Overexpression of phytoene synthase gene from Salicornia europaea alters response to reactive oxygen species under salt stress in transgenic Arabidopsis

A phytoene synthase gene SePSY was isolated from euhalophyte Salicornia europaea L. The 1655 bp full-length SePSY has an open reading frame of 1257 bp and encodes a 419-amino acid protein. The overexpression of SePSY enhanced the growth of transgenic Arabidopsis. When the plants were exposed to 100 mM NaCl, the photosynthesis rate and photosystem II activity (Fv/Fm) increased from 92% to 132% and from 9.3% to 16.6% in the transgenic lines than in the wild-type, respectively. The transgenics displayed higher activities of SOD and POD and lower contents of H(2)O(2) and MDA than the WT. In conclusion, the transgenic lines showed higher tolerance to salt stress than WT plants by increased photosynthesis efficiency and antioxidative capacity. This is the first report about improving the salt tolerance by genetic manipulation of carotenoid biosynthesis.     

Chloroplastic NADPH-dependent alkenal/one oxidoreductase contributes to the detoxification of reactive carbonyls produced under oxidative stress

Lipid peroxide-derived reactive carbonyls (RCs) can cause serious damage to plant functions. A chloroplastic NADPH-dependent alkenal/one oxidoreductase (AOR) detoxifies RCs, but its physiological significance remains unknown. In this study, we investigated the biological impacts of AOR using an AOR-knock out Arabidopsis line (aor). Methyl viologen treatment, mainly to enhance photosystem (PS) I-originated reactive oxygen species (ROS) production, caused more severe damage to aor than wild type (Col-0). In contrast, the high light treatment used to enhance PSII-originated ROS production resulted in no difference in PSII damage between Col-0 and aor. In conclusion, AOR can contribute to detoxify stromal RCs produced under oxidative stress.     

Endogenous glutamate contributes to the maintenance of glutathione level under oxidative stress in isolated nerve terminals

Exposure of isolated nerve terminals to hydrogen peroxide (25-500 microM) for 10 min produced a partially reversible decrease in the total and reduced glutathione level. No release and resynthesis of glutathione by the oxidant was involved in this effect. Loss of reduced glutathione was associated with elimination of H(2)O(2), which was very quick with >70% of the oxidant eliminated within 5 min. Recovery of both total and reduced glutathione was pronounced after 10 min when the majority of H(2)O(2) was eliminated. Previously we have reported that glutamate metabolism under oxidative stress contributes to the operation of the Krebs cycle, thus to the production of NAD(P)H [J. Neurosci. 20 (2000) 8972]. In the present study we addressed whether metabolism of endogenous glutamate plays a role in the maintenance of glutathione level in nerve terminals. Glutamine and beta-hydroxybutyrate (5mM), alternative metabolites in synaptosomes, were able to decrease the loss of total and reduced glutathione induced by hydrogen peroxide. Metabolic consumption of glutamate was reduced at the same time. In addition an increased demand on the glutathione system by the catalase inhibitor aminotriazole augmented the metabolic consumption of glutamate. It is concluded that under oxidative stress glutamate metabolism contributes to the maintenance of glutathione level, thus to the antioxidant capacity of nerve terminals.     

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.     

Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco

Aluminum (Al) inhibits plant growth partly by causing oxidative damage that is promoted by reactive oxygen species and can be prevented by improving antioxidant capacity. Ascorbic acid (AsA), the most abundant antioxidant in plants, is regenerated by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We investigated the role of MDAR and DHAR in AsA regeneration during Al stress using transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis cytosolic MDAR (MDAR-OX) or DHAR (DHAR-OX). DHAR-OX plants showed better root growth than wild-type (SR-1) plants after exposure to Al for 2 weeks, but MDAR-OX plants did not. There was no difference in Al distribution and accumulation in the root tips among SR-1, DHAR-OX, and MDAR-OX plants after Al treatment for 24 h. However, DHAR-OX plants showed lower hydrogen peroxide content, less lipid peroxidation and lower level of oxidative DNA damage than SR-1 plants, whereas MDAR-OX plants showed the same extent of damage as SR-1 plants. Compared with SR-1 plants, DHAR-OX plants consistently maintained a higher AsA level both with and without Al exposure, while MDAR-OX plants maintained a higher AsA level only without Al exposure. Also, DHAR-OX plants maintained higher APX activity under Al stress. The higher AsA level and APX activity in DHAR-OX plants contributed to their higher antioxidant capacity and higher tolerance to Al stress. These findings show that the overexpression of DHAR, but not of MDAR, confers Al tolerance, and that maintenance of a high AsA level is important to Al tolerance.     

Responses of transgenic poplar (Populus tremula x P. alba) overexpressing glutathione synthetase or glutathione reductase to acute ozone stress: visible injury and leaf gas exchange

Untransformed hybrid poplar (Populus tremula x P. alba) and transgenic lines overexpressing glutathione synthetase (GshS) in the cytosol (200-300-fold) or glutathione reductase (GR) either in the cytosol 5-fold) or in the chloroplast (150-200-fold) were exposed to 0 (control), 100, 200 or 300 nl l^-1 ozone for 3 d for 7 h d^-1. Following acute ozone stress treatments, wild-type and transgenic poplar suffered from visible foliar injury consisting of dark brown necrotic lesions on the laminae. Necrotic lesions were sharply separated from photosynthetically active cells by a band of red-violet discoloured cell lines showing yellow autofluorescence by blue light, and blue autofluorescence by UV-light excitation. When plants were exposed to 100 nl l^-1 ozone, leaf injury was in general negligible, but when 200 and 300 nl l^-1 ozone was applied, in both untransformed poplar and transgenic lines overexpressing GshS or GR up to 60% and 80%, respectively, visible injury developed on mature leaves. The mean percentage of injured leaf area amounted to 20-30% (200 nl l^-1) and 40-60% (300 nl l^-1). Irrespective of transformation, young leaves of poplar trees were only slightly affected by ozone treatments. In support of these observations, net CO2 assimilation rates of mature leaves were decreased by up to 65% (300 nl l^-1 ozone) in wild-type and transformed poplar, whereas net photosynthesis of young leaves remained unaffected even under severe stress conditions. Leaf conductance was significantly decreased by all ozone treatments, but was in the same range in young and mature leaves, and in wild-type and transformed poplar, pre- and post-exposure to ozone. It can therefore be assumed that the ozone doses effectively taken up into the leaf tissue were not dependent on leaf development and that the strength of the ozone stress exerted was similar in all types of poplar trees investigated in this study.From these data it is concluded that: (i) elevated foliar activities of glutathione synthetase or glutathione reductase alone are not sufficient to improve tolerance of hybrid poplar to acute ozone stress, and (ii) the sensitivity of poplar leaves to acute ozone stress is controlled by unknown factors closely related to leaf development rather than by foliar activities of glutathione synthetase and glutathione reductase, or leaf conductance.     

Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress

Chloroplast glutathione reductase (GR) plays an important role in protecting photosynthesis against oxidative stress. We used transgenic tobacco (Nicotiana tabacum) plants with severely decreased GR activities by using a gene encoding tobacco chloroplast GR for the RNAi construct to investigate the possible mechanisms of chloroplast GR in protecting photosynthesis against chilling stress. Transgenic plants were highly sensitive to chilling stress and accumulated high levels of H?O? in chloroplasts. Spectroscopic analysis and electron transport measurements show that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrate that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses illustrate that PSII protein accumulation was decreased greatly in transgenic plants. Our results suggest that chloroplast GR plays an important role in protecting PSII function by maintaining the electron transport in PSII acceptor side and stabilizing PSII complexes under chilling stress. Our results also suggest that the recycling of ascorbate from dehydroascorbate in the ascorbate-glutathione cycle in the chloroplast plays an essential role in protecting PSII against chilling stress.     

Enhancement of phenol stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants overexpressing glutathione S-transferase

Plant glutathione S-transferases (GSTs) are important for protecting plants against oxidative damage. We studied the function of a glutathione S-transferase family protein in Arabidopsis, AtGSTF2. Our results indicate the transgenic plants showed increased tolerance to oxidative stress caused by application of phenol. Under phenol stress, the lipid hydroperoxidation [the production of malondialdehyde (MDA)] of the leaves in overexpressing lines was suppressed compared with that of control plants. The antioxidative enzyme activities (SOD and POD) were higher in transgenic plants than in control. Furthermore, the residual phenol in medium was decreased more in transgenic plants than in control plants. These results indicate overexpressing GST protein reduce the damage of lipid hydroperoxidation and oxidative damage caused by phenol. Our findings also provide a suitable remediation strategy for sites contaminated by phenol.     

CAX1 suppresses Cd‐induced generation of reactive oxygen species in Arabidopsis halleri

The molecular analysis of metal hyperaccumulation in species such as Arabidopsis halleri offers the chance to gain insights into metal homeostasis and into the evolution of adaptation to extreme habitats. A prerequisite of metal hyperaccumulation is metal hypertolerance. Genetic analysis of a backcross population derived from Arabidopsis lyrata × A. halleri crosses revealed three quantitative trait loci for Cd hypertolerance. A candidate gene for Cdtol2 is AhCAX1, encoding a vacuolar Ca²⁺/H⁺ antiporter. We developed a method for the transformation of vegetatively propagated A. halleri plants and generated AhCAX1‐silenced lines. Upon Cd²⁺ exposure, several‐fold higher accumulation of reactive oxygen species (ROS) was detectable in roots of AhCAX1‐silenced plants. In accordance with the dependence of Cdtol2 on external Ca²⁺ concentration, this phenotype was exclusively observed in low Ca²⁺ conditions. The effects of external Ca²⁺ on Cd accumulation cannot explain the phenotype as they were not influenced by the genotype. Our data strongly support the hypothesis that higher expression of CAX1 in A. halleri relative to other Arabidopsis species represents a Cd hypertolerance factor. We propose a function of AhCAX1 in preventing a positive feedback loop of Cd‐elicited ROS production triggering further Ca²⁺‐dependent ROS accumulation.     

NMDA receptor dysfunction contributes to impaired brain‐derived neurotrophic factor‐induced facilitation of hippocampal synaptic transmission in a Tau transgenic model

While the spatiotemporal development of Tau pathology has been correlated with occurrence of cognitive deficits in Alzheimer's patients, mechanisms underlying these deficits remain unclear. Both brain‐derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB play a critical role in hippocampus‐dependent synaptic plasticity and memory. When applied on hippocampal slices, BDNF is able to enhance AMPA receptor‐dependent hippocampal basal synaptic transmission through a mechanism involving TrkB and N‐methyl‐d‐Aspartate receptors (NMDAR). Using THY‐Tau22 transgenic mice, we demonstrated that hippocampal Tau pathology is associated with loss of synaptic enhancement normally induced by exogenous BDNF. This defective response was concomitant to significant memory impairments. We show here that loss of BDNF response was due to impaired NMDAR function. Indeed, we observed a significant reduction of NMDA‐induced field excitatory postsynaptic potential depression in the hippocampus of Tau mice together with a reduced phosphorylation of NR2B at the Y1472, known to be critical for NMDAR function. Interestingly, we found that both NR2B and Src, one of the NR2B main kinases, interact with Tau and are mislocalized to the insoluble protein fraction rich in pathological Tau species. Defective response to BDNF was thus likely related to abnormal interaction of Src and NR2B with Tau in THY‐Tau22 animals. These are the first data demonstrating a relationship between Tau pathology and synaptic effects of BDNF and supporting a contribution of defective BDNF response and impaired NMDAR function to the cognitive deficits associated with Tauopathies.     

Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod‐influenced responses to ozone using Arabidopsis knockout mutants

Oxidative stress caused by ozone (O₃) affects plant development, but the roles of specific redox‐homeostatic enzymes in O₃ responses are still unclear. While growth day length may affect oxidative stress outcomes, the potential influence of day length context on equal‐time exposures to O₃ is not known. In Arabidopsis Col‐0, day length affected the outcome of O₃ exposure. In short‐days (SD), few lesions were elicited by treatments that caused extensive lesions in long days (LD). Lesion formation was not associated with significant perturbation of glutathione, ascorbate, NADP(H) or NAD(H). To investigate roles of two genes potentially underpinning this redox stability, O₃ responses of mutants for cytosolic NADP‐isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) were analysed. Loss of ICDH function did not affect O₃‐induced lesions, but slightly increased glutathione oxidation, induction of other cytosolic NADPH‐producing enzymes and pathogenesis‐related gene 1 (PR1). In gr1, O₃‐triggered lesions, salicylic acid accumulation, and induction of PR1 were all decreased relative to Col‐0 despite enhanced accumulation of glutathione. Thus, even at identical irradiance and equal‐time exposures, day length strongly influences phenotypes triggered by oxidants of atmospheric origin, while in addition to its antioxidant function, the GR‐glutathione system seems to play novel signalling roles during O₃ exposure.     

Acyl‐CoA‐binding protein 2 binds lysophospholipase 2 and lysoPC to promote tolerance to cadmium‐induced oxidative stress in transgenic Arabidopsis

Lysophospholipids are intermediates of phospholipid metabolism resulting from stress and lysophospholipases detoxify lysophosphatidylcholine (lysoPC). Many lysophospholipases have been characterized in mammals and bacteria, but few have been reported from plants. Arabidopsis thaliana lysophospholipase 2 (lysoPL2) (At1g52760) was identified as a protein interactor of acyl‐CoA‐binding protein 2 (ACBP2) in yeast two‐hybrid analysis and co‐immunoprecipitation assays. BLASTP analysis indicated that lysoPL2 showed ～35% amino acid identity to the lysoPL1 family. Co‐localization of autofluorescence‐tagged lysoPL2 and ACBP2 by confocal microscopy in agroinfiltrated tobacco suggests the plasma membrane as a site for their subcellular interaction. LysoPL2 mRNA was induced by zinc (Zn) and hydrogen peroxide (H₂O₂), and lysoPL2 knockout mutants showed enhanced sensitivity to Zn and H₂O₂ in comparison to wild type. LysoPL2‐overexpressing Arabidopsis was more tolerant to H₂O₂ and cadmium (Cd) than wild type, suggesting involvement of lysoPL2 in phospholipid repair following lipid peroxidation arising from metal‐induced stress. Lipid hydroperoxide (LOOH) contents in ACBP2‐overexpressors and lysoPL2‐overexpressors after Cd‐treatment were lower than wild type, indicating that ACBP2 and lysoPL2 confer protection during oxidative stress. A role for lysoPL2 in lysoPC detoxification was demonstrated when recombinant lysoPL2 was observed to degrade lysoPC in vitro. Filter‐binding assays and Lipidex competition assays showed that (His)₆‐ACBP2 binds lysoPC in vitro. Binding was disrupted in a (His)₆‐ACBP2 derivative lacking the acyl‐CoA‐binding domain, confirming that this domain confers lysoPC binding. These results suggest that ACBP2 can bind both lysoPC and lysoPL2 to promote the degradation of lysoPC in response to Cd‐induced oxidative stress.     

Overexpression of wheat alternative oxidase gene Waox1a alters respiration capacity and response to reactive oxygen species under low temperature in transgenic Arabidopsis

Under low temperature conditions, the cytochrome pathway of respiration is repressed and reactive oxygen species (ROS) are produced in plants. Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for the cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. To study functions of wheat AOX genes under low temperature, we produced transgenic Arabidopsis by introducing Waox1a expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis thaliana. The enhancement of endogenous AOX1a expression via low temperature stress was delayed in the transgenic Arabidopsis. Recovery of the total respiration activity under low temperature occurred more rapidly in the transgenic plants than in the wild-type plants due to a constitutively increased alternative pathway capacity. Levels of ROS decreased in the transgenic plants under low temperature stress. These results support the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.     

Overexpression of bacterioferritin comigratory protein (Bcp) enhance viability and reduced glutathione level in the fission yeast under stress

The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCPlO. The bcp ⁺ mRNA level in the pBCPlO-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCPIO exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.     

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1‐mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA‐dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA‐dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA‐responsive genes, such as PR1 and PR2. Furthermore, other SA‐accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.     

Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.     

拟南芥CEO1基因在氯化镉诱导的氧化胁迫中的作用

以拟南芥ceo1、突变体为材料,研究CEO1（clone eight-one）在镉胁迫条件下作用的结果表明,与野生型植株相比,150μmol·L^-1的CdCl2处理10d后,拟南芥ceo1突变体表现为植株生长矮小,叶片卷曲发黄,根系短小。镉处理后,拟南芥突变体幼苗叶中H2O2的积累较多;镉处理1h后的突变体中抗坏血酸过氧化物酶（APX）活性明显上升,至2h时又开始下降,而镉处理2h后,野生型APX活性才开始增加。镉处理2h后的野生型的谷胱甘肽还原酶（GR）显著增加,而突变体无明显变化。两种类型拟南芥的超氧化物歧化酶（SOD）与过氧化氢酶（CAT）的活性没有明显差异。