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; [email protected] ).