Co-overexpression of SWEET sucrose transporters modulates sucrose synthesis and defence responses to enhance immunity against bacterial blight in rice.

Enhancing carbohydrate export from source to sink tissues is considered to be a realistic approach for improving photosynthetic efficiency and crop yield. The rice sucrose transporters OsSUT1, OsSWEET11a and OsSWEET14 contribute to sucrose phloem loading and seed filling. Crucially, Xanthomonas oryzae pv. oryzae (Xoo) infection in rice enhances the expression of OsSWEET11a and OsSWEET14 genes, and causes leaf blight. Here we show that co-overexpression of OsSUT1, OsSWEET11a and OsSWEET14 in rice reduced sucrose synthesis and transport leading to lower growth and yield but reduced susceptibility to Xoo relative to controls. The immunity-related hypersensitive response (HR) was enhanced in the transformed lines as indicated by the increased expression of defence genes, higher salicylic acid content and presence of HR lesions on the leaves. The results suggest that the increased expression of OsSWEET11a and OsSWEET14 in rice is perceived as a pathogen (Xoo) attack that triggers HR and results in constitutive activation of plant defences that are related to the signalling pathways of pathogen starvation. These findings provide a mechanistic basis for the trade-off between plant growth and immunity because decreased susceptibility against Xoo compromised plant growth and yield.

Biochemical and structural characterization of a robust and thermostable ascorbate recycling monodehydroascorbate reductase (MDHAR) from stress adapted pearl millet.

Ascorbate (AsA) is a crucial antioxidant in plants, and its recycling is necessary for protecting cells from oxidative damage and imparting stress tolerance. The monodehydroascorbate reductase (MDHAR) enzyme of the ascorbate-glutathione pathway plays a vital role in recycling AsA from monodehydroascorbate (MDHA) radical. Pennisetum glaucum (Pg), also known as pearl millet, is known to be more tolerant to abiotic stress than other food crops, such as rice. However, the contribution of MDHAR from this sessile plant to its unique stress tolerance mechanism is not well understood. In this study, we isolated a gene encoding the MDHAR enzyme from heat stress-adapted pearl millet and characterized it using enzyme kinetics, thermal stability assays, and crystal structure determination. Our results indicate that PgMDHAR is a more robust enzyme than its rice counterpart (Oryza sativa; Os). We solved the crystal structure of PgMDHAR at 1.8 Å and found that the enzyme has a more compact structure and greater stability than OsMDHAR. Using hybrid quantum mechanics and molecular mechanics calculations, we demonstrate that the structure of PgMDHAR contributes to increased stability towards bound FAD. Overall, the higher structural stability and affinity for NADH demonstrated by PgMDHAR are expected to impart improved stress tolerance. Our findings suggest that transgenic food crops expressing MDHAR from stress-adapted pearl millet may exhibit better tolerance to oxidative stress in the unpredictable climatic conditions prevalent today.

Plant dehydroascorbate reductase moonlights as membrane integrated ion channel.

Plant dehydroascorbate reductases (DHARs) are only known as soluble antioxidant enzymes of the ascorbate-glutathione pathway. They recycle ascorbate from dehydroascorbate, thereby protecting plants from oxidative stress and the resulting cellular damage. DHARs share structural GST fold with human chloride intracellular channels (HsCLICs) which are dimorphic proteins that exists in soluble enzymatic and membrane integrated ion channel forms. While the soluble form of DHAR has been extensively studied, the existence of a membrane integrated form remains unknown. We demonstrate for the first time using biochemistry, immunofluorescence confocal microscopy, and bilayer electrophysiology that Pennisetum glaucum DHAR (PgDHAR) is dimorphic and is localized to the plant plasma membrane. In addition, membrane translocation increases under induced oxidative stress. Similarly, HsCLIC1 translocates more into peripheral blood mononuclear cells (PBMCs) plasma membrane under induced oxidative stress conditions. Moreover, purified soluble PgDHAR spontaneously inserts and conducts ions in reconstituted lipid bilayers, and the addition of detergent facilitates insertion. In addition to the well-known soluble enzymatic form, our data provides conclusive evidence that plant DHAR also exists in a novel membrane-integrated form. Thus, the structure of DHAR ion channel form will help gain deeper insights into its function across various life forms.

Expression and functional analysis of various structural domains of tobacco topoisomerase II: To understand the mechanistic insights of plant type II topoisomerases.

In contrast to bacterial, yeast and animal systems, topoisomerases (topo) from plants have not been well studied. In this report, we generated four truncated topoisomerase II (Topo II) cDNA fragments encoding different functional domains of Nicotiana tabacum topo II (NtTopoII). Each of these recombinant polypeptides was expressed alone or in combination in temperature-sensitive topoisomerase II yeast mutants. Recombinant NtTopoII with truncated polypeptides fails to target the yeast nuclei and does not rescue the temperature-sensitive phenotype. In contrast complementation was achieved with the full-length NtTopoII, which localized to the yeast nucleus. These observations suggested the presence of a potent nuclear localization signal (NLS) in the extreme C-terminal 314 amino acid residues of NtTopoII that functioned effectively in the heterologous yeast system. Biochemical characterization of purified recombinant full-length and the partial NtTopoII polypeptides revealed that the ATP-binding and hydrolysis region of NtTopoIIwas located at 413 amino acid N-terminal region and this ATPase domain is functional both when it is expressed as a separate polypeptide or as part of the holoenzyme. The present findings also revealed that all NtTopoII truncated polypeptides were detrimental for in vitro supercoiled DNA relaxation and/or DNA nicking and ligation activity. Further, we discuss the possible disruption of coordinated macromolecular interface movements and the dimer interactions in truncated NtTopoII that are required for functional topoisomerase activity.

Antifungal and defense elicitor activity of Potassium phosphite against fungal blast disease on ptxD-OE transgenic indica rice and its acceptor parent.

In rice farming, the blast disease caused by Magnaporthe oryzae (T.T. Hebert) M.E. Barr. is one of the primary production constraints worldwide. The current blast management options such as blast-resistant varieties and spraying fungicides are neither durable nor commercially and environmentally compatible. In the present study, we investigated the antifungal and defense elicitor activity of potassium phosphite (Phi) against M. oryzae on elite rice cultivar BPT5204 (popularly known as Samba Mahsuri in India) and its transgenic rice variant (ptxD-OE) over-expressing a phosphite dehydrogenase enzyme. The Phi was evaluated both preventively and curatively on rice genotypes where the preventive spray of Phi outperformed the Phi curative application with significant reductions in both rice blast severity (35.67-60.49%) and incidence (22.27-53.25%). Moreover, the application of Phi increased the levels of photosynthetic pigments (Chlorophyll and Carotenoids) coupled with increased activity of defense enzymes (PAL, SOD, and APx). Besides, Phi application also induced the expression of defense-associated genes (OsCEBiP and OsPDF2.2) in the rice leaf. Furthermore, the Phi application reduced the reactive Malondialdehyde (lipid peroxidation) to minimize the cellular damage incited by Magnaporthe in rice. Overall, the present study showed the potential of Phi for blast suppression on rice as an alternative to the current excessive use of toxic fungicides.

Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plants and humans.

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (HsCLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (PgDHAR). In comparison, HsCLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.

Pyramiding ascorbate-glutathione pathway in Lycopersicum esculentum confers tolerance to drought and salinity stress.

KEY MESSAGE: Stacking Glutathione-Ascorbate pathway genes (PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR) under stress inducible promoter RD29A imparts significant tolerance to drought and salinity stress in Solanum lycopersicum. Although the exposure of plants to different environmental stresses results in overproduction of reactive oxygen species (ROS), many plants have developed some unique systems to alleviate the ROS production and mitigate its deleterious effect. One of the key pathways that gets activated in plants is ascorbate glutathione (AsA-GSH) pathway. To demonstrate the effect of this pathway in tomato, we developed the AsA-GSH overexpression lines by stacking the genes of the AsA-GSH pathway genes isolated from Pennisetum glaucoma (Pg) including PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR under stress inducible promoter RD29A. The overexpression lines have an improved germination and seedling growth with concomitant elevation in the survival rate. The exposure of transgenic seedlings to varying stress regiments exhibited escalation in the antioxidant enzyme activity and lesser membrane damage as reflected by decreased electrolytic leakage and little accumulation of malondialdehyde and H2O2. Furthermore, the transgenic lines accumulated high levels of osmoprotectants with increase in the relative water content. The increased photosynthetic activity and enhanced gaseous exchange parameters further confirmed the enhanced tolerance of AsA-GSH overexpression lines. We concluded that pyramiding of AsA-GSH pathway genes is an effective strategy for developing stress resistant crops.

Mitochondria-Targeted SmsHSP24.1 Overexpression Stimulates Early Seedling Vigor and Stress Tolerance by Multi-Pathway Transcriptome-Reprogramming.

Among the diverse array of heat shock proteins across the three domains of life, mitochondria-targeted small heat shock proteins (sHSPs) are evolved in the plant lineage. However, they remained mysterious and understudied. In this study, we reported a systematic study of a novel mitochondria-targeted nuclear sHSP from eggplant (Solanum melongena L.; SmsHSP24.1). Differential expression of SmsHSP24.1 indicated its positive role exerted during stress conditions. Escherichia coli-BL21 cell line overexpressing the SmsHSP24.1 showed excellent thermo-tolerance ability, tolerating up to 52°C. Spectrometry and electron microscopy revealed a multimeric structure of the protein which acted as a molecular chaperone at high temperatures. Overexpression of SmsHSP24.1 significantly enhanced resistance against heat, drought, and salt stresses and showed rapid germination in constitutively overexpressed eggplant lines. RNA-seq analysis reveals an apparent upregulation of a set of reactive oxygen species (ROS) scavenging enzymes of the glutathione (GHS) pathway and mitochondrial electron transport chain (ETC). Significant upregulation was also observed in auxin biosynthesis and cell-wall remodeling transcripts in overexpressed lines. qPCR, biochemical and physiological analysis further aligned with the finding of transcriptome analysis and suggested an essential role of SmsHSP24.1 under various stress responses and positive physiological influence on the growth of eggplants. Therefore, this gene has immense potential in engineering stress-resilient crop plants.

CRISPR-Cas9 mediated mutation in GRAIN WIDTH and WEIGHT2 (GW2) locus improves aleurone layer and grain nutritional quality in rice.

Enhancing crop productivity and their nutritional quality are the key components and primary focus of crop improvement strategy for fulfilling future food demand and improving human health. Grain filling and endosperm development are the key determinants of grain yield and nutritional quality. GRAIN WIDTH and WEIGHT2 (GW2) gene encodes a RING-type E3 ubiquitin ligase and determines the grain weight in cereal crops. Here we report GW2 knockout (KO) mutants in Indica (var. MTU1010) through CRISPR/Cas9 genome editing. The endosperm of GW2-KO mutant seed displays a thick aleurone layer with enhanced grain protein content. Further the loss of function of OsGW2 results in improved accumulation of essential dietary minerals (Fe, Zn, K, P, Ca) in the endosperm of rice grain. Additionally, the mutants displayed an early growth vigour phenotype with an improved root and shoot architecture. The hull morphology of GW2-KO lines also showed improved, grain filling thereby promoting larger grain architecture. Together, our findings indicate that GW2 may serve as a key regulator of improved grain architecture, grain nutritional quality and an important modulator of plant morphology. The study offers a strategy for the development of improved rice cultivars with enriched nutritional quality and its possible implementation in other cereals as well.

Antifungal activity of glyphosate against fungal blast disease on glyphosate-tolerant OsmEPSPS transgenic rice.

Weeds, pests, and pathogens are among the pre-harvest constraints in rice farming across rice-growing countries. For weed management, manual weeding and herbicides are widely practiced. Among the herbicides, glyphosate [N-(phosphonomethyl) glycine] is a broad-spectrum systemic chemical extensively used in agriculture. Being a competitive structural analog to phosphoenolpyruvate, it selectively inhibits the conserved 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme required for the biosynthesis of aromatic amino acids and essential metabolites in eukaryotes and prokaryotes. In the present study, we investigated the antifungal and defense elicitor activity of glyphosate against Magnaporthe oryzae on transgenic-rice overexpressing a glyphosate-resistance OsEPSPS gene (T173I + P177S; TIPS OsmEPSPS) for blast disease management. The glyphosate foliar spray on OsmEPSPS transgenic rice lines showed both prophylactic and curative suppression of blast disease comparable to a blasticide, tricyclazole. The glyphosate displayed direct antifungal activity on Magnaporthe oryzae as well as enhanced the levels of antioxidant enzymes and photosynthetic pigments in rice. However, the genes associated with phytohormones-mediated defense (OsPAD4, OsNPR1.3, and OsFMO) and innate immunity pathway (OsCEBiP and OsCERK1) were found repressed upon glyphosate spray. Altogether, the current study is the first report highlighting the overexpression of a crop-specific TIPS mutation in conjugation with glyphosate application showing potential for blast disease management in rice cultivation.

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

Glyphosate is a popular, systemic, broad-spectrum herbicide used in modern agriculture. Being a structural analog of phosphoenolpyruvate (PEP), it inhibits 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) which is responsible for the biosynthesis of aromatic amino acids and various aromatic secondary metabolites. Taking a lead from glyphosate-resistant weeds, two mutant variants of the rice EPSPS gene were developed by amino acid substitution (T173I + P177S; TIPS-OsEPSPS and G172A + T173I + P177S; GATIPS-OsEPSPS). These mutated EPSPS genes were overexpressed in rice under the control of either native EPSPS or constitutive promoters (maize ubiquitin [ZmUbi] promoter). The overexpression of TIPS-OsEPSPS under the control of the ZmUbi promoter resulted in higher tolerance to glyphosate (up to threefold of the recommended dose) without affecting the fitness and related agronomic traits of plants in both controlled and field conditions. Furthermore, such rice lines produced 17%-19% more grains compared to the wild type (WT) in the absence of glyphosate application and the phenylalanine and tryptophan contents in the transgenic seeds were found to be significantly higher in comparison with WT seeds. Our results also revealed that the native promoter guided expression of modified EPSPS genes did not significantly improve the glyphosate tolerance. The present study describing the introduction of a crop-specific TIPS mutation in class I aroA gene of rice and its overexpression have potential to substantially improve the yield and field level glyphosate tolerance in rice. This is the first report to observe that the EPSPS has role to play in improving grain yield of rice.

Development of transgenic cotton (Narasimha) using triple gene Cry2Ab-Cry1F-Cry1Ac construct conferring resistance to lepidopteran pest.

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they are recalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing three Cry genes driven by three different promoters conferring insect resistance. The meristematic region of embryo axis explants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100 vector with three Cry gene cassettes (alpha-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt II as a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds of kanamycin selectionmedium.About 2551T1 seedswere collected from111T0 plants and these seeds screened again with kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR, lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showed amplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as 395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteins accumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry genes were accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effective resistance againstHelicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentage of insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgenic cotton plants carrying triple Cry genes could be an excellent germplasmresource for the breeders for introgressions.

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.

Co-expression of P173S Mutant Rice EPSPS and igrA Genes Results in Higher Glyphosate Tolerance in Transgenic Rice.

Weeds and their devastating effects have been a great threat since the start of agriculture. They compete with crop plants in the field and negatively influence the crop yield quality and quantity along with survival of the plants. Glyphosate is an important broad-spectrum systemic herbicide which has been widely used to combat various weed problems since last two decades. It is very effective even at low concentrations, and possesses low environmental toxicity and soil residual activity. However, the residual concentration of glyphosate inside the plant has been of major concern as it severely affects the important metabolic pathways, and results in poor plant growth and grain yield. In this study, we compared the glyphosate tolerance efficiency of two different transgenic groups over expressing proline/173/serine (P173S) rice EPSPS glyphosate tolerant mutant gene (OsmEPSPS) alone and in combination with the glyphosate detoxifying encoding igrA gene, recently characterized from Pseudomonas. The molecular analysis of all transgenic plant lines showed a stable integration of transgenes and their active expression in foliar tissues. The physiological analysis of glyphosate treated transgenic lines at seed germination and vegetative stages showed a significant difference in glyphosate tolerance between the two transgenic groups. The transgenic plants with OsmEPSPS and igrA genes, representing dual glyphosate tolerance mechanisms, showed an improved root-shoot growth, physiology, overall phenotype and higher level of glyphosate tolerance compared to the OsmEPSPS transgenic plants. This study highlights the advantage of igrA led detoxification mechanism as a crucial component of glyphosate tolerance strategy in combination with glyphosate tolerant OsmEPSPS gene, which offered a better option to tackle in vivo glyphosate accumulation and imparted more robust glyphosate tolerance in rice transgenic plants.

Phosphite: a novel P fertilizer for weed management and pathogen control.

The availability of orthophosphate (Pi) is a key determinant of crop productivity because its accessibility to plants is poor due to its conversion to unavailable forms. Weed's competition for this essential macronutrient further reduces its bio-availability. To compensate for the low Pi use efficiency and address the weed hazard, excess Pi fertilizers and herbicides are routinely applied, resulting in increased production costs, soil degradation and eutrophication. These outcomes necessitate the identification of a suitable alternate technology that can address the problems associated with the overuse of Pi-based fertilizers and herbicides in agriculture. The present review focuses on phosphite (Phi) as a novel molecule for its utility as a fertilizer, herbicide, biostimulant and biocide in modern agriculture. The use of Phi-based fertilization will help to reduce the consumption of Pi fertilizers and facilitate weed and pathogen control using the same molecule, thereby providing significant advantages over current orthophosphate-based fertilization.

Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation.

KEY MESSAGE: The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

Abiotic Stress Tolerance in Plants: Myriad Roles of Ascorbate Peroxidase.

One of the most significant manifestations of environmental stress in plants is the increased production of Reactive Oxygen Species (ROS). These ROS, if allowed to accumulate unchecked, can lead to cellular toxicity. A battery of antioxidant molecules is present in plants for keeping ROS levels under check and to maintain the cellular homeostasis under stress. Ascorbate peroxidase (APX) is a key antioxidant enzyme of such scavenging systems. It catalyses the conversion of H2O2 into H2O, employing ascorbate as an electron donor. The expression of APX is differentially regulated in response to environmental stresses and during normal plant growth and development as well. Different isoforms of APX show differential response to environmental stresses, depending upon their sub-cellular localization, and the presence of specific regulatory elements in the upstream regions of the respective genes. The present review delineates role of APX isoforms with respect to different types of abiotic stresses and its importance as a key antioxidant enzyme in maintaining cellular homeostasis.

Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut.

Eukaryotic translational initiation factor 4A belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. The eIF4A gene was isolated and cloned from semi-arid cereal crop of Pennisetum glaucum. In present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium tumefaciens (LBA4404) carrying rd29A:PgeIF4A construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T0 plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T0 transgenic plants through Southern blot analysis. Predicted Mendelian ratio of segregation (3:1) was noted in transgenic plants at T1 generation. The T2 homozygous lines (L1-5, L8-2, and L16-2) expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length, and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. The quantitative real time-PCR results confirmed higher expression of PgeIF4A gene in L1-5, L8-3, and L16-2 plants imposed with drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging, and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.

Ectopic expression of PgRab7 in rice plants (Oryza sativa L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress.

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200 mM NaCl) and drought stress (up to 12 days after withdrawing water). The protection against salt and drought stress may be by regulating Na+ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89-96 % of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33-53 % decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.

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.