Abstract
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.
Highlights
Glutamine synthetase (GS; L-glutamate-ammonia ligase; electrical conductivity (EC) 6.3.1.2) is a key enzyme involved in nitrogen (N) metabolism of plants, as it catalyzes the critical incorporation of inorganic ammonium into glutamine in an ATP-dependent manner (Miflin and Habash, 2002; Bernard and Habash, 2009)
Our results demonstrate that the increase in expression of the GS isoforms in the overexpress the cytosolic GSl;1 (OsGS1);1/one gene encodes the plastidic GS2 (OsGS2) co-overexpressing transgenic Nipponbare rice, led to a corresponding increase in GS protein as well as total GS enzymatic activity (Figures 2C–E)
Our results show that the enhanced agronomic yield parameters in the OsGS1;1/OsGS2 co-overexpressing transgenic rice plants correlated with the increased photosynthetic rates of transgenic lines in comparison to control plants, which suggests that the co-overexpression of OsGS1;1/OsSG2 provided efficient photo-oxidative protection to the transgenic rice plants under reproductive stage drought and salinity stress
Summary
Glutamine synthetase (GS; L-glutamate-ammonia ligase; EC 6.3.1.2) is a key enzyme involved in nitrogen (N) metabolism of plants, as it catalyzes the critical incorporation of inorganic ammonium into glutamine in an ATP-dependent manner (Miflin and Habash, 2002; Bernard and Habash, 2009). GS is responsible for the re-assimilation of NH+4 , produced during various cellular metabolic processes including, photorespiration and protein degradation, which are further enhanced during stress or senescence (Bernard and Habash, 2009). Two isoforms of GS, the cytosolic GS1, and the chloroplastic GS2 are generally present in higher plants. The smaller cytosolic isoform GS1 is responsible for the primary assimilation of inorganic N availed from the soil in the form of nitrate or ammonia, and the re-assimilation of NH+4 released by protein degradation in senescing leaves (Bernard and Habash, 2009). A multigene family encodes the cytosolic GS1, while only a single gene encodes the chloroplastic GS2. Due to its central role in N metabolism, GS is considered a Abbreviations: Ai, Active ingredient; BCIP, 5-Bromo-4-chloro-3-indolyl phosphate; DAB, 3,3-Diaminobenzidine; DIG, Digoxigenin; EC, Electrical conductivity; GS, Glutamine synthetase; MDA, Malondialdehyde; MW, Molecular Weight; NBT, Nitroblue tetrazolium; NUE, Nitrogen use efficiency; PAGE, Polyacrylamide gel electrophoresis; PBS, Phosphate buffered saline; PEG, Poly Ethylene Glycol; PPT, Phosphinothricin; QTL, Quantitative trait loci; ROS, Reactive oxygen species; RWC, Relative water content; SDS, Sodium dodecyl sulfate; WUE, Water use efficiency; YS, Yoshida hydroponics nutrient solution
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