Environ. Sci. Technol. 1994, 28, 812-815
Does Atmospheric Hydrogen Peroxide Contribute to Damage to Forest Trees? Andrea Polle’’t and Wolfgang Junkermann* Albert-Ludwigs-Universitat Freiburg, Institut fur Forstbotanik und Baumphysiologie, Professur fur Baumphysiologie, Am Flughafen 17, D-79085 Freiburg, Germany, and Fraunhofer Institut fur Atmospharische Umweltforschung, Kreuzeckbahnstrasse 19, D-82467 Garmisch-Partenkirchen, Germany H2O2 has two faces
in plants: it is toxic at low
concen-
trations in the chloroplasts, and it is a necessary cosubstrate for the production of biopolymers in the apoplastic compartment. Plants have evolved an antioxidant system that enables them to cope with high intrinsic production rates of H2O2. Measurements of H2O2 in air at a rural, forested site were used to calculate the influx of atmospheric H2O2 into spruce needles. The estimated uptake rates were compared with the capacity of protective systems present in the aqueous matrix of the cell wall and inside the cells in the symplastic space. Evidence is presented that the rate of H2O2 detoxification exceeds its uptake up to 106-fold. Therefore, it is unlikely that atmospheric H2O2 in the absence of synergistic effects of other air pollutants can overwhelm the intrinsic protection of mesophyll cells, thereby contributing to damage to the spruce trees.
Introduction Hydrogen peroxide (H2O2) plays an important role as oxidant in the chemistry of the troposphere. It can be formed in the gas phase by photooxidative reactions of ozone with volatile hydrocarbons from biogenic or anthropogenic origin (1-4). Ambient H2O2 concentrations range from about 0.02 to 6 ppb (5, 6). The presence of H2O2 in air has been considered a source of concern because H2O2 is a strong oxidant which can impair metabolic processes in living tissues. H2O2 can inactivate enzymes by the oxidation of SH groups or cause lipid peroxidation in the presence of metal catalysts (7,8). It has been suggested that H2O2 is an important factor contributing to forest decline (1, 9). However, exposure of tree seedlings to acid mist containing 147-235 ^M H2O2 yielded conflicting results, i.e., leaf injury in Norway spruce and beech (10) and no effect in red spruce (11). When red spruce trees were exposed in short-term chamber experiments to gaseous H2O2 at concentrations ranging from 0.18 to 3.9 ppb, dry deposition rates to needles ranged from 22 to 423 pmol nr2 s_1 (12,13). The significance of dry deposition of ambient H2O2 concentrations for damage to tree species is not known. At rural forested sites, mean H2O2 gas-phase concentrations of 0.9 ppb were observed in the summer (2,14). Because of its high water solubility [Henry constant of 1.65 X 105 M_1 s-* (15)], 0.9 ppb H2O2 would correspond to an equilibrium concentration of 148 mM H2O2 in the aqueous phase. As plant tissues contain about 50-90% water and photosynthesis is already severely inhibited at 1
*
Corresponding author; Telephone: +761-808368, Fax: +761-
808349. 1
t
812
Albert-Ludwigs-Universitat Freiburg. Fraunhofer Institut fur Atmospharische Umweltforschung. Environ. Sci. Technol., Vol. 28, No. 5, 1994
10 jtM H2O2 (8), plant life would not be possible, even at fairly low atmospheric H2O2 concentrations, if barriers and detoxification mechanisms were absent. In this paper, we will discuss the significance of protective measures for H2O2 present in plants with special emphasis on spruce (Picea sp.), which is one of the most important forest tree species in Europe and North America. External and Internal Sources of H2O2 in Leaf Tissue. The vegetation is an important sink for air pollutants. In order to estimate the maximum portion of ambient H2O2 that might be expected to enter the leaf interior, it was assumed that H2O2 has to pass the same diffusion barriers as other air pollutants such as ozone or SO2 (16,17). Hydrophilic gaseous compounds enter plant
leaves predominantly via the stomata. The possibility that H2O2 might mainly deposit on the surface of needles
and the limitation to the application of the multiple resistance model (employed below) for conifer needles are neglected (13,18,19). The H2O2 influx (Fh2o2) can then be calculated according to the following flux equation (Fick’s law): =
“
£H202(cout
^H202
(*)
cin)
the conductance for H2O2 (m s_1), cout is the concentration of H2O2 in air, and Cjn is the concentration of H2O2 in the leaf (mol m-3). The conductance for H2O2 (£h2o2) is determined according to (19):
£h2o2 is
+
=
#H202
Ra, Rh, and Rc are
+
*.)
1
®
aerodynamic, boundary, and canopy
resistances (s nr1), respectively. Aerodynamic and boundary resistances are small in a well-mixed atmosphere. In the example shown in Figure 1, the mean wind speed was about 3 m s_1, and therefore, R& and R^ were negligible. The canopy resistance is a composite resistance taking into account the transfer of a gas to different surfaces such as vegetation, water, and soil (19). Among different
components comprising the canopy resistance, stomatal and mesophyll resistances are important in order to determine the portion of H2O2 that might enter the leaves and reach the mesophyll cells. Because of the high water solubility of H2O2 and its rapid degradation in the tissue (see below), the mesophyll resistance was assumed to be zero. Under these conditions, the uptake of H2O2 into the leaves is determined by the stomatal resistance (r,-1 = £h2o2)- In a first approximation, gn2o2 is proportional to the stomatal conductance for water (D#h2o) corrected for the lower diffusivity of H2O2 in air than H2O in air [D = (molwtofH20/molwtof H202)1/2 = 0.714; (20)]. Under these assumptions, the influx of H2O2 into the leaves (eq 1) reads as follows: =
•^h2o2
0.714gH2O cout
0013-936X/94/0928-0812$04.50/0
©
(3)
1994 American Chemical Society
1. Hydrogen Peroxide Related Systems in Total Needles and Cell Walls of Norway Spruce (Picea abies, L.) Needles*
Table
CO
E
E E
o E a
n o.
3
0N I
oN
o>
X
N
X • I
•
ll
Time of day (hours) Figure 1. Diurnal variation of the gas-phase mixing ratio and flux of H202 into spruce needles. The concentration of H202 in air was determined on June 27, 1991, at a meteorological station located at Wank mountain (1175 m a.s.l.) close to a spruce forest. Mean temperature, air pressure, and wind speed were (± standard deviation) 18.8 ± 3.2 °C, 886.7 ± 1.4 bar, and 3.1 ± 2.1 m s-1, respectively, in the measuring period of 24 h. Measurements of total peroxide were carried out fluorometrically in the presence and absence of catalase (48). The amount of H202 was obtained after subtraction of the catalaseresistant part of the signal. In order to calculate the flux of H202 into spruce needles, stomatal conductances for H20 found under similar weather conditions were taken from refs 16 and 21. The H202 flux (F) was calculated as explained in the text and is indicated in pmol nr2 leaf area s-1.
Figure 1 shows diurnal fluctuations of ambient H2O2 and stomatal conductance for water vapor of spruce needles on a clear summer day. These data were used to calculate H2O2 flux rates (according to eq 3) for the course of a day. As maximum gaseous H2O2 concentrations of 2 ppb occurred when the stomatal conductance was also at its maximum, the maximum rate of influx, 70 pmol m-2 s-1 (leaf area basis), was also the highest possible influx (Figure 1). The actual flux of H2O2 may even be lower because of the special structural features of conifer needles discussed by Claiborn et al. (18). It is also important to note that stomatal conductances of conifer needles on cloudy days or under laboratory conditions are about 2-3fold lower than in the above example (cf. refs 13,16, and 21). This would cause further reductions in H2O2 influx
into needles. According to this model, broadleaf trees and herbaceous plants with about 5-10 times higher stomatal conductances than conifers would be expected to show correspondingly higher H2O2 uptake. It is obvious that high nighttime concentrations of H2O2 occasionally observed at high elevation (14, 22, 23) are not important to plants which close their stomata at night. In addition to H2O2 present in ambient air, plants are also exposed to internal sources of H2O2 such as photosynthesis and photorespiration (24,25). About 5-20% of photosynthetically produced electrons are transferred to dioxygen (26). The resulting 02*' radicals are removed by the activity of superoxide dismutase at an almost diffusioncontrolled reaction rate [2 X109 M-1 s-1 (24)]. The product of this reaction is H2O2. The intracellular H2O2 production rate in spruce can be assessed as follows: In the light each reaction center (RC) produces 100 electrons/s (27). Since chloroplasts contain about 1 RC/1000 chlorophyll molecules, spruce needles with about 1000 Mg of chlorophyll (g
parameter
needle
surface area (m2 (g of fresh wt)-1) water content (mL (g of fresh wt)-1) ascorbate (mM) ascorbate (Mmol m-2) peroxidase (mM s-1) peroxidase (Mmol m-2 s-1) NADH oxidase (mM s-1) NADH oxidase (Mmol m-2 s-1)
3.5 X 10-3 451 23.03 3708 20.4 2628 10.57 1360
cell wall
nd 67 1.95 41 1.67 35 0.67 12
0 Data from Polle et al. (41) and Chakrabarti et al. (46) were related to surface area and water content of the apoplastic and symplastic space of spruce needles. According to Gross and Koch (47), it was assumed that the apoplastic water content accounted for 13 % of the total water content of the needles. Peroxidase activity was determined with coniferyl alcohol, a specific substrate of lignifying peroxidases. The contamination of the apoplastic washing fluid with symplastic components amounted to less than 0.02% as estimated from measurements of glutathione in both fractions, nd = not determined.
of fresh wt)-1 contain 1 nmol of RC (g of fresh wt)-1. If 10% of photosynthetically generated electrons are used for O2*- production, a H2O2 production rate of 5 nmol of H2O2 s-1 (g of fresh wt)-1 can be inferred. If this figure is related to the specific leaf area of spruce needles (Table 1), the photosynthetic H2O2 production rate amounts to 1.6 Mmol m-2 s-1. Photorespiration is also an important source of H2O2 in plant cells. There is evidence that about 30-50 % of photosynthetically produced reductant is used in this pathway (28). Photorespiration would result in an additional generation of H2O2 of 2.3-3.5 ywmol m-2 s-1. Apparently, the cellular production rate of H2O2 is 5 orders of magnitude greater than the maximum influx of external H2O2 of 70 pmol m-2 s-1 estimated for a peak concentration of 2 ppb H2O2 in the air and at least 4 orders of magnitude greater than deposition rates of H2O2 to needle surfaces [22-423 pmol m-2 s-1 (12,13)]. Apoplastic and Cellular Protection from H2O2. Because of high intracellular production rates of H2O2, plant cells need effective means to prevent an accumulation of H2O2. The subcellular distribution of protective systems found in mesophyll cells is indicated in Figure 2. Ascorbate and ascorbate peroxidase are the most important components for the removal of H2O2 in chloroplasts and the cytosol, whereas catalase is predominantely localized in peroxisomes (29,30). In mature spruce needles, the mean concentration of ascorbate ranged from 15 to 25 mM, assuming a homogeneous distribution in the cellular fluid (Table 1, cf. ref 31). Ascorbate peroxidase activity was in the range from 300 to 600 nmol (g of fresh wt)-1 s-1 and catalase in the range from 0.4 to 5 Mmol (g of fresh wt)-1 s-1 (31-33), i.e., equivalent to H2O2 decomposition rates of 100-1400 Mmol m-2 s-1. The actual protection from H2O2 in cytosol and chloroplasts is probably higher because they comprise less than 10% of the total volume but contain 80-90 % of the antioxidant substrates and enzymes. This estimate shows that the intrinsic protection from H2O2 is extremely powerful. Therefore, the putative influx of external H2O2 is unlikely to be sensed in the symplastic compartment on top of internal H2O2 turnover. It has been suggested that the primary targets for reactive air pollutants such as ozone are localized outside the symplastic space in the aqueous matrix of the cell wall, which is called apoplastic space (34, cf. Figure 2). Is this concept also valid for H2O2? The apoplastic space of Environ. Soi. Technol., Vol. 28, No. 5, 1994
813
Apoplastic space
Plasma membrane
Symplastic space
Cliloronlast Cytosol Glutathione Ascorbate Superoxide dismutase Ascorbate peroxidase Monodehydroascorbate
Chloroplast
reductase
Nucleus
Dehydroascorbate reductase Glutathione reductase "Unspecific" peroxidase
reductase
Dehydroascorbate reductase Glutathione reductase
©
Vacuole
ot-Tocopherol Carotenoids Glutathione Ascorbate Superoxide dismutase Ascorbate peroxidase Monodehydroascorbate
©
Mitochondria
Vacuole ,
Glutathione (?) Ascorbate "Unspecific" peroxidase
Cell wall
Glutathione Superoxide dismutase Glutathione reductase
Cell wall
Peroxisomes
Ascorbate
Superoxide dismutase
Catalase "Unspecific" peroxidase Figure 2. Subcellular localization of antioxidants and protective enzymes in a typical mesophyll cell.
plants contains high activities of “unspecific” peroxidases that use various phenolic substrates such as ferulic acid and coniferyl alcohol for the removal of H202 (35). The physiological function of these peroxidases is thought to be the production of biopolymers such as lignin, suberin, or extensin. Typical activities of coniferyl alcohol peroxidase extracted from cell walls of mature spruce needles were greater than 30 Mmol H202 m-2 s~x (Table 1). Cell walls of different plant species contain ascorbate at concentrations ranging from about 1 to 3 mM (32,3638; Table 1). However, the “unspecific” peroxidases in the apoplastic space of spruce needles have only little specificity for ascorbate (32), and the nonenzymatic reduction of H202 by ascorbate is slow [Jf = 6 M'x s_1 at pH 5.3 (39)]. Still, apoplastic ascorbate might be important for H202 degradation. Circumstantial evidence suggests that the aqueous phase of cell walls contains free phenolics that are oxidized to phenoxy radicals in the presence of H202 and peroxidases (38). Phenoxy radicals can be reduced nonenzymatically by ascorbate at a high rate [K = 4-20 X 10® M_1 s_1 (40)]. Thereby, ascorbate can recycle the substrate for peroxidase and mediate protection from H202. Apparently peroxidase, phenolics, and ascorbate provide an extracellular detoxification system for H202. The efficacy of this system depends on the regeneration of reduced ascorbate in the apoplastic space. The regeneration mechanism for apoplastic ascorbate is not known. But even if it is operating slowly, at a time scale of hours to days, accessible sites such as the plasma membrane would be well-protected from external H202 in spruce needles because of high concentrations of apoplastic ascorbate and low uptake rates of H202. Is H202 Influx Affected by Resistances? The discussion so far has only considered maximum influx of H202, i.e., a situation in which the internal H202 concentration was presumed to be negligible and in which the diffusion of H202 was hindered by the same resistances as water vapor. A significant flux reduction—or at its extreme, even an exhalation of H202—may be expected if internal sources cause H202 accumulation. This possibility can be considered theoretically because the apoplastic space contains enzymatic activities and NADH 814
Environ. Scl. Technol., Vol. 28, No. 5, 1994
oxidases, which can produce H202 (41). H202 is a necessary cosubstrate for lignification and has been detected in plant tissues in phases of active lignification (42). The capacity for H202 production of NADH oxidases extracted from the apoplastic space of mature spruce needles amounted to 12 Mmol H202 m-2 s_x (Table 1). However, in fluids extracted from the apoplastic space of mature, i.e., fully lignified spruce needles, H202 was not detected (detection limit 0.5 nM; Polle and Seifert, unpublished results). In addition to apoplastic enzymic reactions, the possibility of H202 production in the gaseous intercellular space by terpenes with ozone may also be envisaged. In laboratory experiments, H202 was produced in gas-phase reactions in the presence of ozone and cyclic monoterpenes such a- and jS-pinene or limonene, i.e., components emitted from coniferous species (1,2,43). At an alkene:ozone ratio of 1:2, the yield of H202 amounted to 15% of the initial terpene concentration (2). Deposition rates of H202 determined with micrometeorological methods suggested the presence of H202 sources in the crown region of spruce forests (44,45). Since spruce needles emitted terpenes at rates of about 200 pmol m 2 s_1 in the summer (21), ozone alkene reactions were suspected to be the reason of the apparent H202 formation. The production of H202 in the gas phase inside the leaves seems at least as likely as outside
in their atmospheric environment. Thus, the influx of ambient ozone concentrations into needles, which is in the range of 1 nmol irr2 s_1 at 60 ppb ozone (17), might sustain H202 production in the intercellular system, thereby generating a resistance to the uptake of external H202.
Fluxes of H202 determined in branches and young seedlings of red spruce showed no day to night variations. It is, therefore, possible that H202 deposition is limited to the surface of needles (12, 13). Claiborn et al. (18) suggested that the wax-filled stomatal antechamber may provide an extra resistance to the diffusion of water-soluble gases as compared to water vapor. The presence of internal and external resistances to H202 uptake cannot be ruled out and needs to be investigated in order to understand biosphere/atmosphere interactions. However, regardless of whether spruce trees have to cope with internal or
external sources of H2O2, the needles appear to be overprotected from this oxidant by several orders of magnitude. Therefore, it is unlikely that gaseous H2O2 as a single factor can contribute to damage to spruce trees. Acknowledgments We would like to thank Prof. Dr. H. Rennenberg for his constant support and for his critical reading of the manuscript. Financial support by the Bayerisches Staatsministerium fur Landesentwicklung und Umweltfragen is gratefully acknowledged.
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Received for review June 4,1993. Revised manuscript received December 29,1993. Accepted January 26, 1994.9 ®
Abstract published in Advance ACS Abstracts, March 1,1994.
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