Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system

Ascorbic acid has numerous and diverse roles in plant metabolism. We have used the vtc-1 mutant of Arabidopsis, which is deficient in ascorbate biosynthesis, to investigate the role of ascorbate concentration in growth, regulation of photosynthesis, and control of the partitioning of antioxidative enyzmes. The mutant possessed 70% less ascorbate in the leaves compared with the wild type. This lesion was associated with a slight increase in total glutathione but no change in the redox state of either ascorbate or glutathione. In vtc-1, total ascorbate in the apoplast was decreased to 23% of the wild-type value. The mutant displayed much slower shoot growth than the wild type when grown in air or at high CO(2) (3 mL L(-1)), where oxidative stress is diminished. Leaves were smaller, and shoot fresh weight and dry weight were lower in the mutant. No significant differences in the light saturation curves for CO(2) assimilation were found in air or at high CO(2), suggesting that the effect on growth was not due to decreased photosynthetic capacity in the mutant. Analysis of chlorophyll a fluorescence quenching revealed only a slight effect on non-photochemical energy dissipation. Hydrogen peroxide contents were similar in the leaves of the vtc-1 mutant and the wild type. Total leaf peroxidase activity was increased in the mutant and compartment-specific differences in ascorbate peroxidase (APX) activity were observed. In agreement with the measurements of enzyme activity, the expression of cytosolic APX was increased, whereas that for chloroplast APX isoforms was either unchanged or slightly decreased. These data implicate ascorbate concentration in the regulation of the compartmentalization of the antioxidant system in Arabidopsis.     

Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica

Ramonda sp. (Gesneriaceae) is an endemic and relic plant ina very small group of poikilohydric angiosperms that are ableto survive in an almost completely dehydrated state. Senescence-and drought-related changes in the activity of peroxidase (POD;EC 1.11.1.7 [EC] ), ascorbate peroxidase (EC 1.11.1.11 [EC] ), and superoxidedismutase (SOD; EC 1.15.1.1 [EC] ) were determined in leaves of differentage and relative water content. The results indicate that differentPOD isoforms were stimulated during senescence and dehydration.Two of the soluble POD isoforms were anionic with pI 4.5, andtwo were cationic with pI 9.3 and 9.0. The activity of ascorbateperoxidase remained unchanged either by drought or senescence.For the first time, SOD isoforms have now been determined inthis resurrection plant. Several SOD isoforms, all of the Mntype, were found to be anionic with pI 4 and a few others hadpI from 5 to 6, while one band of FeSOD with a lower molecularweight was neutral. Rehydration brought about a remarkable decreaseover the first hour in the activity of all the antioxidant enzymesexamined but activity recovered 1 d after rehydration. The resultsconfirmed that dehydration and senescence caused disturbancein the redox homeostasis of Ramonda leaves, while inducing differentPOD isoforms. A physiological role of peroxidase reaction withhydroxycinnamic acids in conservation and protection of cellularconstituents of desiccated Ramonda leaves is suggested. Key words: Desiccation, peroxidase, Ramonda, senescence, superoxide dismutase     

UV responses of Lolium perenne raised along a latitudinal gradient across Europe: a filtration study

Lolium perenne (cv. AberDart) was grown at 14 locations along a latitudinal gradient across Europe (37-68°N) to study the impact of ultraviolet radiation (UV) and climate on aboveground growth and foliar UV-B absorbing compounds. At each location, plants were grown outdoors for 5 weeks in a replicated UV-B filtration experiment consisting of open, UV-B transparent (cellulose diacetate) and UV-B opaque (polyester) environments. Fourier transform-infrared spectroscopy was used to compare plant metabolite profiles in relation to treatment and location. UV radiation and climatic parameters were determined for each location from online sources and the data were assessed using a combination of anova and multiple regression analyses. Most of the variation in growth between the locations was attributable to the combination of climatic parameters, with minimum temperature identified as an important growth constraint. However, no single environmental parameter could consistently account for the variability in plant growth. Concentrations of foliar UV-B absorbing compounds showed a positive trend with solar UV across the latitudinal gradient; however, this relationship was not consistent in all treatments. The most striking experimental outcome from this study was the effect of presence or absence of filtration frames on UV-absorbing compounds. Overall, the study demonstrates the value of an European approach in studying the impacts of natural UV across a large latitudinal gradient. We have shown the feasibility of coordinated UV filtration at multiple sites but have also highlighted the need for open controls and careful interpretation of plant responses.     

Peroxide processing in photosynthesis: antioxidant coupling and redox signalling

Photosynthesis has a high capacity for production of hydrogen peroxide (H2O2), but the intracellular levels of this relatively weak oxidant are controlled by the antioxidant system, comprising a network of enzymatic and non-enzymatic components that notably includes reactions linked to the intracellular ascorbate and glutathione pools. Mutants and transformed plants with specific decreases in key components offer the opp ortunity to dissect the complex system that maintains redox homeostasis. Since H2O2 is a signal-transducing molecule relaying information on intracellular redox state, the pool size must be rigorously controlled within each compartment of the cell. This review focuses on compartment-specific differences in the stringency of redox coupling between ascorbate and glutathione, and the significance this may have for the flexibility of the control of gene expression that is linked to photosynthetic H2O2 production.     

Detection of Monodehydroascorbic Acid Radical in Sulfite-Treated Leaves and Mechanism of its Formation

The aim of the present study is to detect the monodehydroascorbicacid (MDA) radical in broad bean (Vicia faba L.) leaves whichwere treated by vacuum-infiltration in Na₂SO₃ solution and subsequentcentrifugation (sulfite-treated leaves). When sulfite-treatedleaves were illuminated with white light, the electron spinresonance (ESR) signal of MDA radical was observed. The levelof the MDA radical depended on the concentration of sulfitethat was used for vacuum-infiltration and on the light intensityof illumination. The formation of the MDA radical in sulfite-treatedleaves was inhibited by DCMU or by replacement of air with N₂.Glycolaldehyde also inhibited the formation of MDA radical insulfite-treated leaves. Catalase activity was decreased by thesulfite treatment without affecting significantly the activitiesof ascorbate peroxidase (AA-POX) and of peroxidase which preferentiallyoxidizes phenolics (PhOH-POX). From these results, we concludethat the formation of the MDA radical in sulfite-treated leavesis catalyzed by peroxidases using the H₂O₂ which is generatedby photorespiration and the Mehler reaction. ¹On leave from the Center for Multidisciplinary Studies, Universityof Belgrade, Yugoslavia.     

Inhibition of photosynthesis,acidification and stimulation of zeaxanthin formation in leaves by sulfur dioxide and reversal of these effects

Leaves of Pelargonium zonale L. and Spinacia oleracea L. were fumigated with high concentrations of SO₂ for very short periods of time with the aim of first producing acute symptoms of damage and then observing repair. The response of different photosynthetic parameters to SO₂ was monitored during and after fumigation. The following results were obtained: (1) Inhibition of CO₂ assimilation in the light was accompanied by increased reduction of the quinone acceptor, Q_A, of photosystem II and by increased oxidation of the electrondonor pigment P₇₀₀ of photosystem I. Increased control of photosystem II activity in the SO₂-inhibited state was also indicated by increased light scattering and by increased non-photochemical quenching of chlorophyll fluorescence. Both are indicators of chloroplast energization. Apparently, SO₂ did not decrease but rather increased energization of the chloroplast thylakoid system by light. (2) Accumulation of dihydroxyacetone phosphate, fructose-1,6-phosphate and ribulose-1,5-phosphate and a decrease of 3-phosphoglycerate and hexosephosphate indicated that SO₂ inhibited enzymes of the Calvin cycle. (3) Stimulated postillumination CO₂ evolution suggested that when photosynthesis declined respiration increased to provide energy for repair reactions. (4) Increased leaf absorbance at 505 nm indicated increased stimulation of zeaxanthin formation in thylakoid membranes under the influence of SO₂. A similar increase in 505-nm absorbance could be induced by high concentrations of CO₂. In darkened leaves, SO₂ did not produce changes in 505-nm absorbance. (5) While zeaxanthin formation was stimulated, changes in the fluorescence of the pH-indicating dye pyranine, which had been fed to the leaves, indicated acidification of the cytoplasm of leaf cells by SO₂. Maximum acid production by SO₂ required light. In contrast, cytoplasmic acidification of leaf cells by CO₂ was similar in the light and in the dark. (6) Since zeaxanthin formation is known to depend on the acidification of the thylakoid lumen, SO₂-dependent zeaxanthin formation indicated SO₂-dependent acidification of the thylakoid lumen as the indirect result of cytoplasmic acidification by SO₂. (7) Inhibition of photosynthesis and other effects of SO₂ were fully reversible in the light. Detoxification of SO₂ and reactivation of the photosynthetic apparatus were slow or absent in the dark. Light had a dual effect on the action of SO₂. Transiently, it first increased the extent of inhibition of assimilation, but, finally, it reversed inhibition. Sulfur dioxide was inhibitory as a consequence of the chemical reactivity of its hydration products rather than as a result of cellular acidification by the produced acid. The initial acidification was followed by an appreciable alkalisation demonstrating the action of the pH-stat mechanism. (8) The data are discussed in relation to SO₂ toxicity under field conditions when plants are chronically exposed to polluted air.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - F6P fructoce-6-phosphate - F, Fm, Fm, Fo, Fo chlorophyll fluorescence levels - PGA 3-phosphoglycerate - P₇₀₀ primary donor of photosystem I - Q_A primary quinone acceptor of photosystem II - q_p photochemical quenching of chlorophyll fluorescence - NPQ non-photochemical quenching of chlorophyll fluorescence - RuBP ribulose-1,5-bisphosphate Dedicated to Professor O.L. Lange on the occasion of his 65th birthdayOn leave from the Centre for Multidisciplinary Sciences, University of Belgrade, YugoslaviaThis work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 251 of the University of Würzburg. S. V.-J. acknowledges support by the Leibniz program of the Deutsche Forschungsgemeinschaft and by the Fonds for Science of the Republic of Serbia (contract no. 8604). We are grateful to Drs. Z.-H. Yin, U. Takahama and K.-J. Dietz (Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, FRG) for cooperation and helpful discussions.     

Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves

Resurrection plants are able to dehydrate/rehydrate rapidly without cell damage by a mechanism, the understanding of which may be of ecological importance in the adaptation of crop plants to dry conditions. The o -diphenol oxidase in Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, was characterized in respect to different isoforms, preferable substrates and specific inhibitors. Two anionic isoforms with pI 4.6 and 4.7 were separated from turgid leaves. Three additional anionic isoforms (pI 5.1, 5.3 and 5.6) and three neutral isoforms (pI from 6.8 to 7.4) were induced in desiccated leaves. Based on apparent K_m values, the affinity for reducing substrates decreased as follows: methyl catechol > chlorogenic acid > 3,4-dihydroxyphenylalanine > caffeic acid > pyrogallol. Polyphenol oxidase (PPO) activity was specifically sensitive to diethyldithiocarbamate and also inhibited by KCN, DTT and salicylic hydroxamic acid but with no inhibitory effect of Na₃N. Plants were subjected to drought-to-near complete water loss (approximately 2% relative water content, RWC) and several fold higher PPO activity was detected in desiccated leaves. Ramonda leaves contain high levels of phenolics, which decreased during drought. Rehydration of dry leaves from 2% RWC to 95% RWC led to transient inhibition of PPO in the first few hours. Within a day, the levels completely recovered to those determined in desiccated leaves. The finding of desiccation-induced high activity of PPO and new isoforms, which were also present in rehydrated turgid leaves, indicates a substantial role for PPO in the adaptation mechanism of resurrection plants to desiccation and also to the oxidative stress during rehydration.