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 oxygen to water. In contrast to the mitochondrial terminal oxidase, cytochrome c oxidase, AOX is nonprotonmotive and thus allows the dramatic drop in free energy between ubiquinol and oxygen to be dissipated as heat. Using reverse transcription-polymerase chain 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 AmAOX1e transcripts appears to predominate in thermogenic appendices among the seven AmAOXs. Interestingly, the amino acid sequence 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-liquid chromatography-tandem mass spectrometry analysis of mitochondrial proteins from thermogenic appendices. Further functional analyses with mitochondria prepared using a yeast heterologous expression system demonstrated that AmAOX1e is insensitive to stimulation by pyruvate. These data suggest that a QNT type of pyruvate-insensitive AOX, AmAOX1e, plays a crucial role in stage- and organ-specific heat production in the appendices of A. maculatum.

Figure 1.

Developmental features and temperature profile of the inflorescences in A. maculatum. A, Images depicting morphological development. The developmental stages are indicated at top left. The inside of the floral chamber is shown in the inset of the δ-stage image. The positions of the male (M) and female (F) flowers are indicated by arrows. B, Continuous temperature measurements of the appendix (Tapp), floral chamber (Tfc), and ambient air (Ta) over a 48-h period in the field. Developmental stages from β- to ε-stages are indicated at the top of the graph. Infrared thermal imaging of heat production at the δ-stage is also shown.

Figure 2.

Comparison of deduced amino acid sequences of AmAOX proteins with previously reported AOXs. A, Sequence alignment of deduced amino acid sequences of E/DNV-, QDC-, and QD/NT/S-type AOX proteins. Seven amino acid residues that are potentially involved in the regulation of AOX activity are shaded either in black or green. Residues shaded in green are elements that are related to α-keto acid responsiveness. Regions 1 to 4 are designated in accordance with a previous report (Crichton et al., 2005). B, Unrooted dendrogram of various plant AOX proteins in monocots and dicots. Seven AmAOX proteins from A. maculatum are underlined. Boldface lettering denotes AOXs from thermogenic plants. Abbreviations and data sources are as follows: AmAOX1a, A. maculatum AOX1a (AB565465); AmAOX1b, A. maculatum AOX1b (AB565466); AmAOX1c, A. maculatum AOX1c (AB565467); AmAOX1d, A. maculatum AOX1d (AB565468); AmAOX1e, A. maculatum AOX1e (AB565469); AmAOX1f, A. maculatum AOX1f (AB565470); AmAOX1g, A. maculatum AOX1g (AB615377); AtAOX1a, A. thaliana AOX1a (NP_188876); AtAOX1b, A. thaliana AOX1b (NP_188875); AtAOX1c, A. thaliana AOX1c (NP_189399); AtAOX2, A. thaliana AOX2 (NP_201226); DvAOX, D. vulgaris AOX (BAD51465); GmAOX1, Glycine max AOX1 (AAAC35354); GmAOX2a, G. max AOX2a (AAB97285); GmAOX2b, G. max AOX2b (AAB97286); LeAOX1a, Solanum lycopersicum (formerly Lycopersicon esculentum) AOX1a (AAK58482); LeAOX1b, S. lycopersicum AOX1b (AAK58483); NnAOX1a, N. nucifera AOX1a (AB491175); NnAOX1b, N. nucifera AOX1b (AB491176); OsAOX1a, Oryza sativa AOX1a (BAA28773); OsAOX1b, O. sativa AOX1b (BAA28771); OsAOX1c, O. sativa AOX1c (BAB71945); SgAOX, S. guttatum AOX (P22185); SrAOX, S. renifolius AOX (BAD83866); StAOX1a, Solanum tuberosum AOX1a (BAE92716); TaAOX1a, Triticum aestivum AOX1a (BAB88645); ZmAOX1a, Zea mays AOX1a (AAR36136).

Figure 3.

Expression levels of AmAOXs, AmUCP, and AmPorin transcripts in various tissues in thermogenic δ-stage A. maculatum. A, Expression levels of AmAOX and AmUCP transcripts. The top panel shows expression levels of AmAOX transcripts analyzed by qRT-PCR. The levels are depicted as expression ratios relative to EF1α. The total level of AmAOX expression was analyzed with common primers (termed AmAOXallFW and AmAOXallRV and shown in Supplemental Table S4) that detect all types of transcripts (AmAOX1a to -1g; indicated by “all”). Transcripts for AmAOX1a, -1b, -1c, -1d, and -1f were analyzed with specific primers for each gene. Transcripts for AmAOX1e and -1g were coamplified during the analysis (indicated as “1e+1g”). Primers used are listed in Supplemental Table S4 with the combinations shown in Supplemental Table S5. The bottom panel shows expression analysis by cycleave PCR. Transcripts for the QNT- and QDT-type AmAOXs were coamplified in the analysis (QNT+QDT). An infrared thermal image is inset, and the positions of the appendix (A), leaf (L), and spathe (S) are indicated by arrows. B, Expression levels of AmPorin transcripts analyzed by qRT-PCR. These levels were depicted as expression ratios relative to EF1α. “Hot” indicates the expression level in the thermogenic appendices. Values with different letters in the graph indicate that they are statistically significantly different (n = 3; P < 0.05).

Figure 4.

Expression levels for AmAOX and AmUCP transcripts in β-, δ-, and єε-stage A. maculatum. A, Infrared thermal images of intact A. maculatum grown outdoors. Positions of the appendices (A) and the male (M) and female (F) flowers are indicated by arrows. Temperature scales are shown below the images. B, Expression levels of the AmAOX and AmUCP transcripts in the appendix (i and ii), male flowers (iii and iv), and female flowers (v and vi). The y axis represents the expression ratio to EF1α. The expression levels analyzed by qRT-PCR and cycleave PCR are shown in the left and right columns, respectively. Data are means ± sd (n = 3). Values with different letters in the graph are statistically significantly different (P < 0.05).

Figure 5.

Identification of an AmAOX1e-specific peptide among the mitochondrial proteins from thermogenic appendices of A. maculatum. A, Western-blot analysis of mitochondrial protein. Ten micrograms of protein was separated by SDS-PAGE, visualized by the Coomassie Brilliant Blue (CBB) stain, transferred to a polyvinylidene difluoride membrane, and incubated with AOA monoclonal antibodies. A band corresponding to the position of the AOX protein was excised and analyzed by nano-LC-MS/MS. A weak signal at around 19 kD that reacted with the AOA antibody is a degradation product of AmAOX in the mitochondria. Molecular mass standards (MW) are shown on the left of the panel. B, MS/MS spectrum of an AmAOX1e-specific peptide fragment (DIDSGAIQNTPAPAIALDYWR) derived by tryptic digestion of the protein sample. Additional information regarding AmAOX1e-specific peptides is displayed in Table II. C, Sequence coverage of the AmAOX1e protein analyzed by nano-LC-MS/MS. Amino acid sequences identified from a MASCOT database search are indicated in red lettering. An AmAOX1e-specific peptide is underlined.

Figure 6.

Functional analysis of AmAOX proteins expressed in S. pombe. Mitochondria were preincubated in the presence of 2 mm KCN and 1 μm carbonyl cyanide m-chlorophenylhydrazone, and 2 mm NADH, 4 mm dithiothreitol (DTT), 8 mm pyruvate (Pyr), and 0.3 mm n-propyl gallate (nPG) were subsequentially added. Respiration rates were then measured. Data are means ± sd (n = 9). Values with different letters in the graph are statistically significantly different (P < 0.05).