Karl-Josef Dietz

The relationship between metal toxicity and cellular redox imbalance

The relationship between cellular redox imbalances leading to oxidative stress and metal toxicity in plants has been studied intensely over the past decades. This interdependency was often considered to reflect a rather indirect metal effect of cellular disregulation and progressive secondary damage development. By contrast, recent experiments revealed a clear relationship between metal stress and redox homeostasis and antioxidant capacity. Analysis of plants expressing targeted modifications of components of the antioxidant system, the comparison of closely related plant species with different degrees of toxic metal sensitivity and effector studies with, for instance, salicylic acid have established a link between the degree of plant tolerance to metals and the level of antioxidants.

Salicylic Acid Alleviates the Cadmium Toxicity in Barley Seedlings

Salicylic acid (SA) plays a key role in plant disease resistance and hypersensitive cell death but is also implicated in hardening responses to abiotic stressors. Cadmium (Cd) exposure increased the free SA contents of barley (Hordeum vulgare) roots by a factor of about 2. Cultivation of dry barley caryopses presoaked in SA-containing solution for only 6 h or single transient addition of SA at a 0.5 mmconcentration to the hydroponics solution partially protected the seedlings from Cd toxicity during the following growth period. Both SA treatments had little effect on growth in the absence of Cd, but increased root and shoot length and fresh and dry weight and inhibited lipid peroxidation in roots, as indicated by malondialdehyde contents, in the presence of Cd. To test whether this protection was due to up-regulation of antioxidant enzymes, activities and transcript levels of the H2O2-metabolizing enzymes such as catalase and ascorbate peroxidase were measured in control and SA-treated seedlings in the presence or absence of 25 μmCd. Cd stress increased the activity of these enzymes by variable extent. SA treatments strongly or completely suppressed the Cd-induced up-regulation of the antioxidant enzyme activities. Slices from leaves treated with SA for 24 h also showed an increased level of tolerance toward high Cd concentrations as indicated by chlorophyll a fluorescence parameters. The results support the conclusion that SA alleviates Cd toxicity not at the level of antioxidant defense but by affecting other mechanisms of Cd detoxification.

pTAC2, -6, and -12 Are Components of the Transcriptionally Active Plastid Chromosome That Are Required for Plastid Gene Expression

Transcription in plastids is mediated by a plastid-encoded multimeric (PEP) and a nuclear-encoded single-subunit RNA polymerase (NEP) and a still unknown number of nuclear-encoded factors. By combining gel filtration and affinity chromatography purification steps, we isolated transcriptionally active chromosomes from Arabidopsis thaliana and mustard (Sinapis alba) chloroplasts and identified 35 components by electrospray ionization ion trap tandem mass spectrometry. Eighteen components, called plastid transcriptionally active chromosome proteins (pTACs), have not yet been described. T-DNA insertions in three corresponding genes, ptac2, -6, and -12, are lethal without exogenous carbon sources. Expression patterns of the plastid-encoded genes in the corresponding knockout lines resemble those of Δrpo mutants. For instance, expression of plastid genes with PEP promoters is downregulated, while expression of genes with NEP promoters is either not affected or upregulated in the mutants. All three components might also be involved in posttranscriptional processes, such as RNA processing and/or mRNA stability. Thus, pTAC2, -6, and -12 are clearly involved in plastid gene expression.

Free Radicals and Reactive Oxygen Species as Mediators of Heavy Metal Toxicity in Plants

In most plants, exposure to elevated concentrations of heavy metals results in growth inhibition (see Chap. 8). After prolonged metal exposure, sensitive plants develop visible symptoms of toxicity such as chlorosis and necrotic lesions. During the past two and half decades, symptoms of metal toxicity and deficiency have been described extensively (Marschner 1995). However, our knowledge of the biochemical basis of metal toxicity symptoms in plants is still sketchy. It is known that heavy metals can bind to functionally important domains of biomolecules and thereby inactivate them. The result is, for instance, the inhibition of an enzymatic reaction and a disturbance of metabolism (Van Assche and Clijsters 1986). Furthermore, heavy metals have been demonstrated to stimulate formation of free radicals (FR) and reactive oxygen species (ROS), either by direct electron transfer involving metal cations, or as a consequence of metal-mediated inhibition of metabolic reactions (Halliwell and Gutteridge 1984; Elstner 1990). If the plant cell cannot match the increased rate of FR and ROS formation with an increased activity of the antioxidative machinery, uncontrolled oxidation and radical chain reactions will result in oxidative stress. Therefore, the degree of cell damage under heavy-metal stress depends on the rate of FR and ROS formation, and on the efficiency and capacity of detoxification and repair mechanisms. A comparison of closely related metal-sensitive and -tolerant species or ecotypes suggested that an enhanced oxidative defence is not a particular trait conferring heavy-metal tolerance to plants (De Vos et al. 1991). Metal-tolerant plants are usually efficient in avoiding the occurrence of toxic concentrations of of free heavy metal cations in plasmatic compartments, and thereby avoid the development of oxidative stress (Woolhouse 1983).

S-nitrosylation of peroxiredoxin II E promotes peroxynitrite-mediated tyrosine nitration

Nitric oxide (NO) is a free radical product of cell metabolism that plays diverse and important roles in the regulation of cellular function. S-Nitrosylation is emerging as a specific and fundamental posttranslational protein modification for the transduction of NO bioactivity, but very little is known about its physiological functions in plants. We investigated the molecular mechanism for S-nitrosylation of peroxiredoxin II E (PrxII E) from Arabidopsis thaliana and found that this posttranslational modification inhibits the hydroperoxide-reducing peroxidase activity of PrxII E, thus revealing a novel regulatory mechanism for peroxiredoxins. Furthermore, we obtained biochemical and genetic evidence that PrxII E functions in detoxifying peroxynitrite (ONOO−), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and O2− that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO− detoxification activity of PrxII E, causing a dramatic increase of ONOO−-dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO−, indicating that the PrxII E modulation of ONOO− bioactivity is biologically relevant. We conclude that NO regulates the effects of its own radicals through the S-nitrosylation of crucial components of the antioxidant defense system that function as common triggers for reactive oxygen species– and NO-mediated signaling events.

Divergent light-, ascorbate-, and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Arabidopsis.

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q andprx II E, decreased upon application of ascorbate.prx Q also responded to peroxides and diamide treatment.prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (Em′) of Prx II B, Prx II C, and Prx II E were around −290 mV and, thus, less negative than Em′ of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (−307 to −322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.

The plant-specific function of 2-Cys peroxiredoxin-mediated detoxification of peroxides in the redox-hierarchy of photosynthetic electron flux

The 2-cysteine peroxiredoxins (2-Cys Prx) constitute an ancient family of peroxide detoxifying enzymes and have acquired a plant-specific function in the oxygenic environment of the chloroplast. Immunocytochemical analysis and work with isolated intact chloroplasts revealed a reversible binding of the oligomeric form of 2-Cys Prx to the thylakoid membrane. The oligomeric form of the enzyme was enhanced under stress. The 2-Cys Prx has a broad substrate specificity with activity toward hydrogen peroxides and complex alkyl hydroperoxides. During the peroxide reduction reaction, 2-Cys Prx is alternatively oxidized and reduced as it catalyzes an electron flow from an electron donor to peroxide. Escherichia coli thioredoxin, but also spinach thioredoxin f and m were able to reduce oxidized 2-Cys Prx. The midpoint redox potential of −315 mV places 2-Cys Prx reduction after Calvin cycle activation and before switching the malate valve for export of excess reduction equivalents to the cytosol. Thus the 2-Cys Prx has a defined and preferential place in the hierarchy of photosynthetic electron transport. The activity of 2-Cys Prx also is linked to chloroplastic NAD(P)H metabolism as indicated by the presence of the reduced form of the enzyme after feeding dihydroxyacetone phosphate to intact chloroplasts. The function of the 2-Cys Prx is therefore not confined to its role in the water–water cycle pathway for energy dissipation in photosynthesis but also mediates peroxide detoxification in the plastids during the dark phase.

Peroxiredoxins in Plants and Cyanobacteria

Peroxiredoxins (Prx) are central elements of the antioxidant defense system and the dithiol-disulfide redox regulatory network of the plant and cyanobacterial cell. They employ a thiol-based catalytic mechanism to reduce H2O2, alkylhydroperoxide, and peroxinitrite. In plants and cyanobacteria, there exist 2-CysPrx, 1-CysPrx, PrxQ, and type II Prx. Higher plants typically contain at least one plastid 2-CysPrx, one nucleo-cytoplasmic 1-CysPrx, one chloroplast PrxQ, and one each of cytosolic, mitochondrial, and plastidic type II Prx. Cyanobacteria express variable sets of three or more Prxs. The catalytic cycle consists of three steps: (i) peroxidative reduction, (ii) resolving step, and (iii) regeneration using diverse electron donors such as thioredoxins, glutaredoxins, cyclophilins, glutathione, and ascorbic acid. Prx proteins undergo major conformational changes in dependence of their redox state. Thus, they not only modulate cellular reactive oxygen species- and reactive nitrogen species-dependent signaling, but depending on the Prx type they sense the redox state, transmit redox information to binding partners, and function as chaperone. They serve in context of photosynthesis and respiration, but also in metabolism and development of all tissues, for example, in nodules as well as during seed and fruit development. The article surveys the current literature and attempts a mostly comprehensive coverage of present day knowledge and concepts on Prx mechanism, regulation, and function and thus on the whole Prx systems in plants.

The Mitochondrial Type II Peroxiredoxin F Is Essential for Redox Homeostasis and Root Growth of Arabidopsis thaliana under Stress

Peroxiredoxins (Prx) have recently moved into the focus of plant and animal research in the context of development, adaptation, and disease, as they function both in antioxidant defense by reducing a broad range of toxic peroxides and in redox signaling relating to the adjustment of cell redox and antioxidant metabolism. At-PrxII F is one of six type II Prx identified in the genome of Arabidopsis thaliana and the only Prx that is targeted to the plant mitochondrion. Therefore, it might be assumed to have functions similar to the human 2-Cys Prx (PRDX3) and type II Prx (PRDX5) and yeast 1-Cys Prx that likewise have mitochondrial localizations. This paper presents a characterization of PrxII F at the level of subcellular distribution, activity, and reductive regeneration by mitochondrial thioredoxin and glutaredoxin. By employing tDNA insertion mutants of A. thaliana lacking expression of AtprxII F (KO-AtPrxII F), it is shown that under optimal environmental conditions the absence of PrxII F is almost fully compensated for, possibly by increases in activity of mitochondrial ascorbate peroxidase and glutathione-dependent peroxidase. However, a stronger inhibition of root growth in KO-AtPrxII F seedlings as compared with wild type is observed under stress conditions induced by CdCl2 as well as after administration of salicylhydroxamic acid, an inhibitor of cyanide-insensitive respiration. Simultaneously, major changes in the abundance of both nuclear and mitochondria-encoded transcripts were observed. These results assign a principal role to PrxII F in antioxidant defense and possibly redox signaling in plants cells.

THE RELATIONSHIP BETWEEN THE REDOX STATE OF QA AND PHOTOSYNTHESIS IN LEAVES AT VARIOUS CARBON-DIOXIDE, OXYGEN AND LIGHT REGIMES

The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor QA of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O2 and CO2 concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of QA. Both at low and high fluence rates, the redox state of QA was little altered when the CO2 concentration was reduced from saturation to about 600 μl·l-1 although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO2 content the amount of reduced QA increased appreciably even at low fluence rates where light limited CO2 reduction. Both in the presence and in the absence of CO2, a more reduced QA was observed when the O2 concentration was below 2%. QA was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, QA was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of QA changed more during a transition from air containing 350 μl·l-1 CO2 to CO2-free air than in sun leaves. The results are discussed with respect to the energy status and the CO2-fixation rate of the leaves.