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

AbstractC4photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations in spite of sharing key biochemical modules.Portulacais a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4, but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4+CAM. It has been hypothesized that C4+CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly ofP. amilisand sampled mRNA fromP. amilisandP. oleraceaduring CAM induction. Gene co-expression network analyses identified C4and CAM gene modules shared and unique to bothPortulacaspecies. 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 theP. amilisgenome. Preservation of this co-expression module regardless of water status suggests thatPortulacaconstitutively operate a weak CAM cycle that is transcriptionally and post-transcriptionally upregulated during drought. C4and CAM mostly used mutually exclusive genes for primary carbon fixation and it is likely that nocturnal CAM malate stores are shuttled into diurnal C4decarboxylation pathways, but we find evidence that metabolite cycling may occur at low levels. C4likely evolved inPortulacathrough co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.