Respiratory and C-4-photosynthetic NAD-malic enzyme coexist in bundle sheath cell mitochondria and evolved via association of differentially adapted subunits

In plant mitochondria, nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) has a housekeeping function in malate respiration. In different plant lineages, NAD-ME was independently co-opted in C-4 photosynthesis. In the C-4 Cleome species, Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-ME alpha, NAD-ME beta 1, and NAD-ME beta 2) were affected by C-4 evolution and are expressed at higher levels than their orthologs in the C3 species Tarenaya hassleriana. In T. hassleriana, the NAD-ME housekeeping function is performed by two heteromers, NAD-ME alpha/beta 1 and NAD-ME alpha/beta 2, with similar biochemical properties. In both C-4 species, this role is restricted to NAD-ME alpha/beta 2. In the C-4 species, NAD-ME alpha/beta 1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. Gynandropsis gynandra NAD-ME alpha/beta 1 (GgNAD-ME alpha/beta 1) exhibits high catalytic efficiency and is differentially activated by the C-4 intermediate aspartate, confirming its role as the C-4-decarboxylase. During C-4 evolution, NAD-ME beta 1 lost its catalytic activity; its contribution to the enzymatic activity results from a stabilizing effect on the associated alpha-subunit and the acquisition of regulatory properties. We conclude that in bundle sheath cell mitochondria of C-4 species, the functions of NAD-ME as C-4 photosynthetic decarboxylase and as a housekeeping enzyme coexist and are performed by isoforms that combine the same alpha-subunit with differentially adapted beta-subunits.