A classification scheme for alternative oxidases reveals the taxonomic distribution and evolutionary...

Mitochondrion xxx (2014) xxx–xxx

MITOCH-00913; No of Pages 12

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Mitochondrion

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A classification scheme for alternative oxidases reveals the taxonomicdistribution and evolutionary history of the enzyme in angiosperms☆

José Hélio Costa a,⁎, Allison E. McDonald b, Birgit Arnholdt-Schmitt c, Dirce Fernandes de Melo a

a Department of Biochemistry and Molecular Biology, Federal University of Ceara, 60455-760 Fortaleza, Ceará, Brazilb Department of Biology, Wilfrid Laurier University, Science Building, 75 University Avenue West, Waterloo, Ontario N2L 3C5, Canadac EU Marie Curie Chair, ICAAM, University of Évora, Apartado 94, 7002-554 Évora, Portugal

Abbreviations: AOX, Alternative oxidase; BLAST, basicsequence harmony.☆ Authorship credit: JH Costa and AEMcDonald provideCosta conducted thedata analyses. B Arnholdt-Schmitt cooper. DF deMelo suggested the development of a bioinformof AOX sequences and an analysis of the evolutionary histAE McDonald wrote the paper with input and suggestionDF de Melo.⁎ Corresponding author. Tel.: +55 85 3366 9825; fax: +

E-mail address: [email protected] (J.H. Costa).

http://dx.doi.org/10.1016/j.mito.2014.04.0071567-7249/© 2014 Elsevier B.V. and Mitochondria Resear

Please cite this article as: Costa, J.H., et al., Ahistory of the enzyme in angi..., Mitochondri

a b s t r a c t
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Article history:Received 15 December 2013Received in revised form 23 March 2014Accepted 11 April 2014Available online xxxx

Keywords:Alternative oxidaseFlowering plantsTaxonomic distributionClassificationEvolution

A classification scheme based on protein phylogenies and sequence harmonymethodwas used to clarify the tax-onomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data setanalyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a–c/1e,AOX1d, AOX2a–c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamilywas not detected in monocots, the AOX1 subfamily has expanded (AOX1a–e) in the large majority of theseplants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a–c)were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in theFabales order (soybean, cowpea) was divergent from AOX2a–c showing some specific amino acids withAOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative andmu-tually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based onthe data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specificareas where further research would be most beneficial.

© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction

Alternative oxidase (AOX) was discovered in plants and is responsi-ble for the phenomenon of cyanide-resistant respiration (Bendall andBonner, 1971). AOX is a terminal quinol oxidase found in themitochon-drial electron transport chain that introduces a branch-point at the levelof ubiquinol (McDonald, 2008; Moore et al., 2013). AOX is of researchinterest for studying the phenomenon of retrograde regulation betweenthemitochondrion and the nucleus and due to its role in the acclimationof plants to a variety of environmental stressors (Giraud et al., 2009;McDonald, 2008). In the last several years, AOX has become of centralinterest as a gene candidate for functional marker development relatedto breeding of plants with efficient stress responses to various environ-mental conditions (Arnholdt-Schmitt, 2009; Arnholdt-Schmitt et al.,

local alignment search tool; SH,

d the raw sequence data and JHrdinated the concept for thepa-atics approach for the collectionory of the protein. JH Costa ands from B Arnholdt-Schmitt and

55 85 3366 9829.

ch Society. All rights reserved.

classification scheme for alteon (2014), http://dx.doi.org/1

2006; Frederico et al., 2009; Polidoros et al., 2009; Santos Macedoet al., 2009).

It is hypothesized that AOX arose in prokaryotes and entered theeukaryotic lineage via the primary endosymbiotic event that led to theorigin of mitochondria (Atteia et al., 2004; Finnegan et al., 2003;McDonald et al., 2003). This hypothesis is supported by the limited dis-tribution of AOX in the proteobacteria and itswidespread distribution inmany eukaryotic lineages including a wide array of protists, fungi, andanimals (McDonald, 2008). AOX has been most well studied in theplant kingdom and in particular in angiosperms (i.e. flowering plants).

The first discovery and cloning of a plant AOX gene occurred inthe thermogenic arum Sauromatum guttatum (Schott) (Rhoads andMcIntosh, 1991). AOX genes have been identified in various plant or-ders in monocots and eudicots (McDonald et al., 2002) and in plantsAOX is often encoded in the nuclear genome by a multigene familyconsisting of 3 to 5 members (Clifton et al., 2006; Thirkettle-Wattset al., 2003). The naming of AOX genes originally occurred in the orderof their discovery in a species (e.g. AOX1, AOX2, andAOX3). Asmore se-quences became available and experiments were performed, an initialanalysis of 18 full length and 30 partial plant AOX sequences confirmedthat 2 subfamilies exist (AOX1 and AOX2) (Considine et al., 2002). Thegeneral trend observed at that time was that AOX1 is often induced bystress stimuli, while AOX2 is usually constitutively or developmentallyexpressed (Considine et al., 2002). This led to the hypothesis that each

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subfamily might serve a specific function (i.e. differing physiologicalroles for AOX1 vs. AOX2). The AOX1 subfamily was composed of 2groups; one that contained monocot AOX1s and one that containedAOX1s from eudicots (Considine et al., 2002; Costa et al., 2004). Theeudicot AOX1s could be further separated based on the plant orders ofRosidae (e.g. Arabidopsis thaliana) and members of the Caryophyllidaeand Asteridae orders (e.g.Mesembryanthemum crystallium and tomato)(Considine et al., 2002). Evidence for the AOX2 subfamily was onlyfound in eudicots (in the Rosidae and Asteridae orders), despite exam-ining the available resources in wheat, barley, maize, and sorghum(Considine et al., 2002). It therefore appeared that the AOX2 subfamilywas absent from monocots. AOX2 sequences were not recovered fromseveral dicot species as well (e.g. potato), however, this was hypothe-sized to be due to a lack of data rather than the gene being absent(Considine et al., 2002). A second analysis of AOX genes (using 47plant and fungal AOXs) supported the existence of the AOX1 andAOX2 subfamilies and indicated the potential existence of a third sub-family AOX3 (Borecky et al., 2006). Unrooted phylogenies provided ev-idence for 4 AOX groups: AOX1 (one class of mostly monocot AOXs, asecond class of mostly eudicot AOXs), AOX2 from eudicots (no AOX2from monocots was found), and AOX3 from eudicots (Borecky et al.,2006). Although in practice the AOX3 designation has not been utilizedby the scientific community, three different clades within the AOX1subfamily later emerged after the Considine et al.'s (2002) classification.

These studies represented an important milestone in AOX researchand served as a catalyst for several research groups in identifyinggenes and unraveling their role within the context of members ofmultigene families in different species. It has been observed that themajority of studied angiosperm species have expanded the AOX1 sub-family, while only soybean and cowpea have expanded AOX2 (AOX2aand AOX2b; Costa et al., 2004). With regard to AOX gene expression,an examination of orthologous genes between soybean and Arabidopsisdid not show similar expression profiles (Thirkettle-Watts et al., 2003).For example, in soybean, AOX2a and AOX2b are the predominantlyexpressed genes in a variety of organs at different growth stages, where-as in Arabidopsis, AOX1a and AOX1c display the highest expressionlevels (Thirkettle-Watts et al., 2003). The paradigm that AOX1 is thegene related to stress responses is also beginning to be challenged.AOX2b in the leguminous plants Vigna unguiculata (Costa et al., 2010)and Medicago sativa (Cavalcanti et al., 2013) and AOX2 in Arabidopsis(Clifton et al., 2005) have been shown to be stress-responsive.

The above results raise the question of whether each AOX gene (andits associated protein product) may have a specific physiological role,and therefore whether the evolutionary divergence of AOX subfamiliesacross plant species might have implications for physiological function(Borecky et al., 2006; Considine et al., 2002). That is, can a link betweenphylogenetic relationships and gene expression and functionality bemade?

We argue that answering this question requires an initial step: i) anidentification of all currently available angiosperm AOX sequences; ii) arobust classification scheme for AOX subfamilies that provides a consis-tentmeans of annotation; and iii) an analysis of the taxonomic distribu-tion of each subfamily in angiosperms in order to detect major trendsand investigate the evolutionary history of the enzyme.

In this paper, we have collected a dataset of AOX sequences from anexhaustive search of all available databases (including 65 angiospermgenomes and GenBank databases). The goal was to utilize the availablefull-length AOX sequences in order to generate a new, robust classifica-tion scheme. We generated a classification scheme using protein phy-logenies and specific amino acids found in each subfamily. We foundthat AOX1 and AOX2 could be subdivided into 4 major phylogeneticclades: AOX1a–c/1e, AOX1d, AOX2a–c and AOX2d. In this way, it waspossible to annotate almost all partial sequences found in public data-bases aswell as to adjust or correct the classification of previously anno-tated sequences. These data were also used to identify major trends inAOX subfamily and class distribution in different plant species. The

Please cite this article as: Costa, J.H., et al., A classification scheme for altehistory of the enzyme in angi..., Mitochondrion (2014), http://dx.doi.org/1

analysis revealed high diversity in AOX family compositions; namelyplantswith single copies of both AOX1 andAOX2; plantswhich expand-ed the AOX1 family, but not the AOX2 family; plants which expandedAOX2, but not AOX1; plant species that have expanded both AOX1and AOX2 subfamilies; and some plants that contain either the AOX1or AOX2 subfamily, but not both. Hypotheses about the evolutionaryhistory of AOX genes in flowering plants have been generated and thepotential impact of the classification scheme on plant research isdiscussed.

2. Materials and methods

New plant alternative oxidase sequences were identified by BLAST(Altschul et al., 1997) searches against the nucleotide collection (nr),reference mRNA sequences (refseq_rna), reference genomic sequences(refseq_genomic), NCBI genomes (chromosome), expressed sequencetags (est), genomic survey sequences (gss), high throughput genomicsequences (htgs), whole-genome shotgun reads (wgs) and TSA (tran-scriptome shotgun assembly) GenBank databases (http://www.ncbi.nlm.nih.gov) as well as against several sequenced plant genomes atthe Phytozome database (http://www.phytozome.net).We are gratefulto all the Consortiums involved in the sequencing of the angiosperm ge-nomes used in this study (Supplemental Table S1) for giving us the op-portunity to search the whole genomes for individual AOX genes thusmaking it possible to draw a general picture of the distribution ofthese genes in angiosperms.

In addition, data from transcript and genomic libraries [SequenceRead Archive (SRA)] available at GenBank (http://www.ncbi.nlm.nih.gov) were used to retrieve AOX genes. Data from EST and/or SRA se-quences were assembled into contigs using the CAP3 Sequence Assem-bly Program (Huang and Madan, 1999).

Gene or cDNA annotation [determination of exons/introns or ORF(open reading frame)] was done manually for all sequences comparingthe identified sequences against annotated AOX cDNAs from nr orrefseq_rna databases (NCBI) with the help of the BLASTn. DeducedcDNAs (from genomic DNA) or cDNAs (from EST, TSA, SRA data) identi-fied in this study were translated into amino acid sequences using thetranslate tool at the ExPASy web server (http://web.expasy.org/translate). All deduced proteins were checked against the AOX proteinsequences available in the GenBank using the BLASTp tool.

The AOX protein phylogenetic tree was constructed using theneighbor-joiningmethod (Saitou andNei, 1987) in theMEGA5 program(Tamura et al., 2011). The alignmentwas performed using the “blosum”

substitution matrix with standard gap penalties of pairwise (gap open:10; gap extension: 0.1) andmultiple (gap open: 10; gap extension: 0.2)alignments and 30% of delay divergent cutoff. The phylogenetic treewascarried out using the following parameters: Bootstrap method with1000 replication, “number of differences” as the substitution modeland “complete deletion” for gaps/missing data treatment.

Specific sites that distinguish each AOX subfamily/type from eachother were determined using the “sequence harmony” (SH) methodol-ogy (Feenstra et al., 2007). The SH method was designed to identifysubfamily-specific functional sites. Entropies (E) are calculated for Aand B separately and together. SH values are calculated as SH = 1/2(EA + B − EA − EB), with EA + B = −∑(pA + pB) log (pA + pB)i.e. using the sum of the normalized frequencies of A and B. SH valuesrange from ‘zero’, for completely non-overlapping residue composi-tions, to ‘one’ for identical compositions. Below a SH score of 0.2, sitesare rated as exhibiting relevant differences between A and B. Addition-ally, stretches of neighboring selected sites are identified and the size ofeach of these stretches is assigned to the sites as the ‘Rank’. Finally, se-lected sites are sorted on i) increasing SH, ii) decreasing rank and iii) in-creasing entropy. This sorted list of selected sites is the primary result ofthe SH algorithm (Feenstra et al., 2007). For the present analyses, align-ments of the deduced amino acids were generated by Clustal X(Thompson et al., 1997) and two AOX subfamilies/classes (A and B)


http://www.ncbi.nlm.nih.gov


http://www.phytozome.net



http://web.expasy.org/translate

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were selected at a time for comparison. All alignments used in SHmethod are available in the Supplementary data (Supplementarymate-rial 1 to 4).

3. Results and discussion

3.1. Identification of angiosperm alternative oxidase sequences

Supplemental Table S2 shows a large dataset of AOX genes in angio-sperms obtained from an exhaustive basic local alignment search tool(BLAST) search of genomic and cDNA databases. In total, 627 AOX se-quences were identified, of which 80 corresponded to annotated se-quences present in the non-redundant (nr) database of GenBank(NCBI). Another 15 AOX genes from the EST and/or genomic databasesof Lotus japonicus, Sorghum bicolor, Populus trichocarpa (Costa et al.,2009b), Centaurea maculosa and Triphysaria pusilla (Campos et al.,

Fig. 1. Representative AOX phylogenetic tree of angiosperms with 132 full-length alternativeAOX2d. Two AOXs [AOX0A (accession number: XP_001694605); AOX0B (accession number: XThe phylogenetic tree was constructed following the parameters described in the Material andtogether in the bootstrap test (1000 replicates) are shownnext to the branches. Branches are draimprove the visualization). Abbreviations are as follows: Ac (Allium cepa), Ach (Actinidia chinens(Camptotheca acuminata), Cr (Catharanthus roseus), Cs (Citrus sinensis), Dv (Dracunculus vulgavesca), Gm (Glycine max), Gr (Gossypium raimondii), Lj (Lotus japonicus), Ma (Musa acuminatanucifera), Ns (Nicotiana sylvestris), Os (Oryza sativa), Pb (Philodendron bipinnatifidum), Pp (Pr(Symplocarpus foetidus), Sg (Sauromatum guttatum), Sl (Solanum lycopersicum), So (Saccharum(Vitis vinifera), Zm (Zea mays), Zom (Zostera marina).


2009) were also previously annotated and classified. Thus, the majorityof these AOX sequences (532) are presented here for the first time.

3.2. Identification of angiosperm alternative oxidase subfamilies

The full-length AOX sequences were recovered and used for phylo-genetic analysis of the deduced proteins. In order to generate the classi-ficationwe have used not only the protein phylogeny (Fig. 1) to identifydifferent AOX subfamilies, but also the specific amino acids identifiedusing the SHmethod (see below) and the known evolutionary relation-ships between different angiosperm plants. The results confirm that an-giosperm AOXs can clearly be divided into 2 major subfamilies: AOX1and AOX2 supporting the earlier work of Considine et al. (2002) inwhich the sequences were annotated as AOX1a–d and AOX2a/b. How-ever, in the present study we detected new trends in AOX sequencesthat led us to propose an adjustment in the classification of some AOXproteins. These included the discovery of a new AOX1 subtype found

oxidases showing the 4 major phylogenetic clades: AOX1a–c/1e, AOX1d, AOX2a–c andP_001703329)] from the green algae Chlamydomonas reinhardtii were used as out group.methods section. The percentage of replicate trees in which the associated taxa clusteredwn in proportion to genetic distance (except for algal AOX branchwhichwas shortened tois), Ad (Agave deserti), At (Arabidopsis thaliana), Ate (Agave tequilana), Br (Brassica rapa), Caris), Eg (Eucalyptus grandis), Egu (Elaeis guineensis), Ev (Ensete ventricosum), Fv (Fragaria), Md (Malus domestica), Mg (Mimulus guttatus), Mt (Medicago truncatula), Nn (Nelumbounus persica), Pt (Populus trichocarpa), Pvi (Panicum virgatum), Sb (Sorghum bicolor), Sfofficinarum), St (Solanum tuberosum), Tp (Triphysaria pusilla), Vu (Vigna unguiculata), Vv




in monocots that has been named as AOX1e. In addition the AOX1b and1d subtypes originally found in monocots such as sugarcane and maizewere found to be orthologous to the eudicot AOX1d subtype (i.e.Arabidopsis AOX1d) (Fig. 1), and were therefore renamed asAOX1d1 and 1d2, respectively. Several recent AOX2 duplications similarto those found in Arabidopsis AOX1a–c were identified in eudicots andthese AOXs were classified as AOX2a, 2b, and 2c (e.g. Actinidiachinensis AOX2a, 2b, 2c). The AOX2b originally identified ineudicots in the Fabales order (soybean, cowpea) was more diver-gent and was therefore renamed as AOX2d.

Therefore, in our analyses, all angiosperm AOX1s and AOX2s couldbe subdivided into 4 major phylogenetic clades: AOX1a–c/1e, AOX1d,AOX2a–c and AOX2d (Fig. 1). AOX1a–c/1e proteins are found in a vari-ety of basal angiosperms, magnoliids, and well-studied eudicots andmonocots. AOX2a–c proteins were identified in basal angiosperms, inmagnoliids, and in eudicots. Both the AOX1a–c/1e and AOX2a–c cladescontain proteins exhibiting high sequence similarity that is suggestiveof recent gene duplications (e.g. Arabidopsis AOX1a, 1b, 1c in theAOX1a–c/1e clade and A. chinensis AOX2a, 2b, 2c in the AOX2a–cclade). AOX1d includes a limited number of proteins from both mono-cot and eudicot species. AOX2d is limited to the core eudicot ordersFabales, Fagales, Malvales, and Rosales, and the basal eudicot ordersProteales and Ranunculales. Many of these proteins were previouslynamed AOX2b (Fig. 1, Supplemental Table S2).

3.3. Identification of amino acids specific to each AOX subfamily andsubtype

In order to detect AOX1 or AOX2 subfamily and type specific sites,the entropy-based method ‘sequence harmony’ (SH) was applied(Feenstra et al., 2007), which utilized multiple sequence alignmentsfrom divergent AOX sequences. Divergent sites are indicated by i) in-creasing SH, ii) decreasing rank, and iii) increasing entropy. Thus, themost trusted sites for differentiating between different AOX subfamiliesare denoted by the lowest SH value (zero); highest rank values, andlowest entropy (Feenstra et al., 2007).

In our analyses it was not possible to find specific sites to differenti-ate each AOX type within the AOX1 (a, b, c, e) or AOX2 (a, b, c) cladeswhen taking into account species from different orders. For example,Arabidopsis (Brassicales) and P. trichocarpa (Malpighiales) haveAOX1a, 1b and 1c, but these genes are highly similarwithin each speciesand it was not possible to determine exactly the orthologous pair and/orspecific sites when comparing proteins. In this context, the classificationof AOXwithin AOX1a–c/1e or AOX2a–c was performed considering thephylogenetic relationship with previously annotated AOX sequences inclosely related species (generally species belonging to the same order).For example, in the Brassicales, Brassica rapa AOX1bwas classified basedon its orthology to Arabidopsis AOX1b. Similarly, B. rapa AOX1a1 andAOX1a2 were classified based on their high sequence similarity withArabidopsis AOX1a in comparison to Arabidopsis AOX1b and AOX1c.Thus, our analyses of specific sites were concentrated in differentiatingthe AOXs within the 4 major phylogenetic clades: AOX1a–c/1e, AOX1d,AOX2a–c and AOX2d (Fig. 1).

In Fig. 2, specific amino acids that can be used for the reliable classi-fication of AOX sequences into AOX subfamilies are shown. Initially, thetwo largest and divergent AOX classes were compared (i.e. AOX1a–c/1evs. AOX2a–c) and specific amino acids that could be used to discrimi-nate between these two groups were identified (SupplementalTable S3). Next, AOX1d was compared to AOX1a–c/1e and AOX2d wascompared to AOX2a–c to determine specific sites for each AOX subtype(Supplemental Tables S4–S6). Once these specific amino acids wereidentified, any AOX sequence could be classifiedwith regard to subfam-ily and subtype using the full-length or partial amino acid sequence. InFig. 3, we demonstrate the use of the classification scheme using theOryza sativa AOX1b sequence, which was reclassified as an AOX1d pro-tein. The first step of the classification scheme is to determine whether


the AOX protein belongs to the AOX1 or AOX2 subfamily (Fig. 3). Oncethis has been determined, if the protein is an AOX1, it can be assignedto either the AOX1a–c/1e or AOX1d subtype (Fig. 3). Similarly, if theprotein is an AOX2, it can be assigned to either the AOX2a–c or AOX2dsubtype. If the protein is AOX1a–c/1e (absence of AOX1d specificsites) or AOX2a–c (absence of AOX2d specific sites) it could be furtherclassified based on the phylogenetic relationship with annotated pro-teins of closely related species as described above.

3.3.1. AOX1a–c/1e vs. AOX2a–cIn general, to have a good classification scheme more than one spe-

cific site should be considered. AOX1a–c/1e and AOX2a–c can be differ-entiated by 13 amino acid positions, with 5 of them presenting strongreliability parameters (SH=0) (Supplemental Table S3, Fig. 2). Accord-ing to these findings, AOX1a–c/1e proteins differ from AOX2a–c pro-teins primarily in amino acids (positions 229, 233, 241) in the vicinityof the highly conserved glutamate (E222) and histidine (H225) resi-dues, which are known to be involved in the co-ordination of the di-iron center of AOX (Andersson and Nordlund, 1999; Berthold et al.,2000), and secondarily in amino acids (positions 112; 124; 129) in theneighborhood of the regulatory cysteine residue CysI (C127) which isimportant for the post-translational regulation of many angiospermAOXs (Crichton et al., 2005; Umbach et al., 2006). Previously, specificsites that could be used to discriminate between AOX1 and AOX2 sub-families had been detected at positions 124, 129 (Costa et al., 2009a),199, 229, 233, 286, and 311 (Frederico et al., 2009), however, usingthe present larger dataset it can be concluded that the amino acid resi-dues at positions 199, 311 and 286 are not reliable for the differentiationof AOX1 and AOX2 and care should be taken when considering position129 since it presents SH values N 0.

3.3.2. AOX1a–c/1e vs. AOX1dTo identify AOX1d specific amino acids, AOX1d sequences from

monocots and eudicots were compared separately to AOX1a–c/1e pro-teins in order to find specific differences.

Monocot AOX1d differed from AOX1a–c/1e proteins in 11 positionswith 5 of them showing strong reliability parameters in SH (Fig. 2, Sup-plemental Table S4). The eudicot AOX1d appeared more divergent anddiffered from AOX1a–c/1e proteins in 14 positionswith 12 of them pre-senting strong reliability parameters in SH (Fig. 2; SupplementalTable S5). Three specific amino acids (positions 175, 178, 181) withstrong reliability parameters in SH were found in both monocot andeudicot AOX1d, but other amino acidswere also well conserved at posi-tions 142, 230 and 277 (Fig. 2). In general, the main amino acid differ-ences of AOX1d in relation to AOX1a–c/1e occur in the vicinity of thesecond conserved cysteine, Cys II (C177).

3.3.3. AOX2a–c vs. AOX2dAOX2d proteins were found in eudicot species of the Fabales,

Fagales, Malvales, Rosales, Proteales, and Ranunculales orders. Whenall AOX2d proteins were compared to AOX2a–c proteins, they differedin 3 positions with 2 showing strong reliability parameters in SH (167and 178). When AOX2d proteins from the Fagales, Malvales, andRanunculales orders were excluded from the analysis, the differenceswere greater, revealing 9 differing positions with 5 of them presentingthe strong reliability parameters in SH (162, 166, 167, 170, and 178)(Fig. 2; Supplemental Table S6). AOX2d proteins can therefore be classi-fiedby examiningpositions 167 and 178first, followed by positions 162,166, and 170 if required.

Considering all of the AOX2d proteins analyzed here, the proteins ofmembers of the Fabales are the most divergent from AOX2a–c. Ouranalysis revealed 20 different sites with 17 of them presenting strongreliability parameters (data not shown). In general, the data revealsthat AOX2d proteins contain changes in relation to AOX2a–c proteinsmostly in the region encoded by exon 2 in the vicinity of the second con-served cysteine, Cys II (C177).



Fig. 2. Amino acids specific to each AOX subfamily and subtype in angiosperms. An alignment of the consensus sequences of AOX1a–c/1e and AOX2a–c appears with specific amino acids(representedwith different colors) to each subfamily/type. Red represents residues found inAOX1a–c/1e proteins (as compared to AOX2a–c); yellow represents residues found inAOX2a–c (as compared to AOX1a–c/1e); bright green represents residues found in monocots AOX1d (as compared to AOX1a–c/1e); green represents residues found in eudicot AOX1d (as com-pared to AOX1a–c/1e) and blue represents residues found in AOX2d (as compared to AOX2a–c). Capital letters at the specific sites represent the more frequently seen amino acids. Con-sensus symbols used in the alignment are as follows: ! is I (Isoleucine), or V (valine); $ is L (leucine), or M (methionine); % is F (phenylalanine), or Y (tyrosine); # is N (asparagine),D (aspartic acid), Q (glutamine), E (glutamic acid), or Bz (aromatic amino acids). E (glutamate) and H (histidine) amino acid residues involved in iron-binding (Andersson and Nordlund,1999; Berthold et al., 2000) are indicated by filled black circles. Numbers give the position of the amino acid sequence aligned to the Arabidopsis thaliana AOX1a protein (NM_113135).


Please cite this article as: Costa, J.H., et al., A classification scheme for alternative oxidases reveals the taxonomic distribution and evolutionaryhistory of the enzyme in angi..., Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.007


Fig. 3. A flowchart outlining how to use the classification scheme using an AOX protein from Oryza sativa.


3.4. Classification scheme and reclassification of previously annotated AOXs

In addition to themonocot AOX1b and 1d proteins thatwere respec-tively renamed as AOX1d1 and 1d2 aswell as the AOX2b proteins (orig-inally found in fabales species) that were renamed as AOX2d, otherchanges in annotation were needed. Following the classification systemdescribed above, it was necessary to rename 27 of 80 annotated AOXsequences (Supplemental Table S2). In some cases, AOX genes havebeen incorrectly annotated in terms of subfamily (e.g. somegenes anno-tated as AOX2 proteins are actually AOX1 proteins). For example, Nico-tiana attenuataAOX2 (AY422689)was reclassified as an AOX1a protein,and Corylus heterophylla AOX1a was reclassified as an AOX2 protein. Inaddition, Mangifera indica AOX1c, Vitis vinifera AOX1b, and Solanumlycopersicum AOX1b were renamed as AOX1d in correspondence withthe orthologous Arabidopsis AOX1d. Furthermore, we detected anAOX1 (gi|1588565) incorrectly annotated as an AOX protein from


Solanum tuberosum (potato). This sequence is not found in the recentlysequenced potato genome and an examination of the protein indicatesthat it corresponds to a monocot AOX protein (AOX1) from theAlismatales order. The above situations serve to illustrate the need fora robust and comprehensive scheme to classify AOX proteins.

3.5. Diversity and evolutionary history of the alternative oxidase multigenefamily in angiosperms

All classified AOX sequencesweremapped onto an angiospermphy-logeny by subfamily and subtype (Supplemental Table S2; Fig. 4) ac-cording to the guidelines established by the Angiosperm PhylogenyGroup (APG III, 2009). Specific characteristics and patterns could be de-duced for the AOXs of basal angiosperms, magnoliids, monocots andeudicots.



Fig. 4. The distribution of AOX gene subfamilies in angiosperms mapped onto a phylogeny of flowering plants. The angiosperm phylogeny is presented according to the guidelinesestablished by the Angiosperm Phylogeny Group (APG III, 2009). Red denotes members of the AOX1a–c/e clade, green denotes members of the AOX1d clade, yellow denotes membersof the AOX2a–c clade, and blue denotes members of the AOX2d clade. (For interpretation of the references to color in this figure legend, the reader is referred to the web version ofthis article.)


3.5.1. Basal angiosperms and magnoliidsRecently, AOX1 and AOX2 subfamilies were found in the gymno-

sperm (Frederico et al., 2009; Neimanis et al., 2013). Thus, it was notparticularly surprising to find both subfamilies present in basal angio-sperms (e.g. within the Amborellales and Nymphaeales) (Fig. 4). Thisindicates the presence of the AOX2 subfamily prior to the divergenceof gymnosperms and angiosperms and its successful transmission tothe angiosperm lineage from the last common ancestor. AOX1 andAOX2 proteins from the species placed at the base of the angiospermphylogeny appear to be most similar to AOX1a–c and AOX2a–c,


respectively and these might therefore be considered the ancestralAOX proteins in angiosperms (Figs. 1 and 4).

AOX1 subfamily duplicationswere detected in the basal angiospermorder Nymphaeales and the magnoliid orders Magnoliales and Lauralessuggesting that the AOX1 subfamily gene expansion was an early eventin angiosperm evolution (Fig. 4). However, due to high sequence simi-larity among duplicated AOX1s identified in these species we suspectthat these genes are derived from recent gene duplication. No AOX1d(the more divergent form of AOX1) was detected, although this genehas been found in monocots and eudicots. A recent duplication of the




AOX2 subfamily was also detected, but only in a single species withinthe Piperales (i.e. Pipermethysticum). No evidence of AOX2dwas detect-ed. To enable a better resolution of the evolutionary history, futureworkshould investigate whether AOX proteins similar to AOX1d and AOX2dare present or absent in these species.

3.5.2. MonocotsThe majority of accessible data presented here correspond to AOX

sequences of monocot and eudicot species. With regard to the new se-quences identified, no AOX2 proteins were detected in monocots, ashas been previously observed (Considine et al., 2002). However, alarge amount of thedata available formonocots correspond tomembersof the Poales order, with some data available for the Zingiberales,Arecales, Asparagales, Dioscoreales, and Alismatales orders (Fig. 4; Sup-plemental Table S2). It therefore appears premature to state that AOX2is completely absent frommonocot species considering that no data arecurrently available for 6 of the 12 monocot orders (Fig. 4). Future re-search should determinewhether species belonging to the basal mono-cot orders, Alismatales and Acorales, contain the AOX2 subfamily.

The full complement of AOX genes can be determined in severalmembers of the Poales due to the availability of fully sequenced ge-nomes for species in this order. It has been previously shown that theAOX1 subfamily expanded in the Poales to 3 genes (AOX1a, 1c, 1d2) inmaize (Karpova et al., 2002), 3 genes (AOX1a, 1c, 1d1) (Ito et al., 1997;Saika et al., 2002) or 4 genes (AOX1a, 1c, 1d, 1e) in rice (Considineet al., 2002) and 4 genes in sugarcane (AOX1a, 1c, 1d1, 1d2) (Boreckyet al., 2006). Recently, 4 genes (AOX1a, 1c, 1d1, 1d2) were found in sor-ghum, and a fourth AOX gene (AOX1d1) was identified in maize (Zeamays) (Costa et al., 2009b). Here, we report additional species of Poalescontaining 4 AOX genes (AOX1a, 1c, 1d1, 1d2) such as in Brachypodiumdistachyon, Hordeum vulgare, Setaria italica and Triticum aestivum andinterestingly some Poales species containing 5 genes (AOX1a, 1c, 1d1,1d2, 1e) as in Panicum virgatum andHordeumpubiflorum (SupplementalTable S2). Thus, these findings reveal that AOX in the large majority ofinvestigated Poales is encoded by 4 or 5 AOX1 genes (Fig. 4; Supplemen-tal Table S2).

The AOX1e gene found in the Poales appears to be present in fewspecies, besides P. virgatum and H. pubiflorum this gene was detectedin rice (AOX1d, renamed as AOX1e). Previously, this rice AOX genewas identified only in genomic DNA (Considine et al., 2002) suggestingthat it might be a pseudogene, however, our analysis detected severalESTs (Supplemental Table S2) only in shoots and roots of germinatingseeds indicating perhaps a specific function in germination. Here, wefound that AOX1e is also present in some plants of the Zingiberales,Arecales (it was duplicated), Asparagales and Alismatales orders.(Fig. 4; Supplemental Table S2).

Apart from rice, which has a single AOX1d protein, other species(B. distachyon, H. vulgare, P. virgatum, Saccharum officinarum, S. italica,S. bicolor, T. aestivum and Z. mays) within the Poales have duplicatedthis gene resulting in the presence of AOX1d1 and 1d2. In O. sativa,Z. mays, S. bicolor, S. italica and B. distachyon, where the genomes havebeen sequenced, a similar gene arrangement along chromosomes isfound, revealing a common tandem position of the AOX1d2, AOX1d1and AOX1a genes (except in O. sativa which lacks AOX1d2) on thesame chromosome while AOX1c is located on a different chromosome.Within these five species, intron 2 of the AOX1d1 and 1d2 genes hasbeen deleted resulting in a peculiar gene structure of 3 exons and 2 in-trons. In our analyses, AOX1d was also detected in some plants outsideof the Poales order, in the Asparagales (Agave genus) and Zingiberales(Costus pictus) orders, but only EST data were available.

Overall, monocots typically show a variable expansion of the AOX1subfamily (AOX1a–e) (Fig. 4). Our results indicate that the AOX2 sub-family may have been lost early in monocot evolution. A search for theAOX2 subfamily in the Ceratophyllales order will be useful to better un-derstand the evolutionary history, since it is placed between themono-cots and dicots in the phylogeny of angiosperms (Fig. 4).


3.5.3. EudicotsIn Eudicot species, we have detected several patterns of AOX family

composition and distribution. Unlike the situation in monocots, fewexamined eudicots have expanded the AOX1 by recent gene duplication(AOX1a–c). We observed that AOX1 expansion has occurred in basaleudicots in the Ranunculales and Proteales orders, and in thecore eudicot orders of Celastrales, Malpighiales, Brassicales, Apiales,Sapindales, Solanales, and Asterales (Fig. 4). The eudicot AOX1d pro-teins have a broad distribution within the Rosid and Asterid clades ofthe core eudicots, but are not found in any other eudicot groups(Fig. 4). Curiously, plants that contain an AOX1d gene appear to lackthe AOX2d gene and vice versa (Fig. 4).

AOX2 subfamily expansion by AOX2 duplication is more commonthan was previously recognized in eudicots. We have detected severalAOX2 duplications in the Rosids and Asterids (Fig. 4). While Rosid spe-cies typically contain AOX2a and AOX2d proteins (in the Fagales,Rosales, Fabales and Malvales orders), in some cases recent duplicationof the AOX2 gene (AOX2a–b) was confirmed (Fig. 4). The release of thePrunus persica and Prunusmume (Rosales), Cicer arietinum (Fabales) andGossypium raimondii (Malvales) genomes allowed the retrieval of 4 AOXgenes that correspond to AOX1, AOX2a, AOX2b and AOX2d (Supple-mental Table S2). Also, in species of the Malpighiales order, similarAOX2 duplications (AOX2a–b) have been found in Hevea brasiliensis,Euphorbia peplus, and Linum usitatissimum (Supplemental Table S2). InAsterids, while no AOX2d was found, recent AOX2 duplications(AOX2a–b or AOX2a–c) were extensively detected in 7 of 8 orders ana-lyzed. Only in the Solanaleswas this duplication not found (Supplemen-tal Table S2).

AOX2d proteins are restricted to Rosid species in the orders Fagales,Rosales, Fabales, Malvales, and to the most basal eudicot ordersProteales and Ranunculales (Fig. 4). This may indicate that the originof AOX2d proteins was an early event in eudicot evolution (Fig. 4). Inorder to test this hypothesis, the presence of AOX2d proteins shouldbe investigated in the Ceratophyllales and other Rosid orders.

Overall, the eudicots have at least one AOX1 (subtype 1a–c) and oneAOX2 (subtype 2a–c) while AOX1d and AOX2d proteins appear to bemutually exclusive and species specific. However, the sequencing ofsome plant genomes has helped us to find other important trends in an-giosperm AOX families. We have identified species with complete dele-tions of the AOX1 or AOX2 subfamilies in eudicots.

Our investigation has revealed that some Rosids lack the AOX1 sub-family. BLAST searches on the sequenced genome of Cucumis sativus (aCucurbitaceae) cultivars, Chinese Long (Huang et al., 2009) andBorszczagowski (Wóycicki et al., 2011), revealed a single AOX2 (sub-type 2a-c) gene. Similarly, searches in the available genomes (NCBI) ofother Cucurbitaceae as Cucumis melo and Citrullus lanatus also showonly the same AOX2 gene. In addition, BLAST searches on the sequencedgenomes of Euphorbiaceae such as Jatropha curcas, Manihot esculentaand Ricinus cummunis also revealed only a single AOX2 (subtype 2a–c)gene. Several EST sequences (NCBI) indicate that it is expressed insome species (Supplemental Table S2). Searches in the draft genomeof H. brasiliensis, another Euphorbiaceae, also failed in detecting anyAOX1 gene, revealing two AOX2 (subtype 2a–b) genes. EST data avail-able for other Euphorbiaceae (i.e. Euphorbia esula, Euphorbia tirucalli)(see Supplemental Table S2) also indicate expression of only Aox2 (sub-type 2a–b) genes. Thus, for the first time, eudicot plant species that lackAOX1 were identified.

In addition to all studied monocots, the dicot P. trichocarpa (orderMalpighiales; family Salicaceae) also lacks an AOX2 in its genome(Costa et al., 2009b). Here, we have also observed the absence ofAOX2 in the genome of Salix purpurea, another Salicaceae species (Sup-plemental Table S2). Thus, it is possible that this AOX2 deletion mayhave extended throughout several other species of the Salicaceae fami-ly. To date, this represents the only case of an AOX2 deletion in eudicots.

Applying the new AOX classification scheme revealed that eudicotplants can be categorized by differences in their AOX family composition




into plants that have: 1) a single copy of AOX1 and AOX2 (usually AOX1aand AOX2a); 2) an expanded AOX1 family (AOX1a–c); 3) an expandedAOX2 family (AOX2a–c or AOX2d1, 2d2, e.g.Medicago truncatula); 4) ex-panded AOX1 and AOX2 families, such as Aquilegia coerulea (AOX1a–b;AOX2a, 2d); and 5) a lack of AOX2 (e.g. P. trichocarpa). For the firsttime, plant species that lack any gene of the AOX1 subfamily e.g. inthe Cucurbitales (C. lanatus, C. sativus and C. melo) and in someMalpighiales (M. esculenta, R. cummunis, J. curcas and H. brasiliensis)were discovered.

Fig. 5. A model for the evolutionary history of AOX subfamilies and types in an


3.6. The evolutionary history of AOX in angiosperms

Due to the fact that members of both the gymnosperms and angio-sperms contain AOX1 and AOX2 subfamilies, we hypothesize that thelast common ancestor of these lineages contained both of these subfam-ilies (Fig. 5). Due to the presence of AOX1 (subtype 1a–c) and AOX2(subtype 2a–c) genes in basal angiosperms we believe that these arelikely the ancestral forms of the enzyme in angiosperm plants (Fig. 5).However, in spite of the fact that no AOX1d has been found in basal

giosperm plants based on taxonomic distribution and AOX classification.




angiosperms, the presence of this gene in both monocots and eudicotsindicates that it may also be an ancestral gene. Based on the currentdata, it appears that monocots experienced a loss of the AOX2 geneearly in their evolution as no monocot examined to date contains thissubfamily (Fig. 5). As mentioned previously, an examination of theAOX genes present in a member of the Ceratophyllales could help to re-solve this question. Monocots experienced variable AOX1 duplication(AOX1a–e) including AOX1d (AOX1d1, 1d2) that is duplicated in themajority of Poales examined (Fig. 5, Supplemental Table S2). TheAOX2d appears to have its origin somewhere between the basal angio-sperms and the evolution of eudicots, since monocots do not containthis gene (Fig. 5). Loss of AOX1d/AOX2d seemed to be common sinceboth genes are facultative and mutually exclusive. Within the coreeudicots, while some Rosids have duplicated the AOX2d gene, it appearsto have been lost in the Asterids. Conversely, the loss of AOX1dwas seenmainly in some Rosids. Some AOX1 (subtype 1a–c) duplications oc-curred in both Rosids and Asterids while AOX2 (type 2a–c) duplicationswere more common in the Asterids. Interestingly, all examinedCucurbitales and some Malpighiales (Euphorbiaceae family) have lostAOX1 (subtype 1a–c), AOX1dandAOX2d (Fig. 5). The loss of AOX2, sim-ilar to that found in monocots, was also found in eudicot species of theSalicaceae family (Fig. 5).

3.7. AOX1d and AOX2d — a hypothesis of convergent gene evolution

One of the most interesting findings of our study was the commonspecific sites found between AOX1d and AOX2d proteins. In spite ofthe large divergence among AOX2d (within eudicots) and AOX1d (inmonocots and eudicots) (Fig. 1), we have found four specific sites incommon (at positions 167, 178 and 277, 278) where the amino acidsdiffered between AOX2a–c and AOX1a–c/1e proteins, but were similaramong these other proteins (Fig. 2). In addition, these differences inboth AOX2d and AOX1d occurred mainly in the vicinity of the secondconserved cysteine, Cys II (C177) suggesting that both proteins couldhave similar post-translational regulation.

Some facts lend support for a hypothesis that invokes a convergentevolution of these genes such as: 1) Monocot AOX1d appears to haveundergone a duplication event (except in rice) and exists in a tandemarrangement with AOX1a (in an identical gene order: AOX1d2,AOX1d1, AOX1a) in all investigated Poales; 2) Eudicot AOX1d appearsin a tandem arrangement with AOX1a in S. lycopersicum, S. tuberosumand Solanum phureja (Asterids), V. vinifera and Eucalyptus grandis(Rosids), despite the fact that these genes appear in separate chromo-somes in A. thaliana and P. trichocarpa; and 3) AOX2d appears in a tan-dem arrangement with AOX2a in the Fabales (Glycine max and Lotusjaponicas) and in the Rosales (P. persica). Considering that AOX1d andAOX2d can appear in a tandem arrangement with AOX1a and AOX2a,respectively, this may indicate that both genes evolved from a duplica-tion of a specific AOX subfamily/class and later converged in sequenceresulting in the existing genes.

With regard to expression analyses, in spite of the limited data avail-able evidence suggests that AOX1d and AOX2d could play a complemen-tary role with AOX1(a) in stress conditions. In monocots, experimentaldata are found for O. sativa and Z. mays. In O. sativa, different studiesshowed that AOX1a and AOX1b (renamed as AOX1d) were induced byseveral stress conditions including chilling, drought, and high salt,while the AOX1c gene was not responsive to these stresses (Fenget al., 2013; Li et al., 2013; Ohtsu et al., 2002). In Z. mays, aox2(AOX1a) and aox3 (AOX1d2) were induced in normal maize seedlingsby specific inhibitors of respiratory complexes (Karpova et al., 2002).No data for AOX1d1 is available.

In eudicots, two species presenting AOX1d in their genomes werewell studied: Arabidopsis and S. lycopersicum. In Arabidopsis, besidesAOX1a, the most studied AOX stress-responsive gene (Clifton et al.,2005, 2006; Giraud et al., 2008; Ho et al., 2008; Saisho et al., 1997),AOX1d also appears among the most stress-responsive genes encoding


mitochondrial proteins (Clifton et al., 2006), while AOX1c has beenlinked to plant development (Ho et al., 2007). In S. lycopersicum, recentworks have focused on AOX1a revealing its role in stress and fruit ripen-ing using transgenic plants (Ma et al., 2011; Xu et al., 2012). However,studies focusing on all AOX genes (AOX1a, AOX1b (renamed as AOX1d),AOX1c and AOX2) also show that AOX1b (AOX1d) together with AOX1aare the most AOX1 responsive genes to tobacco mosaic virus infection(Fu et al., 2010; Liao et al., 2012) and to cold-treatment of fruit(Holtzapffel et al., 2003).

For AOX2d (previously named AOX3/AOX2b) the data are limited tothe leguminous plantsG.max,V. unguiculata and species of theMedicagogenus. The stress-induced expression of both AOX1 and AOX2b (AOX2d)was first found in V. unguiculata (Costa et al., 2010). In G. max, althoughonly AOX1 was stress-responsive in the Stevens cultivar (Djajanegaraet al., 2002; Millar et al., 1997; Thirkettle-Watts et al., 2003), bothAox1 and Aox2b (AOX2d) were induced in response to salicylic acid inthe Cresir cultivar (Matos et al., 2009). Recently, Cavalcanti et al.(2013) identified that duplicated AOX2b genes (renamed as AOX2d1and2d2) were differentially responsive to several stress conditions togetherwith AOX1 in two species of the Medicago genus (M. sativa and M.truncatula). Both AOX1 and AOX2bs (AOX2d1 and 2d2) were located onthe same chromosome corroborating with the idea that commontrans-acting elements could be involved in AOX1 and AOX2d inductionunder stress conditions. Similarly for AOX1a and AOX1d, the stress-induced co-expression could be linked to gene proximity. Except forArabidopsis in which AOX1a and AOX1d are on different chromosomes(Clifton et al., 2006), S. lycopersicum, O. sativa and Z. mays AOX1a/AOX1d are in tandem arrangement on the same chromosome.

4. Conclusions and future directions

Wehave generated a classification scheme for angiospermAOXpro-teins using a large data set of deduced amino acid sequences. This newclassification is based on the phylogenetic analyses of protein se-quences, the analysis of specific amino acid sites found to differ betweenAOX subfamilies and subtypes, and the known evolutionary history ofangiosperms. It confirmed the presence of two subfamilies, AOX1 andAOX2, and indicated that these are distributed into 4major phylogenet-ic clades: AOX1a–c/1e, AOX1d, AOX2a–c and AOX2d. In spite of thelarge data set of sequences used here we are aware that the classifica-tion scheme will need to be continuously updated. For instance, someBrassicales AOX2 clustered away from the core AOX2a–c subtype inthe phylogenetic tree (Fig. 1). This fact appeared to be due to the addi-tion of two amino acids between positions 110 and 111 not found inother AOX1 or AOX2 proteins as well as a change in a key position(230) of the main specific sites of AOX2 (Supplemental Fig. S1). Thus,a suitable AOX classification could also consider other gene features asintron/exon structure and/or expression profile. However, for AOX, ge-nomic data are still limited and expression data are not yet available forgreater than 90% of all genes presented here.

The gene number of AOX in angiosperms is very diverse rangingfrom 1 to 6 genes and is comprised of variable combinations of differentAOX subfamilies and types among species (Supplemental Table S7). Ourresults confirmed the previous finding that monocots appear to lack theAOX2 subfamily (Considine et al., 2002). Despite the fact that a feweudicots appear to have lost the AOX1 or AOX2 subfamily, the generalprofile of AOX families in eudicots is one that contains at least onecopy of AOX1 (subtype 1a–c) and AOX2 (subtype 2a–c)with a variationin the presence/absence of AOX1d and AOX2d. Thus, it is suggested thatAOX1 (subtype 1a–c) and AOX2 (type 2a–c) appear more frequentlydue to a requirement in eudicot metabolism, while the AOX1d andAOX2d proteins (which appear to be mutually exclusive) might be in-volved in acclimations and adaptations to environmental stressorsthat are species specific. It remains curious as to why eudicot plantsthat have AOX1d lack AOX2d and vice versa. For the first time, it wasconfirmed that in some eudicot species AOX is encoded by a single




gene, generally an AOX2, establishing that AOX is not always encodedby multigene family.

AOX families/types differ from each other mainly in the region sur-rounding the di-iron binding sites co-ordinated by conserved glutamate(E222) and histidine (H225) residues and/or in the vicinity of conservedcysteines, Cys I (C127) and Cys II (C177). As these regions are importantin the activity and post-translational regulation of the enzyme, thesedifferences may be functionally important.

AOX1d and AOX2d share common specific amino acids primarily inthe vicinity of the second conserved cysteine, Cys II (C177), suggestingthat they could have a common regulation in spite of the fact thatthey are from different subfamilies. In addition, available expressiondata indicate a complementary role of these genes with a AOX1(a) instress conditions. These findings suggest that both AOXs apparentlyevolved through convergent evolution.

We anticipate that the new classification systemwill have an impacton the identification of trends in AOX functionality across species. Animproved knowledge of the AOX family composition in different angio-sperm species will provide a better chance to gain insight into AOX reg-ulation and physiological function. For instance, comparing the twoBrassicales species, Arabidopsis (AOX1a, 1b, 1c, 1d andAOX2) andpapa-ya (AOX1 and AOX2) does not appear to be a good choice to study func-tional similarities due to the differences in the complement of AOXproteins present. It may be more appropriate to compare Arabidopsiswith poplar (AOX1a, 1b, 1c, 1d), a Malpighiales, which shows similarAOX1 gene duplication, in order to address questions about AOX1 func-tionality. Similarly, a comparison between papaya AOXgenes to those inCannabis sativa (AOX1 and AOX2)would perhaps be an effectiveway tostudy both AOX subfamilies. In this context, a major challengewill be tounderstand why some plants that are more phylogenetically divergenthave similar multigene families, while more closely related specieshave such large differences in AOX family composition. In addition,since AOX1 is known as the main stress-responsive AOX gene, whatwould be the implication of its absence in species from Cucurbitalesand some Malpighiales orders? The role of AOX2 duplication (AOX2a–c) which has occurred mainly in asterids needs to be clarified. In the fu-ture, AOX gene expression studies involvingdifferent species containingdifferent AOX families and types will help to advance research intoquestions about the physiological significance of different AOX typesin angiosperms. It remains to be determined whether the classificationscheme that we have developed here for angiosperms will be of use ininvestigating the recently described AOXs of non-angiosperm plants(Neimanis et al., 2013). Finally, the classification system might be ofhelp to identify potential functional marker candidates for plant breed-ing across diverse species (Arnholdt-Schmitt et al., 2006; Cardoso andArnholdt-Schmitt, 2013).

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.mito.2014.04.007.

Acknowledgments

The authors thank CNPq (Brazilian Government Organization forgrant aid and fellowship to Brazilian researchers) for the productivityfellowships granted to J. H. Costa and D. Fernandes de Melo. BirgitArnholdt-Schmitt was supported by the Portuguese Foundation for Sci-ence and Technology (FCT) with a personal grant and through funds ofthe research center ICAAM/University of Évora, as well as projects byFCT and the European Commission. Work in AE McDonald's lab isfunded by start-up funds fromWilfrid Laurier University and the Natu-ral Sciences and Engineering Research Council of Canada.

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A classification scheme for alternative oxidases reveals the taxonomic distribution and evolutionary...

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