Abstract C 4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations in spite of sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C 4 , but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C 4 +CAM. It has been hypothesized that C 4 +CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of P. amilis and sampled mRNA from P. amilis and P. oleracea during CAM induction. Gene co-expression network analyses identified C 4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase (PEPC) to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and post-transcriptionally upregulated during drought. C 4 and CAM mostly used mutually exclusive genes for primary carbon fixation and it is likely that nocturnal CAM malate stores are shuttled into diurnal C 4 decarboxylation pathways, but we find evidence that metabolite cycling may occur at low levels. C 4 likely evolved in Portulaca through co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C 4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.

Gene co-expression reveals the modularity and integration of C4and CAM inPortulaca