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

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.

Keywords: Glufosinate; Multi-Round Gateway technology; abiotic stress; glutamine synthetase; herbicide tolerance; in vitro gene pyramiding.

Figure 1

A simplified schematic diagram of the LR-Recombination based Multi-Round Gateway™ technology used for in vitro pyramiding of the OsGS1;1 and OsGS2 genes. The OsGS1;1 gene was cloned into the Entry vector 1 (EV-1) by restriction enzyme based cloning under the rice Actin 2 promoter (PAct2) and Act2 terminator (Act2T) whereas the OsGS2 gene was cloned into the entry vector 2 (EV-2) under the rice Actin 1 promoter (PAct1) and Act1 terminator (Act1T). In the first round of LR cloning, EV-1 was recombined with the destination vector (pMDC99) to obtain the first LR recombined product. Subsequently, EV-2 was recombined with the first LR product in a second round of LR cloning to obtain the final gene pyramided pMDC99 construct containing both OsGS1;1 and OsGS2 genes to be used for plant transformation (For a detailed scheme see Chen et al., 2006).

Figure 2

Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2.(A) PCR amplification of hygromycin phosphotransferase (hpt), OsGS1;1 and OSGS2 genes using specific primers in wild type (wt), null segregant (ns), and five positive T₂ transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T₂ transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt. The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) (D; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt as assayed by a modified semi-biosynthetic assay (Singh and Ghosh, 2013). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).

Figure 3

Phenotype of OsGS1;1/OsGS2 co-overexpressing transgenic rice under moderate osmotic and salinity stress at seedling stage. Phenotype of 2-week-old seedlings of wt, ns and three transgenic rice lines (L1, L4, and L5), grown hydroponically in (A) normal Yoshida solution (untreated control) or (B) Yoshida solution supplemented with 15% PEG (osmotic stress) or (C) 150 mM NaCl (EC ~ 12 dS/m) (salinity stress) before and after 7 days of treatment. Visual phenotypic variation amongst seedlings of wt, ns and three transgenic rice lines (L1, L4, and L5), following 4 days of recovery after being grown hydroponically for 4 days in (D) Yoshida solution supplemented with 20% PEG (osmotic stress) or (E) 200 mM NaCl (EC ~ 19 dS/m) (salt stress). Scale bar = 1 cm.

Figure 4

Various biochemical and physiological parameters of 2-week-old seedlings of wt, ns, and three OsGS1;1/OsGS2 co-overexpressing transgenic rice lines (L1, L4, and L5) assessed after 2 days of osmotic (20% PEG) or salinity stress (200 mM NaCl) treatments as compared to untreated control conditions. (A) Fresh weight (FW) (in g). (B) Total chlorophyll content (in mg/g FW). (C) Relative water content (RWC) (in %). (D) Proline content (in μmol/g FW). (E) Electrolyte leakage (in %) (F) Malondialdehyde (MDA) content (in nmol/g FW). All data represented are means ± SD (n = 3). Asterisks above bars indicate significant differences from wt (*p-value ≤ 0.05 and **p-value ≤ 0.01).

Figure 5

Assessment of tolerance of OsGS1;1/OsGS2 co-overexpressing transgenic rice to methyl viologen (MV) induced photo-oxidative stress. (A; top panel) Leaf strips of wt, ns, and three T₂ transgenic lines (L1, L4, and L5) after photo-oxidative stress treatment (incubation in 10 μM MV). Untreated (–MV) wt was used as a control (A; middle panel) Histochemical assessment of in vivo H₂O₂ formation following MV treatment by DAB staining. (A; bottom panel) In vivo generation of O${}_2^{-}$ in leaf strips after MV treatment as detected by NBT staining. (B) Total chlorophyll contents (in mg/g FW) after MV treatment as compared to untreated controls. Data represented are means ± SD (n = 3). Asterisks above bars indicate significant differences from wt (*p-value ≤ 0.05 and **p-value ≤ 0.01).

Figure 6

Agronomic and physiological performance of OsGS1;1/OsGS2 co-overexpressing transgenic rice plants under abiotic stresses at reproductive stage. (A) Phenotypes of wild type (wt), null segregant (ns), and three transgenic lines (L1, L4, and L5) at reproductive stage under untreated control conditions. (B) Phenotypes after recovery for 15 days following drought stress treatment imposed by water withdrawal for 12 days post panicle initiation. (C) Phenotypes after recovery following moderate salinity stress imposed on ~2-month-old plants by irrigating pots every fortnight with water supplemented with 50 mM NaCl (EC~6 dS/m) until booting stage. Various physiological parameters such as (D) net photosynthetic rate (P_N) (in μmol CO₂/m²/s) (E) chlorophyll content (in SPAD values) and (F) chlorophyll fluorescence (F_v/F_m) assessed under control, drought and salinity stress conditions. All data represented are means ± SD (n = 3). Asterisks above bars indicate significant differences from wt (*p-value ≤ 0.05 and **p-value ≤ 0.01). Spider plots of agronomic traits of three independent T₂ transgenic lines (L1, L4, and L5) and corresponding ns and wt controls under (G) untreated control (H) drought and, (I) salinity stress conditions respectively. Data plotted are percentages of mean values (n = 5). Mean values from wt plants were set at 100% as reference. (J) Grain filling phenotypes in wt, ns, and three transgenic lines (L1, L4, and L5) after recovery from drought and salinity stress as compared to untreated wt control.

Figure 7

Assessment of tolerance of OsGS1;1/OsGS2 co-overexpressing transgenic rice to phosphinothricin (PPT). (A) Phenotypes of wt, ns, and three T₂ transgenic (L1, L4, and L5) rice seedlings after 0.5% (v/v) Basta herbicide (Glufosinate/PPT) spraying. Survival rates (SR) after spraying are indicated as percentages. (B) Mature leaves of wt, ns, and three T₂ transgenic lines (L1, L4, and L5) painted with a solution of 0.5% Basta (v/v) (Bayer, 13.5% ai) supplemented with 0.01% Tween-20. (C) Mean NH${}_4^{+}$ liberation from leaves before and after PPT treatment. Data represented are the means ± SD (n = 3). Different letters above bars indicate significant difference among means of PPT treated group (Tukey-Kramer tests, p-value < 0.05).

Figure 8

Putative mechanistic roles of GS in abiotic stress tolerance. Overexpression of GS alleviates hyper-ammonia toxicity caused due to proteolysis associated with various abiotic stresses. Since GS is considered the rate limiting step for photorespiration, GS overexpression can increase photorespiratory capacities and thereby ensuring photoprotection of the photosynthetic machinery via reduced reactive oxygen species (ROS) generation. GS overexpression has been reported to enhance production of the amino acids glutamine and glutamate, which is necessary for production of proline and polyamines which provide osmoprotection, as well as the anti-oxidant glutathione which can modulate anti-oxidant enzyme responses and thereby alleviate oxidative stress. Higher glutamine content is reported to induce expression of stress related transcription factors. Overexpression of GS is also reported to increase photosynthetic rates and is likely to improve N recycling efficiency, thereby leading to better yield under abiotic stress.