Characterisation and mode of in vitro replication of pea chloroplast OriA sequences

A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication. When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo

Power System State Estimation using Microsoft Excel

This paper presents the design and development of a Microsoft Excel based tool for Power System Static State Estimation. This tool can be effectively used to understand the process of state estimation and its real-time application. The tool contains Newton-Raphson load flow that provides system measurements, which are used as inputs to the state estimator that uses the popular weighted least square (WLS) algorithm. The spreadsheet has features for corrupting the data to simulate wide ranging scenarios of data errors. With the user-friendly screens, this can be a versatile desktop tool for learning and experimenting with the Power System State Estimation. Different system loading and operating configurations and wide range of corrupted measurements can be simulated for obtaining the reliable state estimator. All intermediate numerical results are made available for verification purposes. For illustration IEEE 14 bus system is considered

Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco

Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants

Simultaneous expression of regulatory genes associated with specific drought‐adaptive traits improves drought adaptation in peanut

Adaptation of crops to drought-prone rain-fed conditions can be achieved by improving plant traits such as efficient water mining (by superior root characters) and cellular-level tolerance mechanisms. Pyramiding these drought-adaptive traits by simultaneous expression of genes regulating drought-adaptive mechanisms has phenomenal relevance in improving stress tolerance. In this study, we provide evidence that peanut transgenic plants expressing Alfalfa zinc finger 1 (Alfin1), a root growth-associated transcription factor gene, Pennisetum glaucum heat-shock factor (PgHSF4) and Pea DNA helicase (PDH45) involved in protein turnover and protection showed improved tolerance, higher growth and productivity under drought stress conditions. Stable integration of all the transgenes was noticed in transgenic lines. The transgenic lines showed higher root growth, cooler crop canopy air temperature difference (less CCATD) and higher relative water content (RWC) under drought stress. Low proline levels in transgenic lines substantiate the maintenance of higher water status. The survival and recovery of transgenic lines was significantly higher under gradual moisture stress conditions with higher biomass. Transgenic lines also showed significant tolerance to ethrel-induced senescence and methyl viologen-induced oxidative stress. Several stress-responsive genes such as heat-shock proteins (HSPs), RING box protein-1 (RBX1), Aldose reductase, late embryogenesis abundant-5 (LEA5) and proline-rich protein-2 (PRP2), a gene involved in root growth, showed enhanced expression under stress in transgenic lines. Thus, the simultaneous expression of regulatory genes contributing for drought-adaptive traits can improve crop adaptation and productivity under water-limited conditions

Characterization of phoA, a Bacterial Alkaline Phosphatase for Phi Use Efficiency in Rice Plant

Fertilizers and herbicides are two major components in the agriculture system for achieving crop productivity. Massive use of orthophosphate fertilizers and herbicides poses threats to phosphate reserves and aids the evolution of herbicide tolerant weed biotypes. Phosphite (Phi), a phosphate analog, has been proposed as more beneficial than traditionally used phosphate fertilizers and herbicides in the agriculture. We developed phoA overexpressing transgenic rice that minimizes the phosphate loss and contributes to weed management in the agriculture. The phoA rice lines showed improved root, shoot length and total biomass production under phosphite conditions. Additionally, the complete phenotype and productivity of phoA lines under the phosphite treatment attained was similar to that of plants under phosphate sufficient condition. The Phi metabolizing properties of the phoA overexpressed lines improved under the Phi application and phi treatment enabled controlling of weeds without compromising the yield of transgenic rice plants. Our results indicated that phoA alone or in combination with other Phi metabolizing gene(s) can possibly be used as an effective ameliorating system for improving crop plants for phi-based fertilization and weed management strategy in the agriculture

Transcriptional Downregulation of Rice rpL32 Gene under Abiotic Stress Is Associated with Removal of Transcription Factors within the Promoter Region

Background: The regulation of ribosomal proteins in plants under stress conditions has not been well studied. Although a few reports have shown stress-specific post-transcriptional and translational mechanisms involved in downregulation of ribosomal proteins yet stress-responsive transcriptional regulation of ribosomal proteins is largely unknown in plants. Methodology/Principal Findings: In the present work, transcriptional regulation of genes encoding rice 60S ribosomal protein L32 (rpL32) in response to salt stress has been studied. Northern and RT-PCR analyses showed a significant downregulation of rpL32 transcripts under abiotic stress conditions in rice. Of the four rpL32 genes in rice genome, the gene on chromosome 8 (rpL32_8.1) showed a higher degree of stress-responsive downregulation in salt sensitive rice variety than in tolerant one and its expression reverted to its original level upon withdrawal of stress. The nuclear run-on and promoter:reporter assays revealed that the downregulation of this gene is transcriptional and originates within the promoter region. Using in vivo footprinting and electrophoretic mobility shift assay (EMSA), cis-elements in the promoter of rpL32_8.1 showing reduced binding to proteins in shoots of salt stressed rice seedlings were identified. Conclusions: The present work is one of the few reports on study of stress downregulated genes. The data revealed that rpL32 gene is transcriptionally downregulated under abiotic stress in rice and that this transcriptional downregulation i

Identification of salt-induced genes from Salicornia brachiata, an extreme halophyte through expressed sequence tags analysis

Salinity severely affects plant growth and development causing crop loss worldwide. We have isolated a large number of salt-induced genes as well as unknown and hypothetical genes from Salicornia brachiata Roxb. (Amaranthaceae). This is the first description of identification of genes in response to salinity stress in this extreme halophyte plant. Salicornia accumulates salt in its pith and survives even at 2 M NaCl under field conditions. For isolating salt responsive genes, cDNA subtractive hybridization was performed between control and 500 mM NaCl treated plants. Out of the 1200 recombinant clones, 930 sequences were submitted to the NCBI database (GenBank accession: EB484528 to EB485289 and EC906125 to EC906292). 789 ESTs showed matching with different genes in NCBI database. 4.8% ESTs belonged to stress-tolerant gene category and approximately 29% ESTs showed no homology with known functional gene sequences, thus classified as unknown or hypothetical. The detection of a large number of ESTs with unknown putative function in this species makes it an interesting contribution. The 90 unknown and hypothetical genes were selected to study their differential regulation by reverse Northern analysis for identifying their role in salinity tolerance. Interestingly, both up and down regulation at 500 mM NaCl were observed (21 and 10 genes, respectively). Northern analysis of two important salt tolerant genes, ASR1 (Abscisic acid stress ripening gene) and plasma membrane H<SUP>+</SUP>ATPase, showed the basal level of transcripts in control condition and an increase with NaCl treatment. ASR1 gene is made full length using 5' RACE and its potential role in imparting salt tolerance is being studied

Glutathione Peroxidase of <i>Pennisetum glaucum</i> (PgGPx) Is a Functional Cd²⁺ Dependent Peroxiredoxin that Enhances Tolerance against Salinity and Drought Stress

<div><p>Reactive oxygen species (ROS) arise in the plant system due to inevitable influence of various environmental stimuli. Glutathione peroxidases are one of the important ROS scavengers inside the cell. A glutathione peroxidase (<i>PgGPx</i>) gene was previously found from <i>Pennisetum glauccum</i> abiotic stressed cDNA library. Enzyme kinetics data revealed that PgGPx possessed preference towards thioredoxin rather than glutathione as electron donor and thus belongs to the functional peroxiredoxin group. Moreover, its activity was found to be dependent on divalent cations, especially Cd²⁺ and homology model showed the presence of Cd²⁺ binding site in the protein. Site directed mutagenesis study of PgGPx protein revealed the vital role of two conserved Cysteine residues for its enzymatic activity and structural folding. Expression analysis suggested that <i>PgGPx</i> transcript is highly up-regulated in response to salinity and drought stresses. When expressed ectopically, <i>PgGPx</i> showed enhanced tolerance against multiple abiotic stresses in prokaryotic <i>E</i>. <i>coli</i> and model plant, rice. Transgenic rice plants showed lesser accumulation of MDA and H₂O₂; and higher accumulation of proline as compared to wild type (WT) plants in response to both salinity and drought stresses that clearly indicates suppression of lipid peroxidation and ROS generation in transgenic lines. Moreover, transgenic plants maintained better photosynthesis efficiency and higher level of antioxidant enzyme activity as compared to WT plants under stress conditions. These results clearly indicate the imperative role of PgGPx in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance towards oxidative stress.</p></div

Confirmation of the ectopically expressed <i>PgGPx</i> transgenic rice plants and their stress tolerance potential.

<p>(A) Schematic illustration of the pMDC99-PgGPx expression vector used to overexpress <i>PgGPx</i> in rice plants. Putative transgenic lines were initially screened by PCR using <i>PgGPx</i> gene specific primers (B) and further confirmed by Southern blot analysis using full length <i>PgGPx</i> cDNA as probe (C). To evaluate the expression of <i>PgGPx</i> transcript in the 14 days old WT and five single copy T₁ transgenic lines (L-1, L-3, L-6, L-8 and L-10) under normal condition, Reverse transcription PCR (RT-PCR) was carried out (D) and compared with house keeping gene, <i>Tubulin</i>. (E) Total glutathione peroxidase activity was determined from WT and transgenic plants (L-1, L-3, L-6, L-8 and L-10). Rapid leaf strips senescence assay of transgenic vis-a-vis WT plants was performed, to assess the stress tolerance under 200 mM NaCl or 150 mM mannitol. Leaf strips floated on water served as the experimental control (G). Total chlorophyll contents were estimated from the corresponding control, salt and mannitol treated leaf strips of WT as well as transgenic lines. The data represent means±standard deviation (STD) of three biological replicates (n = 3). Statistically significant differences were determined using two-tailed paired Student’s t-test as compared with WT plants of similar conditions and indicated by ^a,P < 0.001 or ^b,P < 0.01 or ^c,P < 0.05.</p

Transgenic tobacco expressing Entamoeba histolytica calcium binding protein exhibits enhanced growth and tolerance to salt stress

Calcium has been recognized as an important signal in many abiotic stresses. Our earlier work indicated the presence of homologues of a novel calcium binding protein from Entamoeba histolytica (EhCaBP) and its binding proteins with kinase activity in different plants. In this paper, we demonstrate the transfer of EhCaBP cDNA into tobacco via Agrobacterium mediated transformation. The transgenic plants expressing EhCaBP proteins were identified using western blots and immuno-precipitation of the labeled EhCaBP protein. The seeds obtained from the transgenic plants exhibited enhanced rate of germination and increased growth and had 20-37% more dry weight in 3 weeks old seedlings compared to wild type controls. The seeds of transgenic plants were also able to germinate and grow on 200 mM NaCl. The dry weight of different EhCaBP expressing plants maintained on NaCl was 55-100% more as compared to wild type plants. The increased growth and tolerance to salinity conditions were correlated with the expression of EhCaBP protein in the transgenic plants