Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses

Glutamine synthetase (GS) is a key enzyme involved in the nitrogen metabolism of higher plants. Abiotic stresses have adverse effects on crop production and pose a serious threat to global food security. GS activity and expression is known to be significantly modulated by various abiotic stresses. However, very few transgenic overexpression studies of GS have studied its impact on abiotic stress tolerance. GS is also the target enzyme of the broad spectrum herbicide Glufosinate (active ingredient: phosphinothricin). In this study, we investigated the effect of concurrent overexpression of the rice cytosolic GS1 (OsGS1;1) and chloroplastic GS2 (OsGS2) genes in transgenic rice on its tolerance to abiotic stresses and the herbicide Glufosinate. Our results demonstrate that the co-overexpression of OsGS1;1 and OsGS2 isoforms in transgenic rice plants enhanced its tolerance to osmotic and salinity stress at the seedling stage. The transgenic lines maintained significantly higher fresh weight, chlorophyll content, and relative water content than wild type (wt) and null segregant (ns) controls, under both osmotic and salinity stress. The OsGS1;1/OsGS2 co-overexpressing transgenic plants accumulated higher levels of proline but showed lower electrolyte leakage and had lower malondialdehyde (MDA) content under the stress treatments. The transgenic lines showed considerably enhanced photosynthetic and agronomic performance under drought and salinity stress imposed during the reproductive stage, as compared to wt and ns control plants. The grain filling rates of the transgenic rice plants under reproductive stage drought stress (64.6 ± 4.7%) and salinity stress (58.2 ± 4.5%) were significantly higher than control plants, thereby leading to higher yields under these abiotic stress conditions. Preliminary analysis also revealed that the transgenic lines had improved tolerance to methyl viologen induced photo-oxidative stress. Taken together, our results demonstrate that the concurrent overexpression of OsGS1;1 and OsGS2 isoforms in rice enhanced physiological tolerance and agronomic performance under adverse abiotic stress conditions, apparently acting through multiple mechanistic routes. The transgenic rice plants also showed limited tolerance to the herbicide Glufosinate. The advantages and limitations of glutamine synthetase overexpression in crop plants, along with future strategies to overcome these limitations for utilization in crop improvement have also been discussed briefly

Cys residues are decisive for the thioredoxin dependent PgGPx activity.

<p>Homology model of PgGPx (B) was built based on Poplar GPx structure (2p5q) as template (A). (C) Superimposed model of both the structures onto each other. PgGPx active site possess a cadmium ion (pink color ball), coordinated to the peroxidatic Cys 42, Trp 131 and Glu 77 residues. (D) Specific activities were calculated using WT and all other muteins in response to thioredoxin dependent system. Note that all the muteins become almost inactive except C72. (E) Lineweaver-Burk plot was generated to determine different enzyme kinetic parameters for C72 protein. (F) Effects of redox potential on the conformation of PgGPx. Wild type (WT) proteins along with all muteins were either oxidized (lanes 1, 3, 5, 7, 9, and 11) or reduced (lanes 2, 4, 6, 8, 10, and 12) with 30 mM H₂O₂ or 30 mM DTT, respectively and separated on 14% SDS- PAGE.</p

Transgenic plants ectopically expressing <i>PgGPx</i> maintained physiological balance under salinity and drought stresses.

<p>(A-C) Mature transgenic as well as WT rice plants grown in normal condition (A) or in presence 200 mM NaCl (B) or 150 mM mannitol (C). Spider plot (D) was generated by comparing various photosynthetic parameters of three independent transgenic (average of three transgenic lines) plants and corresponding WT under control, salinity and drought stresses. Mean values from WT controls were set at 100% as a reference. (E, F) Agronomic traits for <i>PgGPx</i> rice plants grown in the field under normal and stress conditions. Spider plots of agronomic traits of three independent T1 transgenic lines (average of three transgenic lines) and corresponding WT controls under salinity (E) and drought (F) conditions, respectively, were drawn using mean values from WT control as 100%.</p

Assessment of salinity and drought tolerance of <i>PgGPx</i> overexpressing T₁ transgenic seedlings.

<p>Growth of the transgenic as well as WT rice plants grown under control condition (A) or in the presence 200 mM NaCl (B) or 150 mM mannitol (C). Comparison of shoot length (D) and fresh weight (E) of transgenic vis-a-vis WT plants under salinity and drought stresses showed that transgenic plants had better physiological growth. Analysis of cellular damage in response to salinity and drought were measured by electrolytic leakage (F). Relative H₂O₂ and O^2- accumulation in the transgenic and WT plants under salinity and drought stresses were assessed by histochemical staining. Total H₂O₂ and O^2- levels were visualized by 3,3 0-diaminobenzidine (DAB) staining (G) and nitro blue tetrazolium (NBT) staining (H), respectively.</p

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

Biochemical analysis of T1 <i>PgGPx</i> transgenic lines under salinity and drought stresses.

<p>Different biochemical parameters such as proline, MDA and H₂O₂ was determined in transgenic vis-a- vis WT plants, under salinity (A-C) and drought (D-F) stresses at different time points (0 to 24 hr.). Alterations in the level of proline accumulation (A, D), lipid peroxidation expressed in terms of Malondialdehyde (MDA) content (B, E), and hydrogen peroxide (H₂O₂) content (C, F) were measured from the the transgenic and WT plants. Data represent the average of three replicates ± Standard deviation. 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

Comparison of enzyme kinetic parameters of recombinant GPx proteins from different species.

<p>Comparison of enzyme kinetic parameters of recombinant GPx proteins from different species.</p

Stress responsive expression analysis of <i>PgGPx</i>.

<p>Alteration of <i>PgGPx</i> transcripts were determined by qRT-PCR analysis after exposing <i>Pennisetum gluccum</i> seedlings with 200 mM NaCl for salinity stress or 150 mM manitol for drought stress for 48 h. Histograms depicts the fold change in expression of <i>PgGPx</i> under salinity (A) and drought (B) stresses. Steady-state mRNA level of <i>PgGPx</i> was normalized with respect to house keeping gene <i>tubulin</i> and fold change in expression were calculated as compared to control sample (0 h). Values are presented as mean ± standard deviation of three independent experiments. Statistical significance was determined using two-tailed paired Student’s t-test and are represented with different letters (p < 0.05).</p

Overexpression of improved EPSPS gene results in field level glyphosate tolerance and higher grain yield in rice

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