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average daytime concentration of O3 increased from 1985 to 1999, at a rate of 0.33 nmol mol－1 year－1 (Ohara and Sakata, 2003). Since the ambient levels of O3 adversely affect the growth and physiological functions, such as photosynthesis, of forest tree species, this gas is considered to be one of the most important factors relating to forest decline and tree dieback in the USA, Europe and Japan (Sandermann et al., 1997; Bytnerowicz et al., 2004; ADORC, 2006). Because a forest plays important conservation roles for soil and head waters, decline in forests increases the risks of

Effects of Ozone on the Growth and Photosynthesis of Castanopsis sieboldii Seedlings Grown under Different Nitrogen Loads

Makoto WATANABE*†, Masahiro YAMAGUCHI*, Hideyuki MATSUMURA**, Yoshihisa KOHNO**, and Takeshi IZUTA***

*United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183–8509, Japan

**Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, Abiko, Chiba, 270–1194, Japan

***Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183–8509, Japan

AbstractTo obtain basic data for evaluating the critical levels of ozone (O3) for protecting Japanese evergreen

broad-leaved tree species, we investigated the effects of O3 on the growth and net photosynthesis of Castanopsis sieboldii seedlings grown under different nitrogen (N) loads. Two-year-old seedlings were grown in 12 experimental treatments, which were comprised of four gas treatments (charcoal-filtered air and three levels of O3 at 1.0, 1.5 and 2.0 times the ambient concentration) in combination with three N treatments (0, 20 and 50 kg ha－1 year－1) in open-top chambers during the two growing seasons. The net photosynthetic rate and the whole-plant dry mass of the seedlings were reduced by exposure to O3, while were increased by N load. No significant interaction between O3 and the N load for the whole-plant dry mass of the seedlings was observed. The N load increased the sensitivity of net photo-synthetic rate to O3 during the first and second growing seasons. However, the increase in the area of second-flush leaves during the second growing season compensated for the O3-induced reduction in the net photosynthetic rate of the seedlings grown under relatively high N load. We concluded that the N load to the soil at ≤50 kg ha－1 year－1 has no influence on the sensitivity of the whole-plant dry mass of C. sieboldii seedlings to O3. Based on the relationship between AOT40 (accumulated exposure over a threshold of 40 nmol mol－1) of O3 during the daylight hours from April to September and the whole-plant dry mass increment per one growing season, the critical level of O3 for C. sieboldii, which was expressed as AOT40 for one growing season and corresponding to a 5％ reduction in the whole-plant dry mass increment, was evaluated as 22 µmol mol－1 h.

Keywords: Castanopsis sieboldii, Growth, Net photosynthesis, Nitrogen load, Ozone.

Received; March 21, 2008.

Accepted; May 20, 2008.† Present address: JSPS Research Fellowship, Research

Faculty of Agriculture, Hokkaido Uni-

versity, Sapporo 060-8589, Japan

1. Introduction

Ozone (O3) in the troposphere is recognized as a widespread phytotoxic air pollutant and its concentra-tions have been increasing in the Northern Hemisphere (Akimoto, 2003; ADORC, 2006). In Japan, the annual

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Full Paper J. Agric. Meteorol. (農業気象) 64 (3): 143–155, 2008

soil erosion, water pollution and groundwater depletion (Inoue et al., 2003). Therefore, it is necessary to clarify the effects of O3 on growth and physiological functions, such as the photosynthesis of Japanese forest tree species.

The atmospheric deposition of nitrogen (N) to terrestrial ecosystems has been increasing with elevated anthropogenic emissions of N (Galloway et al., 2003; Ohara et al., 2007). In general, N is a limiting resource of tree growth in forest ecosystems, and atmospheric N deposition acts as a fertilizer (Bobbink et al., 2003; Magnani et al., 2007). However, increasing anthropogenic N deposition from the atmosphere induces over-nutrition in some terrestrial ecosystems (Schulze, 1989; Fenn et al., 1996; Baba and Okazaki, 1998). In Europe, the threshold value of N load for the occurrence of N-saturation and the critical N load for protecting forest ecosystems are considered to be approximately 10 kg ha－1 year－1 and 10 to 30 kg ha－1 year－1, respectively (Wright et al., 1995; WHO Regional Office for Europe, 2000). In the forested area of Japan, N input by wet deposition (bulk precipitation) and that by throughfall and stemflow ranged from 10 to 25 kg ha－1 year－1 (Baba and Okazaki, 1998; Baba et al., 2001; Okochi and Igawa, 2001) and from 10 to 40 kg ha－1 year－1 (Kobayashi et al., 1995; Baba and Okazaki, 1998; Baba et al., 2001), respectively. Furthermore, the phenomena of N-saturation in several Japanese forest ecosystems have been already reported (Baba and Okazaki, 1998; Okochi and Igawa, 2001). Generally, excessive N deposition from the atmosphere to forest ecosystems causes soil acidification, reduction in the growth of fine roots, degradation of nutrient status in leaves, decline of frost hardiness and tolerance of trees to parasite attacks, and changes the sensitivity of trees to other environmental stresses such as gaseous air pollutants (Nihlgård, 1985; Schulze, 1989; Izuta, 2001; Bobbink et al., 2003). Therefore, N deposition from the atmosphere may induce negative impacts on Japanese forest ecosystems.

In Europe, critical levels of gaseous air pollutants for vegetation have been developed (Wieser and Tausz, 2007). Current critical levels of gaseous air pollutants are defined as “concentration, cumulative exposure or cumulative stomatal flux of atmospheric pollutants above which direct adverse effects on sensitive vegeta-tion may occur according to present knowledge” (Mills, 2004). There is a possibility of Japanese forest tree species being affected, not only by O3, but also by

N deposition. To evaluate the critical levels of O3 for protecting Japanese forests tree species, therefore, it is necessary to clarify the effects of O3 on growth and physiological functions, such as photosynthesis under different N loads. Watanabe et al. (2006) and Häikiö et al. (2007) reported that increasing N availability can confer greater resistance to O3 in the seedlings of Larix kaempferi and Populus tremula×Populus tremuloides, respectively. However, opposite responses have been reported in the seedlings of Pinus sylvestris and Fagus crenata by Utriainen and Holopainen (2001a) and Yamaguchi et al. (2007a), respectively. Pell et al. (1995) reported that the greatest O3-induced reduction in the whole-plant growth of Populus tremuloides seedlings was observed under the condition of N supply that induced the highest tree growth rate. These results suggest that there are considerable differences in the combined effects of O3 and N load among tree species. However, no information is available on the combined effects of O3 and N deposition on growth and physiological functions such as the photosynthesis of Japanese evergreen broad-leaved tree species.

Castanopsis sieboldii is a representative evergreen broad-leaved tree species, which is distributed in a warm-temperate zone in Japan (Murata, 2004). This tree species is most widely distributed among evergreen broad-leaved tree species in Japan (Kohno, 2004). Therefore, to obtain basic data for evaluating the critical levels of O3 for protecting evergreen broad-leaved tree species, we investigated the effects of O3 on the growth and net photosynthesis of C. sieboldii seedlings grown under different N loads.

2. Materials and Methods

2.1 Plant materialsAndisol was collected from the A soil horizon of an

oak-pine forest in Maebashi, Gunma Prefecture, Japan. The soil was passed through a 2-mm sieve after the collection. The total N concentration and ratio of the carbon (C) concentration to the N concentration (C/N ratio) of the soil on the basis of the soil dry weight were 3.7 mg g－1 and 15.5 g g－1, respectively. On 8 March, 2004, two-year-old seedlings of C. sieboldii, which were obtained from a commercial nursery in Ibaraki Prefecture, Japan, were individually planted in 12 L pots filled with the soil. The average values ± standard deviation of the whole-plant dry mass, plant height and stem base diameter of the seedlings at the beginning of the experiment were 11.9±2.9 g,

J. Agric. Meteorol. (農業気象) 64 (3), 2008

144

53.5±1.8 cm and 4.43±0.1 mm, respectively. All the seedlings were grown until 15 April, 2004 under field conditions at an experimental field of the Akagi Testing Center of the Central Research Institute of Electric Power Industry (Gunma Prefecture, Japan, 36º28´ N, 139º11´ E, 540 m above sea level). On 16 April, 2004, the seedlings were transferred into 12 open-top chambers (OTC, 13.0 m2 of growth space and 2.4 m in height) located at the experimental field and were then grown for 564 days until 1 November, 2005. The average air temperature, average absolute air humidity and average photosynthetic photon flux density in the OTCs from April to October were 20.2℃, 15.4 g m－3 and 18.6 mol m－2 day－1 in 2004 and 19.7℃, 15.3 g m－3 and 17.0 mol m－2 day－1 in 2005, respectively.2.2 Ozone exposure and nitrogen treatment

The present experiment had a split-plot factorial design and employed the randomized block method. The whole-plot treatment was comprised of 4 levels of O3 with three chamber replications for a total of 12 OTCs to analyze the data, including the variance among the 12 OTCs. The sub-plot treatment consisted of 3 levels of N treatment in each OTC. Four seed-lings per OTC were assigned to each O3-N-chamber combination for a total of 144 seedlings. From 16 April, 2004 to 1 November, 2005, the seedlings were exposed to charcoal-filtered air or three levels of O3 at 1.0, 1.5 or 2.0 times the ambient concentration, these O3 treatments being designated as CF, O3×1.0, O3×1.5 and O3×2.0, respectively. Ozone was generated from oxygen-enriched dry air by an electrical discharge O3 generator (Oz-24-UA, Ebara Corporation, Japan) and injected into 9 OTCs for O3-exposure through a water trap to remove nitrogen by-products such as N2O5, produced by the O3 generator (Brown and Roberts, 1988). The concentrations of O3 in the 12 OTCs at 90 cm above the floor were continuously monitored at intervals of 12 minutes with a UV absorption O3 analyzer (ML9810, Monitor Labs, USA). The ambient concentration of O3, which is the standard concentration to regulate the O3 concentration in the OTCs, was also continuously monitored using the same system at the experimental field. The AOT40 (accumulated exposure over a threshold of 40 nmol mol－1, Kärenlampi and Skärby, 1996) of O3 during the growing season from April to September were calculated based on the monitoring data of O3 concentration in the OTCs. The AOT40 is the sum of the differences between the hourly mean O3 concentration and 40 nmol mol－1

for each hour when the O3 concentration exceeded 40 nmol mol－1. The AOT40 of O3 was accumulated for 24 hours and during the daylight hours with solar irradiation above 50 W m－2.

Ammonium nitrate (NH4NO3) was used for the N load to the soil in the present study. The current molar ratio of NH4

＋ to NO3－ in precipitation has

been reported to be 1.0 in Japan (Network Center for EANET, 2006). From April to September in 2004 and 2005, 500 ml of 1.17 or 2.92 mM of NH4NO3 solution was added to the surface of the potted soil at 1-month intervals. The total amounts of N added to the potted soil were 20 and 50 kg ha－1 year－1 on the basis of the potted soil surface area, these soil treatments being designated as N20 and N50, respectively. The control soil (N0) was supplemented with deionized water instead of NH4NO3 solution.2.3 Soil solution analyses

Soil solutions of the potted soil were collected using a soil moisture sampler (Eijklkamp Co., The Netherlands) in July and November 2004 and April, July and October 2005. The concentration of NH4

＋ of the soil solution was determined by the method of indophenol blue colorimetry (Tsuzuki, 1999), while the concentration of NO3

– of the soil solution was determined with an ion chromatograph (IC7000, Yokogawa, Japan). The pH of the soil solution was determined with a pH meter (D-24, Horiba, Japan). Because neither a significant effect of the N load nor significant interaction between O3 and the N load for the pH and inorganic N concentrations of soil solution was observed, these data were pooled across

Table 1. The pH and concentrations of NO3－ and

NH4＋ of soil solution.

N0 N20 N50pHNO3

－ (μM)NH4

+ (μM)

5.60.63.5

5.62.43.7

5.615.43.9

n.s.*

n.s.The N was supplied to the potted soil as NH4NO3 at 0 (N0), 20 (N20) and 50 kg ha－1 year－1 (N50).The values are the means of five analyses of soil solution collected in July and November 2004, and April, July and October 2005.Because no significant effect of O3 nor significant in-teraction between O3 and N load were detected, these data were pooled across the gas treatments.The significance of treatment effect is shown as an as-terisk (Friedman test: *p＜0.05; n.s. not significant).

M. Watanabe et al. : Effects of Ozone and Nitrogen Load on Castanopsis sieboldii Seedlings

145

the gas treatments. Table 1 indicates the mean values of the pH and concentrations of NO3

－ and NH4＋ of

soil solutions collected from the potted soil in July and November 2004 and April, July and October 2005. There was no significant effect of the N load on the pH of the soil solutions. Although the N load significantly increased the concentration of NO3

－ of the soil solutions, no significant effect of N load on the concentration of NH4

＋ of the soil solutions was observed. This phenomenon may be due to the nitrification of the ammonium (Izuta, 2001; Bobbink et al., 2003). Similar results were reported by Nakaji et al. (2001) and Izuta et al. (2005).2.4 Measurements of plant growth

On 1 November, 2005, all the seedlings were harvested in order to determine the leaf area and dry mass of the plant organs. The seedlings were separated into leaves having emerged in 2004 ('04 leaves), leaves emerged in May 2005 ('05 first-flush leaves), leaves emerged from July to October 2005 ('05 second-flush leaves) and stems and roots. The plant organs were dried at 80℃ for 1 week and weighed.2.5 Measurements of the leaf gas exchange rates

The net photosynthetic rates of the leaves at 380 μmol mol－1 CO2 (A380) were measured with a portable infrared gas analyzer system (LI-6400, Li-Cor Inc., Lincoln, NE, USA) on 13 July and 27 October, 2004 and 7 June, 19 July and 13 October, 2005. In each measurement, the A380 of the 2 seedlings per treat-ment-chamber combination (6 seedlings per treatment) was determined. During the measurements of A380, the atmospheric CO2 concentration, air temperature and relative air humidity in the leaf cuvette were maintained at 380±5 μmol mol－1, 24.0±0.1℃ and 60±5％, respectively, while the photosynthetic photon flux density (PPFD) was maintained at approximately 1500 μmol m－2 s－1 at the leaf surface. The A380 was determined on the basis of the leaf area. Before the measurements of A380, the seedlings were taken from OTCs into an experimental room and placed under dark conditions for at least 12 h. After acclimation of the seedlings to the dark conditions, the A380 was measured in the room from 5:00 to 17:00 under the aforementioned conditions. The leaf clipped in the leaf chamber of LI-6400 was adapted to the conditions for approximately 30 min. to obtain a steady state. During the present study, we defined the steady state as a condition with stable A380 for 1–2 min. When the steady state was obtained, the A380 was measured.

2.6 Calculations of the ozone exposure-response relationships

The relationship between daylight AOT40 and the whole-plant dry mass increment were investigated. The mean daylight AOT40 of the two growing seasons from April to September in 2004 and 2005 was used in the calculation. The whole-plant dry mass increment was the difference between the whole-plant dry mass on 16 April, 2004 and that on 1 November, 2005. A regression line was obtained from the relationship between AOT40 of O3 and the whole-plant dry mass increment. The theoretical whole-plant dry mass increment at zero AOT40 was determined to be the y-axis intercept of the regression line. The theoretical whole-plant dry mass increment at zero AOT40 was used as a reference (100％) in order to calculate the relative whole-plant dry mass increment for each gas treatment. The calculated value is the relative whole-plant dry mass increment during the two growing seasons. The whole-plant dry mass increment per one growing season was calculated as the square root of the relative whole-plant dry mass increment during the two growing seasons. The aforementioned procedure was conducted separately for each N treatment. The regression line was obtained from the relationship between AOT40 of O3 and the relative whole-plant dry mass increment to calculate the slope and coefficient of determination values (R2).2.7 Statistical analyses

All statistical analyses were performed with the SPSS statistical package. The Friedman test was used to test the effects of N load on the chemical properties of the soil solution, while analysis of the variance (ANOVA) was used to test the effects of O3 and N load on the growth and leaf gas exchange parameters of the seedlings (Tjoelker and Luxomre, 1991). When a significant interaction of O3 and N load was detected, Tukey's HSD test was performed to identify significant differences among the 12 treatments.

3. Results

Table 2 shows the concentration and AOT40 of O3 during the growing seasons of C. sieboldii seedlings from April to September in 2004 and 2005. The values of O3 exposure indices in 2004 were similar to those in 2005 except for the maximum 1-h peak concentrations. The average 24-h concentration of O3 in the CF, O3×1.0, O3×1.5 and O3×2.0 treatments during the two growing seasons were 11.7, 42.6, 63.3


146

Table 3. Leaf number on 1 November 2005 and accumulated number of shed leaves of Castanopsis sieboldii seedlings during two growing season.

Treatment’04 leaves

’05 leavesShed leaves

Gas N First-flush Second-flushCFO3×1.0O3×1.5O3×2.0

N0N0N0N0

60.3 (4.9)29.3 (17.9)10.8 (6.8)6.6 (3.2)

102.7 (14.8)99.8 (8.9)

108.9 (7.7)81.7 (8.8)

8.0 (7.4) b3.9 (3.6) b

15.1 (4.7) ab15.1 (3.3) ab

23.6 (8.7)61.3 (21.4)88.0 (12.7)

124.2 (45.0)CFO3×1.0O3×1.5O3×2.0

N20N20N20N20

73.7 (23.3)39.3 (4.9)8.3 (3.2)3.6 (0.8)

122.4 (14.2)135.8 (29.6)110.3 (9.3)111.6 (19.1)

3.7 (1.9) b5.8 (5.9) b

18.9 (6.8) ab33.2 (8.9) a

28.5 (3.6)64.8 (35.0)82.9 (21.5)

111.5 (19.1)CFO3×1.0O3×1.5O3×2.0

N50N50N50N50

81.3 (15.1)36.4 (6.5)16.9 (9.1)6.4 (1.7)

149.3 (12.9)170.2 (75.5)175.8 (43.4)127.6 (15.4)

2.6 (3.0) b4.6 (5.0) b

16.1 (18.4) ab36.2 (4.5) a

28.7 (6.1)75.6 (27.1)96.3 (16.0)

135.8 (2.0)ANOVA O3

NO3×N

***n.s.n.s.

***n.s.

**n.s.*

**n.s.n.s.

Each value is the mean of 3 replicates, and the standard deviation is shown in paren-theses.The seedlings were exposed to charcoal-filtered air (CF) or three levels of O3 at 1.0, 1.5 and 2.0 times ambient concentration and grown in the soil supplied N as NH4NO3 at 0 (N0), 20 (N20) or 50 kg ha－1 year－1 (N50).ANOVA: * p＜0.05; ** p＜0.01; *** p＜0.001; n.s. not significant. When the significant interaction of O3 and N was detected, Tukey's HSD test was performed to identify sig-nificant differences among the 12 treatments. Values with different letters are significantly different at p＜0.05.

Table 2. Concentration and AOT40 of O3 in the gas treatments during the growing seasons of Castanopsis Sieboldii seedlings.

Period Treatmenta

Concentration (nmol mol－1) AOT40 (μmol mol－1 h)b

Average 24-h

Average 1-h peak

Maximum1-h peak

24-h Daylight hoursc

2004 CF 13.2 23.5 64.7 1.0 0.816 Apr.-30 Sep. O3×1.0

O3×1.5O3×2.0

43.363.984.7

72.6106.4141.6

159.7234.7312.7

47.0115.6193.8

28.964.9

105.02005 CF 10.3 17.6 77.7 0.7 0.61 Apr.-30 Sep. O3×1.0

O3×1.5O3×2.0

42.062.682.7

68.1100.7133.2

136.0204.3262.3

44.3118.7200.7

27.166.6

107.8a Treatment: CF, charcoal-filtered air; O3×1.0, 1.0 time ambient O3 concentration; O3×1.5, 1.5 times ambient O3

concentration; O3×2.0, 2.0 times ambient O3 concentration.b AOT40 (accumulated exposure over a threshold of 40 nmol mol－1): sum of the differences between the hourly

mean O3 concentration and 40 nmol mol－1 for each hour.c Daylight hours: global radiation ＞50 W m－2.


147

and 83.7 nmol mol－1, respectively.Table 3 indicates the leaf number per plant on 1

November, 2005 and the accumulated number of shed leaves of C. sieboldii seedlings during the two growing seasons. The exposure to O3 significantly reduced the numbers of the '04 leaves and '05 first-flush leaves, while conversely, the number of the '05 second-flush leaves was significantly increased by such exposure. The N load significantly increased the number of the '05 first-flush leaves. There was a significant interaction between O3 and the N load for the number of the '05 second-flush leaves. The exposure to O3 at 2.0 times the ambient concentration significantly increased the number of the '05 second-flush leaves in the N20 and N50 treatments, but not in the N0 treatment. The exposure to O3 significantly increased the accumulated number of shed leaves. Neither a significant effect of the N load nor any significant interaction between O3 and the N load for the accumulated number of shed leaves was observed.

Table 4 shows the leaf area and dry mass of plant organs of C. sieboldii seedlings at the end of the second growing season (1 November, 2005). The exposure to O3 significantly reduced the area of the '04 leaves, dry mass of the '04 leaves, the stem and roots, and the whole-plant dry mass. In contrast, the area and dry mass of the '05 second-flush leaves were significantly increased by the exposure to O3. The N load significantly increased the areas of the '05 first-flush leaves and '05 second-flush leaves, the dry mass of leaf, stem and roots, and the whole-plant dry mass. Significant interactions between O3 and the N load for the area of the '05 second-flush leaves and dry mass of the '05 second-flush leaves and roots were found. The exposure to O3 at 2.0 times the ambient concentration significantly increased the area and dry mass of the '05 second-flush leaves of the seedlings grown in the N20 and N50 treatments, but not in the N0 treatment. The exposure to O3 at 2.0 times the ambient concentration significantly reduced the root dry mass of the seedlings grown in the N0 treatment. In the N20 and N50 treatments, the root dry mass was significantly reduced, not only by the exposure to O3 at 2.0 times the ambient concentration, but also by the exposure to O3 at 1.0 and 1.5 times the ambient concentration in the N20 treatment and O3 at 1.5 times the ambient concentration in the N50 treatment.

Table 5 indicates the net photosynthetic rate at 380 μmol mol－1 CO2 (A380) of C. sieboldii seedlings. The

exposure to O3 significantly reduced the A380 of the '04 leaves during the first and second growing seasons and that of the '05 first-flush leaves on 13 October, 2005. The A380 of the '05 first-flush leaves on 7 June 2005 was significantly increased by the exposure to O3. The N load significantly increased the A380 of the '04 leaves during the first and second growing seasons, and that of the '05 first-flush leaves on 19 July and 13 October, 2005. There were significant interactions between O3 and the N load for the A380 of the '04 leaves on 13 July, 2004 and 19 July, 2005 and that of the '05 first-flush leaves on 13 October, 2005. The O3 concentration that induced a significant reduction in the A380 decreased with an increase in the amount of N added to the potted soil.

4. Discussion

4.1 Growth and photosynthetic responses to ozone and nitrogen loads

Although the exposure to O3 significantly reduced the net photosynthetic rate of the '04 leaves of C. sieboldii seedlings during the first and second growing seasons, net photosynthetic rate of the '05 first-flush leaves in June of the second growing season was significantly increased by the exposure to O3 (Table 5). Greitner et al. (1994) reported that exposure to O3 increased the net photosynthetic rate of younger leaves, but reduced that of older leaves in Populus tremuloides seedlings. This phenomenon is considered to be a compensatory response to the negative effects of O3 on older leaves (Greitner et al., 1994; Bielenberg et al., 2001). In the case of C. sieboldii seedlings, which is an evergreen tree species, the O3-induced increase in the net photosynthetic rate of the '05 first-flush leaves compensated for the O3-induced reduction in that of the '04 leaves. Therefore, we considered this response to be one of the “carry-over effects of O3” (Oksanen and Saleem, 1999; Yonekura et al., 2004). An ozone-induced increase in the number and area of the '05 second-flush leaves were observed during the second growing season (Tables 3 and 4). These results indicate that compensatory responses of C. sieboldii seedlings were apparent, not only for physiological functions, such as the net photosynthetic rate, but also for morphological characteristics. However, these compensatory responses were insufficient to prevent O3-induced negative impacts on the whole-plant dry matter production of the seedlings (Table 4).

The nitrogen load increased the growth and net


148

Tab

le 4

.　L

eaf

area

and

dry

mas

s of

pla

nt o

rgan

s of

Cas

tano

psis

sie

bold

ii se

edlin

gs o

n 1

Nov

embe

r 20

05.

Trea

tmen

tL

eaf

area

(cm

2 )D

ry m

ass

(g)

Gas

N’0

4 le

aves

’05

leav

es’0

4 le

aves

’05

leav

esSt

emR

oot

Who

le-p

lant

Firs

t-flus

hSe

cond

-flus

hFi

rst-fl

ush

Seco

nd-fl

ush

CF

O3×

1.0

O3×

1.5

O3×

2.0

N0

N0

N0

N0

653

(87)

262

(121

)11

5 (9

3) 8

4 (5

3)

562

(42

) 6

00 (

15)

595

(11

3) 5

53 (

75)

31

(27)

c 1

3 (1

1) c

138

(75)

bc

160

(45)

abc

9.5

(1.6

)3.

9 (1

.6)

1.5

(1.2

)1.

1 (0

.7)

8.2

(0.5

)8.

5 (0

.4)

8.4

(1.3

)7.

3 (1

.3)

0.4

(0.3

) b

0.2

(0.1

) b

1.6

(0.8

) ab

2.0

(0.7

) ab

20.2

(3.

4)14

.3 (

1.2)

15.5

(0.

7)13

.1 (

0.6)

26.3

(1.

2) c

d20

.5 (

1.2)

de

20.5

(2.

2) d

e14

.9 (

2.1)

e

64.6

(4.

5)47

.3 (

1.7)

47.4

(3.

4)38

.4 (

1.7)

CF

O3×

1.0

O3×

1.5

O3×

2.0

N20

N20

N20

N20

697

(238

)46

7 (7

3) 8

4 (4

2) 4

1 (1

4)

674

(70

) 6

94 (

82)

704

(69

) 6

98 (

21)

11

(6)

c 6

4 (9

2) c

144

(93)

abc

365

(15)

a

12.0

(3.

7)6.

6 (0

.7)

1.2

(0.6

)0.

5 (0

.2)

10.1

(1.

1)10

.0 (

1.3)

10.0

(1.

3)9.

5 (0

.6)

0.2

(0.1

) b

0.8

(1.1

) b

1.7

(1.0

) ab

4.0

(0.2

) a

22.7

(2.

3)19

.8 (

1.0)

17.7

(2.

3)16

.5 (

0.3)

35.6

(7.

1) a

b26

.9 (

2.6)

cd

22.3

(1.

2) c

de19

.1 (

1.3)

de

80.6

(14

.0)

64.2

(3.

1)52

.8 (

4.1)

49.6

(1.

6)C

FO

3×1.

0O

3×1.

5O

3×2.

0

N50

N50

N50

N50

961

(216

)39

2 (4

3)19

1 (1

12)

61

(20)

898

(15

5) 9

87 (

269)

1025

(26

1) 8

79 (

21)

9

(11)

c 3

5 (5

5) c

116

(109

) bc

323

(61)

ab

14.4

(2.

4)5.

9 (0

.4)

2.9

(1.9

)1.

0 (0

.2)

14.0

(2.

1)15

.8 (

3.0)

16.1

(2.

9)12

.9 (

1.2)

0.1

(0.1

) b

0.4

(0.6

) b

1.9

(2.1

) ab

3.8

(0.9

) a

26.3

(3.

6)26

.7 (

5.5)

24.6

(2.

3)23

.6 (

4.0)

41.0

(3.

1) a

43.1

(3.

6) a

29.8

(1.

8) b

c25

.0 (

1.7)

cd

95.7

(6.

2)91

.8 (

11.1

)75

.2 (

4.8)

66.2

(5.

9)A

NO

VA

O3

N O3×

N

***

n.s.

n.s.

n.s.

***

n.s.

** ***

***

*** * n.s.

n.s.

***

n.s.

** * *

** ***

n.s.

** ***

**

***

***

n.s.

Eac

h va

lue

is t

he m

ean

of 3

rep

licat

es,

and

the

stan

dard

dev

iatio

n is

sho

wn

in p

aren

thes

es.

The

see

dlin

gs w

ere

expo

sed

to c

harc

oal-fi

ltere

d ai

r (C

F) o

r th

ree

leve

ls o

f O

3 at

1.0

, 1.

5 an

d 2.

0 tim

es a

mbi

ent

conc

entr

atio

n an

d gr

own

in t

he s

oil

supp

lied

N a

s N

H4N

O3

at 0

(N

0),

20 (

N20

) or

50

kg h

a－1

year－

1 (N

50).

AN

OV

A:

* p＜

0.05

; **

p＜

0.01

; **

* p＜

0.00

1; n

.s.

not

sign

ifica

nt.

Whe

n th

e si

gnifi

cant

int

erac

tion

of O

3 an

d N

was

det

ecte

d, T

ukey

's H

SD t

est

was

per

form

ed t

o id

entif

y si

gnifi

cant

dif

fere

nces

am

ong

the

12 t

reat

men

ts.

Val

ues

with

dif

fere

nt l

ette

rs a

re s

igni

fican

tly d

iffe

rent

at

p＜0.

05.


149

Tab

le 5

.　N

et p

hoto

synt

hetic

rat

e at

380

μm

ol m

ol－

1 C

O2

(μm

ol m

－2

s－1 )

in t

he l

eave

s of

Cas

tano

psis

sie

bold

ii se

edlin

gs.

Trea

tmen

t'0

4 le

aves

'05

first

-flus

h le

aves

'05

seco

nd-fl

ush

leav

esG

asN

13-J

ul-0

427

-Oct

-04

7-Ju

n-05

19-J

ul-0

513

-Oct

-05

7-Ju

n-05

19-J

ul-0

513

-Oct

-05

13-O

ct-0

5C

FO

3×1.

0O

3×1.

5O

3×2.

0

N0

N0

N0

N0

11.8

(1.

4) b

cd11

.7 (

0.2)

bcd

11.9

(0.

7) b

c6.

8 (1

.8)

e

4.8

(0.6

)3.

1 (0

.7)

2.5

(0.5

)1.

2 (0

.3)

2.7

(0.2

)1.

6 (0

.6)

2.0

(0.3

)1.

8 (0

.1)

2.3

(0.3

) bc

d1.

6 (0

.2)

cde

0.9

(0.1

) e

1.2

(0.6

) e

2.5

(0.1

)1.

5 (0

.5)

– –

3.0

(0.3

)4.

6 (0

.2)

4.1

(0.9

)4.

3 (0

.5)

3.2

(1.4

)4.

0 (0

.1)

4.0

(0.3

)2.

5 (0

.2)

4.7

(0.4

) ab

c5.

5 (0

.4)

ab3.

6 (1

.1)

cd2.

3 (0

.3)

de

– –5.

9 (2

.9)

6.4

(2.0

)C

FO

3×1.

0O

3×1.

5O

3×2.

0

N20

N20

N20

N20

13.3

(0.

9) a

b11

.9 (

0.5)

bc

9.3

(0.7

) cd

e8.

1 (2

.1)

e

6.2

(1.3

)4.

0 (1

.4)

2.2

(0.1

)2.

4 (0

.3)

3.3

(0.5

)3.

3 (0

.6)

2.8

(0.1

)3.

1 (0

.4)

3.3

(0.2

) ab

2.6

(0.4

) ab

c2.

0 (0

.3)

cde

1.0

(0.6

) e

2.8

(0.5

)1.

4 (0

.6)

– –

2.9

(0.4

)3.

4 (0

.9)

5.2

(0.9

)5.

2 (0

.2)

4.0

(0.7

)4.

2 (0

.6)

4.2

(0.4

)2.

8 (0

.5)

4.7

(0.2

) ab

c4.

5 (0

.6)

bc3.

4 (0

.3)

cd1.

3 (0

.4)

e

– –5.

6 (0

.4)

7.8

(0.6

)C

FO

3×1.

0O

3×1.

5O

3×2.

0

N50

N50

N50

N50

16.1

(0.

5) a

13.7

(0.

5) a

b11

.4 (

0.7)

bcd

8.7

(0.9

) de

5.1

(0.2

)3.

1 (0

.6)

2.5

(0.3

)2.

3 (0

.3)

3.6

(0.1

)2.

3 (0

.3)

2.6

(0.4

)2.

6 (0

.2)

3.5

(0.1

) a

1.9

(0.3

) cd

e1.

4 (0

.4)

de1.

7 (0

.6)

cde

3.2

(1.2

)2.

3 (0

.7)

– –

3.6

(0.5

)4.

1 (0

.6)

5.0

(0.9

)4.

7 (0

.2)

5.2

(0.4

)5.

2 (0

.5)

4.7

(0.6

)5.

4 (1

.4)

4.6

(0.1

) ab

c5.

4 (1

.1)

a2.

7 (0

.3)

d2.

4 (0

.6)

de

– –5.

8 (3

.5)

6.6

(3.3

)A

NO

VA

O3

N O3×

N

***

***

**

*** * n.s.

***

***

n.s.

** ***

**

– – –

** n.s.

n.s.

n.s.

***

n.s.

*** * *

– – –E

ach

valu

e is

the

mea

n of

3 r

eplic

ates

, an

d th

e st

anda

rd d

evia

tion

is s

how

n in

par

enth

eses

.T

he s

eedl

ings

wer

e ex

pose

d to

cha

rcoa

l-filte

red

air

(CF)

or

thre

e le

vels

of

O3

at 1

.0,

1.5

and

2.0

times

am

bien

t co

ncen

trat

ion

and

grow

n in

the

soi

l su

pplie

d N

as

NH

4NO

3 at

0 (

N0)

, 20

(N

20)

or 5

0 kg

ha－

1 ye

ar－

1 (N

50).

AN

OV

A:

* p＜

0.05

; **

p＜

0.01

; **

* p＜

0.00

1; n

.s.

not

sign

ifica

nt.

Whe

n th

e si

gnifi

cant

int

erac

tion

of O

3 an

d N

was

det

ecte

d, T

ukey

's H

SD t

est

was

per

-fo

rmed

to

iden

tify

sign

ifica

nt d

iffe

renc

es a

mon

g th

e 12

tre

atm

ents

. V

alue

s w

ith d

iffe

rent

let

ters

are

sig

nific

antly

dif

fere

nt a

t p＜

0.05

.O

n 13

Oct

ober

200

5, n

et p

hoto

synt

hetic

rat

e of

’04

lea

ves

of t

he s

eedl

ings

exp

osed

to

O3

at 1

.5 a

nd 2

.0 t

imes

am

bien

t co

ncen

trat

ion

and

that

of

’05

sec-

ond-

flush

ed l

eave

s of

the

see

dlin

gs e

xpos

ed t

o ch

arco

al-fi

ltere

d ai

r an

d O

3 at

1.0

tim

e am

bien

t co

ncen

trat

ion

wer

e no

t m

easu

red

beca

use

mos

t of

the

lea

ves

wer

e de

folia

ted

and

few

lea

ves

wer

e em

erge

d, r

espe

ctiv

ely.


150

photosynthetic rate of C. sieboldii seedlings during the first and second growing seasons (Tables 4 and 5). Furthermore, the N load did not significantly affect the pH and concentrations of the aluminum and manganese of soil solution (data not shown). In the present study, therefore, N acted as a fertilizer for the growth and net photosynthesis of the C. sieboldii seedlings.

The N load increased the sensitivity to O3 of the net photosynthetic rate of C. sieboldii seedlings during the first and second growing seasons (Table 5). On the other hand, the exposure to O3 significantly increased the '05 second-flush leaves of the seedlings grown under relatively high N load, but not under relatively low N load (Tables 3 and 4). These results suggest that the increase in the area of the '05 second-flush leaves compensated for the O3-induced reduction in the net photosynthetic rate of the seedlings grown under a relatively high N load. The degree of O3-induced reduction in the root dry mass of the seedlings was greater under a relatively high N load than under a relatively low one (Table 4). However, no N load-induced increase in the sensitivity of the whole-plant dry mass of the seedlings to O3 was observed (Table 4). Furthermore, no significant interaction between O3 and N load for the ratio of shoot dry mass to root dry mass of the seedlings was observed (data not shown). Therefore, we conclude that the N load to the soil at ≤50 kg ha－1 year－1 does not influence the sensitivity of the whole-plant dry mass of C. sieboldii seedlings to O3. This result is in agreement with the results obtained from seedlings of Picea abies, Pinus densiflora, Cryptomeria japonica and Quercus serrata (Utriainen and Holopainen, 2001b; Watanabe et al., 2006; Watanabe et al. 2007). We previously reported that the effects of N load on the sensitivity to O3 of the whole-plant dry mass of Q. serrata and F. crenata seedlings was closely related to those on the sensitivity to O3 of the net photosynthetic rate during the growing season, i.e. when the sensitivity of the net photosynthetic rate to O3 was increased by the N load, the negative impact of O3 on the whole-plant dry mass was increased (Watanabe et al,. 2007; Yamaguchi et al., 2007a; Yamaguchi et al., 2007b). In C. sieboldii seedlings, however, the sensitivity of the whole-plant dry mass to O3 did not necessarily correspond to that of the net photosynthetic rate to O3 (Tables 4 and 5). In C. sieboldii seedlings, the compensatory response of the second-flush leaves to O3 was observed under a relatively high N load (Tables 3 and 4). Therefore,

we must take into account not only the physiological functions, such as photosynthesis, but also phenological characteristics, in order to evaluate the effects of N load on the sensitivity to O3 of the whole-plant growth of Japanese forest tree species.4.2 Evaluation of critical levels of ozone for C. sieboldii

The current critical level of O3 for forest trees in Europe is expressed as AOT40 during the daylight hours for a one growing season from April to Sep-tember (Mills, 2004). Therefore, we investigate the relationship between daylight AOT40 of O3 and relative whole-plant dry mass increment per one growing season of C. sieboldii seedlings. Because no significant interaction between O3 and the N load for the whole-plant dry mass was detected at the end of the second growing season, a single regression line across three N treatments was obtained (Fig. 1). In Europe, a critical level of O3 for sensitive forest tree species. such as European beech and birch, was evaluated as 5 μmol mol－1 h of daylight AOT40, which is associated with a 5％ reduction in the growth of the seedlings (Mills, 2004). Therefore, we evaluate the AOT40 of O3 that induced a 5％ reduction in the whole-plant dry mass increment as a critical level of O3. Based on the results obtained from the present study, the critical level of O3

Fig. 1. The relationship between AOT40 (accumu-lated exposure over a threshold of 40 nmol mol－1) of O3 during the daylight hours with a solar irradiation above 50 W m－2 and relative whole-plant dry mass increment per one growing season of Castanopsis sieboldii seedlings (Relative WDMinc). The AOT40 is the means of two growing seasons from April to September in 2004 and 2005.

E

E E

E

C

C

C

C

GG

G

G

60

70

80

90

100

110

0 20 40 60 80 100 120AOT40 of O3 (µmol mol-1 h)

Rel

ativ

e W

DM

inc

(%)

slope: -0.231R2=0.858***

� N0� N20� N50


151

for C. sieboldii was estimated to be 21.7 μmol mol－1 h. This value was relatively low as compared with that for the other representative Japanese forest tree species, as shown in Fig. 2 (Watanabe et al., 2006, Watanabe et al., 2007, Yamaguchi et al., 2007a).

The main habitat of C. sieboldii in Japan is the Kyushu region, in the southern part of Japan. Therefore, we estimated the AOT40 of photochemical oxidant in the Kyushu region during daytime hours (6:00-18:00) from April to September, based on the monitoring data of photochemical oxidant and using the method of Ishii et al. (2007). When photochemical oxidant is regarded as O3, the yearly average AOT40 of O3, detected at 8％ of the monitoring stations in the Kyushu region during the period 2000-2004, exceeded the estimated critical level of O3 for C. sieboldii (21.7 μmol mol－1 h). Furthermore, the yearly average AOT40 of O3 in this area increased from 1985 to 2004, representing an increase rate of 0.41 μmol mol－1 h. Based on the

increase rate of AOT40 of O3 in this area, an AOT40 at approximately 39％ of the monitoring stations in the Kyushu region will exceed the estimated critical level of O3 for C. sieboldii in 2030.

Because there is a possibility that the sensitivity of mature trees to O3 is different from that of the seedlings to O3, we cannot directly apply the results obtained from seedlings to phenomena of mature tree grown in actual field (Nunn et al., 2005). However, our results obtained from the present study indicate the possibility that current ambient levels of O3 negatively affect the growth of C. sieboldii in Japan, and the adverse effects of O3 on the tree species will become serious in the near future.

Acknowledgments

This study was financially supported by the Ministry of the Environment, Japan through the program of Global Environmental Research Fund (C-03-07, 2003-2005). The authors are greatly indebted to M. Iwasaki, J. Naba, N. Matsuo, C. Tabe, R. Yamashita, Y. Shinozaki and M. Tanaka (Tokyo University of Agriculture and Technology), and staff of CERES Inc. for their technical support and discussion. We use the data file of photochemical oxidant from “Numerical database for environment” of National Institute for Environmental Studies.

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leve

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(�m

olm

ol-1

h)

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ta(N

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C. s

iebo

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要約

日本の常緑広葉樹を対象としたオゾンのクリティカルレベルを評価するための基礎的知見を得ることを目的として , 日本の代表的な常緑広葉樹であるスダジイの成長と光合成に対するオゾンの影響を異なる窒素条件下において調べた。2年生のスダジイの苗木を 4段階のガス処理 (浄化空気 , 外気オゾン濃度の 1.0, 1.5および 2.0倍 )と 3段階の土壌窒素処理 (0, 20および 50 kg ha－ 1 year－ 1)を組み合わせた合計 12処理区で 2成長期にわたって育成した。オゾン暴露によって , スダジイ苗の純光合成速度と個体乾重量は有意に低下した。一方 , 土壌への窒素負荷によってスダジイ苗の純光合成速度および個体乾重量は有意に増加した。スダジイ苗の純光合成速度におけるオゾン感受性は土壌への窒素負荷によって増加した。しかしながら , 個体乾重量におけるオゾン感受性

は土壌への窒素負荷の影響を受けなかった。その原因として , 2成長期目に比較的窒素負荷量の多い処理区で認められたオゾン暴露による 2ndフラッシュ葉の有意な増加が純光合成速度の低下を補償したことが考えられる。本研究の結果より , 50 kg ha－ 1 year－ 1までの土壌への窒素負荷はスダジイの個体乾物成長におけるオゾン感受性に影響を与えないことが明らかになった。個体乾重量の増加量を 5％低下させる 1成長期あたりのAOT40 (accumulated exposure over a threshold of 40 nmol mol－ 1)をクリティカルレベルとすると , スダジイのクリティカルレベルは 22 μmol mol－ 1 hと評価された。キーワード：オゾン，純光合成，成長，スダジイ，窒

素負荷

異なる窒素負荷量で育成したスダジイ苗の成長および光合成に対するオゾンの影響

渡辺　誠 *†・山口真弘 *・松村秀幸 **・河野吉久 **・伊豆田　猛 ****東京農工大学大学院連合農学研究科

**(財 )電力中央研究所環境科学研究所

***東京農工大学大学院共生科学技術研究院

†現在：北海道大学大学院農学研究院


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