Identification of a gene for pyruvate-insensitive mitochondrial alternative oxidase expressed in the thermogenic appendices in Arum maculatum

Abstract

Heat production in thermogenic plants has been attributed to a large increase in the expression of the alternative oxidase(AOX). AOX acts as an alternative terminal oxidase in the mitochondrial respiratory chain, where it reduces molecular oxygento water. In contrast to the mitochondrial terminal oxidase, cytochrome c oxidase, AOX is nonprotonmotive and thus allows thedramatic drop in free energy between ubiquinol and oxygen to be dissipated as heat. Using reverse transcription-polymerasechain reaction-based cloning, we reveal that, although at least seven cDNAs for AOX exist (AmAOX1a, -1b, -1c, -1d, -1e, -1f, and-1g) in Arum maculatum, the organ and developmental regulation for each is distinct. In particular, the expression of AmAOX1etranscripts appears to predominate in thermogenic appendices among the seven AmAOXs. Interestingly, the amino acidsequence of AmAOX1e indicates that the ENV element found in almost all other AOX sequences, including AmAOX1a, -1b,-1c, -1d, and -1f, is substituted by QNT. The existence of a QNT motif in AmAOX1e was confirmed by nano-liquidchromatography-tandem mass spectrometry analysis of mitochondrial proteins from thermogenic appendices. Furtherfunctional analyses with mitochondria prepared using a yeast heterologous expression system demonstrated that AmAOX1eis insensitive to stimulation by pyruvate. These data suggest that a QNT type of pyruvate-insensitive AOX, AmAOX1e, plays acrucial role in stage- and organ-specific heat production in the appendices of A. maculatum. © 2011 American Society of Plant Biologists. All Rights Reserved.