*3.2. CAM Development*

The development of CAM, which can be measured by titratable acidity levels and diurnal acid fluctuations, showed a slower development in cotyledons from that of the C4 pathway. The study of CAM development showed comparable titratable acidity levels but slightly lower and comparable to mature leaf tissues under control conditions [20]. One can measure significant total titratable acidity levels in the cotyledons at 10 and 25 days. The acid levels found in the 10 days old cotyledons of *P. grandiflora* are similar but larger than those found in well-watered cotyledons of *Mesembryanthemum crystallinum*, a facultative CAM species [25], and higher than the acid levels in the CAM-cycling species, *Lewisia cotyledon* [26]. The acid levels of *P. grandiflora* were lower than those found in a number of columnar cactus seedlings at 1 day and 7 days old [27]. The acid levels were lower than those for *Opuntia elatior* at 23 days old cotyledons [28]. However, these cactus species primarily utilize the CAM pathway for CO2 uptake, even in cotyledons. The cotyledon anatomy in cactus has succulent anatomy with a spongy parenchyma tissue with little airspace, which is more conducive to CAM photosynthesis [29,30]. The results in the present study indicated *P. grandiflora* cotyledons showed similar total titratable acid levels comparable to other CAM species.

We investigated the potential for CAM activity by water stressing the cotyledons for three and seven days. Our results differed with the age of the cotyledons and duration of the drought. Ten days old cotyledons showed small acid fluctuations indicative of CAM activity after three days of water stress. The 25 days old water-stressed cotyledons showed a small acid fluctuation compared to control cotyledons which showed no acid fluctuation. Continuation of water stress for seven days induced a very large and significant CAM acid fluctuation in both cotyledons and primary leaf tissue. The acid fluctuation measured in the cotyledons was comparable to acid levels in mature leaf tissue. This indicated an induction of CAM similar to previous research on *P. grandiflora* [20] and was indicative of CAM activity.

In addition, enzyme activity, as measured by nocturnal PEPCase activity, was quite high and comparable to C4 rates of PEPCase measured during the day. Water stress only had a slight effect on the overall activity of PEPCase. NADP-ME was affected more by water stressed conditions and this result had been observed previously in *P. grandiflora* [20]. Water stress in other facultative CAM plants, such as *Portulacaria afra*, show the decarboxylase enzymes are more affected than PEPCase [31]. The enzyme activities of PEPCase and NADP-ME reported here in cotyledons of *P. grandiflora* were much higher than those reported for cotyledons of *Salsola* spp. [32] and for mature leaves of *P. grandiflora* [20] and may be related to the lower chlorophyll concentrations.

The physiological and anatomical changes differed with the age. At 10 days, water stressing the cotyledons induced a small acid fluctuation and it lowered both the day and night activity of PEPCase. NADP-ME activity also decreased in 10 days old cotyledons. This indicated a decrease in C4 and CAM activity. It appears that NADP-ME is more sensitive to water stress than Rubisco and PEPCase. Anatomically, the mesophyll CAM tissue showed some shrinkage, which may be due to a redistribution of water from the CAM cells to the C4 metabolic cells. This has been observed in mature leaves of *P. grandiflora* during a 10 days drought [20]. The sensitivity of the enzymes due to drought at 10 days may be due more in part to structural development of the CAM tissue and water storage at this stage. The enzymatic activity appeared earlier prior to full anatomical development of the CAM tissue. This is similar to the development of C4 photosynthesis in *A. rosea*, which showed differential enzyme expression of the C4 photosynthetic enzymes by 4 days [24].

By 20–25 days old, the response to water stress and CAM activity was similar to the 10 days old cotyledons. There was a similar induction of an acid fluctuation in water-stressed plants. Seven days of water stress induced a much larger acid fluctuation indicative of CAM activity and greater than observed after three days of water stress. This acid fluctuation was similar to acid fluctuations in other CAM-cycling species [10,20]. The results indicated an induction of CAM activity similar to control plants of *Lewisia cotyledon* [26]. Induction of water stress did not lower PEPCase activity in 25 days old cotyledons compared to 10 days old cotyledons. NADP-ME did not show a decrease in activity due to water stress and showed a slight increase in activity, which may be due to increased CAM activity of the leaf. The enzyme activity indicated a maintenance of the C4 pathway in the 25 days old cotyledons. This was supported by the JO2 data, which showed no changes in activity during three days of drought. Structurally, the CAM/hydrodermal tissue was more mature in the 25 days old cotyledons and did not appear to show as much water loss as the 10 days old cotyledons. This may explain the ability of older cotyledons to maintain C4 activity when compared to 10 days old cotyledons. The continuation of drought showed a much stronger induction of CAM and was very similar to the response of mature leaves [20]. The CAM tissue in mature leaves recycles CO2, redistributes water to the C4 tissue to aid in the survival of the plant, and may play a similar role in cotyledons [20].

Mature leaves of *P. grandiflora* have a Pilosoid-type anatomy with a ring of bundles surrounding the water storage tissue [2,20]. *P. grandiflora* cotyledons have an Atriplicoid-type anatomy where the vascular bundles are in one plane of the leaf. This arrangement is considered ancestral to the Pilosoid arrangement with the water storage tissue found on both the abaxial and adaxial sides of the leaf [33]. Voznesenskaya et al. [2] reported on the diversity of structure in the *Portulaca* and found the arrangement of an inner water storage, the derived condition found in *P. grandiflora*, *P. pilosa*, and *P. amilis* [2], to be due to the ecological constraints of being found in more semi-arid regions than other *Portulaca* spp. However, they did not report any CAM activity in the cotyledons of the *Portulaca spp*. in their study. Further, we report the presence of glycine decarboxylase, a mitochondrial marker for the C2 photorespiratory pathway in the CAM tissue of cotyledons. Previous research has shown the GDC to be localized in the mitochondria of bundle sheath cells in cotyledons [2]. The presence of GDC adds another dimension to another function of CAM in *P. grandiflora*. Previous work indicated CAM tissue might transfer water from the tissue to the Kranz anatomy to help maintain C4 photosynthetic activity under water stress. Additionally, decarboxylation of the acid may produce CO2 for the adjacent mesophyll and bundle sheath tissue. We now suggest the C2 pathway may provide an additional source of CO2 for the C4 pathway. It was shown the photorespiratory pathway may elevate CO2 levels three-fold in the leaf in *Flaveria pubescens*, (a C3-C4 intermediate) [34]. The photorespiratory pathway in the CAM tissue of *P. grandiflora* may provide an additional source of CO2 for the C4 pathway. Since CAM tissue has reduced airspace, the Atriplicoid arrangement may reduce diffusion of water out of the leaf and maintain CO2 levels around the Kranz anatomy. This may aid in survival of the seedlings. Further work will be needed to investigate the role of photorespiration in the different Kranz types CAM tissue in *P. grandiflora*.

This is the first report of CAM activity in the cotyledons and the results indicated CAM can be induced in cotyledons of *P. grandiflora*. The development of CAM occurred after the development of the C4 photosynthetic pathway and may be due to more of a constraint of leaf anatomy of the CAM tissue in cotyledons. The retention of CAM in the cotyledons of *P. grandiflora* further supports the idea that CAM was an ancient pathway in the genus *Portulaca* including the retention of the C2 pathway. Since the cotyledons have a different type of Kranz anatomy and water storage tissue than the mature leaves in *P. grandiflora*, this leads to questions on the evolution of C4 and CAM within the genus *Portulaca*. Research on the origins of CAM in *Portulaca* using PEPCase have indicated the CAM-specific gene was similar in sequence in other species utilizing CAM [22]. The question that arises: Could C4 photosynthesis have evolved from the CAM cells? Christin et al. [22] have shown the C4 specific PEPCase gene appears to have evolved from a non-photosynthetic form rather than the CAM form. They also suggest the other enzymes required for C4 photosynthesis may not have required new genes but utilization of genes already present in the cell. Since CAM cells in the *Portulaca* retain all features of C3 and CAM photosynthesis including the C2 pathway, it maybe that novel cells types of the mesophyll and bundle sheath may have differentiated from CAM-like cells. During the slow evolution of C4 photosynthesis as suggested by Christin et al. [22], a change in the regulatory sequences of PEPCase and GDC would be required as the cells evolved. Current research does not support the idea but questions remain on the change from a CAM-type leaf structure evolved into a C4 photosynthetic leaf in *Portulaca.*
