Parinita Agarwal

Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance

Dehydration responsive element binding (DREB) factors or CRT element binding factors (CBFs) are members of the AP2/ERF family, which comprises a large number of stress-responsive regulatory genes. This review traverses almost two decades of research, from the discovery of DREB/CBF factors to their optimization for application in plant biotechnology. In this review, we describe (i) the discovery, classification, structure, and evolution of DREB genes and proteins; (ii) induction of DREB genes by abiotic stresses and involvement of their products in stress responses; (iii) protein structure and DNA binding selectivity of different groups of DREB proteins; (iv) post-transcriptional and post-translational mechanisms of DREB transcription factor (TF) regulation; and (v) physical and/or functional interaction of DREB TFs with other proteins during plant stress responses. We also discuss existing issues in applications of DREB TFs for engineering of enhanced stress tolerance and improved performance under stress of transgenic crop plants.

Surfactant-induced coagulation of agarose from aqueous extract of Gracilaria dura seaweed as an energy-efficient alternative to the conventional freeze–thaw process

Surfactant-induced coagulation of agarose from alkali-treated Gracilaria dura seaweed extract (SE) is reported. The new approach, which was suitable for linear galactans with low sulphate content is an alternative to the traditional energy intensive process of “freeze–thaw” cycles employed for product isolation from the extract. Only nonionic surfactants were effective, and detailed studies were undertaken with octyl phenol ethoxylate (Triton X-100). The coagulated product was successively washed with water and water–isopropyl alcohol (IPA) to yield a fine powder of agarose in 13–15% yield (with respect to dry biomass). The product exhibited excellent properties [sulphate content: 0.2% w/w; degree of electro-endosmosis: 0.13; gel strength: 2200 g cm−2 (1% gel, w/v); and gelling temperature: 35 ± 1 °C] essential for demanding molecular biology applications, and the desired gel electrophoretic separation of DNA and RNA was demonstrated. It was further confirmed that there was no degradation of nucleic acids in the gel. The agarose-depleted extract, along with water used for washings, was subjected to reverse osmosis for recovering the surfactant in concentrated form for its subsequent reuse. Energy savings from the improved process were assessed.

Ion homeostasis in a salt-secreting halophytic grass

Salinity adversely affects plant growth and development, and disturbs intracellular ion homeostasis, resulting in cellular toxicity. Glycophytes tolerate salinity up to 40 mM NaCl, however halophytes grow luxuriantly in salt-marshes where the salt concentration is 200 mM NaCl or more. Aeluropus lagopoides secretes salt crystals from the adaxial and abaxial leaf surface through salt glands. At high salt concentrations the level of different organic and inorganic osmolytes is elevated to prevent toxicity and maintain a sustainable K+/Na+ ratio. Different transporter genes AlHKT2;1, HAK, SOS1, NHX1, and V-ATPase expression coordinate to regulate ion homeostasis in A. lagopoides.

Molecular characterization of an MYB transcription factor from a succulent halophyte involved in stress tolerance

Abiotic stresses cause dramatic changes in agricultural productivity. Plants encounter a wide range of biotic stresses and have evolved mechanisms to increase tolerance through both physical adaptations and interactive molecular and cellular changes that begin after the onset of stress. Transcription factors regulate the gene expression associated with both abiotic and biotic stresses, growth, metabolism, and plant development. SbMYB44 is an R2R3 type transcription factor involved in stress regulation in an extreme halophyte, Salicornia brachiata. SbMYB44 binds to the cis elements of a stress responsive promoter and confers stress tolerance. In future, SbMYb44 may prove to be a candidate gene for developing stress tolerance in crop plants.

Overexpression of a novel SbMYB15 from Salicornia brachiata confers salinity and dehydration tolerance by reduced oxidative damage and improved photosynthesis in transgenic tobacco.

AbstractMain conclusionSbMYB15, R2R3-type MYB was induced by the different stresses, and conferred stress tolerance in transgenic tobacco by regulating the expression of stress-responsive genes. MYBs are the master regulators of various metabolic processes and stress responses in plants. In this study, we functionally characterised a R2R3-type SbMYB15 transcription factor (TF) from the extreme halophyte Salicorniabrachiata. The SbMYB15 acts as a transcriptional activator. Transcriptional analysis showed that SbMYB15 transcript was strongly upregulated in red shoots and was also induced by different stresses; however, its expression remained unchanged with ABA. Overexpression of SbMYB15 in tobacco significantly improved salinity and dehydration tolerance. The enhanced tolerance of the transgenic plants was defined by the changes in chlorophyll, malondialdehyde (MDA), proline, total soluble sugar and total amino acid contents. The transgenic plants exhibited a higher membrane stability and reduced electrolyte leakage, H2O2 and O2− content compared to the wild type (WT). With ionic stress, transgenics showed a low Na+ and a high K+ content. In the transgenic plants, the expression of stress-responsive genes such as LEA5, ERD10D, PLC3, LTP1, HSF2, ADC, P5CS, SOD and CAT was enhanced in the presence of salinity, dehydration and heat. Exposure to gradual salinity and dehydration resulted in an increased stomatal conductance, water use efficiency, photosynthesis rate, photochemical quenching and reduced transpiration rate. Thus, SbMYB15 served as an important mediator of stress responses regulating different stress signalling pathways, leading to enhanced stress tolerance.

Insights into the role of seaweed Kappaphycus alvarezii sap towards phytohormone signalling and regulating defence responsive genes in Lycopersicon esculentum

Seaweeds, an integral part of marine coastal environment, are an important food, industrial raw materials and biostimulants for crop growth due to the presence of a number of plant growth stimulants. The sap of an economically important seaweed Kappaphycus alvarezii (K-sap) is gaining momentum for sustainable intensification of agricultural productivity. In the present study, the phytohormone levels and transcript regulation of defence-related genes were studied in tomato seedlings in response to K-sap application alone and in combination with the fungus Macrophomina phaseolina, the causal agent of charcoal rot in tomato. The application of K-sap alone and in combination with fungus M. phaseolina significantly increased the concentration of ABA, IAA, SA and zeatin hormones. The enhanced transcript of pathogenesis related genes (PR-1b1, PR-3 and PR-5) was observed in response to application of K-sap alone and in combination with fungus M. phaseolina in tomato. The transcription factor, Pti4 and mitogen-activated kinase pathway gene, MPK2 also showed transcript accumulation on combined treatment of Macrophomina and K-sap. This is the first report highlighting the differential regulation of defence-related genes in accordance with phytohormone levels on application of seaweed sap.

Ectopic Expression of JcWRKY Transcription Factor Confers Salinity Tolerance via Salicylic Acid Signaling.

Plants, being sessile, have developed intricate signaling network to specifically respond to the diverse environmental stress. The plant-specific WRKY TFs form one of the largest TF family and are involved in diverse plant processes, involving growth, development and stress signaling through auto and cross regulation with different genes and TFs. Here, we report the functional characterization of a salicylic acid -inducible JcWRKY TF. The JcWRKY overexpression confers salinity tolerance in transgenic tobacco, as was evident by increased chlorophyll content and seed germination potential. The transgenic plants showed increased soluble sugar, membrane stability, reduced electrolyte leakage and generation of reactive oxygen species (H2O2 and O2•-) as compared to the wild type. Furthermore, the low SA treatment along with salinity improved the tolerance potential of the transgenics by maintaining ROS homeostasis and high K+/Na+ ratio. The transcript expression of SA biosynthetic gene ICS1 and antioxidative enzymes (CAT and SOD) showed upregulation during stress. Thus, the present study reflects that JcWRKY is working in co-ordination with SA signaling to orchestrate the different biochemical and molecular pathways to maneuvre salt stress tolerance of the transgenic plants.

Molecular Cloning and Characterization of a Group II WRKY Transcription Factor from Jatropha curcas, an Important Biofuel Crop

The WRKY family of transcription factors (TFs) play an intricate role in regulating the stress signaling pathways by autoregulation or may be by cross regulation through interaction with other proteins. Although WRKY TFs are considered to be plant specific, however, their presence has been reported from unicellular algae, slime mould, and gymnosperms. We have isolated the JcWRKY cDNA from an important biofuel crop Jatropha curcas growing in the wastelands of India. The JcWRKY gene has an ORF of 693 bp and encodes a 230 amino acids protein with estimated molecular mass of 25.25 kDa. JcWRKY shows close homology to FaWRKY1 and St-WRKY1. The JcWRKY contains seven potential phosphorylation sites, which might be involved in regulating its function. The transcript analysis revealed that the JcWRKY transcript gets upregulated in response to salinity, dehydration, salicylic acid (SA), methyl jasmonate (MeJa), and collar rot fungus Macrophomina. However, maximum expression is observed under SA, highlighting its role in enhancing systemic acquired resistance for disease tolerance. The JcWRKY recombinant protein showed binding to W-box of pathogenesis related-1 (PR-1) and iso1 (encoding isoamylase1) promoters. Overexpression of JcWRKY in Escherichia coli enhanced the growth of cells in NaCl, KCl, mannitol, sorbitol, SA, and MeJa treatments, indicating that it protects and promotes growth under ionic, osmotic, and chemical stresses. The enhancement in growth can be due to the regulation of stress responsive genes. Therefore, it can be used as an important gene for enhancing abiotic and biotic resistance in plants and to facilitate faster growth of E. coli cells under stress conditions for efficient expression.

Molecular characterization and identification of target protein of an important vesicle trafficking gene AlRab7 from a salt excreting halophyte Aeluropus lagopoides.

The endomembrane system plays an important role during cellular adaptation of the plants with the extracellular environment. The small GTP-binding protein Rab7 located at the vacuolar membrane regulates the vesicle fusion with the vacuole and thereby helps in recycling of the molecules. This is the first report on isolation and characterization of AlRab7 gene from the halophyte plant, Aeluropus that extrudes NaCl through salt glands and grows luxuriantly throughout the year at the Gujarat coast, India. The AlRab7 encodes a protein with 206 amino acids, and a highly conserved effector-binding domain and four nucleotide-binding domains. The in silico analysis predicts the presence of the prenylation site for Rab geranylgeranyltransferase 2 and the Rab escort protein site. The C-terminal two cysteine residues in -XCC sequence are present for membrane attachment. Transcript expression of the AlRab7 gene was differentially regulated by different environmental stimuli such as dehydration, salinity, and hormone abscisic acid (ABA). The recombinant Escherichia coli cells showed improved growth in Luria Bertani medium supplemented with NaCl, KCl, mannitol, ABA, and indole-3-acetic acid. A novel Rab7 interacting partner AlRabring7 was identified by yeast two-hybrid screening.

Improved Shoot Regeneration, Salinity Tolerance and Reduced Fungal Susceptibility in Transgenic Tobacco Constitutively Expressing PR-10a Gene.

Plants in ecosystems are simultaneously exposed to abiotic and biotic stresses, which restrict plant growth and development. The complex responses to these stresses are largely regulated by plant hormones, which in turn, orchestrate the different biochemical and molecular pathways to maneuver stress tolerance. The PR-10 protein family is reported to be involved in defense regulation, stress response and plant growth and development. The JcPR-10a overexpression resulted in increased number of shoot buds in tobacco (Nicotiana tabacum), which could be due to high cytokinin to auxin ratio in the transgenics. The docking analysis shows the binding of three BAP molecules at the active sites of JcPR-10a protein. JcPR-10a transgenics showed enhanced salt tolerance, as was evident by increased germination rate, shoot and root length, relative water content, proline, soluble sugar and amino acid content under salinity. Interestingly, the transgenics also showed enhanced endogenous cytokinin level as compared to WT, which, further increased with salinity. Exposure of gradual salinity resulted in increased stomatal conductance, water use efficiency, photosynthesis rate and reduced transpiration rate. Furthermore, the transgenics also showed enhanced resistance against Macrophomina fungus. Thus, JcPR-10a might be working in co-ordination with cytokinin signaling in mitigating the stress induced damage by regulating different stress signaling pathways, leading to enhanced stress tolerance.