Browsing by Author "Majee, Manoj"

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ABI transcription factors and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE module mediate seed desiccation tolerance and longevity in Oryza sativa
(The Company of Biologists, 2022) Kamble, Nitin Uttam; Majee, Manoj
In contrast to desiccation-tolerant orthodox seeds, recalcitrant seeds are desiccation sensitive and are unable to survive for a prolonged time. Here, our analyses of Oryza species with contrasting seed desiccation tolerance reveals that PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT), an enzyme that repairs abnormal isoaspartyl (isoAsp) residues in proteins, acts as a key player that governs seed desiccation tolerance to orthodox seeds but is ineffective in recalcitrant seeds. We observe that, unlike the orthodox seed of Oryza sativa, desiccation intolerance of the recalcitrant seeds of Oryza coarctata are linked to reduced PIMT activity and increased isoAsp accumulation due to the lack of coordinated action of ABA and ABI transcription factors to upregulate PIMT during maturation. We show that suppression of PIMT reduces, and its overexpression increases, seed desiccation tolerance and seed longevity in O. sativa. Our analyses further reveal that the ABI transcription factors undergo isoAsp formation that affect their functional competence; however, PIMT interacts with and repairs isoAsp residues and facilitates their functions. Our results thus illustrate a new insight into the mechanisms of acquisition of seed desiccation tolerance and longevity by ABI transcription factors and the PIMT module.
Abiotic stress tolerance in plants: Brassinosteroids navigate competently
(MDPI AG, 2022) Chaudhuri, Abira; Halder, Koushik; Abdin, Malik Z; Majee, Manoj; Datta, Asis
Brassinosteroid hormones (BRs) multitask to smoothly regulate a broad spectrum of vital physiological processes in plants, such as cell division, cell expansion, differentiation, seed germination, xylem differentiation, reproductive development and light responses (photomorphogenesis and skotomorphogenesis). Their importance is inferred when visible abnormalities arise in plant phenotypes due to suboptimal or supraoptimal hormone levels. This group of steroidal hormones are major growth regulators, having pleiotropic effects and conferring abiotic stress resistance to plants. Numerous abiotic stresses are the cause of significant loss in agricultural yield globally. However, plants are well equipped with efficient stress combat machinery. Scavenging reactive oxygen species (ROS) is a unique mechanism to combat the deleterious effects of abiotic stresses. In light of numerous reports in the past two decades, the complex BR signaling under different stress conditions (drought, salinity, extreme temperatures and heavy metals/metalloids) that drastically hinders the normal metabolism of plants is gradually being untangled and revealed. Thus, crop improvement has substantial potential by tailoring either the brassinosteroid signaling, biosynthesis pathway or perception. This review aims to explore and dissect the actual mission of BRs in signaling cascades and summarize their positive role with respect to abiotic stress tolerance.
Arabidopsis ABSCISIC ACID INSENSITIVE4 targets PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 in seed
(Springer Nature Publishing AG, 2022) Kamble, Nitin Uttam; Ghosh, Shraboni; Achary, Rakesh Kumar; Majee, Manoj
PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme (PRE) and is implicated in seed vigor and longevity. PIMT has been shown to be induced by ABA, however, its detailed regulation by ABA signaling components is unknown. Herein, we report that ABSCISIC ACID INSENSITIVE4 (ABI4) directly binds to the PIMT1 promoter and regulates its expression in Arabidopsis seeds. AtPIMT1 promoter analysis demonstrated the presence of putative ABI4 binding sites. Our Y1H analysis revealed that AtABI4 transcription factor binds to the AtPIMT1 promoter. Dual luciferase assay also demonstrated the binding of the AtABI4 transcription factor to the AtPIMT1 promoter. Subsequently, we have generated AtPIMT1 promoter GUS lines and revealed that ABA induced expression of GUS in Arabidopsis thaliana. Expression analyses exhibited reduced accumulation of PIMT1 protein and transcript with significant reduction in total PIMT activity in abi4-1 mutants as compared to that of the wild type. The AtPIMT1 promoter GUS expression in abi4-1 mutants was also found to be severely affected in both the control and ABA treatment. Hence, through molecular and genetic evidences we show that the AtABI4 plays a central role in regulating the expression of AtPIMT1 to impart seed vigor and longevity to orthodox seeds.
The Arabidopsis F-box protein SKP1-INTERACTING PARTNER 31 modulates seed maturation and seed vigor by targeting JASMONATE ZIM DOMAIN proteins independently of jasmonic acid-isoleucine
(Oxford University Press, 2023) Varshney, Vishal; Hazra, Abhijit; Rao, Venkateswara; Ghosh, Shraboni; Kamble, Nitin Uttam; Achary, Rakesh Kumar; Gautam, Shikha; Majee, Manoj
F-box proteins have diverse functions in eukaryotic organisms, including plants, mainly targeting proteins for 26S proteasomal degradation. Here, we demonstrate the role of the F-box protein SKP1-INTERACTING PARTNER 31 (SKIP31) from Arabidopsis (Arabidopsis thaliana) in regulating late seed maturation events, seed vigor, and viability through biochemical and genetic studies using skip31 mutants and different transgenic lines. We show that SKIP31 is predominantly expressed in seeds and that SKIP31 interacts with JASMONATE ZIM DOMAIN (JAZ) proteins, key repressors in jasmonate (JA) signaling, directing their ubiquitination for proteasomal degradation independently of coronatine/jasmonic acid-isoleucine (JA-Ile), in contrast to CORONATINE INSENSITIVE 1, which sends JAZs for degradation in a coronatine/JA-Ile dependent manner. Moreover, JAZ proteins interact with the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) and repress its transcriptional activity, which in turn directly or indirectly represses the expression of downstream genes involved in the accumulation of LATE EMBRYOGENESIS ABUNDANT proteins, protective metabolites, storage compounds, and abscisic acid biosynthesis. However, SKIP31 targets JAZ proteins, deregulates ABI5 activity, and positively regulates seed maturation and consequently seed vigor. Furthermore, ABI5 positively influences SKIP31 expression, while JAZ proteins repress ABI5-mediated transactivation of SKIP31 and exert feedback regulation. Taken together, our findings reveal the role of the SKIP31-JAZ-ABI5 module in seed maturation and consequently, establishment of seed vigor.
Arabidopsis Protein L-Isoaspartyl Methyltransferase repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance
(American Society for Biochemistry and Molecular Biology, 2020) Ghosh, Shraboni; Kamble, Nitin Uttam; Verma, Pooja; Salvi, Prafull; Petla, Bhanu Prakash; Roy, Shweta; Rao, Venkateswara; Hazra, Abhijit; Varshney, Vishal; Kaur, Harmeet; Majee, Manoj
Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme Protein L-Isoaspartyl Methyltransferase (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging and thereby protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.
Arabidopsis SKP1-like protein 13 (ASK13) positively regulates seed germination and seedling growth under abiotic stresses
(Oxford University Press, 2018) Rao, Venkateswara; Petla, Bhanu Prakash; Verma, Pooja; Salvi, Prafull; Kamble, Nitin Uttam; Ghosh, Sharboni; Kaur, Harmeet; Saxena, Saurabh C; Majee, Manoj
SKP1 (S-Phase Kinase Associated Protein1) proteins are key members of the SCF (SKP-Cullin-F-box protein) E3 ligase complexes that ubiquitinate the target proteins and play diverse roles in plant biology. However, as compared to other members of the SCF complex, study of SKP1-like proteins is very limited in plants. In the present work, we report that Arabidopsis SKP1-like protein13 (ASK13) is differentially regulated in different organs, during seed development and germination, and is upregulated in response to abiotic stresses. Y2H library screening and subsequent in vivo interaction through BiFC analysis revealed that ASK13 not only interacts with F-box proteins but also with other proteins which are not components of SCF complexes. Biochemical analysis revealed that ASK13 not only exists as a monomer but also as a homo-oligomer or heteromer with other ASK proteins. Functional analysis using ASK13 overexpression and knockdown lines revealed that ASK13 positively influences seed germination and seedling growth particularly under abiotic stresses. Taken together, our data strongly suggests that apart from participation to form SCF complexes, ASK13 interacts with several other proteins and is implicated in different cellular processes distinct from protein degradation. Overall, ASK13 positively regulates seed germination and seedling growth particularly under abiotic stress conditions.
Association of SNP in a novel DREB2-like gene SiDREB2 with stress tolerance in foxtail millet [Setaria italica (L.)]
(Oxford University Press, 2011) Lata, Charu; Bhutty, Sarita; Bahadur, Ranjit Prasad; Majee, Manoj; Prasad, Manoj
The DREB genes code for important plant transcription factors involved in the abiotic stress response and signal transduction. Characterization of DREB genes and development of functional markers for effective alleles is important for marker-assisted selection in foxtail millet. Here the characterization of a cDNA (SiDREB2) encoding a putative dehydration-responsive element-binding protein 2 from foxtail millet and the development of an allele-specific marker (ASM) for dehydration tolerance is reported. A cDNA clone (GenBank accession no. GT090998) coding for a putative DREB2 protein was isolated as a differentially expressed gene from a 6 h dehydration stress SSH library. A 5' RACE (rapid amplification of cDNA ends) was carried out to obtain the full-length cDNA, and sequence analysis showed that SiDREB2 encoded a polypeptide of 234 amino acids with a predicted mol. wt of 25.72 kDa and a theoretical pI of 5.14. A theoretical model of the tertiary structure shows that it has a highly conserved GCC-box-binding N-terminal domain, and an acidic C-terminus that acts as an activation domain for transcription. Based on its similarity to AP2 domains, SiDREB2 was classified into the A-2 subgroup of the DREB subfamily. Quantitative real-time PCR analysis showed significant up-regulation of SiDREB2 by dehydration (polyethylene glycol) and salinity (NaCl), while its expression was less affected by other stresses. A synonymous single nucleotide polymorphism (SNP) associated with dehydration tolerance was detected at the 558th base pair (an A/G transition) in the SiDREB2 gene in a core set of 45 foxtail millet accessions used. Based on the identified SNP, three primers were designed to develop an ASM for dehydration tolerance. The ASM produced a 261 bp fragment in all the tolerant accessions and produced no amplification in the sensitive accessions. The use of this ASM might be faster, cheaper, and more reproducible than other SNP genotyping methods, and thus will enable marker-aided breeding of foxtail millet for dehydration tolerance.
Cellular responses in the pigeonpea wild relative Cajanus platycarpus to Helicoverpa armigera herbivory: The role of methionine sulfoxide reductase B1 (CpMSRB1) in enhanced defense
(American Phytopathological Society, 2025) Rathinam, Maniraj; Dokka, Narasimham; Senthil, Kameshwaran; Mahawar, Shivangi; Tyagi, Shaily; Rengarajan, Dineshkumar; Vijayaraghavareddy, Preethi; Iyyappan, Yuvaraj; YB, Basavaraj; Reddy, Sandeep; T, Vinutha; G, Rama Prashat; Sinha, Subodh Kumar; Dash, Prasanta K.; Sreeman, Sheshshayee; Majee, Manoj; Sreevathsa, Rohini
Understanding key cellular mechanisms leading to improved defense against various stressors is essential for cultivating robust nutritious crops capable of flourishing in diverse environments. We present an in-depth characterization of the defense response in the pigeonpea wild relative Cajanus platycarpus to herbivory by pod borer Helicoverpa armigera. To fight the attacking pest, C. platycarpus strategically activated non-enzymatic reactive oxygen species (ROS) scavengers and unleashed methionine sulfoxide reductases to safeguard the integrity of methionine residues. We unveiled for the first time physical interaction between CpMSRB1 and chorismate mutase (CpCM1.1), a pivotal player in the phenylpropanoid pathway. This association fueled the synthesis of phenylpropanoids and enhanced ROS scavenging crucial for repelling herbivores. Repairing CpCM1.1 also boosted salicylic acid production, coordinating defense signaling with jasmonic acid. Additionally, heterologous expression of CpMSRB1 in tomato improved defense against herbivory by enhanced ROS scavenging and polyphenol production. This study demonstrates the role of CpMSRB1 in protecting a major enzyme in the shikimate pathway, reinforcing defense against H. armigera.
Chromatin-based transcriptional reprogramming in plants under abiotic stresses
(MDPI AG, 2022) Halder, Koushik; Chaudhuri, Abira; Abdin, Malik Z.; Majee, Manoj; Datta, Asis
Plants’ stress response machinery is characterized by an intricate network of signaling cascades that receive and transmit environmental cues and ultimately trigger transcriptional reprogramming. The family of epigenetic regulators that are the key players in the stress-induced signaling cascade comprise of chromatin remodelers, histone modifiers, DNA modifiers and regulatory noncoding RNAs. Changes in the histone modification and DNA methylation lead to major alterations in the expression level and pattern of stress-responsive genes to adjust with abiotic stress conditions namely heat, cold, drought and salinity. The spotlight of this review falls primarily on the chromatin restructuring under severe abiotic stresses, crosstalk between epigenetic regulators along with a brief discussion on stress priming in plants.
Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana
(Springer, 2013) Bhatt, Deepesh; Saxena, Saurabh C.; Jain, Sourabh; Dobriyal, Anoop K.; Majee, Manoj; Arora, Sandeep
Eleusine coracana (finger millet) is a stress-hardy but under-utilized cereal crop that possesses an efficient antioxidant defense system. The plant is capable of enduring long durations of water deficit stress. Experiments were conducted to clone a potent stress responsive isoform of ascorbate peroxidase and validate its role under drought stress. Reverse transcriptase PCR was used to obtain the partial cDNA of apx1 gene, from a meticulously screened drought tolerant genotype of E. coracana (PR202). Using RACE strategy, the full length apx1 cDNA was cloned and sequenced. The cDNA length of the E. coracana apx1 (Ec-apx1) gene is 1,047 bp with a 750 bp ORF, encoding a 250 amino acid protein having a molecular weight of 28.5 kDa. The identity of the amino acid sequence, deduced from the cDNA, with the APX family homologs was about 74-97 %. The full-length apx1 ORF was sub-cloned in a prokaryotic expression vector pET23b. The recombinant fusion protein, Ec-apx1, had high expression level in BL21 strain of E. coli and exhibited APX enzyme activity. The structure-function relationship of the protein was deduced by modelling a three-dimensional structure of Ec-apx1, on the basis of comparative homology using SWISS-MODEL. Real time PCR analysis of Ec-apx1 expression at mRNA level showed that the transcript increased under drought stress, with maximum levels attained 5-days after imposition of stress. Our results suggest that Ec-apx1 has a distinct pattern of expression and plays a pivotal role in drought stress tolerance. Therefore, the cloned isoform of ascorbate peroxidase can be used for developing stress tolerant genotypes of important crops, through transgenic approach.
A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis
(Portland Press, 2021) Salvi, Prafull; Kumar, Bhavnesh; Kamble, Nitin Uttam; Hazra, Abhijit; Majee, Manoj
Galactinol synthase (GolS) catalyzes the key regulatory step in the biosynthesis of Raffinose Family Oligosaccharides (RFOs). Even though the physiological role and regulation of this enzyme has been well studied, little is known about active site amino acids and the structure-function relationship with substrates of this enzyme. In the present study, we investigate the active site amino acid and structure function relationship for this enzyme. Using a combination of three-dimensional homology modelling, molecular docking along with a series of deletion, site directed mutagenesis followed by in vitro biochemical and in vivo functional analysis; we have studied active site amino acids and their interaction with the substrate of chickpea and Arabidopsis GolS enzyme. Our study reveals that the GolS protein possesses GT8 family-specific several conserved motifs in which NAG motif plays a crucial role in substrate binding and catalytic activity of this enzyme. Deletion of entire NAG motif or deletion or the substitution (with alanine) of any residues of this motif results in complete loss of catalytic activity in in vitro condition. Furthermore, disruption of NAG motif of CaGolS1 enzyme disrupts it’s in vivo cellular function in yeast as well as in planta. Together, our study offers a new insight into the active site amino acids and their substrate interaction for the catalytic activity of GolS enzyme. We demonstrate that NAG motif plays a vital role in substrate binding for the catalytic activity of galactinol synthase that affects overall RFO synthesis.
CONSTANS, a key-player connecting day length to seed size
(Elsevier B.V., 2023) Achary, Rakesh Kumar; Majee, Manoj
Plants sense oscillation in the day length as a reliable seasonal cue to drive optimal vegetative and reproductive growth. A recent study by Yu et al. has revealed how day length regulates seed size through CONSTANS. The CONSTANS-APETALA2 module enables plants to optimize their reproductive growth based on their photoperiod response type.
Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum
(Elsevier B.V., 2020) Yadav, Prakarsh K.; Salvi, Prafull; Kamble, Nitin Uttam; Petla, Bhanu Prakash; Majee, Manoj; Saxena, Saurabh C.
Myo-inositol monophosphatase (IMP) is a crucial enzyme in the inositol biosynthetic pathway that dephosphorylates myo-inositol 1-phosphate and other inositol phosphate derivative compounds to maintain the homeostasis of cellular inositol pool. In our previous research, we have biochemically and functionally characterized IMP enzyme from chickpea (CaIMP), which was able to catalyze diverse substrates. We cloned, overexpressed recombinant CaIMP protein and purified it and further characterized the CaIMP with its three main substrates viz. galactose 1-P, inositol 6-P and fructose 1,6-bisP. Homology model of CaIMP was generated to elucidate the factors contributing to the broad substrate specificity of the protein. The active site of the CaIMP protein was analysed with respect to its interactions with the proposed substrates. Structural features such as, high B-factor and flexible loop regions in the active site, inspired further investigation into the static and dynamic behaviour of the active site of CaIMP protein. The electrostatic biding of each of the key substrates was assessed through molecular docking. Furthermore, molecular dynamics simulations showed that these interactions indeed were stable for extended periods of time under physiological conditions. These experiments conclusively allowed us to establish the primary factors contributing to the promiscuity in substrate binding by CaIMP protein.
Deep transcriptome sequencing of wild halophyte rice, Porteresia coarctata, provides novel insights into the salinity and submergence tolerance factors
(Oxford University Press, 2014) Garg, Rohini; Verma, Mohit; Agrawal, Shashank; Shankar, Rama; Majee, Manoj; Jain, Mukesh
Porteresia coarctata is a wild relative of rice with capability of high salinity and submergence tolerance. The transcriptome analyses of Porteresia can lead to the identification of candidate genes involved in salinity and submergence tolerance. We sequenced the transcriptome of Porteresia under different conditions using Illumina platform and generated about 375 million high-quality reads. After optimized assembly, a total of 152 367 unique transcript sequences with average length of 794 bp were obtained. Many of these sequences might represent fragmented transcripts. Functional annotation revealed the presence of genes involved in diverse cellular processes and 2749 transcription factor (TF)-encoding genes in Porteresia. The differential gene expression analyses identified a total of 15 158 genes involved in salinity and/or submergence response(s). The stress-responsive members of different TF families, including MYB, bHLH, AP2-EREBP, WRKY, bZIP and NAC, were identified. We also revealed key metabolic pathways, including amino acid biosynthesis, hormone biosynthesis, secondary metabolite biosynthesis, carbohydrate metabolism and cell wall structures, involved in stress tolerance in Porteresia. The transcriptome analyses of Porteresia are expected to highlight genes/pathways involved in salinity and submergence tolerance of this halophyte species. The data can serve as a resource for unravelling the underlying mechanism and devising strategies to engineer salinity and submergence tolerance in rice.
Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation
(Nature Publishing Group, 2016) Salvi, Prafull; Saxena, Saurabh Chandra; Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kaur, Harmeet; Verma, Pooja; Rao, Venkateswara; Ghosh, Shraboni; Majee, Manoj
Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation.
Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses
(Oxford University Press, 2013) Saxena, Saurabh C.; Salvi, Prafull; Kaur, Harmeet; Verma, Pooja; Petla, Bhanu Prakash; Rao, Venkateswara; Kamble, Nitin; Majee, Manoj
myo-Inositol monophosphatase (IMP) is an essential enzyme in the myo-inositol metabolic pathway where it primarily dephosphorylates myo-inositol 1-phosphate to maintain the cellular inositol pool which is important for many metabolic and signalling pathways in plants. The stress-induced increased accumulation of inositol has been reported in a few plants including chickpea; however, the role and regulation of IMP is not well defined in response to stress. In this work, it has been shown that IMP activity is distributed in all organs in chickpea and was noticeably enhanced during environmental stresses. Subsequently, using degenerate oligonucleotides and RACE strategy, a full-length IMP cDNA (CaIMP) was cloned and sequenced. Biochemical study revealed that CaIMP encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity, although maximum activity was observed with the myo-inositol 1-phosphate and l-galactose 1-phosphate substrates. Transcript analysis revealed that CaIMP is differentially expressed and regulated in different organs, stresses and phytohormones. Complementation analysis in Arabidopsis further confirmed the role of CaIMP in l-galactose 1-phosphate and myo-inositol 1-phosphate hydrolysis and its participation in myo-inositol and ascorbate biosynthesis. Moreover, Arabidopsis transgenic plants over-expressing CaIMP exhibited improved tolerance to stress during seed germination and seedling growth, while the VTC4/IMP loss-of-function mutants exhibited sensitivity to stress. Collectively, CaIMP links various metabolic pathways and plays an important role in improving seed germination and seedling growth, particularly under stressful environments.
Differentially expressed seed aging responsive heat shock protein OsHSP18.2 implicates in seed vigor, longevity and improves germination and seedling establishment under abiotic stress
(Frontiers Media S.A., 2015) Kaur, Harmeet; Petla, Bhanu P.; Kamble, Nitin U.; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Ghosh, Shraboni; Majee, Manoj
Small heat shock proteins (sHSPs) are a diverse group of proteins and are highly abundant in plant species. Although majority of these sHSPs were shown to express specifically in seed, their potential function in seed physiology remains to be fully explored. Our proteomic analysis revealed that OsHSP18.2, a class II cytosolic HSP is an aging responsive protein as its abundance significantly increased after artificial aging in rice seeds. OsHSP18.2 transcript was found to markedly increase at the late maturation stage being highly abundant in dry seeds and sharply decreased after germination. Our biochemical study clearly demonstrated that OsHSP18.2 forms homooligomeric complex and is dodecameric in nature and functions as a molecular chaperone. OsHSP18.2 displayed chaperone activity as it was effective in preventing thermal inactivation of Citrate Synthase. Further, to analyze the function of this protein in seed physiology, seed specific Arabidopsis overexpression lines for OsHSP18.2 were generated. Our subsequent functional analysis clearly demonstrated that OsHSP18.2 has ability to improve seed vigor and longevity by reducing deleterious ROS accumulation in seeds. In addition, transformed Arabidopsis seeds also displayed better performance in germination and cotyledon emergence under adverse conditions. Collectively, our work demonstrates that OsHSP18.2 is an aging responsive protein which functions as a molecular chaperone and possibly protect and stabilize the cellular proteins from irreversible damage particularly during maturation drying, desiccation and aging in seeds by restricting ROS accumulation and thereby improves seed vigor, longevity and seedling establishment.
Ectopic expression of the ABA-inducible dehydration-responsive chickpea L-myo-inositol 1-phosphate synthase 2 (CaMIPS2) in Arabidopsis enhances tolerance to salinity and dehydration stress
(Springer, 2013) Kaur, Harmeet; Verma, Pooja; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C.; Majee, Manoj
Myo-inositol participates in many different aspects of plant physiology and myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate limiting step of inositol biosynthetic pathway. Chickpea (Cicer arietinum), a drought-tolerant leguminous crop plant, is known to accumulate increased inositol during dehydration stress. Previously, we reported two differentially expressed divergent genes (CaMIPS1 and CaMIPS2) encoding two MIPS isoforms in chickpea. In this communication, we demonstrated that CaMIPS2 is an early dehydration-responsive gene and is also rapidly induced by exogenous ABA application, while CaMIPS1 expression is not much influenced by dehydration or ABA. The regulation of expression of these two genes has been studied by examining their promoter activity through GUS reporter gene and differential promoter activity has been observed. Moreover, unlike CaMIPS1 promoter, CaMIPS2 promoter contains CRT/DRE cis-regulatory element which seems to play a key role in dehydration-induced expression of CaMIPS2. Furthermore, CaMIPS1 and CaMIPS2 have been successfully complemented and shown to repair the defect of seedling growth and altered seed phenotype of Atmips1 mutant. Moreover, Arabidopsis transgenic plants overexpressing CaMIPS1 or CaMIPS2 exhibit improved tolerance to salinity and dehydration stresses and such tolerance of transgenic plants is correlated with their elevated level of inositol. Remarkably, CaMIPS2 transgenic lines perform better in all attributes than CaMIPS1 transformants under such stress conditions, due to comparatively unabated production of inositol by CaMIPS2 enzyme, as this enzyme retains significant activity under stress conditions.
Ectopic over-expression of ABA-responsive Chickpea galactinol synthase (CaGolS) gene results in improved tolerance to dehydration stress by modulating ROS scavenging
(Elsevier B.V., 2020) Salvi, Prafull; Kamble, Nitin Uttam; Majee, Manoj
Galactinol synthase (EC: 2.4.1.123) is a crucial enzyme of raffinose family oligosaccharides (RFO’s) biosynthesis, essentially catalyzes the first crucial step in the raffinose biosynthetic pathway. Galactinol and raffinose accumulation along with its higher homologs such as stachyose and verbascose has been considered to participate in plant abiotic stress tolerance. Previously, we reported the function of chickpea galactinol synthase (CaGolS) genes in seed vigor and heat and oxidative stress tolerance. Here, we demonstrate the role and regulation of CaGolS in response to dehydration stress in chickpea. Through transcript accumulation and promoter-gus analyses, we demonstrate that the expression of CaGolS1 and CaGolS2 are positively influenced by dehydration stress and ABA treatment. Further, we have shown that the level of galactinol and raffinose are remarkably enhanced in response to dehydration stress and ABA. Utilizing CaGolS1 and CaGolS2 over-expression Arabidopsis lines, we demonstrate the role of CaGolS in dehydration stress tolerance. The biochemical and physiological analysis revealed that CaGolS over-expressing transgenic lines exhibited improved phenotype with respect to higher number of siliques, plant height, and rosette diameter under dehydration stress. The improved dehydration stress tolerance was corelated with higher chlorophyll retention and relative water content of transgenic lines. Further, lower H2O2, MDA content, and ion-leakage in transgenic lines suggest that CaGolS mediates dehydration stress tolerance by protecting the membrane damage from ROS attack. Collectively, our data highlight the prospect of CaGolS genes in improving dehydration stress tolerance in plants.
Ectopic overexpression of cytosolic ascorbate peroxidase gene (Apx1) improves salinity stress tolerance in Brassica juncea by strengthening antioxidative defense mechanism
(Springer Nature, 2020) Saxena, Saurabh C.; Salvi, Prafull; Kamble, Nitin Uttam; Joshi, Pankaj K.; Majee, Manoj; Arora, Sandeep
Salinity stress is considered to be a key constrain that reduces the crop productivity by impairing plant growth and development. During salt stress condition, an underlying mechanism for reduction in crop yield is increase in ROS level that can potentially harm cellular macromolecules, leading to disruption of essential physiological and biochemical processes. Plants possess a complex antioxidative defense machinery for scavenging these ROS. Ascorbate peroxidase (APX, E.C. 1.11.1.11), is a crucial antioxidant enzyme involved in Ascorbate–Glutathione pathway that primarily detoxifies the negative impact of H2O2 in cell. The efficient scavenging of H2O2 is a prerequisite for enhanced tolerance to salinity stress. Here, we have inspected whether over-expression of APX could provide protection against salinity stress. Cytosolic ascorbate peroxidase (Apx1) gene, isolated from Arabidopsis thaliana, was chosen as the candidate gene for strengthening the antioxidative defense system of Brassica juncea. Physiological parameters were employed to analyze the growth status of transgenic plants. Leaf disc assay was done to evaluate the salinity stress tolerance potential of transgenic plants, using several physiological and biochemical parameters. Under salinity stress, the transgenic plants performed well as compared to their non-transgenic counterparts; as revealed through greater proline accumulation, increased chlorophyll stability index, lower chlorophyll a/b ratio, and higher antioxidative enzyme activities. Further, the lower H2O2 levels were well correlated with lesser membrane damage as measured through MDA content. Collectively, our results clearly depicted that ectopic overexpression of AtApx1 gene was able to confer salinity stress tolerance by strengthening the antioxidative defense system in B. juncea.