*2.3. Electron Transport Rate*/*JO2*

The JO2 rates showed no differences between control and water-stressed plants in 10 days old and 25 days old cotyledons (Figure 5). The JO2 rates were lower in 25 days old cotyledons than in the 10 days old cotyledons.

**Figure 5.** JO2 rates of leaf samples for 10 days and 25 days control and water-stress treatments. Error bars represent one standard error of the mean. N = 12–14 leaves per time point.

The gross rate of photosynthesis (JO2) showed a peak at midday for the well-watered 10 days old cotyledons while the stressed cotyledons showed a peak earlier in the morning (Figure 5). In the 25 days old cotyledons, the JO2 calculated rates were relatively constant over the course of the light period. There was no difference between the control and water-stressed plants in the JO2 rates (Figure 5).

#### *2.4. Leaf Anatomy*

*Portulaca grandiflora* cotyledons were dissected and photographed to display the internal gross anatomy of the tissue (Figure 6). The sections illustrate the Atriplicoid Kranz anatomy in the cotyledon (Figure 6A), and its linear arrangement compared to a mature leaf where the Kranz anatomy shows a radial arrangement (Figure 6B). A low-magnification view of a cotyledon leaf tissue showed a well-developed Atriplicoid Kranz anatomy arranged in a linear fashion (Figure 7). Mesophyll cells were well developed with chloroplast along the cell wall (Figure 7A). The bundle sheath cells were also well developed with chloroplasts located along the periphery of the inner cell wall close to the vascular bundles (Figure 7A). The CAM/hydrodermal tissue showed fewer chloroplasts than the C4 tissue (Figure 7A). At 25 days, the Kranz anatomy was very well developed and the vascular tissue was more prominent. Under water-stress conditions, the CAM/hypodermal tissue at 10 days showed signs of water loss compared to the control cotyledon tissue. The cells appeared to have some shriveling and a more irregular shape. At 25 days, there were less noticeable changes under water-stress conditions in the succulent mesophyll and hypodermal tissue (Figure 7).

**Figure 6.** Cross section of 1.7× magnification of (**A**) ~20 days old cotyledon and (**B**) mature leaf of *P. grandiflora*. The dark green bundles within the leaf tissue are the C4 Kranz anatomy with high levels of chlorophyll. The lighter areas are the CAM/hydrodermal tissue.

**Figure 7.** Light microscope images of cotyledons of *P. grandiflora* at low magnification, (**A**) 10 days control, (**B**) 10 days water stress, (**C**) 25 days control, (**D**) 25 days water stress. Bars = 50 μm, VB = vascular bundle, BS = bundle sheath, M = mesophyll, CAM = hypodermal tissue.

The sections illustrate the Atriplicoid Kranz anatomy in its linear arrangement compared to a mature leaf where the Kranz anatomy shows a radial arrangement (Figure 6B). We performed tissue prints of cotyledons to determine the presence of glycine decarboxylase (GDC, a mitochondrial marker for the photorespiratory pathway). GDC was found to be present in the CAM/hydrodermal tissue of *P. grandiflora* cotyledons (Figure 8B). In mature leaves of *P. grandiflora*, GDC was also located in the inner CAM/hydrodermal tissue (data not shown). For comparison, *Portulacaria afra*, a facultative CAM species, showed the presence of GDC throughout the spongy parenchyma mesophyll tissue (8b).

**Figure 8.** Glycine decarboxylase (GDC) protein presence by tissue printing. (**A**) Cotyledon leaf samples were sectioned and photographed, then printed onto nitrocellulose, incubated with GDC antibody, and visulaized. The arrow points to the bundle sheath tissue which showed more GDC protein. The GDC protein was also found present in the surroounding succulent CAM tissue. (**B**) *Portulacaria afra*, a facultative CAM species, leaf tissue print with the arrow indicating GDC antibody found throughout the spongy parenchyma tissue.
