Graham Noctor

Modification of thiol contents in poplars (Populus tremula x P-alba) overexpressing enzymes involved in glutathione synthesis

Abstract. The hybrid poplar (Populus tremula × P.␣alba) was transformed to express the Escherichia coli gene for γ-glutamylcysteine synthetase (EC 6.3.2.2: γ-ECS) in the cytosol. Four transformed lines of poplar were obtained. These were phenotypically indistinguishable from untransformed poplars. Three lines, ggs28 (Noctor et al. 1996, Plant Physiol 112: 1071–1078), ggs11 and ggs5 possessed high levels of bacterial gene transcripts. Line ggs17 had lower transcript levels. Antisera were prepared against bacterial γ-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS). Using the antiserum prepared against the purified His-tagged E.␣coliγ-ECS, lines ggs28, ggs11 and ggs5 were shown to possess abundant quantities of the bacterial protein, whereas ggs17 contained lower amounts. The antiserum prepared against the purified His-tagged E. coli GS was also effective in screening poplars transformed with the E.␣coli gene coding for this enzyme. Immunoblots of leaf extracts from poplars overexpressing GS using this antibody revealed two bands. The extractable foliar γ-ECS activities of the γ-ECS transformants were in quantitative agreement with the protein levels. Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher γ-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold. The lines strongly overexpressing γ-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), γ-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold). Foliar thiol contents in ggs17 were no different to those of untransformed plants.

Stress-triggered redox signalling: What’s in pROSpect?

Reactive oxygen species (ROS) have a profound influence on almost every aspect of plant biology. Here, we emphasize the fundamental, intimate relationships between light-driven reductant formation, ROS, and oxidative stress, together with compartment-specific differences in redox buffering and the perspectives for their analysis. Calculations of approximate H2 O2 concentrations in the peroxisomes are provided, and based on the likely values in other locations such as chloroplasts, we conclude that much of the H2 O2 detected in conventional in vitro assays is likely to be extracellular. Within the context of scant information on ROS perception mechanisms, we consider current knowledge, including possible parallels with emerging information on oxygen sensing. Although ROS can sometimes be signals for cell death, we consider that an equally important role is to transmit information from metabolism to allow appropriate cellular responses to developmental and environmental changes. Our discussion speculates on novel sensing mechanisms by which this could happen and how ROS could be counted by the cell, possibly as a means of monitoring metabolic flux. Throughout, we place emphasis on the positive effects of ROS, predicting that in the coming decades they will increasingly be defined as hallmarks of viability within a changing and challenging environment.

Light-dependent modulation of foliar glutathione synthesis and associated amino acid metabolism in poplar overexpressing γ-glutamylcysteine synthetase

Abstract. Glutathione (GSH), γ-glutamylcysteine (γ-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula × P. alba) and poplars expressing Escherichia coli genes for γ-glutamylcysteine synthetase (γ-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing γ-ECS, foliar γ-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of γ-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and γ-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate γ-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, γ-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, γ-EC was negligible in the light but increased in the dark. Cysteine content was little affected by light in any of the poplar types. No light-dependent changes in the extractable activities of γ-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing γ-ECS at elevated CO2 or low O2 largely abolished the inverse light-dependent changes in γ-EC and GSH. Low O2 did not affect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for γ-EC synthesis is augmented.

Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling.

Subcellular compartmentation and spatial redox transfer plays a critical role in signaling related to reactive oxygen and antioxidants. Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area.

Oxidative stress and antioxidative systems: recipes for successful data collection and interpretation.

Oxidative stress and reactive oxygen species (ROS) are common to many fundamental responses of plants. Enormous and ever-growing interest has focused on this research area, leading to an extensive literature that documents the tremendous progress made in recent years. As in other areas of plant biology, advances have been greatly facilitated by developments in genomics-dependent technologies and the application of interdisciplinary techniques that generate information at multiple levels. At the same time, advances in understanding ROS are fundamentally reliant on the use of biochemical and cell biology techniques that are specific to the study of oxidative stress. It is therefore timely to revisit these approaches with the aim of providing a guide to convenient methods and assisting interested researchers in avoiding potential pitfalls. Our critical overview of currently popular methodologies includes a detailed discussion of approaches used to generate oxidative stress, measurements of ROS themselves, determination of major antioxidant metabolites, assays of antioxidative enzymes and marker transcripts for oxidative stress. We consider the applicability of metabolomics, proteomics and transcriptomics approaches and discuss markers such as damage to DNA and RNA. Our discussion of current methodologies is firmly anchored to future technological developments within this popular research field.

Interactions Between Carbon and Nitrogen Metabolism

Over the past two decades, many studies have revealed the interdependence of carbon and nitrogen assimilation. Primary carbon metabolism is dependent on nitrogen assimilation, most obviously because much of the nitrogen budget of the plant is invested in the proteins and chlorophyll of the photosynthetic apparatus. Conversely, nitrogen assimilation requires a continuous supply of energy and carbon skeletons. This means that photosynthetic products must be partitioned between carbohydrate synthesis and the synthesis of amino acids. Controls over this partitioning must be flexible, since both external nitrogen availability and internal nitrogen demand may be variable.

Signaling and Integration of Defense Functions of Tocopherol, Ascorbate and Glutathione

Ascorbate, glutathione, and tocopherol are the three major low molecular weight antioxidants of plant cells. While tocopherol is hydrophobic and is found only in lipid membranes, ascorbate and glutathione are hydrophilic, accumulating to high concentrations in the chloroplast stroma and other compartments of the plant cell. Ascorbate and glutathione not only limit photo-oxidative damage but can also act independently as signal-transducing molecules regulating defense gene expression. Both metabolites transmit information concerning oxidative load and redoxbuffering capacity. Ascorbate modifies the expression of chloroplast genes. Net glutathione synthesis during stress restores the cellular redox state and allows orchestration of systemic acquired resistance. The degree of redox coupling between these antioxidants has profound implications for regulation, function, and signaling associated with the two major energy-generating systems, i.e. photosynthesis and respiration. Tocopherol fulfills an essential protective function, counter-acting the harmful effects of singlet oxygen production at photosystem II. Ascorbate reduces and thus regenerates oxidized tocopherol, but flux through this reaction is not sufficient to maintain the reduced tocopherol pool under high light stress. This may be because tocopherol regeneration draws on the ascorbate pool of the chloroplast lumen, which may be depleted under stress. Moreover, while glutathione always reduces oxidized ascorbate (dehydroascorbate), the degree of coupling between the ascorbate and glutathione redox couples is variable. The flexibility of coupling between these antioxidant pools is crucial to differential redox signaling, particularly by ascorbate and glutathione.

High CO2 primes plant biotic stress defences through redox-linked pathways

Growth of Arabidopsis at high CO2 activates the salicylic acid pathway and other defense reactions through pathways that involve cytosolic NADP-linked dehydrogenases. Industrial activities have caused tropospheric CO2 concentrations to increase over the last two centuries, a trend that is predicted to continue for at least the next several decades. Here, we report that growth of plants in a CO2-enriched environment activates responses that are central to defense against pathogenic attack. Salicylic acid accumulation was triggered by high-growth CO2 in Arabidopsis (Arabidopsis thaliana) and other plants such as bean (Phaseolus vulgaris). A detailed analysis in Arabidopsis revealed that elevated CO2 primes multiple defense pathways, leading to increased resistance to bacterial and fungal challenge. Analysis of gene-specific mutants provided no evidence that activation of plant defense pathways by high CO2 was caused by stomatal closure. Rather, the activation is partly linked to metabolic effects involving redox signaling. In support of this, genetic modification of redox components (glutathione contents and NADPH-generating enzymes) prevents full priming of the salicylic acid pathway and associated resistance by high CO2. The data point to a particularly influential role for the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, a cytosolic enzyme whose role in plants remains unclear. Our observations add new information on relationships between high CO2 and oxidative signaling and provide novel insight into plant stress responses in conditions of increased CO2.

Lighting the fuse on toxic TNT

An enzyme that helps control reactive oxidants sensitizes plants to TNT pollution [Also see Research Article by Johnston et al.] Most of the oxygen used by aerobic cells is safely converted to water, but a small part can be transformed into unstable derivatives called reactive oxygen species (ROS). Although dangerous to life, ROS are also important signaling messengers that provide information on the cellular environment. They are implicated in human diseases and in plant development and environmental responses. Cells can live with ROS and exploit them as signals because they use antioxidant systems to regulate their accumulation and so avoid the oxidative stress they can cause. Plant antioxidant networks are complex, and there is keen interest in dissecting the biological roles of the many enzymes and metabolites that may be involved. On page 1072 of this issue, Johnston et al. report a role for a specific member of the antioxidant enzyme family in conferring sensitivity to soil-borne pollutants, including the explosive 2,4,6-trinitrotoluene (TNT), on the model plant Arabidopsis (1).

Climate Change, CO2, and Defense: The Metabolic, Redox, and Signaling Perspectives

Ongoing human-induced changes in the composition of the atmosphere continue to stimulate interest in the effects of high CO2 on plants, but its potential impact on inducible plant defense pathways remains poorly defined. Recently, several studies have reported that growth at elevated CO2 is sufficient to induce defenses such as the salicylic acid pathway, thereby increasing plant resistance to pathogens. These reports contrast with evidence that defense pathways can be promoted by photorespiration, which is inhibited at high CO2. Here, we review signaling, metabolic, and redox processes modulated by CO2 levels and discuss issues to be resolved in elucidating the relationships between primary metabolism, inducible defense, and biotic stress resistance.