TY - JOUR
AU1 - Schwanz, Peter
AU2 - Polle, Andrea
AB - Abstract Maritime pine (Pinus pinaster), a drought‐avoiding species, contained 2–4‐fold lower activities of superoxide dismutase, ascorbate peroxidase, catalase, dehydroascorbate reductase, and glutathione reductase than pendunculate oak (Quercus robur), a drought‐tolerant species. The levels of ascorbate, monodehydroascorbate radical reductase activity, and glutathione in pine needles were similar to those in oak leaves. In both species the development of drought stress, characterized by decreasing predawn water potentials, caused gradual reductions in antioxidant protection, increased lipid peroxidation, increased oxidation of ascorbate and glutathione and in pine also significant loss in soluble proteins and carotenoids. These results support the idea that increased drought‐tolerance in oak as compared with pine is related to increased biochemical protection at the tissue level. To test the hypothesis that elevated CO2 ameliorated drought‐induced injury, young oak and pine trees acclimated to high CO2 were subjected to drought stress. Analysis of plots of enzymatic activities and metabolites against predawn water potentials revealed that the drought stress‐induced decreases in antioxidant protection and increases in lipid peroxidation were dampened at high CO2. In pine, protein and pigment degradation were also slowed down. At high CO2, superoxide dismutase activities increased transiently in drought‐stressed trees, but collapsed in pine faster than in oak. These observations suggest that the alleviation of drought‐induced injury under elevated CO2 is related to a higher stability of antioxidative enzymes and an increased responsiveness of SOD to stressful conditions. This ameliorating mechanism existed independently from the effects of elevated CO2 on plant water relations and is limited within a species‐specific metabolic window. Ascorbate, antioxidants, glutathione, drought stress, climate change, oxidative stress. SOD, superoxide dismutase Introduction Water deficit is probably the most important stress factor determining plant growth and productivity world‐wide. At the same time, water use and growth of plants are strongly influenced by climatic conditions and atmospheric CO2 concentrations (Chaves and Pereira, 1992). Human activities cause increasing atmospheric concentrations of green house gases, including CO2, thus, driving global warming (Hasselman, 1997). The extent of temperature increase is not known, but model predictions suggest changes in precipitation patterns and lower water availability for northern mid‐latitudes, especially during the growth phase (Roeckner, 1992). Growth under elevated atmospheric CO2 concentrations generally resulted in improved performance of plants suffering from drought stress as compared with those grown under ambient CO2 (Hsiao and Jackson, 1999). At the cellular level the biochemical mechanisms underlying CO2‐mediated protection are still poorly understood. It has been shown that the photosynthetic apparatus per se is highly resistant against drought‐induced injury (Kaiser, 1987; Cornic et al., 1989; Quick et al., 1992). Limited water supply results in stomatal closure and restricts net CO2 fixation rates. To protect the photosynthetic apparatus from photo‐oxidative destruction plants must dissipate excess light energy. Protection may be afforded by down‐regulation of the photochemical efficiency via the action of the xanthophyll cycle (Demmig‐Adams et al., 1996) or by maintenance of electron flux involving alternative pathways as ‘redox valves’, for example, photorespiration and the Mehler‐peroxidase reaction (Foyer and Harbinson, 1994; Asada, 1999). Both pathways, the Mehler reaction and photorespiration, imply increased production rates of potentially toxic oxygen species such as \(O_{2}^{{\cdot}{-}}\) and H2O2. In fact, electron spin resonance studies have shown that drought‐stressed plants displayed elevated concentrations and production rates of superoxide radicals (Price and Hendry, 1991; Quartacci and Navaro‐Izzo, 1992). Furthermore, drought stress may cause an uncontrolled release of transition metals like Cu or Fe in cells, thereby, increasing the risk of oxidative injury through Fenton‐type reactions (Moran et al., 1994). It has been suggested that protective mechanisms are less needed under elevated CO2 because of a relatively higher internal availability of CO2 as the terminal electron acceptor of photosynthesis in comparison with plants grown under ambient CO2 (Polle, 1996). Antioxidative systems provide protection against the toxic effects of activated oxygen species. Important components of these protective systems are enzymatic defences such as superoxide dismutases (SODs) and catalases as well as peroxidases which scavenge \(O_{2}^{{\cdot}{-}}\) and H2O2, respectively (Noctor and Foyer, 1998). Metabolites such as ascorbate, glutathione and tocopherol also contribute to control the levels of activated oxygen in plant tissues (Noctor and Foyer, 1998). The maintenance of the reduced, i.e. active form of antioxidants is achieved by the reduction of ascorbate free radicals by the activity of monodehydroascorbate radical reductase and consumption of NAD(P)H (Borracino et al., 1989) or by the operation of the ascorbate‐glutathione cycle (Foyer and Halliwell, 1976). This cycle catalyses the reduction of dehydroascorbate by dehydroascorbate reductase activity and oxidation of glutathione and, in turn, the reduction of glutathione disulphide by glutathione reductase activity and consumption of NADPH (Foyer and Halliwell, 1976). In several herbaceous plant species, antioxidative systems were induced in response to drought stress suggesting that water deficits required increased capacities of protective systems for stress compensation (Smirnoff, 1993). For example, a drought‐tolerant maize strain responded with significant increases in antioxidants to water deficits, whereas a susceptible strain maintained a lower protection from oxidants (Pastori and Trippi, 1992). In drying acorns of Quercus robur a loss in viability was associated with a significant reduction in antioxidative defences and the appearance of free radicals (Hendry et al., 1992). A clear picture has not yet emerged as to whether or not elevated levels or enhanced reactivity of antioxidative systems contribute to the positive effects of elevated CO2 in plants suffering from oxidative stress (Rao et al., 1995; McKee et al., 1997; Niewiadomska et al., 1999). In previous studies with oak and pine seedlings grown under elevated CO2 concentrations of 700 μmol mol−1, significant decreases in SOD activities and variable CO2 responses of other antioxidative systems have been observed (Schwanz et al., 1996a, b; Schwanz and Polle, 1998). When trees grown under elevated CO2 were drought‐stressed, foliar SOD activities increased, whereas they decreased in leaves of trees grown under ambient CO2 (Schwanz et al., 1996a). Based on these observations it was suggested that trees grown under elevated CO2 might have an enhanced metabolic flexibility to encounter drought‐induced oxidative stress (Schwanz et al., 1996a). To generalize such conclusions it would be worthwhile to study stress responses under more than one level of CO2‐enhancement. Futhermore, the metabolic window, in which such positive acclimatory responses may operate, has not been determined nor is it known whether CO2 results in ameliorative adjustments of antioxidants in species differing in their drought susceptibility. The aim of the present studies was to investigate foliar antioxidative systems in two species, pendunculate oak (Quercus robur) and maritime pine (Pinus pinaster) as models for drought‐tolerant and drought‐avoiding trees, respectively (Picon et al., 1996; Epron and Dreyer, 1993a, b). Young trees were grown under ambient (350 μmol mol−1) or elevated CO2 concentrations of 700 or 1200 μmol mol−1, respectively. High CO2 concentrations were chosen to exacerbate potentially positive effects during the gradual development of severe water deficits. To test the hypothesis that high CO2 had ameliorating effects, beside the levels of antioxidative systems, indicators for foliar vitality such as protein, pigment and malondialdehyde concentrations were determined in addition to the relative water content of the leaves and the predawn water potentials of the trees. Materials and methods Growth of plants, drought treatment, and harvest Acorns (Quercus robur, L. provenance Manoncourt) were peeled, soaked in water for 2 d, and planted into a peat‐sand mixture (1/19) containing a complete fertilization (4.5 g l−1 of slow releasing fertilizer, N/P/K 13/13/13+oligoelements, Nutricote 100, France) in 35 cm long tubes (diameter 8 cm). One‐year‐old container‐grown pine seedlings (Pinus pinaster Ait, provenance Les Landes) obtained from a greenhouse (Freiburg, Germany) were planted into 3.0 l pots containing the same soil–fertilizer mixture and were cultivated under the same conditions as the oaks. The pots were transferred to phytochambers (Heraeus Voetsch, Balingen, Germany) and inoculated with mycorrhizal fungi (Laccaria laccata). The plants were grown with a day/night regime of 14/10 h, an air temperature of 20/15 °C, humidity in air of 70/80%, and either ambient (350±30 μmol mol−1) or elevated CO2 concentrations (experiment 1: 700±35 μmol mol−1 and experiment 2: 1200± 308 μmol mol−1). In experiment 1 a total number of 16 oak and 40 pine trees and in experiment 2 a total of 40 oak and 70 pine trees were used. Light was provided by fluorescent (L58W/77 Osram München, Germany) alternating with TDL 58W/95o lamps (Philips Hamburg, Germany) and incandescent lamps (Osram Krypton q428) at PAR of 250 μmol m−2 s−1 (Li 185 B, Quantum Radiometer photometer, LiCor, Lincoln, Nebraska, USA) at pot height. The plants were grown for about 10 weeks under ambient and elevated CO2 concentrations, respectively, before exposure to drought stress started by withholding water. Control trees were watered regularly. During exposure to drought stress up to several weeks (Figs 1, 2), controls and stressed plants were harvested at regular intervals at the end of the night. The plants were immediately used for the determination of the water potential using a Scholander pressure chamber (Soil moisture, Santa Barbara, CA). When severe water stress was indicated by water potentials of −3 to −4 MPa, the plants were rewatered. For biochemical analyses, the youngest needle age class of pine and the oldest leaves of oak were harvested and used either immediately for extractions or were stored at −80 °C until analysis. Biochemical analyses Extraction of enzymes: Fresh oak leaves were ground to a fine powder in liquid nitrogen. Aliquots of 200 mg of frozen leaf powder were transferred into 10 ml of extraction buffer containing 100 mM KH2PO4/K2HPO4, pH 7.8, 2% Triton X‐100, 5 mM ascorbate, and 400 mg insoluble polyvinylpolypyrrolidone, mixed for 1 min (Vibrofix, Janke & Kunkel, IKA Labortechnik, Staufen, Germany) and incubated on ice for 30 min. The homogenate was centrifuged for 30 min (48 400 g, 4 °C). Pine needles were extracted in the same manner as oak leaves but in a buffer containing 1% Triton X‐100 (cf. Schwanz et al., 1996a). Glutathione reductase EC 1.6.4.2, ascorbate peroxidase EC 1.11.1.11, monodehydroascorbate radical reductase EC 1.1.5.4 and dehydroascorbate reductase activities EC 1.8.5.1 were determined in extracts prepared from fresh leaves. The supernatant was gel‐filtered over Sephadex G‐25 columns (PD‐10, Pharmacia, Freiburg, Germany) which had been equilibrated with 100 mM KH2PO4/K2HPO4, pH 7.0 (containing 1 mM ascorbate for the determination of ascorbate peroxidase). Extracts for the determination of catalase EC 1.11.1.6, unspecific guaiacol peroxidase EC 1.11.1.7, superoxide dismutase activities EC 1.15.1.1, and the soluble protein content were gel‐filtered over Sephadex G‐25 (PD‐10 columns, Pharmacia, Freiburg, Germany) with 20 mM KH2PO4/K2HPO4, pH 7.8, 0.5% Triton and stored at −20 °C for analysis. Residual leaf material was frozen in liquid nitrogen and stored at −80 °C until analyses of antioxidants and basic parameters. Fig. 1. View largeDownload slide Effect of drought stress on the predawn water potential (A) and on SOD activities (B) in pine (Pinus pinaster) grown under ambient (•‐•) and elevated CO2 (○‐○) concentrations of 350 and 700 μmol mol−1. The plants were immediately rewatered after predawn water potentials below −3 MPa had been measured (see arrow). Data indicate means (n=3, ±SD). Unstressed controls maintained water potentials and SOD activities similar to those found at day 0 (not shown). Fig. 1. View largeDownload slide Effect of drought stress on the predawn water potential (A) and on SOD activities (B) in pine (Pinus pinaster) grown under ambient (•‐•) and elevated CO2 (○‐○) concentrations of 350 and 700 μmol mol−1. The plants were immediately rewatered after predawn water potentials below −3 MPa had been measured (see arrow). Data indicate means (n=3, ±SD). Unstressed controls maintained water potentials and SOD activities similar to those found at day 0 (not shown). Fig. 2. View largeDownload slide Changes of the predawn water potentials (A, C) and of SOD activities (B, D) in oak (A, B, Quercus robur) and pine (C, D, Pinus pinaster) grown under ambient (•‐•) and high CO2 (○‐○) concentrations of 350 and 1200 μmol mol−1 and subjected to drought stress. The plants were immediately rewatered after predawn water potentials below −3 MPa had been measured (see arrow). Data indicate means (n=3 or 4). For clarity, error bars have been omitted (for statistical treatment of the data cf. Table 2). At each measuring day predawn water potential and SOD activities were also determined in well‐watered trees. To indicate the range of fluctuation the data were taken together and are shown as box plots at the beginning of the curves. Grey box: ambient CO2, White box: elevated CO2. Fig. 2. View largeDownload slide Changes of the predawn water potentials (A, C) and of SOD activities (B, D) in oak (A, B, Quercus robur) and pine (C, D, Pinus pinaster) grown under ambient (•‐•) and high CO2 (○‐○) concentrations of 350 and 1200 μmol mol−1 and subjected to drought stress. The plants were immediately rewatered after predawn water potentials below −3 MPa had been measured (see arrow). Data indicate means (n=3 or 4). For clarity, error bars have been omitted (for statistical treatment of the data cf. Table 2). At each measuring day predawn water potential and SOD activities were also determined in well‐watered trees. To indicate the range of fluctuation the data were taken together and are shown as box plots at the beginning of the curves. Grey box: ambient CO2, White box: elevated CO2. Determination of enzymatic activities: Standard enzymatic assays were performed in a total volume of 1 ml and at 25 °C according to the following methods: ascorbate peroxidase (Nakano and Asada, 1987), monodehydroascorbate radical reductase (Borraccino et al., 1989), dehydroascorbate reductase (Dalton et al., 1986), glutathione reductase (Foyer and Halliwell, 1976), guaiacol peroxidase (Polle et al., 1990), and catalase (Aebi, 1983). Superoxide dismutase activity was determined by the inhibition of the formation of epinephrine at pH 10.4 and 30 °C (Kröniger et al., 1995). One unit of superoxide dismutase activity was defined as the amount of enzyme that inhibited the epinephrine formation by 50%. Extraction and determination of antioxidants: The frozen foliage was ground to a fine powder in liquid nitrogen and extracted in 0.1 N HCl, 4% polyvinylpolypyrrolidone and 5 μM EDTA as described previously (Schwanz et al., 1996a). Ascorbate, ascorbate+dehydroascorbate (referred to as ‘total’ ascorbate), glutathione, and oxidized glutathione were measured by HPLC (Polle et al., 1992; Anderson et al., 1992; Schupp and Rennenberg, 1988; Gorin et al., 1966). Recoveries of antioxidants ranged between 88% and 97%. Basic parameters and statistical analysis The dry matter of leaves was determined after drying for 72 h at 80 °C. Pigments were extracted in 80% acetone and their concentrations were calculated with the extinction coefficients given by Lichtenthaler and Wellburn (Lichtenthaler and Wellburn, 1983). Soluble protein was determined with the bicinchoninic acid reagent (Pierce, München, Germany). Bovine serum albumin served as standard. Malondialdehyde was determined according to Peever and Higgins (Peever and Higgins, 1988). Statistical analysis was performed with the software STATGRAPHICs (STN, St Louis, USA) using multiple analysis of variance followed by an LSD test to evalute significant treatment effects. Linear regression curves, coefficients (R), and significance levels (P) were calculated by Origin® (Microcal, Northhampton, MA). Results Changes of SOD activities in response to drought in pine and oak grown under elevated and ambient CO2 Young pine trees grown at ambient or 700 μmol mol−1 CO2 were subjected to drought stress by withholding water (Fig. 1A, B). The development of drought stress was determined by measuring the predawn water potential of the plants. After 3 weeks the predawn water potential of drought‐stressed pines under ambient CO2 reached about −3 MPa and was 22% lower than that of plants grown under elevated CO2 (Fig. 1A; PDS≤0.001). After rewatering the water potential showed a fast phase of recovery within 1 d and approached levels of well‐watered plants after 1 week (Fig. 1A). The changes in water potential were also accompanied by pronounced changes in the activities of SODs in needles (Fig. 1B; PDS≤0.001). Needles of pine trees grown under ambient CO2 contained higher SOD activities than those of trees grown under elevated CO2 and showed declining enzymes activities when subjected to drought stress (Fig. 1B; PCO2≤0.001). By contrast, in drought‐stressed pine trees grown under elevated CO2, SOD activities showed an almost 3‐fold increase as compared with well‐watered plants and an immediate decrease within 1 d when the trees were rewatered (Fig. 1B; IDSxCO2≤0.001). Similarly, oaks suffering from drought stress, as indicated by water potentials between −2 and −3 MPa, showed elevated or decreased SOD activities as compared with well‐watered trees under elevated CO2 (700 μmol mol−1) or under ambient CO2, respectively (Table 1). To investigate the interactions of CO2‐growth conditions and drought on antioxidative systems in more detail and to find out whether further increases in CO2 had further depressing effects on SOD activities, oak and pine trees were grown under ambient and 1200 μmol mol−1 CO2, subjected to drought stress and analysed for SOD activities (Fig. 2). Withholding water caused a strong decrease in the predawn water potential from about −0.8 MPa in watered oak seedlings to less than −4 MPa in stressed plants grown under ambient CO2 (Fig. 2A). In oak seedlings grown under elevated CO2 the decrease in predawn water potential was slower and the recovery after rewatering faster than in plants grown under ambient CO2 (Fig. 2A). Pine plants showed a slower development of drought stress than oaks but no significant differences between trees grown under elevated as compared with ambient CO2 (Fig. 2C; Table 2). Similar to previous observations in pine and oak (Fig. 1B; Schwanz and Polle, 1998; Schwanz et al., 1996a) and no matter whether the data were expressed on the basis of fresh mass, dry mass or protein, foliage of trees grown under elevated CO2 contained lower SOD activities than that of trees grown under ambient CO2 (in Table 2 data for A and PCO2). In both species drought caused significant increases in SOD activities only in plants grown at high CO2, whereas those grown under ambient CO2 shown showed gradually decreasing SOD activities (Fig. 2B, D). The positive responses of SOD in trees grown under elevated CO2 were, however, not maintained over the whole stress treatment; SOD activities started to decline after about 3 weeks of withholding water in both oak and pine, although at that time the water potentials had decreased to different levels in the two species (Fig. 2B, D). Table 1. Effect of drought stress after withholding water for 4 weeks on predawn water potentials and SOD activities in oak (Quercus robur) grown under ambient or elevated CO2 (700 μmol mol−1) in comparison with well‐watered controls (n=4, ±SD)   Predawn water potential (MPa)     SOD activity (units mg−1 protein)       Ambient CO2   Elevated CO2   Ambient CO2   Elevated CO2   Control  −0.26±0.34  −0.50±0.24  57.1±16.0  16.0±3.6  Stress  −3.20±0.82  −2.18±1.00  14.3±1.7  34.0±4.3    Predawn water potential (MPa)     SOD activity (units mg−1 protein)       Ambient CO2   Elevated CO2   Ambient CO2   Elevated CO2   Control  −0.26±0.34  −0.50±0.24  57.1±16.0  16.0±3.6  Stress  −3.20±0.82  −2.18±1.00  14.3±1.7  34.0±4.3  View Large Table 2. Results of linear regression analysis (y=A+Bx, R=regression coefficient, P=calculated level of significance) and of multiple analysis of variance of the effects of elevated CO2 (PCO2) and drought stress (PDS) and their interactions (ICO2xDS) on physiological parameters in pine (Pinus pinaster) and oak (Quercus robur) The analyses have been performed with the data shown in Figs 3–6. Significant effects at P≤0.05 have been indicated by bold letters, trends with 0.10≥P≥0.05 by italic letters. Parameter  Species  CO2  Regression analysis         Multiple analysis of variance             A   B   Ra   P   (PCO2)   (PDS)   ICO2xDS   PDWPb  Oak  A  –  –  –  –  0.366  0.01  0.254      E  –  –  –  –          Pine  A  –  –  –  –  0.080  0.01  0.971      E  –  –  –  –        SOD  Oak  A  4866  1084  0.992  0.005  0.004  0.098  0.001      E  1776  −680  −0.907  0.001          Pine  A  1588  412  0.738  0.001  0.001  0.001  0.003      E  245  −281  −0.913  0.001        CAT  Oak  A  210  56.9  0.599  0.011  0.162  0.175  0.510      E  217  51.6  0.619  0.005          Pine  A  54  13.6  0.562  0.001  0.027  0.033  0.963      E  34  8.5  0.545  0.001        GPOD  Oak  A  1461  −31  −0.026  0.921  0.730  0.147  0.218      E  1036  −688  −0.470  0.050          Pine  A  4089  −280  −0.393  0.110  0.607  0.753  0.471      E  4713  151  0.125  0.621        APOD  Oak  A  1463  352  0.747  0.003  0.366  0.026  0.059      E  857  207  0.677  0.006          Pine  A  877  203  0.643  0.001  0.003  0.002  0.754      E  701  176  0.549  0.001        MDARR  Oak  A  959  216  0.587  0.022  0.248  0.239  0.903      E  714  140  0.525  0.031          Pine  A  1160  223  0.538  0.006  0.008  0.001  0.648      E  809  147  0.489  0.001        DHAR  Oak  A  36.1  −0.5  −0.074  0.837  0.989  0.949  0.612      E  30.1  −4.3  −0.419  0.227          Pine  A  12.3  4.2  0.536  0.100  0.546  0.001  0.684      E  8.7  2.8  0.294  0.051        GR  Oak  A  124  12.4  0.568  0.086  0.197  0.079  0.041      E  85  −1.9  −0.254  0.481          Pine  A  65  8.0  0.396  0.006  0.070  0.004  0.393      E  52  4.9  0.313  0.035        ASCtot  Oak  A  25.0  4.4  0.244  0.341  0.001  0.402  0.157      E  10.2  −6.7  −0.542  0.065          Pine  A  30.5  3.6  0.316  0.190  0.451  0.012  0.788      E  30.3  5.6  0.804  0.001        rASC  Oak  A  69  11.8  0.701  0.008  0.246  0.233  0.292      E  59  9.0  0.565  0.035          Pine  A  93  12.0  0.812  0.001  0.208  0.189  0.464      E  93  10.5  0.799  0.001        GSH  Oak  A  1616  215  0.297  0.291  0.840  0.048  0.735      E  1013  −80  −0.111  0.700          Pine  A  1821  −53.5  0.086  0.750  0.141  0.595  0.094      E  1762  178  0.323  0.281        rGSSG  Oak  A  2.0  −5.0  −0.811  0.001  0.844  0.001  0.593      E  2.4  −4.2  −0.832  0.001          Pine  A  −0.5  −5.2  −0.839  0.001  0.003  0.078  0.153      E  4.5  −4.6  −0.660  0.007        RWC  Oak  A  68  12.7  0.605  0.010  0.200  0.024  0.267      E  75  6.3  0.541  0.013          Pine  A  75  7.9  0.672  0.001  0.056  0.010  0.431      E  65  4.8  0.592  0.001        MDA  Oak  A  18.9  −56.6  −0.925  0.001  0.888  0.088  0.210      E  22.1  −37.3  −0.897  0.001          Pine  A  54.2  −44.1  −0.705  0.001  0.318  0.001  0.617      E  61.2  −27.9  0.055  0.001        PRO  Oak  A  108  12.9  0.217  0.295  0.196  0.067  0.278      E  98  7.2  0.198  0.352          Pine  A  107  16.1  0.643  0.001  0.001  0.003  0.633      E  78  11.7  0.527  0.001        Parameter  Species  CO2  Regression analysis         Multiple analysis of variance             A   B   Ra   P   (PCO2)   (PDS)   ICO2xDS   PDWPb  Oak  A  –  –  –  –  0.366  0.01  0.254      E  –  –  –  –          Pine  A  –  –  –  –  0.080  0.01  0.971      E  –  –  –  –        SOD  Oak  A  4866  1084  0.992  0.005  0.004  0.098  0.001      E  1776  −680  −0.907  0.001          Pine  A  1588  412  0.738  0.001  0.001  0.001  0.003      E  245  −281  −0.913  0.001        CAT  Oak  A  210  56.9  0.599  0.011  0.162  0.175  0.510      E  217  51.6  0.619  0.005          Pine  A  54  13.6  0.562  0.001  0.027  0.033  0.963      E  34  8.5  0.545  0.001        GPOD  Oak  A  1461  −31  −0.026  0.921  0.730  0.147  0.218      E  1036  −688  −0.470  0.050          Pine  A  4089  −280  −0.393  0.110  0.607  0.753  0.471      E  4713  151  0.125  0.621        APOD  Oak  A  1463  352  0.747  0.003  0.366  0.026  0.059      E  857  207  0.677  0.006          Pine  A  877  203  0.643  0.001  0.003  0.002  0.754      E  701  176  0.549  0.001        MDARR  Oak  A  959  216  0.587  0.022  0.248  0.239  0.903      E  714  140  0.525  0.031          Pine  A  1160  223  0.538  0.006  0.008  0.001  0.648      E  809  147  0.489  0.001        DHAR  Oak  A  36.1  −0.5  −0.074  0.837  0.989  0.949  0.612      E  30.1  −4.3  −0.419  0.227          Pine  A  12.3  4.2  0.536  0.100  0.546  0.001  0.684      E  8.7  2.8  0.294  0.051        GR  Oak  A  124  12.4  0.568  0.086  0.197  0.079  0.041      E  85  −1.9  −0.254  0.481          Pine  A  65  8.0  0.396  0.006  0.070  0.004  0.393      E  52  4.9  0.313  0.035        ASCtot  Oak  A  25.0  4.4  0.244  0.341  0.001  0.402  0.157      E  10.2  −6.7  −0.542  0.065          Pine  A  30.5  3.6  0.316  0.190  0.451  0.012  0.788      E  30.3  5.6  0.804  0.001        rASC  Oak  A  69  11.8  0.701  0.008  0.246  0.233  0.292      E  59  9.0  0.565  0.035          Pine  A  93  12.0  0.812  0.001  0.208  0.189  0.464      E  93  10.5  0.799  0.001        GSH  Oak  A  1616  215  0.297  0.291  0.840  0.048  0.735      E  1013  −80  −0.111  0.700          Pine  A  1821  −53.5  0.086  0.750  0.141  0.595  0.094      E  1762  178  0.323  0.281        rGSSG  Oak  A  2.0  −5.0  −0.811  0.001  0.844  0.001  0.593      E  2.4  −4.2  −0.832  0.001          Pine  A  −0.5  −5.2  −0.839  0.001  0.003  0.078  0.153      E  4.5  −4.6  −0.660  0.007        RWC  Oak  A  68  12.7  0.605  0.010  0.200  0.024  0.267      E  75  6.3  0.541  0.013          Pine  A  75  7.9  0.672  0.001  0.056  0.010  0.431      E  65  4.8  0.592  0.001        MDA  Oak  A  18.9  −56.6  −0.925  0.001  0.888  0.088  0.210      E  22.1  −37.3  −0.897  0.001          Pine  A  54.2  −44.1  −0.705  0.001  0.318  0.001  0.617      E  61.2  −27.9  0.055  0.001        PRO  Oak  A  108  12.9  0.217  0.295  0.196  0.067  0.278      E  98  7.2  0.198  0.352          Pine  A  107  16.1  0.643  0.001  0.001  0.003  0.633      E  78  11.7  0.527  0.001        Parameter  Species  CO2  Regression analysis         Multiple analysis of variance             A   B   Ra   P   (PCO2)   (PDS)   ICO2xDS   CHL  Oak  A  10.2  0.96  0.599  0.014  0.790  0.158  0.605      E  9.8  1.00  0.352  0.202          Pine  A  4.78  0.55  0.567  0.001  0.010  0.050  0.500      E  3.33  0.31  0.448  0.003        CAR  Oak  A  1.69  0.09  0.342  0.193  0.920  0.095  0.536      E  1.63  0.10  0.241  0.408          Pine  A  0.85  0.06  0.332  0.022  0.010  0.040  0.630      E  0.53  0.02  0.121  0.424        Parameter  Species  CO2  Regression analysis         Multiple analysis of variance             A   B   Ra   P   (PCO2)   (PDS)   ICO2xDS   CHL  Oak  A  10.2  0.96  0.599  0.014  0.790  0.158  0.605      E  9.8  1.00  0.352  0.202          Pine  A  4.78  0.55  0.567  0.001  0.010  0.050  0.500      E  3.33  0.31  0.448  0.003        CAR  Oak  A  1.69  0.09  0.342  0.193  0.920  0.095  0.536      E  1.63  0.10  0.241  0.408          Pine  A  0.85  0.06  0.332  0.022  0.010  0.040  0.630      E  0.53  0.02  0.121  0.424        aRegression analysis for SOD under elevated CO2 only before drought‐stress induced collapse. bPDWP, predawn water potential; SOD, superoxide dismutase; CAT, catalase; GPOD, guaiacol peroxidase; APOD, ascorbate peroxidase; MDARR, monodehydroascorbate radical reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; ASCtot, total ascorbate; rASC, ascorbate/(total ascorbate)×100; GSH, glutathione; rGSSG, GSSG/GSH×100; PRO, protein; RWC, relative water content; CAR, carotenoids; CHL, chlorophyll; MDA, malondialdehyde. View Large Relationship between antioxidative systems and water potential under ambient and high CO2 Elevated CO2 affects water use efficiencies in a different way in pine and oak (Guehl et al., 1994; Picon et al., 1996). For the comparison of drought responses in the two species as affected by high CO2, it is necessary to analyse the relationship between the activities of antioxidative enzymes on a common basis representative of the internal water status of the plant. For this purpose SOD, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, and glutathione reductase activities as well as the redox status of the ascorbate and glutathione pools were plotted against the corresponding data for water potential (Figs 3, 4, 5). The data were fitted by linear curves except for SOD (Fig. 3). Activities of unspecific peroxidases were also measured using guaiacol as the substrate (not shown). The results of the statistical analysis of the data have been summarized in Table 2. The activities of SOD showed opposite behaviour in foliage of trees grown at high CO2 as compared with those grown under ambient CO2 (Fig. 3A, C). This response was highly significant in both species, oak and pine (Table 2). However, when the predawn water potential of pine trees dropped below −2 MPa SOD activities started to decrease in pine grown at high CO2, whereas they continued to increase in drought‐stressed oak grown at high CO2 until the water potential dropped below −4 MPa (Fig. 3A, C). It was not possible to determine the threshold for the collapse of SOD in oak exactly because water potentials of −4 MPa exceeded the scale of the instrument. The differential responses of SOD indicating significant interactions between drought stress and CO2 exposure were, however, not typical for other components of antioxidative systems (Figs 3B, D, 4, 5). Catalase activities were highly susceptible to drought stress and dropped to low levels when the water potential decreased below −1 MPa in oak and pine regardless of the growth CO2 concentrations (Fig. 3B, D). In pine, catalase activities were approximately 40% lower at elevated CO2 as compared with ambient CO2. By contrast, in oak catalase remained unaffected by the CO2 conditions (Table 2). Ascorbate peroxidase activities were less inhibited by drought than catalase (Figs 5A; 3B, D). The general response pattern to CO2 and drought was, however, similar to that observed for catalase (Fig. 4A, E). Ascorbate peroxidase activities decreased with decreasing water potential in both pine and oak. But only in pine high growth CO2 caused some reduction in ascorbate peroxidase activities (−20%, Table 2). In contrast to ascorbate peroxidase and catalase, unspecific peroxidase activities were neither affected by drought stress nor by growth CO2 concentrations (Table 2). Among the enzymes responsible to maintain the antioxidants ascorbate and glutathione in their reduced forms, only monodehydroascorbate radical reductase consistently showed linear correlations between water potentials and enzyme activities in oak and pine and declined approximately 1.5‐fold slower in foliage from high as compared with that from ambient CO2 (Fig. 4B, F; Table 2). There were no clear relationships between dehydroascorbate reductase activities and CO2 (Fig. 4C, G). Apparently dehydroascorbate reductase was extremely sensitive against beginning water limitation in pine, because significant enzyme activities (>5 nkat g−1 dry mass) were only found at water potentials above −1 MPa and no activities in most cases below this threshold (Fig. 4G). In oak this enzymes was not affected by drought (Table 2). Gutathione reductase activities also showed no consistent response to drought stress in the two species (Fig. 4D, H). Glutathione reductase tended to slightly higher activities in drought‐stressed oaks than in well‐watered trees (Fig. 4D). In pine, it decreased faster in response to drought stress (1.7‐fold) in needles from trees grown at ambient as compared with those from high CO2 (Fig. 4H). The loss of protective enzyme activities resulted in declining redox ratios of the ascorbate and glutathione pools in both stressed pines and oaks (Fig. 5). In well‐watered trees the redox state of the ascorbate pool [defined as ascorbate/(ascorbate+dehydroascorbate)× 100] ranged between 60% and 70% in oak and was 93% in pine, respectively (Table 2, data for A). When the trees were drought‐stressed these ratios declined at similar rates in both species by about 10% per units of water potential (Δ=1 MPa, Table 2; Fig. 5A, C). Interestingly, oak leaves from elevated CO2 contained 2.4‐fold lower ascorbate and 1.6‐fold lower glutathione concentrations than those from ambient CO2 (Table 2). Since pine did not show such CO2 responses, it retained higher ascorbate and glutathione concentrations than oak at high CO2 (Table 2). In well‐watered trees, glutathione disulphide was low in the two species accounting for about 2% of the GSH pool (Table 2). Drought stress resulted in relative increases in GSSG of about Δ=5% per 1 MPa (Table 2; Fig. 5B, D). Drought‐induced injury under ambient and high CO2 Drought stress resulted in decreases in the relative water content of the foliage at 1.3‐1.6‐fold higher rates in oak than in pine (Table 2). Under elevated CO2 the loss of foliar water was 2‐fold and 1.6‐fold slower in oak and pine, respectively, than under ambient CO2 (Table 2). Water loss and increasing water potentials were accompanied by increases in malondialdehyde indicating lipid peroxidation (Fig. 6A, E). The increase in malondialdyde was about 1.5‐fold higher in foliage of trees grown under ambient as compared with high CO2 (Fig. 6A, E). Elevated CO2 had no significant effects on the malondialdehyde concentrations in tissue of well‐watered pine and oak trees. In pine but not in oak other basic cellular constituents such as protein, chlorophyll and carotenoids were diminished at high as compared with ambient CO2 (protein: −25%, chlorophyll: −30%, carotenoids: −38%, Table 2, cf. Fig. 6). In oak, there was only a trend towards decreasing protein and carotenoids with increasing drought stress and no clear CO2 effect (Fig. 6B, C, D; Table 2). By contrast, in pine increasing drought‐stress resulted in significant loss in protein, chlorophyll and carotenoid contents under ambient CO2 (Fig. 6F, G, H; Table 2). Under high CO2, these decreases were dampended by factors of 1.4, 1.8 and 3 (Table 2). Fig. 3. View largeDownload slide The relationship between SOD (A, C) and CAT (B, D) activities and the predawn water potential in oak (A, B, Quercus robur) and pine (C, D, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (…. ambient CO2, — high CO2, see Table 2) exept for SOD in pine: y=125−645×−175x2, P≤0.001. Abbreviations and calculated statistical parameters are shown in Table 2. Note the difference in the scale. Fig. 3. View largeDownload slide The relationship between SOD (A, C) and CAT (B, D) activities and the predawn water potential in oak (A, B, Quercus robur) and pine (C, D, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (…. ambient CO2, — high CO2, see Table 2) exept for SOD in pine: y=125−645×−175x2, P≤0.001. Abbreviations and calculated statistical parameters are shown in Table 2. Note the difference in the scale. Fig. 4. View largeDownload slide The relationship between APOD (A, E), MDARR (B, F), DHAR (C, G) and GR (D, H) activities and the predawn water potential in oak (A–D, Quercus robur) and pine (E–H, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (— ambient CO2, — high CO2) and significant fits have been shown. Abbreviations and calculated statistical parameters are shown in Table 2. Fig. 4. View largeDownload slide The relationship between APOD (A, E), MDARR (B, F), DHAR (C, G) and GR (D, H) activities and the predawn water potential in oak (A–D, Quercus robur) and pine (E–H, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (— ambient CO2, — high CO2) and significant fits have been shown. Abbreviations and calculated statistical parameters are shown in Table 2. Fig. 5. View largeDownload slide The relationship between the redox state of the ascorbate (A, C) and glutathione (B, D) pools and the predawn water potential of oak (A, B, Quercus robur) and pine (C, D, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (— ambient CO2, — high CO2). Abbreviations and calculated statistical parameters are shown in Table 2. Fig. 5. View largeDownload slide The relationship between the redox state of the ascorbate (A, C) and glutathione (B, D) pools and the predawn water potential of oak (A, B, Quercus robur) and pine (C, D, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (— ambient CO2, — high CO2). Abbreviations and calculated statistical parameters are shown in Table 2. Fig. 6. View largeDownload slide The relationship between the malondialdehyde (A, E), protein (B, F), Chlorophyll (C, G), and carotenoid (D, H) concentrations and the predawn water potential of oak (A–D, Quercus robur) and pine (E–H, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (— ambient CO2, — high CO2). Abbreviations and calculated statistical parameters are shown in Table 2. Fig. 6. View largeDownload slide The relationship between the malondialdehyde (A, E), protein (B, F), Chlorophyll (C, G), and carotenoid (D, H) concentrations and the predawn water potential of oak (A–D, Quercus robur) and pine (E–H, Pinus pinaster) trees grown under ambient (•‐•) and high CO2 (○‐○). Curves were fitted by linear regression analysis (— ambient CO2, — high CO2). Abbreviations and calculated statistical parameters are shown in Table 2. Discussion Drought responses of antioxidative systems in pine and oak under ambient CO2 concentrations Maritime pine and pendunculate oak are drought‐avoiding and drought‐tolerant species, respectively, which differ in morphological structures and in stomatal behaviour (Picon et al., 1996; Epron and Dreyer, 1993a, b). The present results suggest that the differences in drought susceptibility may additionally be related to different levels of biochemical protection in the foliage, since pine needles displayed inherently lower activities of H2O2 and O2·− scavenging enzymes and of enzymes driving the ascorbate–glutathione cycle than oak leaves. If the enzyme activities in unstressed controls of oaks were set as 100%, the following reductions were observed in unstressed controls of pine needles: SOD −70%, catalase −75%, ascorbate peroxidase −40%, dehydroascorbate reductase −75%, and glutathione reductase −50% (cf. Table 2, data for A). The concentrations of antioxidative metabolites, ascorbate and glutathione, and the activity of monodehydroascorbate radical reductase were similar in pine to levels found in oak (Table 2; Fig. 4). Under ambient CO2, both species showed drought‐related decreases in the activities of SOD and other antioxidative enzymes. This result may indicate that both species do not have any intrinsic reserves or activation mechanisms to cope with increasing levels of oxidative stress. Such an observation may be alarming, if it was true under field conditions. In contrast to the two tree species analysed here, in herbaceous plants drought generally induced increases in antioxidative systems (Smirnoff, 1993; Polle, 1997). For instance, in drought‐stressed peas an increased production of mRNA for ascorbate peroxidase and SOD as well as enhanced enzymatic activity of these proteins were found; catalase activity was also increased (Mittler and Zilinskas, 1994). These observations lend support to the idea that both the Mehler reaction and photorespiration are important metabolic pathways for the dissipation of light energy when the flux of CO2 into the leaf is limited under drought conditions (Foyer and Harbinson, 1994). However, it is doubtful whether these pathways are important protective mechanisms in drought‐stressed oak or pine trees because the key enzymes involved in these reactions declined in both species with increasing water deficits (Figs 3, 4). In pine, the initial decline in catalase was faster and resulted in lower residual activities than in oak. This also points to an enhanced stress susceptibility of pine since compensatory increases, for example, that of ascorbate peroxidase or of unspecific peroxidases were not observed. Pine needles maintained, however, a higher redox ratio of the ascorbate pool than oak leaves (Fig. 5). This was surprising because dehydroascorbate reductase activities in pine were extremely sensitive to drought stress and not affected in oak. Since monodehydroascorbate radical reductase activities were only moderately suppressed, these data suggest that in drought‐stressed trees monodehydroascorbate radical reductase is more important in keeping ascorbate in its functional state than the operation of the ascorbate–glutathione cycle. Comparisons of drought‐susceptible and ‐tolerant cultivars of herbaceous species suggested that increased tolerance correlated with induction of higher levels or higher responsiveness of antioxidative defences to stress (Sairam et al., 1998; Jagtap and Bhargava, 1995; van Rensburg and Krüger, 1994). At the same time tolerant species maintained lower concentrations of O2·− than drought‐susceptible species, thereby diminishing the risk of oxidative injury (Quartacci and Navaro‐Izzo, 1992; Quartacci et al., 1994). Although stress did not result in transient increases in antioxidative protection in oak and pine, both species were less drought‐sensitive than herbaceous species, in which significant protein and chlorophyll degradation were observed at water potentials below −0.5 MPa (Smirnoff and Colombé, 1988; Baisak et al., 1994; Moran et al., 1994). In both oak and pine the relative drought‐induced degradation of chlorophyll was small accounting to −10% per −1 MPa (Fig. 6). Despite significantly higher activities of antioxidative enzymes in oak, both species seemed to suffer to a similar extent from oxidative stress as indicated by similar accumulation rates of glutathione disulphide and malondialdehyde (Figs 5, 6). Still, oak leaves were apparently more drought‐tolerant than pine needles because even severe stress had only moderate or no significant effects on soluble protein or carotenoid concentrations, but caused marked decreases in these components in pine needles (Fig. 6). Ameliorative effects of high CO2 in drought‐stressed pine and oak The present results corroborate observations that growth under elevated CO2 may cause reductions in activities of protective enzymes (Figs 3, 4) and of antioxidative metabolites (oak, Table 2, cf. also Polle, 1996). Superoxide dimutase activity was consistently diminished under elevated CO2 regardless of the species analysed here (Figs 1, 2). Such depressing effects of high CO2 on protective enzymes are not confined to trees but also occurred, for example, in barley, in which significant increases in SOD and catalase activities were observed when plants grown at high CO2 were transferred to ambient CO2 (Azevedo et al., 1998). These observations led to the suggestion that plants grown under elevated CO2 are exposed to decreased intrinsic levels of oxidative stress (Polle, 1996; Azevedo et al., 1998). The present results furthermore confirm the observation that drought‐stressed foliage of oak and pine under ambient CO2 contained lower and that from elevated CO2 higher SOD activities compared with their respective controls (Figs 1, 2). Based on such findings, it has previously been suggested that growth under elevated CO2 may lead to an increased metabolic flexibility to encounter oxidative stress (Schwanz et al., 1996a). Rao et al. found that negative effects of ozone were ameliorated under elevated CO2 and that this protection was accompanied by increased antioxidative defences (Rao et al., 1995). However, the potentially positive effects of elevated CO2 with respect to ozone injury may also be related to diminished ozone uptake (McKee et al., 1997) and have not been observed in all cases (Niewiadomska et al., 1999). In the present study the observed oxidation of the ascorbate and glutathione pools as a result of water deficits is a clear indication that the tissues suffered from oxidative stress (Figs 5, 6). The injurious consequences of this stress were apparently diminished under high CO2 because of smaller accumulation rates of malondialdehyde in both species and a lower loss of protein and pigments in pine (Fig. 6). Improved water relations, reduced chlorophyll degradation, delayed degradation of the small subunit of ribulose‐1,5‐bisphosphate carboxylase, and higher membrane stability have been observed in herbaceous species exposed to drought stress or chilling‐induced drought under high CO2 (Boese et al., 1997; Sgherri et al., 1998; Vu et al., 1999). There is also some evidence that Q. ilex trees grown at naturally CO2‐enriched sites acquire improved protection against drought stress (Chaves et al., 1995). In cherry trees positive effects of elevated CO2 on the water status of the plants have not been found (Centritto et al., 1999). Therefore, the authors concluded that the drought stress tolerance of the seedlings grown under elevated CO2 was not increased. However, the present data show that even when the effects of elevated CO2 on the relative water content or predawn water potentials are only moderate or not significant, the activities of protective enzymes still may show lower rates of drought stress related decline (Table 2). These data strongly support the hypothesis that alleviation of drought‐induced injury under elevated CO2 was related to a higher stability of antioxidative enzymes and an increased responsiveness of SOD to stressful conditions. The metabolic window, in which positive responses of SOD in drought‐stressed foliage under high CO2 were observed, was wider in oak than in pine (collapse of SOD activities at water potentials below −4 MPa and −2 MPa in oak and pine, respectively). In drought‐stressed wheat, in which increases in SOD were found under ambient CO2, the collapse of SOD occurred at water potentials of −1 MPa (Baisak et al., 1994). Despite the prolonged maintenance of high SOD activities in drought‐stressed oak under high CO2, it not clear whether this results in a relative increase in vitality of oak as compared with pine. That the stress tolerance of both species profited from growth at high CO2, but that there was no evidence that oak had an extra benefit, has been discussed above. For example, in both species the production rates of malondialdehyde were diminished to similar extents, i.e. by factors of 1.5 (Table 2). 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TI - Differential stress responses of antioxidative systems to drought in pendunculate oak (Quercus robur) and maritime pine (Pinus pinaster) grown under high CO2 concentrations
JF - Journal of Experimental Botany
DO - 10.1093/jexbot/52.354.133
DA - 2001-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/differential-stress-responses-of-antioxidative-systems-to-drought-in-fjdgjs4Sv5
SP - 133
EP - 143
VL - 52
IS - 354
DP - DeepDyve
ER -