Slow development of leaf photosynthesis in an evergreen broad-leaved tree, Castanopsis sieboldii: relationships between leaf anatomical characteristics and photosynthetic rate

ABSTRACT

Changes in net photosynthetic rate on a leaf area basis and anatomical properties during leaf development were studied in an evergreen broad-leaved tree, Castanopsis sieboldii and an annual herb, Phaseolus vulgaris. In C. sieboldii, surface area of mesophyll cells facing the intercellular air spaces on a leaf area basis (S_mes) was already considerable at the time of full leaf area expansion (FLE). However, surface area of chloroplasts facing the intercellular air spaces on a leaf area basis (S_c), and chlorophyll and Rubisco contents on a leaf area basis increased to attain their maximal values 15–40 d after FLE. In contrast, in P. vulgaris, chloroplast number on a leaf area basis, S_c and S_mes at 10 d before FLE were two to three times greater than the steady-state levels attained at around FLE. In C. sieboldii, the internal CO₂ transfer conductance (g_i) slightly increased for 10 d after FLE but then decreased toward the later stages. Limitation of photosynthesis by g_i was only about 10% at FLE, but then increased to about 30% at around 40 d after FLE. The large limitation after FLE by g_i was probably due to the decrease in CO₂ concentration in the chloroplast caused by the increases in thickness of mesophyll cell walls and in Rubisco content per chloroplast surface area. These results clearly showed that: (1) in C. sieboldii, chloroplast development proceeded more slowly than mesophyll cell expansion and continued well after FLE, whereas in P. vulgaris these processes proceeded synchronously and were completed by FLE; (2) after FLE, photosynthesis in leaves of C. sieboldii was markedly limited by g_i. From these results, it is suggested that, in the evergreen broad-leaved trees, mechanical protection of mesophyll cells has priority over the efficient CO₂ transfer and quick construction of the chloroplasts.

Abbreviations:

A_n

A_on

INTRODUCTION

It is widely accepted that the rate of light-saturated net photosynthesis on a leaf area basis (A_n) attains its maximum at or slightly before full leaf area expansion (for a review, see Šesták 1985). This is the case in many annual crop plants and deciduous tree species. However, there are many other species to which this rule does not apply. For instance, in cocoa (Theobroma cacao, Baker & Hardwick 1973), shade-tolerant species in tropical rainforests (Kursar & Coley 1992a), Syzygium species in tropical rainforests (Woodall, Dodd & Stewart 1998), and in evergreen broad-leaved trees in warm temperate forests (Miyazawa, Satomi & Terashima 1998), A_n is still low at the time of full leaf area expansion (FLE) and increases for a long time thereafter to attain its peak value. In Castanopsis sieboldii and evergreen Quercus species, A_n continued to increase for 20–40 d after FLE (Miyazawa et al. 1998).

Kursar & Coley (1992a) found that some shade-tolerant evergreen species in a tropical rainforest invested substantial amount of nitrogen into the leaf after FLE when the leaf became tough enough. Based on the finding, they proposed a hypothesis that delayed greening would be an effective strategy for minimizing loss of resources caused by a removal of young leaves by herbivory (Kursar & Coley 1992a), and referred to the species showing such a slow photosynthetic development as ‘delayed greening’ species to distinguish them from ‘normal greening’ species that shows the peak of A_n at or slightly before FLE.

In a previous study, we surveyed the published data of leaf development for various plant species and found a clear tendency that maturation of leaf photosynthesis delays with the increase in leaf dry mass per unit area (LMA) of the mature leaf (Miyazawa et al. 1998). Based on this relationship, we pointed out that the difference between normal greening and delayed greening species is not qualitatively distinct but gradual. In other words, delayed greening is a general phenomenon in the species having large LMA. We also pointed out that the delayed greening is not necessarily related to herbivory (Miyazawa et al. 1998).

The leaf longevity and the period taken for photosynthetic maturation increase with the LMA of the mature leaf, and all of these parameters increase in the order of annuals, deciduous trees and evergreen trees (Mooney & Gulmon 1992; Miyazawa et al. 1998). For a first step towards understanding the nature of wide and continuous spectrum, it is useful to compare leaf development between the species from both extremes. One extreme is the annuals and development of the photosynthetic apparatus has been most intensively studied for Phaseolus vulgaris (Čatsk´y, Tichá & Solárová 1976, O'Toole, Ludford & Ozbun 1977; Šesták 1985). Another extreme is the evergreen tree species having leaves of large LMA. However, unlike the annual herbaceous species, functional analyses of leaf development in the evergreen tree species have not been made, with the exception of a few ecological studies (Kursar & Coley 1992a,b; Miyazawa et al. 1998). The aim of the present study is therefore to analyse delayed greening processes in an evergreen tree species in detail.

Ontogenetic changes in photosynthetic capacity of chloroplasts have been already examined for several delayed greening species. These results indicated that Rubisco and nitrogen content on leaf area basis were both very low at FLE, and increased slowly (Kursar & Coley 1992a; Miyazawa et al. 1998). Respiration rate, stomatal conductance, and internal CO₂ transfer conductance also change dramatically during leaf development, and thereby inevitably affect net photosynthetic rate. Although the effects of these factors on photosynthesis have been studied for various plant species (Homann 1975; Miranda, Baker & Long 1981; Leech & Baker 1985; Ticháet al. 1985), few studies have been made for the delayed greening species. Thus, the relationship between these factors and A_n in delayed greening species should be studied.

The LMA increases with leaf area expansion and reaches its maximum value at around FLE in most annual herbs and deciduous trees (Čatsk´y et al. 1976; Šesták 1985; Koike 1990), whereas, in the delayed greening species, LMA continues to increase for a long time after FLE (Jurik 1986; Hanson et al. 1988; Kursar & Coley 1992a; Miyazawa et al. 1998). The LMA values of mature leaves are greater than those of the leaves at FLE by two to threefold (Miyazawa et al. 1998). The increase in LMA suggests that mesophyll thickness and/or cell wall thickness increase after FLE. In Phaseolus vulgaris (Čatsk´y et al. 1976) and Xanthium italicum (Maksymowych 1973), which are annual herbs, the leaf thickness attained its maximum slightly before FLE. The proportion of the intercellular air spaces in X. italicum leaves also reached the maximal value slightly before FLE (Maksymowych 1973). However, the anatomical changes in delayed greening species have not been studied.

Leaf anatomy affects photosynthesis. The surface area of mesophyll cells exposed to the intercellular air spaces on a leaf area basis (S_mes or A_mes/A) is an important parameter that determines potential area for CO₂ diffusion in the liquid phase (Nobel, Zaragoza & Smith 1975; Nobel 1991). If mesophyll thickness increases after FLE, S_mes would also increase. If chloroplast development occurs synchronously with that of mesophyll cells, surface area of chloroplast facing the intercellular air spaces on a leaf area basis (S_c) would also increase. Internal CO₂ transfer conductance (g_i) in mature leaves is strongly correlated with parameters such as S_mes and S_c (Evans et al. 1994; Syvertsen et al. 1995; Hanba, Miyazawa & Terashima 1999), as the increase in S_mes and S_c after FLE may increase g_i, which would lead to a rise in A_n in delayed greening species. On the other hand, increase in cell wall thickness probably decreases g_i through a lengthening of the CO₂ diffusion path in the liquid phase in cell walls (Nobel 1991).

In this study, we followed changes in the CO₂ gas exchange rate, chlorophyll content and in anatomical properties (chloroplast number on a leaf area basis, S_mes and S_c) during leaf development in Castanopsis sieboldii, a delayed greening species and Phaseolus vulgaris, an annual herb. For C. sieboldii the changes in the A–C_i relationships and Rubisco contents during leaf development were further analysed. On the basis of these data, we calculated the potential photosynthetic rate of chloroplasts as well as limitations imposed by respiration, stomatal conductance and g_i throughout the leaf development of C. sieboldii.

MATERIALS AND METHODS

Plant growth conditions

Plants were grown in a naturally lit greenhouse. Phaseolus vulgaris L. cv. Yamashiro-kurosando (Fabaceae) were grown in vermiculite in 1·3 L plastic pots (one plant/pot). Castanopsis sieboldii (Makino) Hatusima ex Yamazaki et Mashiba (Fagaceae) were grown in soil in 5 L vinyl pots (one plant/pot). The C. sieboldii plants were 2–3 years-old saplings and had been grown in an open space and transferred to the greenhouse in late August 1998 when the new leaves (second flush leaves) started to unfold. Each of the P. vulgaris and C. sieboldii plants was fertilized with 100 mL of nutrient solution containing 2 mM KNO₃, 2 mM Ca(NO₃)₂, 0·75 mM MgSO₄, 0·665 mM NaH₂PO₄, 25 μM Fe-EDTA, 5 μM MnSO₄, 0·5 μM ZnSO₄, 0·5 μM CuSO₄, 25 μM H₃BO₄, 0·25 μM Na₂MoO₄, 50 μM NaCl, and 0·1 μM CoSO₄ every two or three days. Plants were watered daily.

Measurement of leaf area

The length and width of the primary leaves of all the P. vulgaris plants were measured every other day. Using the data on 17 primary leaves, we obtained a logistic curve expressing area growth of a single leaf (= length × width) as a function of time. The age of expanding leaves used for CO₂ gas exchange measurement was estimated from their length and width using this logistic equation. In this paper, leaf age ‘zero’ stands for the time when the leaf area was fully expanded.

We used 60 current-year shoots in six C. sieboldii plants. The shoots in the lower positions of the plants were excluded. Each of these current-year shoots had 3–11 leaves. The length and width of all new leaves on these 60 current-year shoots were measured every two or three days. Area expansion of a single leaf was expressed as a function of time by a logistic equation fitted for each plant. The logistic equation was obtained for each of the six plants. The age of expanding leaves used for CO₂ gas exchange measurement was estimated from their length and width using the logistic equation of the plant.

Measurement of photosynthesis and respiration

The CO₂ gas exchange measurements were made on attached leaves with a portable gas exchange system (LI-6400; LI-Cor, Lincoln, NE, USA) from September to October 1998. When the CO₂ concentration of the air entering the chamber was markedly different from that of the ambient air, leakage of CO₂ through the slits between the sealing pads and the leaf occurred. To minimize such CO₂ leak, we routinely used a laboratory-made skirt with which the air once exhausted from the chamber was again blown to the slits from the outside.

Plants were brought into the laboratory in the early morning. We first measured the rate of dark respiration because the rate of dark respiration would increase with the accumulation of photosynthates (Noguchi, Sonoike & Terashima 1996). Then the leaf was irradiated at about 1000 μmol m⁻² s⁻¹ provided by a Björkman-type lamp (Hansatech, Kings Lynn, UK). The CO₂ concentration of the air entering the chamber was maintained at 360 μmol mol⁻¹ and light-saturated net photosynthetic rate on leaf area basis (A_n) was measured. Then the rate of photosynthesis was measured under various ambient CO₂ concentrations to obtain A–C_i relationships (see Fig. 1). The CO₂ concentration entering the chamber was decreased to 50 μmol mol⁻¹ from 360 μmol mol⁻¹, and then increased to 1200 μmol mol⁻¹. Leaf temperature and leaf to air vapour pressure deficit were always kept at 25 °C and less than 1 kPa, respectively. The rate of photosynthesis at each CO₂ condition was recorded at least three times when generating the A–C_i curve.

Figure 1. Determination of the gross photosynthetic rate assuming no stomatal and internal resistances (A_pg), net photosynthetic rate assuming no stomatal and internal resistances (A_pn), and net photosynthetic rate assuming no internal CO₂ transfer resistance (A_on). Closed circles indicate the representative relationship between net photosynthetic rates and C_i (A–C_i curves) in a mature leaf of C. sieboldii. Open circles and squares indicate the relationship between net or gross photosynthetic rate and C_c, which was calculated from the A–C_i curves and the internal CO₂ transfer conductance (g_i). C_a is ambient CO₂ condition of 360 μmol mol⁻¹. C_i and C_c are the CO₂ concentrations in the stomatal cavities and in the chloroplast stroma at C_a, respectively.

Measurement of chlorophyll and Rubisco contents

After the measurement of photosynthesis, leaf discs (0·79 cm², avoiding major veins) were collected. The leaf discs were stored at −80 °C until use. Chlorophyll content was measured in dimethylformamide using the equations of Porra, Thompson & Kriedemann (1989). The remaining leaf parts were also stored at −80 °C for the measurement of Rubisco content.

Rubisco content was measured according to Tissue, Thomas & Strain (1993) with slight modifications. The frozen leaf was ground to a powder in liquid nitrogen and homogenized in an extraction buffer containing 100 mM HEPES-KOH (pH 7·5), 5 mM EDTA, 0·3% (w/v) soluble polyvinylpyrrolidone, 0·7% (w/v) polyethylene glycol 20000, 1% Triton X-100, 1 mM phenylmethyl-sulfonyl-fluoride, 5 mM iodine acetate and 0·3%β-mercaptoethanol. The extract was centrifuged and the supernatant was used for the determination of Rubisco. A portion of the supernatant was mixed with the reagent containing 0·1 M Tris-HCl (pH 6·8), 4% sodium dodecyl sulfate, 12%β-mercaptoethanol, 20% glycerol and 1% bromophenol blue, and kept at room temperature for 1 h. Rubisco was separated by polyacrylamide gel electrophoresis with 12·5% resolving gel and 4·75% stacking gel (Laemmli 1970). The gel was stained with Coomassie Brilliant Blue R-250. The amount of Rubisco large subunit was determined spectrophotometrically with a gel densitometer (FD-A-IV; Fujiox, Tokyo, Japan). Rubisco measurement was replicated two to four times with the same leaf used for photosynthetic measurement.

Estimation for internal CO₂ transfer conductance (g_i)

Internal CO₂ transfer conductance (g_i) was estimated from the initial slope of the A–C_i curve and the measured Rubisco content according to Evans & Terashima (1988) and Terashima & Evans (1988). The value of g_i was determined as

• (1)

where g_m is the initial slope of A–C_i curve, and k is the initial slope of the relationship between net photosynthetic rate and CO₂ concentration in the chloroplast stroma. The value of k was determined as

• (2)

where V_cmax is the maximal carboxylation activity of Rubisco and K_c and K_o are the Michaelis–Menten constants for CO₂ and O₂. Γ* is a CO₂ compensation point in the absence of day respiration rate. The O₂ concentration of the chloroplast stroma was assumed to be 210 mmol mol⁻¹. The values of Γ*, K_c and K_o were assumed to be 44 μmol mol⁻¹, 325 μmol mol⁻¹ and 397 mmol mol⁻¹, respectively, according to the data for spinach Rubisco (Jordan & Ogren 1984). The specific activity of fully activated Rubisco was not very different between herbs and trees (Vu & Yelonosky 1988; Hikosaka et al. 1998). Specific activity of fully activated Rubisco was assumed to be 4·3 × 10⁻⁵ mol CO₂ g⁻¹ protein s⁻¹ (24 mol CO₂ mol⁻¹ enzyme s⁻¹) at 25 °C from the in vitro measurement of spinach Rubisco (Evans & Terashima 1988).

Light and electron microscopy

Small leaf pieces (1 mm × 2 mm) were cut before the CO₂ gas exchange measurements. They were fixed by 2·5% glutaraldehyde in 25 mM sodium cacodylate buffer (pH 7·2) at 4 °C for at least 1 d. The samples were postfixed in 2% osmium tetroxide for 3–5 h. These samples were dehydrated in acetone and propylene oxide series, and embedded in Spurr's resin (Spurr 1969). For light microscopy, sections were cut at 0·8 μm thick with an ultramicrotome (Reichert Ultracut S; Leica, Vienna, Austria) and stained with toluidine blue. Ultra-thin sections for electron microscopy were stained with lead citrate.

Following Syvertsen et al. (1995), the area of chloroplast surfaces facing the intercellular air spaces on a leaf area basis (S_c) was determined with light and electron micrographs of the sections from the same tissue block. The area of mesophyll surfaces directly exposed to the intercellular air spaces on a leaf area basis (S_mes) was also determined using the same light micrographs. Sections for light microscopy were photographed at magnification of × 100 or × 500. Electron micrographs of palisade and spongy tissues were taken at magnification of × 800 or × 1000. The values of S_c and S_mes were determined as

• (3)

• (4)

respectively. First, using electron micrographs or light micrographs taken at × 500, we measured total perimeter length of the mesophyll cells (l′_mes), and the total length of chloroplast envelopes that directly faced the intercellular air spaces (l′_c). Second, using light micrographs of leaf transverse sections at magnification of × 100, we measured the total perimeter length of the mesophyll cells (l_mes) of the leaf transverse section having width, w. We also measured the total perimeter length of the mesophyll cell surfaces that were directly exposed to the intercellular air spaces (l_mes(i)). F is a curvature correction factor. We assumed that the shape of the cells was a cylinder with hemispherical ends (Thain 1983). The curvature correction factors were obtained separately for palisade and spongy mesophyll cells. In C. sieboldii, the F-values of palisade and spongy mesophyll cells were 1·42–1·51 and 1·29–1·40, respectively. In P. vulgaris, those of palisade and spongy mesophyll cells were 1·41–1·52 and 1·32–1·40, respectively.

The number of chloroplasts on a leaf area basis, Z, was obtained using an equation as follows.

• (5)

where z′ is the number of chloroplasts occurring along l′_mes.

Cell wall thickness was determined for C. sieboldii leaves. Castanopsis sieboldii has two cell layers in the palisade tissue. The cell wall thickness of mesophyll cells in the first cell layer was measured on electron micrograph negatives taken at magnifications of × 800 or × 1000 under the binoculars. A straight line was drawn parallel to the upper epidermis through the middle part of the first cell layer and cell wall thicknesses at the intersections between the line and the cell walls were measured using an ocular micrometer. Measurements were not made on narrow cells, because the thicknesses of the cell walls measured on such cells are likely to be overestimated. Cell wall thickness was obtained as the mean of 6–18 measurements for each leaf.

Limitation of potential photosynthetic rate imposed by respiration rate (r_day), stomatal conductance (g_s) and internal CO₂ transfer conductance (g_i)

The rate of respiration in the light (day respiration rate, r_day) was estimated as the rate of CO₂ evolution at C_i of 44 μmol mol⁻¹ from the A–C_i curves (Brooks & Farquhar 1985). This C_i value corresponds to the CO₂ compensation point in the absence of day respiration (Γ*). The relationships between net photosynthetic rates, A and C_i was well fitted by a non-rectangular hyperbolic equation (see Fig. 1). Using estimated g_i, CO₂ concentration in the chloroplast C_c, is expressed as

• (6)

The value of C_c was calculated for each set of A and C_i. The relationships between A and C_c was also well fitted by a non-rectangular hyperbolic equation (see Fig. 1). The net photosynthetic rate on a leaf area basis at C_c of 360 μmol mol⁻¹ (A_pn) was estimated from the non-rectangular hyperbolic curve expressing the relationship between A and C_c. The gross photosynthetic rate at C_c of 360 μmol mol⁻¹ (A_pg) was calculated as A_pn plus r_day. The net photosynthetic rate on a leaf area basis assuming no internal CO₂ transfer resistance (A_on) was estimated with the supply function according to Farquhar & Sharkey (1982). The supply function was determined from g_s as well as the negligible boundary layer conductance. The extent to which the potential rate of gross photosynthesis was limited by r_day (L_r), g_s (L_s) or g_i (L_i) was calculated as:

• (7)

• (8)

and

• (9)

RESULTS

Leaf area expansion

Changes in leaf area with time in leaves of P. vulgaris and C. sieboldii are shown in Fig. 2. Marked difference in area growth pattern was not found. To analyse the difference statistically, the growth area of each leaf was fitted by the logistic equation (n = 17 and 28 for P. vulgaris and C. sieboldii, r² of the fitting ≥ 0·97). The periods needed for leaf area expansion from 30 to 80% of the maximum leaf area that were calculated from the respective logistic equations were 5·0 ± 0·69 and 4·4 ± 0·80 d (mean ± SD) in P. vulgaris and in C. sieboldii, respectively. The difference was statistically significant (P < 0·05 by t-test), but small.

Figure 2. Changes in leaf area with time in P. vulgaris (○) and C. sieboldii (●). Leaf age of zero is assigned to leaves at full area expansion (FLE), negative ages to younger leaves and positive ages to older leaves. Solid lines represent logistic equations fitted to the data with a non-linear least squares method. The fitted logistic equations: a/{1 + b× exp[–c× (x–d)]} + e, where a = 82·7, b = 0·23, c = 0·59, d = –18·3 and e = 17·2 for P. vulgaris, and a = 96·5, b = 0·21, c = 0·50, d = –18·1 and e = 6·74 for C. sieboldii. The logistic equations were obtained based on the area growth of 17 and 28 leaves for P. vulgaris and C. sieboldii, respectively. Symxbols denote the mean values.

Changes in CO₂ gas exchange parameters during leaf development

Changes in net photosynthetic rate at the ambient CO₂ concentration of 360 μmol mol⁻¹ (A_n) during leaf development are shown in Fig. 3a. In P. vulgaris, A_n increased with leaf age and reached the maximum a few days before FLE. By the 15th day after FLE, A_n decreased to less than half the peak value. In contrast, in C. sieboldii, A_n at FLE was about half the peak value, which was attained approximately 15–20 d after FLE. The dark respiration rates decreased with the increase in A_n (Fig. 3b).

Figure 3. Changes in net photosynthetic rate at ambient CO₂ on leaf area basis (A_n), dark respiration rate on leaf area basis, stomatal conductance (g_s) and the CO₂ concentration in the substomatal cavity (C_i) during leaf development for P. vulgaris (○) and C. sieboldii (●).

Stomatal conductance to water vapour (g_s) increased with leaf area expansion and attained the maximal values at around FLE in both species (Fig. 3c). After FLE, g_s decreased considerably in P. vulgaris whereas in C. sieboldii g_s was relatively constant. In C. sieboldii, g_s at and after FLE were 98·9 ± 59·7 mmol m⁻² s⁻¹ (mean ± SD; n = 8) and 139 ± 31·9 mmol m⁻² s⁻¹ (mean ± SD; n = 10), respectively. There was no statistical difference between these values by t-test (P = 0·11). The values of C_i were high in young expanding leaves and decreased to the almost constant level toward the later stages of leaf development in C. sieboldii (Fig. 3d). There was a transient decrease in C_i at 5–7 d before FLE in P. vulgaris.

Changes in chlorophyll content, chloroplast number, mesophyll surface area (S_mes) and chloroplast surface area (S_c) during leaf development

In C. sieboldii, chlorophyll content on a leaf area basis markedly increased during leaf development (Fig. 4a). In contrast, it was nearly constant in P. vulgaris.

Figure 4. Changes in chlorophyll content (a), chloroplast number (b), mesophyll surface area directly exposed to the intercellular air spaces (S_mes) (c) and chloroplast surface area facing the intercellular air spaces (S_c) (d), all expressed on leaf area basis, during leaf development of P. vulgaris (○) and C. sieboldii (●).

In P. vulgaris, the number of chloroplasts on a leaf area basis was large in very young leaves and decreased to the constant level at around FLE (Fig. 4b). In C. sieboldii, chloroplast number per unit leaf area gradually increased throughout leaf development. In some expanding leaves of C. sieboldii, the number of chloroplasts appeared to be larger than those in the leaves at FLE because the young mesophyll cells were densely packed (see Fig. 5d). However, in comparison with P. vulgaris, this effect was not marked.

Figure 5. Light micrographs of transverse sections of the leaves at three leaf developmental stages for P. vulgaris (a, b, c) and C. sieboldii (d, e, f). The ages of the leaves [before (–) and after (+) full leaf area expansion]: a = –7 d, b = 0 d , c = +14 d, d = –8 d, e = 0 d and f = +5 d. Bar = 50 μm.

In P. vulgaris, the area of chloroplast surfaces facing the intercellular air spaces per leaf area, S_c, was highest at early stages of leaf development and tended to decrease toward FLE (Fig. 4d). The mesophyll surface area exposed to the intercellular air spaces (S_mes) changed in a similar manner to S_c in P. vulgaris. On the contrary, S_c showed a significant increase during leaf development in C. sieboldii (Fig. 4d). The value of S_c at FLE was only half the maximum value. However, S_mes, was fairly large at FLE in C. sieboldii and remained nearly constant thereafter (Fig. 4c).

The size of chloroplast was small in the expanding leaves of C. sieboldii (Fig. 6d) compared with that in P. vulgaris (Fig. 6a), although these two electron micrographs were obtained when the leaves were of the similar age. After FLE, large plastoglobuli were observed in the leaves of P. vulgaris (Fig. 6c).

Figure 6. Electron micrographs of palisade tissue chloroplasts at three leaf developmental stages for P. vulgaris (a, b, c) and C. sieboldii (d, e, f). Ages of sample leaves were the same as in Fig. 5. Bar = 1 μm.

Changes in S_c/S_mes during leaf development

Patterns of changes in the ratio of S_c/S_mes were different between P. vulgaris and C. sieboldii (Fig. 7). In P. vulgaris, S_c/S_mes reached around one at the early stage of leaf development because chloroplasts developed considerably and occupied the entire surface of the mesophyll cells (Fig. 5a). In contrast, in C. sieboldii, S_c/S_mes was only 0·4, 10 d before FLE and increased with leaf age. The maximum value (nearly one) was attained 20–30 d after FLE. Chloroplast development proceeded more slowly than the mesophyll cell expansion in C. sieboldii.

Figure 7. Changes in the ratio of chloroplast surface area to mesophyll surface area directly exposed to the intercellular air spaces (S_c/S_mes) during leaf development of P. vulgaris (○) and C. sieboldii (●).

Changes in leaf mesophyll thickness, intercellular air space and mesophyll cell wall thickness during leaf development in C. sieboldii

The mesophyll thickness increased after the completion of leaf area expansion for C. sieboldii (Fig. 8a). Even for the data after FLE, there was a significant positive correlation between mesophyll thickness and leaf age (r² = 0·33; P < 0·01). There was also a linear relationship between S_mes and mesophyll thickness for the data after FLE (r² = 0·45; P < 0·01). However, extent of the increase of mesophyll thickness with leaf age was not great after FLE. The thickness of the upper and lower epidermis did not change after FLE (data not shown).

Figure 8. Changes in mesophyll thickness (a), fraction of mesophyll volume occupied by the intercellular air spaces (b) and mesophyll cell wall thickness (c) during leaf development of C. sieboldii.

The fraction of mesophyll cell volume occupied by the intercellular air spaces increased with leaf area expansion and attained the maximum, 45% on average, at around FLE (Fig. 8b). After FLE, the fraction of the mesophyll occupied by the air spaces was not correlated with leaf age (r² = 0·02; P = 0·45).

During leaf area expansion, thickness of mesophyll cell walls in C. sieboldii was almost constant at about 0·2–0·25 μm (Fig. 8c). Shortly after FLE, wall thickness increased by about 50%.

Changes in Rubisco content, g_i and C_c/C_i during leaf development in C. sieboldii

In C. sieboldii, Rubisco content on a leaf area basis was about one-third of the maximum at FLE, and considerably increased thereafter (Fig. 9). The internal CO₂ transfer conductance (g_i) that was estimated from the initial slope of the A–C_i curve, Rubisco content, and kinetic parameters of Rubisco including specific activity of 24 mol CO₂ mol⁻¹ s⁻¹, agreed well with those values estimated by the on-line carbon isotope discrimination technique (Hanba et al. 1999). There was a correlation between A_n and g_i (Fig. 10). The horizontal bars indicate the ranges of g_i values that were estimated assuming the range of Rubisco-specific activity was between 18 and 32 mol mol⁻¹ s⁻¹. The range covers most of the reported values for C₃ species (Hikosaka et al. 1998). With the increase in the specific activity, values of g_i decreased.

Figure 9. Changes in Rubisco content on leaf area basis during leaf development in C. sieboldii.

Figure 10. Relationship between A_n and the internal CO₂ transfer conductance (g_i) during leaf development in C. sieboldii. g_i is calculated from Rubisco content, kinetic parameters of Rubisco and initial slope of A–C_i curve on the assumption that Rubisco-specific activity of 24 mol mol⁻¹ enzyme s⁻¹ (●). Horizontal bars indicate the ranges of g_i that were estimated assuming the range of Rubisco-specific activities from 18 to 32 mol mol⁻¹ enzyme s⁻¹., value of g_i measured with an on-line carbon isotope system for mature leaves of C. sieboldii (data from Hanba et al. 1999).

The value of g_i increased slightly after FLE, and from 10 d after FLE somewhat decreased (Fig. 11a). The ratio of CO₂ concentration in the chloroplast stroma to that of substomatal cavities, C_c/C_i, decreased with leaf age (Fig. 11b). The C_c/C_i ratios in the leaves 20–30 d after FLE were two-thirds of those at FLE. Effects of the specific activity on the C_c/C_i ratios were not marked and the generally decreasing trend was robust.

Figure 11. Changes in the internal CO₂ transfer conductance (g_i) and the ratio of CO₂ concentration in the chloroplast stroma to that of the stomatal cavity (C_c/C_i) during leaf development in C. sieboldii. The value of g_i is calculated from Rubisco content, initial slope of A–C_i curve and kinetic parameters of Rubisco on the assumption that Rubisco-specific activity is 24 mol mol⁻¹ enzyme s⁻¹ (●). Vertical bars indicate the ranges of g_i or C_c/C_i values which were estimated assuming the range of Rubisco-specific activity from 18 to 32 mol mol⁻¹ enzyme s⁻¹.

Limitations of potential gross photosynthetic rate by respiration rate, stomatal conductance and internal CO₂ transfer conductance in C. sieboldii

From the A–C_c curves calculated from both A–C_i curves and estimated g_i values, we calculated the gross photosynthetic rate at C_c of 360 μmol mol⁻¹ (A_pg). The value of A_pg at FLE was less than half of the peak values (Fig. 12). The changes in gross photosynthetic rate assuming no internal CO₂ transfer resistance (A_on + r_day), and gross photosynthetic rate at ambient CO₂ concentration (A_n + r_day) during leaf development are also shown. The scale for day respiration rate (r_day, the lower panel of Fig. 12) is different from that for other parameters. The value of r_day decreased with leaf age in a similar fashion to the change in dark respiration rate; values of r_day were always lower than the dark respiration rates throughout the leaf development.

Figure 12. Changes in the gross photosynthetic rate assuming no stomatal and internal CO₂ transfer resistances (A_pg, ●), gross photosynthetic rate assuming no internal CO₂ transfer resistance (A_on + r_day, ○), gross photosynthetic rate at ambient CO₂ concentration of 360 μmol mol⁻¹ (A_n + r_day, ●) and day respiration rate (r_day, ● in the lower panel) during leaf development of C. sieboldii.

The changes in the A_n/A_pg and in the extents to which A_pg was limited by r_day, g_s, or g_i are shown in Fig. 13. A_n/A_pg increased with leaf area expansion, reached the maximum value of about 0·6 at around FLE and then decreased (Fig. 13a). The limitation by respiration (L_r) was considerable when leaves of C. sieboldii were expanding. However, L_r at FLE was about 15%, and at 10 d after FLE, it was only less than 5%. After FLE, the limitations by g_s (L_s) and by g_i (L_i) were positively correlated with leaf age at P < 0·05 and P < 0·01, respectively. The value of L_s ranged from 10 to 15% in the expanding stage to FLE (Fig. 13c). After FLE, L_s increased, but remained less than about 20%. The value of L_i changed in a similar fashion to that of L_s. However, L_i was almost always greater than L_s, reaching values that were greater than 30% by about 40 d after FLE (Fig. 13d).

Figure 13. Changes in the A_n/A_pg (a) and in the limitation of potential gross photosynthetic rate by day respiration rate (L_r, b), stomatal conductance (L_s, c) and internal CO₂ transfer conductance (L_i, d) during leaf development in C. sieboldii.

DISCUSSION

Differential development of chloroplasts and mesophyll surface area in C. sieboldii

Both S_mes and proportion of the intercellular air spaces attained their maximum values at around FLE in C. sieboldii (Figs 5 & 8). This indicates that the expansion of mesophyll cells and the epidermal cells was nearly completed at around FLE.

Chloroplast development in C. sieboldii was much slower than that in P. vulgaris, although leaf area expansion of these species proceeded similarly (Figs 2 & 4). In P. vulgaris, the number of chloroplasts on unit leaf area declined with leaf area expansion, which was also reported for Solanum tuberosum L. (Šesták 1985). Thus, substantial materials (i.e. carbon and nitrogen) are used for chloroplast development at the very early stages of leaf area expansion in P. vulgaris and S. tuberosum (normal greening species). On the other hand, in C. sieboldii, S_c increased much later than S_mes as indicated by the increase in S_c/S_mes with leaf age (Fig. 7). Such differential development of S_mes and S_c in C. sieboldii would be important in understanding delayed greening. The low values of S_c/S_mes of young expanding leaves might be effective in protecting the young chloroplasts from stresses from herbivory because such leaves have a large amount of structural compounds (Kursar & Coley 1992a,b).

Limitation of photosynthetic rate by respiration rate, and stomatal and internal CO₂ transfer conductances in C. sieboldii

In the expanding leaves of C. sieboldii, the potential photosynthetic rate (A_pg) was limited by high respiration rate (Fig. 13b). As the leaf tissues were still immature, energy for constructing these tissues would be mainly provided by respiration (Lambers, Szaniawski & de Visser 1983). Small intercellular air spaces (Fig. 8b) of the young leaves would apparently contribute to lowering the internal CO₂ transfer conductance. However, g_i was not a major factor limiting the rate of photosynthesis in the expanding leaves. The high r_day of the young expanding leaves contributed to maintain high C_i and partly obviated the need for CO₂ diffusion from the atmosphere to the chloroplasts (Miranda et al. 1981).

Stomatal conductance to water vapour (g_s) attained the maximum values at around FLE both in P. vulgaris and C. sieboldii (Fig. 3c). The difference in the period taken for stomatal development would be much smaller than the difference in the period for chloroplasts development. After FLE, g_s declined in P. vulgaris, whereas it stayed at a constant level in C. sieboldii. For many herbaceous species, it has been reported that g_s attains the maximal value at around FLE and declines thereafter (Šesták 1985). Although the data are limited, the trend of changes in g_s observed in C. sieboldii could be generally the case in the delayed greening species. In T. cacao, g_s increased sharply with leaf area expansion and attained the maximum near FLE (Abo-Hamed, Collin & Hardwick 1983). In Brachystegia spiciformis, another delayed greening species, g_s did not increase greatly in comparison with the marked increase in net photosynthetic rate after FLE (Choinski & Johnson 1993). The limitation of potential photosynthetic rate by stomatal conductance (L_s) increased after FLE in C. sieboldii (Fig. 13c). This was because g_s did not increase greatly after FLE, although the potential photosynthetic rate increased.

There was a positive relationship between A_n and g_i (Fig. 10). Using the on-line carbon isotope discrimination technique for g_i estimation, Hanba also found a positive relationship between the two factors throughout the leaf emerging to senescing in Alnus and Acer (Dr Y.-T. Hanba, Okayama University; personal communication). The g_i values estimated in the present study agreed very well to those obtained by the on-line discrimination method for the same species (Hanba et al. 1999).

Makino, Mae & Ohira (1983) reported that specific activity of fully activated Rubisco was almost constant from young to senescing leaves in rice. They also claimed that the activation state could not be a major factor responsible for the changes in photosynthetic rate during leaf ontogeny. Thus, the ontogenetic differences in the specific activity and the activation state of the present g_i estimation were not taken into account in evaluating the present g_i estimation. On the other hand, we made sensitivity analyses. When different specific activities of 18 and 32 mol mol⁻¹ s⁻¹ were used for the estimation of g_i, the trend did not change markedly (Fig. 11). On the other hand, when the low specific activity of 15 mol mol⁻¹ s⁻¹ reported for rice (Makino, Mae & Ohira 1988) was used, some g_i values were estimated to be negative and deviated from the values obtained by the on-line method. Judging from the agreement to the values obtained by the on-line method and the results of the sensitivity analyses, the trend of changes in g_i obtained in the present study would be fairly robust. The g_i values estimated with a combination of gas exchange and carbon isotope fractionation were reported for mature leaves of evergreen trees such as Citrus limon, Macadamia integrifolia (Syvertsen et al. 1995), Quercus glauca, Quercus Phillyraeoides and Camellia japonica (Hanba et al. 1999). These values were similar to the present values calculated for mature leaves of C. sieboldii (leaf age 20–35 d after FLE).

Day respiration rate calculated at the intersection of Γ* on the A–C_i curves is underestimated in the presence of internal CO₂ transfer conductance (von Caemmerer et al. 1994). Therefore, the values of r_day estimated in this study might be lower than actual day respiration rates. To estimate the maximum extent to which A_pg values are affected by respiration rate, we also calculated A_pg using dark respiration rate instead of r_day. Dark respiration rate was always greater than day respiration rate as has been reported (Brooks & Farquhar 1985; Villar, Held & Merino 1994). The ratio of L_s or L_i calculated using dark respiration rate to those calculated using r_day ranged from 0·87 to 0·99. Thus, the calculated limitations by g_s or g_i did not differ markedly even though we might underestimate the day respiration rates. Effects of the overestimation on L_r could be considerable because the ratio of L_r calculated using dark respiration rate to those calculated using r_day ranged from 1·1 to 1·9. However, the trend of L_r during leaf development did not change greatly.

We expected that the increase in A_n after FLE in delayed greening species would be partly caused by an increase in g_i. We observed a dramatic increase in S_c after full leaf area expansion (Fig. 4d). The value of g_i would be proportional to S_c if other conditions are unchanged (Evans et al. 1994). However, g_i did not increase proportionally to the increase in S_c (compare Figs 4d & 11a). Moreover, g_i even decreased at the later stages. The internal CO₂ transfer conductance is separated into two components, gas-phase conductance (intercellular air space) and liquid-phase conductance (cell wall, cell membrane, cytosol, chloroplast envelope and stroma). The CO₂ transfer conductance through the air-phase component would decrease after FLE because the increase in mesophyll thickness would result in a longer CO₂ diffusion pathway from the substomatal cavities to the chloroplasts (Fig. 8a). The CO₂ transfer conductance through the air-phase component was calculated from the mesophyll thickness, the fraction of mesophyll cells occupied by the intercellular air spaces and diffusion coefficient of CO₂ in air according to Nobel (1991). The values obtained were very large; 1·6 ± 0·12 mol m⁻² s⁻¹ 1(mean ± SD; n = 4) and 1·4 ± 0·23 mol m⁻² s⁻¹(mean ± SD; n = 8), at and after FLE, respectively. Corresponding resistances were only 3·5 and 5·6% of the total internal resistance to CO₂ diffusion. Thus, the decrease in g_i at the later stages should be attributed to a decrease in liquid-phase conductance. The increase in mesophyll cell wall thickness would contribute to the decrease in g_i through increasing distance in the liquid phase after FLE (Fig. 8c). Judging from the electron micrographs, other factors such as thickness of the cell membrane, cytosol, and chloroplast envelope did not appear to change after FLE. The increase in thickness of chloroplast stroma should not be a major factor leading to low CO₂ transfer conductance when activity of carbonic anhydrase in the chloroplast is high (Cowan 1986). Actually, in the case of mature leaves of oak, carbonic anhydrase activity is considerable (Gillon & Yakir 2000). As the properties of cell wall, cell membrane, cytosol and chloroplast envelope can change with leaf age and will affect liquid-phase conductance, further studies are needed to elucidate the responsible factors.

In C. sieboldii leaves after FLE, the increase in Rubisco content was faster than the increase in S_c (compare Figs 4d & 9). Therefore, Rubisco content per chloroplast surface area would increase toward the later stages of leaf development. Such high Rubisco content would lower C_c/C_i in mature leaves because of the increase in carboxylation rate per chloroplast surface area.

The increase in mesophyll cell wall thickness and high Rubisco content per chloroplast surface area would be mainly responsible for the decrease in C_c/C_i after FLE (Fig. 13d). The decrease in C_c was also observed in senescing leaves of wheat by Loreto et al. (1994), and it was suggested that g_i might limit photosynthetic rate in the course of leaf senescence.

If a leaf is to acquire its photosynthetic functions quickly, available materials should be preferentially invested in chloroplasts to raise photosynthetic capacity. Furthermore, the rapid chloroplast development leads to high S_c, which is essential to efficient photosynthetic CO₂ transfer (Evans et al. 1994; Hanba et al. 1999). However, the leaves of the evergreen broad-leaved tree had low S_c/S_mes when they were young (Fig. 7) and mesophyll cell walls thicken after FLE (Fig. 8c), which suggests that a considerable share of the available materials for leaf construction was preferentially invested in cell wall construction (structural compounds) throughout the leaf development. In the C. sieboldii saplings used in this study, the leaf longevity was about two years. Such long-lived leaves would be valuable enough to be protected by thick cell walls at the expense of an efficient CO₂ transfer and quick construction of leaf photosynthetic functions.

ACKNOWLEDGMENTS

The authors wish to thank Dr H. Muraoka, Dr M. Ohkoshi and Dr A. Ohmiya for the procedure of microscopy and anatomical measurements, Dr K. Ishimaru, Dr K. Ono and Dr K. Hikosaka for the measurement of Rubisco, and members of our laboratory for constructive comments and discussions. This study was financially supported by the Sasakawa Scientific Research Grant from The Japan Science Society and by the Ministry of Education, Science, Sports and Culture (09NP1501).