1. The influence of leaf thickness on internal conductance for CO2 transfer from substomatal cavity to chloroplast stroma (gi) and carbon isotope ratio (δ13C) of leaf dry matter was investigated for some evergreen tree species from Japanese temperate forests. gi was estimated based on the combined measurements of gas exchange and concurrent carbon isotope discrimination.
2. Leaves with thicker mesophyll tended to have larger leaf dry mass per area (LMA), larger surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area (Smes) and smaller volume ratio of intercellular spaces to the whole mesophyll (mesophyll porosity).
3.gi of these leaves was correlated positively to Smes but negatively to mesophyll porosity. The variation in gi among these species would be therefore primarily determined by variation of the conductance in liquid phase rather than that in gas phase.
4.δ13C was positively correlated to mesophyll thickness and leaf nitrogen content on an area basis. However, gi values did not correlate to δ13C. These results suggest that difference in δ13C among the species was not caused by the variation in gi, but mainly by the difference in long-term photosynthetic capacity.
5. Comparison of our results with those of previous studies showed that the correlation between leaf thickness and gi differed depending on leaf functional types (evergreen, deciduous or annual). Differences in leaf properties among these functional types were discussed.
However, the simple theoretical model of the relation between δ13C and Pi/Pa was based on an assumption that CO2 partial pressure at the carboxylation site was equal to Pi (Farquhar et al. 1982). This assumption means that CO2 transfer conductance from the substomatal cavity to the carboxylation site (gi) is infinite, which is clearly inappropriate for thick hypostomatous leaves. Epron et al. (1995) showed that the ratio of CO2 partial pressure of the carboxylation site (Pc) to Pi can go down to 0·6 in some tree species. The low Pc that is caused by small gi may increase leaf δ13C. Then, it would be dangerous to use the leaf δ13C as a simple index of leaf physiological status such as water-use efficiency and leaf photosynthetic capacity in such leaves.
Whether or not leaf thickness should affect gi is controversial. Vitousek, Field & Matson (1990) suggested that the positive relation between leaf dry mass per area (LMA) and leaf δ13C obtained for the leaves of Hawaiian Metrosideros polymorpha collected at various altitudes should be attributed to the reduction in gi with leaf thickness rather than variation in Pi/Pa. Syvertsen et al. (1995) measured gi of grapefruit, lemon, macadamia and peach plants, and showed that thicker leaves tended to have smaller gi. On the other hand, Evans et al. (1994) showed that the reduction of CO2 transfer conductance was not associated with the increase in mesophyll thickness of the leaves of tobacco plants. Lauteri et al. (1997) also showed that thicker leaves had larger gi for the genotypes of the chestnut, Castanea sativa, collected from different locations.
The aim of this work is to examine the effects of leaf thickness on gi, and to clarify whether variations in leaf thickness affect leaf δ13C through variation in gi. We chose some evergreen tree species that have different leaf thicknesses. The gi values were calculated based on combined measurements of gas exchange and carbon isotope discrimination. Anatomical characteristics of these leaves were quantified to investigate the relationships between leaf anatomical characteristics and gi. We also measured leaf nitrogen content, concentration of Rubisco and leaf dry matter δ13C. Correlations between these parameters were examined to elucidate their effects on CO2 transfer conductance and leaf dry matter δ13C.
Materials and methods
Two- to 3 year-old seedlings of evergreen tree species, Quercus glauca Thunb. ex Murray (Fagaceae) and Castanopsis sieboldii (Makino) Hatusima ex Yamazaki et Mashiba (Fagaceae), were grown under field conditions in 5 litre vinyl pots from March to November 1997. All seedlings of C. sieboldii and a seedling of Q. glauca were grown under bright conditions (under the shading cloth transmitting 50–75% sunlight). The other seedlings of Q. glauca were grown under the shaded condition (under the shading cloth transmitting 5% sunlight). Plants were watered sufficiently and fertilized three times a week with the diluted Hoagland nutrient solution containing 2 mM N (Epstein 1972).
Shoots of Quercus phillyraeoides A. Gray (Fagaceae), Cinnamomum camphora (L.) Presl (Lauraceae), Ligustrum lucidum Alt. (Oleaceae) and Camellia japonica L. (Theaceae) were collected from the sunny side of the crown of the trees grown on the campus of the University of Tsukuba in November and December 1997. The shoots were bathed in deionized water immediately after collection and kept in the dark overnight before the gas-exchange measurements (Koike 1986). The variation in carbon-isotope composition in local atmospheric CO2 would have a negligible effect on leaf dry matter δ13C of these potted plants and shoots, because all plants were grown in the open air.
Gas-exchange measurements were made from November to December 1997. We routinely checked the damage to PS II in the leaves with a pulse-amplitude modulated fluorometer (PAM-101, H. Waltz, Effeltrich, Germany). All the leaves used in the present study showed Fv/Fm values greater than 0·7, which indicates that the materials did not suffer from high-light stress.
Two to three leaves of the plants were enclosed in an acrylic chamber (12 cm × 10 cm × 2 cm high). PPFD (350 W halogen lamp light source), which was measured using a quantum sensor (LI-190SA, Li-Cor, NE, USA), was changed from 100 to 400 μmol m–2 s–1 to vary the assimilation rate. Photosynthesis in the leaves of Q. glauca and C. sieboldii was almost light-saturated at 400 μmol m–2 s–1. Leaf temperature, monitored with a copper–constantan thermocouple, was kept at 25 °C. Humidity of the air leaving the chamber was monitored with a dew-point hygrometer (Hygro M4, General Eastern, MA, USA). The CO2 partial pressure, measured with an IR gas analyser (ZRC, Fuji, Tokyo, Japan), was controlled by mixing 10% CO2 in air and CO2-free air with two mass-flow controllers (Model 3910 and 3960, Kofloc, Kyoto, Japan). Gas-exchange measurements were made before and after the gas collection for the carbon isotope analysis, and the parameters were calculated according to von Caemmerer & Farquhar (1981).
CO2 was collected according to the method of von Caemmerer & Evans (1991) with some modification. After leaf photosynthesis reached a steady-state, the air leaving the assimilation chamber was passed through a vacuum line consisting of Pyrex glass at a rate of 500–1000 ml min–1 for 3–5 min to trap the CO2 in a sample tube using cold traps. The carbon isotope ratio of the collected air was little affected by the changes in the flow rate and/or trapping time. The sample tube was combusted with 0·1 g of copper at 400 °C for 3 h to reduce N2O to N2. The carbon isotope ratio of CO2 was measured with a dual inlet mass spectrometer (MAT252, Finnigan MAT, Bremen, Germany).
Leaf discs were punched from the leaves after the gas-exchange measurements, dried at 60 °C for 48 h and then finely ground. Leaf dry matter δ13C was measured for subsamples of 0·1–0·2 mg with a combined system of an elemental analyser (EA1108, Carlo-Erba, Italy) and a stable isotope ratio mass spectrometer (Finnigan MAT 252). The mean reproducibility for the isotope measurements was ± 0·08‰.
CALCULATION OF THE INTERNAL CO2 TRANSFER CONDUCTANCE
The internal CO2 transfer conductance from the substomatal cavity to the carboxylation site (gi) was calculated by the equations given by Evans et al. (1986). The gi values were calculated from the slope of the linear regression line between A/Pi and (Δi–Δ)Pa/Pi as follows. The equation used is,
where Δ is carbon isotope discrimination during photosynthesis, and Δi is the simplified expression of the discrimination when gi is infinite, Pa and Pi are CO2 partial pressures in the ambient air and in the substomatal cavity, respectively, ai and b are discrimination during CO2 diffusion/hydration into water (1·8‰) and through carboxylation by photosynthetic enzymes Rubisco and PEP carboxylase (30‰), e and f represent fractionation associated with day respiration (Rd) and photorespiration, k is carboxylation efficiency of Rubisco and Γ* is the compensation point in the absence of day respiration. Here we assumed that f and e were so small that they did not affect the slope of the above equation according to the previous report by von Caemmerer & Evans (1991).
LEAF NITROGEN AND RUBISCO CONTENT, LIGHT MICROSCOPY
After the measurements of photosynthesis, some leaf discs (0·79 cm2) were punched out and were stored at –85°C. Crude extract of these discs was obtained according to the method of Tissue, Thomas & Strain (1993). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the crude extract was carried out according to the method of Laemmli (1970). The content of Rubisco large subunit was determined spectrophotometrically by scanning the gel at 560 nm with a gel densitometer (CS-900, Shimadzu, Kyoto, Japan). Leaf nitrogen content on a dry-mass basis was measured with an NC analyser (CNC-900, Shimadzu, Kyoto, Japan), with mean reproducibility of ± 0·02%.
For light microscopy, leaf pieces were fixed in 2·5% glutaraldehyde in 100 mM phosphate buffer (pH 7·2) for at least 3 days at 4 °C. They were post-fixed in 2% osmium tetroxide for 3 h at 4 °C, dehydrated in an acetone series and propylene oxide and embedded in Spurr’s resin (Spurr 1969). Sections, 0·8 μm thick, were stained with 0·5% toluidine blue, and photographed under a microscope (BX50/PM30, Olympus, Tokyo, Japan). Micrographs were digitized with a scanner (JX250, SHARP, Osaka, Japan) and analysed with software (NIH Image, National Institute of Health) to measure leaf mesophyll thickness and leaf porosity. We also estimated the surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area (Smes) by the method of Thain (1983), assuming that the mesophyll cells were cylinders with flat ends.
The leaf section of C. sieboldii, which had thin mesophyll, had a relatively loose assemblage of palisade tissue cells and large intercellular airspaces (Fig. 1a). On the other hand, C. japonica, which had thick mesophyll, had three-layered palisade tissue cells and small intercellular airspaces (Fig. 1b).
The internal conductance for CO2 transfer from the substomatal cavity to the carboxylation sites (gi) varied from 0·04 to 0·14 mol m–2 s–1 (Table 1). Leaf dry mass per area (LMA) varied from 78 to 192 g m–2, and the species that had large LMA tended to have thicker leaf mesophyll (Fig. 2a). The volume ratio of intercellular air spaces to the whole mesophyll (porosity), which was relatively small in these evergreen species (0·2–0·4), tended to be smaller in the species with thicker mesophyll (Fig. 2b). There was a linear correlation between the surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area (Smes) and mesophyll thickness (Fig. 2c). Leaf nitrogen content on an area basis (Narea) was positively correlated to mesophyll thickness (Fig. 2d). There was a strong positive correlation between nitrogen and Rubisco contents (Fig. 3).
Table 1. . Assimilation rate at saturating light (400 μmol m–2 s–1) and CO2 transfer conductance from the substomatal cavities to the chloroplasts (gi) for the evergreen tree species. The gi values were calculated from the slope of the regression lines for A/Pi against (Δi–Δ) Pa/Pi. The r2, p and n were for these regression lines. The data presented here were only for the regression lines that were statistically significant (P < 0·05). Note that the values of assimilation rate have some uncertainties because they were measured under different ambient CO2 partial pressures (25–29 Pa)
gi was positively correlated with LMA, leaf mesophyll thickness and Smes (Fig. 4a,b,d). According to Parkhurst (1994), conductance for CO2 diffusion in the intercellular airspaces (gias) would be proportional to the inverse of mesophyll thickness and proportional to porosity. If gias is the major determinant of gi, gi will be positively correlated with porosity and/or the inverse of mesophyll thickness. However, neither was the case (Fig. 4b,c).
Leaf dry matter δ13C was positively correlated with LMA, mesophyll thickness and Narea (Fig. 5a,b,c). However, no significant correlation was obtained between leaf dry matter δ13C and gi (Fig. 5d).
LEAF ANATOMY AND LEAF THICKNESS
The positive correlation between mesophyll thickness and Smes (Fig. 2c) supports the idea of Nobel (1991) and Evans et al. (1994) that thick leaves have large Smes. The larger Smes in the thicker leaves may be caused by the more developed palisade tissues in the thicker leaves (Fig. 1b), because palisade tissue had greater cell surfaces exposed to intercellular air spaces per unit volume than the spongy tissue (Turrell 1936).
The correlation between leaf thickness and LMA as shown in 2Fig. 2a does not hold across different plant functional types. If our results were plotted together with the data of the previous studies (Evans et al. 1994; Syvertsen et al. 1995; Lauteri et al. 1997), LMA values in tobacco and peach leaves were much smaller than those in the other species (Fig. 6a). This might be caused by loosely packed cells and/or thinner cell walls in tobacco and peach leaves. On the other hand, Smes was linearly correlated with mesophyll thickness irrespective of leaf functional types (Fig. 6b), which suggests that dependence of Smes on the leaf thickness holds across the different leaf functional types. The variation in mesophyll porosity against mesophyll thickness was not so distinct across the different leaf functional types (Fig. 6c).
LEAF ANATOMY AND CO2 TRANSFER CONDUCTANCE
CO2 diffuses from the substomatal cavity to the carboxylation site in gas and liquid phases (Evans & von Caemmerer 1996). The conductance of diffusion in gas phase (gias) is largely affected by leaf porosity and distribution pattern of stomata (whether plants have amphistomatous or hypostomatous leaves). If gias determines gi, gi should increase with mesophyll porosity. In the amphistomatous leaves of tobacco the opposite was the case, so Evans et al. (1994) concluded that gias is so large that it is not a major determinant of gi in tobacco leaves. The evergreen tree species in our study had hypostomatous leaves, however, the correlation between leaf porosity and gi was again negative (Fig. 4c). Therefore, gias was not a major determinant of gi in the hypostomatous leaves examined in this study.
If liquid phase diffusion is a major limitation, gi should be positively correlated with the exposed surface area of chloroplast, Sc, assuming constant condactance across cell walls and chloroplast per unit leaf area (Evans et al. 1994). For the species in this study, the mesophyll cell walls facing to the intercellular air spaces were mostly covered with chloroplasts, so Smes would be closely related to Sc (data not shown). Therefore, the positive correlation between Smes and gi (Fig. 4d) suggests that liquid phase diffusion was a major determinant of gi.
When gi values were plotted against some leaf characteristics together with the data of the previous studies (Evans et al. 1994; Syvertsen et al. 1995; Lauteri et al. 1997), the data plots can be divided into two groups (Fig. 7): the gi in tobacco and peach leaves was larger than the leaves of evergreen species. The amphistomatous nature of tobacco leaves (Parkhurst et al. 1988; Terashima et al. 1995), the loosely packed mesophyll cells of peach leaves (Syvertsen et al. 1995) and the difference in thickness of mesophyll cell walls may related to the smaller gi (Fig. 7) in peach and tobacco leaves than the other species. However, the comparison of the absolute values of gi obtained by the different measurements would have some uncertainties, because the treatments the effect of photorespiration on the carbon isotope discrimination differ between the studies (see Scartazza et al. 1998).
DRY MATTER δ13C, CO2 TRANSFER CONDUCTANCE, AND LMA
Vitousek et al. (1990) argued that gi would decrease with the increase in LMA, causing positive δ13C. However, in the present case, increase in δ13C was not accompanied by the decrease in gi (Fig. 5d), nevertheless δ13C was positively correlated to LMA (Fig. 5a), which was consistent with the observation by Lauteri et al. (1997) for chestnut leaves. The apparent discrepancy beween Vitousek et al. (1990) and our study may be partly owing to the dependence of assimilation rate on LMA. In contrast to the case of Vitousek et al. (1990), thicker leaves had larger Narea (Fig. 5c) and larger assimilation rate (Table 1) in the present study. The effects of assimilation rate and gi on δ13C were antagonistic in the present case, because thicker leaves had larger gi (Fig. 4a). The decrease in gi causing positive δ13C appeared to be overcome by the antagonistic effect of the decrease in assimilation rate, which would explain the weak positive (but not negative) dependence of δ13C on gi (Fig. 5d).
The positive correlation between δ13C and Narea (Fig. 5c) suggests that variation in long-term photosynthetic capacity could be a major determinant of leaf δ13C. Previous works have shown that higher nitrogen contents were associated with more positive δ13C in the leaves of forest trees (Ehleringer et al. 1986; Hanba et al. 1997). This was ascribed to the possibility that long-term average of CO2 partial pressure in the intercellular air spaces (Pi) in these leaves was lower because of the higher photosynthetic rates.
However, if we compare leaves of different plant functional types, the difference in dry matter δ13C between species could be partly affected by the difference in gi. The existence of two distinct groups in Fig. 7 suggests that the larger-LMA group had smaller gi than that of the smaller-LMA group, which may give more positive δ13C in the leaves of the larger-LMA group.
In conclusion, the difference in leaf thickness affects the variation in CO2 transfer conductance (gi), but this variation in gi would not cause the difference in leaf dry matter δ13C, as long as the leaves of similar functional types (e.g. evergreen, annual or deciduous) grown under similar elevation are compared. However, the difference in gi can cause the difference in leaf dry matter δ13C between the functional types. Therefore, care must be taken in discussing long-term leaf physiological characteristics (e.g. water-use efficiency) based on the leaf δ13C across the different leaf functional types.
Y. T. Hanba et al.
Y. T. Hanba et al.
This study was supported by National Institute Post-Doctoral Fellowship, JSPS fellowships for Young Scientists to Y.T.H., grants from the Environmental Agency (#B-52·3.2), the Ministry of Education, Science, Sports and Culture (09NP1501), and from Yamada Science Foundation. We appreciate the National Institute of Agro-Environmental Sciences for supporting the measurements of carbon isotope ratios. We thank Dr S. von Caemmerer for her kind suggestion about the technique of concurrent carbon isotope discrimination. We also thank the constructive comments from Drs S. Funayama-Noguchi and K. Noguchi on the manuscript.