*4.6. Tissue Printing*

Cotyledons were cut with a razor and then photographed. The sections were pressed onto nitroceullusoe membranes. Protein localization of Glycine decarboxylase (GDC-H) antibodies was performed according to the method of Guralnick et al. [20]. Rabbit anti-GDC-H antibodies were purchased from Agrisera and diluted to a 1:5000 concentration. The presence of GDC was visualized using a secondary antibody linked to alkaline phosphatase and then photographed. GDC was a mitochondrial marker for the C2 photorespiratory pathway.

## **5. Conclusions**

Research has shown the cotyledons of *Portulaca grandiflora* could be induced to perform CAM similar to mature leaves [20]. The CAM pathway appears to develop later due to anatomical but not physiological aspects of CAM. In the species, *P. grandiflora,* both pathways are present and appear to be operating independently in the cotyledons but CAM still aids the C4 pathway under water-stress conditions. Additionally, the C2 pathway may raise CO2 concentrations inside the cotyledons, aiding in raising the internal CO2 concentrations. More research will be needed to understand the evolution of CAM and C4 photosynthesis in the genus *Portulaca*.

**Author Contributions:** The authors on this manuscript contributed to many portions as undergraduate researchers. Conceptualization, L.J.G.; methodology, L.J.G., K.E.G., D.D., and N.A.; data collection, L.J.G., K.E.G., D.D., and N.A.; data analysis, L.J.G., K.E.G., D.D., and N.A.; writing, original draft preparation, L.J.G. and D.D.; writing, review, L.J.G., K.E.G., and N.A.; supervision and project administration, L.J.G.; funding acquisition, L.J.G., D.D., and N.A. All authors have read and agreed to the published version of the manuscript.

#### **Funding:** The research received no external funding.

**Acknowledgments:** The research was supported in part from an Internal RI-INBRE fellowship (IDeA Networks of Biomedical Research Excellence) and a Center for Economic and Environmental Development (CEED) fund grant awarded to Diana Denio and Nicolas Antico. Part of this research was presented at the CAM 2010 conference held at the Smithsonian Tropical Research Institute in Panama City, Panama, by Diana Denio. The authors would like to thank Tammy Sage and Rowan Sage for their help with the light microscopy. The authors would like to thank Tim Pelletier and Colleen McKnight-Torres for their support with equipment and supplies for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
