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.
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.
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 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
Mesophyll profiles
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).
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