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Tandem Orientation of Duplicated Xanthine Dehydrogenase Genes from Arabidopsis thaliana

2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês

10.1074/jbc.m312929200

1083-351X

Christine Hesberg, Robert Hänsch, Ralf R. Mendel, Florian Bittner,

Plant nutrient uptake and metabolism

Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species. Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species. Xanthine oxidoreductase (XOR) 1The abbreviations used are: XOR, xanthine oxidoreductase; ABA, abscisic acid; AO, aldehyde oxidase; Moco, molybdenum cofactor; XDH, xanthine dehydrogenase; XO, xanthine oxidase; RT, reverse transcriptase; Ni-NTA, nickel-nitrilotriacetic acid. 1The abbreviations used are: XOR, xanthine oxidoreductase; ABA, abscisic acid; AO, aldehyde oxidase; Moco, molybdenum cofactor; XDH, xanthine dehydrogenase; XO, xanthine oxidase; RT, reverse transcriptase; Ni-NTA, nickel-nitrilotriacetic acid. is a ubiquitous metalloflavo enzyme with a central role in purine catabolism where it catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid. The enzyme from higher eukaryotes is active as a homodimer composed of two identical subunits of 150 kDa, each being subdivided into three domains: a N-terminal domain of 20 kDa for binding of two iron-sulfur clusters of the [2Fe-2S] type, a 40-kDa domain harboring a FAD-binding site, and a C-terminal molybdenum cofactor (Moco)-binding domain of 85 kDa. XOR enzymes in mammals are present either as the predominantly existing xanthine dehydrogenase (XDH; EC 1.1.1.204) or as O2-dependent xanthine oxidase (XO; EC 1.1.3.22). Whereas XDH possesses high reactivity toward NAD+ and low reactivity toward O2 as electron acceptor, XO reacts in a strictly O2-dependent manner with negligible reactivity toward NAD+. Both forms can be interconverted reversibly by oxidation of cysteine residues (1Stirpe F. Della Corte E. J. Biol. Chem. 1969; 244: 3855-3863Google Scholar), whereas the conversion of XDH into XO by limited proteolysis is irreversible (2Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Google Scholar). In contrast to mammalian XOR the avian enzyme is exclusively present in the dehydrogenase form (3Sato A. Nishino T. Noda K. Amaya Y. Nishino T. J. Biol. Chem. 1995; 270: 2818-2826Google Scholar). The ability of mammalian XO to produce superoxide and hydrogen peroxide by reducing molecular oxygen (4Hille R. Nishino T. FASEB J. 1995; 9: 995-1003Google Scholar) led to the suggestion that it might play an important role in the pathogenesis of cellular injury (5McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Google Scholar, 6Nishino T. J. Biochem. (Tokyo). 1994; 116: 1-6Google Scholar). Among all XOR enzymes studied so far the bovine enzyme from milk is most exhaustively studied and recently its crystal structure has been determined for both the XDH and the XO form (7Enroth C. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10723-10728Google Scholar).Besides functional characterization of XDH/XO, the corresponding nucleotide and protein sequence information was published for organisms like humans (8Wright R.M. Vaitaitis G.M. Wilson C.M. Repine T.B. Terada L.S. Repine J.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10690-10694Google Scholar), cow (9Berglund L. Rasmussen J.T. Andersen M.D. Rasmussen M.S. Petersen T.E. J. Dairy Sci. 1996; 79: 198-204Google Scholar), rat (2Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Google Scholar), mouse (10Terao M. Cazzaniga G. Grezzi P. Bianchi M. Falciani F. Perani P. Garattini E. Biochem. J. 1992; 283: 863-870Google Scholar), chicken (3Sato A. Nishino T. Noda K. Amaya Y. Nishino T. J. Biol. Chem. 1995; 270: 2818-2826Google Scholar), insects (11Keith T.P. Riley M.A. Kreitman M. Lewontin R.C. Curtis D. Chambers G. Genetics. 1987; 116: 67-73Google Scholar, 12Komoto N. Yukuhiro K. Tamura T. Insect Mol. Biol. 1999; 8: 73-83Google Scholar), fungi (13Glatigny A. Scazzocchio C. J. Biol. Chem. 1995; 270: 3534-3550Google Scholar), and bacteria (14Leimkuehler S. Kern M. Solomon P.S. McEwan A.G. Schwarz G. Mendel R.R. Klipp W. Mol. Microbiol. 1998; 27: 853-869Google Scholar, 15Xi H. Schneider B.L. Reitzer L. J. Bacteriol. 2000; 182: 5332-5341Google Scholar) also. With the exception of the silkworm all organisms analyzed so far possess one XOR gene.In plants the XDH but not the oxidase form was purified from nodules of bean (16Boland M.J. Biochem. Int. 1981; 2: 567-574Google Scholar), from the green algae Chlamydomonas reinhardtii (17Perez-Vicente R. Alamillo J.M. Cardenas J. Pineda M. Biochim. Biophys. Acta. 1992; 11117: 159-166Google Scholar), from wheat leaves (18Montalbini P. Plant Sci. 1998; 134: 89-102Google Scholar) and leaves of legumes (19Montalbini P. J. Plant Physiol. 2000; 156: 3-16Google Scholar), as well as from pea seedlings (20Sauer P. Frebortova J. Sebela M. Galuszka P. Jacobsen S. Pec P. Frebort I. Plant Physiol. Biochem. 2002; 40: 393-400Google Scholar). All plant XDH proteins were found to be homodimers with a molecular mass of ∼300 kDa and showed highest substrate specificity for hypoxanthine and xanthine but were also able to convert purines, pterines, and aldehydes at a much lower rate. Beside purine degradation, plant XDH is supposed to play a role in important cellular processes: (i) plant-pathogen interactions between phytopathogenic fungi, legumes, and cereals (21Montalbini P. J. Phytopath. 1992; 134: 218-228Google Scholar, 22Montalbini P. Plant Sci. 1992; 87: 225-231Google Scholar); (ii) cell death associated with hypersensitive response (23Montalbini P. Physiol. Mol. Plant Pathol. 1995; 46: 275-292Google Scholar, 24Montalbini P. Della Torre G. Physiol. Mol. Plant Pathol. 1996; 48: 273-287Google Scholar); and (iii) natural senescence (25Pastori G.M. Del Rio L.A. Plant Physiol. 1997; 113: 411-418Google Scholar). As all these processes require the formation of reactive oxygen species XDH was supposed to be able to produce superoxide anions and/or hydrogen peroxide (25Pastori G.M. Del Rio L.A. Plant Physiol. 1997; 113: 411-418Google Scholar). Supporting this hypothesis, XDH activity was found to be increased concomitant with superoxide dismutase and other oxygen-related enzymes in senescent pea leaves (25Pastori G.M. Del Rio L.A. Plant Physiol. 1997; 113: 411-418Google Scholar). Although much effort was spent at purification and biochemical characterization of plant XDH neither cloning of the corresponding cDNAs nor molecular data are published so far.In this work we describe the cloning of two XDH cDNAs from Arabidopsis thaliana, their tandem arrangement in the genome, their mRNA expression levels as well as the enzymatic activities at various stresses and treatments, and the recombinant expression of AtXDH1 cDNA in the methylotrophic yeast Pichia pastoris with subsequent purification and characterization of the AtXDH1 protein.EXPERIMENTAL PROCEDURESPlant Material and Plant Growth—A. thaliana Col-O, Ler, and aba3.2 seeds were grown in pots containing low-nutrient soil in an AR-36L A. thaliana growth chamber (Percival Scientific, Perry, IA) at 13 h light/11 h darkness, at 21 °C, and 70% relative humidity for periods as given in the text.Stress Treatment—For drought stress experiments, soil was completely removed from the roots prior to incubation under normal conditions in the chamber for 4 h or as given in the text (loss of fresh weight about 50%). Subsequently, roots and leaves were detached and used for RNA and activity analysis. ABA treatment in the case of plants without drought treatment was performed by spraying plants with 50 μm (±)-cis,trans-ABA in water uniformly onto the leaves for 4 h. In the case of combined ABA and drought treatment plants were sprayed with ABA prior to removal of plants from the soil and 2 h after removal. Treatment also lasted 4 h and control plants were sprayed with water instead of ABA solution. For NaCl treatment, 3-week-old plants were transferred to hydroponic culture 2 days before treatment and then incubated in nutrition solution containing 200 mm NaCl for 6 and 20 h. Cold stress treatment at 4 °C was performed in a chamber with ambient temperature for 6 and 20 h and freezing stress was applied by incubating plants in small chambers precooled to -4 °C. Because longer freezing stress will result in freezing of the soil freezing temperature treatment lasted only 6 h.Preparation of RNA—Total RNA was prepared either as described by Ref. 26Logemann J. Schell J. Willmitzer L. Anal. Biochem. 1987; 163: 16-20Google Scholar or by using the NucleoSpin RNA Plant kit (Macherey & Nagel, Dueren, Germany) according to the manufacturers instructions.Relative Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)—For each RT reaction 2 μgof A. thaliana total RNA was reverse-transcribed with avian myeloblastosis virus-reverse transcriptase (Promega, Madison, WI) and oligo-d(T)18-BamHI primer according to standard procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). RT-PCR was performed on a PCR Express gradient cycler (Hybaid, Heidelberg, Germany) by using the SAWADY Taq DNA polymerase (Peqlab, Erlangen, Germany). AtXDH1-specific primers were AtXDH1+, 5′-CACATTTACTGAGCTAGTA-3′, and AtXDH1-,5′-GTTTCCCCTCTGATGATGTTC-3′; AtXDH2-specific primers were AtXDH2+, 5′-TCTTCTCAAGGGTAATCCA-3′, and AtXDH2-, 5′-TTCTCCCCTCTATTAAAGTTT-3′. The following PCR program was used: 3 min at 94 °C for initial denaturing of templates, 30 cycles including denaturing for 30 s at 94 °C, annealing for 1 min at 56 °C and elongation for 1 min at 72 °C, and a final elongation step for 6 min at 72 °C. RT-PCR generated fragments were directly ligated to pGEM-T Easy (Promega) and sequenced for ascertaining proper amplification.Cloning of AtXDH2 cDNA—Two overlapping cDNA subfragments were generated by PCR from reverse transcribed total RNA of A. thaliana (Col-O) and subsequently fused by PCR according to standard procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The obtained full-length cDNA of AtXDH2 was directly ligated to pGEM-T Easy (Promega) and sequenced. Specific PCR primers for generation of the 5′ subfragment (2080 base pairs) were AtXDH2-ATG, 5′-GTTCAGTGAAGATGGAGCAGAAC-3′, and AtXDH2–2622rev, 5′-GCGACAAGCACACCAATA-3′. Primers for the 3′ subfragment (2085 base pairs) were AtXDH2–2400for, 5′-TTATTTGCTACAGACGTG-3′, and AtXDH2–3′, 5′-TGATCCATCTTTCTCCCC-3′.Generation of AtXDH1 Expressing P. pastoris—The cDNA clone AV548322 coding for full-length A. thaliana XDH1 was obtained from the Kazusa DNA Research Institute (Chiba, Japan). The yeast expression vector pPICZA with C-terminal His6 tag and P. pastors strain KM71 muts were purchased from Invitrogen (Carlsbad, CA). Standard molecular cloning techniques were used for DNA manipulation. The AtXDH1 cDNA was used as template for PCR to remove the 5′-untranslated region and stop codon and to generate a KpnI site at the 5′ end and a ApaI site at the 3′ end. Primers used for introducing restriction sites were AtXDH1 KpnI start, 5′-ATATATGGTACCATGGGTTCACTGAAAAAGGACGGC-3′, and AtXDH1 ApaI stop, 5′-ATATATGGGCCCAACACTAAGATTAGGGTAGAAATCTGA-3′. The resulting PCR fragment containing the total coding region was cloned into pPICZA. P. pastoris was transformed with pPICZA/AtXDH1 and pPICZA (vector only) by electroporation according to the manual (EasySelect Pichia expression kit version A, Invitrogen). The presence of AtXDH1 cDNA in zeocin-resistant colonies was confirmed by PCR on the P. pastoris colonies.Expression and Purification of AtXDH1—Several positive transformants were grown in 25 ml of BMGY (1% yeast extract, 2% peptone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base without amino acids, 0.04% biotin, 1% glycerol, and 100 μg/ml zeocin) in a 250-ml baffled flask for 16–20 h (A600 2–3) at 30 °C and 150 rpm. Cells were collected by centrifugation and resuspended in 10 ml of BMMY (1% yeast extract, 2% peptone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base without amino acids, 0.04% biotin, 0.3 mm sodium molybdate, and 0.5% methanol) in a 100-ml baffled flask and cultured again at 30 °C and 150 rpm. Cells were harvested after 0, 6, 10, 14, 18, 24, and 36 h of methanol induction by centrifugation and resuspended in breaking buffer (50 mm sodium phosphate, pH 7.4, 0.5 mm EDTA, 200 mm NaCl, 0.2 mm phenylmethylsulfonyl fluoride, and 5% glycerol). Cells were broken by vigorous vortexing with equal amounts of acid-washed glass beads (425–600 μm, Sigma) before cell debris and glass beads were removed by centrifugation. In the resulting supernatant XDH activity was examined by activity staining after native PAGE. Strongest intensity was detected 10 h after methanol induction with a gradual decrease until 36 h of incubation. The clone showing the highest XDH activity was selected for a large scale expression culture. Cells were grown in 250 ml of BMGY in a 1-liter baffled flask for 20 h, collected by centrifugation, and resuspended in 50 ml of BMMY in a 500-ml baffled flask. After cultivation for 10 h in BMMY the cells were harvested by centrifugation and resuspended in breaking buffer. Depending on the quantity of cells, they were broken either by vigorous vortexing with an equal volume of glass beads at 4 °C for a total of 30 min in bursts of 30 s alternating with cooling on ice or by three passages through a French press pressure cell with 14,000 p.s.i. operating pressure. After centrifugation the supernatant was used for purification of the His-tagged AtXDH1 protein by affinity chromatography with Ni-nitrilotriacetic acid (Ni-NTA)-superflow matrix (Qiagen, Hilden, Germany) under native conditions at 4 °C according to the manufacturers instructions. The sample was rebuffered to 50 mm Tris/HCl, pH 8.0, 5 mm EDTA, 2.5 mm dithiothreitol. For further purification AtXDH1 was subjected to anion exchange chromatography using a Source™ 15Q column (Amersham Biosciences) equilibrated with 50 mm Tris/HCl, pH 8.0, 5 mm EDTA, 2.5 mm dithiothreitol (buffer A). Protein samples were applied to the column and eluted with buffer A followed by a linear gradient of 0 to 1 m NaCl in buffer A. Final purification and size determination was achieved by chromatography on a Superdex™ 200 HR10/30 size exclusion column (Amersham Biosciences) equilibrated with 50 mm Tris/HCl, pH 8.0, 200 mm NaCl, 1 mm EDTA.Determination of Protein Concentrations—Concentrations of total soluble protein were determined by use of Roti Quant solution (Roth, Karlsruhe, Germany) according to Ref. 28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar.Wavescan of AtXDH1—Absorption spectroscopy was carried out using an Ultrospec 3000® spectrophotometer (Amersham Biosciences).Sequence Analysis—Sequence analysis was performed with the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit on an ABI Prism 310 cycle sequencer (PE Applied Biosystems, Warrington, UK) with a pop 6 polymer.Expression of ABA3 and AOα from A. thaliana—Recombinant molybdenum cofactor sulfurase ABA3 and aldehyde oxidase AOα from A. thaliana were overexpressed and purified as described earlier in Refs. 29Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Google Scholar and 30Koiwai H. Akaba S. Seo M. Komano T. Koshiba T. J. Biochem. (Tokyo). 2000; 127: 659-664Google Scholar.Enzyme Assays—For preparation of plant crude extracts plant material was squeezed at 4 °C in 2 volumes of extraction buffer (100 mm potassium phosphate, 2.5 mm EDTA, 5 mm dithiothreitol, pH 7.5), sonificated and centrifuged, and the supernatant was used for activity assays. XDH activity in plant crude extracts and recombinant AtXDH1 was visualized according to Ref. 31Mendel R.R. Mueller A. Biochem. Physiol. Pflanz. 1976; 170: 538-541Google Scholar, except that native electrophoresis in the absence of SDS was run with 7.5% polyacrylamide gels and staining solution contained 1 mm hypoxanthine as substrate, 1 mm 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 0.1 mm phenazine methosulfate in 250 mm Tris/HCl, pH 8.5. For standard in gel XDH activity assays, each lane were loaded either with 80 μg of plant crude extract protein or with 1 μg of recombinant AtXDH1. The in vitro reconstitution of recombinant AtXDH1 by ABA3 was performed in a total volume of 0.4 ml of 50 mm Tris/HCl, pH 8.0. AtXDH1 (20 μg) was incubated with ABA3 (40 μg) in the presence of 1 mml-cysteine for 1 h at 30 °C, followed by native PAGE with ⅕ volume of the reaction mixture and activity staining with hypoxanthine as substrate. Spectrophotometric determination of XDH activity was measured at 340 nm in a 1-ml reaction mixture containing 1 mm of the respective substrate, 1 mm NAD+, 50 mm Tris/HCl, pH 8.0, 1 mm EDTA and a suitable amount of recombinant AtXHD1. Reaction was started by addition of substrate. Inhibitors were preincubated with the enzyme for 5 min before starting the reaction. The xanthine-O2 reductase activity was measured under the same conditions but without NAD+, and O2-dependent production of uric acid was monitored at 295 nm. The production of superoxide radicals was monitored by following the reduction of cytochrome c at 550 nm. The specificity of O2˙--dependent reduction of cytochrome c was estimated by incorporating an excess of bovine superoxide dismutase in the assay mixture according to Refs. 32McCord J.M. Fridovich I. J. Biol. Chem. 1968; 243: 5753-5760Google Scholar and 33Ballou D. Palmer G. Massey V. Biochem. Biophys. Res. Commun. 1969; 36: 898-904Google Scholar.RESULTSCloning of AtXDH1 and AtXDH2 cDNAs and Their Tandem Arrangement in the Genome—Sequence similarity searches at "The Arabidopsis Information Resource" (TAIR) 2See www.arabidopsis.org. using human and bovine full-length XDH as query sequences detected two putative genes in the A. thaliana genome with higher similarity to XOR than to aldehyde oxidases (AO). Both genes were found in a tandem orientation on chromosome 4 with their reading frames pointing to the same direction (Fig. 1A). Based on the BAC clone AL079347/ATF11I11 as reference both putative XDH genes, annotated as T11I11.130 and T11I11.140 and designated by us as AtXDH1 and AtXDH2, respectively, are located within the center region of this clone with an interspace of only 704 bp. The predicted genes range from position 50,304 (ATG) to 44,057 (TGA) for T11I11.130/AtXDH1 and from position 56,627 (ATG) to 51,009 (TGA) for T11I11.140/AtXDH2, thereby spanning regions of 6,248 and 5,619 base pairs, respectively. For both predicted genes expressed sequence tags were found indicating that both genes actually are transcribed. Although these expressed sequence tags clearly showed that annotation of the putative coding sequences is not fully correct they confirmed the predicted transcription start and stop sites. Neither extended data base searches nor genomic DNA hybridizations (data not shown) revealed more than these two XDH genes within the A. thaliana genome.Complete sequencing of the A. thaliana expressed sequence tag AV548322 has shown that the full-length open reading frame of AtXDH1 is interrupted by 13 introns and contains 4083 base pairs encoding a protein of 1361 amino acids (Fig. 1B) with 47% identity to human and bovine XDH. A full-length cDNA of AtXDH2 with an open reading frame of 4059 base pairs was obtained by reverse transcriptase-PCR using sequence information of the predicted AtXDH2 gene. The AtXDH2 gene also contains 13 introns highly conserved in exon/intron junctions compared with AtXDH1. The encoded AtXDH2 protein exhibits a length of 1353 amino acids with identities of 46% to human and bovine XDH. Both proteins, AtXDH1 and AtXDH2, reveal lower identities of 29–31% to AO proteins from A. thaliana but share overall identities of 93% to each other, indicating that they are similar to AO but functionally divergent. Full-length cDNA and protein sequences were deposited at GenBank™ (AY171562 for AtXDH1; AY518202 for AtXDH2).Comparative primary structure analysis of AtXDH1 and AtXDH2 and XOR proteins from other organisms revealed a three-domain structure for both A. thaliana XDH monomers as is typical for XOR proteins. Like the chicken XDH (2Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Google Scholar) both A. thaliana XDH proteins contain a N-terminal domain including 8 strictly conserved cysteine residues for binding of two non-identical iron-sulfur clusters of the [2Fe-2S] type spanning amino acid positions 19 to 173 in AtXDH1 and 11 to 164 in AtXDH2, respectively. In each protein, the [Fe-S]-binding domain is followed by a FAD-binding domain (amino acids 260 to 440 in AtXDH1 and 252 to 432 in AtXDH2), whereas both domains are separated by hinge regions that are less conserved among all XOR proteins. FAD domains of both XDH proteins contain a FFLGYR motif (amino acids 417 to 422 in AtXDH1 and 409 to 414 in AtXDH2) that is supposed to be responsible for binding the second substrate NAD+ via the invariant tyrosine (2Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Google Scholar, 34Nishino T. Nishino T. J. Biol. Chem. 1989; 264: 5468-5473Google Scholar). The third and C-terminal domain includes the Moco- and substrate-binding sites as well as the dimerization motif (35Romao M.J. Archer M. Moura I. Moura J.J. LeGall J. Engh R. Schneider M. Hof P. Huber R. Science. 1995; 270: 1170-1176Google Scholar). In AtXDH1 it spans amino acid residues 612 to 1272 and in AtXDH2 604 to 1264, respectively, and is separated from the FAD/NAD domain by another hinge region. Within the Moco domain of XOR proteins both a strictly conserved glutamate and arginine residues are supposed to be essential for binding and proper positioning of purine substrates (36Glatigny A. Hof P. Romao M.J. Huber R. Scazzocchio C. J. Mol. Biol. 1998; 278: 431-438Google Scholar). AtXDH1 and AtXDH2 exhibit identical residues at the corresponding positions (Glu-831 and Arg-909 in AtXDH1, Glu-832 and Arg-901 in AtXDH2) indicating that the favored substrates of both proteins should be purines rather than aldehydes.To find out at what evolutionary point XDH gene duplication might have occurred we analyzed the phylogenetic relationships of XDH proteins from various eukaryotic organisms (Fig. 2). Because AO is homologous to XDH but functionally divergent we have chosen three AO proteins from A. thaliana as an outgroup. The sequences used for this analysis show a splitting into three groups, among which plant sequences clearly form their own monophyletic subgroup besides animal and fungi XDH. Therein, A. thaliana XDH gene duplication appears to have happened long after the separation of dicots and monocots. Different from A. thaliana, none of the fully sequenced genomes of rice and C. reinhardtii were found to contain more than one XDH gene. Among the animals, vertebrates, insects, and nematodes group separately. Generally, the phylogenetic tree based on XDH protein similarities mirrors the species phylogeny and gives one more indication that AtXDH1 and AtXDH2 are in fact xanthine dehydrogenases rather than aldehyde oxidases.Fig. 2Phylogenetic neighbor joining tree of XDH proteins. Full-length sequences of XDH proteins and A. thaliana AOs were aligned using ClustalX, and phylogeny of XDH proteins was constructed by use of the neighbor joining method using PAUP 4.0. Numbers in the phylogenetic tree indicate 100 bootstrap replicates. A. thaliana AO1, AO2, and AO3 were used as an outgroup. The accession numbers of the XDH sequences are as follows: AtXDH1 (AY171562), AtXDH2 (AY518202), Oryza sativa (cDNA: AK065099), C. reinhardtii (not available), Emericella nidulans (CAA58034), Neurospora crassa (EAA27223), Homo sapiens (P47989), Rattus norvegicus (P22985), Mus musculus (CAA44705), Bos taurus (CAA58497), Gallus gallus (P47990), Drosophila melanogaster (S07245), Calliphora vicina (JQ0407), B. mori XDH1 (BAA21640), B. mori XDH2 (BAB47183), and Caenorhabditis elegans (NP_502747). Accession numbers for the AOs from A. thaliana are AtAO1 (BAA28624), AtAO2 (BAA28625), and AtAO3 (BAA82672).View Large Image Figure ViewerDownload (PPT)Differential Transcription and Enzyme Activities of AtXDH1 and AtXDH2—Because of the high similarities between the mRNAs of AtXDH1 and AtXDH2 and nearly identical transcript sizes, one cannot distinguish both transcripts by use of mRNA hybridization. Therefore we performed relative quantitative reverse transcriptase-PCR, using specific PCR primers designed for binding within the 3′-regions of each transcript and generating PCR fragments of 693 base pairs for AtXDH1 and 279 base pairs for AtXDH2, respectively. Proper amplification of the corresponding fragments was confirmed by sequencing.As shown in Fig. 3A expression of AtXDH1 and AtXDH2 on the mRNA level can be detected in roots, leaves, stem, flowers, and siliques, indicating that both mRNAs are ubiquitously expressed in A. thaliana, although with varying amounts. Consistent with these findings also XDH activities were found in these organs. Unfortunately, discrimination of two XDH isoforms in non-denaturing polyacrylamide gels was impossible, either because of very similar physicochemical properties of both XDH proteins or because of the fact that only one isoform is actually translated. When analyzing the expression levels of AtXDH1 and AtXDH2 in plants of different age it turned out that mRNA levels of AtXDH1 increased in aging and senescent leaves, whereas AtXDH2 transcript levels remained unaltered (Fig. 3B) thereby simultaneously serving as an internal standard. In the same plants, a strong increase of XDH activity could be observed in senescent leaves but not at any other stage of development (Fig. 3B).Fig. 3Relative AtXDH1 and AtXDH2 mRNA expression and XDH activity at different conditions. A, relative mRNA expression (upper image) and enzymatic activity (lower image) of A. thaliana XDHs in different tissues; B, in leaves of different age (seedlings, 6 days; young, 2 weeks; adult, 3.5 weeks; aging, 6 weeks; senescent, 8 weeks); C, at salinity, cold and freezing stress; D, at drought stress treatment; and E, at ABA treatment in wild types and aba3.2 mutants. After RT-PCR, 2% agarose gels were loaded with 10 μl of the respective PCR reaction (M = 100 base pair ladder, upper band = 1000 base pairs; C = control; D = 4 h drought stressed). For XDH activity measurements, each lane on 7.5% native polya