Ontogenetic differences in mesophyll structure and chlorophyll distribution in Eucalyptus globulus ssp. globulus (Myrtaceae) ^†

Cultivation

Two Eucalyptus globulus ssp. globulus Labill. provenances, exhibiting different rates of vegetative phase change, were grown in a climate-controlled glasshouse. Night and day temperatures varied between ∼15–18°C and 21–27°C, respectively, while humidity ranged from 22 to 51%. Additional lighting extended the photoperiod to 15 h. Plants were watered daily to field capacity, and nutrients (NPK) applied as controlled-release fertilizer. Seven-month-old seedlings from St Marys, Tasmania (CSIRO forest research seedlot provenance 16474 sib. CL002) were producing true juvenile leaves at the time of analysis. Saplings from Wilson's Promontory, New South Wales (provenance 16399 Sib. DFC 219) were 2.5 yr old and had transitional and adult leaf types at the time of analysis.

Leaf measurements

Five representative leaves of each leaf type (juvenile, transitional, and adult) were selected from nodes 18–23, 41–48, and 49–54 from the base of individual plants (N = 3), respectively. Transitional and adult leaves were selected on the basis of petiole length and the ratio of leaf length to width (Table 1). Additional structural characteristics were determined for each of the leaves (Table 1), and stomatal densities were measured (James and Bell, 1995).

Leaf sections of ∼15 mm² were collected at leaf midlength, midway between the midvein and leaf margin, fixed in 3% glutaraldehyde in a 0.005 mol/L phosphate buffer (pH 7.0) for 24 h under vacuum, dehydrated in a graded ethanol series in 10% steps, and embedded in LR White acrylic resin (London Resin Co. Ltd, Reading, UK) within gelatin capsules (polymerized at 70°C for 24 h). Sections of 1 μm thickness were stained with 0.5% toluidine blue in 0.1% sodium carbonate buffer (pH 9.0). Anatomical characteristics were determined for each leaf from transverse sections and computed as an average of five measurements at 300× magnification (Table 1). Oblique-paradermal sections were cut at an angle of 30° – 85° to the surface of the leaf (Fig. 1), and used to determine mesophyll structure.

Mesophyll parameters

The internal anatomy of leaves has previously been determined quantitatively using drawings prepared with the aid of camera lucida (Turrell, 1965; Nobel, Zaragoza, and Smith, 1975; Smith and Nobel, 1978; Thain, 1983). This method generates mesophyll layers drawn from paradermal views, while transverse sections are used to construct a three-dimensional model from which all cell lengths and diameters can be determined. Cell surface areas are calculated assuming the palisade mesophyll cells are cylindrical with hemispheres at each end, while spongy mesophyll cells are spherical with certain correction factors applied. Others have developed stereological techniques to quantitatively measure internal leaf structure (Parkhurst, 1982; Kubinova´, 1991, 1993, 1994). The current study uses a newly developed image analysis technique to determine mesophyll structure within the Eucalyptus leaves.

The ratio of mesophyll surface area (A_mes) to external surface area (A) was determined using an oblique-paradermal section of each embedded leaf (Fig. 1). A consecutive sequence, or transect, of 0.0079 mm² digital images at 750× magnification was collected from the adaxial to abaxial epidermis using Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland) and a Javelin CCTV camera (Javelin electronics, Los Angeles, California) attached to a binocular light microscope. The number of images per leaf ranged from four to 15, depending on the thickness of the leaf and the angle of the oblique paradermal section. A single transect was measured for each leaf (N = 5 per leaf type).

Images were manipulated using Adobe Photoshop 3.0 software (Adobe Systems Inc., Mountain View, California) for a Macintosh computer. The intercellular area within each cell was blackened, and contrast was maximized to produce a sharp black-white image. The area of mesophyll cells (A_m), intercellular airspace (A_a), and the length of mesophyll cell wall exposed to intercellular airspace (P_i) was determined for each image using the Image-Pro Plus software. A_mes/A was then calculated for each image as

and where T_i is the projected thickness of a given section i, T_m is the mesophyll thickness of the leaf determined from transverse sections of the same leaf, L_i is the length of the image, A_p is the projected area of the image, L_p is the projected length of the image, and W_i is the width of the image. C is a conversion factor for P_i, which gives the projected mesophyll perimeter. The sum of L_i for each of the leaf images gives the total transect length of the oblique-paradermal section (L_s), while the sum of L_p gives the projected length of L_s. Where vascular tissue or oil cavities were present within the mesophyll of the image, A_p was computed as a proportion of the projected area of an image containing only mesophyll. Hence, A_mes/A for the leaf was calculated as the sum of the individual values for each image or

(5)

V_mes per unit leaf area (V_mes/A) was calculated for each leaf by substituting P_i with A_m in Eq. 1, and A_mes/A with V_mes/A in Eq. 5. In the same manner, substituting A_m for P_i in Eq. 1 enables calculation for A_mes per unit leaf volume (A_mes/V), or

Similarly, the volume of mesophyll per unit leaf volume (V_mes/V) was calculated by substituting A_m for P_i in Eqs. 6 and 7.

Relationships between anatomical and morphological characteristics were determined by regression analysis using Minitab Release 10.51 (Minitab Inc., State College, Pennsylvania) for a Macintosh computer. Differences between structural characteristics of the three leaf types were determined using one-way and multivariate ANOVA.

Chlorophyll and carotenoid determination

Four of the five leaves selected as representative of each leaf type were also used to determine leaf chlorophyll content. Six to ten leaf disks 31.2 mm² in size were sampled from midway along the leaf and paradermally sectioned into 30 μm (juvenile and transitional) or 40 μm (adult) layers using a cold-stage microtome (Spencer Lens Co., Buffalo, New York). Corresponding layers were pooled in a small vial containing 1 mL of 95% ethanol, plus 0.01 mL of sodium ascorbate as an antioxidant. Because the leaf disks separated from the cold stage before the entire thickness of the disk could be sectioned, adaxial and abaxial surfaces were alternately sectioned and tissues pooled separately. Samples were kept in the dark until analysis, and concentrations of chlorophyll a, b, and carotenoids were determined by measuring absorbance at 470.0, 648.6, and 664.2 nm, minus absorbance at 730 nm with a spectrophotometer (Hewlett Packard model 8453, San Diego, California). Extinction coefficients determined according to Lichtenthaler (1987) were used to calculate pigment concentrations. Total chlorophyll content of the leaves was determined by averaging measurements from two leaf disks from the same region. Leaf, palisade, and epidermal thicknesses were also measured using transverse sections of fresh leaves.

Whole-leaf characteristics

Anatomical and morphological parameters of juvenile, transitional, and adult leaf types of E. globulus were significantly different (Table 1). Juvenile leaves were significantly shorter and broader than adult leaves, while petiole length was least for the juvenile and greatest for the adult leaf type, in correspondence with their horizontal and vertical orientations, respectively. Juvenile leaves were hypostomatous and dorsiventral with a single layer of palisade mesophyll only beneath the adaxial epidermis. In contrast, adult leaves were amphistomatous and isobilateral, with three layers of palisade on both adaxial and abaxial sides. Transitional leaves had characteristics intermediate between the adult and juvenile leaves (Table 1), being less strongly amphistomatous than adult leaves and approaching isobilateral symmetry in whole-leaf structure.

A_mes/A, A_mes/V, and V_mes/A for the entire leaf thickness were least for the dorsiventral, juvenile leaves and greatest for the isobilateral, adult leaf type (Table 2). Values for the transitional leaves were between those of the juvenile and adult leaves. Whole-leaf V_mes/V was no different between the three leaf types.

Total chlorophyll content for whole leaves, expressed on both a unit area and volume basis, was significantly different among the three leaf types (Table 2). Juvenile chlorophyll a + b content was significantly less than that of the transitional and adult leaf types when based on a unit area basis, but greater when expressed per unit volume. The chlorophyll a:b ratio was greatest for the juvenile leaf type, while the transitional and adult leaf types were similar.

Leaf thickness was significantly and positively related to A_mes/A (r² = 0.96, P < 0.0005) and V_mes/A (r² = 0.92, P < 0.0005). However, A_mes/A was even more strongly correlated with the sum of adaxial and abaxial palisade mesophyll thicknesses (r² = 0.99, P < 0.0005). A_mes/A was also positively associated with the ratio of adaxial to abaxial stomatal density (r² = 0.80, P < 0.0005), as was the relationship between greater leaf thickness and a higher ratio of adaxial to abaxial stomatal density (r² = 0.84, P < 0.0005).

Mesophyll profiles

The three leaf types of E. globulus had different patterns in A_mes/A (Fig. 2A) and chlorophyll distribution (Fig. 2B). However, chlorophyll a + b concentrations mirrored A_mes/A distribution for all leaf types. The dorsiventral, juvenile leaves had a single maximum value of A_mes/A at the base of the single adaxial palisade layer (Fig. 2A), which declined steadily through the spongy mesophyll. Correspondingly, the chlorophyll a + b concentration was highest at the base of the adaxial palisade layer and also declined through the spongy mesophyll (Fig. 2B). In contrast, the chlorophyll a:b ratio declined sigmoidally from the adaxial to abaxial leaf surface with the greatest value of chlorophyll a:b found adjacent to the adaxial epidermis (Fig. 3A).

In comparison to the juvenile leaves, the isobilateral adult leaves showed a more bimodal profile in A_mes/A throughout the mesophyll, with maxima occurring within the adaxial and abaxial palisade layers (Fig. 2A). Similarly, the chlorophyll a + b content was highest within the adaxial and abaxial palisade layers (Fig. 2B). A_mes/A and chlorophyll contents between the adaxial and abaxial palisade layers of the adult leaf type were similar. Maximum values of chlorophyll a:b were adjacent to both the adaxial and abaxial epidermes with minimum values occurring within the spongy mesophyll (Fig. 3A).

Profiles of A_mes/A and chlorophyll distributions inside transitional leaves were intermediate to those of the juvenile and adult leaf types (Fig. 2). With palisade mesophyll on both surfaces, a maximum in A_mes/A occurred within each of the adaxial and abaxial palisade layers. However, the maximum value of A_mes/A within the abaxial palisade was lower than that of the adaxial palisade, due to a less developed abaxial palisade. Chlorophyll a + b contents across the transitional leaves were more similar to that of the juvenile leaf type, peaking at the base of the palisade mesophyll layer, and declining to the abaxial epidermis (Fig. 2B). As found for the juvenile leaves, the chlorophyll a:b ratio declined sigmoidally, with a maximum adjacent to the adaxial epidermis and one near the abaxial epidermis (Fig. 3A).

For all leaf types, the lowest values of A_mes/A were measured adjacent to the adaxial and abaxial epidermises (Fig. 2A). Microscopic observation indicated a greater packing density of the cells adjacent to the epidermis, with fewer intercellular airspaces. The epidermises were devoid of chlorophyll in all three leaf types. Thus, minimum values of chlorophyll were also found for these areas (Fig. 2B).

Other leaf and mesophyll associations

As expected, the profile of A_mes/V distribution throughout the leaf interior was similar to A_mes/A for each of the leaf types. In contrast, V_mes/V was greatest adjacent to the adaxial epidermis, minimal within the spongy mesophyll, and increased again at the abaxial epidermis for all three leaf types (Fig. 3B). High values of V_mes/V indicated a low volume of intercellular airspace and greater cell packing. As found for A_mes/A, V_mes/V of the adaxial and abaxial palisade layers of the adult leaf type were similar in value. Also, no pattern was discovered in the distribution of V_mes/A values throughout the mesophyll of the three leaf types (data not shown).

The first adaxial palisade layer and the spongy mesophyll layer, the only tissues common to all three leaf types, were analyzed for each of the three leaf types (Table 3). Chlorophyll a:b and all of the structural parameters measured for the mesophyll were significantly greater in the palisade than spongy mesophyll of all leaf types (P < 0.004), with the exception of V_mes/A (Table 3). No difference was found between leaf types in the length of the first adaxial palisade layer (Table 1), or in A_mes/A, V_mes/A, or A_mes/V of either the palisade or spongy mesophyll (Table 3). Chlorophyll concentration and the chlorophyll a:b ratio within the first palisade layer of each leaf type were also similar. In contrast, the chlorophyll a:b was significantly greater for the spongy mesophyll of juvenile leaves than the transitional and adult leaves. Also, a significantly lower proportion of intercellular airspace, or greater V_mes/V, was found within the spongy mesophyll of the adult leaf type (Table 3).

A_mes/A

Most studies of mesophyll structure have described changes in A_mes/A under different light and stress regimes, or season of the year (Nobel, Zaragoza, and Smith, 1975; Chabot and Chabot, 1977; Smith and Nobel, 1977, 1978; Chabot, Jurik, and Chabot, 1979). Although whole-leaf A_mes/A has been measured often, incremental changes in A_mes/A throughout the leaf mesophyll, or other changes in mesophyll structure, such as V_mes/V, have not been measured. Similarly, while the distribution of chlorophyll within dorsiventral leaves has been measured (Terashima, 1989; Nishio, Sun, and Vogelmann, 1993), no such measurements exist for isobilateral leaves, to our knowledge.

Maximum photosynthesis of numerous species has been found to increase linearly with whole-leaf A_mes/A, often without changes in the intrinsic photosynthetic capacity of the cells (El-Sharkawy and Hesketh, 1965; Nobel, Zaragoza, and Smith, 1975; Chabot and Chabot, 1977; Bjo¨rkman, 1981; Nobel and Walker, 1985). Such increases in A_mes/A have been reported to occur in response to increasing sunlight exposure and the development of the classical “sun” leaf anatomy and morphology (Nobel, 1976). As shown for several other species (Turrell, 1965; Nobel, Zaragoza, and Smith, 1975; Nobel, 1976; Chabot and Chabot, 1977; Osborne and Raven, 1986), a strong relationship was found between A_mes/A and leaf thickness of E. globulus leaves. The value of leaf thickness at zero mesophyll thickness was equivalent to the summed upper and lower epidermises of the eucalypt leaves, as also found for Plectranthus parviflorus (Nobel, Zaragoza, and Smith, 1975). However, the summed thicknesses of adaxial and abaxial palisade layers showed a stronger relationship with A_mes/A than did total leaf thickness. Thus, development of palisade mesophyll appeared more important in increasing A_mes/A than the development of spongy mesophyll, or a change in the size or shape of the individual cells.

Whole-leaf A_mes/A varied between 10 and 40 for leaves of many plant species with strong correlations between low values and less light exposure, both between and within species (Bjo¨rkman, 1981; Nobel and Walker, 1985). The values of A_mes/A found here for the three ontogenetically and structurally different E. globulus leaf types were within the range found for other studies of intraspecific variation among individuals, or within the same individual (El-Sharkawy and Hesketh, 1965; Chabot and Chabot, 1977; Chabot, Jurik, and Chabot, 1979; Bjo¨rkman, 1981). The dorsiventral leaves of Fragaria species, when grown under different light regimes, had a leaf thickness of 74–179 μm, an A_mes/A from 9 to 26, a V_mes/A from 0.08 to 0.021 mm³/mm², and a V_mes/V from 0.27 to 0.56 (Chabot and Chabot, 1977; Chabot, Jurik, and Chabot, 1979). As discussed below, the juvenile leaves of E. globulus, although slightly thicker, had values very similar to these. For a range of dicotyledonous leaves, A_mes/A was found to be low in shade leaves (6.8–9.9), transitional in mesomorphic leaves (11.6–19.2) and high in xeromorphic sun leaves (17.2–31.3) (Turrell, 1936; Esau, 1965). Similarly, Nobel (1991) classified xerophytes as having an A_mes/A from 20 to 50. Therefore, all three types of E. globulus leaves could be classified as being xeromorphic, with the juvenile leaf type being slightly more mesomorphic in character than the transitional or adult leaf types.

A_mes/A has been shown to increase proportionally with increasing total daily PAR during leaf development, accompanied by corresponding increases in leaf thickness, a greater number of cell layers, greater specific leaf mass (due to increased V_mes/A and sclerophylly), a higher proportion of palisade cells, and hence, a higher photosynthetic rate per unit leaf area (Nobel, 1976; Bjo¨rkman, 1981; Nobel and Walker, 1985). Within sun leaves, palisade cells are often longer, with A_mes/A being 2–4 times larger than for shade leaves (Bjo¨rkman, 1981). In E. globulus, A_mes/A was 2.4 times greater in adult than juvenile leaves due to multiple layers of palisade mesophyll within the thicker adult leaf. Although each E. globulus leaf developed under similar light regimes, light interception by individual leaves was largely dictated by leaf orientation. Juvenile leaves were typically oriented horizontally while adult leaves were inclined almost vertically. An inclined leaf orientation decreases radiation absorption at midday and, thus, heat load and possible radiation damage (Nobel and Walker, 1985). Moreover, internal leaf structure may be strongly influenced by the relative incidence of sunlight between the adaxial and abaxial faces, not necessarily the total amount of intercepted sunlight alone (Smith, Bell, and Shepherd, 1997).

Chlorophyll

Within dorsiventral leaves, chlorophyll content has often been found to increase from the adaxial epidermis to the palisade mesophyll, and then decline through the spongy mesophyll (Terashima and Saeki, 1983; Cui, Vogelmann, and Smith, 1991; Nishio, Sun, and Vogelmann, 1993). Chlorophyll concentration of palisade tissue was found to be 1.5–2.5 times that in the spongy mesophyll of Camellia leaves (Terashima and Saeki, 1983), while the chlorophyll concentrations within the palisade cells of E. globulus were about 1.6 times that found in spongy mesophyll cells. Also, a similar chlorophyll content was found within the adaxial palisade of both sun and shade leaves of spinach (Cui, Vogelmann, and Smith, 1991).

Shade leaves tend to have less chlorophyll on an area basis and a lower ratio of chlorophyll a:b (e.g., Nobel, Forseth, and Long, 1993). Also, the chlorophyll a:b ratio has often been found to decline through dorsiventral leaves (Terashima and Inoue, 1984; Terashima, 1989; Cui, Vogelmann, and Smith, 1991; Nishio, Sun, and Vogelmann, 1993; Terashima and Hikosaka, 1995), as reported here for the dorsiventral, juvenile leaves of E. globulus. Light incident on the adaxial leaf surface of dorsiventral leaves has been observed to be rapidly attenuated in the upper 20% of the leaf (Nobel, Forseth, and Long, 1993), corresponding to the decrease in chlorophyll a:b from the adaxial to abaxial epidermis (Terashima, 1989). It has been proposed that the photochemical properties of palisade chloroplasts in dorsiventral leaves may be acclimated to higher light, while those of spongy chloroplasts are acclimated to lower light (Terashima and Inoue, 1984). In contrast, the isobilateral leaves of adult E. globulus had maximum values of the chlorophyll a:b ratio just beneath each of the adaxial and abaxial epidermises, with minima occurring in the spongy mesophyll near the middle of the leaf thickness.

Photosynthetic impact

Structure may be important in the internal processing of light and CO₂ (Terashima and Hikosaka, 1995; Vogelmann, Nishio, and Smith, 1996) and maximization of photosynthesis per unit leaf biomass (Smith et al., 1997; Smith, Bell, and Shepherd, 1998). In this regard, correspondence between greater A_mes/A with increased leaf thickness and number of stomata on the adaxial leaf surface reported here acts to couple internal absorption capability with the suppy of CO₂, respectively (Smith et al., 1997). Also, crucial to this hypothesis is the idea that internal light absorption profiles should correspond to CO₂ absorption capabilities within the mesophyll. The correspondence of A_mes/A with chlorophyll concentrations throughout the mesophyll is further evidence for a functional relationship between leaf structure and photosynthetic performance. There is also some evidence that maximum light absorption within the mesophyll is matched by maximum chlorophyll concentrations (Nishio, Sun, and Vogelmann, 1993; Evans, 1995), possibly the result of light focusing by lens-like epidermal cells and greater light propagation into the leaf interior via columnar, light-channeling palisade cells (Vogelmann and Martin, 1993; Vogelmann, Nishio, and Smith, 1996; Smith et al., 1997). Moreover, the greatest light absorption in E. globulus occurred at the same location in the spongy mesophyll (just below the palisade cell layers) where A_mes/A and chlorophyll concentrations were also maximal.

Palisade cells are usually contiguous with the upper epidermis over an extended area where incident light enters the mesophyll without being interrupted by airspaces (Haberlandt, 1914; Terashima and Hikosaka, 1995). Within E. globulus leaves, A_mes/A was greatest at the base of the first palisade layer rather than directly beneath the adaxial epidermis, especially for the dorsiventral, adult leaves. Similarly, in a preliminary study of spinach leaves, Terashima and Hikosaka (1995) observed that maximum A_mes per unit leaf thickness did not occur in the most adaxial part of the leaf, but near the transition between the first and second palisade cell layers. This is in contrast to the steep light gradient found at the adaxial leaf surface, but might explain the subsurface maximum of Rubisco abundance and the low chlorophyll concentration adjacent to the adaxial epidermis of dorsiventral leaves (Nishio, Sun, and Vogelmann, 1993).

Structural impact

Cell packing, or V_mes/V, in E. globulus increased beneath the adaxial and abaxial epidermises, possibly indicating a structural support function of this first cell layer, rather than acting primarily to enhance photosynthetic performance. That is, the reduction in A_mes/A and chlorophyll, with increased V_mes/V within the first cell layer may reflect a loss in photosynthetic function in favor of an increase in structural support within the leaf. The mechanical strength of parenchyma is maximized when the cells are closely packed together, with little interstitial volume (Niklas, 1992). Palisade mesophyll is generally 10–40% air by volume, while spongy mesophyll is 50–80% air (Nobel, 1991; Nobel, Forseth, and Long, 1993). Palisade mesophyll within the E. globulus leaves was found to have only 15–21% airspace by volume, while spongy mesophyll was 32–44% airspace. Although greater than the palisade mesophyll, the volume of intercellular airspace within the spongy mesophyll is less than that reported for other leaves, possibly indicating a relatively high structural integrity in leaves of E. globulus, potentially an adaptation to water stress. Because the mechanical strength of parenchyma cells also depends on their water content, the stiffness of leaves may be less impacted by tissue dehydration (wilting) when they have a lower volume of spongy relative to palisade mesophyll.

Ecological implications

Many species within the Australian genus Eucalyptus produce leaves that show both structural and orientational changes through ontogenetic development. The advantage of ontogenetic changes in this genus is unclear, but has been widely speculated to be a response to the changing light environment during natural establishment and growth (Cameron, 1970; Stoneman, 1994). The juvenile leaves of E. globulus within this study had a significantly higher chlorophyll a:b ratio and a greater content of chlorophyll on a unit volume basis than the ontogenetically older adult leaf type. Because the orientation of the juvenile leaf is horizontal and that of the adult leaf is vertical, the interception of radiation by the adult leaf type is reduced, resulting in a leaf chlorophyll content more characteristic of a shade leaf. Cameron (1970) also measured the characteristics of E. fastigata leaves with ontogenetic change and found total chlorophyll on a unit leaf area basis to decrease from the juvenile leaf type to the transitional leaf type, although no measurements were given for the adult leaf type. This is in contrast to E. globulus, in which chlorophyll content per unit leaf area increased from the juvenile to adult leaves. Similarly, the chlorophyll a:b ratio increased from the juvenile to transitional leaf types of E. fastigata, but decreased for the E. globulus leaves measured here. Differences in juvenile leaf chlorophyll characteristics between these two species may be due to the light environment under which the two species establish. E. fastigata typically germinates beneath a shady forest canopy (Cameron, 1970), while E. globulus regenerates only after the overstory has been removed (Stoneman, 1994).

Isobilateral leaves are typically amphistomatous and often occur where both leaf surfaces receive substantial levels of light (Nobel and Walker, 1985). In a survey of five Australian plant communities, significant relationships were found between leaf inclination and thickness, the presence of isobilateral palisade, bicoloration, and amphistomy (Smith, Bell, and Shepherd, 1998). Amphistomy was also found to be strongly correlated with leaf thickness, and all of these characteristics were significantly related to differences in total daily sunlight characteristics of the community. Similar relationships were found between leaves of E. globulus, indicating that the difference in leaf anatomy may be most strongly influenced by light availability, which is strongly influenced by leaf orientation. Adult leaves of E. globulus may be better adapted to water stress than the juvenile leaves (Ito and Suzaki, 1990; Chalmers, 1992; Potts and Jordan, 1994). Low soil water potential leads to smaller leaves, smaller mesophyll cells, and often more layers of palisade cells. The greater number of layers of mesophyll cells under drought conditions led to an increase in A_mes/A of 40–50% for a range of species (Nobel and Walker, 1985). Smith and Nobel (1978) also found that, although total daily photosynthetically active radiation was the primary influence on A_mes/A, temperature and, especially, water stress were also important.

Conclusions

The three main leaf types produced by E. globulus (ssp. globulus) follow the model proposed for different leaf types observed for different species based on the generalized stress and light levels of their native habitats (Smith et al., 1997; Smith, Bell, and Shepherd, 1998). As reported here for E. globulus, leaves on the same plant produced at different stages of ontogenetic development also appear to have similar correspondence between leaf orientation and structural characteristics. The observation that maximum values of cell surface area for CO₂ absorption (A_mes/A) and chlorophyll contents overlap inside the leaf, and also at the location of the potentially greatest light availability, provides more mechanistic evidence that internal leaf structure may enhance photosynthetic capability. However, the occurrence of maximum values of mesophyll cell volume per unit leaf volume (V_mes/V) at locations near the epidermises shows that other structural features of the mesophyll may be important for functions such as mechanical support (e.g., sclerophylly).