Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress

To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.     

黄芩过氧化物酶同工酶电泳和抗坏血酸过氧化物酶活性分析

对黄芩（Scutellaria baicalensis Georgi)二倍体和同源四倍体过氧化物酶进行了同工酶电泳分析及抗坏血酸过氧化物酶活性测定。结果表明,黄芩二倍体与同源四倍体过氧化物酶同工酶酶谱一致,但后者的着色程度大于前者,二年生黄芩叶子在快速区Rf为0．494和0．512处出现新的谱带,不同发育阶段和不同组织器官的谱带存在明显差异;根和叶中谱带数最多,花其次,种子最少;试管苗谱带数先减少后增加,并在整个培养过程中出现特征性谱带C。各组织器官抗坏血酸过氧化物酶总活力差异明显,顶芽最高,叶子次之,花最低。黄芩一年生和二年生各个多倍体株系叶子抗坏血酸过氧酶总活力均高于二倍体叶子抗坏血酸过氧化酶总活力。试管苗生长过程中抗坏血酶过氧化物酶活活力的变化与生长趋势一致,表明该酶与植株生长发育紧密相关。     

Growth of tomato (Lycopersicon esculentum) in response to salicylic acid under water stress

Abstract

Plants of Lycopersicon esculentum L. cv. K-25 were subjected to water stress by withholding water for 10 days at 20 (WS I) and 30 (WS II) days after sowing (DAS). Seedlings were sprayed with double distilled water (DDW) or 10^?5M salicylic acid (SA) at 45 DAS. The water stress at earlier stage of growth (20 day stage) was more inhibitory as compared to the later stage (30 day stage). The plants exposed to water stress exhibited a significant (p<0.05) decline in photosynthetic parameters, membrane stability index (MSI), leaf water potential, activity of nitrate reductase (NR), carbonic anhydrase (CA), chlorophyll and relative water content (RWC). A follow-up treatment with SA protected against the stress generated by water and significantly improved the above parameters. However, proline content and antioxidant enzymes increased under drought as well as under SA treatments.     

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.     

Reversibility of the effects of cadmium on the growth and nitrogen metabolism in the tomato(Lycopersicon esculentum)

In order to better understand the effects of heavy metals on the growth of plants, we decided to perform recovering experiments by following both chemical and physiological parameters in cadmium pre-stressed tomato seedlings after cadmium had been removed from the nutrient solution. The work shows that cadmium suppression results in resumption of growth activity. The biomass of leaves and stems rose steadily. The increase in root biomass exceeded those of leaves and stems. At the same time, nitrate content was increased to reach the level obtained with unstressed controls. In all the organs studied, the activities of the enzymes involved in the anabolic nitrogen primary assimilation pathways (nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) soared after that cadmium had been removed. While NAD(+)-dependent glutamate dehydrogenase (GDH-NAD+) activity also rose progressively during the recovering time, the cognate NADH-dependent glutamate dehydrogenase (GDH-NADH) activity decreased. This result allows us to propose that the ammonia produced by the stress-induced protein catabolism is detoxified and re-assimilated by the GDH-NADH isoenzyme. On the basis of these results, we will discuss the ability of the plant to dilute the effects of pollutants during the recovering period. An important outcome of this work is that a transient contamination of the culture medium by pollutants is not necessarily followed by a significant depreciation in product yield or quality.     

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999     

Ultrastructure and lipid alterations induced by cadmium in tomato (Lycopersicon esculentum) chloroplast membranes

The effects of cadmium (Cd) uptake on ultrastructure and lipid composition of chloroplasts were investigated in 28-day-old tomato plants (Lycopersicon esculentum var. Ibiza F1) grown for 10 days in the presence of various concentrations of CdCl2. Different growth parameters, lipid and fatty acid composition, lipid peroxidation, and lipoxygenase activity were measured in the leaves in order to assess the involvement of this metal in the generation of oxidative stress. We first observed that the accumulation of Cd increased with external metal concentration, and was considerably higher in roots than in leaves. Cadmium induced a significant inhibition of growth in both plant organs, as well as a reduction in the chlorophyll and carotenoid contents in the leaves. Ultrastructural investigations revealed that cadmium induced disorganization in leaf structure, essentially marked by a lowered mesophyll cell size, reduced intercellular spaces, as well as severe alterations in chloroplast fine structure, which exhibits disturbed shape and dilation of thylakoid membranes. High cadmium concentrations also affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the fatty acid content and a shift in the composition of fatty acids, resulting in a lower degree of fatty acid unsaturation in chloroplast membranes. The level of lipid peroxides and the activity of lipoxygenase were also significantly enhanced at high Cd concentrations. These biochemical and ultrastructural changes suggest that cadmium, through its effects on membrane structure and composition, induces premature senescence of leaves.     

Redox-mediated decolorization of Direct Red 23 and Direct Blue 80 catalyzed by bioaffinity-based immobilized tomato (Lycopersicon esculentum) peroxidase

The aim of this study was to investigate the role of concanavalin A (Con A)-cellulose-bound tomato peroxidase for the decolorization of direct dyes. Cellulose was used as an inexpensive material for the preparation of bioaffinity support. Con A-cellulose-bound tomato peroxidase exhibited higher efficiency in terms of dye decolorization as compared to soluble enzyme under various experimental conditions. Both Direct Red 23 and Direct Blue 80 dyes were recalcitrant to the action of enzyme without a redox mediator. Six compounds were investigated for redox-mediating property. Immobilized peroxidase decolorized both dyes to different extent in the presence of all the used redox mediators. However, 1-hydroxybenzotriazole emerged as a potential redox mediator for tomato peroxidase catalyzed decolorization of direct dyes. These dyes were maximally decolorized at pH 6.0 and 40 degrees C by soluble and immobilized peroxidase. The absorption spectra of the untreated and treated dyes exhibited a marked difference in the absorption at various wavelengths. Immobilized tomato peroxidase showed a lower Michaelis constant than the free enzyme for both dyes. Soluble and immobilized tomato peroxidase exhibited significantly higher affinity for Direct Red 23 compared to Direct Blue 80.     

外源NO与蔗糖对盐胁迫下番茄(Lycopersicon esculentum Mill)幼苗氧化损伤的保护效应

选取长至6～8片真叶的健康番茄(Lycopersicon esculentum Mill)幼苗,分别进行蔗糖、硝普钠(sodium nitropresside, SNP, 作为外源NO供体)及其体积比例组合(1:1)处理;36h后施以NaCl胁迫,并分别于0h(胁迫前)、24h、48h和72h 取样,进行相关生理生化指标测定.具体5个实验处理如下:A. 蒸馏水(CK); B. 100 mmol/L NaCl;C. 0.1 mmol/L SNP 100 mmol/L NaCl;D. 0.1 mmol/L SNP 1.0mmol/L蔗糖 100 mmol/L NaCl; E. 1.0 mmol/L蔗糖 100 mmol/L NaCl.结果表明:与SNP和蔗糖单独处理相比,二者组合处理对缓解盐胁迫下番茄幼苗的氧化损伤存在正协同效应,主要表现在进一步增强了番茄幼苗超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)的活性;提高了脯氨酸(Pro)的含量,同时膜脂过氧化产物丙二醛(MDA)含量显著降低(P＜0.05).采用聚丙烯酰胺浓度梯度凝胶电泳对盐胁迫24 h和48 h材料的POD同功酶检测表明,当NaCl单独处理时,番茄幼苗叶片POD同功酶第V条带缺失,其它谱带酶量减少,抑制了POD同功酶的表达;SNP和蔗糖单独处理能够保护盐胁迫(24、48h)所导致的POD同功酶条带的完整;而组合处理既保证了POD同功酶条带的完整,又加强了酶量的表达.随着盐胁迫时间的延长,其氧化损伤程度愈烈,SNP和蔗糖组合处理能够更有效地缓解盐胁迫对番茄幼苗植株造成的氧化损伤.     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii

The response of the antioxidative systems of leaf cell mitochondria and peroxisomes of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species Lycopersicon pennellii (Lpa) to NaCl 100 mM stress was investigated. Salt-dependent oxidative stress was evident in Lem mitochondria as indicated by their raised levels of lipid peroxidation and H2O2 content whereas their reduced ascorbate and reduced glutathione contents decreased. Concomitantly, SOD activity decreased whereas APX and GPX activities remained at control level. In contrast, the mitochondria of salt-treated Lpa did not exhibit salt-induced oxidative stress. In their case salinity induced an increase in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione-dependent peroxidase (GPX). Lpa peroxisomes exhibited increased SOD, APX, MDHAR and catalase activity and their lipid peroxidation and H2O2 levels were not affected by the salt treatment. The activities of all these enzymes remained at control level in peroxisomes of salt-treated Lem plants. The salt-induced increase in the antioxidant enzyme activities in the Lpa plants conferred cross-tolerance towards enhanced mitochondrial and peroxisomal reactive oxygen species production imposed by salicylhydroxamic acid (SHAM) and 3-amino-1,2,4-triazole (3-AT), respectively.     

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.     

Salicylic acid increases tolerance to oxidative stress induced by hydrogen peroxide accumulation in leaves of cadmium-exposed flax (Linum usitatissimum L.)

The aim of this study is to investigate the impacts of exogenous salicylic acid (SA) pretreatments on hydrogen peroxide (H₂O₂) accumulation, protein oxidation, and H₂O₂-scavenging enzymes in leaves of Cd-treated flax seedlings. Cd-enhanced H₂O₂ levels were related to increased activities of guaiacol peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11), and were independent of changes in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activities. In control flax seedlings, exogenous SA pretreatments inhibited the activity of CAT, resulted in an enhanced production of H₂O₂ suggesting that SA requires H₂O₂ to initiate an oxidative stress. However, although leaves of Cd-free flax seedlings pretreated with SA accumulated in vivo H₂O₂ by 1.2-fold compared with leaves of Cd-only exposed ones; the damage to growth and proteins after the exposure to Cd was significantly less, indicating that SA can regulate the Cd-induced oxidative stress. Moreover, the Cd-treated seedlings primed with SA exhibited a higher level of total antioxidant capacities and increased activities of H₂O₂-detoxifying enzymes.     

Modeling and phylogenetic analysis of cytosolic ascorbate peroxidase (OsAPX1) from rice reveal signature motifs that may play a role in stress tolerance

Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha helical region that may play a significant role during stress.