Molecular Distinction between Alternative Oxidase from Monocots and Dicots

The alternative oxidase (Aox) is encoded in two discrete gene subfamilies in higher plants. Aox1 is most widely known for its induction by stress stimuli in many tissues and is present in both monocot and eudicot plant species. Aox2, on the other hand, is usually constitutive or developmentally expressed in eudicot species but is absent from the genomes of all monocot species examined to date. This molecular distinction suggests a divergence of Aox across plant families and may even have implications for the role of this enzyme in different plant species. The attention of plant biologists was first drawn to this respiratory oxidase through its pivotal role in the thermogenic climacteric that occurs during the fertilization of flowers from aroid monocot species. The first Aox antibody and gene sequence came from work on the thermogenic spadix of Sauromatum guttatum (Aox1;Elthon et al., 1989; Rhoads and McIntosh, 1991). Subsequent approaches including cDNA library screening, PCR with degenerate primers designed from conserved regions, and complementation methods have identified cDNAs encoding Aox1-type sequences from a wide variety of non-thermogenic monocot and dicot plants, together with fungi and protista. Initial reports indicated a single nuclear gene in many species (Rhoads and McIntosh, 1991; Kumar and Soll, 1992). It has subsequently been shown that a small gene family exists in soybean (Glycine max), tobacco (Nicotiana tabacum), rice (Oryza sativa), Arabidopsis, and mango (Mangifera indica; Whelan et al., 1996; Ito et al., 1997; Saisho et al., 1997; Considine et al., 2001). Studies have revealed differential expression of the soybean genes in response to developmental cues and environmental perturbations (Finnegan et al., 1997; Millar and Day, 1997; McCabe et al., 1998;Tanudji et al., 1999). In soybean, Aox1 expression has only been documented after respiratory inhibition of cell cultures by antimycin A (Finnegan et al., 1998; Tanudji et al., 1999). The two soybean Aox2-type isozymes, Aox2 and Aox3 (now renamed Aox2a and Aox2b), are more prevalent, although differentially regulated (Finnegan et al., 1997; McCabe et al., 1998). Most studies to date have focused on the relationship of Aox1-type gene expression to stress adaptation (Vanlerberghe and McIntosh, 1997, and refs. therein). We present results from our own experimental studies and a review of available genomic and expressed sequence tag (EST) data, showing that whereas Aox2 is found in many eudicots, including soybean, mango, tobacco, tomato (Lycopersicon esculentum), and Arabidopsis, it is absent in all monocots examined to date. The fact that the Aox2 family represents the major form expressed in eudicots studied to date, where it shows tissue and developmental stage specificity but does not respond to stress, has implications for the role and function of Aox proteins in different plant species. Phenograms that show the sequence homology among various alternative oxidase protein sequences (A and B), together with a scale diagram of the intron/exon structure ofAox genes present in rice and Arabidopsis genomes (C). A, Comprising many mature or putatively mature, full-length plant Aox. B, Comprising a 78-amino acid region, corresponding to Met-158 through Met-235 of soybean Aox1. Alignment includes sequences that were amplified by PCR in this study, present in GenBank or SwissProt but not full length, or identified by tBLASTn searches of TIGR EST databases. Sequences were aligned with ClustalX v1.81 using the BLOSUM protein weight matrix (National Center for Biotechnology Information, Bethesda, MD). Using the Bionavigator programs Seqboot, Protdist, Neighbor, and Consense, the alignments were bootstrapped with 2,000 replicates, and data were combined into a consensus phenogram using the unweighted pair group method using arithmetic averages method with the C. albicansAoxA as an outgroup (Entigen Corp., Sunnyvale, CA). Extending the search to other angiosperms, our PCR approach also failed to amplify an Aox2-type clone from wheat (Triticum aestivum) genomic DNA. From wheat, we sequenced 16 exclusivelyAox1-type clones, representing two loci. tBLASTn against TIGR databases for wheat, barley (Hordeum vulgare), maize (Zea mays), and sorghum, which are all monocots, also failed to find Aox2-type ESTs (Fig. 1B). SeveralAox2-type ESTs were retrieved from the TIGR tomato EST database but not from the other eudicot ESTs searched, such as potato (Solanum tuberosum), medic, and ice plant (Mesembryanthemum crystallinum). The deduced sequences of several isozymes did not conform to the plant Aox1/2 subfamily classification, particularly the two barley Aox-like ESTs,Aox0a and Aox0b (data not shown). Their close alignment to the Aox genes of Candida albicans strongly suggests that they represent fungal contamination rather than authentic plant sequences. The combined homology phenogram of available data (Fig. 1B) clearly shows separation of Aox2- and Aox1-type sequences. Some further separation of Aox1 types into groups representing monocots and eudicots can also be observed, consistent with the notion that monocots represent a clade nested within the two major lineages of the dicots, now named eudicots and magnoliid dicots (Daly et al., 2001). Interestingly, the eudicot Aox1 types are found in two groups, one containing species of the order Rosidae (Arabidopsis, mango, and soybean) and the other containing species of the orders Asteridae (tomato and tobacco) and Caryophyllidae (ice plant; Fig. 1B).Aox2-types are found in species from the orders of both Rosidae (soybean, Arabidopsis, mango, and cowpea [Vigna unguiculata]) and Asteridae (tomato and tobacco). The eudicot species potato, ice plant, and medic that lack Aox2-types to date span these three eudicot orders. Thus, the absence of Aox2-types in these plants appears more likely at this stage to be due to limited available data rather than to a very narrow phylogenetic occurrence of Aox2 types among eudicots. Further data will be required to address this issue thoroughly. This combined data set shows that Aox2-type sequences are absent from all five of the monocot species investigated to date but are present in six of the nine dicot species investigated. It is unlikely that the degenerate PCR primers we used were unable to amplify monocot Aox2-type sequences. Aox displays a high degree of homology in discrete regions and PCR primers based on these sequence blocks have been used successfully across the wide phylogenetic gap between plants and fungi. Notably, we have amplified Aox-type sequences from plant tissue that clearly represent fungal contamination (M.J. Considine, J. Whelan, R.C. Holtzapffel, P.M. Finnegan, and D.A. Day, unpublished data). However, because EST data do not give a complete picture of all transcripts from a genome, we searched the complete genomic sequences available for plants. The Arabidopsis genome, a model dicot, was sequenced in 2000 (The Arabidopsis Information Resource, 2000). Also, the extensive but not complete rice genome sequence, a model monocot, was made available in 2001 (Barry, 2001; Monsanto Company, St. Louis). From extensive tBLASTn searches, we have concluded that the rice genome sequence currently available lacks an Aox2, whereas the presence of the well-characterized Arabidopsis Aox2 was detected. An alignment of the intron/exon structure of the Arabidopsis and riceAoxs reveals a large degree of conservation in intron positioning (Fig. 1C). Exceptions include Arabidopsis Aox2, which has four introns, the additional one intervening the first exon of other Aoxs. Also, one Aox of each species lacks an intron at the second “conserved” position, corresponding to the second intron of the rice Aox1a, riceAox1b, and Arabidopsis Aox1d. Among other species, only the soybean Aox genes have been as well characterized, revealing introns in Aox1 andAox2-type sequences at each of the three conserved positions common to most sequences shown in Figure 1C (McCabe, 2001). The presence of two Aox gene subfamilies raises the intriguing question: Do these families differ in their expression or regulation and thereby in their roles in plant respiratory metabolism? Two roles among Aox isozymes may be proposed based on the available expression data summarized in TableI—one a constitutive isozyme required for a generic, “housekeeping” function in respiratory metabolism, and the other related to a particular need under stress conditions. This proposal encompasses the long-held hypothesis that AOX acts as an overflow for carbon metabolism to uncouple anaplerotic functions from ATP production (Lambers, 1982) and the more recent hypothesis that it functions to minimize reactive oxygen species production from electron transport during oxidative stress (Purvis and Shewfelt, 1993; Wagner and Moore, 1997). Summary of tissue- and/or stress-related expression of various Aox isozymes that were publicly available on August 1, 2001 Aox isozymes were classed Aox1 or Aox2 types based on sequence alignment; exceptions were C. albicans, C. reinhardtii, and barley Aox/EST, which did not conform to the classification and were denoted Aox0. In addition, where sequence data were not given, data are denoted Aox0. Italics denote gene expression and plain text denotes protein abundance. Data were gathered from publications or retrieved by tBLASTn search, using soybean Aox1-3 as probes, from an EST database (TIGR). Note: “Aox0” does not preclude multiple Aox-immunoreactive bands in the study referenced. Summary of tissue- and/or stress-related expression of various Aox isozymes that were publicly available on August 1, 2001 Aox isozymes were classed Aox1 or Aox2 types based on sequence alignment; exceptions were C. albicans, C. reinhardtii, and barley Aox/EST, which did not conform to the classification and were denoted Aox0. In addition, where sequence data were not given, data are denoted Aox0. Italics denote gene expression and plain text denotes protein abundance. Data were gathered from publications or retrieved by tBLASTn search, using soybean Aox1-3 as probes, from an EST database (TIGR). Note: “Aox0” does not preclude multiple Aox-immunoreactive bands in the study referenced. The tandem Aox genes in C. albicans,Aox0A and Aox0B, also fit this dual function model. C. albicans Aox0A is constitutively expressed, whereas Aox0B is stress-induced by agents such as antimycin A and herbicides (Table I). Similarly, in the green algaeChlamydomonas reinhardtii, only one of the two characterized Aox isozymes showed mRNA induction in a mutant lacking mitochondrial complex III activity (Dinant et al., 2001). The well-characterized eudicots soybean and Arabidopsis fit this model too. In soybean, expression of Aox1 gene and protein is limited to conditions of extreme environmental stress (Table I). The Aox2-type genes, Aoxa2 and Aox2b, are apparently not stress-induced, Aox2a being abundant only in photosynthetic tissues and Aox2b seemingly ubiquitous. Likewise, in Arabidopsis, several agents that inhibit respiration or ATP synthesis induced the expression of Aox1a, whereas Aox2 was not induced by stress but was expressed during normal seed development (Table I). Both Aox1 and Aox2 sequences in mango are expressed in fruit. Notably, Aox2 peaks early in fruit development, whereas both Aox1s are expressed during late stages of fruit ripening/senescence that may involve oxidative stress. Considering that the total number of unique ESTs available in each of the monocots surveyed here averaged over 28,000, it was intriguing to find no evidence of an Aox2-type (Table I; TIGR, August 1, 2001). The Aox1-types identified through EST databases in both monocots and dicots were typically, but not exclusively, from stress expression-profiling experiments (Table I). We concede that this divide of function, although following a trend, does not fit every piece of the available data (Table I), and there are very likely cases of Aox1-type expression in some plant species after normal growth and developmental rather than stress cues. However, it must also be noted that there is no clear cut distinction between what is truly normal plant growth and development and what physiological states might impose an oxidative stress in plant cells and thus invoke a stress response. In our own research, young seedlings from monocotyledonous rice, maize, and wheat do not contain Aox protein in isolated mitochondria, and Aox activity is only observed after chilling or chemical stress treatments (A.H. Millar, D.A. Day, and J. Whelan, unpublished data). The apparent absence of a gene family member from monocot plants is not unique to Aox. The pyruvate dehydrogenase complex (PDC) contains a central subunit, dihydrolipoamide acetyltransferase, which has been found to differ between monocots and dicots. Two genes, one with a large N-terminal repeat, are found in dicots and both gene products are incorporated into the active PDC enzyme (Millar et al., 1998; Thelen et al., 1999). In contrast, only the smaller of the two proteins has been found in the monocot PDC enzyme and genome searches have suggested that the longer gene lineage is absent from monocots (Thelen et al., 1999). At a genomic level, multigene and multigenome approaches to identify the closest living relatives of flowering plants have confirmed the molecular distinction between dicot and monocot species (Qiu et al., 1999; Soltis et al., 1999; Daly et al., 2001), which was previously based largely on phenotypic traits. The apparent absence of the Aox2 gene family lineage in monocot plant species may have significant implications for researchers investigating the role and regulation of Aox in plants. There is a need to determine whether Aox plays somewhat different roles in plant species separated by the molecular divide between monocots and eudicots. Studies that have concentrated on the stress-induced nature of Aox may be missing part of the story.