Tanushri Kaul

Redox homeostasis, antioxidant defense, and methylglyoxal detoxification as markers for salt tolerance in Pokkali rice

To identify biochemical markers for salt tolerance, two contrasting cultivars of rice (Oryza sativa L.) differing in salt tolerance were analyzed for various parameters. Pokkali, a salt-tolerant cultivar, showed considerably lower level of H2O2 as compared to IR64, a sensitive cultivar, and such a physiology may be ascribed to the higher activity of enzymes in Pokkali, which either directly or indirectly are involved in the detoxification of H2O2. Enzyme activities and the isoenzyme pattern of antioxidant enzymes also showed higher activity of different types and forms in Pokkali as compared to IR64, suggesting that Pokkali possesses a more efficient antioxidant defense system to cope up with salt-induced oxidative stress. Further, Pokkali exhibited a higher GSH/GSSG ratio along with a higher ratio of reduced ascorbate/oxidized ascorbate as compared to IR64 under NaCl stress. In addition, the activity of methylglyoxal detoxification system (glyoxalase I and II) was significantly higher in Pokkali as compared to IR64. As reduced glutathione is involved in the ascorbate–glutathione pathway as well as in the methylglyoxal detoxification pathway, it may be a point of interaction between these two. Our results suggest that both ascorbate and glutathione homeostasis, modulated also via glyoxalase enzymes, can be considered as biomarkers for salt tolerance in Pokkali rice. In addition, status of reactive oxygen species and oxidative DNA damage can serve as a quick and sensitive biomarker for screening against salt and other abiotic stresses in crop plants.

Expression of OsDREB2A transcription factor confers enhanced dehydration and salt stress tolerance in rice (Oryza sativa L.)

Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants improved stress tolerance indicating the DRE/DREB regulon is conserved across plants. We developed 30 transgenic T0 rice plants overexpressing OsDREB2A which were devoid of any growth penalty or phenotypic abnormalities during stressed or non-stressed conditions. Integration of T-DNA in the rice genome and stress inducible overexpression of OsDREB2A had occurred in these transgenic lines. Functional analyses of T1-3 and T1-10 lines revealed significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type. OsDREB2A, thus, confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier.

Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses

Molecular chaperones (Hsps) have been shown to facilitate protein folding or assembly under various developmental and adverse environmental conditions. The aim of this study was to unravel a possible role of heat-shock proteins in conferring abiotic stress tolerance to plants. We isolated a cDNA encoding a cytoplasmic Hsp70 (PgHsc70) from Pennisetum glaucum by screening heat-stress cDNA library. PgHsc70 cDNA encoding 649 amino acids represents all conserved signature motifs characteristic of Hsp70s. The predicted molecular model of PgHsc70 protein suggests that the N-terminus ATP-binding region is evolutionarily conserved, in comparison to C-terminus peptide-binding domains. A single intron in ATPase domain coding region of PgHsc70 exhibited a high degree of conservation with respect to its position and phasing among other plant Hsp70 genes. Recombinant PgHsc70 protein purified from E. coli possessed in vitro chaperone activity and protected PgHsc70 expressing bacteria from damage caused by heat and salinity stress. Nucleotide sequence analysis of 5′ flanking promoter region of PgHsc70 gene revealed a potential heat-shock element (HSE) and other putative stress-responsive transcription factor binding sites. Positive correlation existed between differentially up-regulated PgHsc70 transcript levels and the duration and intensity of different environmental stresses. Molecular and biochemical analyses revealed that PgHsc70 gene was a member of the Hsp70 family and suggested that its origin was from duplication of a common ancestral gene. Transcript induction data, presence of several putative stress-responsive transcription factor-binding sites in the promoter region of PgHsc70 and the presence of a protective in vitro chaperone activity of this protein against damage caused by heat and salinity, when expressed in E. coli, suggest its probable role in conferring abiotic stress tolerance to this plant.

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.

Characterization and Functional Validation of Tobacco PLC Delta for Abiotic Stress Tolerance

The role of plant phospholipase C-mediated signaling has been implicated in various phases of plant growth and development. In this study, we report on the isolation and characterization of phospholipase C from tobacco and demonstrate that transcripts of phospholipase C are up-regulated in responses to drought and salt stress. These responses are likely by abscisic acid (ABA). Transgenic tobacco plants overexpressing the phospholipase C protein were found to tolerate higher levels of drought and also salinity stress. This tolerance could be mediated by the regulation of genes downstream to phospholipase mediated signaling. As a demonstration, when tested the transgenic plants showed higher transcript of heat shock factor NtHSF2, heat shock protein HSP70-3 and an AP2 domain transcription factor. Also the transgenic plants showed higher accumulation of sodium in older leaves compared to the young leaves. The present report is the first to demonstrate the role of phospholipase C in salinity stress tolerance.

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.

Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C4 plant, Pennisetum glaucum

Genes encoding for many β-carbonic anhydrases and their functions in various developmental processes are well established in lower plants, however, similar studies are limited in higher plants. We report the cloning and characterization of cDNA encoding for a β-carbonic anhydrase (PgCA) from Pennisetum glaucum, a C(4) crop plant. cDNA encoding 249 amino acids and its deduced amino acid sequence analysis revealed that is related to other plant β-CA family members with an over all conserved architecture of a typical β-CA protein. Phylogenetic analysis revealed that PgCA is evolutionarily very close to chloroplast β-CA isoform. Signal sequence predicting programs identify a N-terminus putative chloroplast targeting sequence. Heterologous Escherichia coli expression system was utilized to overexpress recombinant PgCA, which showed high thermostability, an alkaline pH optima and dual activity with both reversible CO(2) hydration and esterase activities. The β-CAs studied so far possessed only CO(2) hydration activity with no detectable esterase activity. Recombinant PgCA esterase activity is inhibited by standard CA inhibitors acetazolamide, methazolamide and azide. Subcellular immunostaining studies revealed a chloroplastic localization of PgCA protein. Expression of PgCA transcript is differentially up regulated in response to various abiotic stresses wherein its accumulation in Pennisetum leaves positively correlated with the intensity and duration of stress. Biochemical and transcript analyses suggest that PgCA may play a significant role in plants adaptation to different abiotic stresses in addition to the previously recognized role of replenishing the CO(2) supply within plant cells.

Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: interlinking oxidative and heat-stress responses.

The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 degrees C for 0.5, 1.0 and 24h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Over-expression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 degrees C) being maximum at 24h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance.

Genome-Wide Dissection of Arabidopsis and Rice for the Identification and Expression Analysis of Glutathione Peroxidases Reveals Their Stress-Specific and Overlapping Response Patterns

Excessive generation of reactive oxygen species (ROS) due to environmental stresses critically effects plant development and productivity. Plants efficiently detoxify ROS by both non-enzymatic and enzymatic mechanisms. Plant glutathione peroxidases (GPXs) are non-haeme thiol peroxidases that catalyze the reduction of H2O2 (or organic hydroperoxides) to water or the respective alcohols using reduced glutathione or thioredoxin. Genome-wide analysis of the known GPXs from rice and Arabidopsis genomes revealed their gene structure, conserved motifs, localization and tissue-specific and/or organ-specific expression profiles in response to various abiotic stresses. Among the eight genes that encoded GPX proteins from Arabidopsis, AtGPX3 showed two alternate spliced forms that spread over four chromosomes. Five genes encoded for rice GPX proteins, while OsGPX1 showed three spliced variants that were distributed on five chromosomes. Utilizing the publicly available microarray and massively parallel signature sequencing (MPSS) data, the GPXs revealed stress-responsive, tissue-specific and/or organ-specific expression profiles. Presence of important cis-regulatory elements analyzed in the GPX promoter sequences revealed their overlapping or specific responsiveness to different abiotic stresses. Co-expression data of Arabidopsis GPX genes suggested that various protein kinase family members and stress-responsive proteins co-expressed with the GPX proteins. Transcript profile of rice GPX genes by qRT-PCR validated their functional roles in signal transduction and stress pathways. Results revealed that plant GPXs play a crucial role in response to stress and significantly contribute towards their growth and development.

Advances in Maize Transformation Technologies and Development of Transgenic Maize

Maize is the principal grain crop of the world. It is also the crop where genetic engineering has been employed to a great extent to improve its various traits. The ability to transform maize is a crucial step for application of gene technology in maize improvement. There have been constant improvements in the maize transformation technologies over past several years. The choice of genotype and the explant material to initiate transformation and the different types of media to be used in various stages of tissue culture can have significant impact on the outcomes of the transformation efforts. Various methods of gene transfer, like the particle bombardment, protoplast transformation, Agrobacterium-mediated, in planta transformation, etc., have been tried and improved over years. Similarly, various selection systems for retrieval of the transformants have been attempted. The commercial success of maize transformation and transgenic development is unmatched by any other crop so far. Maize transformation with newer gene editing technologies is opening up a fresh dimension in transformation protocols and work-flows. This review captures the various past and recent facets in improvement in maize transformation technologies and attempts to present a comprehensive updated picture of the current state of the art in this area.