Ethylene signaling triggered by low concentrations of ascorbic acid regulates biomass accumulation in Arabidopsis thaliana.

Ascorbic acid (AA) is a major redox buffer in plant cells. The role of ethylene in the redox signaling pathways that influence photosynthesis and growth was explored in two independent AA deficient Arabidopsis thaliana mutants (vtc2-1 and vtc2-4). Both mutants, which are defective in the AA biosynthesis gene GDP-L-galactose phosphorylase, produce higher amounts of ethylene than wt plants. In contrast to the wt, the inhibition of ethylene signaling increased leaf conductance, photosynthesis and dry weight in both vtc2 mutant lines. The AA-deficient mutants showed altered expression of genes encoding proteins involved in the synthesis/responses to phytohormones that control growth, particularly auxin, cytokinins, abscisic acid, brassinosterioids, ethylene and salicylic acid. These results demonstrate that AA deficiency modifies hormone signaling in plants, redox-ethylene interactions providing a regulatory node controlling shoot biomass accumulation.

Regulation of calcium signalling and gene expression by glutathione.

The glutathione redox couple is an information-rich redox buffer that interacts with numerous cellular components. To explore the role of glutathione in redox signalling, leaf contents were increased either chemically, by feeding reduced glutathione (GSH), or genetically, by over-expressing the first enzyme of the GSH biosynthetic pathway, gamma-glutamylcysteine synthetase (gamma-ECS). Leaf discs were also fed glutathione disulphide (GSSG), leading to increases in both GSH and GSSG. The effects of increases in GSH were compared with non-specific changes in leaf thiol status induced by feeding dithiothreitol (DTT) or the monothiol beta-mercaptoethanol (beta-ME). Photosynthesis measurements showed that none of the feeding treatments greatly disrupted leaf physiology. Transgenic plants expressing aequorin were used to analyse calcium signatures during the feeding treatments. Calcium release occurred soon after the onset of GSH or GSSG feeding, but was unaffected by DTT or beta-ME. Pathogenesis-related protein 1 (PR-1) was induced both in the gamma-ECS overexpressors and by feeding GSH, but not GSSG. Feeding DTT also induced PR-1. Key transcripts encoding antioxidative enzymes were much less affected, although glutathione synthetase was suppressed by feeding thiols or GSSG. It is concluded that modulation of glutathione contents transmits information through diverse signalling mechanisms, including (i) the establishment of an appropriate redox potential for thiol/disulphide exchange and (ii) the release of calcium to the cytosol.

The functions of inter- and intracellular glutathione transport systems in plants.

Glutathione is one of the major redox buffers in most aerobic cells, and it has a broad spectrum of functions in plants. Recent discoveries implicate this thiol peptide in signalling and cellular homeostasis. Glutathione can sense intracellular redox status: perturbations of glutathione reduction state are transduced into changes in gene expression. This central role demands precise control of both the concentration and the reduction state of glutathione in different compartments. In addition to the regulation of glutathione biosynthesis and redox state, attention is now turning to the role of glutathione transporters.

Glutamine and alpha-ketoglutarate are metabolite signals involved in nitrate reductase gene transcription in untransformed and transformed tobacco plants deficient in ferredoxin-glutamine-alpha-ketoglutarate aminotransferase.

Transformed tobacco (Nicotiana tabacum L.) plants with varying activities of the key enzyme of ammonia assimilation, ferredoxin-glutamine-alpha-ketoglutarate aminotransferase (Fd-GOGAT; EC 1.4.7.1), were used to examine the roles of ammonium, glutamine (Gln) and alpha-ketoglutarate (alpha-KG) in the regulation of nitrate reductase (NR; EC 1.6.6.1) transcript abundance. In wild-type leaf discs, NR mRNA abundance was increased following feeding with NO3-, sucrose and alpha-KG and decreased by feeding Gln. In air, leaves with decreased GOGAT accumulated Gln and alpha-KG simultaneously; this was accompanied by increased NR transcripts. The inhibition of NR transcription by Gln observed in leaf-disc experiments was therefore not observed in the low-Fd-GOGAT plants that accumulate Gln in vivo. The results suggest that the negative effect of Gln on NR transcript abundance was offset by high alpha-KG and that the relative amounts of alpha-KG and Gln are more important in controlling NR gene transcription than the concentration of either metabolite alone.

Plant antioxidants: colour me healthy.

Plants make a variety of compounds in response to environmental stress, many of which function as antioxidants when consumed. The plants' own defences against oxidative stress can be used for your benefit, prolonging your life by acquiring their protection. By eating plenty of vegetables and fruit, you may help to significantly reduce the risk of many age-related degenerative diseases.

Prospects for enhancement of the soluble antioxidants, ascorbate and glutathione.

Ascorbic acid (vitamin C) and the tripeptide thiol, glutathione gamma-glutamyl cysteinyl glycine (glutathione) are the major low molecular weight soluble antioxidants in plant cells. The pathway of glutathione biosynthesis is similar in animals and plants while that of ascorbate biosynthesis differs considerably between the two kingdoms. The potential for obtaining substantial constitutive changes in the tissue contents of these antioxidants by manipulation of the biosynthetic enzymes has been demonstrated. Moreover, the concentrations of ascorbate and glutathione are greatly modified in response to a variety of environmental triggers, particularly those that cause increased oxidative stress. It is essential that the signals and associated signal transduction pathways that trigger enhanced antioxidant accumulation are elucidated as these offer an important alternative means of achieving greater nutritional value in edible plant organs.

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.

Antisense suppression of 2-cysteine peroxiredoxin in Arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism.

The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool.

Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves.

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.

Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures.

In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co-ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 degrees C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.

Homeostasis of adenylate status during photosynthesis in a fluctuating environment.

This review describes and assesses pathways likely to influence and stabilize the ATP/reductant balance during whole cell photosynthesis. The sole reductive step of the Calvin cycle occurs during the conversion of 3-phosphoglycerate to triose phosphate. Photophosphorylation linked to this reaction can undoubtedly supply most of the ATP required by the Calvin cycle and other chloroplastic reactions. Small but crucial contributions must come from several other pathways, some of which involve co-operation between the chloroplast and the rest of the cell. Extrachloroplastic compartments can contribute to chloroplastic ATP requirements by supplying ATP directly or, probably more significantly, by accepting reducing equivalents and so supporting ATP synthesis within the chloroplast.

Early H(2)O(2) accumulation in mesophyll cells leads to induction of glutathione during the hyper-sensitive response in the barley-powdery mildew interaction.

H(2)O(2) production and changes in glutathione, catalase, and peroxidase were followed in whole-leaf extracts from the susceptible (AlgS [Algerian/4* (F14) Man.(S)]; ml-a1 allele) and resistant (AlgR [Algerian/4* (F14) Man.(R)]; Ml-a1 allele) barley (Hordeum vulgare) isolines between 12 and 24 h after inoculation with powdery mildew (Blumeria graminis [DC]. Speer [syn. Erysiphe graminis DC] f.sp hordei Marchal). Localized papilla responses and cell death hypersensitive responses were not observed within the same cell. In hypersensitive response sites, H(2)O(2) accumulation first occurred in the mesophyll underlying the attacked epidermal cell. Subsequently, H(2)O(2) disappeared from the mesophyll and accumulated around attacked epidermal cells. In AlgR, transient glutathione oxidation coincided with H(2)O(2) accumulation in the mesophyll. Subsequently, total foliar glutathione and catalase activities transiently increased in AlgR. These changes, absent from AlgS, preceded inoculation-dependent increases in peroxidase activity that were observed in both AlgR and AlgS at 18 h. An early intercellular signal precedes H(2)O(2), and this elicits anti-oxidant responses in leaves prior to events leading to death of attacked cells.

Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-gamma-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.

Simultaneous measurement of foliar glutathione, gamma-glutamylcysteine, and amino acids by high-performance liquid chromatography: comparison with two other assay methods for glutathione.

The recent production of transformed plants with enhanced capacity for glutathione synthesis has highlighted the interactions between foliar glutathione and turnover of free amino acid pools. The development of a convenient method for simultaneous measurement of glutathione, gamma-glutamylcysteine, and 16 amino acids is reported. This method utilizes derivatization of compounds with o-phthalaldehyde in the presence of 2-mercaptoethanol followed by separation using reversed-phase high-performance liquid chromatography. Eluted compounds are detected fluorimetrically. The method was tested using untransformed poplars and poplars in which foliar thiol contents have been enhanced by overexpression of gamma-glutamylcysteine synthetase. Foliar contents of glutathione determined by this method were comparable to those measured in common extracts by two other techniques.

Overexpression of iron superoxide dismutase in transformed poplar modifies the regulation of photosynthesis at low CO2 partial pressures or following exposure to the prooxidant herbicide methyl viologen.

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 microL L-1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 micromol m-2 s-1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6(o)C the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

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

The hybrid poplar (Populus tremula x P. alba) was transformed to express the Escherichia coli gene for gamma-glutamylcysteine synthetase (EC 6.3.2.2: gamma-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 gamma-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS). Using the antiserum prepared against the purified His-tagged E. coli gamma-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 gamma-ECS activities of the gamma-ECS transformants were in quantitative agreement with the protein levels. Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher gamma-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold. The lines strongly overexpressing gamma-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), gamma-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold). Foliar thiol contents in ggs17 were no different to those of untransformed plants.

Synthesis of Glutathione in Leaves of Transgenic Poplar Overexpressing [gamma]-Glutamylcysteine Synthetase.

Internode stem fragments of the poplar hybrid Populus tremula x Populus alba were transformed with a bacterial gene (gshl) for [gamma]-glutamylcysteine synthetase ([gamma]-ECS) targeted to the cytosol. Lines overexpressing [gamma]-ECS were identified by northern analysis, and the transformant with the highest enzyme activity was used to investigate the control of glutathione synthesis. Whereas foliar [gamma]-ECS activity was below the limit of detection in untransformed plants, activities of up to 8.7 nmol mg-1 protein min-1 were found in the transformant, in which the foliar contents of [gamma]-glutamylcysteine ([gamma]-EC) and glutathione were increased approximately 10- and 3-fold, respectively, without affecting either the reduction state of the glutathione pool or the foliar cysteine content. A supply of exogenous cysteine to leaf discs increased the glutathione content from both transformed and untransformed poplars, and caused the [gamma]-EC content of the transformant discs to increase still further. The following conclusions are drawn: (a) the native [gamma]-ECS of untransformed poplars exists in quantities that are limiting for foliar glutathione synthesis; (b) foliar glutathione synthesis in untransformed poplars is limited by cysteine availability; (c) in the transformant interactions between glutathione synthesis and cysteine synthesis operate to sustain the increased formation of [gamma]-EC and glutathione; and (d) the foliar glutathione content of the transformant is restricted by cysteine availability and by the activity of glutathione synthetase.

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO2 fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.