ALBERT

Notes: Abstract To cope with environmental fluctuations and to prevent invasion by pathogens, plant metabolism must be flexible and dynamic. Active oxygen species, whose formation is accelerated under stress conditions, must be rapidly processed if oxidative damage is to be averted. The lifetime of active oxygen species within the cellular environment is determined by the antioxidative system, which provides crucial protection against oxidative damage. The antioxidative system comprises numerous enzymes and compounds of low molecular weight. While research into the former has benefited greatly from advances in molecular technology, the pathways by which the latter are synthesized have received comparatively little attention. The present review emphasizes the roles of ascorbate and glutathione in plant metabolism and stress tolerance. We provide a detailed account of current knowledge of the biosynthesis, compartmentation, and transport of these two important antioxidants, with emphasis on the unique insights and advances gained by molecular exploration.

Notes: Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l-galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.

Notes: While the chemical nature of reactive oxygen species (ROS) dictates that they are potentially harmful to cells, recent genetic evidence suggests that in planta purely physicochemical damage may be much more limited than previously thought. The most potentially deleterious effect of ROS under most conditions is that at high concentrations they trigger genetically programmed cell suicide events. Moreover, because plants use ROS as second messengers in signal transduction cascades in processes as diverse as mitosis, tropisms and cell death, their accumulation is crucial to plant development as well as defence. Direct ROS signal transduction will ensue only if ROS escape destruction by antioxidants or are otherwise consumed in a ROS cascade. Thus, the major low molecular weight antioxidants determine the specificity of the signal. They are also themselves signal-transducing molecules that can either signal independently or further transmit ROS signals. The moment has come to re-evaluate the concept of oxidative stress. In contrast to this pejorative or negative term, implying a state to be avoided, we propose that the syndrome would be more usefully described as ‘oxidative signalling’, that is, an important and critical function associated with the mechanisms by which plant cells sense the environment and make appropriate adjustments to gene expression, metabolism and physiology.

Notes: The effects of salicylic acid (SA) and hydrogen peroxide (H2O2) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4-week-old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H2O2 were estimated at the following harvest points: stage (a) 4-week-old in vitro plants treated with either 0.1 mM SA or 5 mM H2O2; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4-h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H2O2 accumulation only in Atlantic at stage (a); (4) H2O2 enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H2O2 treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H2O2 accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H2O2 accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA-induced tolerance in Alpha was not accompanied by H2O2 accumulation. H2O2 was able to induce freezing tolerance only in Atlantic, even though H2O2 accumulated in both cultivars following this treatment.

Notes: The light-dependent generation of active oxygen species is termed photooxidative stress. This can occur in two ways: (1) the donation of energy or electrons directly to oxygen as a result of photosynthetic activity; (2) exposure of tissues to ultraviolet irradiation. The light-dependent destruction of catalase compounds the problem. Although generally detrimental to metabolism, superoxide and hydrogen peroxide may serve useful functions if rigorously controlled and compartmentalised. During photosynthesis the formation of active oxygen species is minimised by a number of complex and refined regulatory mechanisms. When produced, active oxygen species are eliminated rapidly by efficient antioxidative systems. The chloroplast is able to use the production and destruction of hydrogen peroxide to regulate the thermal dissipation of excess excitation energy. This is an intrinsic feature of the regulation of photosynthetic electron transport. Photoinhibition and photooxidation only usually occur when plants are exposed to stress. Active oxygen species are part of the alarm-signalling processes in plants. These serve to modify metabolism and gene expression so that the plant can respond to adverse environmental conditions, invading organisms and ultraviolet irradiation. The capacity of the antioxidative defense system is often increased at such times but if the response is not sufficient, radical production will exceed scavenging and ultimately lead to the disruption of metabolism. Oxidative damage arises in high light principally when the latter is in synergy with additional stress factors such as chilling temperatures or pollution. Environmental stress can modify the photooxidative processes in various ways ranging from direct involvement in light-induced free radical formation to the inhibition of metabolism that renders previously optimal light levels excessive. It is in just such situations that the capacity for the production of active oxygen species can exceed that for scavenging by the antioxidative defense systems. The advent of plant transformation, however, may have placed within our grasp the possibility of engineering greater stress tolerance in plants by enhancement of the antioxidative defence system.

Notes: Plants adapt to environmental stresses through specific genetic responses. The molecular mechanisms associated with signal transduction, leading to changes in gene expression early in the stress response, are largely unknown. It is clear, however, that gene expression associated with acclimatory responses is sensitive to the redox state of the cell. Of the many components which contribute to the redox balance of the cell, two factors have been shown to be crucial in mediating stress responses. Thiol/disulphide exchange reactions, particularly involving the glutathione pool and the generation of the oxidant H2O2, are central components of signal transduction in both environmental and biotic stresses. These molecules are multifunctional triggers, modulating metabolism and gene expression. Both are able to cross biological membranes and diffuse or be transported long distances from their sites of origin. Glutathione and H2O2 may act alone or in unison, in intracellular and systemic signalling systems, to achieve acclimation and tolerance to biotic and abiotic stresses.

Notes: The terminal step of glutathione (GSH) synthesis is the condensation of γ-glutamyl-cysteine (γ-EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ-EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ-EC and GSH was studied in excised leaves and leaf discs from untransformed poplar (Populus tremula×P. alba) and poplar overexpressing γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2). Poplars strongly overexpressing γ-ECS (ggs28) had enhanced levels of γ-EC and GSH compared to untransformed poplars. The relationship between γ-EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50-fold higher than γ-EC. On darkening, γ-EC accumulated markedly and GSH declined, so that the GSH:γ-EC ratio was close to 1. These dark-induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ-EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ-EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ-EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.

Notes: Development-dependent changes in fruit antioxidants were examined in the exocarp (epidermal and hypodermal tissues) of the monogenic recessive tomato (Lycopersicon esculentum L.) mutant high pigment (hp-1) and its wild-type parent ‘Rutgers’ grown under non-stress conditions in a greenhouse. The hp-1 mutant was chosen for this study because the reportedly higher lycopene and ascorbic acid (AsA) contents of the fruit may alter its tolerance to photooxidative stress. Throughout most of fruit development, reduced AsA concentrations in the exocarp of hp-1 were 1.5 to 2.0 times higher than in ‘Rutgers’, but total glutathione concentrations were similar in both genotypes. Only in ripe red fruit were reduced AsA and total glutathione concentrations lower in hp-1 than in ‘Rutgers’. The redox ratios (reduced : reduced + oxidized) of AsA in hp-1 and ‘Rutgers’ exocarps were similar and usually 〉 0.9, however, the redox ratio of glutathione was lower in hp-1 than in ‘Rutgers’ throughout development. Lycopene concentrations in ripe red fruit were about 5 times higher in hp-1 than in ‘Rutgers’. Large increases in the specific enzyme activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) occurred during fruit development in both genotypes, with an inverse relationship between the activities of these enzymes and chlorophyll content. Glutathione reductase (EC 1.6.4.2) and MDHAR-specific activities were higher in hp-1 than ‘Rutgers’ only at the later stages of fruit development. Dehydroascorbate reductase (EC 1.8.5.1) activities, however, were usually higher in ‘Rugters’ than in hp-1. Catalase (CAT, EC 1.11.1.6) activities increased with fruit development until the fruit were orange/light red, when CAT was higher in ‘Rutgers’ than in hp-1, but then declined in the ripe red fruit of both genotypes. These results suggest that elevated AsA in the exocarp of hp-1 fruit early in fruit development may increase the tolerance of hp-1 fruit to photooxidative injury at that time, but the increasing activities of antioxidant enzymes appear to be developmentally associated with fruit ripening.

Notes: Chloroplasts and mitochondria are the powerhouses of photosynthetic cells. The oxidation-reduction (redox) cascades of the photosynthetic and respiratory electron transport chains not only provide the driving forces for metabolism but also generate redox signals, which participate in and regulate every aspect of plant biology from gene expression and translation to enzyme chemistry. Plastoquinone, thioredoxin and reactive oxygen have all been shown to have signalling functions. Moreover, the intrinsic involvement of molecular oxygen in electron transport processes with the inherent generation of superoxide, hydrogen peroxide and singlet oxygen provides a repertoire of additional extremely powerful signals. Accumulating evidence implicates the major redox buffers of plant cells, ascorbate and glutathione, in redox signal transduction. The network of redox signals from energy-generating organelles orchestrates metabolism to adjust energy production to utilization, interfacing with hormone signalling to respond to environmental change at every stage of plant development.

Notes: We studied photoinhibition in two cultivars of tobacco (Nicotiana tabacum L.) expressing the bacterial gor gene in the cytosol and in four lines of poplar (Populus tremula×P. alba) expressing the FeSOD gene of Arabidopsis thaliana in the chloroplast. The respective total activities of glutathione reductase (EC 1.6.4.2) in leaves of gor tobaccos and superoxide dismutase (EC 1.15.1.1) in the FeSOD poplars were 5–8 times higher than in the respective untransformed control plants. Leaves of control and transformed plants were subjected to high-light stress at 20°C, and photoinhibition of photosystem II (PSII) was measured by oxygen evolution and chlorophyll fluorescence. The leaves were illuminated both in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis. In both cases, the time course of loss of PSII activity was identical in plants overproducing superoxide dismutase (SOD) and in the untransformed controls, suggesting that the ability to convert superoxide to hydrogen peroxide is not a limiting factor in protection against photoinhibition, or in the repair of photoinhibitory damage or that the site of O2− production is not accessible to the transgene product. The rate constant of photoinhibition, measured in lincomycin-treated leaves, was smaller in glutathione reductase (GR) overproducing tobacco cv. Samsun than in the respective wild-type, but this difference was not seen in cv. Bel W3. The steady-state level of PSII activity measured when the PSII repair cycle was allowed to equilibrate with photoinhibitory damage under high light was not higher in the GR overproducing cv. Samsun, suggesting that the repair of photoinhibitory damage was not enhanced in plants overproducing GR in the cytosol.