransferase6 is involved in growth and resistance against pathogens via ethylene and lignin production in Arabidopsis
(John Wiley & Sons, 2024) Singh, Pooja; Kumari, Aprajita; Khaladhar, Vemula Chandra; Singh, Namrata; Pathak, Pradeep Kumar; Kumar, Vinod; Kumar, Ritika Jantu; Jain, Priyanka; Thakur, Jitendra K.; Fernie, Alisdair R.; Bauwe, Hermann; Raghavendra, A.S.; Gupta, Kapuganti Jagadis
Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one-carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens.
Transcriptome-wide identification and expression analysis of expansin genes in big and small leaf-morphotypes of Adhatoda vasica
(Springer Nature Publishing AG, 2025) Soundararajan, Prabhakaran; Singh, Pooja; Letro, Awele; Singh, Gourav; Manivannan, Abinaya
In the present study the expansin genes were identified from Adhatoda vasica, a potential medicinal plant. It consists of two morphotypes S (small leaf bearing plants with low alkaloid content) and B (large leaf bearing plants with high alkaloid content). The difference in the leaf size influence the alkaloid content and it act as the economically important trait for this medicinal plant. Therefore, exploring the expression of expansins will facilitate the molecular regulation of leaf expansion in A. vasica. For the identification of expansins, Hidden Markov model (HMM) profiles of double-psi beta-barrel (DPBB) and pollen allergen domains were utilized and searched against the genomes of Arabidopsis thaliana, Catharanthus roseus and Camellia sinensis and confirmed with BLAST search against published expansins of A. thaliana. The resulted expansins were used for homology-based identification of expansin transcripts in A. vasica transcriptome assembly. A total of 22 expansin transcripts were identified in A. vasica leaf transcriptome. The phylogenetic tree illustrated that the expansins were clustered into four subfamily EXPA, EXPB, EXPLA, and EXPLB. Interactome analysis revealed that the expansins interacted with genes involved in cell wall modification. In addition, leaf transcriptome analysis of S and B morphotypes revealed that majority of the expansins were upregulated in B morphotype than S morphotype. Further, qPCR analysis of selected expansin genes from transcriptome were validated in the young and mature leaf tissues of both morphotypes. Overall, the outcomes of the present study will facilitate the understanding the expansins based molecular regulation of leaf size in A. vasica which will aid in the higher production of pharmaceutically important alkaloids.
Using different forms of nitrogen to study hypersensitive response elicited by avirulent Pseudomonas syringae
(Springer Nature Publishing AG, 2020) Singh, Namrata; Singh, Pooja; Pathak, Pradeep Kumar; Gupta, Kapuganti Jagadis
Nitrate, ammonium, or a combination of both is the form of N available for nitrogen assimilation from soil by the plants. Nitrogen is an important and integral part of amino acids, nucleotides, and defense molecules. Hence it is very important to study the role of nitrate and ammonium nutrition in plant defense via hypersensitive response (HR). Shifting plants from ammonium nitrate Hoagland solution to nitrate Hoagland nutrition slightly enhances root length and leaf area. HR phenotype is different in nitrate and ammonium grown plants when challenged with avirulent Pseudomonas syringae DC3000 avrRpm1. HR is also associated with increased production of reactive oxygen species (ROS) and nitric oxide (NO). Hence to understand HR development it is essential to measure HR lesions, cell death, ROS, NO, and bacterial growth. Here we provide a stepwise protocol of various parameters to study HR in Arabidopsis in response to nitrate and ammonium nutrition.
Using foldscope to monitor superoxide production and cell death during pathogen infection in arabidopsis under different nitrogen regimes
(Springer Nature Publishing AG, 2020) Arora, Reena; Singh, Pooja; Kumari, Aprajita; Pathak, Pradeep Kumar; Gupta, Kapuganti Jagadis
Nitrogen nutrition plays a role in plant growth development and resistance against biotic and abiotic stress. During pathogen infection various signal molecules such as reactive oxygen species, calcium, reactive nitrogen species, salicylic acid, and ethylene plays an important role. The form of nitrogen nutrition such as nitrate or ammonium plays a role in production of these molecules. Under nitrate nutrition NO is predominant. The produced NO plays a role in reacting with superoxide to generate peroxynitrite to induce cell death during hypersensitive response elicited by avirulent pathogens. Excess of ROS is also detrimental to plants and NO plays a role in regulating ROS. Hence it is important to observe superoxide production during infection. By using an avirulent Pseudomonas syringae and Arabidopsis differential N nutrition we show superoxide production in leaves using a paper microscope called Foldscope, which can be applied as a simple microscope to observe objects. The data also compared with root system infected with pathogenic Fusarium oxysporum. Taken together here we show that Foldscope is a cost-effective and powerful technique to visualize superoxide and cell death in plants during infection.