Effects of sulfur dioxide on growth and net photosynthesis of six Japanese forest tree species grown under different nitrogen loads

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1 Trees (2012) 26: DOI /s y ORIGINAL PAPER Effects of sulfur dioxide on growth and net photosynthesis of six Japanese forest tree species grown under different nitrogen loads Masahiro Yamaguchi Makoto Watanabe Chikako Tabe Junichi Naba Hideyuki Matsumura Yoshihisa Kohno Takeshi Izuta Received: 26 February 2012 / Revised: 21 June 2012 / Accepted: 26 June 2012 / Published online: 21 July 2012 Ó Springer-Verlag 2012 Abstract We examined the growth and photosynthetic responses of Japanese forest tree species to sulfur dioxide (SO 2 ) under different nitrogen (N) loads to soil. We grew Quercus serrata, Fagus crenata, Castanopsis sieboldii, Larix kaempferi, Pinus densiflora, and Cryptomeria japonica seedlings in Andisol supplemented with N as NH 4 NO 3 solution at 0, 20, and 50 kg ha -1 year -1. Seedlings were exposed daily to charcoal-filtered air or SO 2 at 10, 20, and 40 nl l -1 for two growing seasons. Except for C. japonica seedlings, exposure to SO 2 at a relatively low concentration stimulated whole-plant growth, especially under a relatively high N load. The effects of N load on the negative impact of SO 2 on whole-plant growth were synergistic in Q. serrata, F. crenata, C. sieboldii, and Communicated by H. Rennenberg. Electronic supplementary material The online version of this article (doi: /s y) contains supplementary material, which is available to authorized users. M. Yamaguchi C. Tabe J. Naba Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo , Japan masah_ya@cc.tuat.ac.jp M. Watanabe Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido , Japan H. Matsumura Y. Kohno Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, Abiko, Chiba , Japan T. Izuta (&) Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo , Japan izuta@cc.tuat.ac.jp P. densiflora, counteractive in L. kaempferi, and additive in C. japonica. InQ. serrata, F. crenata, C. sieboldii, and P. densiflora seedlings, the different responses of wholeplant growth to SO 2 among the N treatments were because of the effect of N load on the response of the net photosynthetic rate to SO 2. L. kaempferi seedlings showed N load-induced tolerance of whole-plant growth to SO 2.This was explained by the effect of N load on the responses of photosynthesis and development of assimilative organs to SO 2. The different growth responses to SO 2 among the N treatments were explained by the effects of N load on the SO 2 uptake rate (evaluated by stomatal diffusive conductance) or the accumulated SO 2 uptake (evaluated by foliar S concentration). Keywords Sulfur dioxide Nitrogen Japanese forest tree species Growth Net photosynthesis Introduction In East Asia, sulfur dioxide (SO 2 ) emissions are increasing and relatively high concentrations of SO 2 have been detected in the atmosphere (Larssen et al. 2006; Network Center for EANET 2012). SO 2 is a major air pollutant that adversely affects not only human health, but also vegetation (Kondo 2002; Nouchi 2002). It is absorbed into the leaves predominantly via stomata (Laisk et al. 1988; Rennenberg and Polle 1994; Hänsch et al. 2007). Once absorbed into leaves, SO 2 dissolves in the aqueous phase of the cell surface yielding bisulfite, sulfite ions, and protons. Because SO 2 can enter the sulfate assimilation pathway (Rennenberg et al. 2007), it can contribute to sulfur nutrition and stimulate plant growth at low concentrations (Van der Kooij et al. 1998; De Kok et al. 2005; Yang et al.

2 1860 Trees (2012) 26: ). It has been suggested that the oxidation of SO 2-3 to SO 2-4 is the major detoxification pathway of SO 2 among the reactions in the sulfate assimilation pathway (Hänsch et al. 2007; Lang et al. 2007; Randewig et al. 2012). However, when uptake rates or accumulated uptake of SO 2 exceed the rate of detoxification, phytotoxic effects of SO 2 will occur due to tissue acidification, high endogenous levels of toxic sulfite, and/or intracellular superoxidemediated free radical chain oxidation of sulfite to sulfate in the chloroplasts (De Kok and Tausz 2001; Kondo 2002; Nouchi 2002). Consequently, physiological processes such as photosynthetic activity are negatively affected by exposure to SO 2, resulting in the reduction in growth of plants including forest tree species (Keller 1985; Kozlowski et al. 1991; Kondo 2002; Nouchi 2002; Randewig et al. 2012). Atmospheric deposition of nitrogen (N) to terrestrial ecosystems has increased with higher anthropogenic emissions of N since the industrial revolution (Galloway et al. 2003). In terrestrial ecosystems, especially from temperate to boreal forest ecosystems, increased N input into forest ecosystems generally stimulates tree growth, since N is a limiting nutrient for plant growth (Vitousek and Howarth 1991). However, many researchers have suggested that excessive deposition of N from the atmosphere to forest ecosystems has negative effects such as soil acidification, modifications to tree nutrient status, and increased sensitivity of trees to other environmental stresses such as gaseous air pollutants (Nihlgård 1985; Nakaji et al. 2001; Nakaji and Izuta 2001; Bobbink et al. 2003; Izuta et al. 2005). In Europe, therefore, empirical N critical loads of kg ha -1 year -1 for deciduous and coniferous tree species have already been set (Bobbink et al. 2003). In East Asia, the average rate of wet and dry deposition of N from the atmosphere was estimated to be 22 kg ha -1 year -1, and its maximum rate would be [ 50 kg ha -1 year -1 (Kohno et al. 2005). Therefore, it is possible that Asian forest tree species will be adversely affected by excessive N deposition from the atmosphere. There are already high concentrations of SO 2 in the atmosphere in Asia, and it is projected that SO 2 emissions will increase (Ohara et al. 2007). In Japan, there are relatively low annual mean concentrations of SO 2 (below 10 nl l -1 ) (Network Center for EANET 2012), but it has been suggested that SO 2 emitted in the Asian continent is transported to Japan (Lu et al. 2010). N deposition is expected to increase in East Asia, including Japan (Dentener et al. 2006). Therefore, Japanese forest tree species may or will be adversely affected by SO 2 and excessive N deposition. However, there is no information on the combined effects of SO 2 and N deposition on growth and net photosynthesis of Japanese forest tree species. Total atmospheric N deposition is the sum of dry, wet, and occult deposition of N (e.g., Fowler and Smith 2004). Although mimicking N loads to soil using NH 4 NO 3 solution does not exactly simulate total atmospheric N deposition, it is difficult to conduct controlled experiments on the combined effects of SO 2 and total atmospheric N deposition. In the present study, therefore, we investigated the effects of SO 2 on growth and net photosynthesis of seedlings of six Japanese representative forest tree species grown under different soil N loads adjusted using NH 4 NO 3 solution. Materials and methods Plant materials Seedlings of Quercus serrata (deciduous broad-leaved tree), Fagus crenata (deciduous broad-leaved tree), Castanopsis sieboldii (evergreen broad-leaved tree), Larix kaempferi (deciduous conifer), Pinus densiflora (evergreen conifer), and Cryptomeria japonica (evergreen conifer) were used as plant materials in the present study. On 8 March 2004, seedlings were individually planted in 12-l pots filled with Andisol (volcanic ash soil). The total N concentration and carbon/nitrogen (C/N) concentration ratio of the soil was 3.5 mg g -1 (dry weight basis) and 15.6, respectively. From April 2004 to November 2005, all the seedlings were grown in 12 rectangular open-top chambers (OTCs, 13.0 m 2 growth space and 2.4 m in height) at the Akagi Testing Center of the Central Research Institute of Electric Power Industry (Maebashi, Gunma Prefecture, Japan), located on the south slope of Mt. Akagi at 540 m above sea level. The average values for the plant height, stem base diameter, and the whole-plant dry mass of the seedlings at the beginning of the experiment are shown in Table S1 (supplementary material). Sulfur dioxide fumigation and soil nitrogen treatment We used a split-plot factorial design and a randomized block method. The whole-plot treatment comprised four levels of SO 2 with three chamber replications (total of 12 OTCs) to analyze the data, including variance among the 12 OTCs. The sub-plot treatment consisted of three levels of N treatment in each OTC. In each treatment, four seedlings of each tree species per OTC were assigned to each SO 2 N chamber combination, making a total of 144 seedlings per species. From 16 April 2004, the seedlings were exposed to charcoal-filtered air (CF) or SO 2 at 10, 20, or 40 nl l -1 (ppb). The SO 2 gas (99.9 %) was diluted with charcoalfiltered dry air and injected into the nine OTCs using mass flow controller (SEC-4400MO-CRP, HORIBA STEC, Japan) to achieve the target SO 2 concentrations of 10, 20,

3 Trees (2012) 26: or 40 ppb. The concentrations of SO 2 at 90 cm above the floor in the 12 OTCs were continuously monitored at intervals of 3 min with a UV fluorescence SO 2 analyzer (ML9850, Monitor Labs, Englewood, CO, USA). The average concentration and sum of all hourly concentrations (SUM00) of SO 2 during the experimental period were calculated from SO 2 monitoring data from the OTCs. The SUM00 was calculated as the sum of the hourly mean SO 2 concentration. The average concentration and SUM00 of SO 2 during the growing seasons of the seedlings from April to November in 2004 and 2005 are shown in Table S2 (supplementary material). The average 24-h concentrations of SO 2 in the CF, 10, 20, and 40 ppb SO 2 treatments during the two growing seasons were 0.2 ± 0.1, 9.6 ± 0.4, 18.2 ± 0.5, and 34.7 ± 1.4 ppb, respectively. Three soil N treatments were established to simulate atmospheric N deposition. In East Asia, the average rate of wet and dry deposition of N from the atmosphere was estimated to be 22 kg ha -1 year -1 and its maximum rate greater than 50 kg ha -1 year -1 (Kohno et al. 2005). In the present study, therefore, the total N loads were 0, 20, and 50 kg ha -1 year -1 on the basis of potted soil surface area; these soil treatments were designated as N0, N20, and N50,? respectively. Since the molar concentration ratio of NH 4 - to NO 3 in precipitation was reported to be 1.04 in Japan (Network Center for EANET 2012), the N load was supplied using NH 4 NO 3 solution, which approximates, but does not exactly simulate, total atmospheric N deposition. During the experimental period from April to September 2004 and 2005, 500 ml of 1.17 or 2.92 mm NH 4 NO 3 solution was added to the surface of the potted soil at 1-month intervals. Soil solution was taken from the potted soil using a soil moisture sampler (Eijkelkamp, The Netherlands) in October The ph of the soil solution was determined with a ph meter (D-24, Horiba, Japan). The concentration of NH? 4 in the soil solution was determined by the indophenol blue colorimetry method - (Tsuzuki 1999). The concentration of NO 3 in the soil solution was determined by ion chromatography (IC7000, Yokogawa, Japan). At the beginning of the experiment, the soil solution had a ph of 5.89, an NO - 3 concentration of 2.43 mm, and an NH? 4 concentration of 0.09 mm. At the end of the experiment, because we detected neither significant effects of SO 2 nor significant interactions between SO 2 and N load in terms of the ph and inorganic N concentrations of soil solution, these data were pooled across SO 2 treatments. At the end of the experiment, there were no significant effects of N load on ph (ranging from 5.34 to ) and concentrations of NO 3 (ranging from n.d. to 4.61 lm) or NH? 4 (ranging from 2.85 to 4.16 lm) in soil solutions of any of the tree species. These experiments were conducted concurrently with those reported by Watanabe et al. (2006, 2007, 2008) and Yamaguchi et al. (2007). Because the CF treatment in the present study is part of their studies, the values for dry mass and gas exchange rates in the CF treatment reported in their studies are referred to in the present study as the control for the gas treatment. Measurements of plant growth Seedlings were harvested on 18 October 2005 for Q. serrata, 25 October 2005 for F. crenata, 1 November 2005 for C. sieboldii, 10 October 2005 for L. kaempferi, 11 November 2005 for P. densiflora and 4 November for C. japonica, and were used to determine leaf area per plant and dry mass of plant organs. The harvested seedlings were separated into leaves, branches, stems, and roots. Leaf area was measured with an area meter (LI-3100, Li-Cor Inc., Lincoln, NE, USA). Plant organs were dried at 80 C ina forced air oven for 1 week and then weighed. Measurement of leaf or needle gas exchange rates The leaf or needle gas exchange rate of the seedlings was measured with a portable infrared gas analyzer system (LI- 6400, Li-Cor Inc., Lincoln, NE, USA) on 7 July 2005 for Q. serrata, 5 July 2005 for F. crenata, 19 July 2005 for C. sieboldii, 12 July 2005 for L. kaempferi, and 22 July 2005 for C. japonica. Two seedlings per treatment chamber combination were randomly selected to measure leaf or needle gas exchange rates (six measurements per treatment). Net photosynthetic rate (A sat ) and stomatal diffusive conductance to H 2 O(g s ) were determined under the following conditions: 24 ± 0.1 C, 380 lmol CO 2 mol -1, relative air humidity of 60 ± 5 %, and a photosynthetically active photon flux density of 1,500 lmol m -2 s -1. Determination of S concentration in leaves or needles The dried leaves or needles harvested at the end of the experiment were ground to a fine powder with a vibrating sample mill. The powder was digested with HNO 3 and H 2 O 2 and then diluted with 100 mm HCl. The concentration of S in the sample solution was determined by sequential-type inductively coupled plasma-atomic emission spectrometer equipped with vacuum monochromators (ICPS-8100, Shimadzu, Japan). Calculation of dose response relationship The mean 24-h SUM00 of SO 2 in each gas treatment during two growing seasons was used as an SO 2 exposure index to calculate dose response relationships. The wholeplant dry mass at the end of the second growing season in the CF treatment was used as a reference (1.0) to calculate

4 1862 Trees (2012) 26: the relative value of the whole-plant dry mass in each SO 2 treatment. The whole-plant dry mass of seedlings exposed to charcoal-filtered air in the N0, N20, and N50 treatments is shown in Table S3 (supplementary material). Statistical analyses All statistical analyses were performed with IBM SPSS Advanced Statistics 19. Analysis of variance (ANOVA) was used to test the effects of SO 2 and N load on growth, gas exchange parameters, and S concentration in the leaves or needles. Variance for SO 2 treatments was partitioned into linear, quadratic, and cubic components and variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts. When there were significant interactions between SO 2 and N load for the whole-plant dry mass and net photosynthetic rate, orthogonal contrasts using fixed levels of N across linear, quadratic, or cubic components of SO 2 were developed to determine the response to SO 2 in each N treatment. In the case of the response of stomatal conductance to water vapor and S concentration in the leaves or needles, orthogonal contrasts using fixed levels of SO 2 across linear or quadratic components of N were developed to determine the effect of N load on the parameters in each gas treatment. Because it is difficult to consider the cubic responses of growth and gas exchange rates to increases in SO 2 concentration, we discuss significant linear and quadratic responses to increases in SO 2 concentration in the present study. The relationships between the SUM00 of SO 2 and relative whole-plant dry mass were analyzed by linear regression analysis or quadratic curve fitting based on the results of orthogonal polynomial contrasts. Results and discussion Figure 1 shows the relationships between 24-h SUM00 of SO 2 during the experimental period and relative values of whole-plant dry mass (DM W ) of the seedlings at the end of the experiment. A regression analysis was performed based on the results of two-way ANOVA shown in Table 1 and Fig. 1. Because low concentrations of SO 2 contribute to sulfur nutrition (De Kok et al. 2005), we determined increments of root dry mass (DM R ) of the seedlings (Table 2), as this is relevant to the sulfur nutrient requirements of the plant. To clarify the causes of growth responses to SO 2 under different N loads, we determined the leaf area per plant (LA) and needle dry mass per plant (DM N ) (Table 3), and light-saturated net photosynthetic rate (A sat ) in leaves or needles (Table 4). In general, the uptake rate of SO 2 is directly dependent on the degree of stomatal aperture, because the internal (mesophyll) resistance for SO 2 gas flux is very low (Pfanz et al. 1987; Rennenberg and Polle 1994; Van der Kooij and De Kok 1998; De Kok and Tausz 2001; Randewig et al. 2012). The absorbed SO 2 is accumulated as S compounds such as sulfate (De Kok and Tausz 2001; Kondo 2002; Randewig et al. 2012), suggesting that the SO 2 -induced increase in the foliar S concentration reflects the accumulated uptake of SO 2. To discuss different growth responses among N treatments, therefore, we determined the effects of SO 2 and N load on stomatal conductance to H 2 O(g s ) (Table 5) and S concentrations in the leaves or needles of the seedlings (Table 6). Measurements of gas exchange rates were conducted five times during the experimental period (July and September 2004, and May, July, and September 2005). The gas exchange rates measured in summer tended to be higher than those in the other seasons, and the amount of assimilative organs per plant was greater in the second season than in the first growing season. Because the gas exchange rates measured in the summer of the second growing season were considered to be appropriate to explain responses of whole-plant growth to SO 2 under different N loads, A sat and g s measured in July 2005 are reported. Quercus serrata seedlings At the end of the experiment, there was a significant interaction between SO 2 (quadratic) and N load (linear) for DM W of the seedlings (Table 1). A significant quadratic response of DM W to increased SO 2 concentration was observed in the N50 treatment, but not in the N0 and N20 treatments (Fig. 1a). In the N50 treatment, DM W increased in response to 10 ppb SO 2, but decreased in response to 20 and 40 ppb SO 2. A significant interaction was found between SO 2 (quadratic) and N load (linear) for the increment in DM R (Table 2). A linear reduction in the increment of DM R was observed in the N50 treatment, but not the N0 and N20 treatments. These results indicated that the sensitivity of growth of Q. serrata seedlings to SO 2 increased with increasing N load to the soil. SO 2 did not affect LA significantly in any N treatment (Table 3). However, a significant interaction was detected between SO 2 (quadratic) and N load (linear) for A sat (Table 4). A significant quadratic response in A sat to increased SO 2 concentration was observed in the N50 treatment, but not in the N0 and N20 treatments. In the N50 treatment, A sat increased in response to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. These results suggested that, under relatively high N load, the quadratic response of the whole-plant growth to increase in SO 2 concentration was mainly due to the quadratic response of net photosynthetic rate to SO 2. In all the SO 2 treatments, the g s of leaves showed a significant linear increase with increasing N load (Table 5).

5 Trees (2012) 26: (a) Q. serrata (b) F. crenata (c) C. sieboldii Relative value of DMW N0 : n.s. N20: n.s. N50: Q (d) L. kaempferi N0 : Q N20: Q N50: Q (e) P. densiflora N0 : n.s. N20: n.s. N50: Q (f) C. japonica N0 : N20: N50: N0 : L N0 : Q No significant interaction N20: n.s. N20: Q between SO2 and N load N50: Q N50: Q h SUM00 of SO2 (ppm h) Fig. 1 Relationships between 24-h SUM00 of SO 2 during experimental period and relative value of whole-plant dry mass (DM W, CF = 1.0) in November Each symbol shows mean ± standard deviation of three chamber replicates. Two-way ANOVA was used to identify interactions between SO 2 and N load. When significant interactions were observed, orthogonal contrasts using fixed levels of N across linear, quadratic, or cubic components of SO 2 were developed. L and Q indicate significant linear and quadratic responses to SO 2 concentration (P \ 0.05), respectively. n.s. not significant. Regression analyses were performed according to results of orthogonal contrasts. C. japonica seedlings showed a significant linear reduction in DM W in response to exposure to SO 2 without a significant interaction between SO 2 and N load (Table 1); therefore, linear regression analysis was performed using four plots pooled across the N treatments Table 1 The F-values and levels of significance of two-way analysis of variance (ANOVA) for effects of SO 2 and N load on whole-plant dry mass of Q. serrata, F. crenata, C. sieboldii, L. kaempferi, P. densiflora, and C. japonica seedlings Source of variation Q. serrata F. crenata C. sieboldii L. kaempferi P. densiflora C. japonica SO *** 10.97** 13.18** *** 6.92* Linear (L) 8.47* 78.18*** 22.22** 37.04*** *** 19.56** Quadratic (Q) *** *** 0.84 N load *** 82.92*** 67.77*** 4.11* *** Linear (L) *** *** *** *** Quadratic (Q) 17.06*** 5.35* * SO 2 9 N load 4.27** 5.65** * 3.88* 1.90 SO 2 (L) 9 N load (L) * SO 2 (L) 9 N load (Q) SO 2 (Q) 9 N load (L) 10.03** 14.63** 7.44* SO 2 (Q) 9 N load (Q) ** 4.71* * 1.18 Variance for SO 2 treatments was partitioned into linear, quadratic, and cubic components. Variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts L linear, Q quadratic Significance of ANOVA denoted as follows: * P \ 0.05, ** P \ 0.01, *** P \ 0.001

6 1864 Trees (2012) 26: Table 2 Effects of SO 2 on root dry mass increment (g) of seedlings of six Japanese forest tree species grown under different N loads during the experimental period, and F-values and levels of significance of two-way analysis of variance (ANOVA) Treatment Q. serrata F. crenata C. sieboldii L. kaempferi P. densiflora C. japonica N load Gas N0 CF 79.7 (9.5) 26.4 (5.1) 21.8 (1.2) 33.0 (6.0) 58.5 (5.7) 37.0 (2.3) 10 ppb SO (2.8) 28.4 (2.5) 19.3 (1.0) 30.6 (2.5) 54.8 (6.9) 36.2 (0.4) 20 ppb SO (4.6) 28.8 (6.2) 21.4 (2.7) 31.4 (3.2) 48.1 (7.1) 30.4 (2.0) 40 ppb SO (6.8) n.s (1.5) L, Q 21.9 (1.5) n.s (2.6) 42.5 (4.1) 29.1 (0.4) N20 CF 80.7 (5.3) 34.4 (3.1) 31.2 (7.1) 33.5 (7.3) 52.3 (2.6) 43.1 (6.7) 10 ppb SO (9.1) 34.2 (4.4) 26.0 (1.9) 32.3 (1.9) 54.4 (11.2) 39.5 (2.9) 20 ppb SO (4.8) 30.2 (2.2) 25.6 (2.9) 27.3 (2.8) 50.0 (0.8) 37.8 (1.4) 40 ppb SO (7.6) n.s (1.4) L 30.1 (8.0) n.s (2.7) 40.8 (2.4) 29.8 (2.6) N50 CF (7.0) 38.5 (2.9) 36.5 (3.1) 35.4 (2.6) 59.6 (5.6) 45.1 (2.1) 10 ppb SO (3.5) 44.2 (5.0) 40.4 (5.0) 33.2 (3.2) 53.3 (8.6) 40.7 (2.0) 20 ppb SO (3.5) 44.6 (3.7) 41.0 (1.1) 29.6 (2.5) 49.0 (11.2) 44.5 (8.1) 40 ppb SO (5.2) L 24.1 (1.1) L, Q 32.0 (4.1) Q 31.1 (2.0) 42.2 (3.3) 39.3 (2.6) Two-way ANOVA SO *** ** 6.37* Linear (L) 9.01* 57.92*** ** 35.30** 18.07** Quadratic (Q) *** N load 67.33*** 48.73*** 58.30*** *** Linear (L) *** 96.16*** *** *** Quadratic (Q) 13.73** SO 2 9 N load 4.76** 2.78* SO 2 (L) 9 N load (L) 4.51* SO 2 (L) 9 N load (Q) SO 2 (Q) 9 N load (L) 5.56* 5.71* 6.87* SO 2 (Q) 9 N load (Q) * 7.65* Values are mean of three chamber replications. Standard deviation is shown in parentheses. Variance for SO 2 treatments was partitioned into linear, quadratic, and cubic components. Variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts. When there was a significant interaction between SO 2 and N load, nine orthogonal contrasts using fixed levels of N across linear, quadratic, or cubic component of SO 2 were developed to determine the response to SO 2 in each N treatment L linear, Q quadratic, n.s. not significant Significance of ANOVA denoted as follows: * P \ 0.05, ** P \ 0.01, *** P \ However, N load did not affect the S concentration in the leaves in any of the SO 2 treatments (Table 6). These results indicated that Q. serrata seedlings showed an N loadinduced increase in sensitivity of growth and net photosynthesis to SO 2. This could be explained by the N load-induced increase in the uptake rate of SO 2 in the leaves, rather than by the effect of N load on the accumulated uptake of SO 2. Fagus crenata seedlings A significant interaction between SO 2 (quadratic) and N load (quadratic) was detected for DM W of the seedlings at the end of the experiment (Table 1), and a significant quadratic response of DM W to increased SO 2 concentration was observed in all the N treatments (Fig. 1b). In the N20 treatment, DM W was reduced by exposure to SO 2 without an SO 2 -induced increase in DM W at low concentrations. In the N0 and N50 treatments, DM W increased in response to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. We detected a significant interaction between SO 2 (quadratic) and N load (quadratic) for increment of DM R (Table 2). In the N0 and N50 treatments, the increment of DM R increased in response to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. In the N20 treatment, the increment in DM R decreased linearly in response to exposure to SO 2. As compared with the CF, the extent of 40 ppb SO 2 -induced reduction in DM W was 28, 29, and 35 % in the N0, N20, and N50 treatments, respectively. The extent of 40 ppb SO 2 -induced reduction in the increment in DM R was similar among the three N treatments; 36, 36, and 37 % in the N0, N20, and N50 treatments,

7 Trees (2012) 26: Table 3 Effects of SO 2 on leaf area per plant of Q. serrata, F. crenata, and C. sieboldii seedlings and needle dry mass per plant of L. kaempferi, P. densiflora, and C. japonica seedlings, and F- values and levels of significance of two-way analysis of variance (ANOVA) Treatments Leaf area per plant (cm 2 ) Needle dry mass per plant (g) N load Gas Q. serrata F. crenata C. sieboldii L. kaempferi P. densiflora C. japonica Current-year 1-year old Current-year 1-year old Current-year 1-year old N0 CF 2119 (264) 1257 (217) 594 (62) 653 (87) 32.8 (4.7) 63.5 (4.0) 32.1 (3.7) 15.0 (1.3) 22.2 (1.2) 10 ppb SO (82) 1357 (113) 466 (30) 618 (105) 26.7 (3.2) 67.3 (4.4) 26.6 (4.4) 15.5 (0.8) 22.2 (3.3) 20 ppb SO (404) 1346 (122) 586 (119) 663 (120) 27.7 (1.8) 66.8 (3.0) 22.7 (7.2) 12.5 (2.5) 17.9 (2.2) 40 ppb SO (41) 897 (223) 635 (61) 678 (54) 19.4 (2.7) 63.6 (4.9) 14.2 (2.3) 14.3 (1.3) 19.3 (1.4) N20 CF 2448 (168) 1509 (157) 685 (72) 697 (238) 27.0 (5.0) 61.1 (2.2) 33.0 (2.5) 20.5 (3.6) 24.5 (4.6) 10 ppb SO (484) 1408 (115) 655 (19) 734 (44) 28.4 (8.5) 62.6 (4.3) 28.2 (3.2) 19.9 (3.7) 21.4 (0.9) 20 ppb SO (97) 1394 (45) 672 (37) 764 (93) 23.3 (2.5) 73.7 (9.4) 24.0 (2.7) 20.4 (2.4) 22.6 (3.0) 40 ppb SO (268) 1023 (152) 726 (139) 738 (89) 24.6 (4.2) 60.1 (5.8) 8.1 (6.6) 19.2 (6.3) 17.0 (3.2) N50 CF 2752 (74) 1580 (45) 906 (150) 961 (216) 28.4 (3.6) 65.3 (1.7) 33.0 (6.7) 27.4 (3.1) 24.8 (2.1) 10 ppb SO (196) 1863 (94) 861 (164) 961 (107) 33.1 (4.7) 70.3 (1.6) 32.0 (4.5) 25.3 (3.1) 24.0 (2.3) 20 ppb SO (124) 1701 (121) 867 (58) 1059 (172) 27.9 (3.5) 70.6 (3.5) 23.5 (4.3) 26.6 (2.7) 25.9 (0.9) 40 ppb SO (386) 1172 (346) 951 (29) 693 (180) 24.5 (0.7) 63.3 (2.6) 15.3 (6.6) 27.2 (0.8) 23.6 (1.5) Two-way ANOVA SO *** * 54.78*** ** Linear (L) *** * *** ** Quadratic (Q) *** ** 8.82* N load 42.01*** 14.57*** 42.71*** 9.55** *** 8.90** Linear (L) 81.17*** 28.02*** 82.96*** 18.14*** *** 16.25*** Quadratic (Q) SO 2 9 N load SO 2 (L) 9 N load (L) SO 2 (L) 9 N load (Q) SO 2 (Q) 9 N load (L) SO 2 (Q) 9 N load (Q) Values are mean of three chamber replications. Standard deviation is shown in parentheses. Variance for SO2 treatments was partitioned into linear, quadratic, and cubic components. Variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts L linear, Q quadratic Significance of ANOVA denoted as follows: * P \ 0.05, ** P \ 0.01, *** P \ 0.001

8 1866 Trees (2012) 26: Table 4 Effects of SO 2 on light-saturated net photosynthetic rate (A sat ) in leaves or needles of seedlings of six Japanese forest tree species grown under different N loads in July 2005, and F- values and levels of significance of two-way analysis of variance (ANOVA) Treatment Q. serrata (lmol m -2 s -1 ) F. crenata (lmol m -2 s -1 ) C. sieboldii (lmol m -2 s -1 ) L. kaempferi (lmol kg -1 s -1 ) P. densiflora (lmol kg -1 s -1 ) C. japonica (lmol kg -1 s -1 ) N load Gas Current-year 1-year old Current-year 1-year old Current-year 1-year old N0 CF 5.91 (0.29) 5.13 (0.12) 2.94 (0.73) 2.33 (0.27) (4.3) 73.2 (5.9) 68.5 (1.8) 26.1 (0.9) 20.1 (1.9) 10 ppb SO (0.49) 5.33 (0.34) 2.92 (0.18) 2.56 (0.11) (10.0) 85.4 (3.7) 56.1 (1.2) 30.7 (2.9) 21.9 (1.2) 20 ppb SO (0.65) 6.07 (0.62) 2.35 (0.76) 2.31 (0.29) (12.1) 65.0 (6.0) 59.5 (6.1) 24.2 (0.6) 17.2 (4.1) 40 ppb SO (0.55) n.s (1.33) n.s (0.29) n.s (0.30) n.s (24.8) 65.2 (4.7) L 53.0 (13.9) 25.9 (0.7) 16.0 (2.2) L N20 CF 4.85 (0.78) 7.86 (0.51) 3.95 (0.75) 3.23 (0.08) (6.5) 68.3 (2.1) 69.9 (4.2) 37.2 (2.7) 24.3 (1.2) 10 ppb SO (1.97) 8.11 (0.57) 2.72 (0.28) 2.60 (0.32) 97.6 (4.3) 69.8 (7.5) 72.0 (10.9) 38.9 (3.1) 13.9 (2.1) 20 ppb SO (0.19) 7.55 (0.10) 3.99 (0.86) 2.22 (0.15) 84.9 (7.6) 65.7 (21.6) 59.6 (9.3) 37.8 (2.1) 19.4 (3.6) 40 ppb SO (0.57) n.s (0.15) L, Q 5.12 (1.19) Q 3.32 (0.47) Q 66.6 (6.6) 73.4 (9.3) n.s (4.9) 39.3 (2.2) 15.2 (1.5) L N50 CF 6.57 (0.54) (1.14) 5.41 (0.10) 3.48 (0.13) 97.8 (23.9) 92.1 (7.0) 67.6 (5.7) 44.3 (2.9) 24.3 (6.0) 10 ppb SO (0.46) 7.15 (0.39) 5.02 (0.26) 3.94 (0.50) 86.6 (4.4) 73.3 (7.0) 58.6 (6.5) 42.9 (1.9) 24.3 (6.2) 20 ppb SO (0.58) 9.17 (1.05) 4.55 (0.53) 3.22 (0.14) 85.1 (9.7) 82.1 (7.1) 50.0 (9.3) 43.6 (6.1) 22.4 (5.4) 40 ppb SO (0.58) Q 6.36 (0.44) L 4.68 (1.03) n.s (0.49) L, Q 92.5 (8.6) 56.4 (4.0) L 44.4 (4.1) 38.5 (3.8) 13.1 (1.8) L Two-way ANOVA SO * 6.05* 9.14* 11.83** ** 7.77* 11.59** Linear (L) * ** 34.35** 8.31* 30.58** 9.48* 31.15** Quadratic (Q) 7.87* ** * 1.13 N load 5.44* 50.46*** 25.08*** 26.36*** * 75.67*** 2.16 Linear (L) 6.72* 95.76*** 50.04*** 50.87*** *** 2.42 Quadratic (Q) * * 11.02** 1.89 SO 2 9 N load *** *** ** * SO 2 (L) 9 N load (L) *** ** SO 2 (L) 9 N load (Q) * 13.58** ** SO 2 (Q) 9 N load (L) 5.31* * SO2 (Q) 9 N load (Q) *** * Values are mean of three chamber replications. Standard deviation is shown in parentheses. Variance for SO 2 treatments was partitioned into linear, quadratic, and cubic components. Variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts. When there was a significant interaction between SO 2 and N load, nine orthogonal contrasts using fixed levels of N across linear, quadratic, or cubic component of SO 2 were developed to determine the response to SO 2 in each N treatment L linear, Q quadratic, n.s. not significant Significance of ANOVA denoted as follows: * P \ 0.05, ** P \ 0.01, *** P \ 0.001

9 Trees (2012) 26: respectively. These results suggested that in F. crenata seedlings, the sensitivity of growth to negative effects of SO 2 increased with increasing N load to the soil. LA showed a significant quadratic response to increased SO 2 concentration without an interaction between SO 2 and N load (Table 3). In all the N treatments, LA increased with exposure to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. We detected a significant interaction between SO 2 (linear) and N load (linear) for A sat (Table 4). Among the N treatments, A sat showed a quadratic response to increasing SO 2 concentration in the N20 treatment, a linear response to increasing SO 2 concentration in the N50 treatment, and no response in the N0 treatment. In the N20 treatment, A sat increased in response to 10 and 20 ppb SO 2, and decreased in response to 40 ppb SO 2. In contrast, in the N50 treatment, A sat linearly decreased with increasing SO 2 concentration. These results indicated that, in F. crenata seedlings, the quadratic response of whole-plant growth to increasing SO 2 concentration was mainly due to the quadratic response of the amount of assimilative organs. The different responses of whole-plant growth to SO 2 among the N treatments were due to differences in net photosynthetic rate. The g s of leaves showed a significant N load-induced linear increase with increasing SO 2 concentration (Table 5). There was a significant reduction in S concentration with increasing N load above 20 kg N ha -1 year -1 in leaves in all of the gas treatments (Table 6). In F. crenata seedlings, therefore, the N load-induced increase in the sensitivity of growth and net photosynthesis to the negative effects of SO 2 could be explained by the N loadinduced increase in the uptake rate of SO 2 in the leaves, rather than the effect of N load on the accumulated uptake of SO 2. Castanopsis sieboldii seedlings A significant interaction existed between SO 2 (quadratic) and N load (quadratic) for DM W of the seedlings at the end of the experiment (Table 1). We observed a significant quadratic response of DM W to increasing SO 2 concentration in the N50 treatment, but not in the N0 and N20 treatments (Fig. 1c). In the N50 treatment, DM W increased in response to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. There was a significant interaction between SO 2 (quadratic) and N load (quadratic) for the increment of DM R (Table 2), but SO 2 did not affect the increment of DM R in the N0 and N20 treatments. In the N50 treatment, the increment in DM R increased in response to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. These results indicated that the sensitivity of growth of C. sieboldii seedlings to SO 2 increased with increasing N load to the soil. SO 2 did not affect LA in any of the N treatments (Table 3). On the other hand, there were significant interactions between SO 2 (quadratic) and N load (linear) for A sat in current-year leaves and between SO 2 (quadratic) and N load (quadratic) for A sat in 1-year-old leaves (Table 4). In the N50 treatment, A sat showed a significant quadratic response to increased SO 2 concentration in 1-year-old leaves, but not current-year leaves. The A sat in 1-year-old leaves of the seedlings in N50 treatment increased in response to 10 ppb SO 2, but decreased in response to 20 and 40 ppb SO 2. These results suggested that, under relatively high N load, the quadratic response of whole-plant growth to increased SO 2 concentration was mainly due to the quadratic response of net photosynthetic rate in 1-yearold leaves to SO 2. There were significant interactions between SO 2 (linear) and N load (quadratic) for g s in current-year leaves and between SO 2 (quadratic) and N load (quadratic) for g s in 1-year-old leaves (Table 5). The N load significantly increased g s in current-year leaves in all the SO 2 treatments and for g s in 1-year-old leaves in the CF and 20 ppb SO 2 treatments. In contrast, N load did not significantly affect S concentration in current-year leaves in any of the SO 2 treatments (Table 6). In 1-year-old leaves, a significant interaction was observed between SO 2 (linear) and N load (quadratic) for S concentration. An N load-induced significant reduction in S concentration occurred in the CF, 10 ppb SO 2, and 20 ppb SO 2 treatments, but not in the 40 ppb SO 2 treatment. These results suggested that, in seedlings of C. sieboldii, the N load-induced increase in the sensitivity of growth and net photosynthesis to SO 2 could be explained by the N load-induced increase in the uptake rate of SO 2 in the leaves, rather than by the effect of N load on the accumulated uptake of SO 2. Larix kaempferi seedlings A significant interaction was found between SO 2 (linear) and N load (linear) for DM W at the end of the experiment (Table 1). In the N0 treatment, DM W linearly decreased with increasing SO 2 concentration (Fig. 1d). In the N20 treatment, SO 2 did not affect DM W. In the N50 treatment, DM W increased in response to 10 ppb SO 2, but decreased in response to 20 and 40 ppb SO 2. This result indicated that the sensitivity of whole-plant growth of L. kaempferi seedlings to the negative effects of SO 2 decreased with increasing N load to the soil, whereas the increment of DM R linearly decreased in all N treatments (Table 2). There were no significant interactions between SO 2 and N load for DM N and A sat (Tables 3, 4). However, we observed significant counteractive effects of SO 2 and N load on DM W (Fig. 1d). Although the A sat and DM N decreased linearly with increasing SO 2 concentration in all

10 1868 Trees (2012) 26: Table 5 Effects of SO 2 on stomatal conductance to H 2 O(g s ) in leaves or needles of seedlings of six Japanese forest tree species grown under different N loads, and F-values and levels of significance of two-way analysis of variance (ANOVA) Treatment Q. serrata (mol m -2 s -1 ) F. crenata (mol m -2 s -1 ) C. sieboldii (mol m -2 s -1 ) L. kaempferi (mol kg -1 s -1 ) P. densiflora (mol kg -1 s -1 ) C. japonica (mol kg -1 s -1 ) Gas N load Current-year 1-year old Current-year 1-year old Current-year 1-year old CF N (0.014) (0.018) (0.010) (0.018) (0.159) (0.120) (0.078) (0.112) (0.015) N (0.016) (0.055) (0.018) (0.007) (0.379) (0.200) (0.185) (0.057) (0.028) N (0.015) (0.101) (0.045) L (0.005) L (0.553) n.s (0.129) (0.031) (0.014) L (0.116) n.s. 10 ppb SO2 N (0.013) (0.023) (0.007) (0.011) (0.098) (0.217) (0.083) (0.101) (0.088) N (0.042) (0.044) (0.019) (0.015) (0.264) (0.155) (0.093) (0.038) (0.018) N (0.010) (0.082) (0.006) L (0.023) n.s (0.107) L (0.120) (0.075) (0.053) L (0.115) n.s. 20 ppb SO 2 N (0.003) (0.043) (0.011) (0.013) (0.212) (0.077) (0.245) (0.107) (0.105) N (0.037) (0.023) (0.029) (0.004) (0.468) (0.301) (0.122) (0.109) (0.046) N (0.013) (0.009) (0.011) L (0.007) L, Q (0.162) n.s (0.246) (0.117) (0.075) L (0.057) n.s. 40 ppb SO2 N (0.009) (0.034) (0.016) (0.005) (0.361) (0.172) (0.039) (0.062) (0.119) N (0.029) (0.050) (0.023) (0.027) (0.391) (0.475) (0.077) (0.129) (0.050) N (0.030) (0.060) (0.032) L (0.020) n.s (0.088) n.s (0.428) (0.143) (0.064) Q (0.031) n.s. Two-way ANOVA SO2 6.43* * 6.64* 11.33** * * Linear (L) 8.14* ** 27.40** ** ** Quadratic (Q) 11.05* ** N load * 26.59*** 17.40*** * 12.63*** 0.38 Linear (L) 6.34* 9.05** 52.75*** 34.71*** *** 0.22 Quadratic (Q) ** SO2 9 N load ** SO2 (L) 9 N load (L) * SO 2 (L) 9 N load (Q) * * 0.05 SO 2 (Q) 9 N load (L) * SO2 (Q) 9 N load (Q) ** * Values are mean of three chamber replications. Standard deviation is shown in parentheses. Variance for SO 2 treatments was partitioned into linear, quadratic, and cubic components. Variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts. When there was a significant interaction between SO 2 and N load, eight orthogonal contrasts using fixed levels of SO 2 across either linear or quadratic component of N were developed to determine the response to N load in each gas treatment L linear, Q quadratic, n.s. not significant Significance of ANOVA denoted as follows: * P \ 0.05, ** P \ 0.01, *** P \ 0.001

11 Trees (2012) 26: Table 6 Effects of SO 2 on total S concentration in leaves or needles (lmol g -1 DW) of seedlings of six Japanese forest tree species grown under different N loads, and F-values and levels of significance of two-way analysis of variance (ANOVA) Treatment Q. serrata F. crenata C. sieboldii L. kaempferi P. densiflora C. japonica Gas N load Current-year 1-year old Current-year 1-year old Current-year 1-year old CF N (4.9) 24.7 (1.2) 26.0 (12.6) 21.2 (1.2) 26.1 (11.9) 29.3 (12.1) 32.9 (1.1) 42.5 (14.0) 18.2 (0.6) N (1.3) 24.3 (4.5) 26.6 (11.6) 18.0 (1.7) 34.1 (7.4) 33.4 (13.6) 23.3 (1.0) 42.2 (10.6) 19.3 (0.9) N (5.2) 26.7 (1.6) 21.7 (15.0) 17.6 (0.7) L 30.6 (5.0) n.s (10.2) 27.2 (1.1) L, Q 45.4 (1.8) 20.1 (2.1) n.s. 10 ppb SO 2 N (0.9) 43.3 (6.9) 61.5 (20.5) 44.3 (3.6) (18.8) 48.9 (5.8) 28.5 (0.6) 73.8 (12.7) 26.9 (0.4) N (7.6) 50.8 (5.7) 54.7 (8.0) 42.6 (1.6) (3.1) 45.6 (6.3) 28.5 (1.2) 76.3 (10.6) 27.5 (0.7) N (4.4) 42.4 (1.4) 68.5 (28.8) 38.3 (2.4) L (13.1) n.s (10.3) 28.9 (1.5) n.s (13.3) 27.0 (1.6) n.s. 20 ppb SO 2 N (20.0) 97.6 (3.9) (31.0) 85.1 (5.4) (43.3) 49.1 (13.6) 31.0 (2.1) (14.6) 50.5 (4.1) N (2.4) 99.2 (7.1) (4.1) 76.7 (4.0) (22.1) 55.2 (1.6) 31.9 (1.1) (16.9) 44.8 (4.0) N50.2 (9.2) 84.7 (7.8) (13.0) 70.0 (1.2) L (4.2) n.s (3.9) 31.0 (2.5) n.s (20.2) 49.7 (9.6) n.s. 40 ppb SO2 N (26.3) (25.3) (23.1) (22.5) (89.1) 72.6 (13.8) 53.3 (0.9) (13.8) 79.6 (3.1) N (14.2) (11.5) (76.7) (13.0) (80.0) 85.5 (18.1) 53.3 (2.7) (23.5) 94.4 (3.0) N (44.9) (7.8) (47.8) (14.4) n.s (31.2) L 89.0 (10.6) 50.5 (8.6) n.s (27.6) 85.8 (3.4) Q Two-way ANOVA SO *** 676.9*** 264.3*** 148.3*** 54.3*** 81.0***.5*** 169.1*** *** Linear (L) 908.3*** *** 739.0*** 429.5*** 157.3*** 221.8*** 264.8*** 481.2*** *** Quadratic (Q) 64.0*** 224.5*** 53.0*** 15.3** * 93.1*** 24.5** 454.7*** N load * ** 16.0*** Linear (L) ** 31.0*** Quadratic (Q) * SO 2 9 N load *** * SO 2 (L ) 9 N load (L) *** SO2 (L) 9 N load (Q) * 8.9** ** SO2 (Q) 9 N load (L) *** * SO 2 (Q) 9 N load (Q) * * Values are mean of three chamber replications. Standard deviation is shown in parentheses. Variance for SO 2 treatments was partitioned into linear, quadratic, and cubic components. Variance for N load treatments was partitioned into linear and quadratic components using orthogonal polynomial contrasts. When there was a significant interaction between SO 2 and N load, eight orthogonal contrasts using fixed levels of SO2 across either linear or quadratic component of N were developed to determine the response to N load in each gas treatment L linear, Q quadratic, n.s. not significant Significance of ANOVA denoted as follows: * P \ 0.05, ** P \ 0.01, *** P \ 0.001

12 1870 Trees (2012) 26: Table 7 Mean SO 2 concentration (ppb) during experimental period corresponding to 10 % reduction in whole-plant dry mass of six Japanese forest tree species Tree species N load to soil (kg N ha -1 year -1 ) N treatments, the extent of the SO 2 -induced reduction tended to decrease with increasing N load to the soil (Tables 3, 4). These results suggested that, in L. kaempferi seedlings, the N load-induced tolerance of whole-plant growth to SO 2 could be explained by the effect of N load on the responses to SO 2 of both net photosynthesis and development of assimilative organs. We detected significant interactions between SO 2 (quadratic) and N load (linear) for g s and between SO 2 (quadratic) and N load (quadratic) for S concentration in the needles (Tables 5, 6). A significant linear reduction in the g s of needles with increasing N load was observed in the 10 ppb SO 2 treatment, but not in the CF, 20 ppb SO 2, and 40 ppb SO 2 treatments. In the 40 ppb SO 2 treatment, there was a significant N load-induced reduction in S concentration in the needles, suggesting that the N load reduced the accumulated uptake of SO 2. Therefore, the counteractive effect of SO 2 and N load on the whole-plant growth of L. kaempferi could be explained by the N load-induced reduction in the accumulated uptake of SO 2 in the needles. Pinus densiflora seedlings Q. serrata 32.5 F. crenata C. sieboldii 34.6 L. kaempferi P. densiflora C. japonica a 28.3 No significant effect of SO 2 on whole-plant dry mass a Calculation was performed by linear regression analysis using four plots pooled across N treatments, because SO 2 showed a significant main effect (linear) without significant interactions between SO 2 and N load on whole-plant dry mass At the end of the experiment, there was a significant interaction between SO 2 (quadratic) and N load (quadratic) for DM W of the seedlings (Table 1). A significant quadratic response of DM W to increased SO 2 concentration was observed in all the N treatments (Fig. 1e). In the N0 and N50 treatments, DM W was reduced by exposure to SO 2 without an SO 2 -induced increase in DM W at low concentrations. In the N20 treatment, DM W increased in response to exposure to SO 2 up to 20 ppb, and decreased at concentrations greater than 20 ppb. The extent of the reduction in DM W induced by 40 ppb SO 2, as compared to the DM W in the CF, differed among the N0, N20, and N50 treatments: 17, 20, and 21 % in the N0, N20, and N50 treatments, respectively. These results suggested that the sensitivity of whole-plant growth of P. densiflora seedlings to the negative effects of SO 2 increased with increasing N load to the soil, whereas the increment in DM R decreased linearly in all the N treatments (Table 2). We detected significant quadratic responses to increased SO 2 concentration in the DM N of current-year and 1-yearold needles in all the N treatments (Table 3). In current-year needles, the DM N increased in response to exposure to 10 and 20 ppb SO 2, but decreased in response to 40 ppb SO 2. In 1-year-old needles, DM N was reduced by the exposure to SO 2 without an SO 2 -induced increase in DM N at low concentrations. On the other hand, A sat showed a significant linear decrease in 1-year-old needles in all the N treatments and in current-year needles in N0 and N50 treatments (Table 4). In the current-year needles, the extent of the reduction in A sat in response to 40 ppb SO 2 was greater in the N50 treatment (39 %) than in the N0 treatment (11 %). These results indicated that, in P. densiflora seedlings, the SO 2 -induced reduction in whole-plant growth was mainly due to reductions in the amount of assimilative organs and the net photosynthetic rate, and the different responses of whole-plant growth to SO 2 among the N treatments were due to differences in net photosynthetic rate. The g s showed a significant decrease with increasing N load above 20 kg N ha -1 year -1 in the 1-year-old needles, but not in the current-year needles in any gas treatments (Table 5). Although N load did not affect S concentration in current-year and 1-year-old needles in the SO 2 treatments, the N load tended to increase the S concentration in the current-year needles of the seedlings exposed to 40 ppb SO 2 (Table 6). Therefore, the N load-induced increase in the sensitivity of growth and net photosynthesis of P. densiflora seedlings to the negative effects of SO 2 could be explained by the N load-induced increase in the accumulated uptake of SO 2 in the needles. Cryptomeria japonica seedlings SO 2 showed a significant main linear effect on DM W (Table 1). Because there was no significant interaction between SO 2 and N load on DM W (Table 1), we conducted linear regression analysis using four plots pooled across the N treatments (Fig. 1f). The DM W of C. japonica seedlings decreased linearly with increasing SO 2 concentration in all the N treatments (Fig. 1f). The increment of DM R linearly decreased in response to exposure to SO 2 (Table 2). These results indicated that the growth sensitivity of C. japonica seedlings to SO 2 was not affected by N load to the soil. Exposure to SO 2 significantly reduced the DM N of 1-year-old needles, but not that of current-year needles