CAX3 (cation/proton exchanger) mediates a Cd tolerance by decreasing ROS through Ca elevation in Arabidopsis

Key message

Over-expression of CAX3 encoding a cation/proton exchanger enhances Cd tolerance by decreasing ROS (Reactive Oxygen Species) through activating anti-oxidative enzymes via elevation of Ca level in Arabidopsis

Abstract

CAXs (cation/proton exchangers) are involved in the sequestration of cations such as Mn, Li, and Cd, as well as Ca, from cytosol into the vacuole using proton gradients. In addition, it has been reported that CAX1, 2 and 4 are involved in Cd tolerance. Interestingly, it has been reported that CAX3 expressions were enhanced by Cd in Cd-tolerant transgenic plants expressing Hb1 (hemoglobin 1) or UBC1 (Ub-conjugating enzyme 1). Therefore, to investigate whether CAX3 plays a role in increasing Cd tolerance, CAX3 of Arabidopsis and tobacco were over-expressed in Arabidopsis thaliana. Compared to control plants, both transgenic plants displayed an increase in Cd tolerance, no change in Cd accumulation, and enhanced Ca levels. In support of these, AtCAX3-Arabidopsis showed no change in expressions of Cd transporters, but reduced expressions of Ca exporters and lower rate of Ca efflux. By contrast, atcax3 knockout Arabidopsis exhibited a reduced Cd tolerance, while the Cd level was not altered. The expression of Δ90-AtCAX3 (deletion of autoinhibitory domain) increased Cd and Ca tolerance in yeast, while AtCAX3 expression did not. Interestingly, less accumulation of ROS (H₂O₂ and O₂^?) was observed in CAX3-expressing transgenic plants and was accompanied with higher antioxidant enzyme activities (SOD, CAT, GR). Taken together, CAX3 over-expression may enhance Cd tolerance by decreasing Cd-induced ROS production by activating antioxidant enzymes and by intervening the positive feedback circuit between ROS generation and Cd-induced spikes of cytoplasmic Ca.

     

Generation of selectable marker-free transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system

The aim of this research was to generate selectable marker-free transgenic tomato plants with improved tolerance to abiotic stress. An estradiol-induced site-specific DNA excision of a selectable marker gene using the Cre/loxP DNA recombination system was employed to develop transgenic tomato constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase gene from Arabidopsis thaliana. Transgenic tomato plants containing a selectable marker were also produced as controls. The expression of AtIpk2β conferred improved resistance to drought, cold and oxidative stress in both sets of transgenic tomato plants. These results demonstrate the feasibility of using this Cre/loxP-based marker elimination strategy to generate marker-free transgenic crops with improved stress tolerance.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

Overproduction of an Arabidopsis aldo–keto reductase increases barley tolerance to oxidative and cadmium stress by an in vivo reactive aldehyde detoxification

In this work, we expressed an Arabidopsis thaliana-coded protein (AKR4C9) in transgenic barley to study its enzymatic activity and to enhance the reactive aldehyde neutralizing capacity (part of the oxidative stress tolerance) of transgenic plants. Total leaf protein was extracted from transgenic plants expressing either C or N-terminally His-tagged aldo–keto reductase (AKR) enzyme and purified by affinity chromatography. The Arabidopsis-coded enzyme showed moderate activity against the synthetic reactive aldehyde, glutaraldehyde, and low but detectable enzyme activity against fructose with a low Michaelis–Menten constant (K_m value). Activity of the C and the N-terminally His-tagged AKRs were found to be in the same range. Glutaraldehyde was also tested in vivo by spraying onto the leaves of the plants. The reactive aldehyde tolerance of both wild type and transgenic plants, as well as the general physiological effects of this reactive aldehyde treatment were evaluated. The growth rate was found to decrease in all (both wild type and transgenic) plants. The high AKR-expressing transgenic plants showed a lower respiratory rate, and they also showed higher fresh weight, higher chlorophyll content and photosynthetic activity, indicating a higher reactive aldehyde tolerance. Cadmium (Cd) treatment was also performed to validate this result. Cd caused strong lipid peroxidation; however, the Arabidopsis enzyme lowered the reactive aldehyde content as expected. This is the first report in which kinetic parameters of the fructose reduction by the stress inducible plant AKR enzyme are presented. Furthermore, data on the effects of a reactive aldehyde treatment on intact plants are also provided.     

Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana

Aims

Metallothioneins are cysteine-rich, metal-binding proteins, but their exact functions are not fully understood. In this study, we isolated two metallothionein genes, BcMT1 and BcMT2 from Brassica campestris to increase our understanding of metal tolerance mechanisms in Brassica plants.

Methods

Semi-quantitative RT-PCR was used to analyze expression of the two BcMTs genes. BcMT1 and BcMT2 were ectopically expressed in Arabidopsis thaliana. Quantitative real-time RT-PCR and GUS-staining method were used to select transgenic Arabidopsis plants. Cd and Cu concentrations were analyzed by flame atomic absorption spectrometry. Histochemical detection of H₂O₂ and O₂ ^?? were conducted by 3,3-diaminobenzidine and nitroblue tetrazoliu-staining methods.

Results

BcMT1 is expressed predominantly in roots, whereas BcMT2 is expressed mainly in leaves of B. campestris. Expression of BcMT1 was induced by both Cd and Cu, but expression of BcMT2 was enhanced only by Cd. Ectopic expression of BcMT1 and BcMT2 in Arabidopsis thaliana enhanced the tolerance to Cd and Cu and increased the Cu concentration in the shoots of the transgenic plants. Transgenic Arabidopsis accumulated less reactive oxygen species (ROS) than wild-type plants.

Conclusions

BcMT1 and BcMT2 increased Cd and Cu tolerance in transgenic Arabidopsis, and decreased production of Cd- and Cu-induced ROS, thereby protecting plants from oxidative damage.     

Overexpression of AaPal,a peptidoglycan-associated lipoprotein from Alkalomonas amylolytica,improves salt and alkaline tolerance of Escherichia coli and Arabidopsis thaliana

The outer membrane lipoprotein, Pal, plays a major role maintaining the integrity of outer membrane and cell morphology in Gram-negative bacteria. Here, we represent A novel role of AaPal in tolerance to salt and alkaline stresses. The cell density of Escherichia coli expressing AaPal was approx. three times as that of control strain when grown in the presence of 1 M NaCl or at pH 9.0 for 14 h, and transgenic Arabidopsis thaliana grew taller and stronger than wild-type plants when subjected to 200 mM NaCl or pH 9.0 stress. This tolerance was attributed to higher concentrations of K⁺ and lower concentrations of Na⁺ in the transgenic organism. Our study provides a potential use of AaPal in the improvement of salt and alkaline tolerance in bacteria and plants.     

Altered somatic mutation level and DNA repair gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to ultraviolet C,salt, and cadmium stresses

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.     

SNM-dependent recombinational repair of oxidatively induced DNA damage in Arabidopsis thaliana

Two different roles for SNM (sensitive to nitrogen mustard) proteins have already been described: the SNM1/PSO2-related proteins are involved in the repair of the interstrand crosslink (ICL) and the ARTEMIS proteins are involved in the V(D)J recombination process. Our study shows that an Arabidopsis SNM protein, although structurally closer to the SNM1/PSO2 members, shares some properties with ARTEMIS but also has novel characteristics. Arabidopsis plants defective for the expression of AtSNM1 did not show hypersensitivity to the ICL-forming agents but to the chemotherapeutic agent bleomycin and to H(2)O(2). AtSNM1 mutant plants are delayed in the repair of oxidative damage and did not show enhancement of the frequency of somatic homologous recombination on exposure to H(2)O(2) and to the bacterial elicitor flagellin, both inducing oxidative stress, as observed in the control plants. Therefore, our results suggest the existence, in plants, of a novel SNM-dependent recombinational repair process of oxidatively induced DNA damage.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.     

Transgenic tobacco and Arabidopsis plants expressing the two multifunctional sorghum cytochrome P450 enzymes, CYP79A1 and CYP71E1, are cyanogenic and accumulate metabolites derived from intermediates in Dhurrin biosynthesis

Novel cyanogenic plants have been generated by the simultaneous expression of the two multifunctional sorghum (Sorghum bicolor [L.] Moench) cytochrome P450 enzymes CYP79A1 and CYP71E1 in tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis under the regulation of the constitutive 35S promoter. CYP79A1 and CYP71E1 catalyze the conversion of the parent amino acid tyrosine to p-hydroxymandelonitrile, the aglycone of the cyanogenic glucoside dhurrin. CYP79A1 catalyzes the conversion of tyrosine to p-hydroxyphenylacetaldoxime and CYP71E1, the subsequent conversion to p-hydroxymandelonitrile. p-Hydroxymandelonitrile is labile and dissociates into p-hydroxybenzaldehyde and hydrogen cyanide, the same products released from dhurrin upon cell disruption as a result of pest or herbivore attack. In transgenic plants expressing CYP79A1 as well as CYP71E1, the activity of CYP79A1 is higher than that of CYP71E1, resulting in the accumulation of several p-hydroxyphenylacetaldoxime-derived products in the addition to those derived from p-hydroxymandelonitrile. Transgenic tobacco and Arabidopsis plants expressing only CYP79A1 accumulate the same p-hydroxyphenylacetaldoxime-derived products as transgenic plants expressing both sorghum cytochrome P450 enzymes. In addition, the transgenic CYP79A1 Arabidopsis plants accumulate large amounts of p-hydroxybenzylglucosinolate. In transgenic Arabidopsis expressing CYP71E1, this enzyme and the enzymes of the pre-existing glucosinolate pathway compete for the p-hydroxyphenylacetaldoxime as substrate, resulting in the formation of small amounts of p-hydroxybenzylglucosinolate. Cyanogenic glucosides are phytoanticipins, and the present study demonstrates the feasibility of expressing cyanogenic compounds in new plant species by gene transfer technology to improve pest and disease resistance.     

Overexpression of <Emphasis Type="Italic">ThGSTZ1</Emphasis> from <Emphasis Type="Italic">Tamarix hispida</Emphasis> improves tolerance to exogenous ABA and methyl viologen

Key message

Molecular analysis of a zeta subfamily GST gene from T. hispida involved in ABA and methyl viologen tolerance in transgenic Arabidopsis and Tamarix.

Abstract

Glutathione S-transferase (GST) genes are important for the improvement of plant abiotic stress tolerance, and our previous study demonstrated that the ThGSTZ1 gene from Tamarix hispida improves plant salt and drought tolerance. To further understand the role of ThGSTZ1 in the response of plants to abscisic acid (ABA) and oxidative stress, three ThGSTZ1-overexpressing transgenic Arabidopsis thaliana lines were analyzed in the current study. The results showed that the transgenic lines exhibited higher biomass accumulation, higher activities of GST and other protective enzymes, and less reactive oxygen species (ROS) and cell damage than wild-type (WT) plants under ABA and methyl viologen (MV) stress. In addition, the analysis of a transgenic T. hispida line transiently expressing ThGSTZ1 confirmed these results. The activities of GST, glutathione peroxidase, and superoxide dismutase were markedly higher in the ThGSTZ1-overexpressing lines compared with the control lines under both ABA and MV treatments, and the transgenic lines also exhibited a lower degree of electrolyte leakage (EL) and a decreased H₂O₂ content. All these results suggested that ThGSTZ1 can also improve plant ABA and oxidation tolerance by regulating ROS metabolism and that ThGSTZ1 represents an excellent candidate gene for molecular breeding to increase plant stress tolerance.

     

Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to abiotic and biotic stresses in transgenic Arabidopsis

Plant growth and crop production are limited by environmental stress. We used a large population of transgenic Arabidopsis expressing rice full-length cDNAs to isolate the rice genes that improve the tolerance of plants to environmental stress. By sowing T2 seeds of the transgenic lines under conditions of salinity stress, the salt-tolerant line R07047 was isolated. It expressed a rice gene, OsSMCP1, which encodes a small protein with a single C2 domain, a Ca²⁺-dependent membrane-targeting domain. Retransformation of wild-type Arabidopsis revealed that OsSMCP1 is responsible for conferring the salt tolerance. It is particularly interesting that R07047 and newly constructed OsSMCP1-overexpressing Arabidopsis showed enhanced tolerance not only to high salinity but also to osmotic, dehydrative, and oxidative stresses. Furthermore, R07047 showed improved resistance to Pseudomonas syringae. The OsSMCP1 expression in rice is constitutive. Particle-bombardment-mediated transient expression analysis revealed that OsSMCP1 is targeted to plastids in rice epidermal cells. It induced overexpression of several nuclear encoded genes, including the stress-associated genes, in transgenic Arabidopsis. No marked morphological change or growth retardation was observed in R07047 or retransformants. For molecular breeding to improve the tolerance of crops against environmental stress, OsSMCP1 is a promising candidate.