Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance

Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na⁺/H⁺ antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L^?1 Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops.     

Characterization and Expression Analysis of a Glutathione Reductase Gene from Antarctic Moss Pohlia nutans

Glutathione reductase (GR) is a flavoprotein oxidoreductase and plays an important role in response to oxidative stresses in plants. A cDNA-encoding cytosolic GR [GenBank accession number GACA01029426, designated as Pohlia nutans glutathione reductase gene (PnGR)] was successfully cloned from Antarctic moss P. nutans. The full-length PnGR cDNA has 1,654 bp nucleotides with an open reading frame of 1,494 bp, encoding 497 amino acid residues. The deduced amino acid sequence of PnGR had 87.0 % identity with GR in Physcomitrella patens subsp. patens. The phylogenetic analysis showed that PnGR is clustered together with known cytosolic GR in other plants. In addition, the subcellular localization analysis by observing the transient expression of PnGR–green fluorescent protein fusion protein in Arabidopsis thaliana mesophyll protoplasts also revealed PnGR targeting to cytosol in plant cells. The expression patterns of PnGR under different abiotic stresses were determined by real-time PCR. Compared to the normal condition, the maximal mRNA accumulation of PnGR increased 3.82-fold at 4 °C, 2.92-fold at 10 °C, 4.14-fold with 200 mM NaCl, and 3.17-fold with drought stress, respectively. Together, our results suggested that the inducible PnGR might play an important role in Antarctic moss P. nutans acclimatizing to polar environment.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

A Putative PP2C-Encoding Gene Negatively Regulates ABA Signaling in Populus euphratica

A PP2C homolog gene was cloned from the drought-treated cDNA library of Populus euphratica. Multiple sequence alignment analysis suggested that the gene is a potential ortholog of HAB1. The expression of this HAB1 ortholog (PeHAB1) was markedly induced by drought and moderately induced by ABA. To characterize its function in ABA signaling, we generated transgenic Arabidopsis thaliana plants overexpressing this gene. Transgenic lines exhibited reduced responses to exogenous ABA and reduced tolerance to drought compared to wide-type lines. Yeast two-hybrid analyses indicated that PeHAB1 could interact with the ABA receptor PYL4 in an ABA-independent manner. Taken together; these results indicated that PeHAB1 is a new negative regulator of ABA responses in poplar.     

CDPK1 from Ginger Promotes Salinity and Drought Stress Tolerance without Yield Penalty by Improving Growth and Photosynthesis in Nicotiana tabacum

In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) – PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.     

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23^fyp and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.     

2-Cys peroxiredoxin responds to low temperature and other cues in Caragana jubata,a plant species of cold desert of Himalaya

A 2-Cys peroxiredoxin cDNA (CjPrx) was isolated and characterized from Caragana jubata, a temperate/alpine plant species of high altitude cold desert of Himalaya and Eurasia. The cDNA obtained was 1,064 bp long consisting of an open reading frame of 789 bp encoding 262 amino acids. The calculated molecular mass of the mature protein was 28.88 kDa and pI was 5.84. Deduced amino acid sequence of CjPrx shared a high degree homology with 2-CysPrx proteins from other plants. CjPrx had both the PRX_type 2-Cys domain and thioredoxin-like superfamily domains. CjPrx contained 26.72 % α-helices, 6.87 % β-turns, 20.61 % extended strands and 45.80 % random coils, and was a hydrophilic protein. Expression of CjPrx was modulated by low temperature, methyl jasmonate (MJ), salicylic acid and drought stress, but no significant change was observed in response to abscisic acid treatment. Among all the treatments, a strong up-regulation of CjPrx was observed in response to MJ treatment.     

Ectopic expression of PgRab7 in rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> 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.