Publications of NIPGR Scientists

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Author: search.filters.author.Majee, ManojSubject: search.filters.subject.Chickpea

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    Identification, genomic organization, and comprehensive expression analysis reveals the implication of Cicer arietinum SKP1-like genes in abiotic stress
    (Springer Nature Publishing AG, 2023) Varshney, Vishal; Hazra, Abhijit; Majee, Manoj
    Ubiquitin-mediated proteolysis is a central pathway that controls protein turnover. Ubiquitin–proteasome pathway works sequentially to ubiquitinate appropriate substrates that subsequently lead to its degradation via 26S proteasome. Among the several classes of ubiquitin E3 ligases, the SKP1-Cullin-F-box class is generally the most common and widely explored. SKP1-like proteins in plants have gained less attention than other SCF complex components, although they have a larger involvement in controlling wide aspects of vascular plants. Several studies have shown that SKP1-like proteins regulate abiotic stress tolerance on their own, in addition to working in the SCF complex. However, the identification and characterization of SKP1-like genes in chickpea are missing. In the present study, we have identified 15 SKP1-like genes in the chickpea genome that have been categorized into three types type Ia, type Ib, and type II based on the structure and sequence. The evolutionary conservation of the chickpea SKP1 family with dicots and monocots was discovered utilizing phylogenetic analysis. The presence of hormone, plant growth and development, and various stress-related cis-regulatory elements in all chickpea SKP1-like gene promoters showed that SKP1-like genes have a potential role in functions in hormone and various abiotic stress signaling in chickpea. According to the qRT-PCR expression study, most chickpea SKP1-like genes are differently expressed under three abiotic stresses; namely drought, salt, and oxidative stress. As a result, the current work offers up new possibilities for leveraging SKP1-related data to better understand the role of abiotic stress tolerance in the chickpea plant.
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    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.
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    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.
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    Stress inducible galactinol synthase of chickpea (CaGolS) implicates in heat and oxidative stress tolerance through reducing stress induced excessive reactive oxygen species accumulation
    (Oxford University Press, 2018) Salvi, Prafull; Kamble, Nitin Uttam; Majee, Manoj
    Raffinose Family Oligosaccharides (RFOs) participates in various aspects of plant physiology and galactinol synthase (GolS, EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress induced accumulation of RFOs particularly galactinol and raffinose has been reported in few plants; however their precise role and mechanistic insight in stress adaptation remain elusive. In present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. Insilco analysis revealed several common yet distinct cis-acting regulatory elements in their 5' upstream regulatory sequences. Further, in vitro biochemical analysis revealed that CaGolS1 enzyme functions better in stressful conditions than CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 not only exhibits significantly increased galactinol but also raffinose content and display better growth responses than wild type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines are associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared to control plants.Collectively, our data implies that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants.
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    PROTEIN L- ISOASPARTYL METHYLTRANSFERASE2 gene is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins
    (American Society of Plant Biologists, 2013) Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C.; Majee, Manoj
    PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.
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    PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 (CaPIMT1) from chickpea mitigates oxidative stress-induced growth inhibition of Escherichia coli
    (Springer, 2010) Verma, Pooja; Singh, Ajeet; Kaur, Harmeet; Majee, Manoj
    PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs deleterious L-isoaspartyl residues synthesized spontaneously in proteins due to aging or stressful environments and is widespread in living organisms including plants. Even though PIMT activity has been detected from various plant sources, detailed studies are limited to a few species. Our present study on a chickpea (Cicer arietinum) PIMT reveals that apart from seed, PIMT activity is present in other organs and noticeably enhanced during stressful conditions. Using degenerate oligonucleotides and RACE strategy, a full length cDNA (CaPIMT1) was cloned and sequenced. The cDNA is 920 bp in length and contains only one open reading frame of 690 bp encoding 229 amino acids. Genomic structure reveals that the CaPIMT1 gene spans about 2,050 bp in length and contains four exons and three introns. By quantitative real-time RT-PCR, we demonstrate that the transcript of CaPIMT1 is distributed across the organs with maximum levels in seed and is also enhanced under various environmental stress conditions. Purified bacterially expressed protein is further characterized for its catalytic properties. The activity is found to be elevated towards high temperature and pH conditions. Escherichia coli expressing CaPIMT1 show greater tolerance to oxidative stress than E. coli without CaPIMT1. Taken together, our results suggest that PIMT from chickpea shows a distinct pattern of expression and may have a specific role in stress adaptation apart from seed.