Tanushri Kaul - Academia.edu (original) (raw)
Papers by Tanushri Kaul
Plant Molecular Biology Reporter, Apr 8, 2014
Globally, weeds and sap-sucking insects are major constrains in rice production. We have combined... more Globally, weeds and sap-sucking insects are major constrains in rice production. We have combined two desired traits for resistance to glyphosate and sapsuckers by transgenic overexpression of mutated rice 5-enolpyruvylshikimate3phosphate synthase (Os-mEPSPS) and garlic leaf lectin (Allium sativum leaf agglutinin, ASAL) in a high yielding IR-64 rice cultivar. Polymerase chain reaction (PCR), southern and genome walking analyses showed stable integration of transgenes in genomes of transgenic lines. The molecular analysis of transgenic lines revealed effective expression of both Os-mEPSPS and ASAL transcripts and their respective proteins and imparted resistance to glyphosate and brown planthopper. In planta brown hopper assays in the progenies of independent rice transgenic lines exhibit enhanced ASAL expression that imparts substantial level of resistance, reduced survival and fecundity of brown hoppers by~74 and~68 %, respectively. The glyphosate and sap-sucking insect pestresistant transgenic rice lines showed normal phenotype and no growth penalty or fitness costs. The double transgenic traits co-segregated as a single unit in the subsequent generations. In spite of the presence of several herbicide-and insect-resistant GM rice crops in the market, there is a need to develop new transgenic rice lines by selecting the transgenes from homologous and/or edible crop sources that circumvent the biosafety issues raised by regulatory bodies regarding the acceptance of these products for human consumption. In this scenario, the transgenic rice varieties generated in this study serve as a novel genetic resource in rice breeding aimed at durable resistance to glyphosate and sap-sucking insect pests.
Analytical Biochemistry, Oct 1, 2005
We have designed a simple and eYcient polymerase chain reaction (PCR)-based cDNA subtraction prot... more We have designed a simple and eYcient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of diVerentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-ampliWable Wrst-strand cDNA population was synthesized by priming oligo-dT primer with a deWned 5Ј heel sequence and ligating another speciWed single-stranded oligonucleotide primer on the 3Ј ends of Wrst-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5Ј biotinylated forward primer during PCR ampliWcation to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unampliWed Wrst strand (antisense) of the interrogating cDNA population was hybridized with a large excess of ampliWed sense strands of control cDNA. We used magnetic bead technology for the eYcient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR ampliWcation for the enrichment and sequence abundance normalization of diVerentially expressed genes. Construction of a subtracted and normalized cDNA library eYciently eliminates common abundant cDNA messages and also increases the probability of identifying clones diVerentially expressed in low-abundance cDNA messages. We used this method to successfully isolate diVerentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.
Biotechnology Letters, Apr 29, 2011
Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates th... more Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants improved stress tolerance indicating the DRE/DREB regulon is conserved across plants. We developed 30 transgenic T 0 rice plants overexpressing OsDREB2A which were devoid of any growth penalty or phenotypic abnormalities during stressed or non-stressed conditions. Integration of T-DNA in the rice genome and stress inducible overexpression of OsDREB2A had occurred in these transgenic lines. Functional analyses of T 1 -3 and T 1 -10 lines revealed significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type. OsDREB2A, thus, confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier.
Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cell... more Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cells towards embryo development that is crucial for cellular totipotency in higher plants. It is a prototype for comprehension of the physiological, biochemical and molecular biological events transpiring during plant embryo development. In this study, we analyzed the role of exogenous calcium and auxin during somatic embryogenesis in hypocotyl explants of Albizzia lebbeck L. Light microscopic studies revealed that anterior and posterior ends of hypocotyl explants had entirely different patterns of differentiation. Scanning electron microscopic analysis of cultured hypocotyl explants exhibited globular-, heart-and torpedo-shaped somatic embryos developing from the anterior cut surface and gradually proceeding towards the other end. Surface texture of these somatic embryos showed uni-or bi-celled trichomes. Presence of exogenous calcium (1-4mM) in hormone-free B5 medium is prerequisite for induction of somatic embryos in hypocotyl explants derived from 7-d-old, light grown seedlings of Albizzia lebbeck L. A two-fold increase in calcium concentration from 2mM (B5 medium) to 4mM led to doubling of somatic embryogenic response whereas, embryogenic callus was induced in explants raised on medium containing 10 or 20mM calcium. Depletion or high dosage in the medium (20 mM) induce nonembryogenic callus in explants. A similar response induced on hypocotyl explants upon treatments with NPA and PCIB, (auxin transport blockers). Results indicate that polar auxin transport or basipetal movement of IAA triggers acropetal transport of calcium. This combination of inverse fluxes acts as the primary signal for embryogenic response at the cut ends proximal to shoot apex and callusing at the cut ends distal to shoot apex of explants. Moreover, a correlation exists between NPA and PCIB-induced disruption of basipetal IAA transport and the inhibitory effect of NPA and PCIB on calcium translocation in the induction of similar morphogenetic responses.
Molecular Biology Reports, Oct 10, 2014
The generation of excess reactive oxygen species (ROS) is one of the most common consequences of ... more The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of *53.5 kDa. The PgGR peptide exhibits 54-89 % sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal-and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.
Acta Physiologiae Plantarum, 2015
Mutiple abiotic stresses lead to generation of oxidative stress or reactive oxygen species (ROS) ... more Mutiple abiotic stresses lead to generation of oxidative stress or reactive oxygen species (ROS) in cells propounding as a major limiting factor in plant productivity. ROS is ubiquitously produced in cells at low levels during normal growth conditions. However, in stress metabolic perturbations are concomitant with high ROS production and the innate anti-oxidative machinery is in capable to scavenge the excess ROS. Excessive ROS causes peroxidation of membrane lipids, membrane disintegration and eventually cell death. In plants, crucial ROS scavenging enzymes of ascorbate glutathione pathway are instrumental in combating oxidative stress by deactivation of ROs released during multiple redox reactions. In order to eliminate or alleviate excess ROS production during adverse stress conditions, we propose to over express the crucial enzyme of ascorbate glutathione pathway that include Superoxide dismutase (SOD), Ascorbate Peroxidase (APX), Mono dehydroascorbate reductase (MDHAR), Dehydr...
![Research paper thumbnail of A Simple and Efficient Method for High Quality DNA Extraction from Sweet Sorghum [Sorghum bicolor (L.) Moench]](https://attachments.academia-assets.com/47456481/thumbnails/1.jpg)
](A An nn na al ls s o of f P Pl la an nt t S Sc ci ie en nc ce es s
Applied biochemistry and biotechnology
The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gen... more The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg−1 obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74 % of its original activity after incubation at 95 °C for 10 min. In the presence of Ca2+, the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is...
Phytases are phosphohydrolases that facilitate the sequential removal of phosphate from phytate. ... more Phytases are phosphohydrolases that facilitate the sequential removal of phosphate from phytate. Monogastric species such as swine, poultry and fish including humans need external phytase to digest phytate, the major form of phosphorus in plant-derived food products or feed. Eventually, dietary phytases are dispensed to liberate feed phytate-phosphorus to reduce the supplementation of inorganic phosphorus and to alleviate their phosphorus excretion. Phytases have emerged as one of the most efficient and productive feed additives. However, addition of commercially available phytase enzyme to the animal feed is expensive and laborious. To overcome these problems, development of genetically engineered plants for heterologous production of phytases improves the phosphorus and mineral bioavailability. Additionally, it reduces the phytic acid excretion and phosphate load on agricultural ecosystem and thereby alleviates eutrophication of aquatic environment. This review aims to summarize the recent information on plant-based phytase development for identifying an ideal phytase for transgenic plant production and its potential applications. It targets crucial and synergistic analyses on the global impression, innovative application and future prospects of phytases in promoting sustainable animal and human health.
Biochemistry & Physiology: Open Access, 2015
CAREs play an important role in plant stress tolerance by interacting with transcription factors ... more CAREs play an important role in plant stress tolerance by interacting with transcription factors and controlling the expression of many stress related potential genes. Recognition of promoters and their regulatory elements is one of the crucial challenges in biotechnology. In this study, we examine cis acting regulatory element in 5' upstream regions (~1 kb) of Asc-Glu pathway genes such as SOD, APX, MDHAR, DHAR, and GR. The evolutionary relationships amongst these sequences were deciphered using MEGA v. 6.0. The promoter region these genes contain various cis acting regulatory elements such as MBS, DRE/C repeat, W box, HSE, TCA element, LTR, ABRE box, ARE box, Wun and DRE that have significant role in stress tolerance Asc-Glu promoter sequences analysis revealed their specific responsiveness or overlapping in various environment stress and significantly contribute toward plant growth and development.
![Research paper thumbnail of A Simple and Efficient Method for High Quality DNA Extraction from Sweet Sorghum [Sorghum bicolor (L.) Moench]](https://attachments.academia-assets.com/47456484/thumbnails/1.jpg)
](A An nn na al ls s o of f P Pl la an nt t S Sc ci ie en nc ce es s
Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cell... more Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cells towards embryo development that is crucial for cellular totipotency in higher plants. It is a prototype for comprehension of the physiological, biochemical and molecular biological events transpiring during plant embryo development. In this study, we analyzed the role of exogenous calcium and auxin during somatic embryogenesis in hypocotyl explants of Albizzia lebbeck L. Light microscopic studies revealed that anterior and posterior ends of hypocotyl explants had entirely different patterns of differentiation. Scanning electron microscopic analysis of cultured hypocotyl explants exhibited globular-, heart-and torpedo-shaped somatic embryos developing from the anterior cut surface and gradually proceeding towards the other end. Surface texture of these somatic embryos showed uni-or bi-celled trichomes. Presence of exogenous calcium (1-4mM) in hormone-free B5 medium is prerequisite for induction of somatic embryos in hypocotyl explants derived from 7-d-old, light grown seedlings of Albizzia lebbeck L. A two-fold increase in calcium concentration from 2mM (B5 medium) to 4mM led to doubling of somatic embryogenic response whereas, embryogenic callus was induced in explants raised on medium containing 10 or 20mM calcium. Depletion or high dosage in the medium (20 mM) induce nonembryogenic callus in explants. A similar response induced on hypocotyl explants upon treatments with NPA and PCIB, (auxin transport blockers). Results indicate that polar auxin transport or basipetal movement of IAA triggers acropetal transport of calcium. This combination of inverse fluxes acts as the primary signal for embryogenic response at the cut ends proximal to shoot apex and callusing at the cut ends distal to shoot apex of explants. Moreover, a correlation exists between NPA and PCIB-induced disruption of basipetal IAA transport and the inhibitory effect of NPA and PCIB on calcium translocation in the induction of similar morphogenetic responses.
Molecular Biology Reports, 2014
The generation of excess reactive oxygen species (ROS) is one of the most common consequences of ... more The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of *53.5 kDa. The PgGR peptide exhibits 54-89 % sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal-and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.
Plant Molecular Biology Reporter, 2015
Excessive generation of reactive oxygen species (ROS) due to environmental stresses critically ef... more Excessive generation of reactive oxygen species (ROS) due to environmental stresses critically effects plant development and productivity. Plants efficiently detoxify ROS by both non-enzymatic and enzymatic mechanisms. Plant glutathione peroxidases (GPXs) are non-haeme thiol peroxidases that catalyze the reduction of H 2 O 2 (or organic hydroperoxides) to water or the respective alcohols using reduced glutathione or thioredoxin. Genome-wide analysis of the known GPXs from rice and Arabidopsis genomes revealed their gene structure, conserved motifs, localization and tissue-specific and/or organ-specific expression profiles in response to various abiotic stresses. Among the eight genes that encoded GPX proteins from Arabidopsis, AtGPX3 showed two alternate spliced forms that spread over four chromosomes. Five genes encoded for rice GPX proteins, while OsGPX1 showed three spliced variants that were distributed on five chromosomes. Utilizing the publicly available microarray and massively parallel signature sequencing (MPSS) data, the GPXs revealed stress-responsive, tissue-specific and/or organspecific expression profiles. Presence of important cisregulatory elements analyzed in the GPX promoter sequences revealed their overlapping or specific responsiveness to different abiotic stresses. Co-expression data of Arabidopsis GPX genes suggested that various protein kinase family members and stress-responsive proteins co-expressed with the GPX proteins. Transcript profile of rice GPX genes by qRT-PCR validated their functional roles in signal transduction and stress pathways. Results revealed that plant GPXs play a crucial role in response to stress and significantly contribute towards their growth and development.
Molecular Biotechnology, 2015
Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked s... more Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked subunits chain A and B, which loses its sweetness on denaturation. In this study, we validated the expression of a synthetic monellin gene encoding a single polypeptide chain covalently linking the two subunits under T 7 and fruit-ripening-specific promoters in Escherichia coli and tomato fruits, respectively. Purified recombinant monellin protein retained its sweet flavour at 70°C and pH 2. We developed 15 transgenic T 0 tomato plants overexpressing monellin, which were devoid of any growth penalty or phenotypic abnormalities during greenhouse conditions. T-DNA integration and fruit-specific heterologous expression of monellin had occurred in these transgenic tomato lines. ELISA revealed that expression of monellin was 4.5 % of the total soluble fruit protein. Functional analyses of transgenic tomatoes of T 2 -5 and T 2 -14 lines revealed distinctly strong sweetness compared with wild type. Monellin a potential non-carbohydrate sweetener, if expressed in high amounts in fruits and vegetables, would enhance their flavour and quality.
Plant biotechnology journal, 2014
Despite the declarations and collective measures taken to eradicate hunger at World Food Summits,... more Despite the declarations and collective measures taken to eradicate hunger at World Food Summits, food security remains one of the biggest issues that we are faced with. The current scenario could worsen due to the alarming increase in world population, further compounded by adverse climatic conditions, such as increase in atmospheric temperature, unforeseen droughts and decreasing soil moisture, which will decrease crop yield even further. Furthermore, the projected increase in yields of C3 crops as a result of increasing atmospheric CO2 concentrations is much less than anticipated. Thus, there is an urgent need to increase crop productivity beyond existing yield potentials to address the challenge of food security. One of the domains of plant biology that promises hope in overcoming this problem is study of C3 photosynthesis. In this review, we have examined the potential bottlenecks of C3 photosynthesis and the strategies undertaken to overcome them. The targets considered for po...
Applied biochemistry and biotechnology, Jan 15, 2015
The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gen... more The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74 % of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activit...
Bioinformation, 2014
Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cyc... more Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha heli...
Plant Molecular Biology Reporter, Apr 8, 2014
Globally, weeds and sap-sucking insects are major constrains in rice production. We have combined... more Globally, weeds and sap-sucking insects are major constrains in rice production. We have combined two desired traits for resistance to glyphosate and sapsuckers by transgenic overexpression of mutated rice 5-enolpyruvylshikimate3phosphate synthase (Os-mEPSPS) and garlic leaf lectin (Allium sativum leaf agglutinin, ASAL) in a high yielding IR-64 rice cultivar. Polymerase chain reaction (PCR), southern and genome walking analyses showed stable integration of transgenes in genomes of transgenic lines. The molecular analysis of transgenic lines revealed effective expression of both Os-mEPSPS and ASAL transcripts and their respective proteins and imparted resistance to glyphosate and brown planthopper. In planta brown hopper assays in the progenies of independent rice transgenic lines exhibit enhanced ASAL expression that imparts substantial level of resistance, reduced survival and fecundity of brown hoppers by~74 and~68 %, respectively. The glyphosate and sap-sucking insect pestresistant transgenic rice lines showed normal phenotype and no growth penalty or fitness costs. The double transgenic traits co-segregated as a single unit in the subsequent generations. In spite of the presence of several herbicide-and insect-resistant GM rice crops in the market, there is a need to develop new transgenic rice lines by selecting the transgenes from homologous and/or edible crop sources that circumvent the biosafety issues raised by regulatory bodies regarding the acceptance of these products for human consumption. In this scenario, the transgenic rice varieties generated in this study serve as a novel genetic resource in rice breeding aimed at durable resistance to glyphosate and sap-sucking insect pests.
Analytical Biochemistry, Oct 1, 2005
We have designed a simple and eYcient polymerase chain reaction (PCR)-based cDNA subtraction prot... more We have designed a simple and eYcient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of diVerentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-ampliWable Wrst-strand cDNA population was synthesized by priming oligo-dT primer with a deWned 5Ј heel sequence and ligating another speciWed single-stranded oligonucleotide primer on the 3Ј ends of Wrst-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5Ј biotinylated forward primer during PCR ampliWcation to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unampliWed Wrst strand (antisense) of the interrogating cDNA population was hybridized with a large excess of ampliWed sense strands of control cDNA. We used magnetic bead technology for the eYcient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR ampliWcation for the enrichment and sequence abundance normalization of diVerentially expressed genes. Construction of a subtracted and normalized cDNA library eYciently eliminates common abundant cDNA messages and also increases the probability of identifying clones diVerentially expressed in low-abundance cDNA messages. We used this method to successfully isolate diVerentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.
Biotechnology Letters, Apr 29, 2011
Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates th... more Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants improved stress tolerance indicating the DRE/DREB regulon is conserved across plants. We developed 30 transgenic T 0 rice plants overexpressing OsDREB2A which were devoid of any growth penalty or phenotypic abnormalities during stressed or non-stressed conditions. Integration of T-DNA in the rice genome and stress inducible overexpression of OsDREB2A had occurred in these transgenic lines. Functional analyses of T 1 -3 and T 1 -10 lines revealed significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type. OsDREB2A, thus, confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier.
Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cell... more Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cells towards embryo development that is crucial for cellular totipotency in higher plants. It is a prototype for comprehension of the physiological, biochemical and molecular biological events transpiring during plant embryo development. In this study, we analyzed the role of exogenous calcium and auxin during somatic embryogenesis in hypocotyl explants of Albizzia lebbeck L. Light microscopic studies revealed that anterior and posterior ends of hypocotyl explants had entirely different patterns of differentiation. Scanning electron microscopic analysis of cultured hypocotyl explants exhibited globular-, heart-and torpedo-shaped somatic embryos developing from the anterior cut surface and gradually proceeding towards the other end. Surface texture of these somatic embryos showed uni-or bi-celled trichomes. Presence of exogenous calcium (1-4mM) in hormone-free B5 medium is prerequisite for induction of somatic embryos in hypocotyl explants derived from 7-d-old, light grown seedlings of Albizzia lebbeck L. A two-fold increase in calcium concentration from 2mM (B5 medium) to 4mM led to doubling of somatic embryogenic response whereas, embryogenic callus was induced in explants raised on medium containing 10 or 20mM calcium. Depletion or high dosage in the medium (20 mM) induce nonembryogenic callus in explants. A similar response induced on hypocotyl explants upon treatments with NPA and PCIB, (auxin transport blockers). Results indicate that polar auxin transport or basipetal movement of IAA triggers acropetal transport of calcium. This combination of inverse fluxes acts as the primary signal for embryogenic response at the cut ends proximal to shoot apex and callusing at the cut ends distal to shoot apex of explants. Moreover, a correlation exists between NPA and PCIB-induced disruption of basipetal IAA transport and the inhibitory effect of NPA and PCIB on calcium translocation in the induction of similar morphogenetic responses.
Molecular Biology Reports, Oct 10, 2014
The generation of excess reactive oxygen species (ROS) is one of the most common consequences of ... more The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of *53.5 kDa. The PgGR peptide exhibits 54-89 % sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal-and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.
Acta Physiologiae Plantarum, 2015
Mutiple abiotic stresses lead to generation of oxidative stress or reactive oxygen species (ROS) ... more Mutiple abiotic stresses lead to generation of oxidative stress or reactive oxygen species (ROS) in cells propounding as a major limiting factor in plant productivity. ROS is ubiquitously produced in cells at low levels during normal growth conditions. However, in stress metabolic perturbations are concomitant with high ROS production and the innate anti-oxidative machinery is in capable to scavenge the excess ROS. Excessive ROS causes peroxidation of membrane lipids, membrane disintegration and eventually cell death. In plants, crucial ROS scavenging enzymes of ascorbate glutathione pathway are instrumental in combating oxidative stress by deactivation of ROs released during multiple redox reactions. In order to eliminate or alleviate excess ROS production during adverse stress conditions, we propose to over express the crucial enzyme of ascorbate glutathione pathway that include Superoxide dismutase (SOD), Ascorbate Peroxidase (APX), Mono dehydroascorbate reductase (MDHAR), Dehydr...
A An nn na al ls s o of f P Pl la an nt t S Sc ci ie en nc ce es s
Applied biochemistry and biotechnology
The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gen... more The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg−1 obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74 % of its original activity after incubation at 95 °C for 10 min. In the presence of Ca2+, the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is...
Phytases are phosphohydrolases that facilitate the sequential removal of phosphate from phytate. ... more Phytases are phosphohydrolases that facilitate the sequential removal of phosphate from phytate. Monogastric species such as swine, poultry and fish including humans need external phytase to digest phytate, the major form of phosphorus in plant-derived food products or feed. Eventually, dietary phytases are dispensed to liberate feed phytate-phosphorus to reduce the supplementation of inorganic phosphorus and to alleviate their phosphorus excretion. Phytases have emerged as one of the most efficient and productive feed additives. However, addition of commercially available phytase enzyme to the animal feed is expensive and laborious. To overcome these problems, development of genetically engineered plants for heterologous production of phytases improves the phosphorus and mineral bioavailability. Additionally, it reduces the phytic acid excretion and phosphate load on agricultural ecosystem and thereby alleviates eutrophication of aquatic environment. This review aims to summarize the recent information on plant-based phytase development for identifying an ideal phytase for transgenic plant production and its potential applications. It targets crucial and synergistic analyses on the global impression, innovative application and future prospects of phytases in promoting sustainable animal and human health.
Biochemistry & Physiology: Open Access, 2015
CAREs play an important role in plant stress tolerance by interacting with transcription factors ... more CAREs play an important role in plant stress tolerance by interacting with transcription factors and controlling the expression of many stress related potential genes. Recognition of promoters and their regulatory elements is one of the crucial challenges in biotechnology. In this study, we examine cis acting regulatory element in 5' upstream regions (~1 kb) of Asc-Glu pathway genes such as SOD, APX, MDHAR, DHAR, and GR. The evolutionary relationships amongst these sequences were deciphered using MEGA v. 6.0. The promoter region these genes contain various cis acting regulatory elements such as MBS, DRE/C repeat, W box, HSE, TCA element, LTR, ABRE box, ARE box, Wun and DRE that have significant role in stress tolerance Asc-Glu promoter sequences analysis revealed their specific responsiveness or overlapping in various environment stress and significantly contribute toward plant growth and development.
A An nn na al ls s o of f P Pl la an nt t S Sc ci ie en nc ce es s
Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cell... more Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cells towards embryo development that is crucial for cellular totipotency in higher plants. It is a prototype for comprehension of the physiological, biochemical and molecular biological events transpiring during plant embryo development. In this study, we analyzed the role of exogenous calcium and auxin during somatic embryogenesis in hypocotyl explants of Albizzia lebbeck L. Light microscopic studies revealed that anterior and posterior ends of hypocotyl explants had entirely different patterns of differentiation. Scanning electron microscopic analysis of cultured hypocotyl explants exhibited globular-, heart-and torpedo-shaped somatic embryos developing from the anterior cut surface and gradually proceeding towards the other end. Surface texture of these somatic embryos showed uni-or bi-celled trichomes. Presence of exogenous calcium (1-4mM) in hormone-free B5 medium is prerequisite for induction of somatic embryos in hypocotyl explants derived from 7-d-old, light grown seedlings of Albizzia lebbeck L. A two-fold increase in calcium concentration from 2mM (B5 medium) to 4mM led to doubling of somatic embryogenic response whereas, embryogenic callus was induced in explants raised on medium containing 10 or 20mM calcium. Depletion or high dosage in the medium (20 mM) induce nonembryogenic callus in explants. A similar response induced on hypocotyl explants upon treatments with NPA and PCIB, (auxin transport blockers). Results indicate that polar auxin transport or basipetal movement of IAA triggers acropetal transport of calcium. This combination of inverse fluxes acts as the primary signal for embryogenic response at the cut ends proximal to shoot apex and callusing at the cut ends distal to shoot apex of explants. Moreover, a correlation exists between NPA and PCIB-induced disruption of basipetal IAA transport and the inhibitory effect of NPA and PCIB on calcium translocation in the induction of similar morphogenetic responses.
Molecular Biology Reports, 2014
The generation of excess reactive oxygen species (ROS) is one of the most common consequences of ... more The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of *53.5 kDa. The PgGR peptide exhibits 54-89 % sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal-and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.
Plant Molecular Biology Reporter, 2015
Excessive generation of reactive oxygen species (ROS) due to environmental stresses critically ef... more Excessive generation of reactive oxygen species (ROS) due to environmental stresses critically effects plant development and productivity. Plants efficiently detoxify ROS by both non-enzymatic and enzymatic mechanisms. Plant glutathione peroxidases (GPXs) are non-haeme thiol peroxidases that catalyze the reduction of H 2 O 2 (or organic hydroperoxides) to water or the respective alcohols using reduced glutathione or thioredoxin. Genome-wide analysis of the known GPXs from rice and Arabidopsis genomes revealed their gene structure, conserved motifs, localization and tissue-specific and/or organ-specific expression profiles in response to various abiotic stresses. Among the eight genes that encoded GPX proteins from Arabidopsis, AtGPX3 showed two alternate spliced forms that spread over four chromosomes. Five genes encoded for rice GPX proteins, while OsGPX1 showed three spliced variants that were distributed on five chromosomes. Utilizing the publicly available microarray and massively parallel signature sequencing (MPSS) data, the GPXs revealed stress-responsive, tissue-specific and/or organspecific expression profiles. Presence of important cisregulatory elements analyzed in the GPX promoter sequences revealed their overlapping or specific responsiveness to different abiotic stresses. Co-expression data of Arabidopsis GPX genes suggested that various protein kinase family members and stress-responsive proteins co-expressed with the GPX proteins. Transcript profile of rice GPX genes by qRT-PCR validated their functional roles in signal transduction and stress pathways. Results revealed that plant GPXs play a crucial role in response to stress and significantly contribute towards their growth and development.
Molecular Biotechnology, 2015
Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked s... more Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked subunits chain A and B, which loses its sweetness on denaturation. In this study, we validated the expression of a synthetic monellin gene encoding a single polypeptide chain covalently linking the two subunits under T 7 and fruit-ripening-specific promoters in Escherichia coli and tomato fruits, respectively. Purified recombinant monellin protein retained its sweet flavour at 70°C and pH 2. We developed 15 transgenic T 0 tomato plants overexpressing monellin, which were devoid of any growth penalty or phenotypic abnormalities during greenhouse conditions. T-DNA integration and fruit-specific heterologous expression of monellin had occurred in these transgenic tomato lines. ELISA revealed that expression of monellin was 4.5 % of the total soluble fruit protein. Functional analyses of transgenic tomatoes of T 2 -5 and T 2 -14 lines revealed distinctly strong sweetness compared with wild type. Monellin a potential non-carbohydrate sweetener, if expressed in high amounts in fruits and vegetables, would enhance their flavour and quality.
Plant biotechnology journal, 2014
Despite the declarations and collective measures taken to eradicate hunger at World Food Summits,... more Despite the declarations and collective measures taken to eradicate hunger at World Food Summits, food security remains one of the biggest issues that we are faced with. The current scenario could worsen due to the alarming increase in world population, further compounded by adverse climatic conditions, such as increase in atmospheric temperature, unforeseen droughts and decreasing soil moisture, which will decrease crop yield even further. Furthermore, the projected increase in yields of C3 crops as a result of increasing atmospheric CO2 concentrations is much less than anticipated. Thus, there is an urgent need to increase crop productivity beyond existing yield potentials to address the challenge of food security. One of the domains of plant biology that promises hope in overcoming this problem is study of C3 photosynthesis. In this review, we have examined the potential bottlenecks of C3 photosynthesis and the strategies undertaken to overcome them. The targets considered for po...
Applied biochemistry and biotechnology, Jan 15, 2015
The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gen... more The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74 % of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activit...
Bioinformation, 2014
Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cyc... more Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha heli...