Effects of nitrogen additions on mesophyll and stomatal conductance in Manchurian ash and Mongolian oak.

The response of plant CO2 diffusion conductances (mesophyll and stomatal conductances, gm and gsc) to soil drought has been widely studied, but few studies have investigated the effects of soil nitrogen addition levels on gm and gsc. In this study, we investigated the responses of gm and gsc of Manchurian ash and Mongolian oak to four soil nitrogen addition levels (control, low nitrogen, medium nitrogen and high nitrogen) and the changes in leaf anatomy and associated enzyme activities (aquaporin (AQP) and carbonic anhydrase (CA)). Both gm and gsc increased with the soil nitrogen addition levels for both species, but then decreased under the high nitrogen addition level, which primarily resulted from the enlargements in leaf and mesophyll cell thicknesses, mesophyll surface area exposed to intercellular space per unit leaf area and stomatal opening status with soil nitrogen addition. Additionally, the improvements in leaf N content and AQP and CA activities also significantly promoted gm and gsc increases. The addition of moderate levels of soil nitrogen had notably positive effects on CO2 diffusion conductance in leaf anatomy and physiology in Manchurian ash and Mongolian oak, but these positive effects were weakened with the addition of high levels of soil nitrogen.

Introduction

Nitrogen (N) is an important nutrient for plant photosynthesis because it alters N allocation between photosynthetic components[1], but excessive nitrogen depositions break the soil nitrogen balance and have a strongly negative effect on photosynthesis[2,3]. With global climate change, nitrogen deposition has increased dramatically worldwide[4,5], and its effects on CO2 diffusion conductance (both mesophyll and stomatal conductances, gm and gsc) in photosynthesis have attracted considerable attention in global change, physiological ecology, plant physiology and other fields[6-8]. In contrast to gsc, gm has often been neglected in previous studies, with its supply being assumed to be unlimited[9,10]; the importance of gm is only being highlighted in recent decades with the advent of advanced instruments and measuring technologies, and studies of gm have also increased correspondingly[1,11-14].

The relationship of gsc with soil nitrogen has been widely explored; gsc increased with soil nitrogen additions overall[15-17], and gm also showed a positive correlation with moderate soil nitrogen supplementation in general[1,6,7]. However, excessive nitrogen application resulted in a decrease in the ability to scavenge reactive oxygen species (ROS) in wheat[18]. Many changes under excessive nitrogen application may also occur in photosynthesis electron transport rate (Jf) or actual photochemical efficiency of photosystem II (ΦPS II). In addition, several recent papers also reported that high N conditions reduced evapotranspiration, which resulted in constraining N uptake in almond trees[18-20], and this seemed related to the slight decrease in gsc and leaf N content under high nitrogen condition. Hence, it has not been determined how gm and gsc responded to excessive nitrogen additions (≥69 kg N ha−1 a−1). Besides, the changes in leaf anatomy and associated physiological traits were both considered to be important mechanisms in determining gm and gsc[14,21-29].

Xiong et al.[14] revealed that leaves with larger gm in high nitrogen supplement had a larger leaf thickness (Tleaf) and mesophyll surface area exposed to intercellular space per unit leaf area (Smes) than those in low nitrogen supplementation, and Zhu et al.[30] also showed that Tleaf and mesophyll cell thickness (Tmes) both imposed a positive effect on gm recovery in Manchurian ash and Mongolian oak, while Smes had a negative effect on this parameter. In addition, Xiong et al.[29] and Zhu et al.[30] both revealed the determination of stomatal features (size and density) and opening status (SS) to gsc, and Zhu et al.[30] suggested a positive correlation between SS and gsc in Manchurian ash and Mongolian oak, which was also supported by the study of Xu and Zhou (2008)[31]. Besides, the changes of gm and gsc would also be significantly affected by aquaporin (AQP) and carbonic anhydrase (CA) activities[14,21]. AQP mediates the genes in the plasma membrane intrinsic protein (PIP) aquaporin family, and its increase in activity strongly promotes the expression of PIPs and improves CO2 membrane permeability[32-34]. CA regulates gm mainly by changing the dynamics of CO2 to −HCO3[28]. However, it has not been determined how leaf anatomies and AQP and CA activities would change in the Changbai Mountains with the addition of various levels of soil nitrogen.

Manchurian ash (Fraxinus mandshurica Rupr.) and Mongolian oak (Quercus mongolica Fish. ex Ledeb), two species in Oleaceae and Fagaceae, respectively, are widely distributed in the Changbai Mountains, China. Their responses of CO2 diffusion conductance to soil nitrogen additions have rarely been reported; and the mechanisms governing gm and gsc in leaf anatomy and physiology have not been determined. Hence, we measured both gm and gsc and their related anatomical and physiological traits, including leaf N content and AQP and CA activities, to explore the responses of CO2 diffusion conductance to the addition of various levels of soil nitrogen. The present study will advance our mechanistic understanding of global nitrogen deposition impacts on carbon cycling in tree species.

Results

Effects of soil nitrogen additions on gm

Considerable changes in gm were observed in both species after soil nitrogen addition compared with the control values (CK) (Fig. 1a,b). The gm gradually increased with the addition of various levels of soil nitrogen, reaching the maximum with the medium level of 46 kg N ha−1 a−1 (MN) and then decreasing with the addition of 69 kg N ha−1 a−1 (HN) while remaining greater than the gm observed for the CK group. Overall, gm showed significant differences among the four nitrogen-addition treatments in both species, in which it was significantly larger in the MN treatment than in the other treatments (P < 0.05). In addition, the gm in August was slightly lower than that in July in both species overall.

Figure 1

Changes of plant gm, gsc and An with soil N additions in Manchurian ash (I) and Mongolian oak (II) in July and August. Values were means ± SE (n = 5), and different lowercase letters (a–c) indicated significant difference at P < 0.05. CK, the control; LN, low nitrogen addition; MN, medium nitrogen addition; HN, high nitrogen addition.

Effects of soil nitrogen additions on gsc

Concurrently, soil nitrogen addition also resulted in a significant increase in gsc, with a different trend being observed between Manchurian ash and Mongolian oak (Fig. 1c,d). In Manchurian ash, gsc continued to increase with nitrogen addition in July and August, but it increased from low nitrogen (LN, 23 kg N ha−1 a−1) to MN and then decreased with the addition of HN in Mongolian oak. The gsc with the nitrogen addition treatments was significantly larger than that of the control values in July and August in both species (P < 0.05). Overall, the gsc in Manchurian ash was considerably larger than that in Mongolian oak. In addition, influenced by the changes in gm and gsc, leaf An also increased first and then decreased with the addition of progressively higher levels of soil nitrogen (Fig. 1e,f), indicating that plant photosynthetic capacities could also be considerably strengthened by soil nitrogen additions.

Effects of soil nitrogen addition on leaf anatomical characteristics

Effects on mesophyll anatomical traits

The addition of soil nitrogen caused some changes in mesophyll anatomical traits (Table 1), including leaf (Tleaf) and mesophyll cells (Tmes) thicknesses, the surface area of mesophyll cells exposed to intercellular space (Smes) and the ratio of mesophyll surface (Ames) to total leaf surface area (AT) corrects for the actual area available for CO2 diffusion (AT/Ames). Tleaf, Tmes, Smes and AT/Ames all increased after soil nitrogen addition in overall without Tleaf in HN and AT/Ames in LN treatment and were lower than the CK values. When nitrogen supplementation was no more than 69 kg N ha−1 a−1, Tleaf and Smes increased with nitrogen addition in both species, and Tmes also showed increases with the addition of soil nitrogen in Manchurian ash and reached a maximum in the MN treatment, while it reached a maximum in the LN treatment in Mongolian oak. Significant differences between nitrogen addition treatments were observed in Tleaf and Tmes, while no significant differences were shown for Smes and AT/Ames overall in both species (P < 0.05).

Table 1

Values of leaf anatomical characteristics for different nitrogen addition treatments in Manchurian ash and Mongolian oak.

	Manchurian ash						Mongolian oak
	Tleaf (μm)	Tmes (μm)	Smes (μm2 μm−2)	AT/Ames	gias (10−2 m s−1)	gliq (10−4 m s−1)	Tleaf (μm)	Tmes (μm)	Smes (μm2 μm−2)	AT/Ames	gias (10−2 m s−1)	gliq (10−4 m s−1)
CK	130.1 ± 11.0bc	116.3 ± 5.1bc	12.1 ± 2.0a	1.91 ± 0.08ab	4.47 ± 0.37d	1.08 ± 0.06b	206.8 ± 3.2bc	158.1 ± 6.7c	12.9 ± 1.3b	1.81 ± 0.11a	5.41 ± 0.33c	1.14 ± 0.06b
LN	135.9 ± 5.1b	122.9 ± 4.2ab	14.2 ± 3.8a	1.74 ± 0.11b	11.93 ± 0.79a	1.19 ± 0.14ab	213.7 ± 4.7b	185.9 ± 3.5a	13.0 ± 1.5ab	1.67 ± 0.40a	6.21 ± 0.42b	1.24 ± 0.12ab
MN	148.2 ± 7.3a	130.2 ± 2.5a	15.2 ± 2.6a	2.35 ± 0.53a	10.13 ± 0.74b	1.28 ± 0.05a	227.4 ± 6.0a	171.2 ± 8.0b	16.0 ± 2.0a	1.85 ± 0.34a	7.22 ± 0.22a	1.32 ± 0.07a
HN	122.9 ± 3.6c	112.9 ± 3.3c	18.0 ± 5.0a	2.43 ± 0.16a	8.11 ± 1.03c	1.06 ± 0.07b	202.9 ± 2.4c	167.1 ± 4.1bc	14.4 ± 1.7ab	1.78 ± 0.22a	5.76 ± 0.59bc	1.17 ± 0.04b

All data were means ± SE (n = 5). Different lowercase letters (a, b, c) indicated significant differences between nitrogen addition treatments at P < 0.05. CK, the control; LN, low nitrogen addition; MN, medium nitrogen addition; HN, high nitrogen addition.

Changes of leaf gias and gliq

Changes of Tmes and AT/Ames to soil nitrogen additions also directly caused changes in leaf gas-phase (gias) and liquid-phase CO2 diffusion conductance (gliq) inside mesophyll cells (Table 1). Both gias and gliq gradually increased with MN and then decreased under HN treatment. A significant difference between nitrogen addition treatments was observed in gias, while gliq did not change significantly overall (P < 0.05).

Effects on stomatal parameters

Correspondingly, soil nitrogen addition also caused considerable changes in stomatal parameters, mainly including stomatal pore length and width at the centre of the stoma (PL and PW), stomatal opening status (SS) and density (Ds) (Table 2). As two important components to SS, PL and PW both increased with the MN treatment and were similar in both species, but in the HN treatment, they both fell below the CK values. Consequently, leaf SS gradually increased with soil nitrogen addition and then significantly decreased to a minimum under HN treatment. Besides, significant increases also happened to leaf Ds after soil nitrogen additions (P < 0.05) (Table 2), in which Manchurian ash showed a gradually increased Ds with soil nitrogen addition, reaching the maximum under HN treatment, while Ds increased first then decreased in Mongolian oak, reaching its maximum under LN treatment.

Table 2

Values of stomatal parameters for different nitrogen addition treatments in Manchurian ash and Mongolian oak. All data were means ± SE (n = 64).

	Manchurian ash				Mongolian oak
	PL (μm)	PW (μm)	SS (μm2)	Ds (10−2 N cm−2)	PL (μm)	PW (μm)	SS (μm2)	Ds (10−2 N cm−2)
CK	7.01 ± 0.70b	3.04 ± 0.97ab	17.57 ± 1.72b	6.32 ± 0.45b	6.36 ± 0.52a	2.47 ± 0.58b	12.78 ± 1.15b	9.48 ± 0.45b
LN	7.26 ± 0.14ab	3.18 ± 0.27a	18.10 ± 1.43ab	8.28 ± 0.94a	6.53 ± 0.19a	2.54 ± 0.35b	13.06 ± 2.20b	11.89 ± 0.69a
MN	7.66 ± 0.19a	3.28 ± 0.27a	19.74 ± 2.11a	8.43 ± 1.14a	6.71 ± 0.30a	3.13 ± 0.12a	16.52 ± 1.42a	11.14 ± 0.26a
HN	7.35 ± 0.05ab	2.73 ± 0.13b	15.74 ± 0.68b	9.93 ± 0.90a	6.25 ± 0.29a	2.40 ± 0.19b	11.81 ± 1.51b	9.63 ± 0.69b

Different lowercase letters (a, b, c) indicated significant differences between nitrogen addition treatments at P < 0.05. SS, stomatal opening status; PL and PW, stomatal pore length and width at the centre of the stoma; Ds, stomatal density. CK, the control; LN, low nitrogen addition; MN, medium nitrogen addition; HN, high nitrogen addition.

Effects of soil nitrogen additions on leaf N content

Changes in leaf N content in both species during soil nitrogen additions are shown in Table 3. The leaf N content was enlarged by soil nitrogen addition and increased with medium nitrogen addition to MN and then decreased under HN treatment. Overall, no significant differences in leaf N content between soil nitrogen addition treatments were observed in both species (P < 0.05), and Mongolian oak saplings maintained a higher leaf N content in nitrogen addition treatments than did Manchurian ash saplings.

Table 3

Values of leaf N content (g kg−1) during soil nitrogen additions in July and August in both species.

	Manchurian ash		Mongolian oak
	July	August	July	August
CK	13.1 ± 0.88ab	10.4 ± 0.31b	13.0 ± 1.22b	10.2 ± 0.93b
LN	14.4 ± 0.83b	11.8 ± 0.55a	16.4 ± 3.05ab	15.7 ± 2.73a
MN	15.2 ± 0.46a	11.9 ± 0.32a	19.1 ± 1.12a	15.5 ± 0.47a
HN	15.0 ± 1.85ab	11.1 ± 1.10ab	16.0 ± 1.48ab	12.8 ± 0.93ab

All values were means ± SE (n = 3). Different lowercase letters (a, b, c) indicated significant differences between nitrogen addition treatments at P < 0.05. CK, the control; LN, low nitrogen addition; MN, medium nitrogen addition; HN, high nitrogen addition.

Effects of soil nitrogen addition on AQP and CA activities

Soil nitrogen addition also had great effects on leaf AQP and CA activities in both species (Table 4). In this study, the activities of AQP and CA were both enlarged after soil nitrogen addition, and they also gradually strengthened with soil nitrogen added to MN and then weakened with the HN treatment. Similarly, the activities of these two enzymes also did not show significant differences between the four nitrogen addition treatments overall (P < 0.05).

Table 4

Values of leaf AQP and CA activities during soil nitrogen additions in both species.

	Manchurian ash		Mongolian oak
	AQP (U g−1)	CA (U g−1)	AQP (U g−1)	CA (U g−1)
CK	5.57 ± 0.29b	1.54 ± 0.09a	5.57 ± 0.15c	1.42 ± 0.10ab
LN	5.78 ± 0.09ab	1.57 ± 0.05a	6.00 ± 0.11b	1.40 ± 0.03ab
MN	5.92 ± 0.04a	1.64 ± 0.08a	6.80 ± 0.06a	1.51 ± 0.02a
HN	5.91 ± 0.13a	1.61 ± 0.09a	6.17 ± 0.31b	1.38 ± 0.03b

All values were means ± SE (n = 3). Different lowercase letters (a, b, c) indicated significant differences between nitrogen addition treatments at P < 0.05. CK, the control; LN, low nitrogen addition; MN, medium nitrogen addition; HN, high nitrogen addition.

Discussion

Increases in gm and gsc mostly resulted from the improvement of leaf N content

Our results showed that both gm and gsc were enlarged by soil nitrogen addition; specifically, these values increased with nitrogen addition from LN to MN and then decreased with the addition of HN. Concurrently, leaf N content also presented a similar change with soil nitrogen addition (See Table 3). We believed that the increases of gm and gsc were largely related to the enlargement of leaf N content in this study because the relationships between leaf N content and gm and gsc strongly supported the promotion of leaf N content to gm and gsc increases, in which gm and gsc both showed a positive correlation with leaf N content, even though they were highly significant in Mongolian oak (P < 0.01) (Fig. 2). This finding was supported by the positive links between leaf N content and CO2 diffusion conductance (both gm and gsc) in studies of Li et al.[6] and Yamori et al.[1].

Figure 2

The correlations between gm, gsc and leaf N content in Manchurian ash (a,b) and Mongolian oak (c,d). The coefficient of correlation (r) and significant correlation were at P < 0.05 and **P < 0.01.

The positive correlation between leaf N content and gm found in the current study supported earlier observations that if leaves were treated individually, leaf N content explained 32% of measured variability in An but only 11% of that in gm, while variation in gm explained 33% of variation in An[47]. With three-quarters of leaf N associated with photosynthesis (mostly Rubisco and chlorophyll), the relationship between N and gm might simply reflect the relationship between An and gm[48]. In this study, leaf N content increased under soil nitrogen addition conditions (Table 3), this made great improvements to leaf photosynthetic capacity and gm (from CK to MN, see Fig. 1), but the excessive N supply (high N addition of 69 kg N ha−1 a−1, HN) would lower Rubisco activity and content, gm and Cc[49].

On the other hand, the release of photorespiratory CO2 occurred in the bundle-sheath mitochondria might make an enriched CO2 partial pressure in the bundle-sheath chloroplast in C3 species and result in an increased amount of CO2 to be refixed by Rubisco[50-53]. The amount of (photo)respiratory CO2 that was refixed by Rubisco can be calculated from the diffusion resistances[54]. The gm, as an apparent conductance, could also be strongly affected by respiratory and photorespiratory CO2 diffusing towards the chloroplasts from the mitochondria[54]. In this study, both CO2 compensation point in the absence of respiration (Г*) and the mitochondrial respiration in the light (Rd) differed among nitrogen addition levels in Manchurian ash and Mongolian oak (Table S2). This difference might enrich CO2 partial pressure in the bundle-sheath chloroplast in Manchurian ash and Mongolian oak, and increase the amount of CO2 refixed by Rubisco, finally making large influences on gm responding to soil nitrogen additions. However, Г* and Rd did not show significant declines under high nitrogen addition condition as other gas exchange parameters (Table S2). This might be due to the bias of the Laisk method in the estimation Γ*. The Laisk method was pointed out that what it estimated is the intercellular CO2 partial pressure (Ci*), rather than the true CO2 photocompensation point[14,54]. In other words, the Г* measured in the current study might be the Ci* actually, and this would lead to an enlarged CO2 photocompensation point under high nitrogen addition condition, but this only held under the assumption of combined resistance of chloroplast envelope and stroma (rch) being negligible. If rch made up a significant portion of mesophyll resistance (rm), the Ci* was no longer necessarily smaller than the true Г*; it might be equal to Г*, or even greater[54]. Hence, the Г* value estimated using the classical Laisk method and its response to soil N supplement might exist uncertainties under high or excessive nitrogen addition condition.

Since a positive relationship between leaf N content and CO2 assimilation was reported in numerous studies in which N increased Rubisco content and activity[1,6,55-59], the positive effect of leaf N content on gm and gsc might also be achieved, as it improved the activities of related enzymes, such as AQP and CA[28,30,33], which had been implicated in regulation of gm[48,60]. In this study, the activities of AQP and CA were strengthened by soil nitrogen addition (Table 4). This would largely promote the expression of genes greatly in the plasma membrane intrinsic protein (PIP) aquaporin family[34]. Furthermore, the diffusion of CO2 in mesophyll cells was not only a complexly physical process, but also a chemical reaction process[10]. The improvements of AQP and CA activities under nitrogen addition could stimulate the chemical reactions by improving the conversion of CO2 to −HCO3 and the membrane permeabilities to CO2 at plasma membrane, cytosol and chloroplast stroma in mesophyll cells[28,32,61]. Additionally, nitrogen addition could also promote AQP gene expressions, like the PIP2 aquaporin gene family[62,63], which benefited CO2 transmembrane transport and consequently increase gm[14]. Hence, the nitrogen addition in this study might result in an increased CO2 permeability in oocytes membranes expressing NtAQP1[64], eventually leading to the enhancements of gliq and gm.

Strong promotion of soil nitrogen addition on gm and gsc, as explained in leaf anatomy

In this study, leaf anatomical structures were adjusted by soil nitrogen additions from the changes in anatomical characteristics (See Tables 1 and 2), which exhibited notably positive effects on gm and gsc, as leaf anatomical structures were tightly correlated with gm and gsc[14,26,56,61-63]. Furthermore, as two stages in CO2 diffusion, the gas- and liquid-phase diffusional processes were largely strengthened in nitrogen addition leaves, as suggested by the increases of gias and gliq (See Table 1). However, gias and gliq in the high nitrogen addition treatment were lower than those in the low and medium nitrogen addition treatments (LN and MN) in Manchurian ash and Mongolian oak, which was in keeping with the results obtained for rice in Li et al.[6].

Both Tleaf and Tmes increased with soil nitrogen added to MN level and then decreased with the addition of HN (See Table 1); these treatments enlarged total leaf (AT) and mesophyll surface areas (Ames) for the actual areas available for CO2 diffusion (data not shown), widened CO2 flow pathways[44,56], and correspondingly enlarged gias. However, gias was often suggested to be the minor component to gm, while gliq was the major one[38,42,64]. Hence, suggested by our previous conclusion that the recovery of gm after soil rewatering was mainly resulted from the increase of gliq in Zhu et al.[30], we believed the important promotion of soil nitrogen addition on gm increase in this study would also be reached from the enlargement in gliq. The gliq is tightly related to Smes, cell wall thickness (Tcw) and the chloroplast surface facing the intercellular space per unit leaf area (Sc)[24,26,42,65]. The increase of Smes in this study (See Table 1) enlarged the touching area between CO2 and mesophyll cells and then improved the efficiency of CO2 transmembrane diffusion. Although we did not measure Tcw and Sc, the Sc should be enlarged, while Tcw decreased, by soil nitrogen addition in this study, as Sc had been revealed to increase with nitrogen supply in rice[14], and Tcw was negative with both Sc and gm[66,67]. The changes in Sc and Tcw would also strongly promote increased gm. Finally, the increase of AT/Ames caused by mesophyll anatomical changes directly resulted in the enlargement of gliq. In addition, leaf PL and PW were enlarged by soil nitrogen additions in both species (See Table 2), which resulted in the improvement of stomatal opening status (SS) and broadened CO2 diffusion pathway from leaf surface to substomatal cavities. Since Xiong et al.[29] suggested that the change of SS would affect gsc in leaf anatomy, the enlargement of SS in this study would largely promote the increase of gsc in both species.

In summary, our data showed a different effect of soil nitrogen addition levels on leaf anatomical characteristics in this study, which reached the maximum under MN treatment and decreased under the addition of HN, indicating a decreased positive effect from high soil nitrogen addition on plant metabolism. In addition, soil nitrogen addition could decrease the distance between the intercellular space and catalytic site of Rubisco (DI-R) and markedly could increase chloroplast size, finally facilitating CO2 diffusion in the liquid phase of mesophyll cells[6,68]. This phenomenon would be another important aspect to explain the effect of leaf anatomy on gm during soil nitrogen addition.

Conclusion

Soil nitrogen additions could enlarge CO2 diffusion conductance (both gm and gsc) markedly in Manchurian ash and Mongolian oak in Changbai Mountains, but its promotion was dependent on the addition levels. Moderate soil nitrogen additions (≤46 kg N ha−1 a−1) increased gm and gsc, and they reached their maximum under the addition of 46 kg N ha−1 a−1, but this positive effect was weakened with the addition of high level of 69 kg N ha−1 a−1. The effects of soil nitrogen addition on gm and gsc mostly resulted from improvements in physiological traits, such as leaf N content and the activities of AQP and CA, and adjustments in anatomical characteristics, including Tleaf, Tmes, Smes and stomatal opening status.

Material and methods

Plant material and experimental design

Five-year-old potted saplings of similar size in Manchurian ash and Mongolian oak were selected as the materials at the National Research Station of Changbai Mountain Forest Ecosystems of the Chinese Academy of Sciences located in Jilin province of northeast China (128°06′E, 42°24′N), and they were transplanted into individual pots in 2015, filled with 27 l soils collected from a broad-leaved Korean pine forest with a mean annual nitrogen forest deposition of 23 kg N ha−1 a−1. The roots in the pots were blocked from the outside soil by the pedestals placed under the pots. The volume of the pots was 29.28 L with a height of 30.0 cm and a diameter of 34.3 cm.

Four nitrogen addition levels were used to simulate nitrogen deposition intensities with the addition of no nitrogen (CK), low nitrogen (LN, 23 kg N ha−1 a−1), medium nitrogen (MN, 46 kg N ha−1 a−1) and high nitrogen (HN, 69 kg N ha−1 a−1). Urea solutions with different concentrations of nitrogen were sprayed into the pots once every other month from May to October in 2017. Five replicates were designed for each treatment, and all potted saplings were thoroughly watered daily to avoid water deficit. All measurements were carried out during summer growth season (July and August) to explore the changes in effects of soil nitrogen addition on gm and gsc.

Simultaneous gas exchange and chlorophyll fluorescence measurements

We simultaneously measured gas exchange and chlorophyll fluorescence on newly and fully expanded, sun-exposed leaves using an open-flow gas exchange system (Li-6400XT; Li-Cor Inc., Lincoln, NE, USA) equipped with an integrated fluorescence leaf chamber (Li-6400-40; Li-Cor). Leaves were fully light adopted under a saturated photosynthetic active photon flux density (PPFD) of 1200 µmol m−2 s−1 provided by Li-6400 with a 10: 90 blue: red light for 30 minutes, while leaf temperature and relative humidity and CO2 concentration in the leaf chamber were maintained at 25 °C and 60 ± 5% and 400 µmol CO2 mol−1 with a CO2 mixture, respectively. In addition, the gas flow rate was controlled at 300 μmol s−1 to ensure the adequate gas exchange. After stabilization to a steady state, gas exchange parameters, steady-state fluorescence (F) and maximum fluorescence (F′) were recorded.

The actual photochemical efficiency of photosystem II (ΦPS II) was calculated according to Genty et al.[35]:

The photosynthesis electron transport rate (Jf) was calculated as follows:where α is the total leaf absorptance and β is the partitioning of absorbed quantum between PS II and PS I.

In this study, light response curves (An-PPFD curve) for controlled and nitrogen-addition saplings were also measured under a low O2 concentration (<1%) condition to correct the αβ, as it was equal to the slope of the relationship between ΦPS II and 4ΦCO2 (the quantum efficiency of CO2 fixation)[36]. Values of αβ for different nitrogen treatments were shown in Table S1.

The mesophyll conductance (gm) was calculated using the ‘variable J method’ described in Harley et al.[37]:where An is the net photosynthetic rate, Ci is the intercellular CO2 concentration, and these values were directly obtained from gas exchange measurements; Г* represents the CO2 compensation point in the absence of respiration and Rd represents the mitochondrial respiration in the light.

Г* and Rd were measured using the Laisk method, namely, 5 initial slopes of An-Ci curves under low light and low CO2 concentrations were measured for Г* and Rd estimations in this study[38,39]. In theory, three CO2 response curves obtained by varying CO2 concentrations from 150 to 40 µmol CO2 mol−1 under three PPFDs (150, 100 and 50 µmol m−2 s−1) would intersect with each other at a point, and the intersection point at x-axis and y-axis were considered to Г* and Rd, respectively. But in practice, these three linear regressions of the intersected An-Ci curves formed a triangle range rather than a single point[40] (Fig. S1), and the Г* and Rd were calculated as the barycenter of the triangle formed by the intersection of the three lines at x-axis and y-axis, respectively, according to our previous published method[40]. The values of Г* and Rd during soil nitrogen additions were shown in Table S2.

Stomatal conductance to CO2 (gsc, mol CO2 m−2 s−1) was calculated from the ratio of stomatal conductance to water (gsw, mol CO2 m−2 s−1) to 1.6 (i.e., gsc = gsw/1.6), as gsw was 1.6 times larger than gsc[41].

Measurement of leaf anatomical characteristics

After gas exchange measurements, we cut fifteen small leaf samples (4.0 mm × 1.5 mm) from five replicated leaves per treatment and fixed them in FAA (alcohol: formaldehyde: glacial acetic acid = 90: 5: 5) to measure leaf anatomical characteristics, mainly including leaf thickness (Tleaf, μm), mesophyll and stomatal anatomical characteristics, such as the thickness of mesophyll cells between the two epidermal layers (Tmes, μm) and the surface area of mesophyll cells exposed to intercellular space per unit leaf area (Smes, μm2 μm−2) calculated using formula (4), stomatal pore length (PL, μm) and width (PW, μm) at the centre of the stoma, which were detailed described in Zhu et al.[30]. Besides, we counted the number of stomata per unit leaf area to calculate the stomatal density (Ds, N cm−2).where the length of mesophyll cells exposes to intercellular space (Lmes, μm) and the cross-sectional width (W, μm) are measured with Image J software. The curvature correction factor (F) was measured using the method described in the study of Evans et al.[42] and Thain (1983)[43], which was shown in Table S3.

We also calculated the gas- and liquid-phase CO2 diffusion conductance in mesophyll cells (i.e., gias and gliq) with leaf anatomical characteristics according to formulas (5) and (6) to assess their relationships with gm.where Da (m2 s−1) is the diffusion coefficient for CO2 in the gas phase, ΔLias (μm) is taken as half Tmes[26,44], ϛ is the diffusion path tortuosity (m m−1) and fias (%) is the fraction of mesophyll volume occupied by the intercellular airspace. gcw, gpl, gct, gen and gst are the partial conductance for the cell wall, plasmalemma, cytosol, chloroplast envelope and chloroplast stroma (m s−1), respectively. We used an estimation of 0.0035 m s−1 for the gpl and gen according to the previous studies[42,45], and estimated gcw, gct and gst using the formula of in the studies of Tomás et al.[26] and Niinemets and Reichstein (2003)[44], where gi (m s−1) is either gcw, gct or gst, rf is the dimensionless coefficient, Dw (m2 s−1) is the aqueous-phase volatile diffusion coefficient for CO2 (1.79 × 10−9 m2 s−1 at 25 °C), ΔLi (m) is the diffusion path length, and p (m3 m−3) is the effective porosity. According to the previous studies of Tomás et al.[26] and Niinemets and Reichstein (2003)[44], rf was valued as 1 for cell wall, and an estimate of rf of 0.294 for gct and gst in this study, p was taken as 1 for gct and gst, and 0.3 for cell walls, ΔLi was valued as 5.0 × 10−7 (for cell wall, ΔLcw), 9.7 × 10−8 (for cytosol, ΔLct) and 1.65 × 10−6 (for chloroplast stroma, ΔLst). Ames (μm2) and AT (μm2) are mesophyll surface area and total leaf surface area corrected for the actual area available for CO2 diffusions, respectively, calculated from the light microscope.

Measurement of leaf N content

Leaves were picked and over-dried at 75 °C for 24 h to constant weight during gas exchange measuring periods and then ground using a mixer oscillating mill homogenizer (MM400, Retsch, Germany). Approximately 5.0 mg leaf samples were taken to measure leaf nitrogen content per area using a C N element analyser (Elementar vario MACRO, Element, Germany).

Measurements of leaf aquaporin and carbonic anhydrase activities

For analysing the physiological mechanism of gm and gsc responses to soil nitrogen additions, we sampled fifteen fresh leaves per treatment to measure the activities of aquaporin (AQP) and carbonic anhydrase (CA) using enzyme-linked immunosorbent assay (ELISA)[46,47]. Solid-phase antibody was made using purified plant AQP (or CA) antibody. Then, combined with antibody labelled with horseradish peroxidase (HRP), AQP (or CA) was added to microtiter plate wells to become an antibody-antigen-enzyme-antibody complex. This complex became blue with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate solution after complete washing. The optical density (OD) values were measured spectrophotometrically at a wavelength of 450 nm to compare with the standard curves to determine the activity of AQP (or CA) in the samples.

Statistical analysis

SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for one-way statistical analysis of normality and homogeneity of variance between nitrogen addition treatments (one-way ANOVA). Furthermore, regression analysis between gm and leaf N content was also performed. Mean values were compared using the least significant difference (LSD) multiple comparison test at the 0.05 and 0.01 probability levels (P < 0.05 and P < 0.01) with Tukey′s honest significant difference (HSD) test.

Supplementary information.