the arabidopsis and rice formaldehyde dehydrogenase genes

Copyright 0 1997 by the Genetics Society of America

Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant ADH Enzymes

R. Dolferus,**+ J. C. Osterman,I W. J. Peacock* and E. S. Dennis*

"CSIRO, Division of Plant Industry, Canbema A C T 2601, Australia, tCooperative Research Centre for Plant Science, Canbema A C T 2601, Australia and :School of Biolopcal Sciences, Univerisity of Nebraska, Lincoln, Nebraska 68588

Manuscript received January 9, 1997 Accepted for publication March 28, 1997

ABSTRACT This article reports the cloning of the genes encoding the Arabidopsis and rice class I11 ADH enzymes,

members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol- active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class I11 genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class I11 enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway.

A LCOHOL dehydrogenase (ADH: alcohol: NAD+ oxidoreductase; E.C. 1.1.1.1.) belongs to the me-

dium-chain dehydrogenase/reductase protein superfamily, which consists of proteins with a wide variety of catalytic activities and functions, ranging from eye lens <-crystallins to a variety of dehydrogenases, like polyol, threonine, and alcohol dehydrogenases (JORNVALL et al. 1993; PERSON et al. 1994). ADH is present in most life forms, but its metabolic function is variable. ADH is, together with pyruvate decarboxylase (PDC, E.C. 4.1 .l. 1 .) , part of the alcoholic fermentation pathway, a two-step pathway converting pyruvate via acetaldehyde into ethanol. This pathway is present in yeast, higher plants and in some bacteria and gives selective growth advantage to microorganisms such as yeast in the presence of high sugar concentrations (GANCEDO and SER- RANO 1989). In higher plants, alcohol fermentation is essential for survival under some environmental stress situations. Both the Adh and Pdc genes are induced strongly under low oxygen conditions (HACEMANN and FLESHER 1960; SACHS et al. 1980; KELLEY 1989; ANDREWS

et al. 1994), and the Arabidopsis ADHl gene is also induced by low temperature and osmotic stress (DOL- FERUS et al. 1994; DE BRUXELLES et al. 1996).

The first enzyme of the alcoholic fermentation pathway PDC is not present in all life forms and is present in only a few species of bacteria (SCRUTTON 1971; FINN et al. 1984), such as the obligately fermentative bacte- rium Zymomonas mobilis (BRAU and SAHM 1986), yeast

Curresponding author: Rudy Dolferus, CSIRO, Division of' Plant In- dustry, P.O. Box 1600, Canberra ACT 2601, Australia. E-mail: [email protected]

Genetics 146: 1131-1141 (July, 1997)

and plants. Eschm'chia coli and many other bacteria cir- cumvent the PDGcatalyzed reaction by producing ethanol from acetyl-coA by means of acetaldehyde-CoA dehydrogenase and ADH. Animals use lactic acid fermentation instead of ethanol fermentation under anaerobic conditions (GARLICK et al. 1979; HOCHACHKA and MOMMSEN 1983), and ADH, due to its broad substrate specificity, plays a role in detoxification of alcohols and aldehydes in the liver (YOSHIDA et al. 1991). Humans contain a complex set of ADH enzymes subdivided in several classes; class I, the predominant liver enzyme, and class I11 have been the focus of most research (EKLUND et al. 1987, 1990; DANIELSSON et al. 1994).

The human class I11 ADH (ADH5 or X-ADH) has an extremely low affinity for ethanol, is insensitive to the classical ADH inhibitor pyrazole, has a high isoelectric pH and is present in most tissues (KAISER et al. 1989). Class I11 ADH was shown to be the same enzyme as glutathione-dependent formaldehyde dehydrogenase (GSH-FDH; formaldehyde: NAD+ oxidoreductase, glutathione formylating; E.C. 1.2.1.1; KO~WSALO et al. 1989; HOLMQUIST and VALLEE 1991). Class I11 ADH has been identified in a wide variety of organisms, ranging from E. coli to Drosophila. The high degree of amino acid sequence conservation of this enzyme suggested that class I11 ADH is an ancient form, from which other classes of ADH enzymes were derived by gene duplica- tions and acquisition of new substrate specificities (KAI- SER et al. 1988, 1989; DANIELSSON and JORNVALL 1992; DANIELSSON et al. 1992; GUTHEIL et al. 1992; FERNANDEZ et al. 1993; LUQUE et al. 1994). GSH-FDH activity has also been demonstrated in plants (UOTILA and KO~WSALO 1979; GIESE et al. 1994). Recently, the pea and Arabi-

1132 R. Dolferus et al.

dopsis FDH proteins were sequenced and provided sup- portive evidence that class I11 enzymes could be the ancestor of plant class P, ethanol-active ADH enzymes (MARTINEZ et al. 1996; SHAFQAT et al. 1996).

In this paper we report the cloning of plant class I11 ADH-encoding genes from Arabidopsis (a dicotyledon; EDHI) and r ice (a monocotyledon; FDHI). A comparison of the DNA sequences and position of introns allows us to draw further conclusions about the derivation of plant Adh genes from the class I11 ancestor gene and the acquisition of alcoholic fermentation as a vital pathway in plants for survival of stress.

MATERIALS AND METHODS

Plant material and growth conditions: In this study Arabi- dopsis thaliana ecotypes C24, Columbia ('20-0) and Landsberg erecta (Ler), Wassilewskija (Ws-0) and ROO2 (ADH null mutant in Bensheim, Be-0, background; JACOBS et al. 1988) were used. Seeds were sterilized and sown on solid MS medium (MURASHICE and SKOOC 1962). After overnight incubation at 4" to break seed dormancy, the plates were incubated for 1 - 2 wk at 22" (16 hr light/8 hr dark, 200 pE/s/mz) and then transferred to fresh plates for another 2 wk (30-50 plants per plate). Stress treatments were completed as described previously (DOLFERUS et al. 1994). Briefly, plants were transferred to plates containing 15 ml liquid MS medium. Low oxygen treatment was carried out in anaerobic jars, flushed with a 5% oxygen/nitrogen gas mixture. Dehydration treatment was carried using 0.6 M mannitol as osmoticum. For low-temperature treatment, plants were incubated at 4". Un- less stated otherwise, all stress treatments were carried out for 24 hr.

Protein electrophoresis and enzymatic assays: Native (without SDS) and denaturing (+SDS) PAGE were carried out using the buffer system of LAEMMLI (1970). Proteins were extracted in two volumes (w/v) 0.1 M TES buffer (TES, pH 7.8,0.2 M NaCl, 1 mM EDTA, 1 mM PMSF, 2% 2-mercaptoethanol) for SDS electrophoresis or in 0.1 M Tris (pH 8.0), 10% glycerol, 0.5% 2-mercaptoethanol for native gels. For denaturing SDSPAGE, crude protein extracts were denatured by boil- ing for 5 min, after addition of 0.25 volume of denaturation buffer (0.5 TrisHC1, pH 7.6, 8% SDS, 40% glycerol, 16% 2- mercaptoethanol). After electrophoresis, gels were stained for total protein in 50% methanol/lO% acetic acid/O.l% C o e massie Brilliant Blue R250. ADH activity staining on native acrylamide gels was carried out in a solution of 100 mM Tris- HCl (pH 8.0), 0.1 mM NAD+, 0.1 mM nitroblue tetrazolium (NBT), 0.1 mM phenazine methosulfate (PMS) and 0.6% ethanol. Octanol dehydrogenase activity staining was carried out in essentially the same mixture, using octanol instead of ethanol as substrate. For GSH-FDH activity staining, gels were equilibrated first for 15 sec in 0.1 M Na-phosphate buffer (pH 7.0), prior to staining in 0.1 M Na-phosphate buffer (pH 7.0), 0.1 mM NAD', 0.1 mM NBT, 0.1 mM PMS, 1 mM formaldehyde and 1 mM glutathione. All chemicals used in activity stainings were of the highest purity.

Plaque, Southern and Northern blot hybridizations: RNA extractions and Northern blot hybridizations using RNA probes were completed as described previously (DOLFERUS et al. 1994). Quantification of Northern blot hybridization sig- nals was carried out using a phosphorimager (Molecular Dy- namics, Sunnyvale, CA). Plant genomic DNA was extracted from whole plants or callus material using the method of TAYLOR and POWELL (1982). Five micrograms of DNA was digested with restriction enzymes, separated on 0.7% agarose

gels and blotted on Hybond-N membranes (Amersham, UK). DNA probes were made using a random primed labeling system (Megaprime, Amersham, UK). Filters were hybridized in 5X SSPE (2OX SSPE = 3.6 M NaCl, 0.2 M Na-phosphate, pH 7.7, 0.02 M EDTA), 5X Denhardt's solution, 0.5% SDS, 100 pg/ml salmon sperm DNA, 1 mM EDTA, at 65". Washing was at room temperature in 2X SSC/O.5% SDS (2 X 20 min) and in 0.1X sSC/O.l% SDS (2 X 20 min; 20X SSC = 3 M NaC1, 0.3 M Na3 citrate). Plaque hybridizations were carried out according to standard procedures (SAMBROOK et al. 1989), using the same hybridization conditions as described for Southern blot hybridizations.

Cloning and sequencing methods: All cloning procedures were according to standard procedures (SAMBROOK et al. 1989). The Arabidopsis genomic library used to clone the FDHl gene was constructed from partially digested C24 genomic DNA (Sau3A) using the lambda replacement vector EMBL3 (FRISCHAUF et al. 1983). Two positive lambda clones containing the Arabidopsis FDHl gene were isolated, from which the gene was subcloned as two overlapping EcoRI fragments containing the 5' and 3' ends of the gene. A 2266base pair (bp) BamHI subclone (5' end) and a 2495-bp BamHI/ EcoRI fragment (3' end) were sequenced. A rice genomic library, prepared from Sau3A partially digested @yza sativa (L. indica, var. IR36) genomic DNA cloned in lambda phage vector EMBL3 SP6/T7, was obtained from Clonetech (Palo Alto, CA). This library was screened under low stringency conditions (28" in the presence of 50% formamide), using the Arabidopsis FDHl gene as a probe. The rice FDHl gene was subcloned from positive lambda clones as BamHI [3 kilo- base pair (kbp) ] and EcoRI fragments (5 kbp) . BamHI and EcoRI both appeared to cut within the coding region of the rice FDHl gene, and the subclones contained the 3' and 5' end of the gene, respectively. PCR cycle sequencing was carried out using the dye primer and dye terminator sequencing kits from Applied Biosystems. 5' deletions for sequencing were generated using an exonuclease I11 nested deletion kit (Pharmacia). Synthetic oligonucleotides used in sequencing reactions were prepared on an Applied Biosystems synthesizer (Foster City, CA) . All nucleotide and amino acid sequence analyses were carried out using the Pileup, Bestfit, Distances and Growtree programs of the University of Wisconsin GCG software package. GenBank accession numbers for the Arabi- dopsis and rice FDHl genes are U63931 and U77637, respectively. In vitro and in vivo expression experiments of Arabidopsis

FDHl: The 5' end of the EDHl cDNA (EST cDNA clone 80E7 T7) was modified by PCR, in order to introduce an EcoRI site just upstream the ATG translation start codon. The entire cDNA was then subcloned as an EcoRI/HindIII fragment into the corresponding sites of expression vector pKK223-3 (Phar- macia; BROSNIUS and HOLY 1984). The ATG codon was 11 bp 3' of the ribosome-binding site of the vector. The resulting plasmid (pKKFDH1) was used for in vitro transcription/translation experiments using cell-free E. coli S30 extracts (Pro- mega). De novo synthesized proteins were labeled by '%methionine, and the reaction mix was loaded on either native or SDS polyacrylamide gels according to the protocol provided by the supplier. Native gels were stained for GSH-FDH or ADH activity as described above. In vivo expression studies were carried out by transforming plasmid pKKFDHl into E. coli strain JM109 (YANNISCH-PERRON et al. 1985). Protein extracts were prepared from induced (2 mM IPTG) and nonin- duced cultures grown at 37", as well as from nontransformed control JM109 cultures, using sonication. Extracts were loaded on polyacrylamide gels, and enzyme activities were detected as described above.

Mapping chromosomal locations using Arabidopsis recom-

Cloning of the Arabidopsis and Rice Class 111 Adh Genes 1133

Kb:

11 5-

5.1-

2.8-

1.2-

0.8 -

PROBE:

65°C 65°C 55°C - ADHl FDH 1

Kb: 11.5

5.1

2.8

1.7

1.2

0.8 I l l I l l I l l H E B H E B H E B ADHl FDH 1 FDH 1

FIGURE 1 .-Southern blot hybridization results using ADHl and l D H 1 as a probe. Five micrograms of Arabidopsis genomic DNA was digested with Hind111 (H) , EcoRI (E) or BnmHI (B); separated on a 0.7% agarose gel and blotted on Hybond- N (Amersham, UK) filters. Hybridizations with the FDHl probe were carried out under stringent (65') and medium- stringent conditions (55"), which normally detect cross-hybridization between the maize and Arabidopsis ADHl genes (R. DOLFERUS, unpublished result).

binant inbred lines: Recombinant inbred lines were kindly supplied by C. DEAN (LISTER and DEAN 1993). Columbia (Co- 0) and Landsberg (Ler) genomic DNA was analyzed first by Southern blot hybridizations, using different restriction enzyme digests. A polymorphism was identified for the restriction enzyme XDnI. Genomic DNA of recombinant inbred lines was digested with this enzyme, and the polymorphism was identified by Southern blot hybridizations. Linkage analysis was carried out using the MapMaker V1 .O mapping program for the Macintosh computer.

RESULTS AND DISCUSSION

Cloning of the Arabidopsis and rice FDHl genes: An Arabidopsis equivalent of a class 111 ADH (GSH-FDH) gene, full-length EST-cDNA clone 80E7T7 (GenBank no. T45182; NEWMAN et al. 1994), was identified in the EST cDNA database. This clone was similar but not identical to the Arabidopsis ADHl gene (CHANG and MEYEROWITZ 1986). The sequence is 63.1% similar at the nucleotide level (78.6% at amino acid level) to Ara- bidopsis ADHI and has a Southern blot hybridization pattern different to ADHl (Figure 1). We mapped 80E7T7 to chromosome 5, between RFLP marker g4028 (10.2 cM) and the DFR marker (9.8 cM), while ADHl is located on chromosome I (CHANG and MEXROWITZ 1986). The cDNA clone was used as a probe to isolate the corresponding FDHI gene from a lambda genomic library, and the gene was sequenced.

The general occurrence of the class 111 type ADH

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 FIGURE Z.-Southern blot hybridization showing cross-hy-

bridization under medium stringency conditions between Ar- abidopsis ADHl and l D H I probes and genomic DNA isolated from various plant species. Ten micrograms of genomic DNA of Arabidopsis (lane l ) , rice (lane 2), Brassica (lane S), flax (lane 4), cotton (lane 5 ) , lupin (lane 6), pea (lane 7) and barley (lane 8) was digested with EcoRI and separated on a 0.7% agarose gel. The Southern blot filter was hybridized under medium-stringency conditions (55"), using the hybridization mix described in MATERIAIS AND METHOIX

enzymes (GSH-FDH) in the plant kingdom was demonstrated by Southern blot hybridization experiments using genomic DNA of a variety of higher plant species, ranging from monocots to dicots. Filters were hybridized under medium stringency conditions with both the Arabidopsis ADHI and FnHI genes. A different band- ing pattern is obtained with each probe and they hybridized equally well to the genomic DNA of a number of plant species (Figure 2). Both ADH and GSH-FDH activity are present in callus extracts from Arabidopsis, maize, rice and cotton (data not shown). Based on the high degree of sequence homology between plant class 111 genes, we cloned the monocot rice FnHl gene by screening a rice genomic library under low stringency conditions, using the Arabidopsis IDHI cDNA (80E7T7) as a probe. The rice FDHI gene is 64.5% and 61.6% similar at nucleotide level and 77.6% and 76.0% similar at amino acid level to rice ADHl and ADH2, respectively. Homology is higher with the Arabidopsis (78.4% homology at nucleotide level; 95.0% at amino acid level) and pea (95.0% homology in amino acid sequence) FDH enzymes.

Intron positions in Arabidopsis and rice FDHl are identical: Arabidopsis FDHI has eight introns (Figure 3). The positions of six of these introns are identical to those of the six Arabidopsis ADHl introns. The two additional introns are located at the positions of introns in the maize Adhl and Adh2 genes (DENNIS et al. 1984,


Maize AdhZ,2 : ( 3 7 9 1 3 7 9 a a )

Rice AdhZ,2 : ( 3 7 6 1 3 7 0 a a )

Barley AdhZ,2,3 : ( 3 7 9 1 3 7 3 1 3 7 9 a a )

Wheat AdhZ : ( 3 7 9 aa)

Pearl Millet AdhZ : ( 3 8 0 a a )

Petunia AdhZ : ( 3 8 1 a a )

Pea AdhZ : ( 3 8 1 a a )

Ath ADHZ : ( 3 7 9 a a )

Ath FDHZ : ( 3 7 9 a a )

Rice FDHZ : ( 3 8 1 a a )

Human ADHS :

- 1 2 3 4 5 6 7 8 9

I ’ I ‘ I I I

I I

I I I I I I

I J CLASS P

I I

I I 9 1 1 V I

I I 4 1 ; 15 : : f l 9 V I I

I 6 I I

( 3 7 4 a a )

FIGURE 3.-Comparison of intron positions (numbered triangles) between plant class P ADH genes and the Arabidopsis and rice class 111 genes. The length of the different protein sequences is shown between brackets. While the Arabidopsis ADHl is missing maize Adhl introns 4, 5 and 7, the Arabidopsis F21Hl gene is missing only maize intron 7. Maize intron 7 is also missing from the rice HIHl gene. Maize intron 7 is present in both monocot and dicot class P genes, including palm Adh genes (palm Adh sequences are incomplete and not shown in the figure; GAUT et al. 1996). The intron positions in the human ADH5 class 111 gene are completely different than in the plant Adh genes.

1985; Figure 3). The maize (DENNIS et al. 1984, 1985), rice (XIE and WU 1989, 1990), pea (LLEWELLYN et al. 1987) and Petunia (GREGERSON et al. 1991) Adh genes all have nine introns, while the barley (TRICK et al. 1988) and wheat (MITCHELL. et al. 1989) Adh genes lack the last intron (intron 9). Two incomplete palm ADH sequences also contain an intron in the same position as maize intron 7 (GAUT et al. 1996). The Arabidopsis FDHl gene lacks intron 7 of maize Adhl and Adh2 (Fig- ure 3). The rice FDHl gene contains eight introns in identical positions as in the Arabidopsis FDHl gene, also lacking intron 7 of the rice and maize Adhl and Adh2 genes (Figure 3 ) .

Arabidopsis and rice FDHl are class I11 ADH enzymes: The Arabidopsis and rice FDHl proteins are both 95.0% identical to pea ADHIII (SHAFQAT et al.

1996) and are generally more related to the class I11 type ADH enzymes from humans (ADH5), rat, Dro- sophila and Atlantic hagfish than to any of the plant ethanol-active ADH enzymes (Figures 4 and 5 ) . The maize ADH1, Arabidopsis ADHl and FDHl and pea class I11 GSH-FDH enzymes have exactly the same length (379 AA), and regions of high sequence similar- ity are obvious throughout all the sequences shown (Figure 5). All ligands to both the catalytic and noncata- lytic zinc atoms are strictly conserved between all plant enzymes (FIGURE 5; BMNDEN et aZ. 1975; EKLUND et at. 1987).

A detailed biochemical and structural analysis of the Arabidopsis FDHl enzyme has recently been published (MARTINEZ et al. 1996). The amino acid residues distin- guishing class I11 ADH enzymes from the ethanol-active

Cloning of the Arabidopsis and Rice Class I11 Adh Genes 1135

L

POTATO ADHl (X53242)

TOMATO ADH2 (M86724) 1 SOLANACEAE I

PETUNIA ADH2 ( 5 5 8 2 8 2 )

€ TOBACCO ADH (X81853) PETUNIA ADH1 (X54106)

1 SOLANACEAE I I

-COTTON ADH2 (X53242) MALVACEAE - ARABlDOPSlS ADHl (M12196) CRUCIFERAE

PEA ADHl (X06281) TRIFOLIUM ADHl (X14826) FABACEAE

PHASEOLUS ADHl (223170) STRAWBERRY ADH (X1

1 CAPPLE ADH (P48977)

MAIZE ADHl (X04050) PENNISETUM ADHl (X16547) BARLEY ADHl (X07774)

RICE ADHl (X16296)

POACEAE I

WHEAT ADHl BARLEY ADH3 (X1 2734)

BARLEY ADH2 (X1 2733) POACEAE II RICE ADH2 ( M 3 6 4 6 9 )

MAIZE ADH2 (X02915)

DICOTS

MONOCOTS

CANDIDA FDH (M58332) YEAST FDH (P32771)

DROSOPHILA FDH (U0764 1 )

MOUSE ADH2 (P28474) RAT ADH2 (P12711)

HORSE ADHll l (P19854)

HUMAN ADH5 (M30471)

UROMASTYX ADHll l (P80467) HAGFISH ADHll l (P80360)

CAENORHABDITIS ADH (U18781)

% RICE FDHl (U77637) ARABlDOPSlS F D H l (U63931) PEA FDH (P80572)

classes are mainly located in the substrate pocket of the enzyme, and affect size, shape and polarity of the substrate cleft, and hence substrate-specificity and ac- cessibility (EKLUND et al. 1990; HOOG et al. 1992; ENGE- LAND et al. 1993; DANIELSSON et al. 1994; ESTONIUS et al. 1994; HURLEY et al. 1994; Figure 5 ) . Two amino acids (Asp-57 and Arg-115) were determined to be contact points with the substrate S-hydroxymethylglutathione in the substrate pocket of class 111 enzymes, and both are conserved in the Arabidopsis and rice FDHl enzymes (Figure 5 ) . Except for position 116, which is different in Arabidopsis FDHl , Arabidopsis and rice FDHl have all the amino acids in the substrate pocket typical for class 111 ADH enzymes (GSH-FDH) .

Arabidopsis FDHl has GSH-FDH activity: The avail- ability of a full-length cDNA clone for Arabidopsis EDHI enabled us to study the enzymatic activity of the en- coded enzyme. Plasmid pKKFDH1, containing the EDHI cDNA under the control of the tac promoter, was expressed in vitro using E. coli S30 cell-free extracts in the presence of "%methionine (Figure 6A). The main protein band labeled in the extracts has a molecular

CLASS 111 TYPE ADH

(GSH-FDH)

FIGURE 4.-Dendrogram showing the evolutionary relationship between different plant ethanol-active ADH enzymes and class I11 ADH enzymes from a variety of organisms. GenBank or Swiss Protein Bank accession numbers are given between brackets. Distance analysis was done on aligned protein sequences using the K " R A (1981) protein distance option of the GCG Dis- tances program. The phylogram was constructed with the Growtree program, using the neighbor-joining method. The length of the lines from the nodes indicate evolutionary distances. Reliability was tested by com- paring the phylograms obtained from the complete amino acid sequence, with the ones obtained for the first 250 amino acids (starting from residue 9), and the rest of the sequence (250-end) for each sequence.

weight of 43 kD on SDS gels, similar to the expected subunit molecular weight of plant ADH enzymes (FREE- LING 1973; FREELING and SCHWARTZ 1973; DOLFERUS and JACOBS 1984, 1991). The transcribed protein has GSH-FDH activity, but no ADH activity, and has the same migration properties as the FDH enzyme in plant extracts (Figure 6A). In addition, the FDH activity- stained band corresponds with a co-migrating labeled band in native gels of the E. coli expressed gene (data not shown).

In vivo expression of pKJWDHl in E. coli resulted in high levels of GSH-FDH activity after induction with IPTG (Figure 6B). Activity was detected after a 2-hr staining period. The protein expressed in E. coli and the plant protein have the same migration properties. The FDHl enzyme has only GSH-FDH activity and does not show any dehydrogenase activity after a 20-hr staining period with ethanol as substrate (Figure 6B). We were not able to show activity with octanol, although MARTINEZ et al. (1996) demonstrated a 20-fold lower activity of Arabidopsis FDH using octanol. The Dro- sophila GSH-FDH enzyme was originally identified as


1 60 Ath-ADH1 . . . . . . . . . . . . . . . . MSTT GQIIRCKAAV AWEAGKPLVI EEVEVAPPQK HEVRIKILFT ZM-ADH1-S . . . . . . . . . . . . . . . . MATA GKVIKCKAAV AWEAGKPLSI EEVEVAPPQA MEVRVKILFT Human-ADH5 . . . . . . . . . . ........MA NEVIKCKAAV AWEAGKPLSI EEIEVAPPKA HEVRIKIIAT Rat-ADHIII . . . . . . . . . . . . . . . . . . . A NQVIRCKAAV AWEAGKPLSI EEIEVAPPQA HEVRIKIIAT Pea-ADHIII . . . . . . . . . . . . . . . . MATQ GQVITCKAAV AWEPNKPLTI EDVEVAPPQA NEVRIQILFT Rice-FDH1 . . . . . . . . . . . . . . MASSTQ GQVITCKAAV AWEANKPMTI EDVQVAPPQA GEVRVKILFT

61 120

Ath-FDH1 . . . . . . . . . . . . . . . . MATQ GQVITCKAAV AYEPNKPLVI EDVQVAPPQA GEVRIKILYT

Ath-ADH1 SLCWl?DVYEW EAKGQTPLFP RIFGHEAGGI VESVGEGVTD LQPGDHVLPI ETGECGDCRH ZM-ADH1-S SLCHTDWEW EAKGQTPVFP RIFGHEAGGI IESVGEGVTD VAPGDHVLPV ETGECKECPH Human-ADHS AVCHTDAYTL SGADPEGCFP VILGHEGAGI VESVGEGVTK LKAGDTVIPL YIPQCGECKF Rat-ADHIII AVCHTDAYTL SGADPEGCFP VILGHEGAGI VESVGEGVTK LKAGDTVIPL YIPQCGECKF Pea-ADHIII ALCHTDAYTL GGKDPEGLFP CILGHEAAGI VESVGEGVTD VKPGDHVIPS YQAECGECKF

Rice-FDH1 AFCHTDHYTW SGKDPEGLFP CILG-GI VESVGEGVTE VQPGDHVIPC YQAECRECKF 46. *47,48 051 9 68 93. 97. -100 121 180

Ath-ADH1 CQSEESNMCD LWNTERGG MIHDGESRFS INGKPIYHFL GTSTFSEYTV VHSGQVAKIN ZM-ADH1-S CKSAESNMCD LLRINTDRGV MIGDGKSRFS INGKPIYHFV GTSTFSEYTV MHVGCVAKIN

Ath-FDH1 ALCHTDAYTW SGKDPEGLFP CILGHEAAGI VESVGEGVTE VQAGDHVIPC YQAECRECKF

Human-ADH5 CLNPKTNLCQ KIPNTQGKG. LMPDGTSRFT CKGKTILHYM GTSTFSEYTV VADISVAKID Rat-ADHIII CLNPKTNLCQ KIRVTQGKG. LMPDGTSRFT CKGKPILHEM GTSTFSEYTV VADISVAKID Pea-ADHIII CKSPKTNLCG KVRAATGVGV "ADRKSRFS VKGKPIYHEM GTSTFSQYTV VHDVSVAKIH

Rice-FDH1 CKSGJSTNLCG KVRAATGVGV "NDRKSRFS INGKPIYHEM GTSTFSQYTV VHDVSVAKIN 0103 111. 115**116 140-*141 181 240

Ath-FDH1 CKSGKTNLCG KVRSATGVGI "NDRKSRFS VNGKPIYHM GTSTFSQYTV VHDVSVAKID

Ath-ADH1 PDAPLDKVCI VSCGLSTGLG ATLNVAKPKK GQSVAIFGLG AVGLGAAEGA RIAGASRIIG ZM-ADH1-S PQAPLDKVCV LSCGISTGLG ASINVAKPPK GSTVAVEGLG AVGLAAAEGA RIAGASRIIG Human-ADH5 PLAPLYKVCL LGCGISTGYG AAVNTAKLEP GSVCAVFGLG GKLAVIMGC KVAGASRIIG Rat-ADHIII PSAPLDKVCL LGCGISTGYG AAVNTAKVEP GSTCAVFGLG GVGLAVIMGC KVAGASRIIG Pea-ADHIII PDAPLDKVCL LGCGVPTGLG AVWNTAKVEP GSIVAIFGLG TVGLAVAEGA KSAGASRIIG

Rice-FDH1 PQAPLDKVCL LGCGVSTGLG AVWNTAKVEA GSIVAIFGLG TVGLAVAEGA KSAGASRIIG

241 300

Ath-FDH1 PTAPLDKVCL LGCGVPTGLG AVWNTAKVEP GSNVAIFGLG TVGLAVAEGA KTAGASRIIG

114*178* 201. *-202,203

Ath-ADH1 VDFNSKRFDQ AKEFGVTECV NPKDHDKPIQ QVIAEMTDGG VDRSVECTGS VQAMIQAFEC ZM-ADH1-S VDLNPSRFEE ARKFGCTEFV NPKDHNKPVQ EVLAEMTNGG VDRSVECTGN INAMIQAFEC Human-ADH5 VDINKDKFAR AKEFGATECI NPQDLSKPIQ EVLIEMTDGG VDYSFECIGN VKVMRAALEA Rat-ADHIII IDINKDKFAK AKEFGATECI NPQDFSKSIQ EVLIEMTDGG VDFSEECIGN VKVMRSALEA Pea-ADHIII IDIDSNKYDT AKNFGVTERI NPKDHEKPIQ QVIIDLTDGG VDYSFECLGN VSVMRSALEC

Rice-FDH1 IDIDSKKFDV AKNFGVTEFV NPKDHDKPIQ QVIVDLTDGG VDYSFECIGN VSVMRSALEC

301 360

Ath-FDH1 IDIDSKKYET AKKFGVNEFV NPKDHDKPIQ EVIVDLTDGG VDYSFECIGN VSVMRAALEC

0223 0228

Ath-ADH1 VHDGWGVAVL VGVPSKDDAF KTHPMNFLNE RTLKGTFFGN YKPKTDIPGV VEKYMNKELE ZM-ADH1-S VHDGWGVWL VGVPHKDAEF KTHPMNFLNE RTLKGTFFGN YKPRTDLPNV VELYMKKELE Human-ADH5 CHKGWGVSW VGVAASGEEI ATRPEQLVTG RTWKGTAFGG WKSVESVPKL VSEYMSKKIK Rat-ADHIII AHKGWGVSW VGVAASGEEI STRPEQLVTG RTWKGTAFGG WKSVESVPKL VSEYMSKKIK Pea-ADHIII CHKGWGTSVI VGVAASGQEI STRPEQLVTG RVWKGTAFGG FKSRSQVPWL VEKYLKKEIK

Rice-FDH1 CHKGWGTSVI VGVAASGQEI STRPEQLVTG RVWKGTAFGG FKSRSQVPWL VEKYLNKEIK Ath-FDH1 CHKGWGTSVI VGVAASGQEI STRPEQLVTG RVWKGTAFGG FKSRTQVPWL VEKYMNKEIK

294. 306. *309 318. 361 396

Ath-ADH1 LEKFITHTVP FSEINKAFDY MLKGESIRCI ITMGA ZM-ADH1-S VEKFITHSVP FAEINKAFNL MAKGEGIRCI IRMEN Human-ADH5 VDEFVTHNLS FDEINKAFEL MHSGKSIRTV VKI.. Rat-ADHIII VDEF'VTGNLS FEQINKAFDL MHSGNSIRTV LKL.. Pea-ADHIII VDEYITHNLT LLEINKAFDL LHEGQCLRCV LAV..

Rice-FDH1 VDEYVTHSMN LTDINKAFDL LHEGGCLRCV LATDK Ath-FDH1 VDEYITHNLT LGEINKAFDL LHEGTCLRCV LDTSK

362 -369

FIGURE 5.-Protein sequence alignment between the Arabidopsis ADHl (Ath-ADH1) and maize ADHl (ZM-ADH1- S) class P enzymes and the class I11 protein sequences from Arabidopsis (Ath- FDHl), rice (rice-FDHl), pea FDH, human ADH5 and rat ADHIII. Number- ing of the amino acid residues is with respect to the horse liver ADH-E enzyme. Critical amino acids that are part of the substrate or coenzyme binding domain are shown in bold and are numbered below the sequence alignment (EKLUND et al. 1990; HOOG et al. 1992; HURLEY et al. 1994). All ligands to the catalytic (Cys46; His- 68; Cys-174) and noncata- lytic zinc atoms (Cys-97;

are strictly conserved for all classes of ADH enzymes. Comparison of substrate pocket amino acid residues between plant class P and the pea and Arabidopsis class I11 enzymes has been dis- cussed in detail before (MARTINEZ et al. 1996; SHAFQAT et al. 1996). The rice FDHl enzyme has all the amino acid residues typical for class 111 ADH enzymes, while Arabi- dopsis FDHl differs only in a Ser residue (instead of Val) at position 116.

cys-100; cys-103; cys-111)

octanol dehydrogenase (LLJQUE et al. 1994). Both the SHAFQAT et al. 1996). A high degree of substrate ambi- pea and Arabidopsis class I11 GSH-FDH enzyme have a guity is a property of all ethanol-active ADH enzymes. high K, for ethanol as substrate (MARTINEZ et al. 1996; Arabidopsis ADHl has affinity for octanol (Figure 6B)

Cloning of the Arabidopsis and Rice Class 111 Adh Genes

MW

A. . ' "" '

~ ( K W

1137

1 2

B. 2H

20H

1 2

-97.4 -66.2 -55.0 -42.7 '40.0

-31 .O

-21.5 -14.4

3 4 1 2 3 4 5

ADH

3 4 5 6 7 1 2 3 4 5 6 7 FDH

FIGURE 6.- (A) In vitroand (B) in vivo expression studies of the Arabidopsis full-length FDHl cDNA, using plasmid pKKFDH1. (A) Autoradiographs of '"Smethionine-labeled in vitro synthesized proteins (E. coli S30 extracts) separated on SDS gels (left). Lane 1, no DNA control; lane 2, positive control using a luciferase containing expression vector (100 ng plasmid used per assay); lanes 3 and 4, the autoradiograph shows a major labeled protein band of -43 kD, which corresponds to the expected subunit molecular weight of ADH enzymes (1 pg pKKFDHl per assay mix). The right panel shows that it was possible to demonstrate on a native gel that the low amount of in vitro synthesized protein had GSH-FDH activity. Lanes 1 and 2, in vitro transcription/ translation of plasmid pKKFDH1; the arrow indicates the position of the FDHl protein product, which has the same migration as the enzyme present in a whole plant extract from Arabidopsis ecotype C24 (lane 5). Lanes 3 and 4 represent negative controls of in vitro transcription/translation samples containing the luciferase expression vector and no DNA, respectively. The higher intensity faster migrating band on the gel represents the E. coli FDH enzyme, which is present in the S30 extracts. Staining was for 20 hr; no staining of the E. coli ADH enzyme was detected under these GSH-FDH staining conditions, while ADH was detected readily using ethanol as a substrate. (B) In vivo expression studies show that the Arabidopsis FDHl enzyme, expressed by the full-length cDNA plasmid pKKFDHl in E. coli, has the same migration properties as the enzyme present in plant extracts. Sample positions: lane 1, JM109 without plasmid DNA; lane 2, JMlOS/pKKFDHl, not induced with IF'TG, lanes 3 and 7, JM109/ pKKFDH1, induced with IFTG; lanes 4-6, callus extracts of ecotype C24; the ADHl null mutant ROO2 and Ws-0, respectively. Callus extracts were chosen because of the high levels of ADH activity (DOLFERUS et al. 1985). The lower band in lanes 1-3 and 7 show activity of the E. coli FDH enzyme (see also A, right). Prolonged staining for 20 hr showed that the callus extracts of ecotype C24 also has GSH-FDH activity at the position where ADHl migrates (see arrowhead). This is confirmed by the absence of this activity in callus extracts of the null mutant and Ws-0, which has an ADHl enzyme with considerably lower affinity for ethanol. No E. coli endogenous ADH activity is observed even after 20 hr of staining, suggesting there is no ethanol contamination in the staining mix. ADH and ODH indicate activity stainings (carried out for 20 hr) using ethanol and octanol as substrates, respectively. ADHl shows activity for both stainings, while FDHl did not show a trace of activity. The slowest migrating band in lanes 1-3 and 7 indicates the position of the E. coli endogenous ADH activity, which is present in none of the GSH-FDH stainings.

1138 R. Dolferus et al,

CLASS 111 ANCESTOR

~ n t r o n s ? GSH-FDH ACTIVE

v v ANIMAL CLASS I l l ANCESTOR

GSH-FDH ACTIVE X tntrons

v YEAST CLASS 111 ANCESTOR GSH-FDH ACTIVE

no lntruns PLANT CLASS 111 ANCESTOR

GSH-FDH ACTIVE X or 9 introns '!

'0

ANIMAL CLASS 111 ADH A N I M A L CLASS I & I1 ADH GSH-FDH ACTIVE

GSH-FDH ACTIVE no Introns

ETHANOL-ACTIVE n o Introns ETHANOL ACTIVE

X introns

v 7

X o r 9 Introns 1 X intruns '!

0

ETHANOL ACTIVE ETHANOL ACTIVE R o r 9 introns 6 . X or 9 introns

FIGURE 7.-Model showing the evolution of plant genes encoding ADH, by gene duplication from a class 111 ADH type ancestor. Gene duplication events are indicated by crossed circles.

and a variety of other alcohols, including allyl alcohol, n-butanol, n-propanol and iso-propanol (DOLFERUS and JACOBS 1984).

Prolonged staining for FDH activity (20 hr) revealed a second band, present only in callus extracts of C24 but not of ADHl null mutant ROO2 (JACOBS et al. 1988; DOLFERUS et al. 1990) or ecotype Ws-0, which has a less active ADHl isozyme than the C24 ecotype (R. DOL- FERUS, unpublished data); this suggests the second band is due to ADHl activity (Figure 6B). We were not able to show this for rice ADHl and ADH2, due to the similar migration of the rice ADH enzymes and the GSH-FDH activity. Arabidopsis ADHl has the conserved Arg-115 residue, typical for the substrate pocket of class I11 enzymes (KAISER et al. 1988; ENGELAND et al. 1993; DAN- IELSSON et al., 1994; ESTONIUS et al. 1994; Figure 5), and maybe this amino acid is responsible for the GSH-FDH activity. This result stresses the close relationship between plant class I11 and class P enzymes compared to the animal counterparts. While human class I and I11 ADH enzymes are only moderately similar (63% identical at amino acid level), plant class P and class I11 enzymes are generally more similar: Arabidopsis ADHl and FDHl (78.6% identical), rice ADHl and FDHl (77.6% identical) and the pea ADHl and FDH enzymes (77.4% identical). Comparison of amino acid residues in the substrate pocket of plant class I11 and class P

enzymes led to the same conclusions (MARTINEZ et nl. 1996; SHAFQAT et al. 1996).

Evolution of plant ADH enzymes: By providing the genomic sequence of two plant class I11 ADH represen- tatives, one from a dicot and one from a monocot plant, we have shown that class I11 ADH enzymes are wide- spread in the plant kingdom and that they may have preceded the evolution of ethanol-active ADH enzymes (KAISER et al. 1989; DANIELSON and JORNVALL 1992; DANIELSSON et nl. 1992). The class I11 ADH genes from both Arabidopsis and rice have eight introns in identical positions, suggesting that the plant class I11 ancestor gene had these same eight introns (Figure 7). All the human ADH genes (class I, I1 and 111) have eight introns, all in exactly the same positions. Although intron positions in both human and plant ADH genes have been correlated with the separation of functional domains in the ADH protein (BRANDEN et al. 1984; DUES TER et al. 1986b), none of the human ADHgene introns are in the same position as in plant ADHgenes (Figure 3; DUESTER et al. 1986a; YOKOYAMA et al. 1990; VON BAHR-LINSTROM et al. 1991; HUR and EDENBERC 1992; GKECERSON et al. 1993). This could indicate that introns were inserted in a phylum-specific way in the class I11 ancestral genes after the separation of plants and animals, supporting the theory that introns were acquired by insertion after the separation of prokaryotes and


eukaryotes (ROGERS 1989; CAVALIER-SMITH 1991). Alter- natively, the different intron positions in plants and animals could be the result of intron sliding (GILBERT et al. 1986; GILBERT 1987).

Plant class 111 intron positions are maintained in the ethanol-active class P genes (Figures 3 and 7), except that many plant class P genes contain one more intron. It is likely that an additional intron (intron 7 in the maize Adhl gene; Figures 3 and 7) was inserted after the duplication of class 111 genes, as it is present in all plants examined, except Arabidopsis ADHI, which appears to have lost intron 7. Nine introns are generally present in class P genes of both monocots and dicots: maize Adhl,2 (DENNIS et al. 1984, 1985); rice Adhl,2 (XIE and Wu 1989, 1990); pea Adh (LLEWELLYN et al. 1987); Petunia Adhl (GREGERSON et al. 1991; Figure 7). The barley and wheat Adh genes have eight introns (lacking maize intron 9, but containing intron 7; Fig- ure 3) .

The gene duplication event giving rise to ethanol- active ADH has apparently occured several times inde- pendently (Figure 7). The yeast ethanol-active ADH enzyme was derived by gene duplication from a yeast class I11 ancestor without introns and does not have a common origin with the human class I (DANIELSSON and JORNVALL 1992) or plant class P ADH enzymes (Figure 7). The monocot and dicot class P enzymes have a single origin, but within the grass family there is a further duplication (Figure 7). Maize, rice ( @ y a sativa), barley (Hordeum vulgare) and Pennisetum americanum have an Adhl gene, together with other copies (maize, rice, barley Adh2 and barley Adh?). Throughout the monocots, the ethanol-active genes are closer to each other than to the class 111 type Adh genes, suggesting that they were derived by a more recent gene duplication than the duplication event from the class 111 ancestor.

The dicot ADH sequences available belong to six plant families that are all of different orders, but all monocot ADH sequences belong predominantly to the grasses (Poaceae) and the palms. The two incomplete palm Adh gene sequences (GAUT et al. 1996) could not be included in this analysis. Strong clustering is observed for species from the same family (Figure 4). Only in the Solanaceae, where different gene copies were available from one species (Petunia, Adhl,2 Potato, Adhl,2), is a split into two groups observed.

The Arabidopsis ADHl gene is induced predominantly in roots by environmental stresses such as low oxygen, dehydration, low temperature and by the phy- tohormone ABA (DOLFERUS et al. 1994; DE BRUXELLES et al. 1996). In contrast, IDHI gene expression is not affected by any stress treatment (Figure 8). For both Arabidopsis and rice HIHI, expression levels are similar in roots and leaves of the plant and are on average about three times higher than ADHl expression under uninduced conditions. Human class 111 ADH, in con-

ADHl mRNA EXPRESSION

d 16000 n * A

3 12000 B 5

- u)

E 8000

2 4000 PL E

0 C AN D CD

FDH7 mRNA EXPRESSION

3000 1 I 1

C AN D CD

FIGURE 8,"Expression of the Arabidopsis ADHl gene compared to the m H I gene. Total RNA (25 kg) isolated from leaves (H) and roots (m) of control (C), hypoxically treated ( A N ) , dehydration-treated (D) and low-temperature-treated (CD) plants was used in Northern blot hybridizations, using equal quantities of Arabidopsis ADHl and I B H l probes. Sig- nal strength was measured as described in MATERIALS AND METHODS and plotted as absolute expression levels for both probes. m H 1 expression is much lower than ADHI expression levels and is not affected by stress treatments. The different expression patterns of ADHl and FzlHl demonstrate differences in metabolic functions of both enzymes. Standard error bars are shown for three independent experiments.

trast to the liver-specific class I and I1 enzymes, is found in most tissues (KAISER et nl. 1989).

The gene duplication events that led to the evolution of ethanol-active class P ADH enzymes in plants must have been associated with the acquisition of a promoter with new tissue-specific and environmental stress re- sponsive expression. This expression pattern had to suit the new metabolic function of ADH in alcohol fermentation. Animal ADH enzymes evolved a metabolic function different from that in plants.

Physiological role of plant GSH-FDH enzymes: The exact role of glutathione-dependent formaldehyde de-


hydrogenase is unknown. One possible role is as a scav- enger of toxic formaldehyde, which could be either a by-product of metabolic pathways (GIESE et al. 1994) or of xenobiotic origin (UOTILA and KO~WSALO 1989; SANDERMANN 1992; JORNVALL et al. 1993; GIESE et al. 1994). In plants, glutathione conjugates, which are stored in the vacuole, are used to detoxify many organic compounds (WOLF et al. 1996). The constitutive expression pattern of Arabidopsis HIHI (Figure 8) is consis- tent with a role of GSH-FDH in protection and detoxification of toxic formaldehyde of either cellular or xenobiotic origin (SANDERMANN 1992; GIESE et al. 1994). Candida maltosa FDH confers resistance to formaldehyde when transferred to yeast (SASNAUSKAS et al. 1992; WEHNER et al. 1993). Antisense and overexpression technology in transgenic plants will allow us to study the role of GSH-FDH in plants and to identify any selective advantage of a functioning GSH-FDH activity in the presence of elevated formaldehyde concentrations.

The authors thank S. STOPS and J. NORMAN for excellent technical assistance and Drs. C. LISTER and M. STAMMERS (AFRC Institute of Plant Science Research, Cambridge Laboratory, John Innes Centre, Nonvich, UK) for their assistance in mapping the chromosomal location of IDHI.

LITERATURE CITED

ANDREWS, D. L., D. M. MA&.PINE, J. R. JOHNSON, P. M. KELLEY, B. G. CORR et al., 1994 Differential induction of mRNAs for the glyco- lytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings. Plant Physiol. 106: 1575-1582.

BRANDEN, GI., H. JORNVAIJ. and B. FURUGREN, 1975 Alcohol dehydrogenase, pp. 103-190, in The Enzymes, Vol. XI, edited by P. D. BOER, Academic Press, New York.

BRANIxN,C-I.,H.EKLLIND,C.CAMBILLAU~~~A. J . P R Y o R , ~ ~ ~ ~ Corre- lation of exons with structural domains in alcohol dehydrogenase. EMBO J. 3: 1307-1310.

BRAU, B., and H. SAHM, 1986 Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli. Arch. Microbiol. 144: 296-301.

BROSNIUS, J., and A. HOLY, 1984 Regulation of ribosomal RNA promoters with a synthetic lac promoter. Proc. Natl. Acad. Sci. USA

CAVAI.IER-SMITH, T., 1991 Intron phylogeny: a new hypothesis. Trends Genet. 7: 145-148.

CHANG, C., and E. M. MEYEROWITZ, 1986 Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene. Proc. Natl. Acad. Sci. USA 8 3 1408-1412.

DANIEISSON, O., and H. JBw.u.IA, 1992 “Enzymogenesis”: classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line. Proc. Natl. Acad. Sci.

DANIEISSON, O., H. EKL.UND and H. JORN~AI.I., 1992 The major pi- scine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenase of classes I and 111. Biochemistry 31: 3751-3759.

DUARTE et al,, 1994 Fundamental molecular differences between alcohol dehydrogenase clases. Proc. Natl Acad. Sci. USA 91: 4980-4984.

DE BRLTXEILES, G. L., W. J. PEACOCK, E. S. DENNIS and R. DOLFERUS, 1996 Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis. Plant Physiol. 111: 381-391.

DENNIS, E. S., W. L. GERLZCH, A. J. PRYOR, J. L. BENNETZEN and A. INGLIS, 1984 Molecular analysis of the alcohol dehydrogenase (Adhl) gene of maize. Nucleic Acids Res. 12: 3983-4000.

DENNIS, E. S., M. M. SACHS, W. L. GERIACH, E. J. FINNEGAN and W. J.

81: 6929-6933.

USA 89: 9247-9251.

DANIELSSON, O., S. ATRIAN, T. LUQUE, L. HBLMQVIST, R. GONSALEZ-

PEACOCK, 1985 Molecular analysis of the alcohol dehydrogenase 2 (Adh2) gene of maize. Nucleic Acids Res. 13: 727-743.

DOLFERUS, R., and M. JACOBS, 1984 Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L.)Heynh.: genetical and biochemical characterisation. Biochem. Genet. 2 2 817-838.

DOLFERUS, R., and M. JACOBS, 1991 Another ADH system . . . from Arabidopsis thaliana. Maydica 36: 169- 187.

DOI.FERUS, R., G. MARBAIX and M. JACOBS, 1985 Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plants and tissue cultures. Mol. Gen. Genet. 199: 256-264.

DOLFERUS, R., D. VAN DEN BOSSCHE and M. JACOBS, 1990 Sequence analysis of two Adh null-mutant alleles of the single Arabidopsis Adh locus. Mol. Gen. Genet. 224: 297-302.

DOLFERCS, R., M. JACOBS, W. J. PFACOCK and E. S. DENNIS, 1994 Dif- ferential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiol. 105: 1075-1087.

DUESTER, G., M. SMITH, V. BIIANCHONE and G. W. HATFIEI-D, 1986a Molecular analysis of the human class I alcohol dehydrogenase gene family and the nucleotide sequence of the gene encoding the subunit. J. Biol. Chem. 261: 2027-2033.

DUESTER, G., H. JORNVAI.~. and G. W. HATFIEID, 1986b Intronde- pendent evolution of the nucleotide-hinding domains within alcohol dehydrogenase and related enzymes. Nucleic Acids Res. 14: 1931-1941.

EKI.UND, H., E. HOR~ALES, B. L. VALLEE and H. JORNVAI.~., 1987 Com- puter-graphics interpretations of residue exchanges between the a, p , and y subunits of human-liver alcohol dehydrogenase class I isozymes. Eur. J. Biochem. 167: 185-193.

EKINND, H., P. MOLI.ER-WII.L.E, E. HORIALES, 0. FUTER, B. HOLMQUIST et al., 1990 Comparison of three classes of human liver alcohol dehydronase. Eur. J. Biochem. 193: 303-310.

ENGEIAND &, J-0. HOOG, B. HOI.MQUIST, M. ESTONIUS, H. JORNVALI. et al., 1993 Mutation of Arg-115 of human class 111 alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation. Proc. Natl. Acad. Sci.

ESTONICS, M., J-0. HOOG, 0. DANIELSON and H. JORNVAI.I., 1994 Residues specific for class 111 alcohol dehydrogenase. Sitedi- rected mutagenesis of the human enzyme. Biochemistry 33:

FERNANDEZ, M., H. JORNVAII., A. MORENO, R. KAISER and X. PARES, 1993 Cephalopod alcohol dehydrogenase: purification and enzymatic characterisation. FEBS Lett. 328: 235-238.

FINN, R. K., S. BRINGER and H. SAHM, 1984 Fermentation of arahi- nose to ethanol by Sarcinia ventriculi. Appl. Microbiol. Biotech- nol. 19: 161-166.

FREEI~ING, M. 1973 Simultaneous induction by anaerobiosis or 2,4D of multiple enzymes specified by two unlinked genes: differential Adhl-AdhPexpression in maize. Mol. Gen. Genet. 127: 215-227.

FREELING, M., and D. SCIIWARTZ, 1973 Cenetic relationships between the multiple alcohol dehydrogenases of maize. Biochem. Genet. 8: 27-36.

FRISCHAUF, A”., H. LEHRACH, A. POUSTKA and N. MURRAY, 1983 Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol. 170: 827-842.

GANCEDO, C., and R. SERRANO, 1989 Energy yielding metabolism, pp. 205-259, in The Yeasts: Metabolism and Physiology of Yeasts, Vol. 3, edited by A. H. ROSE, and J. S. STUART. Academic Press, London.

GARI.ICK, P. B., G. K. RADDA and P. J. SEELEY, 1979 Studies of acido- sis in the ischaeimic heart by phosphorus nuclear magnetic reso- nance. Biochem. J. 184: 547-554.

GAU’I‘, B. S., B. R. MORTON, B. C. M c W c ; and M. T. CLEGG, 1996 Substitution rate comparisons between grasses and palms: synon- ymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl. Acad. Sci. USA 93: 10274-10279.

GIESE, M., U. BAUER-DORANTH, C. LANGEBARTELS and H. SAN- DERMANN, 1994 Detoxification of formaldehyde by the spider plant (Chlorophytum comosum L.) and soybean (Glycine max. L,.) cell-suspension cultures. Plant Physiol. 104: 1301 -1309.

GIL.BERT, W., 1987 The exon theory of genes. Cold Spring Harbor Symp. Quant. Biol. 52: 901-905.

GIIBERT, W., M. ~ ~ A R C H I O N N I and G. MCKNIGHT, 1986 On the antiq- uity of introns. Cell 46: 151-154.

GREGERSON, R. G., M. MCLEAN, M. BF.I.D, A. G. M. GERATS and J. N.

USA 90: 2491-2494.

15080-15085.


STROMMER, 1991 Structure, expression, chromosomal location, and product of the gene encoding ADHl in Petunia. Plant Mol. Biol. 17: 37-48.

GREGERSON, R. G., L. CAMERON, M. MCLEAN, P. DENNIS and J. N. STROMMER, 1993 Structure, expression, chromosomal location and product of the gene encoding ADH2 in Petunia. Genetics

GUTHEIL, W. G., B. HOLMQUIST and B. L. VALLEE, 1992 Purification, characterisation, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Eschachia coli: a class I11 alcohol dehydrogenase. Biochemistry 31: 475-481.

HAGEMANN, R. H., and D. FLESHER, 1960 The effect of anaerobic environment on the activity of alcohol dehydrogenase and other enzymes in corn seeds. Arch. Biochem. Biophys. 87: 203-209.

HOCHACHKA, P. W., and T. P. MOMMSEN, 1983 Protons and anaerobiosis. Science 219 1391-1397.

HOLMQUIST, B., and B. L. VALLEE, 1991 Human liver class I11 alcohol and glutathione dependent formaldehyde dehydrogenase are the same enzyme. Biochem. Biophys. Res. Commun. 178 1371-1377.

HOOG, J-O., H. EKLUND and H. JORNVALI., 1992 A single-residue exchange gives human recombinant pp alcohol dehydrogenase y y isozyme properties. Eur. J. Biochem. 205: 519-526.

HUR, M-W., and H. J. EDENBERG, 1992 Cloning and characterisation of the ADH5 gene encoding human alcohol dehydrogenase 5, formaldehyde dehydrogenase. Gene 121: 305-311.

HURLEY, T. D., W. F. BOSRON and C. L. STONE, 1994 Structures of human p alcohol dehydrogenase variants. Correlations with their functional differences. J. Mol. Biol. 239 415-429.

JACOBS, M., R. DOLFERUS and D. VAN DEN BOSSCHE, 1988 Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase mutants of Arabidqpsis thaliana (L.)Heynh. Biochem Genet. 26: 105-122.

KAISER, R., B. HOLMQUIST, J. HEMPEI., B. L. VALLEE and H. JORNVALL, 1988 Class I11 human liver alcohol dehydrogenase: a novel structural type equidistantly related to class I and class I1 enzymes. Biochemistry 27: 1132-1140.

KAISER, R., B. HOLMQUIST, B. L. VALLEE and H. JORNVALI., 1989 Char- acteristics of mammalian class I11 alcohol dehydrogenase, an enzyme less variable than the traditional liver enzyme of class I. Biochemistry 28: 8432-8438.

KEI.I.EY, P. M., 1989 Maize pyruvate decarboxylase mRNA is induced anaerobically. Plant Mol. Biol. 13: 213-222.

KIMURA, M. 1981 Estimation of evolutionary distances between ho- mologous nucleotide sequences. Proc. Natl. Acad. Sci. USA 78:

KOIWJSALO, M., M. BAUMANN, and L. UOTILA, 1989 Evidence for the identity of glutathionedependent formaldehyde dehydrogenase and class 111 alcohol dehydrogenase. FEBS Lett. 257: 105-109.

LAEMMLI, U. K., 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

LISTER, C., and C. DEAN, 1993 Recombinant inbred lines for m a p ping RFLP and phenotypic markers in Arabidqpsis thaliana. The Plant Journal 4: 745-750.

LLEWELLYN, D. J.. E. J. FINNEGAN, J. G. ELLIS, E. S. DENNIS and W. J. PEACOCK, 1987 Structure and expression of an alcohol dehydrogenase 1 gene from Pisum sativum (cv. "Greenfast".). J. Mol. Biol.

LUQUE, T., S. ATRIAN, 0. DANIEI.SSON, H. JORNVALL and R. GONSALEZ- DUARTE, 1994 Structure of Drosvphila mlanogmter glutathione- dependent formaldehyde dehydrogenase/octanol dehydrogenase gene (class 111 alcohol dehydrogenase). Eur. J. Biochem.

MARTINEZ, M. C., H. ACHKOR, B. PERSSON, M. R. FERNANDEZ, J. S w .

133 999-1007.

454-458.

195: 115-123.

225: 985-993.

QAT et aL, 1996 Arabidopsis formaldehyde dehydrogenase. Mo- lecular properties of plant class I11 alcohol dehydrogenase provide further insights into the origins, structure and function of plant class P and liver class I alcohol dehydrogenase. Eur. J. Biochem. 241: 849-857.

MITCHELI., L. E., E. S. DENNIS and W. J. PEACOCK, 1989 Molecular

analysis of an alcohol dehydrogenase (Adh) gene from chromosome 1 of wheat. Genome 32: 349-358.

MURASHIGE, T., and F. SKOOG, 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15 473-497.

NEWMAN, T., F. J. DE BRUIJN, P. GREEN, K. KEEGSTRA and H. KENDE, 1994 Genes galore: a summary of methods for accessing results from largescale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 106: 1241-1255.

PERSON, B., J. S. ZIGLER and H. JORNVALL, 1994 A super-family of mediumchain dehydrogenases/reductases (MDR). Eur. J. Bio- chem. 226: 15-22.

ROGERS, J. H., 1989 How were introns inserted into nuclear genes? Trends Genet. 5: 213-216.

SACHS, M. M., M. FREELINC and M. OKIMOTO, 1980 The anaerobic proteins of maize. Cell 20 761-767.

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Clon- ing; A Laboratory Manual, Ed. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

SANDERMANN, H., 1992 Plant metabolism of xenobiotics. Trends Biochem. Sci. 17: 82-84.

SASNAUSKAS, K, R. JOMANTIENE, A. JANUSKA, E. LEBEDIENE, J. LEBEDYS et al., 1992 Cloning and analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cereuisiae. Gene 122: 207-211.

SCRUTTON, M. C., 1971 Assay of enzymes of COY metabolism. Meth- ods Microbiol. 6A: 512-513.

SHAFQAT J., E.-A. MUSTAFA, 0. DANIEISSON, B. PERSSON, X. PARES et al., 1996 Pea formaldehyde-active class 111 alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (class I and P). Proc. Natl. Acad. Sci. USA 93: 5595-5599.

TAYLOR, B., and A. POWELL, 1982 Isolation of plant DNA and RNA. BRL Focus 4: 4-6.

TRICK, M., E. S. DENNIS, K J. R. EDWARDS and W. J. PEACOCK, 1988 Molecular analysis of the alcohol dehydrogenase gene family of barley. Plant Mol. Biol. 11: 147-160

UOTILA, L., and M. KOIWSALO, 1979 Purification of formaldehyde and formate dehydrogenase from pea seeds by affinity chroma- tography, and identification of Sformylglutathione as the inter- mediate of formaldehyde metabolism. Arch. Biochem. Biophys. 196: 33-45.

UOTILA, L., and M. KOIWSALO, 1989 Glutathione: chemical, bic- chemical, and medical aspects, part A, pp. 517-551, in Coazyms and Cofactors, Vol. 3, edited by D. DOLPHIN, R. POUI.SON and 0. AVRAMOVIC. John Wiley & Sons, New York.

VON BAHR-LINDSTROM, H., H. JORNVALL and J-0. HOOG, 1991 Clon- ing and characterisation of the human ADH4 gene. Gene 103:

WEHNER, E. P., E. RAO and M. BRENDEL, 1993 Molecular structure and genetic regulation of SFA, a gene responsible for resistance to formaldehyde in Saccharomyces cermisiae, and characterisation of its protein product. Mol. Gen. Genet. 237: 351-358.

WOLF, A. E., K-J. DIETZ and P. SCHRODER, 1996 Degradation of glutathione Sconjugates by a carboxypeptidase in the plant vacuole. FEBS Lett. 384: 31-34.

XIE, Y., and R. WU, 1989 Rice alcohol dehydrogenase genes: anaerobic induction, organ specific expression and characterisation of cDNA clones. Plant Mol. Biol. 13: 53-68.

XIE, Y., and R. WU, 1990 Molecular analysis of an alcohol dehydrogenase-encoding genomic clone (AdhP) from rice. Gene 87: 185-191.

YANNISCH-PERRON, C., J. VIEIRA and J. MESSING, 1985 Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33: 103-119.

YOKOYAMA, s., R. YOKOYAMA, C. S. KINIAW and H. DE, 1990 Molecu- lar evolution of the zinc-containing long-chain alcohol dehydrogenase genes. Mol. Biol. Evol. 7: 143-154.

YOSHIDA, A., L. C. Hsu and M. YASUNAMI, 1991 Genetics of human alcohol-metabolising enzymes, pp. 255-287, in Progress in Nucleic Acids Research, Vol. 40, edited by E. C. WAI.DO, and K MOI.DAVE. Academic Press, New York.

269-274.

Communicating editor: V. SUNDARESAN

the arabidopsis and rice formaldehyde dehydrogenase genes

Documents