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Keywords:

  • air pollution;
  • dew;
  • OH radical;
  • NO radical;
  • stomata;
  • photosynthesis;
  • Pinus densiflora (Japanese Red pine).

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  • • 
    The effects of free radicals, ·OH and ·NO, generated in polluted dew water on needles of Pinus densiflora (Japanese Red pine) were investigated.
  • • 
    ·OH-generating solutions (HOOH with Fe(III) and oxalate ion; ·OH treatment) and ·OH- and ·NO-generating solutions (NO2; ·OH/·NO treatment) were regulated at 25, 50 and 100 µmol and pH 4.4. HOOH only (HOOH treatment) was used as a control solution. Solutions were applied as a mist to the needle surface of P. densiflora seedlings before dawn twice a week for 3 months.
  • • 
    Within a month, net photosynthesis at near saturating irradiance (Pn) and stomatal conductance (gl) of ·OH-treated needles decreased with increasing solution concentration. The HOOH treatment had no effects on any of the measured parameters. Therefore, ·OH in the artificial dews caused the decreases in Pn and gl. In ·OH/·NO-treated needles, gl increased during the experiment, but Pn was unchanged. In all experiments, the characteristics of PSII were not significantly altered.
  • • 
    Free radicals in polluted dew water have harmful effects on the photosynthesis of P. densiflora and compound effects of ·OH and ·NO are different.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Evaluation of the effects of secondary pollutants generated from NOx on terrestrial ecosystems is an important problem. The possibility of negative effects of nitrogen oxides (NOx) on vegetation has been suggested, but there has been little research aimed specifically at the effects of atmospheric NOx (Berrang et al., 1995). A significant correlation has been observed between atmospheric NOx concentration (which is on average lower than 30 ppb) and tree decline (Naemura et al., 1997). However, when various plants were directly exposed to NO2 in short-term experiments, at least 100 ppb NO2 was needed to detect any effects (Natori & Totsuka, 1984). Even under exposure to 500 ppb NO2 for 6 h d−1 for 66 d, Pinus sylvestris did not show visible injury (Oleksyn et al., 1988). On the other hand, atmospheric nitrogen oxides generates ozone and other peroxides through photochemical reactions. Heber et al. (1995) concluded that the indirect toxic action of NO2 via ozone formation is much more damaging than the direct action of NO2.

The forests of Pinus densiflora on Mt. Gokurakuji (34°23′ N, 132°19′ E, 693 m asl) have severely declined over the last decade. The atmospheric NO2 concentration on the seaward side (an area of forest decline) of Mt. Gokurakuji was significantly higher than that on the inland side (an area free of decline). In the area where decline has occurred, significant correlations were found between the mean concentration of atmospheric NO2 and the mortality of the pines (Naemura et al., 1997) and the ethylene emission from pine needles (Kume et al., 2001). Soil acidification (pH, total N content) was not correlated with the decline (Kume et al., 2000). Kume et al. (2000) showed that maximum net photosynthesis (Pn) in the areas displaying decline was about 30% lower than that in the other areas, and that the lower Pn could be largely explained by the decrease in maximum stomatal conductance (gl). The causes of physiological disorders of needles were speculated to be caused by NOx-related substances but not O3 (Kume et al., 2000, 2001). However, in the most polluted areas of Mt. Gokurakuji, the maximum NOx concentration was about 200 ppb and the annual average NO2 concentration was at most 20 ppb. Therefore, the effects of secondary pollutants or other associated pollutants should be considered.

On Mt. Gokurakuji, the atmospheric NO2 is concentrated at lower altitudes because of the climatic inversion layer that frequently occurs below 300 m (Naemura et al., 1996). The highly humid climate in the Seto Inland Sea area is prone to dew from the night to the morning of clear days throughout the year. The frequency of natural dew formation in the low altitude areas is about 20–30% in summer and 30–50% in winter mornings. Under such conditions, not only do the gaseous pollutants stagnate in the low altitudinal range, but also secondary pollutants are generated from NOx and are accumulated in the morning dews and/or fogs. Peroxides and HNO3 are likely to be deposited on leaf surfaces because of their high solubility in water (Cape, 1997).

Photochemical reactions in the liquid-phase form various oxidants, such as HOOH, HO2, singlet molecular oxygen, and OH (Faust & Allen, 1992; Faust et al., 1993; Anastasio et al., 1994; Arakaki & Faust, 1998). NO2, NO3, HOOH, Fe(OH)2+, and the photo-Fenton reaction (a reaction between photochemically reduced iron and HOOH) are all known OH sources in the liquid-phase (Zellner & Herrmann, 1990; Faust & Hoigné, 1990; Zepp et al., 1992). Arakaki et al. (1998, 1999b) observed that OH and NO radicals (·OH and ·NO) were formed from the photolysis of nitrite ions (NO2) in dew water and estimated ·OH photoformation rate constants (Arakaki et al., 1999a). They suggested that the photolysis of aqueous-phase N(III) (HNO2 and NO2) plays a significant role in initiating oxidation reactions in dew water. Arakaki et al. (1999b) reported that the average rate of ·OH formation in dew collected on a Teflon sheet (1.25 µM h−1) was 3.6 times greater than that in the rainwater (0.35 µM h−1) during the period 1997–1998 at Higashi-Hiroshima, Japan under conditions of the same solar irradiation. Nakatani et al. (2001) determined ·OH formation rates in dew on the needle surface in the declining pine forest of Mt. Gokurakuji during October–November 1999. They found that the mean rate (3.36 µM h−1) and highest rate (5.18 µM h−1) were much higher than the values reported by Arakaki et al. (1999b). Dew dissolves deposits on the surface of the pine needles, and subsequent evaporation after dawn concentrates the ·OH forming compounds. It is probable that the ·OH formation potential is much greater on the surface of the pine needles than in the dew collected before sunrise. Therefore, free radicals generated in the morning dew on needle surfaces might cause some of the ecophysiological disorders observed in the pine needles at Mt. Gokurakuji.

Another important fact is that the mean and range of pH values in dew formed on the pine needles were 4.54 and 4.23–5.96, and these were same levels as those of rain (Nakatani et al., 2001). Therefore, the pH of dew was not low enough to induce harmful effects on the pine needles.

We investigated the effects of polluted dew water, especially ·OH and ·NO on pine needles because ·OH is considered the most potent oxidant in the atmosphere (Warneck, 1988; Wayne, 1991; Thompson, 1992; Finlayson-Pitts & Pitts, 2000) and ·NO is potentially more phytotoxic than NO2 (Wellburn, 1990). Few studies have reported the effects of ·OH on tree decline, probably due to the lower concentrations (1–10 × 106 molecules cm−3, Prinn et al., 1987) and difficulties in detecting ·OH in the atmosphere (Mount & Eisele, 1992).

We focused on wet deposition, in which gaseous and dry phase substances are transformed to the liquid phase, as a factor affecting the ecophysiological traits of pine needles.

In order to verify the effects of ·OH and ·NO on the needles, we conducted simulated morning dew experiments for various liquid phase oxidants, and the effects on photosynthesis, stomatal conductance, and the light reaction of needles were investigated.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Growing areas

The experiments were conducted in three arched metal frame shelters on the Hiroshima University campus (34°24′ N, 132°44′ E, 210 m asl), the purpose of which was to block the fall of dew. They were of semicylindrical shape (horizontal axis) and covered an area of approx. 8 m × 12 m. The upper half of the frame structure (not including the two ends) was covered with a 0.06-mm-thick Tefzel film (Dupont, Wilmington, DE, USA) that is transparent to both visible and UV light. Thus, the structures were well ventilated and did not disturb natural sunlight. Eight areas were established under each structure (i.e. total of 24 areas), with each area containing five ceramic 30-l pots. Under these open glasshouses, two areas were used for one treatment (total of 11 treatments as shown in Table 1) and four areas were used for control experiments. The two areas were allocated randomly in different glasshouses in order to cancel the effects of glasshouse and of position in the glasshouse.

Table 1.  Chemical composition of the pseudo morning dew
 HOOH µMNO2µMFe(III) µMOxalate µM·OH µM h−1·NO µM h−1
  1. The solution pH was adjusted to 4.4 by adding sulfuric acid. Photochemical formation of OH radical was determined by following Arakaki et al. (1998). All the OH radical formation rates were normalized to clear-sky, solar noon conditions of 34-degree north for the sake of comparison. The normalization clearly over predicts OH radical photoformation for morning dew conditions, but enables comparison of data among rain and dew water samples reported Arakaki et al. (1998).

OH-25 25  025  7.4 0
OH-50 50  025 10.2 0
OH-100100  025 14.3 0
NO-25  0 2500  4.4 4.4
NO-50  0 5000  8.8 8.8
NO-100  010000 17.617.6
HOOH-25 25  000  0.3 0
HOOH-50 50  000  0.6 0
HOOH-100100  000  1.3 0
Fe  0  020< 0.1 0
Control  0  000< 0.1 0

Plant and soil materials

Two-year-old seedlings of P. densiflora, which had been grown at Okayama City, about 120 km east of Hiroshima University campus, were planted in the pots (one seedling per pot) on 20 March 1998. The pots were filled with a mixture of weathered granite, which is widely distributed in Hiroshima prefecture and called ‘masado’. The soil surface was covered with organic materials containing F and H horizons from a P. densiflora stand on Hiroshima University campus, accounting for approx. 10% of the total volume. The pots were irrigated automatically, maintaining the soil water potential above –0.01 Mpa.

New needles were fully expanded by the middle of July. On 14 July 1998, the seedlings were 63 ± 6.5 cm in height and 9.7 ± 0.9 mm (mean ± SD) in stem diameter at a height of 5 cm.

Morning dew experiment

·OH and/or ·NO generating solutions and control solutions (Table 1, c. 40 ml per seedling) were sprayed onto the pine needles as a mist twice a week before dawn from 15 July to 15 October 1998. The frequency was similar to that of natural dew formation during the summer period (25–30%) in the lower altitudinal areas of Mt. Gokurakuji. The natural dew usually starts forming from midnight till a few hours after dawn, but the solutions were sprayed immediately before dawn to prevent the surfaces becoming dry during the night in the glasshouse. The soil was prevented from direct contact with the spray solution as well as from mutual interaction among different spray solutions. All the solutions were prepared with distilled water and the pH was adjusted to 4.4 with sulfuric acid. Hydroxyl radical-generating solutions (OH-25, -50, -100, Table 1) were formulated utilizing the photo-Fenton reaction (Zepp et al., 1992). Solar energy reduces Fe(III) to Fe(II), and HOOH reacts with Fe(II) to generate ·OH in the solution. Oxalate was added to the solution to facilitate the reduction of Fe(III) by forming Fe(III)-oxalate complexes, which are photochemically more active (Zuo & Hoigné, 1992). When Fe(III) and oxalate were not added (HOOH-25, -50, -100, Table 1), generation of ·OH from HOOH was below 1.3 µM h−1, compared with 7.4, 10.2, and 14.3 µM h−1 at the same solar intensity, respectively. A solution with only Fe(III) adjusted to pH 4.4 was also applied (Fe, Table 1), and the solution only adjusted to pH to 4.4 with sulfuric acid (control, Table 1) was treated as the control.

Fluorescence analysis and photosynthesis measurement

Physiological measurements was conducted on current needles. In each pot of the glasshouse, the minimal fluorescence (F0) and the photochemical efficiencies of PSII in the dark (Fv/Fm) were measured by a photosystem yield analyser (MINI-PAM and Leaf-clip holder 2030B, Heinz Walz GmbH, Effeltrich, Germany). F0 is indicative of open reaction centres of PSII due to a fully oxidized state of the primary electron acceptor (Schreiber et al., 1995). Fv/Fm indicates the potential maximal PSII quantum yield (Björkman, 1987) and the value of healthy leaves is about 0.8 (Björkman & Demmig, 1987). At night, from 22:00 to 2:00 in each month, Fv/Fm and F0 were measured for each seedling.

On a sunny morning from 07:00 am to 10:00 am, net photosynthesis (Pn), stomatal conductance (gl) and intercellular CO2 concentration (Ci) were measured for each seedling. The seedlings were exposed to direct sunlight during the measurement period. Pn, gl and Ci at 1000 µmol m−2 s−1 PFD at a needle temperature of 25°C were measured with an open-flow infrared gas analyser with a light and temperature control system (LI-6400, Li-Cor Inc., Lincoln, NE, USA). The range of ambient CO2 concentration was between 390 ± 5 µl l−1 and VPD was less than 1 kPa. The width and length of needles were also measured with a digital caliper (CD-15, Mitutoyo Co., Kanagawa, Japan). We used half of the surface area of the needles as the effective leaf area for photosynthesis (Kume et al., 2000). These measurements were conducted at approx. 30-d intervals. At each time, different needles were chosen. Initial values of gl, Pn, Ci, F0, Fv/Fm are shown in Table 2.

Table 2.  Initial values of measured characteristics. (n = 100)
 Mean ± SD
gl (mmol H2O m−2 s−1)   68 ± 15
Pn (µmol CO2 m−2 s−1) 10.5 ± 1.5
Ci (µmol CO2 mol−1)  214 ± 20
F0  268 ± 28
Fv/Fm0.819 ± 0.010

Statistics

Data were transformed to relative values of the initial values (before treatment) of each plant, and were tested by ANOVA and posthoc tests. Tukey's HSD test (Zar, 1996) was applied for the post hoc tests. Data were presented as the mean ± SE and were compared between control and treated plants. Before the treatments, there were no significant differences among the districts in all measured values. All statistical analyses were carried out using the StatView 5.0 (SAS Institute Inc., Cary, NC, USA).

Results and Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Effects of ·OH

Effects of gaseous oxidants on plant leaves have been well studied, but the effects of oxidants in the liquid phase, especially polluted dew on the leaf surface, which may directly affect guard cells and the apoplast, were not considered. Our results clearly showed that ·OH-generating solutions, which simulated polluted dew, had some effects on the stomatal responses of P. densiflora. After 1 month of the mist treatment, Pn and gl decreased significantly with the increase of the concentration of ·OH solutions (OH-25 to OH-100), and these tendencies were maintained over the three-month experimental period (Fig. 1). F0 tended to decrease with time but not significantly, and Fv/Fm did not change significantly (data not shown, P > 0.05). Ci was not significantly lower than that of the control, but all of the Ci values were lower than that of initial values.

image

Figure 1. The patterns of photosynthesis (Pn), stomatal conductance (gl), intercellular CO2 concentration (Ci) and minimal florescence of PSII (F0) 30, 60 and 90 d after the morning dew experiment. Data were expressed as values relative to the initial values of each seedling. Vertical lines indicate SE (n = 10). The data were compared between control and treated plants. *P < 0.05. There was no significant difference in Fv/Fm (data not shown, P > 0.05). Closed columns, -100; heavily dashed columns, -50; lightly dashed columns, -25.

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It is well known that HOOH by itself could not be a major problem for plant growth because it is normally already present at concentrations above 100 µM in many tissues (Schopfer, 1994). In fact, there were no significant differences in all these properties in hydrogen peroxide-treated seedlings (HOOH-100), where ·OH generation was quite low. Furthermore, Fe-only solution did not cause significant differences in all these properties (data not shown, P > 0.05). Though oxalate-only solution was not tested, oxalate by itself is less likely to induce significant physiological changes, considering the low concentration (i.e. 5 µM) that we applied. Therefore, we conclude that ·OH in dew on the needles caused the decreases in Pn and gl.

Compound effects of ·OH and ·NO

In the decline areas of Mt Gokurakuji, the major source of ·OH generation was attributed to NO2 (Arakaki et al., 1999b). Therefore, evaluation of compound effects of ·OH and ·NO is important. Regardless of the concentration of the ·OH/·NO-generating solution (NO-25 to NO-100), gl and Ci increased with time. However, Pn, F0 and Fv/Fm did not change significantly. These results showed that ·OH/·NO treatment did not affect the light reaction process of photosynthesis in chloroplasts, but it affected the process of stomatal control when even weak solutions were sprayed. In spite of the increase in Ci, Pn did not increase but rather tended to decrease. Therefore, the activity in the dark reaction of photosynthesis was lowered. It is well known that NO2 is assimilated from the plant surface and utilized for nitrogen resources (Morikawa et al., 1998). Thus NO2 is not only harmful for plant growth. However, Mehlhorn et al. (1990) suggested that ·NO in polluted air is most probably responsible for generating the observed signals. Wellburn (1990) also pointed out that ·NO is potentially more phytotoxic than NO2. The water solubility of ·NO is low, but we demonstrated that ·NO generation in the polluted dew water with sunlight caused a significant increase in gl. Because ·NO has various effects on various regulation systems of organisms (Halliwell & Gutteridge, 1999), it is not surprising that ·NO has some effects on stomatal aperture.

Neighbour et al. (1988) showed that after one month of fumigation of SO2 (40 ppb) + NO2 (40 ppb), gl of Betula pubescens increased significantly both in the day and at night because of damage to the stomata. In order to examine the conditions of the stomata, we measured gl at midnight (Fig. 2). In NO-100, gl at midnight, about 30 mmol H2O m−2 s−1, was three times larger than it was in the other treatments. The value at midnight was about 30% of that in the daytime. This result suggested that many stomata in the ·OH/·NO treatment were not able to close completely. The ·OH/·NO treatment generated as much ·OH as the ·OH treatment. Therefore, coexisting ·NO had a strong effect on stomata of the pine.

image

Figure 2. Stomatal conductance (gl) measured at 11:00 pm 3 months after the experiment. Vertical lines indicate SE (n = 10).

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It must be pointed out that in our solutions (pH = 4.4) approx. 7% of NO2 exists as HNO2 (NO2 + H+[LEFT RIGHT ARROW] HNO2 or HONO, pKa = 3.27 (Arakaki et al., 1999a)). Using Henry's law constant for HNO2 (49 M atm−1 (Finlayson-Pitts & Pitts, 2000)), HONO concentrations in the gas-phase are estimated to be 151 ppb at 100 µM NO2, 76 ppb at 50 µM NO2, and 38 ppb at 25 µM NO2. Because HONO concentrations are typically below 10 ppb (Finlayson-Pitts & Pitts, 2000), we cannot preclude a possibility that the observed ·OH/·NO effects could have been induced by gaseous HONO (directly or by subsequent photolysis). Further study is clearly needed to better understand the effects of HONO/NO2.

The pattern of declining photosynthesis observed on Mt Gokurakuji and the current experimental results of compound effects of ·OH and ·NO were not consistent. On Mt Gokurakuji, the major part of the decrease in Pn can be explained by the decrease of gl (Kume et al., 2000). In the ·OH treatment, Pn also decreased with the decrease of gl and Ci. On the other hand, in the ·OH/·NO treatment, gl increased to one and a half times the initial values but Pn did not increase, that is not only did the responses of the stomata change but the activity of the dark reaction of photosynthesis was reduced. The responses of stomata to the stress of air pollution are not simple. Maier-Maercker (1998) pointed out the importance of soil moisture content when considering the sensitivity of stomatal response to O3 exposure. The difference in the stomatal responses between the field and the experimental conditions might be due to the higher water content in the cultivated pots where the soil was saturated with water.

Further studies are needed to determine the potential effects of prolonged exposure of pine trees to low concentrations of ·OH. Furthermore, the effects of diesel exhaust particles (DEP) and other fine particles on free radical generation (Sagai et al., 2000) should be considered under field conditions, because the estimated ·OH radical formation from NO2 and NO3 is only about 40% (Nakatani et al., 2001) and the rest of the source for OH radical formation was still unknown. However, in the current study, we demonstrated the possibility that free radicals in polluted dew water have harmful effects on the stomatal responses of P. densiflora.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

We thank Dr T. Kobayashi of the Japan Science and Technology Corporation (JST) and Dr J. N. Cape of the Centre for Ecology & Hydrology (CEH) for advice and comments on the manuscript. We also thank the members of the Nakane Laboratory. This study is a part of a larger project to understand the process of forest decline in Japan, funded by the Core Research for Evolutional Science and Technology (CREST) of JST.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
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