Anatomically induced changes in rice leaf mesophyll conductance explain the variation in photosynthetic nitrogen use efficiency under contrasting nitrogen supply

Background The ratio of CO2 mesophyll conductance (gm) to Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) content has been suggested to positively affect photosynthetic nitrogen use efficiency (PNUE). The anatomical basis of gm has been quantified, but information on the relationship between cell-level anatomies and PNUE is less advanced. Here, hydroponic experiments were conducted in rice plants supplied with ammonium (NH4+) and nitrate (NO3−) under three N levels (low, 0.71 mM; intermediate, 2.86 mM; high, 7.14 mM) to investigate the gas exchange parameters, leaf anatomical structure and PNUE. Results The results showed a lower PNUE in plants supplied with high nitrogen and NH4+, which was positively correlated with the gm/Rubisco ratio. A one-dimensional within-leaf model revealed that the resistance to CO2 diffusion in the liquid phase (rliq) dominated the overall mesophyll resistance (rm), in which CO2 transfer resistance in the cell wall, cytoplasm and stroma were significantly affected by nitrogen supply. The chloroplast surface area exposed to intercellular space (Sc) per Rubisco rather than the gm/Sc ratio was positively correlated with PNUE and was thus considered a key component influencing PNUE. Conclusion In conclusion, our study emphasized that Sc was the most important anatomical trait in coordinating gm and PNUE with contrasting N supply. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-020-02731-7.


Background
Photosynthetic nitrogen use efficiency (PNUE), determined as the ratio of photosynthesis rate (P n ) to leaf organic nitrogen content [1], is a key component of nitrogen use efficiency (NUE) and an indicator of the relationship between leaf nitrogen (N) and P n . Under the present atmosphere, the unsaturated CO 2 concentration in C3 leaves influences the carboxylation of Ribulose-1,5-disphosphate (RuBP) and results in a finite P n , which fails to match the increase in leaf N and induces a decrease in PNUE [2]. By using an "evolutionary" algorithm, the partitioning of photosynthetic enzymes was altered based on a fixed total amount of protein-nitrogen for maximizing P n , and the result showed that an increase in Ribulose-1, 5-bisphosphate carboxylase/ oxygenase (Rubisco) was required to maximize P n [3]. It was also well documented that higher leaf N allocation into Rubisco was linked with an enhancement in PNUE [4].
Numerous studies have clarified that the enhancement in Rubisco activity is another favorable candidate for improving RuBP carboxylation efficiency and P n because of its poor catalytic ability under ambient conditions due to the low CO 2 concentration and the low affinity for CO 2 [1,5]. As the substrate of Rubisco, CO 2 concentration in the chloroplast (C c ), which is determined by stomatal conductance (g s ) and mesophyll conductance (g m ), plays a dominant role in regulating Rubisco activity [6,7]. It has been demonstrated that g m induces 40% of the total decrease in CO 2 concentration between the atmosphere and the carboxylation sites of Rubisco [8]. In a previous study, Li et al. [5] argued that an increase in g m was not sufficient to meet the carboxylation demand of the increased Rubisco content and eventually resulted in a decreased PNUE. Therefore, it is speculated that factors affecting g m would influence Rubisco activity and the relationship between P n and leaf N content.
Evidence is now mounting that g m is largely dependent on leaf anatomical characteristics, including leaf thickness, cell wall thickness and chloroplast morphology [9,10]. Higher leaf density and thicker mesophyll cell walls contribute to a reduction in g m [9,[11][12][13][14], and mesophyll and/or chloroplast surface areas exposed to the intercellular space, S mes and S c , respectively, are positively correlated with g m [15]. The overall importance of different anatomical traits in the restriction of g m varies [16]. For gymnosperms, the strongest sources of g m are cell wall and chloroplast thickness, variation in chloroplast shape and size, and S c [9]. In lycophytes and bryophytes, the highest CO 2 diffusive resistance is mainly driven by extremely high cell wall thickness and low S c [17]. Even though the anatomical factors influencing g m have been widely studied, the role of these anatomical factors in influencing PNUE and their relative contribution in rice plants are still largely unknown.
Leaf anatomy is remarkably influenced by N nutrition; for example, decreasing leaf thickness and smaller chloroplasts with no starch granules have been detected in nitrogendeficient leaves, while high-N leaves have more large chloroplasts with well-developed grana, stroma lamellae and starch granules per mesophyll cell [5,[18][19][20]. For different nitrogen forms, increased leaf thickness and a doubling of chloroplast volume with a larger internal membrane length have been found in NH 4 + -fed plants compared with NO 3 − -fed plants [21][22][23]. In this study, we examine the responses of leaf anatomical characteristics, including leaf thickness, mesophyll cell size, chloroplast length and thickness, chloroplast number per mesophyll cell under NH 4 + and NO 3 − nutrition with different N levels; moreover, we discuss the implications for understanding leaf trait variation with changes in N nutrition along the PNUE. Our objectives of the present study were as follows: (1) Fig. 1a, b). The PNUE was 21, 17 and 14% higher in LNN, MNN and HNN than in LAN, MAN and HAN, respectively (Table 1). Positive correlations existed between PNUE and both the C c /Rubisco ratio and the g m /Rubisco ratio (Fig. 1c, d).

Effects of nitrogen supply on leaf anatomical properties
With increasing leaf N supply levels, leaf thickness (T L ) and mesophyll cell thickness (T m ) increased in NH 4 + -fed plants but decreased in NO 3 − -fed plants ( Supplementary  Fig. S1, Fig. 2a, b). Leaf dry mass per area (M A ), leaf density (D L ) and mesophyll cell wall thickness (T mc ) were increased by high N supply either with NH 4 + or NO 3 − , and were lower under NO 3 − nutrition than under NH 4 + nutrition. Mesophyll surface area exposed to intercellular airspace (S mes ) and the chloroplast surface area facing intercellular airspace (S c ) were upregulated significantly by increasing the nitrogen supply level (Fig. 2b). The S mes increased by 22-37 and 21% under intermediate and high N supply conditions in NH 4 + and NO 3 − nutrition, respectively, and the corresponding S c increased by 22-38% and 21-24%, both compared with their respective low N supply conditions. No obvious differences in S c between NH 4 + and NO 3 − under low N levels were observed, but the S c decreased by 11 and 20% under MNN and HNN, respectively, compared to that under MAN and HAN (Fig. 2b).
We further analyzed the chloroplast number per mesophyll cell (N c ), chloroplast length (L c ), chloroplast thickness (T c ), chloroplast surface area (Sur c ), chloroplast volume (Vol c ) and chloroplast section area (Sec c ).

Anatomical limitations of mesophyll conductance
The values of g m calculated according to the methods of Harley et al. [24] and Tomas et al. [16] were strongly positively linearly correlated ( Supplementary Fig. S2, R 2 = 0.936). Further quantitative analysis showed that both the resistance in the gas phase (r ias ) and proportion of gasphase limitations (l ias ) of g m had little impact on the overall mesophyll resistance (Fig. 3), and that the liquid phase resistance (r liq ) was responsible for the limited g m majority, among which stroma played a dominant role. High N supply significantly increased the resistance in the cell wall (r cw ) and stroma (r st ); compared with low N supply, g m limited by the stroma (l st ) was increased by 9-10% under moderate N supply and by 9-13% under high N supply (Fig. 3b). Consistent with the absolute cytoplasm resistance, g m limited by the cytoplasm (l cyt ) and cell wall (l cw ) were downregulated under high N supply and NO 3 − nutrition, respectively (Fig. 3). Among all the components, r cw was the primary component affected by N forms and was 19, 23 and 16% higher under NH 4 + nutrition than under NO 3 − nutrition in low N, intermediate N and high N supply, respectively (Fig. 3a).

Discussion
Effects of N supply on the g m /Rubisco ratio and photosynthetic nitrogen use efficiency (PNUE) Decreased PNUE under high N supply has been reported in previous and present studies ( Table 1, Fig. 1) [5,[25][26][27]; referring to N forms, higher PNUE under NO 3 − nutrition than NH 4 + nutrition in the present study is consistent with results in barley (Hordeum vulgare L.) [28], pine [29], and cucumber [30]. Leaf nitrogen allocation is an important factor influencing PNUE. Onoda et al. [31] indicated that a higher fraction of photosynthetic nitrogen in electron transport and Rubisco would contribute to increased PNUE in leaves with lower dry mass per area (M A ), while in leaves Table 1 Effects of different nitrogen supply levels on rice biomass (g), leaf area (cm 2 ), leaf nitrogen content (N L , g m − 2 ), Rubisco content (g m − 2 ), stomatal conductance (g s , mol CO 2 m − 2 s − 1 ), mesophyll conductance (g m , mol CO 2 m − 2 s − 1 ), chloroplast CO 2 concentration (C c , μmol mol − 1 ), photosynthesis rate (P n , μmol m − 2 s − 1 ), and photosynthetic nitrogen use efficiency (PNUE, μmol CO 2 mmol − 1 N s − 1 ). Rice plants ("Zhendao 11") were supplied with NH 4 + (AN) or NO 3 − (NN) under 3 different amounts, low N (0.71 mM, LAN and LNN), intermediate N (2.86 mM, MAN and MNN), and high N (7.14 mM, HAN and HNN). Data are presented as the means ± SD of four replications. Significant differences (P < 0.05) between treatments are indicated by different letters with higher M A , the over-investment of nitrogen in photosynthetic nitrogen and/or cell walls would reduce PNUE [1]. The effect of the proportion of Rubisco in leaf N content on PNUE can be expressed based on Eq. (6): Our study detected that the Rubisco allocation ratio was increased under high nitrogen supply but decreased under NO 3 − nutrition compared with NH 4 + nutrition; however, the portion of Rubisco in leaf N content was not associated with PNUE (Fig. 4a). These results implied that Rubisco activity, rather than its content, played a dominant role in regulating PNUE [1,5,32].
An increased Rubisco allocation ratio requires increased CO 2 partial pressure at the carboxylation site (C c ) to meet carboxylation demands; however, the extent of the increase in C c was less than that in Rubisco content, which resulted from the finite stomatal conductance (g s ) and mesophyll conductance (g m ). Li et al. [5] demonstrated that the smaller increases in g m relative to Rubisco content resulted in relatively lower CO 2 levels in chloroplasts and PNUE (Fig. 1c, d), which implied that the g m /Rubisco ratio rather than the absolute value of g m was the key factor that regulates PNUE. We further compared the gap between estimated g m and C c proposed by Harley et al. [24] (Eq. 5, 6) and theoretical C c (C c-Theoretical ) and g m (g m-Theoretical ), which were calculated as follows based on Ding et al. [27], to evaluate the equilibrium state of g m and Rubisco under different N nutrition conditions: Fig. 1 The relationship between leaf N content (N L ) and the photosynthetic rate (P n ) (a) and photosynthetic nitrogen use efficiency (PNUE) (b) and the relationship between PNUE and the ratio of chloroplast CO 2 concentration to Rubisco (C c /Rubisco) (c) and the ratio of mesophyll conductance to Rubisco (g m /Rubisco) (d). Each point represents one replicate (four replicates per treatment). The lines represent the following regression equations: a y = − 1.8756 × 2 + 11.3310x + 13.0290, R 2 = 0.6228, P < 0.05; b y = − 0.0763x + 0.3565, R 2 = 0.7886, P < 0.05; c y = 0.0017x + 0.0774, R 2 = 0.8295, P < 0.01; d y = 1.5663x + 0.0733, R 2 = 0.4215, P < 0.01 As shown in Fig. 5, both theoretical and estimated C c and/or g m , as well as the differences between them, increased obviously with increasing leaf N content, and the gap between theoretical and estimated C c and/or g m under NH 4 + nutrition was larger than that under NO 3 − nutrition. These results confirmed that the balance between Rubisco content and C c and/or g m was weaker when high N and NH 4 + were supplied, and the relatively lower C c failed to meet the carboxylation demands of the increased Rubisco content, resulting in decreased PNUE (Fig. 1c, d).
Overall importance of leaf anatomy in determining g m and PNUE When leaf nitrogen content was expressed on a leaf dry mass basis, no significant differences in leaf N content between NH 4 + and NO 3 − nutrition were obtained. Therefore, the discrepancies in PNUE between different N forms were primarily caused by the difference in M A (Fig. 2), which resulted from leaf anatomy characteristics such as leaf density (D L ), leaf thickness (T L ) and cell wall thickness (T mc ). In NO 3 − -fed plants, the lower M A was the ultimate result of lower D L and T mc . However, a lower M A was not always related to a lower g m , as observed in the present study; Hassiotou et al. [8] found a negative relationship between M A and g m in the range of 100-500 g m − 2 of M A , while Hanba et al. [33] clarified a positive relationship in the same range of M A . These contrasting results were explained by different ways to , mesophyll surface area exposed to intercellular airspace. S c (μm 2 μm − 2 ), chloroplast surface area exposed to intercellular airspace. N c , chloroplast number per mesophyll cell. L c (μm), chloroplast length. T c (μm), chloroplast thickness. Sec c (μm 2 ), chloroplast section area. Vol c (μm 3 ), chloroplast volume. Sur c (μm 2 ), chloroplast surface area. The error bars indicate the standard deviation and at least 15 replicates were conducted for each parameter enhance M A , as the increases in D L and T L were associated with higher g m , while the opposite conclusion would be obtained if the increase in M A was a result of a thickened cell wall [8]. Our positive correlation between M A and g m implied that the contributions of D L and/or T L compensated for the inhibitory effect of T mc on g m .
To qualify the relative importance of each leaf anatomy trait in explaining g m , a one-dimensional within-leaf model was calculated to clarify the limitation of g m in each process [16]. The results showed that more than 90% of the total limitation of g m came from L liq , which was a consequence of limitation in the cell wall (L cw ), plasma membrane (L pl ), envelope (L en ), cytoplasm (L cyt ), and stroma (L st ) (Fig. 3) [34]. The decreased contribution of cytoplasmic resistance to g m under high N resulted from the decreased distance between adjacent chloroplasts, rather than the distance between the cell wall and chloroplasts [16], and the increase in chloroplast thickness (T c ) extended the transport path for CO 2 from the chloroplast membrane to the carboxylation site in the interior of chloroplasts and resulted in an increasing in r st [35,36]. Except for the resistance of each part, the chloroplast surface area exposed to intercellular airspace (S c ) was a paramount factor affecting CO 2 liquid resistance (r liq ). However, the decreased r cyt and increased S c partially compensated for the increased r st under high N supply and resulted in an increased g m , although the effects were weak and did not match the increase in Rubisco content.
Considering the dominant role of S c in determining g m , the g m /Rubisco in Eq. (1) was replaced by the product of g m /S c and S c /Rubisco to demonstrate the effect of leaf anatomies and g m on PNUE, which can be expressed as follows: According to the formula above and Terashima et al. [37], a positive correlation would be summarized between PNUE and g m /S c , which emphasized the potential role of leaf anatomical characteristics except for S c , as well as the activity of carbonic anhydrase (CA) in contributing to PNUE [37]. However, the weak negative relationship between g m /S c and PNUE and the significant positive correlation between the S c /Rubisco ratio and PNUE suggested the dominant role of S c in influencing PNUE. Due to the limited knowledge of the relationship between PNUE and the S c /Rubisco ratio, Onoda et al. [14] did not take this component into consideration when they analyzed the physiological and structural tradeoffs underlying the leaf economics spectrum, and they argued that S c per Rubisco may not correlate strongly with M A or PNUE. However, we detected that S c per Rubisco was a critical parameter associated with PNUE in rice plants supplied with different N nutrition levels. Similar results were well documented in a review by Terashima et al. [15] and Terashima et al. [37], in which they speculated that, from the perspective of Rubisco and nitrogen use efficiency, thicker leaves with larger S c were advantageous because the increased ratio of S c to Rubisco would increase chloroplast CO 2 concentration. The increased S c /Rubisco ratio in NO 3 − and high N-fed plants partially resulted from the lower leaf density, which allowed more chloroplast surface area to be exposed to intercellular airspace (Fig. 2a).

Conclusions
In conclusion, we demonstrated that PNUE is decreased in rice plants supplied with high N and ammonium nutrition, which results from unbalanced increases in g m and Rubisco content. Nitrogen-induced variation in g m is associated with leaf anatomical traits, especially chloroplast surface area exposed to intercellular airspace (S c ). We further concluded that the S c /Rubisco ratio is directly related to the response of PNUE to N supply and that its increase is advantageous to the increase in PNUE. Fig. 4 The relationship between photosynthetic nitrogen use efficiency (PNUE) and the ratio of Rubisco to leaf N content (Rubisco/N L ) (a), the difference between intercellular CO 2 concentration and chloroplast CO 2 concentration (C i -C c ) (b), the ratio of mesophyll conductance to chloroplast surface area exposed to intercellular airspace (g m /S c ) (c), and the ratio of chloroplast surface area exposed to intercellular airspace to Rubisco (S c /Rubisco) (d). Data represent the mean of 4 replicates for PNUE, Rubisco, N L , C i , C c , g m and at least 15 replicates for S c . The line in the figure represents the following regression equation: y = 0.0299x -0.0019, R 2 = 0.9700, P < 0.01

Plant material and growth conditions
Rice seeds (Oryza sativa L., ssp. japonica inbred, cv. 'Zhendao 11') were purchased from Mingtian Seed Company (Nanjing, China), disinfected with 10% H 2 O 2 for 30 min and germinated in 2.0 mM CaSO 4 at 25°C. The rice seedlings were transferred to 6 L rectangular containers (30 × 20 × 10 cm) when the seedlings developed 2.5 visible leaves, and one quarter-strength mixture of NH 4 + and NO 3 − nutrient solution (for composition, see below) was supplied. Three days later, the seedlings were transferred to a one half-strength nutrient solution. After 6 days, the seedlings were supplied with full-strength nutrient solution for 1 week, after which the seedlings were supplied with either (NH 4 ) 2 SO 4 (AN) or Ca (NO 3   . The nutrient solutions were changed every 3 days, and the pH was adjusted to 5.50 ± 0.05 each day with 0.1 M HCl or NaOH. All the treatments were replicated 5 times with a completely randomized design. The temperature in the greenhouse was maintained at 30°C during the day and 18°C at night. Light was supplied by SON-TAGRO 400 W bulbs, and the distance between the light and the rice plants was approximately 60 cm. The light intensity was maintained at a minimum of 1000 μmol photons m − 2 s − 1 at the leaf level using a 14-h photoperiod.

Measurement of biomass and leaf N content
After all the measurements were completed, plant dry weight was determined after oven-drying at 105°C for 30 min and then at 70°C to a constant weight. Pictures of the leaves used for the measurement of P n were taken with a camera along with a benchmark to calibrate, and the leaf area was obtained by ImageJ Pro Plus, after which the leaves were dried and digested with H 2 SO 4 -H 2 O 2 at 260-270°C. The leaf N concentration was determined using a digital colorimeter (AutoAnalyzer 3; Bran+Luebbe).

Gas exchange measurements
Twenty days after treatments, a Li-Cor 6400 infrared gas analyzer was used for the simultaneous measurement of light-saturated photosynthesis (P n ) and chlorophyll fluorescence on the newly expanded leaves from 9: 00 to 15:00. Leaf temperatures were 25°C, the relative humidity was 45%, and photosynthetic photon flux density (PPFD) was 1500 μmol m − 2 s − 1 for all measurements. After equilibration to a steady state, P n was recorded and the photosynthetic nitrogen use efficiency (PNUE) was calculated as the ratio of P n to the leaf nitrogen content per leaf area. The fluorescence (F s ) was also measured simultaneously, and a 0.8 s saturating pulse of light (approx. 8000 μmol m − 2 s − 1 ) was applied to measure the maximum fluorescence (F m ′). The efficiency of photosystem II (Φ PSII ) was calculated as Φ PSII = 1-F s /F m ′. The total electron transport rate (J T ) was calculated as J T = Φ PSII × PPFD×α leaf × β, where α leaf and β were leaf absorption and the proportion of quanta absorbed by photosystem II, respectively. In this study, α leaf was also assumed to be 0.85, and β was assumed to be 0.5 [38]. The following equations proposed by Harley et al. [24] were used to calculate the CO 2 mesophyll conductance (g m ) and chloroplast CO 2 concentration (C c ): where C i is the intercellular CO 2 concentration, Γ* is the CO 2 compensation point and R d is the mitochondrial respiration rate in the light. In the present experiment, Γ* and R d were measured on newly expanded leaves according to the method of Li et al. [39]. PPFDs in the cuvette were controlled as the series of 150, 300, and 600 μmol m − 2 s − 1 . At each PPFD, the ambient CO 2 concentration in the cuvette was adjusted as the series of 25, 50, 75 and 100 μmol CO 2 mol − 1 . Thirty minutes prior to initiating measurements, leaves were placed in the cuvette with a PPFD of 600 μmol photons m − 2 s − 1 and a C a of 100 μmol CO 2 mol − 1 .

Anatomical analysis
For the anatomical analysis, approximately 1-2 mm 2 leaf sections were cut and fixed in FAA (95% ethanol: glacial acetic acid: formalin: distilled water = 10:1:2:7), dehydrated in ethanol series, and embedded in paraffin. After cutting into 6 μm transverse sections with a microtome and mounting on glass, the glass was stained with Safranin O and fast green and then mounted in DPX mounting medium. Images of each section were obtained with a light microscope (BX 53, Olympus) with a CCD camera (eXcope X3, DIX, Korea). Leaf thickness (T L ), mesophyll thickness (T m ), leaf density (D L ), and the volume fraction of intercellular air space (f ias ) were measured and/or calculated from at least 5 sections from four different leaves, and at least 5 different fields of view were observed for a given section of images. D L and f ias were calculated as: where M A is the specific leaf weight (g m − 2 ), ΣS m is the total sectional area of mesophyll cells, and W is the width of the section.
For the transmission electron microscope (TEM) analysis, approximately 1-2 mm 2 leaf sections were cut from the middle of newly expanded leaves using two razor blades, fixed in 2.5% glutaraldehyde (0.1 mol L − 1 phosphate buffer, pH 7.0) and postfixed with 2% osmium tetroxide. Specimens were dehydrated in a graded acetone series and embedded in Epon 812. Ultrathin crosssections of 90 nm for transmission electron microscopy (TEM) were cut with a Power Tome-XL ultramicrotome, stained with 2% uranyl acetate, and examined with an H-7650 transmission electron microscope. For each sample, 15 cross-sections were chosen to measure mesophyll cell wall thickness (T mc ) and total length of the mesophyll cells (L mes ) and chloroplasts (L ch ) facing the intercellular air space. At least 40 chloroplasts from TEM were observed to measure the chloroplast traits, including chloroplast length (L c ), chloroplast thickness (T c ), chloroplast section area (Sec c ), distance between two neighbor chloroplasts (ΔL chl ), and chloroplast distance from the cell wall (ΔL cyt ). The surface area of mesophyll cells to the intercellular air-spaces (S mes ), the surface area of chloroplasts exposed to intercellular airspace (S c ), the chloroplast surface area (Sur c ) and volume (Vol c ) were calculated by using the following formula: where W is the width of the measured section, and F is the curvature correction factor and taken as 1.55 [40].
where a = L c /2, and b = T c /2. The chloroplast number per mesophyll cell (N c ) was determined according to the method of Pyke [41]. Briefly, the leaves were cut into 1-5 mm widths with a scalpel or razor blade, submerged in 3.5% (v/v) glutaraldehyde in a tube and kept in the dark at room temperature for 1 h. The glutaraldehyde solution was then replaced with 0.1 M Na-EDTA (pH 9), and the leaf discs were heat-blocked at 60°C for 12 h and incubated overnight in the dark at 4°C. To view chloroplasts in individual cells, a piece of tissue was removed from the tube with fine forceps and placed on a microscope slide in a drop of water. A scalpel handle was used to tap and macerate the tissue fairly vigorously, and a Leica DM2700 M microscope with DIC/Nomarski optics was used to image and count chloroplast numbers with changing focus to avoid duplicate and uncounted chloroplasts (Fig. S3).

The qualification of the anatomical limitations of mesophyll conductance
The one-dimensional gas diffusion model of Tomas et al. [16] was applied in our present study to determine the anatomical limitations of mesophyll conductance, which was given as: where g ias and g liq are the gas phase conductance and liquid phase conductance, respectively. R is the gas constant (8.31 Pa m 3 K − 1 mol − 1 ), T k is the absolute temperature, and H is Henry's law constant (2943.3 Pa m 3 K − 1 mol − 1 for CO 2 ). The g ias , was calculated as: where r ias is the resistance of the gas phase to CO 2 , D a is the diffusion coefficient for CO 2 in the gas phase and is set to 1.51 × 10 − 5 m 2 s − 1 at 25°C, f ias is the volume fraction of intercellular air space, ΔL ias was taken as half of the mesophyll thickness, and ς is the diffusion path tortuosity (1.57 m m − 1 ).
The g liq was determined by different components in the cell, including the conductance in the cell wall (g cw ), plasma membrane (g pl ), cytosol (g cyt ), chloroplast envelope (g en ), and stroma (g st ). Eventually, g liq was calculated as: g liq ¼ S c r cw þr pl þr cyt þr en þr st À Á ð15Þ where r cw , r pl , r cyt , r en , r st are the reciprocal terms of g cw , g pl , g cyt , g en and g st , respectively. We used an estimate of 0.0035 m s − 1 for the g pl and g en as Tomas et al. [16] suggested. In addition, g cw , g cyt , and g st were calculated as: where i stands for cell wall, cytosol, or stroma conductance. r f,i accounted for the reduction in the aqueous phase diffusion coefficient for CO 2 (D w , 1.79 × 10 − 9 m 2 s − 1 at 25°C) and was taken as 1.0 for cell walls and 0.3 for cytosol and stroma, respectively. p i was the effective porosity (m 3 m − 3 ) and was taken as 1.0 for the cytosol and stroma and 0.28 for the cell walls. ΔL i (m) is the diffusion path length in the corresponding component of the diffusion pathway.
The proportion of g m determined by limited gas-phase conductance (l ias ) was calculated as: The share of g m by different components of the cellular phase conductance (l i ) was determined as: Statistical analysis One-way ANOVA was applied to assess the differences in each parameter among the treatments with the SPSS 16.0 statistical software package. Significant differences (P < 0.05) among treatments are indicated by different letters using the least significant difference test.