Effects of sucrose supply on growth and paraquat tolerance of the late-flowering gi-3 mutant

Mutations at the GI locus in Arabidopsis are pleiotropic: gi mutants are late-flowering, tolerant to the toxicity of the herbicide paraquat, and have an increased starch content. We tested the effects of exogenous sucrose supply on the level of paraquat tolerance and on growth and development of the gi-3 mutant. Paraquat tolerance was the highest in gi-3 seedlings grown on medium containing 1% sucrose. As expected, all measured growth parameters (root length, fresh weight, anthocyanin, and sucrose content) were influenced by the sucrose dose, but in a number of assays (effect on fresh weight and developmental characteristics) the sucrose-dependent response in gi-3 was heterochronic. Additionally, the late-flowering phenotype of the gi-3 mutant was reverted to wild type after prolonged growth in darkness on sucrose-containing media.     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Mechanism of paraquat tolerance in perennial ryegrass

Abstract The mechanism of paraquat tolerance was investigated in lines of perennial ryegrass (Lolium perenne L.) which had been selected for resistance to the herbicide. Uptake, metabolism and translocation of paraquat were studied. Susceptible cultivars and a tolerant line were not found to differ in uptake of radioactive paraquat applied to the leaf surface or supplied to the cut ends of excised leaves. Distribution of herbicide within leaf tissue was similar in tolerant and susceptible plants and no metabolites of ¹⁴C-paraquat were detected in tolerant or susceptible plants treated with sub-lethal concentrations of the herbicide. Autoradiography and quantitative determinations showed much variation in translocation of ¹⁴C-paraquat out of treated leaves of intact plants, but the variation was not related to the degree of susceptibility to the herbicide. It is concluded that paraquat tolerance in perennial ryegrass is unlikely to depend upon reduced uptake, enhanced metabolism or altered translocation of the herbicide.     

The Effect of Paraquat and Hypothermia on Norflurason-Resistant Mutants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and Derived Cell Cultures

A tolerance to paraquat (PQ) of plants and cell cultures of Arabidopsis thaliana mutants, nfz18 and nfz24, obtained by chemical mutagenesis and selected by their tolerance to norflurason was demonstrated. This tolerance to PQ was manifested in less active peroxidation of lipids (POL), which was assessed by the content of thiobarbituric acid-reactive substances, and in a less degree of plant and callus damages, which was accompanied by a higher activity of superoxide dismutase and other antioxidant enzymes. A capability of norflurason-tolerant mutants to cross-adaptation toward PQ and activation of antioxidant enzymes indicate a genetically determined activation of the antioxidant systems, resulting in improved mutant tolerance to these inducers of oxidative stress. The nfz24 mutant was much more sensitive to hypothermia than wild-type plants and nfz18 mutants, which was expressed in a higher level of POL in plants and calluses and in a more rapid decrease in the suspension cell viability of this mutant. A similarity in the responses of plants and derived heterotrophic cultures to PQ and hypothermia indicates that, in these A. thaliana mutants, adaptation to these types of stresses occurs mainly at the cellular level. Possible reasons of increased sensitivity to hypothermia of the nfz24 mutant, which was more tolerant to the inducers of oxidative stress, PQ and norflurason, are discussed.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 421–429.Original Russian Text Copyright © 2005 by Volkova, Burgutin, Soldatova, Ezhova, Lapshin.     

Ameliorative effects of squash (<Emphasis Type="Italic">Cucurbita moschata</Emphasis> Duchesne ex Poiret) leaf extracts on oxidative stress

This study screened paraquat-tolerant plants among 10 plant species, including monocots and dicots angiosperms. Squash (Cucurbita moschata Duchesne ex Poiret) and kidney bean (Phaseolus vulgaris L.) plants exhibited the highest photooxidation-tolerant phenotypes upon a foliar treatment with paraquat. A foliar treatment with paraquat pre-mixed with leaf water extracts from the squash plant significantly alleviated paraquat-induced oxidative damage in maize, but this was not the case after a treatment with the hydrophobic phase of the leaf extracts. In particular, the water extract from young leaves (4th true leaf) of squash plants conferred tenfold higher tolerance to oxidative damage in paraquat-treated leave tissues compared to paraquat-only treatment. This tolerance was tightly linked not only to the increased amounts of ascorbic acid and dehydroascorbate antioxidants in the damaged leaves, but also to the reduced chlorophyll loss, lipid peroxidation, and cellular electrolyte leakage. Moreover, the protective effects of the water extract were apparent when using another bipyridyl herbicide, diquat, but not with a diphenyl-ether herbicide, oxyfluorfen. On the other hand, pre-treatment with the extract prior to the onset of drought or cold stress had no significant antioxidative effect on the treated tissues.     

Flavodoxin overexpression confers tolerance to oxidative stress in beneficial soil bacteria and improves survival in the presence of the herbicides paraquat and atrazine

Aim

To determine whether expression of a cyanobacterial flavodoxin in soil bacteria of agronomic interest confers protection against the widely used herbicides paraquat and atrazine.

Methods and Results

The model bacterium Escherichia coli, the symbiotic nitrogen‐fixing bacterium Ensifer meliloti and the plant growth‐promoting rhizobacterium Pseudomonas fluorescens Aur6 were transformed with expression vectors containing the flavodoxin gene of Anabaena variabilis. Expression of the cyanobacterial protein was confirmed by Western blot. Bacterial tolerance to oxidative stress was tested in solid medium supplemented with hydrogen peroxide, paraquat or atrazine. In all three bacterial strains, flavodoxin expression enhanced tolerance to the oxidative stress provoked by hydrogen peroxide and by the reactive oxygen species‐inducing herbicides, witnessed by the enhanced survival of the transformed bacteria in the presence of these oxidizing agents.

Conclusions

Flavodoxin overexpression in beneficial soil bacteria confers tolerance to oxidative stress and improves their survival in the presence of the herbicides paraquat and atrazine. Flavodoxin could be considered as a general antioxidant resource to face oxidative challenges in different micro‐organisms.

Significance and Impact of the study

The use of plant growth‐promoting rhizobacteria or nitrogen‐fixing bacteria with enhanced tolerance to oxidative stress in contaminated soils is of significant agronomic interest. The enhanced tolerance of flavodoxin‐expressing bacteria to atrazine and paraquat points to potential applications in herbicide‐treated soils.     

Morphological changes and increase of resistance to oxidative stress by overexpression of the <Emphasis Type="Italic">LebZIP2</Emphasis> gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

The tomato bZIP2-encoding gene was inserted into the Nicotiana benthamiana genome using Agrobacterium-mediated transformation to characterize resistance to oxidative stress and two herbicides, glyphosate and paraquat. We produced transgenic tobacco plants using the LebZIP2 gene, which were then utilized to examine salt stress and herbicide resistance through oxidative mechanisms. Transgenic LebZIP2-overexpressing plants were examined using specific primers for selection marker genes (PCR using genomic DNA) and target genes (RT-PCR). Based on microscopic examination, we observed an increase in leaf thickness and cell number in transgenic plants. The electrolyte leakage of leaves suggested that LebZIP2-overexpressing lines were weak tolerant to NaCl stress and resistant to methyl viologen. During our analysis, transgenic lines were exposed to different herbicides. Transgenic plants showed an increased tolerance based on visual injury, as well as an increased biomass. Based on these results, the LebZIP2 gene may be involved in oxidative stress tolerance and cell development in plants.     

Great promoting effect of high irradiance from germination on flowering in Arabidopsis thaliana — a process of photo-acclimation

Arabidopsis thaliana L., ch1-1 (chlorophyll b-less mutant), gi-1 (GI deficient mutant), cry2-1 (blue-light-photoreceptor CRY2 deficient mutant), and Columbia (Col; wild ecotype) were grown under broad range of irradiances (I) from the beginning of germination and the effect of I on the survival, development, and flowering was studied. Under low and moderate I (<300 μmol m⁻² s⁻¹), flowering time and plant size at flowering showed great variations among ch1-1, gi-1, cry2-1, and Col, whereas under higher I (>500 μmol m⁻² s⁻¹), these characteristics were almost the same. Hence under high I, development and flowering of ch1-1, gi-1, cry2-1, and Col converged to almost the same state. Flowering time was negatively correlated with I, and under high I acclimation in A. thaliana was associated with a decrease in chlorophyll (Chl) content and increases in xanthophyll cycle pool and membrane-bound APX activity (EC 1.11.1.11) suggesting that an increase in oxidative stress induces earlier flowering. The plants of gi-1 and cry2-1 survived but Col and ch1-1 died under 1 000 μmol m⁻² s⁻¹, showing that mutants deficient in GI or CRY2 are more photo-stress-tolerant than Col and the Chl b-less mutant. Hence high I promotes in plants of Arabidopsis raised from germination till flowering the development and flowering time involving modulation of the photosynthetic apparatus, and this promoting effect is independent of the functions of flower-inducing GI or CRY2 gene. This can be regarded as photo-acclimation of A. thaliana for survival and reproduction under high I.     

A role for SPINDLY gene in the regulation of oxidative stress response in Arabidopsis

SPINDLY (SPY) gene encodes a putative O-linked N-acetyl-glucosamine transferase, and yeast two-hybrid assay identified GIGANTEA (GI) as a SPY-interacting partner in Arabidopsis. GIGANTEA gene was previously shown to be involved in the regulation of oxidative stress response; however, it is unclear whether SPY gene is also involved in oxidative stress response. Here we showed that SPY plays a role in the regulation of the oxidative stress response. The spy-1 mutant was more tolerant to paraquat (PQ)-or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of the spy-1 mutant to PQ-stress was not due to defects in the PQ uptake or the PQ sequestration from its site of action but rather the spy-1 mutation alleviated oxidative damage of plant cells upon PQ stress. Higher constitutive activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in spy-1 are more likely to be due to activation of both CSD2 gene encoding chloroplast Cu/Zn SOD and APX1 gene. Taken together, these results suggest that enhanced tolerance of the spy-1 mutant to oxidative stress is associated, at least in part, with constitutive activation of CSD2 and APX1. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 604–611. The text was submitted by the authors in English.     

Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii

Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake.     

Effect of 2-aminoethanol pretreatment on the antioxidant enzyme activity in <Emphasis Type="Italic">Zea mays</Emphasis> under oxidative stress

Exposure of plants to stress may result in liberation of 2-aminoethanol as an inducer of alarm reaction that activates cellular resistance and tolerance mechanisms. In the present study, the effect of exogenous 2-aminoethanol on stimulation of tolerance mechanisms was investigated in maize (Zea mays L.) plants treated with the herbicide paraquat. Maize shoots were pretreated with 2-aminoethanol. Two days later the shoots were treated with paraquat. Pretreatment with 2-aminoethanol increased catalase and guaiacol peroxidase activities and cysteine content and decreased MDA and hydrogen peroxide content in maize plants treated with paraquat. Based on the results of this study, it can be suggested that strengthening of the antioxidant mechanisms by plant pretreatment with amino alcohol could be helpful in the development of a broad tolerance to adverse stress conditions.     

Exogenous 2-aminoethanol can diminish paraquat induced oxidative stress in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Effects of unfavourable environmental conditions (stresses) induce stressor specific and unspecific short- and long-term responses in plants. Long-term responses depend on intensity and duration of the stress. Short-term effects comprise the accumulation of reactive oxygen species (ROS), membrane damages by the oxidation of fatty acids, and the release of amino alcohols. They can incite higher stress tolerance in plants. In the present study, shoots of barley (Hordeum vulgare) were pre-treated with 2-aminoethanol, and, 2 days later, with the oxidative stress inducing herbicide, paraquat. Pre-treatments with 2-aminoethanol increased the stress tolerance in barley by the stabilization of the cell membranes, the enhanced production of superoxide dismutase and catalase, and the stimulation of glutathione metabolism (GSH, GST). These mechanisms of stress tolerance activation by 2-aminoethanol are discussed.     

Integration of the cyanobacterial <Emphasis Type="Italic">DesA</Emphasis> gene for Δ12-acyl-lipid desaturase improves potato tolerance to paraquat-induced oxidative stress

The effect of potato plant (Solanum tuberosum L., cv. Desnitsa) transformation with the desA gene from Synechocystis sp. PCC 6803, encoding Δ12 acyl-lipid desaturase, on the development of plant tolerance to oxidative stress was studied. To initiate oxidative stress, plants were treated with 1 mM paraquat; this treatment enhanced oxidative processes in both wild-type and transformed potato plants via the activation of superoxide anion-radical generation. This resulted in the activated oxidation of membrane lipids and the formation of a great amount of fatty acids with coupled double bonds (conjugated dienes, CD), further breakdown of lipid molecules, and enhanced production of MDA in tissues of wild-type and transformed plants. The characteristics of oxidative stress, including lipid peroxidation, were less pronounced in transformants as compared with wild-type plants. After treatment with paraquat, activities of main antioxidant enzymes (superoxide dismutase, catalase, and peroxidase) were much higher in wild-type than in transformed plants. Thus, expression of inserted heterologous desA gene for Δ12 acyl-lipid desaturase in potato plants resulted in improved tolerance of transformants to oxidative stress due to the more efficient maintenance of stable cell membrane structure functioning, and this permits prevention of electron “jump” to oxygen and, as a result, of accelerated ROS generation. More developed and regularly arranged chloroplast membrane system in transformants may also favor their improved tolerance.     

Differential response to paraquat induced oxidative stress in two rice cultivars on antioxidants and chlorophyll a fluorescence

The responses of antioxidative system and photosystem II photochemistry of rice (Oryza sativa L.) to paraquat induced oxidative stress were investigated in a chilling-tolerant cultivar Xiangnuo no. 1, and a chilling-susceptible cultivar, IR-50. Electrolyte leakage and malondialdehyde (MDA) content of Xiangnuo no. 1 were little affected by paraquat, but they increased in IR-50. After paraquat treatment, superoxide dismutase (SOD) activity remained high in Xiangnuo no. 1, while it declined in IR-50. Activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) declined with oxidative stress in both cultivars, but Xiangnuo no. 1 had higher GR activity than IR-50. Under paraquat induced oxidative stress, ascorbic acid (AsA) and reduced glutathione (GSH) concentrations remained high in Xiangnuo no. 1, but decreased in IR-50. The results indicated that higher activities of SOD and GR and higher contents of AsA and GSH in Xiangnuo no. 1 under paraquat induced oxidative stress were associated with its tolerance to paraquat, while paraquat induced damage to IR-50 was related to decreased activities of SOD, APX and GR and contents of AsA and GSH. F _v/F _m, Φ _PSII, and qP remained high in Xiangnuo no. 1, while they decreased greatly in IR-50 under paraquat induced oxidative stress.     

利用氧化剂甲基紫精筛选拟南芥寿限延长突变体

在模式动物中的研究表明,寿限延长与氧化胁迫耐受能力密切有关;在模式植物拟南芥中也发现晚花突变体具有更强的抗氧化胁迫能力,但迄今为止未见有关拟南芥寿限延长突变体的研究报道。本文建立了用氧化剂甲基紫精筛选寿限延长突变体的方法,并对用该方法从经快中子诱变的拟南芥Columbia生态型M2代群体中筛选获得的突变体SFNA-9—4进行分析,发现该突变体的抗氧化胁迫能力和寿限均显著增加。由此说明,用该方法筛选拟南芥寿限延长突变体是可行的。