Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon

Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

A DESD-box helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. PB1)

The exact mechanism of helicase-mediated salinity tolerance is not yet understood. We have isolated a DESD-box containing cDNA from Pisum sativum (Pea) and named it as PDH45. It is a unique member of DEAD-box helicase family; containing DESD instead of DEAD/H. PDH45 overexpression driven by constitutive cauliflower mosaic virus-35S promoter in rice transgenic [Oryza sativa L. cv. Pusa Basmati 1 (PB1)] plants confers salinity tolerance by improving the photosynthesis and antioxidant machinery. The Na⁺ ion concentration and oxidative stress parameters in leaves of the NaCl (0, 100 or 200 mM) treated PDH45 overexpressing T₁ transgenic lines were lower as compared to wild type (WT) rice plants under similar conditions. The 200 mM NaCl significantly reduced the leaf area, plant dry mass, net photosynthetic rate (P_N), stomatal conductance (gs), intercellular CO₂ (Ci), chlorophyll (Chl) content in WT plants as compared to the transgenics. The T₁ transgenics exhibited higher glutathione (GSH) and ascorbate (AsA) contents under salinity stress. The activities of antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were significantly higher in transgenics; suggesting the existence of an efficient antioxidant defence system to cope with salinity induced-oxidative damage. Yeast two-hybrid assay indicated that the PDH45 protein interacts with Cu/Zn SOD, adenosine-5′-phosphosulfate-kinase, cysteine proteinase and eIF(4G), thus confirming the involvement of ROS scavenging machinery in the transgenic plants to provide salt tolerance. Furthermore, the T₂ transgenics were also able to grow, flower, and set viable seeds under continuous salinity stress of 200 mM NaCl. This study provides insights into the mechanism of PDH45 mediated salinity stress tolerance by controlling the generation of stress induced reactive oxygen species (ROS) and also by protecting the photosynthetic machinery through a strengthened antioxidant system.     

Isolation of a cDNA clone (PcSrp) encoding serine-rich-protein from Porteresia coarctata T. and its expression in yeast and finger millet (Eleusine coracana L.) affording salt tolerance

A 1.4 Kb cDNA clone encoding a serine-rich protein has been isolated from the cDNA library of salt stressed roots of Porteresia coarctata, and designated as P. coarctata serine-rich-protein (PcSrp) encoding gene. Northern analysis and in situ mRNA hybridization revealed the expression of PcSrp in the salt stressed roots and rhizome of P. coarctata. However, no such expression was seen in the salt stressed leaves and in the unstressed tissues of root, rhizome and leaf, indicating that PcSrp is under the control of a salt-inducible tissue-specific promoter. In yeast, the PcSrp conferred increased NaCl tolerance, implicating its role in salinity tolerance at cellular level. Further, PcSrp was cloned downstream to rice Actin-1 promoter and introduced into finger millet through particle-inflow-gun method. Transgenic plants expressing PcSrp were able to grow to maturity and set seed under 250 mM NaCl stress. The untransformed control plants by contrast failed to survive under similar salt stress. The stressed roots of transgenic plants invariably accumulated higher Na⁺ and K⁺ ion contents compared to roots of untransformed plants; whereas, shoots of transgenics accumulated lower levels of both the ions than that of untransformed plants under identical stress, clearly suggesting the involvement of PcSrp in ion homeostasis contributing to salt tolerance.     

Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana

The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12 h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%–48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%–43%), total GS activity (39%–45%) and soluble protein concentration (23%–24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

The CCCH zinc finger protein gene AtZFP1 improves salt resistance in Arabidopsis thaliana

The CCCH type zinc finger proteins are a super family involved in many aspects of plant growth and development. In this study, we investigated the response of one CCCH type zinc finger protein AtZFP1 (At2g25900) to salt stress in Arabidopsis. The expression of AtZFP1 was upregulated by salt stress. Compared to transgenic strains, the germination rate, emerging rate of cotyledons and root length of wild plants were significantly lower under NaCl treatments, while the inhibitory effect was significantly severe in T-DNA insertion mutant strains. At germination stage, it was mainly osmotic stress when treated with NaCl. Relative to wild plants, overexpression strains maintained a higher K⁺, K⁺/Na⁺, chlorophyll and proline content, and lower Na⁺ and MDA content. Quantitative real-time PCR analysis revealed that the expression of stress related marker genes KIN1, RD29B and RD22 increased more significantly in transgenic strains by salt stress. Overexpression of AtZFP1 also enhanced oxidative and osmotic stress tolerance which was determined by measuring the expression of a set of antioxidant genes, osmotic stress genes and ion transport protein genes such as SOS1, AtP5CS1 and AtGSTU5. Overall, our results suggest that overexpression of AtZFP1 enhanced salt tolerance by maintaining ionic balance and limiting oxidative and osmotic stress.     

Effect of NaCl on photosynthesis,salt accumulation and ion compartmentation in two mangrove species, Kandelia candel and Bruguiera gymnorhiza

In a 4-week study, we investigated the effects of increasing soil NaCl (100–400 mM) on photosynthesis, salt uptake and transport, and intracellular compartmentation of Na⁺ and Cl⁻ in 1-year-old seedlings of Kandelia candel (L.) Druce and Bruguiera gymnorhiza (L.) Savigny. Increasing NaCl stress significantly elevated Na⁺ and Cl⁻ in root and shoot tissues (stem + leaf) of both species, but B. gymnorhiza showed a rapid Na⁺ accumulation upon the initiation of salt stress and leaves contained 90% more Na⁺ and 40% more Cl⁻ than K. candel at the end of experiment. Net photosynthetic rate (Pn) declined with increasing salinity, and the most marked reduction occurred after exposure of mangrove seedlings to a severe salinity, 400 mM NaCl. However, the inhibitory effects of severe stress varied with species: Pn decreased by 80% in K. candel whereas in B. gymnorhiza the decline was 60%. The quantum yield (AQY) and carboxylation efficiency (CE) response to severe salinity showed a trend similar to Pn, in which a lesser reduction of AQY and CE was observed in B. gymnorhiza (33–35%), as compared to K. candel (43–52%). X-ray microanalysis of leaf mesophyll cells showed evidence of distinct vacuolar compartmentation of Na⁺ in K. candel but Cl⁻ in B. gymnorhiza after seedlings were subjected to 100 mM NaCl for 7 d. Moreover, Na⁺ within cell wall, cytoplasm, vacuole and chloroplast remained 23–72% lower in stressed B. gymnorhiza as compared to K. candel. In conclusion, B. gymnorhiza exhibited effective salt exclusion from chloroplasts although increasing salt stress caused a rapid and higher build up of Na⁺ and Cl⁻ in the leaves. We suggest that the salt-induced Pn reduction in the two mangrove species is correlated with the ability to exclude Na⁺ and Cl⁻ from the chloroplast, rather than with the bulk leaf salt concentration.     

Gamma irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells

In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50 Gy, delivered at 0.33 Gy min⁻¹) and HDR (total doses 50 and 100 Gy, delivered at 5.15 Gy min⁻¹) γ-ray in the 0–24 h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0–4 h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding.     

Ovexpression of a Vacuolar H<Superscript>+</Superscript>-ATPase c Subunit Gene Mediates Physiological Changes Leading to Enhanced Salt Tolerance in Transgenic Tobacco

H⁺-ATPase subunit c (VHA-c) is involved in the adaptation to environmental stresses, including salt, drought, and heavy metals. However, it remains unclear whether VHA-c can induce a physiological response related to stress tolerance. To investigate this possibility, we generated transgenic tobacco lines overexpressing a V-ATPase subunit c (LbVHA-c1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild-type (WT) tobacco, superoxide dismutase (SOD) and peroxidase (POD) activities in the transgenic plants were significantly enhanced under salt stress conditions. The level of malondialdehyde (MDA) in the transgenic plants was significantly lower than that in WT plants grown under salt stress conditions. Moreover, the transgenic plants displayed obviously better growth than the WT plants under salt stress. These results suggest that LbVHA-c1 may confer stress tolerance through enhancing POD and SOD activities, and by protecting membranes from damage by decreasing lipid peroxidation under salt stress.     

Identification of differentially expressed salt-responsive proteins in roots of two perennial grass species contrasting in salinity tolerance

This study was designed to identify physiological responses and differential proteomic responses to salinity stress in roots of a salt-tolerant grass species, seashore paspalum (Paspalum vaginatum), and a salt-sensitive grass species, centipedegrass (Eremochloa ophiuroides). Plants of both species were exposed to salinity stress by watering the soil with 300 mM NaCl solution for 20 d in a growth chamber. The 2-DE analysis revealed that the abundance of 8 protein spots significantly increased and 14 significantly decreased in seashore paspalum, while 19 and 16 protein spots exhibited increase and decrease in abundance in centipedegrass, respectively. Eight protein spots that exhibited enhanced abundance in seashore paspalum under salinity stress were subjected to mass spectrometry analysis. Seven protein spots were successfully identified, they are peroxidase (POD, 2.36-fold), cytoplasmic malate dehydrogenase (cMDH, 5.84-fold), asorbate peroxidase (APX, 4.03-fold), two mitochondrial ATPSδ chain (2.26-fold and 4.78-fold), hypothetical protein LOC100274119 (5.01-fold) and flavoprotein wrbA (2.20-fold), respectively. Immunblotting analysis indicated that POD and ATPSδ chain were significantly up-regulated in seashore paspalum at 20 d of salinity treatment while almost no expression in both control and salt treatment of centipedegrass. These results indicated that the superior salinity tolerance in seashore paspalum, compared to centipedegrass, could be associated with a high abundance of proteins involved in ROS detoxification and energy metabolism.