Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation

Abstract

Much recent attention in the study of adaptation of organismal form has centered on developmental regulation. As such, the highly conserved respiratory machinery of eukaryotic cells might seem an unlikely target for selection supporting novel morphologies. We demonstrate that a dramatic molecular evolutionary rate increase in subunit I of cytochrome c oxidase (COX) from an active-trapping lineage of carnivorous plants is caused by positive Darwinian selection. Bladderworts (Utricularia) trap plankton when water-immersed, negatively pressured suction bladders are triggered. The resetting of traps involves active ion transport, requiring considerable energy expenditure. As judged from the quaternary structure of bovine COX, the most profound adaptive substitutions are two contiguous cysteines absent in approximately 99.9% of databased COX I sequences from Eukaryota, Archaea, and Bacteria. This motif lies directly at the docking point of COX I helix 3 and cytochrome c, and modeling of bovine COX I suggests the possibility of an unprecedented helix-terminating disulfide bridge that could alter COX/cytochrome c dissociation kinetics. Thus, the key adaptation in Utricularia likely lies in molecular energetic changes that buttressed the mechanisms responsible for the bladderworts' radical morphological evolution. Along with evidence for COX evolution underlying expansion of the anthropoid neocortex, our findings underscore that important morphological and physiological innovations must often be accompanied by specific adaptations in proteins with basic cellular functions.

Fig. 1.

The hypothesized evolution and development of ATP-dependent active pumping in Lentibulariaceae. a, divergence of the flypaper trapping strategy (Pinguicula); b, ancestral Utricularia/Genlisea active-pumping suction pitcher prototype; c, corkscrew trapping strategy (Genlisea) with the loss of active pumping; d, Utricularia maintains ATP driven active pumping, with the development of the trap door innovation, which seals a negative internal pressure required for suction-bladder trapping. Colored branches indicate the ancestral Leu-113-Ser-114 motif (red), with gain of functional changes Cys-113-Cys-114 (blue) in the Utricularia/Genlisea ancestor (with Genlisea represented here by Genlisea hispidula; Table 1) followed by partial loss to Cys-113-Ser-114 (green) in Genlisea aurea or complete reversal to Leu-113-Ser-114 (red) in Genlisea violacea (Table 1). The above hypothesis assumes coevolution of the Cys-113-Cys-114 motif, as suggested by our hypothesis of a vicinal disulfide bridge in COX I helix 3. Numbers below branches represent parsimony jackknife support values (63–100 = strong support), based on analysis of two noncoding plastid DNA regions (10, 12). Images show Pinguicula (Top), with flypaper trapping leaves; Genlisea (Middle) with digestive bulb (uppermost), forked region where corkscrew traps begin, and an individual corkscrew trap; Utricularia (Bottom) with suction-trapping bladders and branched appendages at their trap doors.

Fig. 2.

Protein structural reconstruction of COX I and COX VIIa,c based on the bovine enzyme model (15). Mitochondrially encoded COX I is silver, and hemes and coppers show through in purple and orange, respectively. COX subunit VIIa is shown in dark blue; subunit VIIc is in light blue. We identified COX VIIa in plants, including Utricularia, but COX VIIc remains undiscovered. Residues 113 and 114 of helix 3 of COX subunit I are shown as red space-filling models. Cytochrome c, in green, is shown superimposed by using coordinates from the P. denitrificans oxidase (1ar1.pdb)/cytochrome c docked complex (16). COX I residues 113 and 114 and the C terminus of COX VIIa lie directly at the cytochrome c docking base. The model was visualized by using the application vmd (23).