**1. Introduction**

CO2 concentrating mechanisms have evolved in terrestrial plants in response to changing environmental conditions. Two different mechanisms have evolved that involve a similar suite of enzymes utilized in a different fashion to overcome photorespiration and increased water loss. Photorespiration increases when CO2 becomes limited under high light intensities and high evaporative demand resulting in increased transpirational water loss [1]. The C4 pathway overcomes these limitations with the CO2 being initially captured as HCO3 - by phosphoenolpyruvate carboxylase (PEPCase) and then fixed via the C3 pathway by Rubisco. C4 plants have a spatial separation of the C4 and C3 pathways occurring within two different cell types in the leaf. The C4 pathway, located in the palisade mesophyll cells, is radially arranged around the C3 pathway located in the bundle sheath cells, which surround the vascular tissue. This is typically referred to as Kranz anatomy [1,2]. Research by Voznesenskaya et al. [3,4] has shown that the Kranz anatomy is not essential for terrestrial C4 photosynthesis to occur but it can occur in a single cell with a spatial separation of the C4 and C3 pathways within a single chlorenchyma cell. The C4 pathway concentrates CO2 at the site of Rubisco

and helps to suppress photorespiration in the bundle sheath cells. The C4 pathway has been found in approximately 19 plant families and over 8000 species [1] and has evolved independently many times.

Crassulacean acid metabolism (CAM) is a metabolic and anatomical adaptation that is characterized by net nocturnal carbon dioxide uptake with a temporal separation of the C4 and C3 pathway resulting in decreased transpiration rates and water loss [5,6]. The CO2 is similarly fixed (as in C4 plants) by PEPCase, converted to malate and stored as malic acid in the large central vacuole during the night period. In the subsequent light period, the malate is transported out of the vacuole and then decarboxylated to release CO2 for utilization by Rubisco in the C3 cycle. CAM plants typically have a mesophyll anatomy with primarily spongy parenchyma cells with a large central vacuole, which has the ability to store the increasing accumulation of malic acid during the nocturnal period [7]. CAM plants anatomically have very low mesophyll airspace, so when the stomata are closed the CO2 concentrations inside the leaf can reach very high levels to suppress photorespiration [6,7]. CAM has evolved in at least 34 different plant families including 6 aquatic families and over 20,000 species [1,8].

CAM and C4 photosynthesis have evolved independently multiple times in many different plant families. One might hypothesize both CAM and C4 could have evolved in the same plant families numerous times due to the similarity of the enzymes involved in the pathways. The two pathways have evolved in the same family four times (Aizoaceae, Asteraceae, Euphorbiaceae, Portulacaceae). This raises an interesting question about why the C4 and CAM pathways have only concurrently evolved in these four families. The original circumscription of the Portulacaceae describes the family as containing approximately 29 genera [9]. Guralnick and Jackson [10] have reported the evolution and distribution of C4 and CAM photosynthesis found in this family. It has been observed that some members are C3 plants, while others are C3 plants with attributes of CAM. Others are C4 plants with some CAM characteristics, and the more advanced species are facultative CAM plants [10–12].

The genus *Portulaca* is known to have the only C4 photosynthetic members of the family despite previous reports to the contrary [11]. This revision reveals the genus *Portulaca* (Portulacaceae) contains the only known C4 members that are capable of CAM photosynthesis [13]. *Portulaca spp.* tend to inhabit environments with high light intensities, which periodically become dry. The genus *Portulaca* has succulent stems and leaves and because of the high degree of succulence, there have been reports in the literature that members of the *Portulaca* show a diurnal acid fluctuation characteristic of CAM species. Koch and Kennedy [14,15] showed *Portulaca oleracea* having a diurnal acid fluctuation in both the stems and leaves. They also measured low levels of net nighttime CO2 uptake. Research done by Guralnick and Jackson [10] showed that *Portulaca mundula*, *P. pilosa*, and *P. oleracea* exhibited high acid levels and diurnal acid fluctuations indicative of CAM photosynthesis. Kraybill and Martin [16] showed both *P. oleracea* and *P. grandiflora* both undergo CAM cycling with little or no nocturnal CO2 uptake. Mazen [17] indicated that under water stress conditions that *P. oleracea* had increased levels of PEP (phosphoenolpyruvate) carboxylase protein. Further research showed the genus contains a C3-C4 intermediate species, *Portulaca cryptopetala*, in which recent work showed *P. cryptopetala* induced CAM under water stress conditions [2,18]. Winter et al. [18] extended the findings to additional species in the *Portulaca* and also consider them to be facultative CAM species.

*Portulaca grandiflora* is a small herbaceous annual utilizing the C4 photosynthetic pathway. *P. grandiflora* has small succulent leaves with a Pilosoid-type Kranz leaf anatomy where the C4 tissue in the succulent leaves surround the large water storage tissue [2,19]. *P. grandiflora* is known to maintain high organic acid levels and shows a large diurnal acid fluctuation when water stressed, typical of CAM species [20]. Research has indicated the increase in CAM of this species occurs in the water storage portion of the leaf and the stem during water stressed conditions [20]. *Portulaca grandiflora* is unique because it has both C4 and CAM photosynthetic pathways operating simultaneously in the leaf tissue [20]. This situation is unique due to the proposed incompatibility of both pathways to operate in the same leaf [21]. Phylogenetic analysis has indicated the genus *Portulaca* evolved CAM from a C3 ancestor prior to the appearance of the C4 pathway [10]. The objective of this study was to study cotyledon leaf tissue to determine if both the CAM and C4 pathways were developing and operating

simultaneously. CAM induction in developing cotyledons was monitored by withholding water for 3 and 7 days. An understanding of the developmental process of these pathways will aid in clarifying the evolutionary origins of the CAM and C4 pathways in the Portulacaceae.
