Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast

Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.     

Overexpression of barley nicotianamine synthase 1 confers tolerance in the sweet potato to iron deficiency in calcareous soil

Background and aims

Iron (Fe) is an essential micronutrient for all higher organisms. Fe is sparingly available in calcareous soils and Fe deficiency is a major agricultural problem worldwide. Nicotianamine (NA) is a metal chelator involved in metal translocation in plants. Sweet potato is an attractive crop that can grow in poor soil and thus is useful for planting in uncultivated soil. In addition, the sweet potato has recently been suggested as a source of bioethanol. Our aim is to increase NA concentration in sweet potato to ameliorate Fe deficiency.

Method

Sweet potato plants expressing the barley NA synthase 1 (HvNAS1) gene under the control of CaMV 35S promoter were produced by Agrobacterium-mediated transformation.

Results

The transgenic sweet potato exhibited tolerance to low Fe availability when grown in calcareous soil. The level of tolerance to low Fe availability was positively correlated with the HvNAS1 expression level. The NA concentration of the transgenic sweet potato leaves was up to 7.9-fold greater than that of the non-transgenic (NT) plant leaves. Furthermore, the Fe and zinc concentrations were 3- and 2.9-fold greater, respectively, in transgenic sweet potato than in NT plant leaves.

Conclusions

Our results suggest that increasing the NA concentration of sweet potato by overexpression of HvNAS1 could significantly improve agricultural productivity and energy source.

     

Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.     

Overexpression of CmSOS1 confers waterlogging tolerance in Chrysanthemum

正Plants experiencing hypoxia (a shortage of oxygen)are unable to maintain aerobic respiration, which leads to an energy and carbohydrate deficit. The pervasive and rapid accumulation of ethylene is an early and reliable response to hypoxic stress(Sasidharan and Voesenek 2015), producing an uptick in the accumulation of reactive oxygen species (ROS).This in turn triggers apoptosis in root cortex cells,eventually leading to the formation of lysigenous aerenchyma, a tissue from which ethylene is readily     

Overexpression ofENA1 from yeast increases salt tolerance inArabidopsis

In yeast, the plasma membrane Na⁺/H⁺ antiporter and Na⁺-ATPase are key enzymes for salt tolerance.Saccharomyces cerevisiae Na⁺-ATPase (Enalp ATPase) is encoded by theENA1/PMR2A gene; expression ofENA1 is tightly regulated by Na⁺ and depends on ambient pH. Although Enalp is active mainly at alkaline pH values inS. cerevisiae, no Na⁺-ATPase has been found in flowering plants. To test whether this yeast enzyme would improve salt tolerance in plants, we introducedENA1 intoArabidopsis (cv. Columbia) under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on a medium containing kanamyin. Southern blot analyses confirmed thatENA1 was transferred into theArabidopsis genome and northern blot analyses showed thatENA1 was expressed in the transformants. Several transgenic homozygous lines and wild-type (WT) plants were evaluated for salt tolerance. No obvious morphological or developmental differences existed between the transgenic and WT plants in the absence of stress. However, overexpression ofENA1 inArabidopsis improved seed germination rates and salt tolerance in seedlings. Under saline conditions, transgenic plants accumulated a lower amount of Na⁺ than did the wild type, and fresh and dry weights of the former were higher. Other experiments revealed that expression ofENA1 promoted salt tolerance in transgenicArabidopsis under both acidic and alkaline conditions. These authors contributed equally to this article.     

Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass

The Arabidopsis, abscisic acid responsive element-binding factor 3, ABF3 is known to play an important role in stress responses via regulating the expression of stress-responsive genes. In this study, we introduced pCAMBIA3301 vector harboring the ABF3 gene into creeping bentgrass (Agrostis stolonifera) through Agrobacterium-mediated transformation in order to develop a stress-tolerant variety of turfgrass. After transformation, putative transgenic plants were selected using the herbicide resistance assay. Genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by northern blot analysis. Under drought-stressed condition, the transgenic plants overexpressing ABF3 displayed significantly enhanced drought tolerance with higher water content and slower water loss rate than the control plants. Furthermore, the stomata of the ABF3 transgenic plants closed more than those of wild-type creeping bentgrass plants, under both non-stressed and ABA treatment conditions. In addition, the transgenic plants showed enhanced tolerance to heat stress. These results suggest that the overexpression of the ABF3 gene in creeping bentgrass might enhance survival in water-limiting and high temperature environments through increased stomatal closure and reduced water losses.     

Overexpression of the Arabidopsis NPR1 gene confers enhanced salt tolerance by regulating antioxidant and starch accumulation in citrus

Plant Cell, Tissue and Organ Culture (PCTOC) - The Non-Expresser of Pathogenesis-Related Genes 1 (NPR1) is a principal regulator of plant responses to biotic and abiotic stresses. In this...     

Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2, confers enhanced tolerance to oxidative stress in Arabidopsis plants

Mutant pqr-216 from an Arabidopsis activation-tagged line showed a phenotype of increased tolerance to oxidative stress after treatment with 3 μ m paraquat (PQ). Based on the phenotype of transgenic plants overexpressing the genes flanking the T-DNA insert, it was clear that enhanced expression of a Nudix (nucleoside diphosphates linked to some moiety X) hydrolase gene, AtNUDX2 (At5g47650), was responsible for the tolerance. It has been reported that the AtNUDX2 protein has pyrophosphatase activities towards both ADP-ribose and NADH ( Ogawa et al ., 2005 ). Interestingly, the pyrophosphatase activity toward ADP-ribose, but not NADH, was increased in pqr-216 and Pro _35S :AtNUDX2 plants compared with control plants. The amount of free ADP-ribose was lower in the Pro _35S :AtNUDX2 plants, while the level of NADH was similar to those in control plants under both normal conditions and oxidative stress. Depletion of NAD⁺ and ATP resulting from activation of poly(ADP-ribosyl)ation under oxidative stress was observed in the control Arabidopsis plants. Such alterations in the levels of these molecules were significantly suppressed in the Pro _35S :AtNUDX2 plants. The results indicate that overexpression of AtNUDX2 , encoding ADP-ribose pyrophosphatase, confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from maintenance of NAD⁺ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions.     

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.     

Overexpression of AtMYB52 confers ABA hypersensitivity and drought tolerance

We carried out activation tagging screen to isolate genes regulating abscisic acid (ABA) response. From the screen of approximately 10,000 plants, we isolated ca 100 ABA response mutants. We characterized one of the mutants, designated ahs1, in this study. The mutant is ABA-hypersensitive, and AtMYB52 was found to be activated in the mutant. Overexpression analysis to recapitulate the mutant phenotypes demonstrated that ATMYB confers ABA-hypersensitivity during postgermination growth. Additionally, AtMYB52 overexpression lines were drought-tolerant and their seedlings were salt-sensitive. Changes in the expression levels of a few genes involved in ABA response or cell wall biosynthesis were also observed. Together, our data suggest that AtMYB52 is involved in ABA response. Others previously demonstrated that AtMYB52 regulates cell wall biosynthesis; thus, our results imply a possible connection between ABA response and cell wall biosynthesis.     

Antioxidant defense system and cadmium uptake in barley genotypes differing in cadmium tolerance

Using two barley (Hordeum vulgare) cultivars (cvs. Tokak and Hamidiye) nutrient solution experiments were conducted in order to study the genotypic variation in tolerance to Cd toxicity based on (i) development of leaf symptoms, (ii) decreases in dry matter production, (iii) Cd concentration and (iv) changes in antioxidative defense system in leaves (i.e., superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, ascorbic acid and non-protein SH-groups). Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 15, 30, 60 and 120 micromol/L Cd) for different time periods. Of the barley cultivars Hamidiye was particularly sensitive to Cd as judged by the severity and earlier development of Cd toxicity symptoms on leaves. Within 48 h of Cd application Hamidiye rapidly developed severe leaf Cd toxicity symptoms whereas in Tokak the leaf symptoms appeared only slightly. Hamidiye also tended to show more decrease in growth caused by Cd supply when compared to Tokak. The differences in sensitivity to Cd between Tokak and Hamidiye were not related to Cd concentrations in roots and shoots or Cd accumulation per plant. With the exception of catalase, activities of the enzymes involved in detoxification of reactive oxygen species (ROS) were markedly enhanced in Hamidiye by increasing Cd supply. By contrast, in Tokak there was either only a slight increase or no change in the activities of the antioxidative enzymes. Similarly, levels of ascorbic acid and especially non-protein SH-groups were increased in Hamidiye by Cd supply, but not affected in Tokak. The results indicate the existence of a large genotypic variation between barley cultivars for Cd tolerance. The differential Cd tolerance found in the barley cultivars was not related to uptake or accumulation of Cd in plants, indicating importance of internal mechanisms in expression of differential Cd tolerance in barley. As a response to increasing Cd supply particular increases in antioxidative mechanisms in the Cd-sensitive barley cultivar Hamidiye suggest that the high Cd sensitivity of Hamidiye is related to enhanced production and oxidative damage of ROS.     

Overexpression of alfalfa mitochondrial HSP23 in prokaryotic and eukaryotic model systems confers enhanced tolerance to salinity and arsenic stress

The cloning and characterization of a gene (MsHSP23) coding for a heat shock protein in alfalfa in a prokaryotic and model plant system is described. MsHSP23 contains a 633 bp ORF encoding a polypeptide of 213 amino acids and exhibits greater sequence similarity to mitochondrial sHSPs from dicotyledons than to those from monocotyledons. When expressed in bacteria, recombinant MsHSP23 conferred tolerance to salinity and arsenic stress. Furthermore, MsHSP23 was cloned in a plant expressing vector and transformed into tobacco, a eukaryotic model organism. The transgenic plants exhibited enhanced tolerance to salinity and arsenic stress under ex vitro conditions. In comparison to wild type plants, the transgenic plants exhibited significantly lower electrolyte leakage. Moreover, the transgenic plants had superior germination rates when placed on medium containing arsenic. Taken together, these overexpression results imply that MsHSP23 plays an important role in salinity and arsenic stress tolerance in transgenic tobacco. This approach could be useful to develop stress tolerant crops including forage crops.     

Universal stress protein in Malus sieversii confers enhanced drought tolerance

Journal of Plant Research - Drought is an important environmental factor that can severely affect plant growth and reproduction. Although many genes related to drought tolerance have been studied...