Independent Recruitment of Duplicated β-Subunit-Coding NAD-ME Genes Aided the Evolution of C4 Photosynthesis in Cleomaceae.

In different lineages of C plants, the release of CO by decarboxylation of a C acid near rubisco is catalyzed by NADP-malic enzyme (ME) or NAD-ME, and the facultative use of phosphoenolpyruvate carboxykinase. The co-option of gene lineages during the evolution of C-NADP-ME has been thoroughly investigated, whereas that of C-NAD-ME has received less attention. In this work, we aimed at elucidating the mechanism of recruitment of for its function in the C pathway by focusing on the eudicot family Cleomaceae. We identified a duplication of in vascular plants that generated the two paralogs lineages: α and β. Both gene lineages were retained across seed plants, and their fixation was likely driven by a degenerative process of sub-functionalization, which resulted in a NAD-ME operating primarily as a heteromer of α- and β-subunits. We found most angiosperm genomes maintain a 1:1 β/α (β/α) relative gene dosage, but with some notable exceptions mainly due to additional duplications of β-NAD-ME subunits. For example, a significantly high proportion of species with C-NAD-ME-type photosynthesis have a non-1:1 ratio of β/α. In the Brassicales, we found C species with a 2:1 ratio due to a β duplication (β and β); this was also observed in the C and Brassica crops. In the independently evolved C species, and , all three genes were affected by C evolution with α- and β1- driven by adaptive selection. In particular, the β1-NAD-MEs possess many differentially substituted amino acids compared with other species and the β2-NAD-MEs of the same species. Five of these amino acids are identically substituted in β1-NAD-ME of and , two of them were identified as positively selected. Using synteny analysis, we established that β duplications were derived from ancient polyploidy events and that α is in a unique syntenic context in both Cleomaceae and Brassicaceae. We discuss our hypotheses for the evolution of NAD-ME and its recruitment for C photosynthesis. We propose that gene duplications provided the basis for the recruitment of NAD-ME in C Cleomaceae and that all members of the gene family have been adapted to fit the C-biochemistry. Also, one of the β gene copies was independently co-opted for its function in the C pathway.