Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum.

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.

Arabidopsis SKP1-like protein13 (ASK13) positively regulates seed germination and seedling growth under abiotic stress.

SKP1 (S-phase kinase-associated protein1) proteins are key members of the SCF (SKP-cullin-F-box protein) E3 ligase complexes that ubiquitinate target proteins and play diverse roles in plant biology. However, in comparison with other members of the SCF complex, knowledge 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 up-regulated in response to abiotic stress. Yeast two-hybrid library screening and subsequent assessment of in vivo interactions through bimolecular fluorescence complementation analysis revealed that ASK13 not only interacts with F-box proteins but also with other proteins that are not components of SCF complexes. Biochemical analysis demonstrated 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 showed that ASK13 positively influences seed germination and seedling growth, particularly under abiotic stress. Taken together, our data strongly suggest 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.

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.

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.

PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

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.

Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana.

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.

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.

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.

Responses to drought induced oxidative stress in five finger millet varieties differing in their geographical distribution.

The study presents the impact of drought stress on five finger millet varieties (PR202, VL146, VL315, PES400 and VR708), representing contrasting areas of Indian sub-continent. Drought stress induced increase in the activity of superoxide dismutase, ascorbate peroxidase and glutathione reductase was higher in PR202 and VL315, while the activity was lower in the varieties PES400 and VR708. Ascorbate peroxidase : superoxide dismutase ratio, which is a crucial factor in alleviating drought stress, was higher in varieties PR202 and VL315, whilst the varieties PES400 and VR708 exhibited a lower ratio under stress. The variety PES400 recorded maximum stress induced damage, as indicated by higher accumulation of malondialdehyde and hydrogen peroxide; whereas the variety PR202 recorded least stress induced cytotoxic damage. The results clearly indicate that better drought tolerance of the variety PR202 is positively related to the capacity of its antioxidant system to scavenge reactive oxygen species, resulting in a reduced incidence of oxidative damage. Ascorbate peroxidase : superoxide dismutase ratio is found to be a critical factor governing the stress tolerance potential of different varieties. Therefore, varieties PR202 and VL315 were found to be tolerant while PES400 was susceptible to drought stress.