2Department of Botany, University of Wyoming, Laramie, Wyoming 82071-3165; and 3Department of Botany, The University of Western Australia, Nedlands, W.A. 6907, Australia
Received for publication January 6, 1998. Accepted for publication June 16, 1998.
Mesophyll structure has been associated with the photosynthetic performance of leaves via the regulation of internal light and CO2 profiles. Differences in mesophyll structure and chlorophyll distribution within three ontogenetically different leaf types of Eucalyptus globulus ssp. globulus were investigated. Juvenile leaves are blue-grey in color, dorsiventral (adaxial palisade layer only), hypostomatous, and approximately horizontal in orientation. In contrast, adult leaves are dark green in color, isobilateral (adaxial and abaxial palisade), amphistomatous, and nearly vertical in orientation. The transitional leaf type has structural features that appear intermediate between the juvenile and adult leaves. The ratio of mesophyll cell surface area per unit leaf surface area (Ames/A) of juvenile leaves was maximum at the base of a single, adaxial palisade layer and declined through the spongy mesophyll. Chlorophyll a + b content showed a coincident pattern, while the chlorophyll a:b ratio declined linearly from the adaxial to abaxial epidermis. In comparison, the mesophyll of adult leaves had a bimodal distribution of Ames/A, with maxima occurring beneath both the adaxial and abaxial surfaces within the first layer of multiple palisade layers. The distribution of chlorophyll a + b content had a similar pattern, although the maximum ratio of chlorophyll a:b occurred immediately beneath the adaxial and abaxial epidermis. The matching distributions of Ames/A and chlorophyll provide further evidence that mesophyll structure may act to influence photosynthetic performance. These changes in internal leaf structure at different life stages of E. globulus may be an adaptation for increased xeromorphy under increasing light exposure experienced from the seedling to adult tree, similar to the characteristics reported for different species according to sunlight exposure and water availability within their native habitats.
Key Words: Ames/A • chlorophyll • Eucalyptus • leaf structure • light • Myrtaceae • palisade mesophyll • Vmes/V
Source: American Journal of Botany. 1999;86:198-207.
Plants can respond to different levels of sunlight exposure by altering characteristics of internal leaf structure such as the amount and distribution of palisade and spongy mesophyll tissue, as well as external leaf structure. Recently, many of these structural changes have been associated with the regulation of light and CO2 profiles inside leaves (Parkhurst and Mott, 1990 ; Parkhurst, 1994 ; Terashima and Hikosaka, 1995 ; Vogelmann, Nishio, and Smith, 1996 ), and it has been proposed that these structural changes have evolved in concert with properties of leaf orientation, maximizing photosynthesis per unit leaf biomass (Smith et al., 1997 ; Smith, Bell, and Shepherd, 1998 ). For example, the total surface area of mesophyll cells exposed to intercellular airspace (Ames) beneath a unit area of one leaf surface (A) has been shown to have a strong influence on photosynthesis per unit leaf area (Nobel, Zaragoza, and Smith, 1975 ; Nobel and Walker, 1985 ), although some species do not appear to have a strong association between Ames/A and photosynthetic gas exchange (Araus et al., 1986 ; Syvertson et al., 1995 ). This ratio of the internal to external leaf area (Ames/A) represents the area available for internal CO2 absorption beneath a unit area of leaf surface available for light absorption and CO2 uptake from the atmosphere (Björkman, 1981 ; Nobel and Walker, 1985 ; Nobel, 1991 ). Ames/A can respond strongly to the amount of incident sunlight (Nobel, 1991 ) and other environmental conditions (Smith and Nobel, 1978 ). Other studies have shown contrasting results (Araus, et al., 1986 ; Buisson and Lee, 1993 ; Syvertsen et al., 1995 ).
Leaf anatomy and morphology in Eucalyptus globulus ssp. globulus Labill (Tasmanian blue-gum) change dramatically with development from the seedling to adult tree. This vegetative phase change consists of a shift from juvenile to adult leaf type and generally begins at 1–3 yr of age. Juvenile leaves of young seedlings ( in structure, hypostomatous, and approximately horizontal in orientation (Johnson, 1926 ; Jacobs, 1955 ; Penfold and Willis, 1956 ; Pryor, 1976 ; FAO, 1979 ). In contrast, adult leaves of mature trees (>5 yr old) are dark green in color, isobilateral, amphistomatous, and pendent. Transitional leaves are produced over a range of nodes and have structural features that are intermediate between juvenile and adult leaves.
The present study measured Ames/A, chlorophyll content, and related structural parameters incrementally throughout the mesophyll of all three, ontogenetic leaf types in E. globulus using light microscopy and image analysis. The correspondence between mesophyll structure, particularly Ames/A, and chlorophyll content at specific locations within the mesophyll should provide further evidence for the evolution of a concerted interaction between leaf orientation and internal leaf structure for photosynthetic enhancement (Smith et al., 1997 ).
Mesophyll anatomy, chlorophyll distribution, and other structural characteristics that could influence photosynthetic CO2 assimilation were evaluated for three ontogenetically and structurally different leaf types found in E. globulus. In addition to Ames/A and chlorophyll concentrations, mesophyll cell surface area and volume per unit volume (Ames/V and Vmes/V, respectively), mesophyll cell volume per unit leaf surface area (Vmes/A), and the chlorophyll a:b ratio were also measured. All of these internal leaf parameters were measured on a whole-leaf basis, as well as incrementally across the entire leaf thickness.
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.
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 300x 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.
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 ; Kubinová, 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 (Ames) 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 750x 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 (Am), intercellular airspace (Aa), and the length of mesophyll cell wall exposed to intercellular airspace (Pi) was determined for each image using the Image-Pro Plus software. Ames/A was then calculated for each image as
and where Ti is the projected thickness of a given section i, Tm is the mesophyll thickness of the leaf determined from transverse sections of the same leaf, Li is the length of the image, Ap is the projected area of the image, Lp is the projected length of the image, and Wi is the width of the image. C is a conversion factor for Pi, which gives the projected mesophyll perimeter. The sum of Li for each of the leaf images gives the total transect length of the oblique-paradermal section (Ls), while the sum of Lp gives the projected length of Ls. Where vascular tissue or oil cavities were present within the mesophyll of the image, Ap was computed as a proportion of the projected area of an image containing only mesophyll. Hence, Ames/A for the leaf was calculated as the sum of the individual values for each image or
Vmes per unit leaf area (Vmes/A) was calculated for each leaf by substituting Pi with Am in Eq. 1, and Ames/A with Vmes/A in Eq. 5. In the same manner, substituting Am for Pi in Eq. 1 enables calculation for Ames per unit leaf volume (Ames/V), or
Similarly, the volume of mesophyll per unit leaf volume (Vmes/V) was calculated by substituting Am for Pi 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.
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.
Ames/A, Ames/V, and Vmes/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 Vmes/V was no different between the three leaf types.
Leaf thickness was significantly and positively related to Ames/A (r2 = 0.96, P Vmes/A (r2 = 0.92, P However, Ames/A was even more strongly correlated with the sum of adaxial and abaxial palisade mesophyll thicknesses (r2 = 0.99, P Ames/A was also positively associated with the ratio of adaxial to abaxial stomatal density (r2 = 0.80, P leaf thickness and a higher ratio of adaxial to abaxial stomatal density (r2 = 0.84, P
The three leaf types of E. globulus had different patterns in Ames/A (Fig. 2A) and chlorophyll distribution (Fig. 2B). However, chlorophyll a + b concentrations mirrored Ames/A distribution for all leaf types. The dorsiventral, juvenile leaves had a single maximum value of Ames/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 Ames/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). Ames/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 Ames/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 Ames/A occurred within each of the adaxial and abaxial palisade layers. However, the maximum value of Ames/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 Ames/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 Ames/V distribution throughout the leaf interior was similar to Ames/A for each of the leaf types. In contrast, Vmes/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 Vmes/V indicated a low volume of intercellular airspace and greater cell packing. As found for Ames/A, Vmes/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 Vmes/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 Vmes/A (Table 3). No difference was found between leaf types in the length of the first adaxial palisade layer (Table 1), or in Ames/A, Vmes/A, or Ames/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 Vmes/V, was found within the spongy mesophyll of the adult leaf type (Table 3).
Most studies of mesophyll structure have described changes in Ames/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 Ames/A has been measured often, incremental changes in Ames/A throughout the leaf mesophyll, or other changes in mesophyll structure, such as Vmes/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 Ames/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 ; Björkman, 1981 ; Nobel and Walker, 1985 ). Such increases in Ames/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 Ames/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 Ames/A than did total leaf thickness. Thus, development of palisade mesophyll appeared more important in increasing Ames/A than the development of spongy mesophyll, or a change in the size or shape of the individual cells.
Whole-leaf Ames/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 (Björkman, 1981 ; Nobel and Walker, 1985 ). The values of Ames/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 ; Björkman, 1981 ). The dorsiventral leaves of Fragaria species, when grown under different light regimes, had a leaf thickness of 74–179 µm, an Ames/A from 9 to 26, a Vmes/A from 0.08 to 0.021 mm3/mm2, and a Vmes/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, Ames/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, 193 6; Esau, 1965 ). Similarly, Nobel (1991) classified xerophytes as having an Ames/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.
Ames/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 Vmes/A and sclerophylly), a higher proportion of palisade cells, and hence, a higher photosynthetic rate per unit leaf area (Nobel, 1976 ; Björkman, 1981 ; Nobel and Walker, 1985 ). Within sun leaves, palisade cells are often longer, with Ames/A being 2–4 times larger than for shade leaves (Björkman, 1981 ). In E. globulus, Ames/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 ).
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.
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 Ames/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 Ames/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 Ames/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, Ames/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 Ames 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 ).
Cell packing, or Vmes/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 Ames/A and chlorophyll, with increased Vmes/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.
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 Ames/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 Ames/A, temperature and, especially, water stress were also important.
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 (Ames/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 (Vmes/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).
1 This research was supported by NSF IBN-4604497 (WKS and TCV) and a Jean Gilmore Bursary awarded to SAJ by The Australian Federation of University Women (South Australia). Plant seed was provided by Department of Conservation and Land Management, W.A. Research was completed in partial fulfilment of PhD (SAJ) from The University of Western Australia.
4 Author for correspondence (Tel: 61 8 9389 9926; FAX: 61 8 9380 1001; firstname.lastname@example.org ).
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Figure Abbrevations: Ad, adaxial; Ab, abaxial; E, epidermis; P, palisade mesophyll; S, spongy mesophyll. Fig. 1. Illustrated view of an oblique-paradermal and transverse leaf section, and their relationship for a dorsiventral leaf type. The oblique-paradermal section has been divided into a series of images and the variables used to determine Ames/A, Ames/V, and Vmes/V are indicated. See text for explanation of variables used in the computation of mesophyll parameters.
Fig. 2. (A) Internal profile of Ames/A and (B) chlorophyll concentrations from the adaxial to abaxial epidermis for juvenile, transitional and adult leaves of Eucalyptus globulus ssp. globulus. Values are means ± SE for 3–7 leaves of each leaf type.
Fig. 3. (A) Internal profile of chlorophyll a:b and (B) the ratio of mesophyll cell volume per unit mesophyll volume (Vmes/V) from the adaxial to abaxial surface of juvenile, transitional, and adult leaves of Eucalyptus globulus ssp. globulus. Values are means ± SE for 3–5 leaves of each leaf type.
Table 1. Morphological and anatomical properties of juvenile, transitional, and adult leaves of Eucalyptus globulus ssp. globulus. Values are mean ± SE for five representative leaves. Statistical differences between the three leaf types for each characteristic are indicated (ANOVA).
Table 2. Total chlorophyll concentrations and mesophyll characteristics of juvenile, transitional, and adult leaf types in Eucalyptus globulus ssp. globulus. Values are mean ± SE for five leaves (mesophyll characteristics) or four leaves (chlorophyll characteristics; five for intermediate leaves). Statistical differences among the three leaf types for each characteristic are indicated (ANOVA).
Table 3. Characteristics of the first adaxial palisade layer and spongy mesophyll layer for the juvenile, transitional, and adult leaf types of Eucalyptus globulus ssp. globulus. Values are mean ± SE for 9–24 measurements. Statistical differences among the three leaf types for each characteristic are indicated (ANOVA).