Experimental Studies on the Effects of Ozone on Growth and Photosynthetic Activity of Japanese Forest Tree Species

Ozone (O3) is a main component of photochemical oxidants, and a phytotoxic anthropogenic air pollutant. In North America and Europe, the current concentration of O3 has been shown to have significant adverse effects on vegetation. In this review, we summarize the experimental studies on the effects of O3 on the growth and photosynthetic activity of Japanese forest tree species to understand the present knowledge and provide sound basis for future research toward the assessment of O3 impacts on Japanese forest ecosystem. Since the 1990s, several Japanese researchers have conducted the experimental studies on the effects of ambient levels of O3 on growth and physiological functions such as net photosynthesis of Japanese forest tree species. Although the sensitivity to O3 of whole-plant growth is quite different among the species, it was suggested that the current ambient levels of O3 in Japan are high enough to adversely affect growth and photosynthetic activity of Japanese forest tree species classified into high O3 sensitivity group such as Japanese beech. The N load to soil has been shown to reduce the sensitivity to O3 of Japanese larch and increase that of Japanese beech. To establish the critical level of O3 for protecting Japanese forest tree species, therefore, it is necessary to take into account the N deposition from the atmosphere. There is little information on the combined effects of O3 and other environmental factors such as elevated CO2 and drought on growth and physiological functions of Japanese forest tree species. Therefore, it is necessary to promote the experimental study and accumulate the information on the combined effects of O3 and any other abiotic environmental factors on Japanese forest tree species.


INTRODUCTION
Ozone (O 3 ) is a main component of photochemical oxidants and produced by photochemical reaction of volatile organic compounds (VOCs) and nitrogen oxide (NO x ) (ADORC, 2006;EPA, 2006a).Ozone adversely affects not only human health but also vegetation (ADORC, 2006;EPA, 2006a).The emissions of precursor for O 3 from Asian countries have rapidly increased since the 1970s and surpassed the emissions from North America and Europe in the mid-1990s (Ohara et al., 2007;Akimoto, 2003).This situation is expected to continue for at least next couple decades (Klimont et al., 2001).In the near future, therefore, the concentration of ground-level O 3 is expected to increase especially in Asian countries including Japan (Yamaji et al., 2008;Dentener et al., 2006;Derwent et al., 2002;Emberson et al., 2001).
Since the ambient levels of O 3 in the USA and Europe negatively affect growth and physiological functions such as photosynthesis of forest tree species, this gas is considered as one of the important factors relating to forest decline and tree dieback in the relevant regions (Bytnerowicz et al., 2004;Chappelka and Samuelson, 1998;Skärby et al., 1998;Sandermann et al., 1997).In Japan, relatively high concentrations of O 3 above 100 nL L -1 (ppb) have been frequently observed from spring to autumn in several mountainous areas (Kohno et al., 2007;Takeda and Aihara, 2007;Aihara et al., 2004;Maruta et al., 1999).Based on the results of the experimental studies and field surveys, it has been suggested that O 3 is an important environmental stress relating to the forest decline in Japan (Kume et al., 2009;Suto et al., 2008;Takeda and Aihara, 2007;Yamaguchi et al., 2007b;Kohno et al., 2005;Aihara et al., 2004;Yonekura et al., 2001a, b;Maruta et al., 1999).
In Japan, pioneer studies on the effects of O 3 on woody plants were published in the 1970s.Nouchi et al. (1973a, b) and Matsushima et al. (1977) mainly focused on O 3 -induced visible injury and changes in the ultrastructural characteristics of leaves of several tree species.Kuno (1980Kuno ( , 1979)), Furukawa et al. (1983), Fujinuma et al. (1987) and Furukawa (1991) reported the effects of O 3 on growth or physiological functions such as net photosynthesis of street trees.Matsumoto et al. (1992) reported the effects of O 3 at remarkably high concentrations on needle gas exchange rates of Japanese cedar (Cryptomeria japonica).Since the 1990s, the experimental studies on the effects of ambient levels of O 3 on growth, phenological characteristics and physiological functions such as net photosynthesis of Japanese forest tree species have started.To provide sound basis for future research toward the assessment of O 3 impacts on Japanese forest ecosystems, in this review, we summarize the experimental studies hitherto reported on the effects of O 3 on growth and photosynthetic activity of Japanese forest tree species.

EFFECTS OF O 3 ON GROWTH OF JAPANESE FOREST TREE SPECIES
Table 1 indicates the summary of experimental studies on the effects of O 3 on Japanese forest tree species.Miwa et al. (1993) reported that the exposure of Japanese cedar seedlings to relatively high O 3 concentration (300 ppb) did not induce significant reduction in the whole-plant dry mass, but induced significant increase in the ratio of the above-ground dry mass to root dry mass (Top/Root ratio).Izuta et al. (1996) reported that the whole-plant dry mass and root dry mass of Japanese beech (Fagus crenata) seedlings were reduced by the exposure to ambient levels of O 3 (75 and 150 ppb).Yonekura et al. (2001a, b) also reported that the exposure to ambient level of O 3 (60 ppb) reduced dry masses of root, leaf and stem, the whole-plant dry mass and annual ring width of Japanese beech seedlings.Nakaji and Izuta (2001) and Nakaji et al. (2004) reported that dry masses of needles and fine roots and the whole-plant dry mass of Japanese red pine (Pinus densiflora) seedlings were reduced by the exposure to ambient level of O 3 (60 ppb).Aforementioned studies were conducted using the steady-state O 3 exposure system.Because there are seasonal and diurnal variations in tropospheric O 3 concentration (Khiem et al., 2010;EPA, 2006a;Yamaji et al., 2006), O 3 exposure system with the variations in the atmospheric concentration of O 3 observed in the fields needs to be used for the evaluation of the realistic effects of O 3 on forest tree species.Matsumura et al. (1996) and Matsumura et al. (1998) conducted experimental studies on the effects of O 3 on the growth of several Japanese forest tree species using an O 3 exposure system with seasonal and diurnal variations in the atmospheric concentration of O 3 .In the study of Matsumura et al. (1996), the seedlings of Japanese cedar, Japanese cypress (Chamaecyparis obtusa) and Japanese zelkova (Zelkova serrata) were exposed to O 3 at 0.4, 1.0, 2.0 and 3.0 times the ambient concentration (12-h (6:00-18:00) average concentration of O 3 : 16, 39, 74 and 114 ppb, respectively).The whole-plant dry mass of Japanese zelkova exposed to 2.0 and 3.0 times the ambient concentration of O 3 and that of Japanese cedar exposed to 3.0 times the ambient concentration of O 3 were significantly lower than those exposed to 0.4 times the ambient concentration of O 3 , while there was no significant effect of O 3 on the whole-plant dry mass of Japanese cypress.In the study of Matsumura et al. (1998), the seedlings of Japanese cedar, Nikko fir (Abies homolepis), Japanese white birch (Betula platyphylla) and Japanese zelkova were exposed to O 3 at 0.4, 1.0, 2.0 and 3.0 times the ambient concentration (12-h (6:00-18:00) average concentration of O 3 : 18, 37, 67 and 98 ppb, respectively).The whole-plant dry mass of Japanese cedar, Japanese white birch and Japanese zelkova were decreased linearly with increasing the concentration of O 3 , while that of Nikko fir was not.These results indicate that the sensitivity to O 3 of the whole-plant growth is quite different among the Japanese forest tree species.
Aforementioned experimental studies were conducted within one growing season.However, there are several reports concerning the carry-over effects of O 3 on perennial plants.For example, the exposure to O 3 during one growing season changes phenological characteristics such as delay in the timing of bud break, and reduces leaf number per bud and growth in the following growing season (Yonekura et al., 2004;Oksanen and Saleem, 1999;Andersen et al., 1997;Pearson and Mansfield, 1994).Therefore, multi-year experiments are crucial to assessing the degree of adverse effects of O 3 on the growth of forest trees (Ashmore, 1993).Matsumura (2001) conducted the multi-year experiments.Young trees of 14 species were exposed to charcoal-filtered air (CF) or non-filtered air (NF) for three growing seasons at two different sites in Kanto districts of Japan (Chiba Prefecture and Gunma Prefecture; 12-h (6:00-18:00) seasonal mean concentration of O 3 (from April to September) during the experimental period in CF treatments: 8 and 12 ppb,

(Abies firma)
No effect on whole-plant dry mass.
Nikko fir 5 yr Decreased root and whole-plant dry mass (Additive).

Japanese cypress 2 yr
Increased shoot and whole-plant dry mass (Additive).
Japanese cedar 1 or 2 yr Decreased root and whole-plant dry mass (Additive).
Japanese white birch 1 yr Decreased shoot, root and whole-plant dry mass (Additive).

yr
No effect on whole-plant dry mass.
Japanese beech 1 yr Decreased root and whole-plant dry mass (Synergetic or Additive).
Japanese zelkova 1 yr Decreased shoot, root and whole-plant dry mass (Additive).respectively; those in NF treatments: 26 and 37 ppb, respectively).The ambient levels of O 3 reduced the whole-plant dry mass of Japanese red pine, Japanese larch (Larix kaempferi), Veitch's silver fir (Abies veitchii), Japanese white birch, Japanese beech and Japanese zelkova at the both sites.Kohno et al. (2005) summarized several results of experimental studies conducted for multiple growing seasons on the effects of O 3 on forest tree species (e.g.Matsumura, 2001;Matsumura and Kohno, 1999).The sensitivity of each tree species to O 3 was classified into 3 groups (high, moderate and low) based on the response of the wholeplant dry mass growth to O 3 ( Recently, Takeda and Aihara (2007) showed that O 3 negatively affects growth and photosynthetic parameters of Japanese beech grown under field condition at Tanzawa Mountains where the decline of Japanese beech forest has been reported.Kume et al. (2009) suggested the possibility that recent increase in the atmospheric concentration of O 3 is an important factor of Japanese beech decline at Mt. Tateyama based on the results of their field survey.These results and those obtained from the experimental studies clearly indicate that current ambient levels of O 3 in Japan are high enough to adversely affect growth of Japanese forest tree species especially in those classified into high O 3 sensitivity group such as Japanese beech.Izuta et al. (1996) and Matsumura et al. (1996) reported that relative growth rates (RGRs) of Japanese beech, Japanese cedar and Japanese zelkova were reduced by the exposure to O 3 .In their studies, the O 3induced reductions in net assimilation rate (NAR) and net photosynthetic rate of the leaves or needles were reported.These results indicate that the O 3 -induced reduction in the growth was mainly due to that in the net photosynthetic rate of the leaves or needles.It was also reported that the exposure to O 3 reduced the net photosynthetic rate of the leaves or needles of Japanese white birch, Japanese red pine, Japanese larch, Q. serrata and C. sieboldii (Watanabe et al., 2008(Watanabe et al., , 2007(Watanabe et al., , 2006;;Nakaji et al., 2004;Nakaji and Izuta, 2001;Matsumura et al., 1998).When net photosynthetic rate was reduced by the exposure to O 3 , simultaneous reductions in the carboxylation efficiency (CE), CO 2saturated net photosynthetic rate (A max ) and/or maximum quantum yield of photosystem (PS) II (F v /F m ), and increase in the stomatal diffusive conductance to water vapor (g s ) in the leaves or needles of Japanese forest tree species were also observed (Yamaguchi et al., 2007a;Watanabe et al., 2005;Nakaji and Izuta, 70 Asian Journal of Atmospheric Environment, Vol.5(2), 65-78, 2011 2001; Yonekura et al., 2001a, b;Matsumura et al., 1998Matsumura et al., , 1996;;Izuta et al., 1996).It was documented that the exposure to O 3 reduced the concentration and activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and chlorophyll concentration in the leaves or needles of Japanese forest tree species (Watanabe et al., 2007(Watanabe et al., , 2005;;Yamaguchi et al., 2007a, b;Nakaji et al., 2004;Nakaji and Izuta, 2001;Yonekura et al., 2001a;Izuta et al., 1996;Miwa et al., 1993).Yonekura et al. (2001b) reported that the O 3 -induced reduction in net photosynthetic rate was firstly due to the reduction in the quantity and/or activity of Rubisco in the leaves of Japanese beech.Therefore, there is a possibility that the exposure to O 3 firstly reduces the capacity of carbon fixation in the chloroplasts resulting in the reduction in net photosynthetic rate of the leaves or needles of Japanese forest tree species.Proteins such as Rubisco in the leaves or needles represent the predominant N fraction (Feller, 2004;Spreitzer and Salvucci, 2002).Ozone exposure has been shown to reduce the concentration of total soluble protein (TSP) in the leaves of Q. serrata and Japanese beech (Watanabe et al., 2007;Yamaguchi et al., 2007a, b).Watanabe et al. (2007) and Yamaguchi et al. (2007b) reported that the exposure to O 3 reduced photosynthetic nitrogen use efficiency (PNUE) in the leaves of Q. serrata and Japanese beech.In the case of Japanese beech, the exposure to O 3 did not significantly affect N concentration in the leaves, suggesting that O 3 induces alterations in foliar N metabolism and also a reduction in the availability of N for photosynthesis in the leaves (Yamaguchi et al., 2007b).There is limited information on the effects of O 3 on N metabolism in the leaves or needles of Japanese forest tree species (Yamaguchi et al., 2010(Yamaguchi et al., , 2007a;;Nakaji et al., 2004).Nakaji et al. (2004) reported that the exposure to O 3 did not significantly affect the activities of nitrate reductase (NR) and nitrite reductase (NiR) and concentrations of inorganic N compounds (NO 3 -, NO 2 and NH 4 + +

EFFECTS OF O 3 ON THE PHOTOSYNTHETIC ACTIVITY AND OTHER RELATED FUNCTIONS OF JAPANESE FOREST TREE SPECIES
) and free amino acid in the needles of Japanese red pine.On the other hand, Yamaguchi et al. (2010Yamaguchi et al. ( , 2007a) ) reported the O 3 -induced inhibition of resorption of N from the leaves in autumn, reductions in the NR activity and the ratio of TSP concentration to N concentration and increase in the concentration of acidic amino acid in the leaves of Japanese beech.At the present time, it is unclear how O 3 affects N metabolism in the leaves of Japanese forest tree species.To clarify the mechanisms underlying the detrimental effects of O 3 on Japanese forest tree species, therefore, further research concerning the effects of O 3 on physiological functions such as foliar N metabolism is required.

COMBINED EFFECTS OF O 3 AND OTHER ABIOTIC ENVIRONMENTAL FACTORS ON JAPANESE FOREST
TREE SPECIES Izuta (2002Izuta ( , 1998) ) and Izuta et al. (2001) reviewed experimental studies on the combined effects of O 3 and simulated acid rain on Japanese forest tree species.Recently, it was pointed out that the interactive effects of O 3 , N deposition, elevated carbon dioxide (CO 2 ) and climate change such as drought stress must be key issues to predict forest future in the changing environment (Paoletti et al., 2010).In this section, we focused on the combined effects of O 3 and N load to soil, elevated CO 2 or drought on growth, photosynthetic activity and other related functions of Japanese forest tree species.

1 Nitrogen Load to Soil
Atmospheric deposition of N to terrestrial ecosystems has been increasing with elevated anthropogenic emissions of N since the industrial revolution (Richter et al., 2005;Galloway et al., 2004Galloway et al., , 2003;;IPCC, 2001).Because N is a limiting nutrient for plant growth in terrestrial ecosystems (Vitousek and Howarth, 1991), an increase in N input to forest ecosystems generally stimulates tree growth.However, many researchers suggested that excessive deposition of N such as nitrate and ammonium from the atmosphere to forest ecosystems might induce soil acidification, modify tree nutrient status and increase the sensitivity of trees to other environmental stresses such as gaseous air pollutants (Aber et al., 1989;Schulze, 1989;Nihlgård, 1985).
Based on the monitoring data and estimations of O 3 concentration and atmospheric N deposition in East Asia (Network Center for EANET, 2011;Yamaji et al., 2006;Kohno et al., 2005), there is the possibility that forest tree species are adversely affected not only by O 3 , but also by excessive N deposition in East Asian countries including Japan.In the experimental studies of Watanabe et al. (2008Watanabe et al. ( , 2007Watanabe et al. ( , 2006) ) and Yamaguchi et al. (2010Yamaguchi et al. ( , 2007b)), seedlings of Q. serrata, Japanese beech, C. sieboldii, Japanese red pine, Japanese larch and Japanese cedar were grown in potted soil supplied with N as NH 4 NO 3 solution at 0, 20 and 50 kg ha -1 year -1 and simultaneously exposed to charcoal-filtered air or O 3 at 1.0, 1.5 and 2.0 times the ambient concentration for two growing seasons (24-h seasonal mean concentration of O 3 (from April to September) during the experimental period: 12, 43, 63 and 84 ppb, respectively).Watanabe et al. (2008Watanabe et al. ( , 2007Watanabe et al. ( , 2006) ) reported the additive effects of O 3 and N load on growth of the seedlings of Q. serrata, C. sieboldii, Effects of O 3 on Japanese Forest Tree Species Japanese red pine and Japanese cedar.On the other hand, significant interactive effects of O 3 and N load on growth were detected in Japanese larch and Japanese beech (Yamaguchi et al., 2007b;Watanabe et al., 2006).The relationships between relative whole-plant dry mass increment of Japanese larch or Japanese beech and daylight AOT40 of O 3 (accumulated exposure over a threshold of 40 ppb during daylight hours (global radiation¤50 W m -2 ), see Fig. 1) were shown in Fig. 2. Daylight AOT40 is the sum of the difference between the hourly mean O 3 concentration and 40 ppb for all daylight hours (shaded area in Fig. 1) within a specified time period (from April to September in this case).The calculation of the relationships was based on the method of Karlsson et al. (2004).The coefficient of determination (R 2 ) obtained from linear regression analysis and the slope of regression line in each N treatment are indicated in Fig. 2. The absolute value of the slope of regression line indicates the sensitivity to O 3 of whole-plant dry mass growth of the seedlings.While the N load to soil reduced the sensitivity to O 3 of whole-plant dry mass growth of Japanese larch (Watanabe et al., 2006), it increased that of Japanese beech (Yamaguchi et al., 2007b).These results indicate that the combined effect of O 3 and N load on growth is quite different among the Japanese forest tree species.Nakaji and Izuta (2001), Nakaji et al. (2004) and Watanabe et al. (2006) reported that the N load to soil did not change the degree of O 3 -induced reduction in net photosynthetic rate in the needles of Japanese red pine and Japanese larch.In contrast, the degrees of O 3 -induced reduction in net photosynthetic rate of Q. serrata, Japanese beech and C. sieboldi became high with increasing the amount of N load to soil (Watanabe et al., 2008(Watanabe et al., , 2006;;Yamaguchi et al., 2007b).To clarify the mechanisms underlying the combined effects of O 3 and N load on net photosynthesis of Japanese forest tree species, Yamaguchi et al. (2010Yamaguchi et al. ( , 2007b) ) investigated the effects of O 3 and N load on the concentration  and activity of Rubisco, enzyme activity of N metabolism and concentrations of amino acid and soluble protein in the leaves of Japanese beech seedlings.In their studies, the seedlings of Japanese beech were grown in the soil supplied with N as NH 4 NO 3 solution at 0, 20 or 50 kg ha -1 year -1 and simultaneously exposed to charcoal-filtered air (CF) or O 3 at 1.0, 1.5 and 2.0 times ambient concentration.The exposure to O 3 significantly reduced the concentration and activity of Rubisco in the leaves of the seedlings grown in relatively high N load treatment, but not in relatively low N load treatment (Fig. 3a).This result indicates that the interactive effect of O 3 and N load on net photosynthetic rate is mainly attributed to the difference in the degrees of O 3 -induced reduction in the amount of Rubisco among the N treatments (Yamaguchi et al., 2007b).Furthermore, the exposure to O 3 reduced the concentration of TSP and the ratio of TSP concentration to leaf N concentration in relatively high N load treatment, but not in relatively low N load treatment (Fig. 3b and c).Therefore, Yamaguchi et al. (2010) concluded that the exposure to O 3 reduced the allocation of N to soluble protein in the leaves of Japanese beech seedlings grown under relatively high N load, but did not in the leaves of the seedlings grown under a relatively low N load.

2 Elevated CO 2 and Drought
Elevated CO 2 and drought are well known to affect the sensitivity of forest tree species to O 3 (e.g.EPA, 2006b).Unfortunately, there is little information on the combined effects of O 3 and elevated CO 2 or soil water stress on Japanese forest tree species (Watanabe et al., 2010(Watanabe et al., , 2005;;Matsumura et al., 2005;Yonekura et al., 2001a, b).Matsumura et al. (2005) reported that the effect of elevated CO 2 on O 3 -induced reduction in growth was counteractive in Japanese white birch seedlings, while not in the Japanese mountain birch (Betula ermanii), Japanese beech, Japanese red pine and Japanese cedar seedlings.This result indicates that the combined effect of O 3 and elevated CO 2 is different among Japanese forest tree species.On the other hand, Watanabe et al. (2010) reported that the simultaneous exposure to O 3 and elevated CO 2 induced marked growth stimulation of Japanese beech seedlings as compared with those exposed to elevated CO 2 .Yonekura et al. (2001a, b) reported the additive effects of O 3 and soil water stress on the growth of Japanese beech seedlings.On the other hand, Watanabe et al. (2005) reported that chronic soil water stress counteracted the negative effects of O 3 on net photosynthesis of the leaves of Japanese beech seedlings.Combined effects of O 3 and other environmental factors such as elevated CO 2 and drought on growth and physiological functions of Japanese forest tree species are still poorly understood.Therefore, it is necessary to promote the experimental study and accumulate the information on the combined effects of O 3 and any other abiotic environmental factors on growth, physiological functions and nutrient N load (kg ha -1 year -1 ) N load (kg ha -1 year -1 ) N load (kg ha -1 year -1 ) CF status of Japanese forest tree species.

CRITICAL LEVEL OF O 3 FOR PROTECTING JAPANESE FOREST TREE SPECIES
The ambient levels of O 3 in Japan have been shown to adversely affect growth and photosynthetic activity of Japanese forest tree species especially in those classified into high O 3 sensitivity group as mentioned above.In Europe, the concept of critical level has been developed to prevent long-term injury and damage of air pollutants to the receptors such as plants (Mills et al., 2010).The critical levels for vegetation are defined as the concentration, cumulative exposure or cumulative stomatal flux of atmospheric pollutants above which direct adverse effects on sensitive vegetation may occur according to present knowledge (Mills et al., 2010).At the present time, to define the concentration-based critical levels for O 3 , AOT40 has been adopted for use within the United Nations Economic Commission for Europe (UNECE) Convention of Long-Range Trans-boundary Air Pollution (CLRTAP) and the European Union (Mills et al., 2010;Ashmore et al., 2004).As a result of much efforts directing to establishing the critical level of O 3 , critical level for forest trees has been defined as 5 μmol mol -1 h (ppm h) of daylight AOT40 accumulated over a six-month growing season (from April to September) associated with a 5% growth reduction per one growing season for sensitive deciduous tree species native to Europe such as European beech (Fagus sylvatica) and European birch (Betula pendula) (Mills et al., 2010;Karlsson et al., 2004).Because the vegetation and climatic condition in Japan is quite different from that in Europe, critical level of O 3 for forest tree species in Europe is not directly applicable to that in Japan (Kohno et al., 2005).Kohno et al. (2005) proposed that provisional critical level of O 3 for Japanese forest tree species classified into the high O 3 sensitivity group such as Japanese larch and Japanese beech is 8-15 ppm h of daylight AOT40 accumulated over one growing season (from April to September) associated with a 10% reduction in the increment of the whole-plant dry mass per one growing season (Table 2).However, N deposition from the atmosphere should be taken into account to evaluate the critical level of O 3 for protecting Japanese forest tree species, because the sensitivities of Japanese larch and Japanese beech to O 3 are influenced by the amount of N load to soil (Yamaguchi et al., 2007b;Watanabe et al., 2006).Furthermore, as indicated by Matsumura et al. (2005) and Watanabe et al. (2010), it is necessary to take into account the en-vironmental factors such as atmospheric CO 2 concentration to evaluate the critical level of O 3 .To establish the critical level of O 3 for protecting Japanese forest tree species, therefore, further research concerning the combined effects of O 3 and other abiotic environmental factors on the growth of Japanese forest tree species is required.
Ozone enters the leaf through the stomata and then injures cellular components such as plasma membrane (Nouchi, 2002).Since the real impacts of O 3 mainly depend on the amount of O 3 reaching the sites of damage within the leaf, cumulative flux or uptake of O 3 through the stomata and associated response functions are suitable for mapping and quantifying impacts of O 3 at the local and regional scale (Mills et al., 2010).Therefore, atmospheric concentration-based critical level of O 3 expressed as AOT40 can be used only for estimating the risk of damage.The approach based on the O 3 flux into leaves or needles requires the development of mathematical models to estimate stomatal O 3 uptake primarily from the knowledge of stomatal responses to environmental factors (e.g.Emberson et al., 2000a, b).At the present time, however, there is limited information on stomatal flux of O 3 into the leaves or needles of Japanese forest tree species (Hoshika et al., 2009).Therefore, it is necessary to promote the research toward the modeling of stomatal flux of O 3 for the final purpose of mapping and quantifying the impacts of O 3 on Japanese forest tree species.

CONCLUSION AND PERSPECTIVES
Based on the results obtained from the experimental studies, the current levels of O 3 in Japan are high enough to adversely affect growth of Japanese forest tree species with relatively high O 3 sensitivity such as Japanese beech.To protect Japanese forest, therefore, we need to establish the critical level of O 3 , primarily using AOT40 index, for Japanese forest tree species with consideration of other abiotic environmental factors affecting the sensitivity to O 3 such as N deposition from the atmosphere.In addition to the estimating the risk of damage of O 3 using AOT40 index, it is necessary to quantify the impacts of O 3 on Japanese forest tree species.For this purpose, it is necessary to promote the research toward the modeling of stomatal flux of O 3 into the leaves or needles of Japanese forest tree species.Furthermore, Kohno et al. (2005) pointed out whether results obtained from experimental studies on the effects of O 3 on the growth of Japanese forest tree species using the seedlings could be applicable to the evaluation of O 3 -induced adverse effects on the growth of mature trees grown under natural conditions or not.To understand and evaluate the actual impacts of O 3 on the growth and physiological functions of Japanese forest tree species grown in the field, therefore, further research is required for the scaling effects of O 3 from seedlings to mature forest trees.
. Decreased dry masses of needle and root and A (Additive).

Fig. 2 .
Fig. 2. The relationships between relative whole-plant dry mass increment (DMI) of Japanese larch and Japanese beech seedlings per one growing season and daylight AOT40 of O 3 .The seedlings were grown in the soil supplied with N as NH 4 NO 3 solution at 0 (N0), 20 (N20) or 50 kg ha -1 year -1 (N50) and simultaneously exposed to charcoal-filtered air or O 3 at 1.0, 1.5 and 2.0 times ambient concentration.Data source: Watanabe et al. (2006) and Yamaguchi et al. (2007a, b).

Fig. 3 .
Fig. 3. Effects of O 3 and N load on activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), concentration of total soluble protein (TSP) and ratio of TSP to leaf N content per unit leaf area (TSP/N) in the leaves of Japanese beech.The standard deviation is given by vertical bar.Two-way ANOVA: *p⁄0.05,***p⁄0.001,n.s.= =not significant.Different letters above the bar indicate significant difference among the 12 treatments (Tukey's HSD test, p⁄0.05).Data source: Yamaguchi et al. (2010, 2007b).

Table 1 .
Summary of experimental studies on the effects of O (Picea abies)No effect on whole-plant dry mass.
A: net photosynthetic rate; A max : CO 2 -saturated net photosynthetic rate; CE: carboxylation efficiency; CF: charcoal-filtered air treatment; Chl: chlorophyll concentration; E: transpiration rate; F v /F m : maximum quantum yield of photosystem II; GS: glutamine synthetase; g s Table 2).For example, Japanese larch and Japanese beech have been classified into high O 3 sensitivity group; Japanese white birch and Nikko fir have been classified into moderate O 3 sensitivity group; Japanese cedar and Japanese cypress have been classified into low O 3 sensitivity group.

Table 2 .
Classification of Japanese forest tree species into the O 3 sensitivity groups (afterKohno et al., 2005, with permission).The whole-plant dry mass increment was significantly reduced by the exposure to ambient level of O 3 .Moderate O 3 sensitivity: The whole-plant dry mass increment was significantly reduced by the exposure to 1.5 or 2.0 times ambient level of O 3 .Low O 3 sensitivity: The whole-plant dry mass increment was not significantly reduced by the exposure to 1.5 or 2.0 times ambient level of O 3 .Daylight AOT40: accumulated exposure over a threshold of 40 ppb O 3 during daylight hours from April to September (6 months).Daylight hour: global radiation ¤50 W m -2 .