Plant Molecular Biology 35: 585–596, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Characterization of the gene family for alternative oxidase from Arabidopsis
, Eiji Nambara
, Satoshi Naito
, Nobuhiro Tsutsumi
, Atsushi Hirai
Laboratory of Radiation Genetics, Graduate School of Agricultural and Life Science, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan (
author for correspondence);
Department of Applied Bioscience,
Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan
Received 21 January 1997; accepted in revised form 24 June 1997
Key words: alternative oxidase, Arabidopsis thaliana, cyanide-insensitive respiration, differential expression, gene
We investigated the copy number of the gene for alternative oxidase (AOX)ofArabidopsis thalianaby ampliﬁcation
by PCR and Southern hybridization. These studies indicated that there are at least four copies of the AOX gene in
Arabidopsis. We isolated genomic clones containing individual copies (designated as AOX1a, AOX1b, AOX1c and
AOX2)oftheAOX genes. Interestingly, two of the AOX genes (AOX1a and AOX1b) were located in tandem in a ca.
5 kb region on one of the chromosomes of Arabidopsis. Comparison between genomic and cDNA sequences of the
four AOX genes showed that all AOX genes are divided by three introns and the positions of the introns in AOX1a,
AOX1b, AOX1c and AOX2 are the same. We examined whether expression of Arabidopsis AOX genes, like the
tobacco AOX1a gene, is enhanced by treatment with antimycin A, an inhibitor of complex III in the mitochondrial
respiratory chain. We found that, in young plants, the amount of Arabidopsis AOX1a mRNA was dramatically
increased by addition of antimycin A, while the transcription of the other three genes (AOX1b, AOX1c and AOX2)
did not respond to antimycin A. Ampliﬁcation by RT-PCR showed that AOX1a and AOX1c were expressed in all
organs examined (ﬂowers and buds, stems, rosette, and roots of 8-week old plants). In contrast, transcripts of
AOX1b were detected only in the ﬂowers and buds, and transcripts of AOX2 were detected mainly in stems, rosette
and roots. These results suggested that transcriptions of the four genes for alternative oxidase of Arabidopsis are
Mitochondria of higher plants contain two quinol-
oxidizing pathways: one is the cyanide-sensitive,
cytochrome pathway, and the other is the cyanide-
insensitive, alternative pathway (see  for review).
Electron ﬂow via the cytochrome pathway is coupledto
ATP synthesis and its terminal oxidase is cytochromec
oxidase, whereas electron transport via the alternative
The nucleotide sequence data reported will appear in the DDBJ,
EMBL and GenBank Nucleotide Sequence Databases under the
accession number D89875 (AOX1b/AOX1a), AB003175 (AOX1c)
and AB003176 (AOX2).
pathway is not coupled to production of ATP and its
terminal oxidase is alternative oxidase (AOX).
In thermogenic plants such as Sauromatum gut-
tatum (voodoo lily), cyanide-insensitive respiration is
involved in thermogenesis . The heat that is pro-
duced during ﬂowering volatilizes a compound that
attracts pollinating insects. In other plant species, it
hasbeen reported thatcyanide-insensitive respirationis
regulated in response to tissue-speciﬁc, developmental
and environmentalsignals (see [7, 21, 31] for reviews).
For example, growth of maize or tobacco at low tem-
perature results in an increase in cyanide-insensitive
respiration [33, 38]. The increase in the capacity of the
alternative pathwayat low temperatureseems to be due
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