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.

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.

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.

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.

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.

Developing dual herbicide tolerant transgenic rice plants for sustainable weed management.

Herbicides are important constituents of modern integrated weed management system. However, the continuous use of a single herbicide leads to the frequent evolution of resistant weeds which further challenges their management. To overcome this situation, alternating use of multiple herbicides along with conventional weed-management practices is suitable and recommended. The development of multiple herbicide-tolerant crops is still in its infancy, and only a few crops with herbicide tolerance traits have been reported and commercialized. In this study, we developed transgenic rice plants that were tolerant to both bensulfuron methyl (BM) and glufosinate herbicides. The herbicide tolerant mutant variant of rice AHAS (Acetohydroxyacid synthase) was overexpressed along with codon optimized bacterial bar gene. The developed transgenic lines showed significant tolerance to both herbicides at various stages of plant development. The selected transgenic lines displayed an increased tolerance against 100 µM BM and 30 mg/L phosphinothricin during seed germination stage. Foliar applications further confirmed the dual tolerance to 300 µM BM and 2% basta herbicides without any significant growth and yield penalties. The development of dual-herbicide-tolerant transgenic plants adds further information to the knowledge of crop herbicide tolerance for sustainable weed management in modern agricultural system.

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.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development.

Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its ß-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

Glutathione reductase a unique enzyme: molecular cloning, expression and biochemical characterization from the stress adapted C4 plant, Pennisetum glaucum (L.) R. Br.

The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of ~53.5 kDa. The PgGR peptide exhibits 54-89% sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal- and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.

Enhancing C3 photosynthesis: an outlook on feasible interventions for crop improvement.

Despite the declarations and collective measures taken to eradicate hunger at World Food Summits, food security remains one of the biggest issues that we are faced with. The current scenario could worsen due to the alarming increase in world population, further compounded by adverse climatic conditions, such as increase in atmospheric temperature, unforeseen droughts and decreasing soil moisture, which will decrease crop yield even further. Furthermore, the projected increase in yields of C3 crops as a result of increasing atmospheric CO2 concentrations is much less than anticipated. Thus, there is an urgent need to increase crop productivity beyond existing yield potentials to address the challenge of food security. One of the domains of plant biology that promises hope in overcoming this problem is study of C3 photosynthesis. In this review, we have examined the potential bottlenecks of C3 photosynthesis and the strategies undertaken to overcome them. The targets considered for possible intervention include RuBisCO, RuBisCO activase, Calvin-Benson-Bassham cycle enzymes, CO2 and carbohydrate transport, and light reactions among many others. In addition, other areas which promise scope for improvement of C3 photosynthesis, such as mining natural genetic variations, mathematical modelling for identifying new targets, installing efficient carbon fixation and carbon concentrating mechanisms have been touched upon. Briefly, this review intends to shed light on the recent advances in enhancing C3 photosynthesis for crop improvement.

Molecular and biochemical characterization of dehydroascorbate reductase from a stress adapted C4 plant, pearl millet [Pennisetum glaucum (L.) R. Br].

KEY MESSAGE: PgDHAR was isolated from Pennisetum glaucum. PgDHAR responded to abiotic stress and exhibited enzyme activity at broad ranges of temperature, pH and substrate concentrations suggesting its role in stress tolerance. ABSTRACT: Dehydroascorbate reductase (EC 1.8.5.1) is a crucial enzyme actively involved in the recycling of ascorbate redox pool in the cellular environment. In this study, the full-length cDNA coding for DHAR polypeptide and its corresponding gene was isolated from Pennisetum glaucum (PgDHAR). PgDHAR encodes a polypeptide of 213 amino acids with a predicted molecular mass of 23.4 kDa and shares 80-75 % sequence homology with DHAR from other plants. The heterologously expressed recombinant PgDHAR protein exhibited activity in a wide range of substrate concentrations. The recombinant PgDHAR is thermostable and retains its activity over a broad pH range. Furthermore, transcript level of PgDHAR is quantitatively up-regulated in response to temperature. On the whole, PgDHAR alone or in combination with other genes of ascorbate-glutathione cycle can be used for the development of stress tolerant as well as nutritionally improved food crop with enhanced ascorbic acid content.

Aluminium induced oxidative stress and DNA damage in root cells of Allium cepa L.

Aluminium (Al) was evaluated for induction of oxidative stress and DNA damage employing the growing roots of Allium cepa L. as the assay system. Intact roots of A. cepa were treated with different concentrations, 0, 1, 10, 50, 100, or 200 microM of aluminium chloride, at pH 4.5 for 4 h (or 2 h for comet assay) at room temperature, 25+/-1 degrees C. Following treatment the parameters investigated in root tissue were Al-uptake, cell death, extra cellular generation of reactive oxygen intermediates (ROI), viz. O(2)(*-), H(2)O(2) and (*)OH, lipid peroxidation, protein oxidation, activities of antioxidant enzymes namely catalase (CAT), superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX); and DNA damage, assessed by comet assay. The findings indicated that Al triggered generation of extra-cellular ROI following a dose-response. Through application of specific enzyme inhibitors it was demonstrated that extra-cellular generation of ROI was primarily due to the activity of cell wall bound NADH-PX. Generation of ROI in root tissue as well as cell death was better correlated to the levels of root Al-uptake rather than to the concentrations of Al in ambient experimental solutions. Induction of lipid peroxidation and protein oxidation by Al were statistically significant. Whereas Al inhibited CAT activity, enhanced SOD, GPX and APX activities significantly; that followed dose-response. Comet assay provided evidence that Al induced DNA damage in a range of concentrations 50-200 microM, which was comparable to that induced by ethylmethane sulfonate (EMS), an alkylating mutagen served as the positive control. The findings provided evidence that Al comparable to biotic stress induced oxidative burst at the cell surface through up- or down-regulation of some of the key enzymes of oxidative metabolism ultimately resulting in oxidative stress leading to DNA damage and cell death in root cells of A. cepa.