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

Abstract

In different lineages of C₄ plants, the release of CO₂ by decarboxylation of a C₄ 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 C₄-NADP-ME has been thoroughly investigated, whereas that of C₄-NAD-ME has received less attention. In this work, we aimed at elucidating the mechanism of recruitment of <i>NAD-ME</i> for its function in the C₄ pathway by focusing on the eudicot family Cleomaceae. We identified a duplication of <i>NAD-ME</i> in vascular plants that generated the two paralogs lineages: α<i>-</i> and β<i>-NAD-ME</i>. 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<i>-NAD-ME</i>/α<i>-NAD-ME</i> (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 C₄-NAD-ME-type photosynthesis have a non-1:1 ratio of β/α. In the Brassicales, we found C₄ species with a 2:1 ratio due to a β<i>-NAD-ME</i> duplication (β<i>1</i> and β<i>2</i>); this was also observed in the C₃ <i>Tarenaya hassleriana</i> and Brassica crops. In the independently evolved C₄ species, <i>Gynandropsis gynandra</i> and <i>Cleome angustifolia</i>, all three genes were affected by C₄ evolution with α- and β1-<i>NAD-ME</i> 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 <i>G. gynandra</i> and <i>C. angustifolia</i>, two of them were identified as positively selected. Using synteny analysis, we established that β<i>-NAD-ME</i> duplications were derived from ancient polyploidy events and that α<i>-NAD-ME</i> 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 C₄ photosynthesis. We propose that gene duplications provided the basis for the recruitment of NAD-ME in C₄ Cleomaceae and that all members of the <i>NAD-ME</i> gene family have been adapted to fit the C₄-biochemistry. Also, one of the β<i>-NAD-ME</i> gene copies was independently co-opted for its function in the C₄ pathway.