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

Abstract

In different lineages of C 4 plants, the release of CO 2 by decarboxylation of a C 4 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 4 -NADP-ME has been thoroughly investigated, whereas that of C 4 -NAD-ME has received less attention. In this work, we aimed at elucidating the mechanism of recruitment of NAD-ME for its function in the C 4 pathway by focusing on the eudicot family Cleomaceae. We identified a duplication of NAD-ME in vascular plants that generated the two paralogs lineages: α - and β -NAD-ME . 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 -NAD-ME /α -NAD-ME (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 4 -NAD-ME-type photosynthesis have a non-1:1 ratio of β/α. In the Brassicales, we found C 4 species with a 2:1 ratio due to a β -NAD-ME duplication (β 1 and β 2 ); this was also observed in the C 3 Tarenaya hassleriana and Brassica crops. In the independently evolved C 4 species, Gynandropsis gynandra and Cleome angustifolia , all three genes were affected by C 4 evolution with α- and β1- NAD-ME 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 G. gynandra and C. angustifolia , two of them were identified as positively selected. Using synteny analysis, we established that β -NAD-ME duplications were derived from ancient polyploidy events and that α -NAD-ME 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 4 photosynthesis. We propose that gene duplications provided the basis for the recruitment of NAD-ME in C 4 Cleomaceae and that all members of the NAD-ME gene family have been adapted to fit the C 4 ...

Understanding C4 photosynthesis in Setaria by a proteomic and kinetic approach

“…According to a 40% identity (on average), NAD-and NADP dependent enzymes share structural motifs, but they have arisen by different evolutionary events and evolved independently (Tronconi et al, 2018). All plant species conserve at least two NAD-dependent isoforms, one α-NAD-ME and one β-NAD-ME, which arose by a gene duplication that occurred late in the evolution of vascular plants (Tronconi et al, 2020). The α-NAD-ME and the β-NAD-ME encoded in the genome of S. viridis, also called NAD-ME1 and NAD-ME2, respectively, were detected in M and BS protein samples, but they are slightly more abundant in BS (FC(BS/M): 1.8 in both cases, Table 1).…”

Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cells mitochondria and evolved via association of differentially adapted subunits

“…The C 4 -NAD-ME turned out to be an exceptional case. Recently, we described that the evolution of NAD-ME in higher plants is marked by sub-functionalization and differences in the frequency of gene duplication the two paralogous α- and β-NAD-ME gene lineages (Tronconi et al, 2020). Most angiosperm genomes maintained a 1:1 α-NAD-ME / β-NAD-ME relative gene dosage, but a significantly high proportion of species with C 4 -NAD-ME-type photosynthesis have a non-1:1 ratio of α-NAD-ME / β-NAD-ME .…”

“…Most angiosperm genomes maintained a 1:1 α-NAD-ME / β-NAD-ME relative gene dosage, but a significantly high proportion of species with C 4 -NAD-ME-type photosynthesis have a non-1:1 ratio of α-NAD-ME / β-NAD-ME . Specifically, some C 3 and C 4 species of Brassicales possess a single NAD-MEα gene and two NAD-MEβ genes ( β1 and β2 ) (Tronconi et al, 2020). In the independently evolved C 4 species Gynandropsis gynandra and Cleome angustifolia , all three genes were affected by C 4 evolution with the encoded NAD-MEβ1 subunits exhibiting several amino acids identically substituted and positively selected in both C 4 species (Tronconi et al, 2020).…”

“…Specifically, some C 3 and C 4 species of Brassicales possess a single NAD-MEα gene and two NAD-MEβ genes ( β1 and β2 ) (Tronconi et al, 2020). In the independently evolved C 4 species Gynandropsis gynandra and Cleome angustifolia , all three genes were affected by C 4 evolution with the encoded NAD-MEβ1 subunits exhibiting several amino acids identically substituted and positively selected in both C 4 species (Tronconi et al, 2020). Likely, these changes provided the basis for the recruitment of NAD-ME and its function in C 4 -physiology.…”

Acetylation of conserved lysines fine-tune mitochondrial malate dehydrogenase activity in land plants

“…While plant mMDH oligomerization state in vivo is not yet definitively established, pig heart mMDH is active as homodimer of ~70 kDa, possessing two equivalent binding sites (Murphey et al, 1967; Noyes et al, 1974; Shore and Chakrabarti, 1976; Gleason et al, 1994). Mitochondrial malate is not only metabolized through mMDH, but also via the NAD-dependent malic enzyme (NAD-ME) (Tronconi et al, 2008; Sweetlove et al, 2010; Maurino and Engqvist, 2015; Tronconi et al, 2020). Both enzymes together provide a remarkable degree of flexibility to plant respiratory metabolism, since they are able to supply mitochondrial carbon metabolism with substrate to respire, but also replenish the TCA cycle with carbon skeletons to maintain its function even when carbon skeletons are withdrawn for biosynthesis, e.g.…”