[Show abstract][Hide abstract] ABSTRACT: Dehydration-responsive element binding (DREB) transcription factor (TF) plays a key role for abiotic stress tolerance in plants. Earlier, we have published the isolation and characterisation of an A-2-type SbDREB2A TF from an extreme halophyte Salicornia brachiata. The SbDREB2A protein lacks potential proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST) sequence which is known to act as signal peptide for protein degradation. In this study, SbDREB2A TF was over-expressed in tobacco plants without any modification in polypeptide sequence. Transgenic plants showed better seed germination and growth characteristics in both hyperionic and hyperosmotic stresses. Transgenic plants exhibited higher water content, membrane stability and less electrolyte leakage in stress conditions. The transgenic plants accumulated less Na(+) and higher K(+) than wildtype (WT) plants. The transgenic plants revealed higher chlorophyll content, water use efficiency (WUE) and net photosynthesis rate. Transgenics exhibited higher level of proline and low amount of MDA and H2O2 under stress conditions. The real-time PCR of transgenics showed higher expression of downstream heat shock genes (Hsp18, Hsp26 and Hsp70), TFs (AP2 domain containing TF, HSF2 and ZFP), signalling components (PLC3 and Ca (2+) /calmodulin) and dehydrins (ERD10B, ERD10D and LEA5) under different abiotic stress treatments.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
DNA and cell biology 04/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pathogenesis related-10 proteins are small proteins with cytosolic localization, conserved three dimensional structures and single intron at 185 bp position. These proteins have a broad spectrum of roles significantly in biotic and abiotic stresses. The RNase activity, ligand binding activity, posttranslational modification (phosphorylation) and phytohormone signaling provide some information into the mechanism of the regulation of PR-10 proteins, however the presence of isoforms makes it difficult to decipher its exact mode of function. The involvement of phosphorylation/dephosphorylation events in its activation is interesting and provides unique and unbiased insights into the complexity of its regulation. Studies on upstream region of different PR-10 genes indicate the presence of cis-acting elements for WRKY, RAVI, bZ1P, ERF, SEBF and Pti4 transcription factors indicating their role in regulating PR-10 promoter. In this review, we discuss in detail the structure and mechanism of regulation of PR-10 proteins.
[Show abstract][Hide abstract] ABSTRACT: Abiotic stresses affect adversely the growth and productivity of crops. Among abiotic stresses, salinity is one of the major factors leading to crop losses. According to the UN Food and Agriculture Organization, more than 800 Mha of land is salt-affected worldwide. The problem of soil salinization is becoming more serious due to scanty rainfall, repetitive sea water invasion, heavy utilization of ground water for agricultural and industrial purposes, and degradation of saline parent rock. The area under cultivation is fast getting depleted and becoming unsuitable for agricultural crops. Soil salinity adversely affects plant growth and development, and disturbs intracellular ion homeostasis, resulting in cellular toxicity. Plant adaptation to salinity stress involves a plethora of genes involved in ion transport and compartmentalization (ion homeostasis), compatible solutes/osmolytes, reactive oxygen species, and antioxidant defense mechanism. Transporters are an important group of genes that play a pivotal role in ion homeostasis in plants (Na+/H+ antiporters like SOS1 and NHX1, and proton pump HKT1). Over the last two decades the major studies on the molecular mechanisms of salt tolerance have concentrated on glycophytes; however, only limited studies have been performed on halophytes. Halophytes have a unique genetic makeup that provides an advantage for the study of salt-tolerance mechanisms. Halophytes maintain a low salt concentration inside the cytosol by sequestration in vacuoles or extrusion of Na+ outside the plasma membrane or secretion of salt outside the plant (bladders, salt glands). Since halophytes are very important for the study of salt-tolerance mechanisms, this chapter is focused on the work carried out on transporter genes from halophytes present at the plasma membrane and tonoplast controlling Na+ homeostasis under salinity.
[Show abstract][Hide abstract] ABSTRACT: The present investigation aimed to evaluate the reliability of in vitro propagation methods for elite genotypes of Jatropha curcas L., that maintain genetic integrity of tissue culture (TC) regenerates among two regeneration systems developed through direct shoot bud regeneration using nodal/apical shoot segments (protocol-A) and in vitro-derived leaves (protocol-B) as explants. Random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), simple sequence repeat (SSR) molecular markers, and flow cytometery (FCM) were employed to evaluate genetic homogeneity in TC-regenerates at different passages of subcultures. RAPD markers showed genetic homogeneity in fifth-generation TC-regenerates of both protocols. ISSR markers showed genetic stability of leaf regenerates (protocol-B) at 10th generation. FCM analysis of TC-regenerates at 10th generation in protocol-B and at 20th generation in both protocols, showed stability of ploidy level. SSR assessment of TC-regenerates at 20th generation in both protocols confirmed genetic homogeneity. The results confirmed the genetic stability of the TC-regenerates and demonstrated the reliability of the regeneration systems developed so far using explants of two different origins, for large-scale multiplication of elite genotypes of Jatropha.
Applied biochemistry and biotechnology 09/2013; · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. RESULTS: The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. CONCLUSIONS: Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other transporters activity indirectly. These results broaden the role of SbSOS1 in planta and suggest that this gene could be used to develop salt-tolerant transgenic crops.
[Show abstract][Hide abstract] ABSTRACT: Salinity severely affects plant growth and development. Plants evolved various mechanisms to cope up stress both at molecular and cellular levels. Halophytes have developed better mechanism to alleviate the salt stress than glycophytes, and therefore, it is advantageous to study the role of different genes from halophytes. Salicornia brachiata is an extreme halophyte, which grows luxuriantly in the salty marshes in the coastal areas. Earlier, we have isolated SbASR-1 (abscisic acid stress ripening-1) gene from S. brachiata using cDNA subtractive hybridisation library. ASR-1 genes are abscisic acid (ABA) responsive, whose expression level increases under abiotic stresses, injury, during fruit ripening and in pollen grains. The SbASR-1 transcript showed up-regulation under salt stress conditions. The SbASR-1 protein contains 202 amino acids of 21.01-kDa molecular mass and has 79 amino acid long signatures of ABA/WDS gene family. It has a maximum identity (73 %) with Solanum chilense ASR-1 protein. The SbASR-1 has a large number of disorder-promoting amino acids, which make it an intrinsically disordered protein. The SbASR-1 gene was over-expressed under CaMV 35S promoter in tobacco plant to study its physiological functions under salt stress. T(0) transgenic tobacco seeds showed better germination and seedling growth as compared to wild type (Wt) in a salt stress condition. In the leaf tissues of transgenic lines, Na(+) and proline contents were significantly lower, as compared to Wt plant, under salt treatment, suggesting that transgenic plants are better adapted to salt stress.
[Show abstract][Hide abstract] ABSTRACT: Genetic engineering of plants for abiotic stress tolerance is a challenging task because of its multifarious nature. Comprehensive studies for developing abiotic stress tolerance are in progress, involving genes from different pathways including osmolyte synthesis, ion homeostasis, antioxidative pathways, and regulatory genes. In the last decade, several attempts have been made to substantiate the role of “single-function” gene(s) as well as transcription factor(s) for abiotic stress tolerance. Since, the abiotic stress tolerance is multigenic in nature, therefore, the recent trend is shifting towards genetic transformation of multiple genes or transcription factors. A large number of crop plants are being engineered by abiotic stress tolerant genes and have shown the stress tolerance mostly at laboratory level. This review presents a mechanistic view of different pathways and emphasizes the function of different genes in conferring salt tolerance by genetic engineering approach. It also highlights the details of successes achieved in developing salt tolerance in plants thus far.
[Show abstract][Hide abstract] ABSTRACT: Salicornia brachiata is one of the extreme salt tolerant plants and grows luxuriantly in coastal areas. Previously we have reported isolation and characterization of ESTs from S. brachiata with large number of unknown gene sequences. Reverse Northern analysis showed upregulation and downregulation of few unknown genes in response to salinity. Some of these unknown genes were made full length and their functional analysis is being tested. In this study, we have selected a novel unknown salt inducible gene SbSI-1 (Salicornia brachiata salt inducible-1) for the functional validation. The SbSI-1 (Gen-Bank accession number JF 965339) was made full length and characterized in detail for its functional validation under desiccation and salinity. The SbSI-1 gene is 917 bp long, and contained 437 bp 3' UTR, and 480 bp ORF region encoding 159 amino acids protein with estimated molecular mass of 18.39 kDa and pI 8.58. The real time PCR analysis revealed high transcript expression in salt, desiccation, cold and heat stresses. However, the maximum expression was obtained by desiccation. The ORF region of SbSI-1 was cloned in pET28a vector and transformed in BL21 (DE3) E. coli cells. The SbSI-1 recombinant E. coli cells showed tolerance to desiccation and salinity stress compared to only vector in the presence of stress.
[Show abstract][Hide abstract] ABSTRACT: Salinity causes multifarious adverse effects to plants. Plants response to salt stress involves numerous processes that function
in coordination to alleviate both cellular hyperosmolarity and ion disequilibrium. A Na+/H+ antiporter NHX1 gene has been isolated from a halophytic plant Salicornia brachiata in this study. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved
in SbNHX1 classify it as a plant vacuolar NHX gene. The SbNHX1 cDNA has an open reading frame of 1,683bp, encoding a polypeptide of 560 amino acid residues with an estimated molecular
mass 62.44kDa. The SbNHX1 shows high amino acid similarity with other halophytic NHX gene and belongs to Class-I type NHXs.
TMpred suggests that SbNHX1 contains 11 strong transmembrane (TM). Real time PCR analysis revealed that SbNHX1 transcript expresses maximum at 0.5M. Transcript increases gradually by increasing the treatment duration at 0.5M NaCl,
however, maximum expression was observed at 48h. The overexpression of SbNHX1 gene in tobacco plant showed NaCl tolerance. This study shows that SbNHX1 is a potential gene for salt tolerance, and can be used in future for developing salt tolerant crops.
[Show abstract][Hide abstract] ABSTRACT: Dehydration-responsive element binding (DREB) transcription factor plays a key role in plant stress signal transduction pathway. In this study, SbDREB2A has been isolated from the halophyte Salicornia brachiata. SbDREB2A cDNA is 1,062 bp long, encoding protein of 353 amino acids with an estimated molecular mass of 39.37 kDa and a pI of 4.98. On the basis of multiple sequence alignment and phylogenetic analysis, SbDREB2A is classified in A-2 group of the DREB family. The genomic organization confirms that SbDREB2A is an intronless gene. Purified recombinant SbDREB2A protein showed similar binding to both DREs (dehydration-responsive element), ACCGAC and GCCGAC. The transcript expression of SbDREB2A was induced by NaCl, drought and heat stress. The role of SbDREB2A in abiotic stress was studied in E. coli BL21 (DE3). The recombinant E. coli cells exhibited better growth in basal LB medium as well as in supplemented with NaCl, PEG and mannitol. The enhanced growth in recombinant E. coli could be due to the regulation of stress regulated functional genes by this transcription factor. This system can be applied in biotechnological applications, where growth of E. coli can be enhanced under salt stress for efficient recombinant protein production in a short span of time.
[Show abstract][Hide abstract] ABSTRACT: The DREB transcription factors comprise conserved ERF/AP2 DNA-binding domain, bind specifically to DRE/CRT motif and regulate abiotic stress mediated gene expression. In this study we show that PgDREB2A from Pennisetum glaucum is a powerful transcription factor to engineer multiple stress tolerance in tobacco plants. The PgDREB2A protein lacks any potential PEST sequence, which is known to act as a signal peptide for protein degradation. Therefore, the transgenic tobacco plants were raised using full-length cDNA without modification. The transgenics exhibited enhanced tolerance to both hyperionic and hyperosmotic stresses. At lower concentration of NaCl and mannitol, seed germination and seedling growth was similar in WT and transgenic, however at higher concentration germination in WT decreased significantly. D15 and D46 lines showed 4-fold higher germination percent at 200 mM NaCl. At 400 mM mannitol seed germination in WT was completely arrested, whereas in transgenic line it was more than 50%. Seedlings of D15 and D46 lines showed better growth like leaf area, root number, root length and fresh weight compared to wild type for both the stresses. The quantitative Real time PCR of transgenic showed higher expression of downstream genes NtERD10B, HSP70-3, Hsp18p, PLC3, AP2 domain TF, THT1, LTP1 and heat shock (NtHSF2) and pathogen-regulated (NtERF5) factors with different stress treatments.
[Show abstract][Hide abstract] ABSTRACT: MAPK cascade is an important intracellular signaling module and function as a convergent point for crosstalk during abiotic stress signaling. In this study SbMAPKK gene has been isolated from Salicornia brachiata, a highly salt tolerant plant growing in costal marshes of Gujarat, India. The SbMAPKK gene is 1,023 bp long, encodes a 340 amino acid protein with an estimated molecular mass of 37.4 kDa. The SbMAPKK shows high sequence identity with NbMKK1 from N. benthamiana, LeMKK4 from Lycopersicon esculentum. SbMAPKK constitutes 11 conserved subdomains of protein kinase. Northern analysis revealed that SbMAPKK transcript expression is induced by different stresses like dehydration, cold and salt, however, maximum expression is observed during cold stress. The phylogenetic analysis and genomic organization confirms that it is an intron less gene belonging 'D' group in MAPKK family.
[Show abstract][Hide abstract] ABSTRACT: Salinity severely affects plant growth and development causing crop loss worldwide. We have isolated a large number of salt-induced genes as well as unknown and hypothetical genes from Salicornia brachiata Roxb. (Amaranthaceae). This is the first description of identification of genes in response to salinity stress in this extreme halophyte plant. Salicornia accumulates salt in its pith and survives even at 2 M NaCl under field conditions. For isolating salt responsive genes, cDNA subtractive hybridization was performed between control and 500 mM NaCl treated plants. Out of the 1200 recombinant clones, 930 sequences were submitted to the NCBI database (GenBank accession: EB484528 to EB485289 and EC906125 to EC906292). 789 ESTs showed matching with different genes in NCBI database. 4.8% ESTs belonged to stress-tolerant gene category and approximately 29% ESTs showed no homology with known functional gene sequences, thus classified as unknown or hypothetical. The detection of a large number of ESTs with unknown putative function in this species makes it an interesting contribution. The 90 unknown and hypothetical genes were selected to study their differential regulation by reverse Northern analysis for identifying their role in salinity tolerance. Interestingly, both up and down regulation at 500 mM NaCl were observed (21 and 10 genes, respectively). Northern analysis of two important salt tolerant genes, ASR1 (Abscisic acid stress ripening gene) and plasma membrane H+ATPase, showed the basal level of transcripts in control condition and an increase with NaCl treatment. ASR1 gene is made full length using 5' RACE and its potential role in imparting salt tolerance is being studied.
Genes & Genetic Systems 05/2009; 84(2):111-20. · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Rab GTPases are important components of endocytic network in plant cells. Endocytosis participates in the cell's reaction to extracellular stimuli by desensitizing, down-regulating or recycling receptors and membrane proteins. Rab7 is a small GTP-binding protein involved in intracellular vesicle trafficking from late endosome to the vacuole. We have isolated Rab7 cDNA from Pennisetum glaucum, a relatively drought-stress tolerant food grain crop grown commonly in India, during cDNA-subtractive hybridization of dehydration-stress treated plants. The PgRab7 ORF, encoding 207 aminoacids, was over-expressed in E. coli. The recombinant PgRab7 protein showed GTP-binding and GTPase activity. Transcript expression of PgRab7 gene was differentially up-regulated by different environmental stimuli such as cold, dehydration and NaCl and also by a plant hormone IAA. Overexpression of PgRab7 gene enhanced tolerance to NaCl and mannitol in transgenic tobacco. Transgenic plants also had increased alkaline phosphatase (ALP) activity. These results show that PgRab7 is a potential candidate gene for developing both salinity and dehydration tolerance in planta.
[Show abstract][Hide abstract] ABSTRACT: Abiotic stress-mediated gene expression is regulated via different transcription factors of which drought-responsive element-binding (DREB) proteins play an important role. There are two types of DREBs. Presently, the function of DREB1 type protein is well studied; however, much less information is available for DREB2. In this study, a cDNA with an open reading frame of 332 amino acids, encoding the transcription activation factor DREB2A, was cloned from Pennisetum glaucum, a stress tolerant food grain crop. Phylogenetic tree revealed that PgDREB2A is more close to DREBs isolated from monocots, though it forms an independent branch. The PgDREB2A transcript was up-regulated in response to drought within 1 h of the treatment, whereas the induction was delayed in response to cold and salinity stress. However, during cold stress, the transcript was induced more as compared to drought and salinity. The recombinant PgDREB2A protein having a molecular mass of 36.6 kDa was purified using Ni-NTA affinity chromatography. Gel mobility shift assays using the purified protein and two cis elements of rd29A (responsive to dehydration 29A) gene promoter of Arabidopsis revealed that PgDREB2A binds to drought-responsive element (DRE) ACCGAC and not to GCCGAC. PgDREB2A is a phosphoprotein, which has not been reported earlier. The phosphorylation of PgDREB2A in vitro by P. glaucum total cell extract occurred at threonine residue(s). The phosphorylated PgDREB2A did not bind to the DREs. The present data indicate that stress induction of genes could occur via post-translational modification by phosphorylation of DREB2A.
Molecular and General Genetics 03/2007; 277(2):189-98. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Soil salinity is one of the major abiotic stresses affecting plant productivity. We report on the isolation and characterization
of a different isoform of vacuolar Na+/H+ antiporter from Pennisetum glaucum (PgNHX1) that conferred high level of salinity tolerance when over expressed in Brassica juncea. PgNHX1 cDNA has an ORF of 1,413bp and a 3′ and 5′ UTR of 350 and 166bp, respectively. In contrast to AtNHX1 and OsNHX1, which have nine transmembrane-spanning domains, PgNHX1 has five transmembrane domains. Real time quantitative RT-PCR analysis revealed up-regulation of PgNHX1 transcript following NaCl and ABA treatments. The 3.3kb PgNHX1 genomic clone has 12 introns spanning the entire ORF. Confocal imaging of the GFP-PgNHX1 fusion construct showed vacuolar localization in yeast cells. The fungal inhibitor, brefeldin A, inhibited this localization.
PgNHX1 functionally complemented yeast mutants defective in endogenous NHX and such transformed yeast could tolerate up to
70mM NaCl. Transgenic B. juncea plants overexpressing PgNHX1 survive, set flowers and normal seeds in presence of up to 300mM NaCl. The transgenic plants accumulated more Na+ in the leaves than in seeds. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants for growing in high-saline areas.