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

In different lineages of Cleomaceae, NAD-malic enzyme (NAD-ME) was independently co-opted to participate in C4 photosynthesis. In the C4 Cleome species Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-MEα, NAD-MEβ1, and NAD-MEβ2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 Cleome species Tarenaya hassleriana. In the latter C3 species, the NAD-ME housekeeping function is performed by two heteromers, NAD-MEα/β1 and NAD-MEα/β2, with similar biochemical properties. In both C4 species analyzed, this role is restricted the NAD-MEα/β2 heteromer. In the C4 species, NAD-MEα/β1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. GgNAD-MEα/β1 exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, GgNAD-MEβ1and CaNAD-MEβ1 lost their catalytic activity; their contribution to enzymatic activity results from a stabilizing effect on the associated α-subunit. We conclude that in bundle sheath cell mitochondria of C4 Cleome species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a tricarboxylic acid cycle-associated housekeeping enzyme coexist and are performed by isoforms that combine the same α subunit with differentially adapted β subunits.