Cloning and Molecular Characterization of Plant Aldehyde Oxidase*

Primary structural information of a plant aldehyde oxidase (AO), which was purified from maize coleoptiles using indole-3-acetaldehyde as a substrate, was obtained by sequencing a series of cleavage peptides, permitting the cloning of the corresponding cDNA (zmAO-1). The complete nucleotide sequence was determined; the deduced amino acid sequence encodes a protein of 1358 amino acid residues of M r 146,681, which is consistent with the size of the AO monomeric subunit. There is a significant similarity with AO from mammals and xanthine dehydrogenases from various sources. The maize AO polypeptide contains consensus sequences for iron-sulfur centers and a putative molybdopterin cofactor-binding domain. In addition, another cDNA (zmAO-2), highly homologous to zmAO-1 at both the nucleotide and amino acid sequence levels, was cloned. zmAO-2 would encode a protein of 1349 amino acid residues of M r 145,173 and has molecular characteristics similar to those of zmAO-1. zmAO-1 was expressed at a high level in roots, which was closely correlated with immunoblotting results using antiserum raised against the purified maize AO protein, whereas zmAO-2 was expressed at a higher level in coleoptiles than in roots. We propose each zmAO may have a unique function during plant development.

Aldehyde oxidases (aldehyde-oxygen oxidoreductase; EC 1.2.3.1; AO) 1 are ubiquitous enzymes that have been extensively investigated in animals and microorganisms. They catalyze the oxidation of a variety of aldehydes and N 2 -containing heterocyclic compounds in the presence of O 2 or certain redox dyes (1)(2)(3). AOs belong to the family of molybdenum-containing protein like xanthine oxidoreductase (xanthine:NAD ϩ oxidoreductase; EC 1.1.1.204; XD) and sulfite oxidase (4), and they possess a short oxidoreductive chain characterized by four ox-idation centers, such as two iron-sulfur clusters, a flavin cofactor, and a molybdopterin cofactor (5). AOs are known to be a homodimer, consisting of two 148-kDa subunits (see Ref. 6 and references therein).
Recently, an AO was purified from bovine liver and the corresponding cDNA was cloned (7). Consensus sequences for iron-sulfur centers and a molybdopterin-binding site in the deduced polypeptide were identified. The deduced amino acid sequence shows significant similarity with that of XD from various sources. Bovine AO is expressed at high levels in the liver and lung and has been implicated in the detoxification of certain types of environmental pollutants and xenobiotic. The animal AO may also play a role in retinoic acid synthesis (7)(8)(9). In addition, a human liver cDNA that had been thought to encode XD is now thought to encode an AO (10).
In plants, only a limited amount of information has been published concerning AO, being focused on its possible involvement in plant hormone biosynthesis, such as indole-3-acetic acid (IAA) (11)(12)(13)(14) and abscisic acid (ABA) (15,16). The last step in their biosynthetic pathways has been thought to be the oxidation of corresponding aldehyde: indole-3-acetaldehyde (IAAld) for IAA and abscisic aldehyde (ABAld) for ABA. However, there have been almost no detailed analyses of the molecular properties of plant AO, and no sequence data of either protein or DNA have been reported. Therefore, the actual function of the plant AO(s) is still obscure. In a previous paper, we described the purification of an AO that could oxidize IAAld to IAA from coleoptiles of maize (Zea mays L.) seedlings (17). The maize AO has an apparent molecular mass of about 300 kDa as estimated by gel filtration and was composed of two 150-kDa subunits. It contains flavin adenine dinucleotide (FAD), iron, and molybdenum as prosthetic groups. This was the first report showing the presence of these cofactors in a plant AO.
In the present paper, we describe the sequencing of peptides obtained by proteolytic cleavage of the purified maize AO and the molecular cloning of the respective cDNA. In addition, another cDNA showing significant similarity with cDNA of the AO was cloned. The structures of both deduced polypeptides were analyzed and compared with mammalian AOs and various XDs. These cDNA probes and a monospecific antiserum raised against the purified enzyme were used to study the tissue distribution of maize AO mRNA and protein.
Purification of AO and Sequencing of Proteolytic Peptides-AO was purified from maize coleoptile tips as described previously (17) and subjected to SDS-polyacrylamide gel electrophoresis for final purification. Coomassie-stained bands of 150 kDa were excised and treated with 0.3 g of Achromobacter protease I (API; a gift from Dr. Masaki, Ibaraki University (19)) at 37°C for 12 h in 0.1 M Tris-HCl (pH 9.0) containing 0.1% SDS. Peptides generated were extracted from the gel * This work was supported in part by Grant-in-aid for Scientific Research 06640846 (to T. K.) from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D88451 (zmAO-1) and D88452 (zmAO-2).
‡ Present address: Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan.
Molecular Cloning and Sequencing of the cDNA Encoding AO and the Homologous cDNA-The amino acid sequence information from API peptide 4 was used for synthesis of the sense oligonucleotide primer (5Ј-GGIGA(A/G)GCIGTITA(T/C)GTIGA(T/C)GA-3Ј) corresponding to amino acids 9 -16. The antisense primer (5Ј-GTCCAIGTICC(A/G)TC(A/ G)TGIATIAC-3Ј) was derived from positions 7-14 of peptide 3. RNA was extracted from maize coleoptile tips according to standard procedures, and the latter primer served to reverse transcribe the total RNA. The resulting cDNA was 100-fold diluted and amplified by polymerase chain reaction (94°C for 1 min, 48°C for 2 min, and 72°C for 3 min) in the presence of both the sense and antisense primers. Fifty cycles of amplification produced cDNA of 1959 bp that was cloned into the pCRII vector using the TA cloning kit (Invitrogen). The inserts were sequenced in both directions using double-stranded DNA as templates with a Taq dye primer cycle sequencing kit (Perkin Elmer). Nucleotide sequences of inserts from 11 independent clones demonstrated the presence of at least two kinds of cDNA fragments ("first PCR" in Fig. 1) for maize AO, both encoding amino acid sequences that match those of the peptides.
Full-length cDNAs were obtained using the Marathon cDNA ampli-fication kit (CLONTECH) in accordance with the supplier's instructions ( Fig. 1). In addition, oligonucleotide 5Ј-GAGTATAGCACAGAAAATCT-GCAGCCA-3Ј served to reverse transcribe the poly(A) ϩ RNA. The first 5Ј extended cDNAs ("RACE-PCR(1)" for zmAO-1 and -2) were obtained by the amplification with the oligonucleotides 5Ј-TGCTTTGCAGCCA-TATTAGCATATCTT-3Ј and 5Ј-CTTTGCCGCCATGTAGGCATACTTC--3Ј identified in the nucleotide sequences of first PCR for zmAO-1 and -2, respectively. The second cDNAs ("RACE-PCR(2)" for zmAO-1 and -2) were obtained with the oligonucleotides 5Ј-ACAGCCTTTTGGAAGC-CACCTGGA-3Ј and 5Ј-TTCCACCTATGGTTGCAGTGTTCC-3Ј synthesized on the basis of the nucleotide sequences of RACE-PCR(1) for zmAO-1 and -2, respectively. The final extended cDNA ("RACE-PCR(3)" for zmAO-1) were obtained with the oligonucleotide 5Ј-ATCGGACTT-GTTGTCGGCCTTGAC-3Ј on the basis of the sequence of RACE-PCR (2) for zmAO-1. The 3Ј region of the cDNAs (zmAO1-5 and zmAO2-4) were obtained by the amplification with oligonucleotides 5Ј-GATTGCT-GAAACACAAAGATATGCTAAT-3Ј and 5Ј-GATTGCTCAAACACA-GAAGTATGCCTAC-3Ј identified in the sequences of first PCR for zmAO-1 and -2, respectively. Each clone was sequenced in both directions using either vector primers or specific oligonucleotides synthesized based on the basis of information obtained from previous sequence analysis. Full-length cDNA sequences were obtained after fusion of corresponding 5Ј-and 3Ј-RACE fragments in accordance with the supplier's instructions. Computer analysis of the DNA sequence was performed with the Genetyx software package (Software Development. Co., Tokyo). Genomic Southern and Northern Blot Analysis-Genomic DNA was prepared from maize seedlings according to the method of Shure et al. (20). The DNA was digested with EcoRI or HindIII, fractionated on a 0.7% (w/v) agarose gel, denatured, and transferred to a Hybond-N ϩ (Amersham) nylon membrane. Hybridization was carried out according to standard conditions (21), using 32 P-radiolabeled full length zmAO-1 or zmAO-2 cDNA as probes. Northern blot analysis was also performed (21), using same probes as Southern hybridization.  Native Polyacrylamide Gel Electrophoresis and Immunoblot Analysis-Freshly isolated maize organs were homogenized in 2 volumes of 50 mM Tris-HCl (pH 7.5) buffer containing 1 mM EDTA, 2 mM dithiothreitol, 10 M FAD, 1 M sodium molybdate, and 5 M leupeptin, and the homogenates were centrifuged at 12,000 ϫ g for 20 min. The supernatants were collected and subjected to native polyacrylamide gel electrophoresis followed by immunoblotting. Native polyacrylamide gel electrophoresis was performed with a 7.5% acrylamide gel in Laemmli buffers (22) in the absence of SDS at 4°C. Immunoblotting was performed using anti-AO mouse serum (17).

Partial Amino Acid Sequences of Purified AO and cDNA
Cloning-Amino-terminal sequencing of the maize AO protein failed. This might be due to blocking as a result of unknown post-translational modifications, as in the case of bovine AO (7). Partial sequence information for maize AO was obtained from protein subjected to cleavage with API. Of many peptides separated by reversed phase chromatography, the four most prominent fragments were sequenced, allowing the cloning of cDNA fragments corresponding to maize AO mRNA. Polymerase chain reaction amplification with a combination of two degenerate oligonucleotides as primers produced two kinds of 1959-bp cDNA fragments (Fig. 1). Comparison of the deduced amino acid sequences of these cDNAs to the amino acid sequences of the corresponding peptides indicated that both cDNA fragments might encode part of AO subunits. The sequences of the remaining 5Ј-and 3Ј-stretches were established by the RACE-polymerase chain reaction technique using nucleotide sequence information from the respective fragments (Fig. 1). The complete sequence of a cDNA was determined from the sequence of several independent clones obtained by 5Јand 3Ј-RACE procedures with oligonucleotide primers to com-plete the sequences as required.
One nearly full-length cDNA (zmAO-1) exhibits a 45-bp 5Ј untranslated region, followed by a 4074-nucleotide open reading frame and a 263-nucleotide 3Ј-untranslated sequence (Fig.  2). Three presumptive polyadenylation site sequences (AATAA) are observed (double underlined) in the 3Ј-untranslated region. The open reading frame of the cDNA predicts a protein of 1358 amino acids of M r 146,681 and a translation product that contains the sequence of the four peptides obtained by API cleavage of the AO protein (see also Table I).
The other nearly full-length cDNA (zmAO-2) consists of a 90-bp 5Ј-untranslated region, followed by a 4047-nucleotide open reading frame and a 196-nucleotide 3Ј-untranslated sequence (data not shown). Two presumptive polyadenylation site sequences (AATAA) are observed in the 3Ј-untranslated region. The open reading frame of the cDNA predicts a protein of 1349 amino acids of M r 145,173 and a translation product that contains a sequences identical to peptide 3, and similar but not identical to peptides 1, 2, and 4 obtained by API cleavage of the AO protein. These cDNAs are highly homologous in respect to both nucleotide (83.8% identity) and amino acid (83.6% identity), excepting the nucleotide sequences of their presumptive 5Ј-and 3Ј-untranslated regions.
Analysis of the Deduced Amino Acid Sequences of zmAOs-zmAO proteins have no sequences that encode presumptive hydrophobic signal peptides, indicating they are cytosolic enzymes. One of two [2Fe-2S] centers is found between amino acid residues 50 and 80 for zmAO-1 protein and between 46 and 76 for zmAO-2 protein, respectively (Fig. 3). This iron-sulfur center is of the same type observed in ferredoxin from a number of photosynthetic organisms, in bacterial fumarate reductase FIG. 2. Nucleotide and deduced amino acid sequences of zmAO-1. The sequence shown was obtained from both DNA strands. Peptides whose sequence has been determined from purified maize AO are underlined. and in eukaryotic succinate dehydrogenase (23,24). The second putative iron-sulfur center is probably located between amino acid residues 120 -123 for zmAO-1 and 116 -119 for zmAO-2, respectively, since they are conserved in two other classes of molybdoflavoproteins, such as bovine AO and several XDs (7,10,25). Sequences conforming to the consensus for the molybdopterin cofactor binding site reside between amino acids 760 -853 (zmAO-1) and 752-845 (zmAO-2), respectively (Fig. 3). An FAD-binding consensus sequence of the type described by Correll et al. (26) cannot be precisely identified in either amino acid sequence. Consensus sequences for binding of NAD are observed between amino acids 49 -54 and 816 -821 for zmAO-1 and 45-50 and 808 -813 for zmAO-2, respectively (Fig. 3). Despite their high homology overall, some regions that are difficult to align occur between amino acids 451-453, 532-549, and 897-902 for zmAO-1 and 447-454, 533-540, and 889 -894 for zmAO-2, respectively (Fig. 3).
A homology search in the SwissProt and EMBL data banks determined that the two zmAOs have a significant level of similarity with animal AOs and XDs from various sources (Fig.  3). The overall level of respective identities with zmAO-1 and zmAO-2 are 30.9 and 31.2% to bovine AO (7), 29.9 and 30.1% to human AO (initially reported as human XD) (10), 31.2 and 32.1% to human XD (27), 31.5 and 30.3% to Drosophila pseudoobscura XD (28), and 30.3 and 30.8% to Aspergillus nidulans XD (25).
Southern Blotting, Immunoblotting, and Northern Blotting-Genomic Southern blotting showed different hybridization patterns with the two cDNA probes employed for Fig. 4 under conditions of very high stringency with no detectable crosshybridization. Immunoblot analysis was performed on crude extracts of various maize organs with anti-AO mouse anti-serum (Fig. 5). The highest levels of AO protein were observed in the root tissue followed by the coleoptile and mesocotyl. Only a faint immunoreactive band was detected in leaf tissues. Northern blot experiments with RNA extracted from the same tissue preparations used for immunoblotting were performed. zmAO-1 was expressed at high level in the roots, whereas zmAO-2 was expressed at higher levels in coleoptiles than in roots (Fig. 6). DISCUSSION We cloned two cDNAs for plant AO. The molecular mass values of predicted proteins for these cDNAs are coincident with that of the purified protein, and one of them (zmAO-1) contains the sequence of four peptides obtained by API cleavage of the purified AO protein. The pattern of tissue-specific expression of zmAO-1 (Fig. 6) is similar to that of the protein as determined by immunoblotting (Fig. 5). In addition, maize AO activity was immunoprecipitated with a combination of the antiserum raised against purified AO and protein A-Sepharose (17). We conclude that zmAO-1 encodes maize AO that had been purified previously. In the case of human AO, the predicted amino acid sequence shows a relatively lower level of homology to their XD (49.3%) than to bovine AO (85.7%). Since the deduced amino acid sequence for the other cDNA (zmAO-2) shows high similarity to zmAO-1 (83.6%), we considered that both encode maize AOs that may have related functions.
As shown in Fig. 3, predicted proteins for zmAOs have a significant level of similarity to those animal AOs and XDs and can be aligned along the whole length of the proteins. zmAOs exhibit unusual clustering of cysteines in the amino-terminal 200 amino acid residues. Nine of these 12 cysteines are conserved in both animal and plant AOs, and eight of them are conserved in both AOs and XDs. Many of them must contribute to the structure of the two Fe-S centers. Li Calzi et al. (7) suggested that one (or both) of the two cysteines, amino acids 149 and 151 for bovine AO located in a strictly conserved stretch of amino acids, were involved in iron ligation in AO and XDs. These cysteines and the conserved region are found in both zmAOs (amino acids 165-176 for zmAO-1), supporting this possibility. In addition, zmAOs also contain sequences that conform to the consensus for the molybdopterin cofactor binding site (amino acids 760 -853 for zmAO-1). These results are helpful for confirmation of the previous report concerning the biochemical characters of purified maize AO (17). As shown in Fig. 3, there are some additional regions conserved to some extent among plant AOs, mammalian AOs, and XDs (amino acids 597-608, 814 -822, 1053-1064, and 1173-1230 for zmAO-1). Thus, plant AOs are similar to related enzymes from phylogenically different organisms, indicating they evolved from a common ancestral gene. The most primitive animal species in which AO activity has been found is the coelenterate Segartia luciae (29).
Two consensus sequences for NAD binding are found in both zmAOs; however, AO does not require the cofactor for its catalytic activity and in fact purified maize AO is devoid of dehydrogenase activity. It is thus unlikely that these structural elements have functional significance. This is also the case for bovine AO (7).
Glatigny and Scazzocchio (25) suggested that the ERXXXH motif (amino acid residues 910 -915 of A. nidulans XD) is involved in determining substrate specificity. These regions are not conserved in animal AO proteins and plant AOs. In addi-tion, no significant similarities are observed in those corresponding regions among animal AO proteins and zmAO-1 and zmAO-2 proteins. This may reflect the different substrate specificities of plant and animal AOs, since the former have affinity for aromatic aldehydes while the latter do not. To analyze the substrate specificity of the two zmAOs, we tried to produce recombinant proteins, but they were almost completely degraded when the cDNA were expressed in Escherichia coli. This problem also occurs with another molybdopterin cofactorcontaining protein, nitrate reductase. 2 Further research will be required for elucidation of the different substrate specificities of the zmAOs.
In plants, AOs are thought to be involved in plant hormone biosynthesis. Molybdopterin cofactor-deficient mutants of barley and tobacco are deficient in AO and XD activities and have impaired ABA production (15,16). This indicates that ABAld oxidase is a molybdenum-containing enzyme which is indispensable for ABA biosynthesis. The ABAld oxidase oxidizes ABAld to ABA, but such an enzyme has not been purified. In the case of IAA biosynthesis, the pathway has been extensively investigated (18, 30 -32), but is still poorly understood. IAAld oxidase could catalyze the final step in the pathway where IAA is produced from tryptophan (11,14,17). However, this has only been studied in a few plants. Recently, a sur mutant that forms an abnormal number of roots has been isolated from Arabidopsis thaliana (33). It was thought that the phenotype was caused by overproduction of IAA, and, in fact, IAA levels are much higher in seedlings of the mutant. We have checked AO activity in the wild-type and mutant seedlings after native polyacrylamide gel electrophoresis, revealing that at least three AO activity bands. One band, showing a substrate preference for indole-3-aldehyde, is much more intense in the mutant seedlings. Three independent cDNA clones have also been isolated from A. thaliana using degenerate primers, and one of these is highly expressed in the sur mutant. 3 FIG. 4. DNA gel blot analysis of maize. Genomic DNA from maize seedlings was digested with EcoRI or HindIII, fractionated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with the 32 P-labeled full-length zmAO-1 or zmAO-2 cDNA probes.

FIG. 5. Tissue distribution of AO protein.
Crude protein supernatants (50 g) obtained from the indicated organs were subjected to native polyacrylamide gel electrophoresis, followed by immunoblot analysis using the AO-specific polyclonal antibodies.
FIG. 6. Tissue distribution of zmAO mRNA. Poly(A) ϩ RNA was obtained from the indicated tissues. RNA was loaded on a 1.2% (w/v) agarose gels that contained 5% (v/v) formaldehyde and subjected to Northern blot analysis. After transfer, the nylon membrane was hybridized with the 32 P-labeled full-length zmAO-1 or zmAO-2 cDNAs.