(Received for publication, August 2, 1996, and in revised form, October 23, 1996)
From the Department of Plant Genetics, Weizmann
Institute of Science, P. O. Box 26, Rehovot, Israel and the
§ Department of Field and Vegetable Crops, The Faculty of
Agriculture and The Otto Warburg Center for Biotechnology, The Hebrew
University of Jerusalem, Rehovot 76100, Israel
Aldehyde oxidase and xanthine dehydrogenase are a group of ubiquitous hydroxylases, containing a molybdenum cofactor (MoCo) and two iron-sulfur groups. Plant aldehyde oxidase and xanthine dehydrogenase activities are involved in nitrogen metabolism and hormone biosynthesis, and their corresponding genes have not yet been isolated. Here we describe a new gene from tomato, which shows the characteristics of a MoCo containing hydroxylase. It shares sequence homology with xanthine dehydrogenases and aldehyde oxidases from various organisms, and similarly contains binding sites for two iron-sulfur centers and a molybdenum-binding region. However, it does not contain the xanthine dehydrogenase conserved sequences thought to be involved in NAD binding and in substrate specificity, and is likely to encode an aldehyde oxidase-type activity. This gene was designated tomato aldehyde oxidase 1 (TAO1). TAO1 belongs to a multigene family, whose members are shown to map to clusters on chromosomes 1 and 11. MoCo hydroxylase activity is shown to be recognized by antibodies raised against recombinant TAO1 polypeptides. Immunoblots reveal that TAO1 cross-reacting material is ubiquitously expressed in various organisms, and in plants it is mostly abundant in fruits and rapidly dividing tissues.
Molybdenum cofactor (MoCo)1 binding is common to a group of ubiquitous enzymes, including nitrate reductase, xanthine dehydrogenase, sulfite oxidase, and aldehyde oxidase (1, 2). These enzymes are involved in various types of oxidative metabolism, and show broad and occasionally overlapping specificities (1). A subset of this enzyme class, MoCo hydroxylases, includes the structurally similar enzymes xanthine dehydrogenase (XD) and aldehyde oxidase (AO). These enzymes contain, in addition to the molybdenum cofactor, FAD and two types of iron-sulfur centers (3). They have been shown to be related to the AO from Desulfovibrio gigas (MOP) for which a crystal structure has recently been described (4).
AO (aldehyde-oxygen oxidoreductases) are widely distributed among various organisms. They are characterized as dimers of two 150-kDa subunits and catalyze the oxidation of N-heterocyclic compounds in the presence of O2, but appear to display a broad range of substrate specificities (5). In humans, AO has been implicated in familial amyotrophic lateral sclerosis (6) and hepatotoxicity of alcohol (7). In plants, AO activities are implicated in the biosynthesis of two plant hormones, abscisic acid (ABA) and indole acetic acid (IAA). The plant hormone ABA is involved in various processes, including the reaction of plants to environmental stresses such as wounding, water stress, seed development, and plant development (8). In the ABA biosynthesis pathway, AO is thought to catalyze the conversion of ABA aldehyde to ABA, which is considered to be the last step in the biosynthesis of ABA. Indeed, MoCo defective tobacco and barley mutants were found to be deficient in ABA synthesis (9, 10). IAA is involved in many aspects of plant growth and development, however, its biosynthetic pathway remains to be elucidated. Recently, an AO activity from maize coleoptiles was shown to efficiently oxidize indole-3-acetaldehyde, a putative precursor of IAA (11). It is unknown whether the broad substrate specificities attributed to AO originate from a single enzyme or from a family of closely related enzymes.
XD is very similar to aldehyde oxidase in cofactor content, molecular weight, and sequence (1, 3, 12). It is a ubiquitous enzyme which is involved in purine metabolism. In plants, XD plays a central role in nitrogen assimilation, and was found to be highly enriched in nitrogen-fixing nodules of legumes of the ureide class (13). Genes encoding XD have been identified in mammals, chicken, flies, and Aspergillus (14, 15, 16, 17, 18, 19, 20).
The distribution and substrate specificities of human AO and XD activities were shown to overlap but to be distinct (5, 21). Comparison of the sequences of recently described AO genes with those of various genes reveals high homology between eukaryotic AO and XD genes, particularly in the sequences involved in the binding of the different cofactors. However, several sequences thought to be involved in NAD binding and substrate specificity are absent from AO, and can be used to differentiate between the two enzymes. The absence of the NAD-binding site is in agreement with the fact that AO does not require NAD for its action, and the lack of substrate binding sequences corresponds to the different specificity range of the two enzymes. Strikingly, a high level of homology was found between eukaryotic XD and AO genes and the bacterial MOP in the domains which participate in the iron-sulfur centers and MoCo binding (4).
The genes which encode either XD or AO have not been described in plants. Their isolation will shed light on the origin of the different attributed activities, and will aid in the elucidation of the biosynthetic pathways in which these enzymes are involved. Here we describe a novel gene family from tomato, tomato aldehyde oxidase (TAO), which is highly homologous to XD and AO genes. Detailed sequence comparisons in the different functional domains suggest that this gene family belongs to the AO rather than the XD type of MoCo containing hydroxylases. We show that this gene family, as well as AO activity, are highly expressed in fruits of various plant species. We genetically map the genes from the family to two gene clusters on two different chromosomes.
The Lycopersicon pennellii introgression lines population (ILs), described by Eshed and Zamir (22) was used to genetically map the different copies of TAO1. RFLP analysis of Southern blots was performed as described previously (23). Physical distance of TAOa from TG105 was established by partial digestion of YAC 340-63. The digests were fractionated on counter-clamped homogeneous electric field gels (Bio-Rad), blotted, and hybridized with probes. The maximal distance between a pair of markers was estimated according to the smallest partial band that contained both markers.
Clones and Sequence AnalysisYAC 340-63, which contains the
RFLP marker TG105A, was generated from the tomato line Rio
Grande-PtoR, and cloned in the vector pYAC 4 (24). The sequence of
TAO1 was obtained from four overlapping clones, as shown in
Fig. 1. Clones TAO1-1 and TAO1-10 were isolated by
using YAC 340-63 for screening of a cDNA library from roots of
Lycopersicon esculentum c.v. Mogeor. Since these cDNA
clones were not complete, the 5 end of TAO1-1 was used to screen
again the same cDNA library, and a longer but still partial
cDNA clone was isolated, TAO1-5. The 5
end of the sequence was
obtained from a genomic clone, TAO1-G7. Overlapping sequence confirmed that the different clones originated from the same gene. Sequence analysis was performed using the sequence analysis software package of
the University Wisconsin, Genetics Computer Group (25). The insert of
cDNA clone TAO1-10 was used as a probe for Southern blot
analysis.
Production of Anti-TAO1 Antibodies and Immunoblotting
Two
segments of TAO1 were overexpressed in bacteria, using two
different types of expression vectors (Fig. 1). For antibody 1, a
500-base pair long EcoRI fragment from the partial cDNA
clone TAO1-10 (containing one internal EcoRI (E) site and
one EcoRI site from the vector (E), was fused to the
glutathione S-transferase protein from Schistosoma
japonicum, using the pGEX expression vector (Pharmacia). The
resulting fusion protein contained amino acids 950-1128 of the TAO1
protein (Fig. 1A, ab 1). For antibody 15, the construct
contained the flanking 800-base pair EcoRI segment from the
same cDNA clone (again containing one internal EcoRI site and one site from the vector), which was fused to a stretch of
histidine residues, using the pRSET expression vector (Invitrogen). The
resulting fusion protein contained amino acids 1129-1315 of the TAO1
protein (Fig. 1A, ab 15). The polypeptides produced in these
clones were purified by glutathione-agarose for the glutathione S-transferase fusion protein and by bound nickel columns for
the histidine-fusion proteins. The purified peptides were injected into
guinea pigs to obtain antibodies ab 1 and ab 15. Both antibodies recognized on immunoblots polypeptides of apparent 78 kDa (Fig. 1B), which were not detected by control antibodies (not
shown).
SDS-PAGE
and immunoblotting were performed as described by Raz and Fluhr (26),
using the ECL system (Amersham). Young green fruits were ground in 20 ml of extraction buffer containing 50 mM Tris-HCl (pH 2.5),
1 mM EDTA, 1 µM sodium molybdate, 2 mM 2--mercaptoethanol, 5 µg/ml leupeptine, 5 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µM FAD, and 0.5 g of polyvinylpyrrolidone, at
4 °C. The extracts were filtered through 4 layers of gauze, and
centrifuged at 10,000 × g for 20 min. The supernatant
was applied to a 2-ml Q-Sepharose column (Pharmacia). The column was
washed with 3 volumes of extraction buffer without
polyvinylpyrrolidone, and proteins were eluted by 1 volume of
extraction buffer containing 0.5 M NaCl. The eluate was
either dialyzed overnight or desalted on a P6 (Bio-Rad) minidesalting
column. Samples (50 µg) were fractionated on a native PAGE and
activity was assayed as described (10).
During a high
resolution mapping study of the I2 Fusarium wilt resistance
gene from tomato, we used a 350-kilobase YAC clone, YAC 340-63, to
screen a root cDNA library. One of the partial cDNAs isolated
was sequenced and found to be closely homologous to the mammalian AO,
not yet described in plants. The gene has been designated tomato
aldehyde oxidase 1 (TAO1). The sequence of
TAO1 was obtained by a combination of overlapping sequence fragments from 3 different clones (see "Experimental Procedures"). The deduced amino acid sequence, of 1315 amino acids, is compared in
Fig. 2 with the deduced amino acid sequences of a
representative aldehyde oxidase, the human xdh2 (20), and a
representative xanthine dehydrogenase, the rat xdh (14), which are the
most similar to TAO1. The human xdh2 was originally defined
as a xanthine dehydrogenase-type (15), but was shown to actually encode
an aldehyde oxidase activity (19, 27). Regions that contain identical amino acids are indicated with boxes. The homology resides
mainly in the NH2 terminus of the protein, which contains
the iron-sulfur centers, and in the COOH-terminal half that contains
the MoCo-binding domain (see below). The region between these two parts
is considered to bear the FAD-binding region and displays less
homology. We wished to more specifically relate TAO1 to
either the XD or AO groups of MoCo hydroxylases. In Fig.
3, we compare conserved functional regions of
TAO1 to the consensus sequence of XD-type genes, and to 3 AO-type genes. Xanthine dehydrogenases contain two iron-sulfur centers,
an FAD-binding domain, an NAD-binding site, a MoCo complexing domain,
and a substrate binding domain (Fig. 3, top; Ref. 19). The
locations of the two iron-sulfur domains have now been pinpointed by
the recently described crystal structure of MOP (4), and shown to be
highly conserved among XD and AO. The consensus sequence of both
iron-sulfur domains is also conserved in TAO1 (Fig. 3, iron sulfur 1 and iron sulfur 2), except for the
substitution of the first cysteine for methionine in the first
iron-sulfur center of TAO1. The cysteines are considered
crucial for the iron-sulfur domain and we are not aware of their
substitutions in other cases. As the 5 sequence of the TAO1
gene was obtained from a genomic clone (see "Experimental
Procedures"), the deduced sequence at that point may reflect an
intron junction rather than the actual first methionine residue.
However, sequencing of an additional 1020-base pair upstream region
showed no evidence for the presence of an additional
cysteine-containing exon, nor do the relevant surrounding nucleotides
give evidence for a clear splicing consensus sequence.
TAO1, as well as MOP, BAO, and HXD2 do not contain the sequence Gly394-Tyr-Arg (underlined in Fig. 2), which in the XD-type enzymes is thought to be involved in NAD cofactor interaction (28). The precise domain involved in the flavin adenine dinucleotide (FAD) binding is not fully established, and cannot be deduced from the crystal structure of MOP as this region is completely absent from MOP. Indeed in this region a relatively low level of homology exists among the different genes (Fig. 2).
The three-dimensional structure of MOP suggests a funnel-like structure which leads from the surface of the protein to a substrate binding pocket, in close proximity to the site that binds MoCo (4). The domains which participate in MoCo complexing and substrate binding are highly conserved among XDs, AO, and TAO1 as shown in Fig. 3, regions 1-5 (4). However, in several positions amino acid substitutions differentiate TAO1 from XDs, and in those positions TAO1 is more similar to MOP, BAO, and HXD2. Thus, glutamate 807 of rat XD, which is completely conserved among all XDs, is substituted with hydrophobic amino acids in BAO, HXD2, MOP, and TAO1, respectively (Fig. 3, region 1). The equivalent position in MOP is occupied by Phe425, which is situated in the substrate binding pocket, and may participate in determining the substrate specificity that differentiates XD and AO activities within the MoCo binding site. Region 4 of the MoCo-binding domain, which was shown by mutational analysis to play a role in substrate specificity of XD,2 is highly conserved among XDs, but is not conserved between XDs, AOs, and TAO1. In addition, the consensus sequence ERXXXH (underlined in Fig. 2) conserved between all XD-type genes, is completely absent from TAO1, as well as from BAO, HXD2/AO, and MOP. This sequence was suggested by mutational analysis to also be involved in determining substrate specificity of XD (19). Thus, the main apparent differences between TAO1 and XD-type enzymes lie in the proposed NAD and substrate-binding domains. The similarity of TAO1 to MOP, BAO, and HXD2 in these regions suggests that TAO1 encodes a MoCo hydroxylase of the AO-type.
TAO1 Cross-reacting Material Is Highly Abundant in Tomato FruitsIn order to examine the expression pattern of
TAO1, two non-overlapping segments from its 3 end were
expressed as fusion proteins in bacteria, and antibodies were raised
against the recombinant fusion proteins (see "Experimental
Procedures"). In immunoblots of tomato tissue a 78-kDa size protein
band was detected (Figs. 1B and 4).
Occasionally two protein bands of similar size can be resolved, which
may represent several related but distinct proteins of the TAO1 family
(see below), or additional successive degradation products. The 78-kDa
size is considerably less than the calculated 144.5-kDa molecular mass
for TAO1. However, as both antibodies, but not control antibody,
reacted on immunoblots with polypeptides of the same apparent 78-kDa
molecular mass, the possibility that the polypeptide detected is
irrelevant is unlikely (Fig. 1B). Thus, the observed size is
probably a physiological or extraction-based degradation product.
Similarly, a degradation of XD proteins to 80-kDa products was detected
in SDS-PAGE analysis of Drosophila proteins (29), and an
aldehyde oxidase from maize was shown to degrade upon SDS-PAGE, giving
a product of 85 kDa (11).
We wished to examine the expression pattern and tissue distribution of proteins from the TAO family in tomatoes. An immunoblot of various tomato organs is shown in Fig. 4. TAO1 cross-reacting material (CRM) is present in all tomato organs. It is most abundant in ovary, in developing fruits, and in dividing tissues, such as the shoot tips, which contain apical meristematic tissue. TAO1 CRM was found to be present at high levels in all fruit parts examined (Fig. 4), and in all stages of fruit development (not shown). An additional, lower molecular weight band is apparent in some lanes. This is probably an additional degradation product and is occasionally observed.
Tomato Fruits Are Enriched in Several Aldehyde Oxidase Activities Which Cross-react with Anti-TAO1 AntibodiesThe relatively high
expression of TAO1 CRM in tomato fruits prompted us to examine directly
aldehyde oxidase and xanthine dehydrogenase activities in these tissues
by a native activity gel assay (Fig. 5). The upper band
in Fig. 5A (asterisk) was substrate independent.
In leaf tissue, a band of xanthine dehydrogenase activity was detected
when hypoxanthine was used as the substrate (Fig. 5, arrow).
In fruits, overlapping bands could be detected, resulting in a
continuous region of stain (bar in Fig. 5A,
panel HX). When the xanthine dehydrogenase-specific inhibitor
allopurinol was added to the reaction, the single XD activity band from
leaves disappeared, as well as the most rapidly migrating band from the fruits. Thus, most activity bands in the fruits were not inhibited by
allopurinol (Fig. 5A, panel HX+AP), moreover allopurinol
itself served as a substrate for these activities (not shown). The
allopurinol-insensitive fruit activities were also detected when the
AO-specific substrate 6-methylpurine was used, indicating that this
tissue is rich in AO activity (5). This correlates with the high
abundance of TAO1 CRM in fruit tissue.
We wished to establish a correlation between the activities observed and the TAO1 CRM, detected in denaturing conditions. To this end, leaf and fruit proteins comigrating with the activity bands shown in Fig. 5A were excised from lanes adjacent to those stained for activity. The proteins were eluted electrophoretically and fractionated by a SDS-PAGE denaturing gel. As shown in Fig. 5B, anti-TAO1 antibodies detected a major band, of apparent molecular mass of 78 kDa. Minor bands are probably degradation products of the 78-kDa band. This result suggests that the smaller than expected 78-kDa CRM originates from enzymatically active proteins. Specific cleavage may be a result of denaturation during SDS-PAGE analysis. Alternatively, the native protein may be processed in vivo but still retain intactness and activity in native gels, and be separated to smaller fragments upon SDS-PAGE, as has been documented for XD during ischemia (14).
TAO1 Cross-reactive Material Is Ubiquitous in Plants and AnimalsTAO1 antibodies were derived from highly conserved
regions in the MoCo binding area (see Figs. 1, 2, 3). This prompted us to
examine the antibodies for cross-species reactivity in a
"zoo-garden-type" immunoblot (Fig. 6). Different
levels of TAO1 cross-reactive material of apparent 78-kDa migration
size could be detected in young fruits of all plants assayed, dicots
and monocots. In petunia seed pods a considerable amount of 140-kDa
apparent molecular mass fragment was also detected. Cross-reacting
material was also detected in liver cells and in Drosophila.
In Drosophila, mutants which do not express XD are well
known (30). We examined one of these mutants,
ry506, null for XD, for the presence of TAO1
cross-reacting material (Fig. 6, compare lanes DROS wt and
DROS 506). A significant level of cross-reacting material
could be detected in the mutant. The findings are consistent with the
observation that the antibodies recognize a broad family of MoCo
hydroxylases, in which TAO1 is related to the AO type.
Genetic Mapping of TAO1
Southern blot analysis revealed that
TAO1 was part of a multigene family (Fig. 7).
To position the members of the TAO family on the tomato
genetic map we used a near isogenic lines (IL) mapping population, in
which single chromosome segments from L. pennellii were
introgressed in 50 ordered lines into a L. esculentum
background (31). The absence of an L. esculentum-type
polymorphic fragment or the presence of a L. pennellii-type
polymorphic fragment in a Southern blot of a specific IL, indicates
that the origin of the particular fragment is from the introgressed
chromosomal segment. The ILs which proved relevant for the mapping of
the TAO family are illustrated in Fig. 7. A high level of
polymorphism was detected between the original parental lines on a
TaqI digest Southern blot, probed with a segment of the
TAO1 gene (Fig. 7, right panel, compare L. pennellii and L. esculentum). Nearly all the ILs, as exemplified by IL 8-1, displayed the L. esculentum-type
fragment pattern (Fig. 7, right panel). However, specific
ILs lacked a subset of L. esculentum-type bands, indicating
that the introgressed region contained a TAO copy. A
TAO gene cluster was localized in this way to the region of
overlap between lines IL 11-3 and IL 11-4, as several L. esculentum-type fragments were absent from both of these ILs
(fragments are indicated with arrows and designated "a" in the right panel of Fig. 7). These
fragments, as well as other nonpolymorphic fragments, were also present
in YAC 340-63, generated from L. esculentum, which was
previously mapped to this region of chromosome 11 (32). This result
confirms the mapping of the "a" fragments to chromosome 11, and
also delineates the additional non-polymorphic bands, mutual to
L. pennellii and L. esculentum, which map to the
same region of chromosome 11. Physical pulse field electrophoretic
mapping of YAC 340-63 has revealed that TAOa is distal of
marker TG105, with a distance of approximately 50 kilobase between
TG105 and TAOa (data not shown). Other
TAO copies were similarly mapped to a second locus,
TAOb, on chromosome 1, in the region of overlap between IL
1-1 and IL 1-2 (Fig. 7, left panel). Although screening the
rest of the 50 introgression lines did not reveal additional mapping
loci, some TAO fragments were not polymorphic between the
parental lines. Many of these could be assigned to YAC 340-63 positioned at the TAOa locus. However, the possibility
exists that the few remaining nonpolymorphic TAO genes map
to loci other than TAOa and TAOb. TAO1, the
representative TAO gene sequenced above, was assigned
unambiguously to the TAOa locus based on direct sequencing of YAC
340-63 subclones.
We describe here the isolation and characterization of a new gene from tomato, TAO1. Highest homology to TAO1 was found among xanthine dehydrogenase (XD) and aldehyde oxidase (AO) genes from several organisms (12, 14, 15, 16, 17, 18, 19, 20). It contains the consensus sequences of the two iron-sulfur domains and the MoCo-binding domains of XD and AO. We classify TAO1 as an AO-type structure, as it lacks sequences indicative of the NAD-binding domain and sequences suggested to be involved in XD substrate specificity. The lack of these particular features is reminiscent of the aldehyde oxidoreductase structure from D. gigas, (4), the bovine AO (12), and HXD2, a human MoCo containing hydroxylase gene (20) which was recently suggested to encode AO-type activity rather than XD-type (19, 27). Isolation of the cognate TAO1 polypeptide and direct examination of its activity will be necessary to verify these observations.
TAO1 belongs to a multigene family. The family members detected may code for other XD- or AO-type activities with variable substrate specificities and expression patterns. This may suggest that broad substrate range of AO activities originates from different, closely related, enzymes, rather than a single enzyme. The TAO family members display an unusually high level of genetic polymorphism detected by RFLP. This is reminiscent of the high level of polymorphism, detected in both DNA and protein, of XD enzymes from Drosophila ecotypes (33, 34). The polymorphism enabled facile mapping of TAO members to two gene clusters, on chromosomes 1 and 11. The clustering of closely related genes from the same gene family is akin to the clustering found in the recently isolated plant resistance genes (35, 36, 37). This phenomenon was also observed by us for the Fusarium wilt disease resistance gene candidate I2C,3 which maps in close genetic and physical proximity to TAO1. Resistance genes are also characterized by their highly polymorphic nature which are thought to be part of the plants adaptability to the changing pathogenic environment. Interestingly, AO activities in the liver carry out biochemical detoxification (12). If a similar detoxification function is played by the plant AO activities, then environmental adaptability may dictate pressure toward maintaining the observed genetic polymorphism in plant AO genes.
Two types of MoCo containing hydroxylase activities have been detected in plant extracts. One activity is inhibited by the XD specific inhibitor allopurinol and is thus classified as XD-type activity. The second type of activity is highly enriched in tomato fruits, is allopurinol insensitive, and can utilize both typical AO substrates such as 6-methylpurine and hypoxanthine, and the XD inhibitor allopurinol. Anti-TAO1 antibodies detect polypeptides in leaf and fruit. Due to the polyclonal nature of the anti-TAO1 antibodies we cannot rule out cross-reaction between AO- and XD-type polypeptides, however, the antibody reactivity appears well correlated with the prominent AO activity detected in fruits. In addition, we have noted that spectral stress induces accumulation of AO activity in leaves which was also correlated with an increase in cross-reacting polypeptide accumulation as detected by TAO1 antibodies.4
TAO1 CRM indicated immunoreactive polypeptides of approximately 78 kDa, which were detected by 2 different antibodies prepared from fusion peptides originating from sequential nonoverlapping carboxyl-terminal regions. In the cases of Drosophila and fruit pods from petunia plants an additional approximately 140-kDa polypeptide was detected, consistent with the predicted molecular mass of AO. In tomato, the 78-kDa molecular mass was detected when enzymatically active proteins were eluted from native gels and refractionated on SDS-PAGE. Based on the deduced amino acid sequence, the terminal 78-kDa part would begin roughly at amino acid 590, and thus contains the MoCo complexing domain but probably not the region containing the iron-sulfur centers. Interestingly, XD is known to undergo a physiologically well known and important proteolytic irreversible conversion from the XD to xanthine oxidase form. This proteolytic and irreversible conversion was shown to result in a "nicked" but active xanthine oxidase, which upon fractionation in denaturing gels yielded fragments of 20, 40, and 85 kDa, of which the latter is the carboxyl-terminal product (14). Thus the possibility exists that the 78-kDa TAO1 CRM is a result of similar proteolysis of the enzyme, which results in a nicked but enzymatically active protein. Such proteolysis may occur either as a physiological process in the plant, or during the extraction process. In either case, the fact that many of the extracts analyzed in cross-species immunoblot analysis yielded similar size polypeptide indicates that such proteolytic processes are ubiquitous.
Here we show that TAO1 immunoreactive polypeptides are abundant in ovaries and fruits of tomato and other plant species. The presence of immunoreactive polypeptide in tomato fruits correlates with the high level of AO-type activity detected in tomato fruits. A biological role for the presence of TAO in apical meristematic tissue and fruit is not known, and it may be directly related to the biosynthetic capacities required for typical plant metabolic "sink" tissues. Alternatively, the expression of AO in those tissues may fulfill the specific needs for the final steps in biosynthesis of two plant hormones, ABA and IAA. Recently zeaxanthin epoxidase activity was shown to be involved in the first step of ABA biosynthesis (38). An AO-type activity has been implicated by mutational analysis to be essential for the last step of ABA biosynthesis which is the conversion of ABA aldehyde to the active carboxylic form of the plant hormone ABA (9, 10). Interestingly, sitiens, one of the tomato ABA-deficient mutants putatively lacking AO activity, maps to the same chromosomal region as TAOb (39). With the help of a high resolution mapping population of that region we are pursuing the possibility that a member of TAOb is involved.5 In maize an AO activity, enriched in the coleoptile apical region, was shown to oxidize indole-3-acetaldehyde into IAA (11, 40). The abundant tissue-specific expression detected by TAO1 antibody in fruit may reflect the role ABA plays in seed maturation and dormancy, while the elevated expression detected in apical meristems is consistent with the role this tissue plays as a known source of auxin biosynthesis. The isolation of a member of the novel TAO gene family from tomato, highly homologous to the AO group of MoCo containing hydroxylases offers a useful starting point for the analysis of this pivotal gene family in plants.
We thank Dr. S. Tanksley for providing tomato libraries used in the work, and Drs. A. Glatigny and S. Scazzocchio for personal communication. We thank Shlomit Bleichmann for excellent technical assistance.