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Keywords:

  • Herbicide;
  • methylviologen;
  • oxidative stress;
  • paraquat;
  • superoxide dismutase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES

The development of beech leaves (Fagus sylvatica L.) was characterized by determination of the pigment and electrolyte concentrations as well as the accumulation of dry mass and specific leaf mass from bud break to senescence. To test the hypothesis that stress tolerance and responsiveness of defences show developmental and/or seasonal changes, leaf discs were either incubated in the absence (control) or presence of paraquat to induce oxidative stress. Controls displayed developmental changes in stress susceptibility ranging from less than 15% of maximum electrolyte leakage in mature leaves to more than 20% leakage in senescent and 36–46% in immature leaves. Paraquat concentrations were chosen to result in about 95% of maximum electrolyte conductivity within 24 h in all developmental stages. Paraquat accumulation was about two-fold lower in senescent as compared with immature leaves, whereas stress susceptibility, as characterized by the kinetics of the increase in relative leakage, was similar in these developmental stages with 50% of maximum electrolyte conductivity (EC50) = 6·5 h in immature and 7·5 h in senescent leaves. In mature leaves with intermediate paraquat accumulation rates, two classes of stress-sensitivity were distinguished, namely stress-resistant and stress-susceptible leaves with EC50= 9·5 and 5·2 h, respectively. Stress-resistance of mature leaves was accompanied by a rapid, approximately two-fold induction of superoxide dismutase activity. Stress-sensitive mature leaves initially contained high superoxide dismutase activities but showed a rapid, more than six- fold loss in activity in 24 h. Correlation of meteorological data with leakage rates suggested that high air temperatures and low precipitation might have been predisposing for loss of resistance against oxidative stress in beech leaves.


Abbreviations
EC50

50% of maximum electrolyte conductivity

SOD

superoxide dismutase

paraquat

1,1′-dimethyl-4,4′-bipyridinium chloride.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES

European beech (Fagus sylvatica L.) is a late-successional species. Without anthropogenic interference it would be the dominant forest-forming tree in middle and western Europe (Ellenberg 1996). In Germany, where about 30% of the land is covered by forests, beech currently contributes less than 25% to forest trees and conifers are the predominant species (66%, BMELF 1997). Recent regional reforestation programmes aim at converting pure spruce cultures into mixed and structured beech–spruce forests (Otto 1992). However, young saplings planted to date or natural regeneration of beech will be exposed to significant changes in climatic conditions during their life-time. Due to rising atmospheric CO2 concentrations and that of other greenhouse gases, air temperatures are likely to increase and precipitation may be less abundant during the growth phase than today (Roeckner 1992; Schimel et al. 1996). In addition, air pollution will probably increase, at least at a regional scale (Chameides et al. 1994). The ability and limits of beech to maintain and adjust cellular homeostasis by acclimation to changing environmental conditions are therefore of significant economic and ecological interest.

Many environmental variables, such as drought, high temperature, ozone, etc., cause oxidative stress in plants (Polle 1997). To compensate for injurious oxidants, plants are equipped with anti-oxidative systems composed of metabolites such as ascorbate, glutathione, tocopherols, carotenoids, etc., as well as protective enzymes such as superoxide dismutases (SODs), peroxidases and catalases (Alscher, Donahue & Cramer 1997; Noctor & Foyer 1998). In beech, the composition and seasonal fluctuations of anti-oxidative systems have been addressed in several investigations (Franke 1965; Kunert & Ederer 1986; Polle & Morawe 1995a, 1995b; Luwe 1996). For example, ascorbate concentrations were low in leaf buds in winter (Polle & Morawe 1995b) and increased after leaf emergence to concentrations up to 20 µmol per g fresh mass in summer (Franke 1965; Polle & Morawe 1995b; Luwe 1996). Activities of enzymes involved in scavenging of reactive oxygen species such as SOD and ascorbate peroxidase also increased during leaf maturation and showed significant fluctuations in mature leaves (Polle & Morawe 1995b). In senescing leaves the constituents of the antioxidative systems, with the exception of tocopherol, decreased in parallel with chlorophyll (Kunert & Ederer 1986; Polle & Morawe 1995b). Premature chlorophyll loss and senescence were observed in beech leaves after exposure to elevated ozone and/or increased UV-B levels (Lippert et al. 1996; Zeuthen et al. 1997; Grams et al. 1999). Such observations suggest that chronic stress exposure overwhelmed the capacity of protective and repair systems. Another, yet unaddressed possibility is that beech leaves show intrinsic changes in stress susceptibility, meaning that the same dose of stress would result in differential effects depending on the developmental stage of the leaf.

The aim the present study was to characterize developmental stages of beech leaves in relation to stress tolerance. For this purpose leaves collected over the whole season were challenged by exposure to paraquat – a herbicide causing superoxide radical generation (Dodge 1971). Superoxide radicals cause membrane injury and, thereby, electrolyte leakage, if the anti-oxidative defence systems are insufficient to cope with increased radical production. In the present investigation, membrane injury was determined as the rate of increase in electrolyte conductivity during 24 h. In addition, SOD activities were determined to find out whether different stress responses observed at different times of the season were related to changes in the magnitude of SOD activity or its inducibility in response to stress.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES

Site and harvest

Leaves from five 20-year-old beech trees, grown in the south-west of a sun-exposed edge of a small beech forest (Fuchslöcher) close to Göttingen (9°58′ E, 51°33′ N, 235 m above sea level), were used for seasonal studies from 19 May to 21 October 1998 and from 6 May to 21 June 1999. Small twigs from the sun-exposed layer were sampled in weekly or more frequent intervals at 0830 h, transported to the laboratory within 10 min, and exposed to stress. Aliquots of the leaves were immediately frozen in liquid nitrogen and stored at −80 °C for further biochemical analyses. Leaves collected on 27 and 31 May, 7 and 28 July, 11 and 25 August and 6 October 1999 were used for further biochemical analyses. Air temperature and humidity were recorded hygrometrically in the stand during the whole investigation period (Fig. 1a & b). Precipitation was determined at a meteorological station located in a forest at a distance of about 2 km ( Fig. 1c).

image

Figure 1. (a) Air temperature, (b) relative humidity of air and (c) precipitation from spring 1998 to summer 1999. Data in (a) and (b) indicate weekly means of maximum (—bsl00043—) and minimum (—bsl00041—) values, respectively. Data in (c) represent weekly sums.

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Stress exposure

Twenty-five discs (diameter: 6 mm) per treatment were cut from leaves of each tree and transferred to Petri dishes containing either 16 mL of distilled water (Serapur; USF Seral, Göttingen, Germany) (‘controls’) or 10 mm paraquat (‘stressed leaf discs’). The relatively high paraquat concentration was chosen because preliminary studies showed that this concentration resulted in a greater than 90% electrolyte leakage of leaf discs within 24 h, whereas leakage from controls remained low (< 15%). Infiltration of the leaf discs with paraquat was also tested but did not yield significantly different results (not shown).

Incubation of controls and stressed leaf discs occurred in a climate-controlled room at a relative humidity of 70%, a temperature of 20 °C and a photosynthetically active radiation of 200 µE m−2 s−1 (lamp type: Osram HQL 250 W; AEG, Springe, Germany) at the height of the leaf discs. Under these conditions the temperature of the incubation medium was 30 °C. Electrolyte conductivity was determined with a conductometer (LF315; WTW, Weilheim, Germany) after transfer of the discs to the incubation medium (ECt0), and after 4, 8, 12, and 24 h (ECt). Duplicate samples of controls and stressed leaf discs were removed after these incubation times and stored frozen at −80 °C for biochemical analyses. After 24 h, the leaf discs and their incubation medium were transferred into sealed vessels and boiled for 30 min. The boiled samples were cooled down to 30 °C before measurement of maximum electrolyte conductivity (ECmax). Preliminary experiments showed that electrolyte conductivity did not increase further after 30 min of boiling. For each sample, the relative electrolyte conductivity was calculated according to the following equation: EC = (ECt − ECt0/ECmax − ECt0) × 100.

Determination of paraquat

To determine the foliar paraquat concentrations, frozen leaf materials of controls and paraquat-treated samples were ground to a fine powder in liquid nitrogen. Aliquots of 25 mg of the powder were incubated for 15 min with 500 µL of 2% m-phosphoric acid and mixed repeatedly. The homogenate was centrifuged twice for 5 min at 4 °C and 15 000 g. The supernatant was diluted as appropriate and used to determine spectrophotometrically the extinction at λ = 257 nm. Data were corrected for the extinction of untreated samples. To calculate foliar paraquat concentrations, standard curves were produced with para- quat in 2%m-phosphoric acid. The recovery of paraquat added to untreated leaves during the extraction was 91 ± 2%.

Determination of SOD activity

Frozen leaf materials were ground to a fine powder in liquid nitrogen. Aliquots of 100 mg of leaf powder were homogenized in 100 mm phosphate buffer, pH 7·8 containing 1% Triton X-100 and 200 mg of insoluble polyvinylpyrrolidone (after Schwanz et al. 1996). After centrifugation (14 000 g, 15 min, 4 °C) and gel filtration over Sephadex G25 (PD-10 column; Pharmacia, Freiburg, Germany) the extracts were used for determination of SOD activity in the cytochrome C assay (after McCord & Fridovich 1969).

Basis parameters

The specific leaf mass (g m−2) was determined by taking the weight of 25 fresh leaf discs corresponding to a total area of 706·8 mm2. The samples were kept at 70 °C for 3 d and used to determine dry mass. Relative dry mass (%) was calculated as: dry mass × 100/fresh mass.

For pigment analysis, aliquots of frozen leaf material were ground to a fine powder in liquid nitrogen. Thirty milligrams of the powder were transferred into 5 mL of 80% acetone, mixed and incubated in darkness for 15 min. After centrifugation the pigments were determined spectrophotometrically at λ= 663, 646 and 470 nm. The concentrations of chlorophyll a and b, and of carotenoids were calculated using the extinction coefficients determined by Lichtenthaler & Wellburn (1983).

Statistical analysis

Data in figures are means from five individual trees analysed per sampling date (± SD). Significant differences between means were determined by multifactorial or monofactorial analysis of variance and a multiple range test (Statgraph, STN, St. Louis, MO, USA). For curve fitting the computer programme Origin (Microcal, St Louis, MO, USA) was used. Significant differences between curves were calculated by Rank analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES

Changes in leaf area, dry mass and pigment concentrations during the life cycle of beech leaves

After bud break, beech leaves started to accumulate dry mass as determined on the basis of specific leaf area as well as on the basis of fresh mass (Fig. 2a). About 6 weeks after bud break (generally in the last week of June), this process was complete and the leaves, which maintained a specific leaf area of 55·5 ± 3·2 g m−2 and a relative dry mass of 47·4 ± 0·8% until the end of September, were considered mature. Thereafter, the specific leaf mass and relative dry mass started to decline; the changes were, however, relatively small, accounting only for about 20% of total mass until end of October (Fig. 2a). In the following year, accumulation of specific leaf mass after bud break was slightly delayed, perhaps because of the relatively, warm and dry weather in this time (Fig. 1a & c).

image

Figure 2. (a) Specific leaf mass (SLM, —bsl00043—) and relative dry mass (—bsl00041—), and (b) chlorophyll (—bsl00041—) and carotenoid (—bsl00043—) concentrations in beech leaves collected from spring 1998 to summer 1999. Data represent means of five individual trees per sampling date (± SD). Vertical dashed lines indicate developmental stages.

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Foliar development was further characterized by changes in chlorophyll and carotenoid concentrations. Similar to the accumulation of dry mass, net synthesis of the pigments was also accomplished by the end of June (Fig. 2b). Mature leaves contained 19·3 ± 3·7 mg of carotenoids and 77 ± 16 mg of chlorophyll per m2 (Fig. 2b) with a chlorophyll a to chlorophyll b ratio of 4·3 (not shown). The fluctuation of pigment concentrations during summer was relatively high. Similar variations have been observed in sun-acclimated beech leaves harvested at the same site on sunny and cloudy days, respectively (Peltzer & Polle 2001).

In senescing leaves, the concentrations of carotenoids and chlorophyll declined by 56 and 93%, respectively, in same period of time in which dry mass declined only by 20%. Apparently, the degradation rate of chlorophyll was about 1·6-times faster than that of carotenoids (Fig. 2b).

Based on the phases of accumulation, maintenance and degradation of cellular constituents (Fig. 2a & b) the following stages of leaf development were defined: young-immature leaves (mid-May until mid-June), mature leaves (end of June until mid-September) and senescent leaves (end of September until end of October).

Changes in stress susceptibility over the life cycle of beech leaves

In our experimental system, the electrolytes (ECmax) present in mature leaves resulted in mean conductivity of 256 ± 40 µ S cm−1(Fig. 3a). In young leaves electrolytes increased almost two-fold in the phase of foliar expansion and accumulation of other constituents such as dry mass and pigments; but in senescing leaves no significant de-crease in electrolytes was found (−9%, Fig. 3a).

image

Figure 3. (a) Maximum electrolyte conductivity, ECmax, and (b) relative electrolyte conductivity of controls and (c) of paraquat-exposed beech leaf discs after incubation times of 4 (—bsl00000—), 8 (——), 12 (—bsl00041—) and 24 h (—bsl00043—), respectively. Foliage was collected over the whole season from May 1998 to June 1999. Data represent means of five individual trees per sampling date (± SD). For clarity error bars have been omitted for t= 8 and 12 h. Vertical dashed lines indicate developmental stages according to Fig. 2. M-I and M-II = mature I and mature-II leaves (for explanation see text).

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To overcome the difficulty of fluctuating foliar electrolyte concentrations, the results of the subsequent stress treatments were expressed as EC relative to the maximum conductivity of the sample and corrected for background conductivity ECt0 caused by cutting leaf discs (tissue wounding) or the ionic nature of paraquat (see Materials and methods). To test fluctuations in ‘basic’ stress tolerance, leaf discs were floated on water under illumination at a temperature of 30 °C, but in the absence of paraquat. After 24 h, the mature leaves consistently showed a loss of 14 ± 4% of total foliar electrolytes (Fig. 3b). By contrast, immature leaves lost 36 ± 21% and senescing leaves 20 ± 11% of total electrolytes in 24 h (Fig. 3b). Young leaves collected in 1999 were even more stress sensitive than those from 1998 and lost 46 ± 13% of electrolytes during 24 h (Fig. 3b). These data show that the ability to maintain cellular integrity was 2·5- to 3·3-fold higher in mature than in young leaves. Furthermore, there was little variation in EC in mature leaves, whereas this para- meter showed significant fluctuations in other stages of leaf development.

To challenge the system by massive oxidative stress, the leaf discs were floated on paraquat solutions and the relative increase in EC was recorded for 24 h (Fig. 3c). Under these conditions, EC increased more rapidly than in the absence of paraquat and reached 95 ± 5% after 24 h in all samples. However, there were interesting differences in the rates at which 95% EC was attained in leaves collected at different dates. Analysis of meteorological variables such as air temperature and precipitation with the extent of leakage revealed that the initial extent of leakage (ECt = 4 h) was significantly correlated with mean weekly maximum temperatures (Fig. 4) and inversely correlated with the sum of weekly precipitation (Pt = 4 h < 0·05, not shown). These relationships disappeared gradually with prolonged paraquat exposure (Fig. 4 and for precipitation Pt = 8 h = 0·469).

image

Figure 4. Relationship between relative electrolyte leakage after paraquat incubation [exposure time: 4 (—bsl00000—), 8 (—×—), 12 (—bsl00041—), and 24 h (—bsl00043—)] with means of weekly maximum temperatures. P-values indicate levels of significance obtained by linear regression analysis.

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To find out whether leaf development was a further factor affecting the velocity at which tissue electrolytes were released, the observed leakage rates were analysed statistically employing the developmental stages ‘young’, ‘mature-I’, ‘mature-II’ and ‘senescent’ as variables (Table 1). The distinction between two classes of mature leaves was introduced because of an obvious difference in the rate of electrolyte leakage (and supported by frequency analysis of distribution) in mature leaves harvested between the end of June until the beginning of August (M-I, Fig. 3c) and those harvested from August to September (M-II, Fig. 3c). Using this approach, significant differences in the extent of paraquat-induced leakage were apparent between the stages ‘mature-I’ and ‘mature-II’ leaves (Table 1). Young and senescent leaves were clearly distinguished from any type of mature leaves but were similar to each other (Table 1).

Table 1.  Results of multivariate analysis of variance for relative electrolyte leakage of leaf discs incubated in the presence or absence of paraquat (controls). Samples were collected over a whole growth phase and analysed according to developmental stage
Exposure time (h)YoungMature-IMature-IISenescentP
  1. For definition of developmental stages see text and Fig. 3. Same letters in rows indicate homogenous groups. P indicates calculated level of significance.

Controls
4aaaa0·247
8baab0·076
12baaab0·041
24baaa0·001
Paraquat-exposed leaf discs
4ababa0·073
8bcacab0·016
12bacb0·001
24aaaa0·359

To determine the kinetics of membrane injury, data for electrolyte leakage were averaged according to developmental stage and plotted against the incubation time with paraquat (Fig. 5). The curves were different with P = 0·016. For comparison, the rate of electrolyte release from controls is also shown. To assess the overall stress tolerance or susceptibility, corrections for this ‘basic’ leakage were not performed. Analysis of the fitted curves yielded 50% ion leakage, EC50, at incubation times of 6·8 (6·5) h for young leaves, 10·0 (9·5) h for ‘mature-I’ leaves, 5·5 (5·2) h for ‘mature-II’ leaves, and 8·6 (7·5) h for senescent leaves (Fig. 5a & b). If corrections for basic leakage were introduced to analyse the paraquat-specific effect, differences in comparison with uncorrected data were small as shown above by the figures in brackets. These data indicate that different developmental stages have different abilities to withstand paraquat and that mature leaves with similar ‘basic’ stress tolerance can be either relatively resistant or highly susceptible to this herbicide.

image

Figure 5. Time-course of electrolyte leakage (EC) in different stages of foliar development: (a) mature leaves (mature-I: —bsl00043—, mature-II: (—bsl00041—); (b) young (—bsl00043—) and senescent leaves (—bsl00041—). Circles represent leakage in the presence, squares in the absence of paraquat. Data are means of EC measurements in intervals of 5–6 weeks in 1998 (±SD). Curves represent dose–response fits employing the Boltzmann function.

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Relationship between stress susceptibility, foliar accumulation of paraquat and SOD activity

To investigate whether differences in stress tolerance were associated with different levels of SOD activity or with structural changes limiting paraquat uptake, leaf tissues from dates corresponding to typical stages of development and electrolyte leakage were selected for further analyses. The highest SOD activities were observed in controls of ‘mature-II’ and senescent leaves (Fig. 6a & b at t = 0). Young leaves contained about 2·5- to four-fold lower SOD activities than mature or senescent leaves (Fig. 6b at t = 0). When leaf tissue was challenged by paraquat, the response patterns of SOD activities showed interesting differences: in ‘mature-I’ leaves characterized by the slowest increase in electrolyte leakage under stress, SOD activities initially increased two-fold and later decreased to levels similar to those present before stress-exposure (Fig. 6a). In contrast, in ‘mature-II’ leaves, which were characterized by the fastest increase in electrolyte leakage, the initially high SOD activities decreased immediately with stress exposure and dropped within 24 h to levels that were six-fold lower than those present before stress exposure (Fig. 6b). The pattern of SOD induction after stress-exposure of young leaves resembled that of ‘mature-I’ leaves but on a generally lower level of total activity (Fig. 6a,b). In senescent leaves exposure to paraquat caused decreases in SOD activities (Fig. 6b) similar to those found in ‘mature-II’ leaves (Fig. 6a). The curves in Fig. 6a & b were different with P  = 0·013.

image

Figure 6. (a,b) Time-dependent changes in SOD activity and (c,d) in foliar paraquat concentrations in response to paraquat incubation. Mature leaves (a,c) were harvested in July (mature-I: —bsl00041—, hatched bar) and in August (mature-II: —bsl00043—, white bar); young and senescent leaves (b,d) were harvested in May (—bsl00041—, hatched bar) and October (—bsl00043—, white bar). Data indicate means of five individual beech trees (n = 5, ± SD). Bars represent SOD activity after 24 h incubation in the absence of paraquat. Different letters indicate significant differences at P ≤ 0·05. For further details, see text.

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Both ‘mature-II’ and senescent leaves showed significant decreases in SOD activities after incubation of leaf discs for 24 h in the absence of paraquat-induced stress (Fig. 6a & b). By contrast, controls of young and ‘mature-I’ leaves maintained or even increased SOD activities during the incubation of time of 24 h (Fig. 6a & b).

To exclude the possibility that the higher lability of SOD and higher rates of leakage in ‘mature II’ in comparison with ‘mature-I’ leaves were caused by different rates of paraquat uptake, the accumulation pattern of this herbi-cide was determined (Fig. 6c & d). Surprisingly, paraquat uptake in the more stress-susceptible, ‘mature-II’ leaves was slower than that observed in the more resistant ‘mature-I’ leaves (Fig. 6c). The most rapid accumulation of paraquat was found in young leaves (Fig. 6d) and the slowest in senescent leaves (Fig. 6d), although these tissues displayed similar leakage rates. The curves in Fig. 6c & d differed with P = 0·024.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES

The major goal of the present study was to analyse changes in the susceptibility for oxidative stress over the life cycle of beech leaves. Unlike many herbaceous species or continuously growing tree species, which generate new leaves during the whole season, beech generally produces only one flush in spring. After bud break the leaves show a characteristic developmental pattern with a relatively slow phase of maturation of about 6 weeks and towards the end of the season a phase of senescence of about 3 weeks (this study, Fig. 2; Polle & Morawe 1995b).

When foliage from young and senescent stages was analysed for electrolyte leakage, a major difference in comparison with mature leaves was that these stages showed significant release of electrolytes already in the absence of additional oxidative stress imposed by paraquat (Figs 3b, 5a & b). This observation clearly indicates significant developmentally induced changes in the levels of stress tolerance.

In the presence of paraquat, we also found significant differences in leakage rates in leaves of different developmental stages (Figs 3 & 5). It must be considered that structural changes might have limited the uptake of paraquat. For example, in Rhemannia glutinosa, the cell walls hindered access of paraquat to target sites (Chun et al. 1997). However, in the present study the time-course of electrolyte leakage of paraquat-stressed leaf discs from immature leaves was similar to that of senescent leaves (Fig. 5b) despite significant structural and physiological differences (e.g. relative dry mass, Fig. 2a). This observation suggests that the leakage rate was not dependent on structural changes in the first place. To further investigate the question of whether or not the leakage rates were related to developmental changes in the access of paraquat to potential target sites, paraquat accumulation patterns were estimated employing a relative unspecific method. We observed that the apparent paraquat accumulation rate was significantly affected by development (Fig. 6b & c) and was slowed down with t½ = 5·3, 6·0, 9·7, and 20·1 h in immature, mature-I, mature-II, and senescent leaves, respectively (Fig. 6b & c). However, stress susceptibility determined as electrolyte leakage rate in different developmental stages was independent of this pattern. Mature-II leaves with a slow paraquat accumulation rate showed the highest leakage rate. In contrast, the mature-I leaves with a higher paraquat accumulation rate showed the lowest leakage rate. This strongly supports the idea that mature leaves undergo a physiological change from a stage of high stress tolerance to a stage of high stress susceptibility.

External environmental factors appear to have modulating effects on the development of high levels of stress tolerance found in mature leaves as young leaves studied in the hot and relatively dry spring of 1999 showed higher rates of electrolyte leakage than those studied in the preceding year, when the spring was cooler and more humid (Fig. 1). Correlation analysis of meteorological parameters with stress-induced leakage revealed that the initial leakage rate was significantly higher in periods of the season with dry and hot weather than under cooler weather conditions (Fig. 4). Recently, we showed that mature beech leaves collected during hot and sunny weather conditions contained significantly lower activities of ascorbate peroxidase and monodehydroascorbate radical reductase than those collected on cloudy days (Peltzer & Polle 2001). As dry and hot weather conditions usually also correlate with high light intensities, the role of individual stress factors (temperature, irradiation, water availability, air pollutants, biotic stresses, etc.) in increasing the stress susceptibility of beech needs to be analysed under controlled conditions. Taken together, the present observations suggest that unfavourable environmental conditions diminish antioxidant defences and, thereby, may render beech leaves more stress susceptible.

It is well established that high paraquat resistance is mediated by elevated levels of antioxidative systems (Foyer, Descourvieres & Kunert 1994; Allen, Webb & Schake 1997; Polle 1997). As paraquat results in an increased production of O2, SODs provide the first line of defence. Transgenic plants overexpressing SOD activities generally showed higher resistance against paraquat than the wild-type (for a review see: Allen et al. 1997). However, additional increases in other components of antioxidative systems further increased paraquat tolerance (Allen et al. 1997; Scandalios 1997). It has been shown for beech that immature and senescent leaves contained lower activities of SOD, ascorbate peroxidase, monodehydroascorbate radical reductase, and glutathione reductase, less ascorbate and a higher concentration of dehydroascorbate to ascorbate than mature leaves (Franke 1965; Kunert & Ederer 1986, Polle & Morawe 1995a, 1995b, Luwe 1996). This suggests that the lower stress tolerance of these developmental stages reported in the present study may have been related to lower antioxidative capacities of both young and senescent leaves.

It should be emphasized, however, that high stress susceptibility and low SOD activities in young leaves are unusual observations reported here for beech and previously for spruce (Polle et al. 1996). In many cases young leaves were more resistant to oxidative stress and contained higher levels of protective systems than mature leaves (e.g. wheat: Price, Lucas & Lea 1990; poplar: Strohm et al. 1999; birch: Polle et al. 2000). As the contrasting developmental patterns of antioxidative systems occurred in leaves of indeterminate and determinate growing species, one may speculate that relationships exist between the life form and stress tolerance. This needs further attention.

The present study shows that mature beech leaves were highly resistant to paraquat as much higher concentrations were required to induce significant injury in comparison with species such as tobacco, poplar, wheat or others (Aono et al. 1995; Allen et al. 1997; Ramiro et al. 1998; Noctor et al. 1998). Which factors contribute to modulate paraquat tolerance in addition to antioxidative systems is not yet understood. In paraquat-resistant Conyza bonariensisAnsellem et al. (1993) found developmental variability in stress tolerance, which could not be explained by antioxidative systems. Recently, Kurepa et al. (1998) reported that late-flowering mutants of Arabidopsis were more tolerant to paraquat than the wild-type. On the basis of these results they suggested a link between longevity and oxidative stress resistance (Kurepa et al. 1998). The present investigation shows that mature beech leaves displayed a sudden and striking change in paraquat tolerance from being the most tolerant (EC50= 9·5 h, between end of June and mid-August) to being the most sensitive tissues (EC50= 5·2 h, between mid-August and the end of September, Figs 5 & 6). The observed changes in stress susceptibility in mature leaves were not accompanied by changes in foliar constituents such as dry mass and pigments (Fig. 2), ‘basic’ electrolyte leakage (Fig. 3) and could neither be explained by differences in paraquat uptake rates (Fig. 6c & d). Unexpectedly, the stress-susceptible mature leaves contained higher SOD activities than stress-resistant mature leaves (Fig. 6a). However, in the sensitive leaves SOD declined immediately in response to paraquat-induced stress, whereas SOD increased transiently in the more stress-resistant leaves (Fig. 6a). A transient induction of SOD has also been observed in stress-resistant leaves of oak, pine, and poplar that were exposed to drought stress, photo-oxidative stress, and paraquat, whereas less resistant leaves showed an immediate decline of antioxidative defences in response to oxidative stress (Schwanz & Polle, 2001a, 2001b). Perhaps, the responsiveness of SOD to oxidative stress is more important to mediate tolerance than the magnitude of enzyme activity. So far, it remains unknown how external environmental or intrinsic developmentally regulated factors cause changes in stress susceptibility. As the sudden change in stress tolerance in beech occurred during a hot and dry phase of the summer and as elevated stress susceptibility in young foliage was also observed in the spring with high temperatures (Fig. 1), it may be speculated that such meteorological conditions predispose beech to oxidative injury. Elevated temperatures and low humidity limit photosynthesis in beech (Overdieck, Kellomäki & Wang 1998). Under such conditions the cellular capacities for repair and defences may be fully required for intrinsic stress compensation and overwhelmed, if additional stresses occur. Another possibility is that magnitude and responsiveness of the defence systems in beech are under developmental control, which ensures maintenance of the leaves during the most productive phase early in summer and which, thereafter, may be gradually lost. Developmental changes in stress responses have been described in herbaceous species (Scandalios 1997).

In conclusion, under moderate stress imposed in controls by exposure of leaf discs to 24 h illumination at 30 °C, mature beech leaves maintained significantly higher membrane integrity than immature or senescent leaves. Correspondingly, young and old leaves showed a fast increase in membrane injury, when challenged by additional oxidative stress. However, mature leaves subjected to additional oxidative stress were either extremely resistant or were even more stress susceptible than young or senescent leaves. The switch, which caused this increase in stress susceptibility in mature leaves, is currently unknown. It is possible that unfavourable environmental conditions were predisposing or that an intrinsic developmental programme renders mature beech leaves per se more stress sensitive towards the end of the season.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES

We are grateful to Dr W. Meseburg (Niedersächsische Forstliche Versuchsanstalt, Göttingen) for communicating the precipitation data.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. REFERENCES
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Received 12 January 2001;received inrevised form 11 April 2001;accepted for publication 11 April 2001