*3.1. C4 Development*

Research has been performed on the physiological and structural development of C4 photosynthesis in cotyledons of the *Portulaca* but little of CAM development in these C4 species [23]. The research presented here indicated it was apparent that the C4 pathway was well developed in 10 days old cotyledons based on the anatomy and physiology. The leaf anatomy of the cotyledons showed a well-developed Kranz C4 anatomy as found previously in the genus *Portulaca* [23]. *P. grandiflora* cotyledons displayed an Atriplicoid-type anatomy where the vascular bundles are in one plane of the leaf. The Atriplicoid Kranz anatomy showed CAM tissues around the periphery of the leaf surrounding the Kranz bundles. This differs from the Pilosoid Kranz anatomy found in mature leaves of *P. grandiflora* with the C4 tissue in a ring surrounding the CAM water storage tissue [2,20].

The enzymes of the C4 pathway were fully functional at 10 days, as shown by a high activity of PEPCase and NADP-ME. This was comparable to the conclusions reached by Dengler et al. [24] for *Atirplex rosea*, which showed accumulation of PEPCase was detected 2–4 days after leaf development and expansion. The expression was limited to mesophyll tissue adjacent to the bundle sheath tissue. The JO2 studies in this study showed high rates of photosynthesis indicative of C4 activity and were comparable to the rates previously measured in mature leaf tissue [20]. The C4 pathway is the primary CO2 acquiring pathway in this genus and it was predictable the C4 pathway developed quickly for seedling establishment.
