**Lonnie J. Guralnick \*, Kate E. Gilbert, Diana Denio and Nicholas Antico**

Department of Biology, Roger Williams University, One Old Ferry Rd., Bristol, RI 02809, USA; kgilbert721@g.rwu.edu (K.E.G.); ddenio335@g.rwu.edu (D.D.); nantico949@g.rwu.edu (N.A.) **\*** Correspondence: lguralnick@rwu.edu; Tel.: +1-401-254-3581; Fax: +1-401-254-3360

Received: 10 December 2019; Accepted: 27 December 2019; Published: 2 January 2020

**Abstract:** *Portulaca grandiflora* simultaneously utilizes both the C4 and Crassulacean acid metabolism (CAM) photosynthetic pathways. Our goal was to determine whether CAM developed and was functional simultaneously with the C4 pathway in cotyledons of *P. grandiflora*. We studied during development whether CAM would be induced with water stress by monitoring the enzyme activity, leaf structure, JO2 (rate of O2 evolution calculated by fluorescence analysis), and the changes in titratable acidity of 10 and 25 days old cotyledons. In the 10 days old cotyledons, C4 and CAM anatomy were evident within the leaf tissue. The cotyledons showed high titratable acid levels but a small CAM induction. In the 25 days old cotyledons, there was a significant acid fluctuation under 7 days of water stress. The overall enzyme activity was reduced in the 10 days old plants, while in the 25 days old plants CAM activity increased under water-stressed conditions. In addition to CAM, the research showed the presence of glycine decarboxylase in the CAM tissue. Thus, it appears both pathways develop simultaneously in the cotyledons but the CAM pathway, due to anatomical constraints, may be slower to develop than the C4 pathway. Cotyledons showed the ancestral Atriplicoid leaf anatomy, which leads to the question: Could a CAM cell be the precursor to the C4 pathway? Further study of this may lead to understanding into the evolution of C4 photosynthesis in the *Portulaca.*

**Keywords:** *Portulaca grandiflora*; C4 photosynthesis; Crassulacean acid metabolism (CAM), evolution; development; PEP carboxylase; Portulacaceae; glycine decarboxylase
