A study of the activity of enzymes of the antioxidant system in ontogenesis ofArabidopsis thaliana mutants tolerant to norflurazone

The activities of superoxide dismutase and guaiacol-dependent peroxidase were studied in the ontogenesis of recessive homozygous mutants ofArabidopsis thaliana Heynh.le-2 andnfz24, which are characterized by two- to threefold increases in tolerance to the herbicide norflurazone. The mutantsle-2 andnfz24 differed from the initial race Dijon in some phenotypic features, duration of ontogenetic stages, and dynamics of the superoxide dismutase and peroxidase activities in ontogenesis. A single treatment of plants with norflurazone induced an accelerated increase in the level of both enzymes in the mutants as compared to the wild type plants. Under the conditions of multiple treatment with norflurazone, the mutantsle-2 andnfz24 displayed a higher tolerance to the bleaching effect of the herbicide and were characterized by a higher level of superoxide dismutase. The data obtained suggest that the superoxide dismutase and peroxidase activities are controlled by both ontogenetic factors and stress signals. Mutations in the linesle-2 andnfz24 increase sensitivity to a stress signal or increase efficiency of an adaptive response due to long-term maintenance of a high level of the antioxidant enzymes under the conditions of stress.     

Effects of salicylic acid on paraquat tolerance in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Foliar spraying ofArabidopsis thaliana (Columbia ecotype) plants with a 1.0-mM salicylic acid (SA) solution significantly improved their tolerance to subsequent paraquat (PQ)-induced oxidative damage. Leaf injuries, including losses of chlorophyll, protein, and fresh weight, were reduced. Our analysis of antioxidant enzymes in the leaves showed that SA pre-treatment effectively retarded rapid decreases in the activities of Superoxide dismutase (SOD), catalase, and ascorbate peroxidase that are normally associated with PQ exposure. In addition, guaiacol peroxidase activity was remarkably increased. In a native gel assay of peroxidase (POD) isozymes, staining activity of the POD1 isozyme, which disappeared in plants exposed only to 10 μM PQ, was significantly recovered by the 1.0-mM SA pre-treatment POD2 isozyme activity was also pronounced in all SA-treated plants compared with the control. A 12-h SA pre-treatment, without subsequent PQ stress, also caused a small increase in the endogenous H₂O₂ content that accompanies the symptoms of mild leaf injuries. This enhanced level occurred in parallel with a slight SOD increase and a catalase decrease. From our results, it can be assumed that, due to the small increase in SOD as well as catalase inactivation via SA pre-treatment, a moderate increase in H₂O₂ levels may occur. In turn, a large induction of guaiacol peroxidase leads to enhanced PQ tolerance inA. thaliana plants.     

The enzymatic activity of the antioxidant system in the ontogeny of mutant norflurazone-tolerant Arabidopsis thaliana

The activities of superoxide dismutase and guaiacol-dependent peroxidase were studied in the ontogenesis of recessive homozygous mutants of Arabidopsis thaliana Heynh. le-2 and nfz24, which are characterized by two- to threefold increases in tolerance to the herbicide norflurazone. The mutants le-2 and nfz24 differed from the initial race Dijon in some phenotypic features, duration of ontogenetic stages, and dynamics of the superoxide dismutase and peroxidase activities in ontogenesis. A single treatment of plants with norflurazone induced an accelerated increase in the level of both enzymes in the mutants as compared to the wild type plants. Under the conditions of multiple treatment with norflurazone, the mutants le-2 and nfz24 displayed a higher tolerance to the bleaching effect of the herbicide and were characterized by a higher level of superoxide dismutase. The data obtained suggest that the superoxide dismutase and peroxidase activities are controlled by both ontogenetic factors and stress signals. Mutations in the lines le-2 and nfz24 increase sensitivity to a stress signal or increase efficiency of an adaptive response due to long-term maintenance of a high level of the antioxidant enzymes under the conditions of stress.     

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.     

Oxidative stress tolerance and longevity in Arabidopsis: the late‐flowering mutant gigantea is tolerant to paraquat

Recent genetic analyses of longevity in animals have revealed that long-lived strains are more tolerant to environmental stresses. To investigate whether extended longevity in Arabidopsis also correlates with an increase in stress tolerance, the response was tested of 11 late-flowering mutants to the superoxide radical-generating herbicide paraquat. A tight correlation between flowering time and paraquat tolerance was found when plants were exposed to low doses of herbicide. Furthermore, the mutant gigantea (gi-3) with the longest delay in flowering time had a high tolerance level to paraquat-induced oxidative stress. All the tested gi alleles had an increased tolerance to paraquat toxicity compared to wild-type, although the actual levels of tolerance differed. In addition, the gi-3 mutant was more tolerant to hydrogen peroxide. These results suggest that the link between longevity and oxidative stress resistance in plants is similar to that found in animals, implying that this phenomenon may be general for all aerobic organisms.     

UV-B预处理诱导拟南芥耐旱性的提高

为了解UV-B提高拟南芥(Arabidopsis thaliana)耐旱性的生理机制,将2周龄的野生型拟南芥(WT)和sto突变体幼苗用不同剂量UV-B预处理1周,再用30%PEG模拟干旱处理24 h,对植株的表型进行统计,并测定类黄酮、脯氨酸和MDA含量。结果表明,低剂量UV-B预处理能够提高拟南芥的耐旱性,植株的类黄酮与脯氨酸含量分别提高了20%～40%和50%～65%,细胞膜受损程度降低,从而提高了保水性。低剂量UV-B提高拟南芥耐旱性的效应在sto突变体中消失,证明这种效应在分子机制上可能与STO蛋白相关。     

Protective Effect of Nitric Oxide against Oxidative Damage in <Emphasis Type="Italic">Arabidopsis</Emphasis> Leaves under Ultraviolet-B Irradiation

Nitric oxide (NO) is a key molecule involved in many physiological processes. To characterize its roles in the tolerance of Arabidopsis thaliana to ultraviolet-B (UV-B), we investigated the effect of a reduced endogenous NO level on oxidative damage to wild-type and mutant (Atnoa1) plants. Under irradiation, hydrogen peroxide was accumulated more in mutant leaves than in the wild type. However, the amounts of UV-B-absorbing compounds (flavonoids and anthocyanin) and the activities of two antioxidant enzymes—catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11)—were lower in leaves of the former. Supplementing with sodium nitroprusside, an NO donor, could alleviate the oxidative damage to mutant leaves by increasing flavonoid and anthocyanin contents and enzyme activities. In comparison, , an inhibitor of nitric oxide synthase, had the opposite effects on oxidation resistance in wild-type leaves. All these results suggest that nitric oxide acts as a signal for an active oxygen-scavenging system that protects plants from oxidative stress induced by UV-B irradiation.     

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.     

A viral RNA silencing suppressor interferes with abscisic acid‐mediated signalling and induces drought tolerance in Arabidopsis thaliana

Cucumber mosaic virus (CMV) encodes the 2b protein, which plays a role in local and systemic virus movement, symptom induction and suppression of RNA silencing. It also disrupts signalling regulated by salicylic acid and jasmonic acid. CMV induced an increase in tolerance to drought in Arabidopsis thaliana. This was caused by the 2b protein, as transgenic plants expressing this viral factor showed increased drought tolerance, but plants infected with CMVΔ2b, a viral mutant lacking the 2b gene, did not. The silencing effector ARGONAUTE1 (AGO1) controls a microRNA‐mediated drought tolerance mechanism and, in this study, we noted that plants (dcl2/3/4 triple mutants) lacking functional short‐interfering RNA‐mediated silencing were also drought tolerant. However, drought tolerance engendered by CMV may be independent of the silencing suppressor activity of the 2b protein. Although CMV infection did not alter the accumulation of the drought response hormone abscisic acid (ABA), 2b‐transgenic and ago1‐mutant seeds were hypersensitive to ABA‐mediated inhibition of germination. However, the induction of ABA‐regulated genes in 2b‐transgenic and CMV‐infected plants was inhibited more strongly than in ago1‐mutant plants. The virus engenders drought tolerance by altering the characteristics of the roots and not of the aerial tissues as, compared with the leaves of silencing mutants, leaves excised from CMV‐infected or 2b‐transgenic plants showed greater stomatal permeability and lost water more rapidly. This further indicates that CMV‐induced drought tolerance is not mediated via a change in the silencing‐regulated drought response mechanism. Under natural conditions, virus‐induced drought tolerance may serve viruses by aiding susceptible hosts to survive periods of environmental stress.     

Lipid peroxidation during low-temperature adaptation of cold-sensitive tobacco leaves and roots

We studied low-temperature adaptation of cold-sensitive tobacco plants in relation to peroxidation of lipids (POL) in their leaves and roots. Experiments were performed with tobacco plants (Nicotiana tabacum L., cv. Samsun). Cold hardening (6 days at 8°C) exerted principally different action on tobacco leaves and roots. In the leaves, the contents of dienoic conjugates and MDA was reduced, and tissue cold tolerance, even to below zero temperatures, was improved. In contrast, in the roots, POL was activated and root cold tolerance decreased. It is suggested that an incapability of the tobacco root system to adapt to low temperature was a limiting factor determining the low potential of this and other cold-sensitive plants to hypothermia.     

Hormone-Dependent Insertional Mutants of Arabodopsis thalianawith Reduced Viability and Fertility

We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thalianahaving drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thalianaby exogenous hormones.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Maize Sep15‐like functions in endoplasmic reticulum and reactive oxygen species homeostasis to promote salt and osmotic stress resistance

In animals, the Sep15 protein participates in disease resistance, growth, and development, but the function of its plant homologues remains unclear. Here, the function of maize Sep15 was analysed by characterization of two independent Sep15‐like loss‐of‐function mutants. In the absence of ZmSep15‐like, seedling tolerance to both water and salinity stress was compromised. The mutants experienced a heightened level of endoplasmic reticulum stress, and over‐accumulated reactive oxygen species, resulting in leaf necrosis. Characterization of Arabidopsis thaliana atsep15 mutant as well as like with ectopic expression of ZmSep15‐like indicated that ZmSep15‐like contributed to tolerance of both osmotic and salinity stress. ZmSep15‐like interacted physically with UDP‐glucose: glycoprotein glucosyltransferase1 (UGGT1). When the interaction was disrupted, the response to both osmotic and salinity stresses was impaired in maize or Arabidopsis. Co‐expressing ZmUGGT1 and ZmUGGT2 enhanced the tolerance of A. thaliana to both stressors, indicating a functional interaction between them. Together, the data indicated that plants Sep15‐like proteins promote osmotic and salinity stress resistance by influencing endoplasmic reticulum stress response and reactive oxygen species level.     

Auxin signaling participates in the adaptative response against oxidative stress and salinity by interacting with redox metabolism in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin regulates gene expression through direct physical interaction with TIR1/AFB receptor proteins during different processes of growth and development in plants. Here we report the contribution of auxin signaling pathway to the adaptative response against abiotic stress in Arabidopsis. Phenotypic characterization of tir1/afb auxin receptor mutants indicates a differential participation of each member under abiotic stress. In particular, tir1 afb2 and tir1 afb3 mutants resulted more tolerant to oxidative stress. In addition, tir1 afb2 showed increased tolerance against salinity measured as chlorophyll content, germination rate and root elongation compared with wild-type plants. Furthermore, tir1 afb2 displayed a reduced accumulation of hydrogen peroxide and superoxide anion, as well as enhanced antioxidant enzymes activities under stress. A higher level of ascorbic acid was detected in tir1 afb2 compared with wild-type plants. Thus, adaptation to salinity in Arabidopsis may be mediated in part by an auxin/redox interaction.     

Phytochelatin is not a primary factor in determining copper tolerance

Phytochelatin (PC) is involved in the detoxification of harmful, non-essential heavy metals and the homeostasis of essential heavy metals in plants. Its synthesis can be induced by either cadmium (Cd) or copper (Cu), and can form stable complexes with either element. This might suggest that PC has an important role in determining plant tolerance to both. However, this is not clearly apparent, as evidenced by a PC-deficient and Cd-sensitiveArabidopsis mutant (cad1-3) that shows no significant increase in its sensitivity to copper. Therefore, we investigated whether the mechanism for Cu tolerance differed from that for Cd by analyzing copper sensitivity in Cd-tolerant transgenics and Cd-sensitive mutants ofArabidopsis. Cadmium-tolerant transgenic plants that over-expressedA. thaliana phytochelatin synthase 1 (AtPCS1) were not tolerant of copper stress, thereby supporting the hypothesis that PC is not primarily involved in this tolerance mechanism. We also investigated Cu tolerance incad2-1, a Cd-sensitive and glutathione (GSH)-deficientArabidopsis mutant. Paradoxically,cad2-1 was more resistant to copper stress than were wild-type plants. This was likely due to the high level of cysteine present in that mutant. However, when the growth medium was supplemented with cysteine, the wild types also exhibited copper tolerance. Moreover,Saccharomyces cerevisiae that expressedAtPCS1 showed tolerance to Cd but hypersensitivity to Cu. All these results indicate that PC is not a major factor in determining copper tolerance in plants.     

Effect of temperature stress on the endogenous cytokinin content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh plants

The levels of three endogenous cytokinin equivalents: zeatin (Z), iso-pentenyladenine (iP) and dihydrozeatin (dZ) in two Arabidopsis thaliana (L.) Heynh genotypes — wild type (wt) and ethylene-insensitive mutant (eti5), were compared using enzyme immunoassay (ELISA). Cytokinin content was measured after exposure to low (4 °C for 24 h in darkness) or high temperature (38 °C for 24 h in darkness). Measurements were performed immediately and 24, 48 and 120 h after treatments. It was found that at normal growth conditions eti5 plants contained more endogenous cytokinins compared to the wild type. At both temperature treatments mutant plants had decreased total cytokinin levels. Wild-type plants treated with high temperature (HT) exhibited reduced total cytokinins (with the exception of rates at 48 h), while low temperature (LT) treatment resulted in elevated total amount of the studied equivalents (except at 24 h). The obtained results suggested that HT had greater effect on cytokinin levels than LT since it caused more profound changes in the total content. We assume that this was due to the natural chilling tolerance of Arabidopsis plants.     

Knockout of AtDjB1, a J‐domain protein from Arabidopsis thaliana,alters plant responses to osmotic stress and abscisic acid

AtDjB1 is a member of the Arabidopsis thaliana J‐protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1‐1) exhibited greater ABA sensitivity compared with the wild‐type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA‐responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1‐1 plants exhibited higher glucose levels and greater glucose sensitivity in the post‐germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1‐1 than WT seedlings. Taken together, higher glucose levels in atj1‐1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.