Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

Planta (1996)198:64-69 P l a n t ~

�9 Springer-Verlag 1996

Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family Maria Santos 1, Helene Gousseau 2' *, Clare Lister 1, Christine Foyer 2" **, Gary Creissen 1, Philip Mullineaux 1

1 John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK Laboratoire de Metabolisme, INRA, Centre de Recherches de Versailles, F-78026, Versailles, France

Received: 9 March 1995/Accepted: 12 April 1995

Abstract. A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated A P X l b ) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al., 1993, FEBS Lett 315: 313-317; here designated A P X l a ) , which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only A P X l a is expressed. A P X l b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a eDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.

Key words: Arabidopsis - Ascorbate peroxidase (cytosolic, plastidial) - Multigene family - Oxidative stress - Pisum

Introduction

Like all aerobic organisms, plant cells are equipped with protective systems consisting of low-molecular-weight

Accession numbers: The APXlb sequence is in the EMBL database under accession number X80036 Present addresses: * M.I.R., University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada ** Institute of Grassland and Environmental Research, Plas Goger- ddan, Aberystwyth, Dyfed SY23 3EB, UK Abbreviations: ATG = methionine translation initiation codon; bp = base pair; cAPX = cytosolic ascorbate peroxidase; pAPX = plastidial ascorbate peroxidase; RFLP = restriction fragment length polymorphism Correspondence to: P. Mullineaux; Fax: 44 (1603) 456844; Tel.: 44 (1603) 452571

antioxidants and protective enzymes, such as superoxide dismutases, catalases, and peroxidases, which have a com- bined action to detoxify oxyradicals (Bowler et al. 1992).

Ascorbate peroxidase (APX, EC 1.11.1.11) is a hydrogen-peroxide-scavenging enzyme found in higher plants, algae and some cyanobacteria (Creissen et al. 1994), which catalyses the reaction:

2 ascorbate + H 2 0 2 ~ 2 monodehydroascorbate + 2H20

Ascorbate peroxidases are functionally and structurally distinct from guaiacol peroxidases (the major family of plant peroxidases) and APXs are unique in having a pref- erence for ascorbate as the reductant (Chen and Asada 1989; Creissen et al. 1994). In higher plants, APX activity is localised both in the chloroplasts (pAPX) and in the cytosol (cAPX) (Chen and Asada 1989; Sch6ner and Krause 1990; Tanaka et al. 1991). The plastidial activity may be further divided into stromal and thylakoid-asso- ciated fractions (Miyake and Asada 1992; Miyake et al. 1993).

The importance of APX in protecting cells from the deleterious effects of excess hydrogen peroxide has been shown by the increase in APX activity in response to several stresses (Tanaka et al. 1985; Gillham and Dodge 1987; Smirnoff and Colomb6 1988; Sch6ner and Krause 1990); however, the mechanism by which APX activity is induced is not known. Recent experiments have shown that following exposure to stress, APX activity is not elevated to the same extent as its mRNA transcript. In contrast to the increases in transcript levels and APX activity, immunodetectable APX levels did not change in response to the applied stresses (Mittler and Zilinskas 1994). Measurements of the transcription rate of pea cAPX by a nuclear run-on assay showed only a slight increase in response to drought stress (Mittler and Zilin- skas 1994), suggesting that the accumulation of APX mRNA is post-transcriptionally regulated.

Plastidial and cytosolic APXs share some molecular properties but they differ from each other in several characteristics. Compared to the cytosolic form, pAPX activity is rapidly inactivated in ascorbate-depleted medium and is

M. Santos et al.: Arabidopsis cytosolic ascorbate peroxidase genes 65

more sensitive to thiols and to suicide inhibitors (Nakano and Asada 1981; Chen and Asada 1989). The relative ease of purifying c A P X has led to the p roduc t ion of ant i -cAPX antisera, which have been used to identify APX c D N A s f rom several plant species, including pea (Mittler and Zilinskas 1991; 1992a), Arabidopsis (Kubo et al. 1992), soybean ( E M B L accession number L10292), spinach ( E M B L accession number L20864) and radish (EMBL accession number X78452). Compar i sons of their deduced amino-acid sequences have revealed that the cAPXs are a closely related group of proteins. To date, no report of the cloning of a p A P X e D N A has appeared. The complete gene sequence for cAPX is known for pea (Mittler and Zilinskas 1992b) and Arabidopsis (Kubo et al. 1993), and evidence has been presented that there is a single copy of the cAPX gene in both genomes.

In this paper we report our a t tempts to clone p A P X c D N A s by immunoscreening a pea e D N A library and then by low-stringency probing of an Arabidopsis genomic l ibrary with a cAPX cDNA. While we did not succeed in recovering p A P X c D N A s by either strategy we report in this paper the discovery of a second cAPX gene in Ara- bidopsis, establishing that cAPX in this species is encoded by a small multigene family.

Materials and methods

cDNA libraries, plasmids, enzymes vectors. The cDNA library prepared from pea leaf poly(A)+RNA has been described previously (Creissen et al. 1992). cDNA and genomic DNA fragments from bacteriophage vectors were subcloned into pBluescript KS + (Stratagene, La Jolla, Calif., USA; Alting-Mees and Short 1989) using standard in-vitro recombination procedures (Sambrook et al. 1989). Restriction enzymes and DNA-modifying enzymes were pur- chased from Boehringer Mannheim (Lewes, UK).

(1 x SSC is 15 mM trisodium citrate, 150 mM NaC1) at 65 ~ Filters were autoradiographed to visualise positive plaques. DNA was prepared from purified recombinant bacteriophage using the Magic Lambda Preps DNA Purification System (Promega).

Sequencing. Inserts in recombinant bacteriophage 9~ were subcloned into pBluescript SKII + and nested unidirectional deletions were made using exonuclease III digestion (Erase-a-Base system; Promega). The deleted inserts were sequenced using an Automated Laser Fluorescent (A.L.F. TM) DNA Sequencer (Pharmacia LKB, Sweden) employing a fluorescein-15-dATP labelling procedure (Voss et al. 1992). The sequence was completed using gene-specific oligonucleotide primers synthesised by the solid-phase triester method on a GeneAssembler Plus (Pharmacia LKB). Plasmid DNA for sequencing was prepared using the Magic Miniprep kit (Promega) according to the manufacturer's instructions. The sequence data were assembled using the assembly programs of Staden (1984). Further analysis of the completed sequence was carried out using programs available in the Wisconsin GCG package (Devereux et al. 1984).

Mapping. The analysis of genomic blotting, identification of a restriction fragment length polymorphism (RFLP) and the use of an RFLP marker to place a sequence on the Arabidopsis linkage map are described by Lister and Dean (1993).

Detection of APX RNA in different organs of Arabidopsis. The method of Woolston et al. (1988) was used to extract RNA from different organs of Arabidopsis (see Results and discussion). Ascor- bate peroxidase RNA was detected using the 3'RACE (rapid amplification of cDNA ends) PCR (polymerase chain reaction) procedure of Frohmann et al. (1988). The primers specific for APXla and APXlb had the sequences 5' CCCTGGAAGAGAGGACAAG- CCCC 3' (Kubo et al. 1993) and 5' TCCTGGTAGACTGGACA- AAGTTG 3' (Fig. 3). The correct PCR products were confirmed by Southern blotting (Sambrook et al. 1989) and probing with the pea cAPX eDNA from pAPX10.1 (see Results and discussion).

Results and discussion

Western blotting and immunoscreening of cDNA libraries. Western blotting of cell-free extracts and extracts from chloroplasts of pea have been described previously (Edwards et al. 1990). Screening of a pea cDNA library with an anti-pAPX antiserum was carried out as described previously for the recovery of glutathione reductase cDNAs (Creissen et al. 1992).

Construction and screening of an Arabidopsis genomic library. The general method described by Schneeberger et al. (1989) was used with the following modifications:

Total DNA was isolated from three-week-old Arabidopsis (eco- type Landsberg erecta), grown under conditions described by Lister and Dean (1993), according to the method of Woolston et al. (1988). The DNA was then further purified by caesium chloride/ethidium bromide isopycnic gradient centrifugation at 140000-g for 12 h at 15 ~ (Sambrook et al. 1989). The purified genomic DNA (125 gg) was then partially digested with Sau3A (0.02 U/gg DNA), size-fractionated on a continuous gradient of 1.2-5 M NaC1 in 10 mM Tris-HC1 (pH 8), 10 mM EDTA subjected to centrifugation at 36000.g for 3.5 h. Fractions of the gradient were retained which contained DNA fragments of 15-23 kb in size. Aliquots (10 1 gg) of the fractionated DNA were ligated into ~.GEMTM-12 XhoI half-site arms (Promega, Wisconsin) and packaged using the Packagene in-vitro Packaging System (Promega), as directed by the manufacturer. The library was screened and recombinant bacteriophage subsequently purified by successive rounds of transferring plaques to nitrocellulose filters and hybridising with a a2polabelled pea cAPX cDNA probe (see Results and discussion). The DNA was labelled according to the method of Feinberg and Vogelstein (1983). After hybridisation, the filters were washed for 30 min (x 4) at 2 x SSC

Immunoscreening o f a pea cDNA library with an anti- p A P X antibody. We set out to clone c D N A s for pAPXs by immunoscreening with an ant iserum raised against the plastidial (stromal) isoenzyme f rom tea (Chen and Asada 1989). In order to confirm that the tea p A P X antiserum cross-reacted with the pea homologue , the p A P X antiserum was used to detect APXs in Western blots of total and plastidial extracts prepared from three-week-old pea leaves (Edwards et al. 1990). The chloroplast fraction was confirmed to be free of contaminants by assaying for enzymes specific for cytosol and mi tochondr ia (Edwards et al. 1990). We compared the anti-tea A P X serum with a known c A P X antiserum from soybean root nodules (Dal ton et al. 1993). The antisera were generous gifts f rom Dr. D. Dal ton (University of Oregon; soybean) and Prof. K. Asada (Kyoto University; tea). The c A P X antiserum did not react with the plastid fraction, but a 30-kDa protein was detected in total leaf extracts (Fig. 1). The tea p A P X antiserum recognized two chloroplast-specific proteins with approximate sizes of 60 k D a and 33 kDa. The 60-kDa band was always distorted by the co-migrat ing large subunit of r ibulose-l ,5-bisphosphate carboxylase- oxygenase (Rubisco) present in large amoun t s in the protein extracts. The failure of the cAPX antiserum to detect any polypeptide in the plastid fraction was consistent with previous data (Mittler and Zilinskas 1991). However, our

66

data showing that the tea pAPX antiserum could detect only chloroplast-specific APX are in contrast to a previous report using this antiserum (Chen and Asada 1989).

Having determined the specificity of the tea pAPX antibody, we used this antiserum to at tempt to recover pAPX cDNAs by screening a pea leaf k -g t l l cDNA library (Creissen et al. 1992). Nineteen recombinant 'phage were purified to homogeneity and classified into six groups based on cross-hybridization experiments. One of the groups was found to be related to APX. The largest cDNA clone (pAPX10.1) had an insert of 1.1 kb. It was partially sequenced and found to be identical to the pub- lished pea cAPX (Mittler and Zilinskas 1992a) except for the presence of an additional three bases at the 5' end and for a conservative substitution in position 161 (A-C). The remaining groups did not encode any polypeptide exhibi- ting motifs characteristic of peroxidases and were prob- ably artefacts of the immunoscreening procedure (data not shown). In the light of the Western blot data (Fig. 1), it is surprising that, using a pAPX antiserum, we recovered only cAPX clones from the cDNA library. Interestingly, none of the cAPX cDNA clones we recovered were fusions to the lacZ gene in )~gt-ll. This is in accordance with Mittler and Zilinskas (1992a) who reported the same phe- nomenon and suggested that the cAPX polypeptide was expressed in E. coli from its own methionine translation initiation codon (ATG). The failure to clone a pAPX may be because there is no fortuitous expression of this cDNA from its own ATG.

Screenin 9 of an Arabidopsis genomic library using low- stringency conditions. In the light of this apparent failure to clone a pAPX cDNA we reasoned that, using low- stringency probing conditions, we would be able to identify pAPX genes directly by using the pea cAPX cDNA, pAPX 10.1, as a probe against Southern blots of Arabidop- sis DNA. This would be particularly useful in the case of Arabidopsis whose small genome size allows the detection

M. Santos et al.: Arabidopsis cytosolic ascorbate peroxidase genes

of weakly hybridising sequences. In fact, Arabidopsis genomic DNA contained weakly hybridizing bands, which were not detected using high-stringency washing conditions. Since Kubo et al. (1992) reported that cAPX was a single-copy gene in Arabidopsis we interpreted the weakly hybridizing bands as possibly representing pAPX genes (data not shown). Therefore, we constructed a genomic library in )~GEM-12 (see Materials and methods). The library (6 x 104 recombinants) was screened using the pea pAPX10.1 cDNA insert as a probe using low-stringency washing conditions (see Materials and methods). Five clones were purified to homogeneity. Re- striction-endonuclease mapping showed that these clones fell into two classes. Class I represented the cytosolic gene previously cloned (Kubo et al. 1993). Class II clones had a different restriction pattern and hence were assumed to represent a different gene.

Sequencing and analysis of a second A P X gene. The largest Class II cDNA clone was subcloned, as two EcoRI fragments, into pBluescript KSII + and sequenced, according to the strategy presented in Fig. 2 and Materials and methods.

In order to locate the APX gene in the complete sequence, an alignment was carried out using the pea and the Arabidopsis cDNAs (data not shown). The gene sequence is presented in Fig. 3, as an EcoRV-SalI fragment. The nucleotide sequence in the flanking regions of the putative methionine translation initiation codon ATG (AAAAATGGT, coordinate 0 in Fig. 3) is in close agree- ment with the plant consensus sequence for initiation of translation (AACAATGGC) (Joshi 1987). The absence of another potential start codon further upstream of this ATG ruled out the presence of a transit peptide sequence, strongly suggesting that this Class II gene codes for a further form of cAPX in Arabidopsis. Consequently we designated Class I and Class II as A P X l a and APXlb , respectively.

Fig. 1A, B. Western blots of 100 ~tg each of total pea leaf protein (lanes 2, 5) and total chloroplast protein (lanes 3, 6), probed with antisera raised against tea pAPX (A) and soybean cAPX (B), after separation in 11% (w/v) SDS-polyacrylamide gels and electroblot- ting to nitrocellulose. Western blotting procedures and antiserum concentrations are as previously described (Edwards et al. 1990). Pre-stained protein molecular-weight markers (BioRad) are in lanes 1 and 4 with their sizes shown to the left of each panel

,.11-'9- EeoRV EeoRI Sail I [ I I l k b I

H~ H~ tl~ Ms~l l 113 EcoiRV H3 M 1 E~oRV _ n I - - I - - l - - I - - I - - l - - I I ~'SalI I I

R F L P I SO0 bp I

Fig. 2. Location of APXlb on an EcoRV-SaII fragment in the )~-genomic clone and the sequencing strategy employed. The APXlb gene was located in the genomic clone by Southern blotting and restriction mapping and is shown as a solid rectangle. The arrows above the rectangle indicate the direction and extent of sequencing. Below the map of the genomic clone is depicted the structure of the gene in relation to key restriction sites used in this study. Solid rectangles indicate the position of exons. The HindlII (H3) fragment marked RFLP was used as a probe to map the APXlb locus (Fig. 4)


-772 GATATC~GT TGAATTATGA C~GTTTCAC TTTTTTACAT CTCCATTTCT TACTTCGCCT TTATCAGTTT -702 CATTTATTTA TeTCTTGCTC CTTCGTTTCC TATGATGATG CTTGTTATGT TATG~CCTA CTTAAAAACA -632 AGeTG~GCT TTCTTTGGTT TACCCCAGCA GAAACATT~ TC~GTTC~ TTGATC~TA TGTC~TTGA -562 TTTACTGTTT AAA~TAG TGTGTCTATC~CCeTT~G TCTTTTTGGT GGATAGTG~ GCTCAAACCC -492 ~TTCAAATG TCGATC~CC CT~eGTTTT TGTGCTCGAT AGTAAA~AT GAGTATAAAA AG~CGTC~ -422 TTCTTCCTAC ~TCACACAG ATCATTTG~ GCTTTGATTA GTTGGTAC~ CATACTCGAG GAATACAG~ -352 TGACACGTGG TGTGTATCTG TTGGATAAAT TTGTATTATT CTTCTTTACT ATGGATGGTT TCTGG~GGC -282 ~CAAGAG TCCTTCTTTT TCCATCTACA ~TCATGACC GTCCATCTCC AAAAG~GAC TGAAACAGTG -212AAACTAG~G CTTCATTTTG ATCATCAGCC AGTTTTAT~ A~GATCCGT ATACTAAAGT ~C~CTACT -142 AGCACTTTCT TCTGT~TCG ATCATTGTGT TTAAACTAAA ~TTTGTATG TTCTTGT~T CTTATT~TC -72 TCGATAGGTT CTCCATTCTC TTTA~TTTT AAA~CTTCA AC~TTTCCA G ~ G ~TTTGAAAA -2AAAT~TG~ G~GAGTTAC CC~GTGA ~G~GAGTA C~GAAAGCT GTTCAGAGAT ~ G A ~

~ " V K K S Y P E V K E E Y K K A V Q R C K R K 68 GCTCCGTGGT CTTATTGCCG AG~ACTG T~TCCCATC GTCCTCCGTC TTGegtaaga tcctctctct

L R G L I A E K H C A P I V L R L A 138 ggttccagtc ttttctgtta ctgtgagtac tga~tgttc gattgatggc ttctcagagc atattttgtt 208 ggcggaaatt tgcagAT~C ACTC~CT~ GACATTTGAT GTGAAGACGA A~AGG ACCGTTTGGG

W H S A G T F D V K T K T G G P F G 278 ACGAT~GGC ATCCCC~GA GCTA~CCAT GATGCC~CA AT~TCTTGA TATTGCCGTT AGGCTTCTTG

T I R H P Q E L A H D A N N G L D I A V R L L D 348 ACCCTATC~ G~GCTGTTC CCTATTCTGT CATAT~TGA CTTCTACCAG gtaaattttc aacaat~ac

P I K E L F P I L S Y A D F Y Q 418 tgagtttttt gacctctaat t~gaagcaa gggatgcatc aagctgta~ aattaggaac ttgaggtaga 488 accatagaat caagggtttt ttgatgcatc a~gattata ccgagagggg aaacatttgg aagggatttt 558 ~tggttggt acagctaagt tatgacttaa aatgg~tct tctctagctt gagcc~cgg ctattggccc 628 tctggtttgt ctgcagtgaa ctagattcac acatgttaga ttgatactga atctaaaagc ttactttgta 698 tttcgcatgg ctttaagggc ttgattcatc t~ttctt g~tact~a gCTAGCT~A GTTGTT~TG

L A G V V A V 768 TTGAGATCAC TGGAG~CCA GAGATTCCAT TTCATCCTGG TAGACTGgtg a~caatatt caacttctca

E I T G G P E I P F H P G R L 838 ~tttctggc taccaattat tagctatctc ttc~tttaa aacgcaactc actgatgatg agacaccact 908 gctaagtttt ttgttcatgt gattaactga accaaacttc atctttcagG ACAAAGTTGA ~CACCTCCT

D K V E P P P 978 G~GGTCGTC TGCCTCAGGC CACCAAAGgt aaagcagata tatgaaaatg aatttagctt gaattcag~

E G R L P Q A T K G 1048 atcagattgt agatacatgt tcataatcat aaattcagGT GTGGATCATC T~GA~TGT GTTTGGTCGG

V D H L R D V F G R 1118 AT~GACTCA ATGACAAAGA TATCGTTGCA TTGTCTGGTG GACACACCTT G~atgtatg ctgcatccag

M G L N D K D I V A L S G G H T L 1188 aaacaaagac ctatgtgctt ttctca~tc tggctcacac cattctttta ctttcagGGT CGGT~CACA

G R C H K 1258 AGGA~GTTC A~ATTCGAG GGTGCATGGA CACCAAACCC GCTCATTTTT GAC~CTCCT ATTTC~gta

E R S G F E G A W T P N P L I F D N S Y F K 1328 agatcagatt ctctgcttct gcttgctcat acttgaattg gacatgagct gaattagaag ctcttgaatc 1398 ttgatg~tg ttaaacagAG AGATACT~G CGGAGAGAAA G~GGACTTC TTC~CTACC ~CC~C~G

E I L S G E K E G L L Q L P T D K 1468 GCTCTTCTTG ATGATCCTCT CTTTCTCCCA TTTGTTGAAA ~TATGCT~ ~taagttgt tcaactcatc

A L L D D P L F L P F V E K Y A A 1538 atttcatgat tctctcagca tacatatatt gtcaatagga taatatcact gactggattg ttactctcag 1608 GATGAGGATG CCTCTTTCGA AGACTATACG G~GCTCATC TAAAGTTGTC AG~CTC~g taagtaacat

D E D A S F E D Y T E A H L K L S E L G 1678 atagaacaat cattctcatc aacattttat ttaactttct ttcctattct cctaatgtag tgtcgtaaac 1748 tttgaatg~ cctcagaaac tcttttcatt tgcaaacact ttgatcataa taacataact tacgctcatg 1818 tcatcatgtc ctttgcagGT TTGCTGAC~ GGAGTAATCT GG~TCTGGT GTCATCTCCT ACCTCCTTGA

F A D K E 1888 TTCTTGTTCC CTCACATTAT TATTGT~TC ATAGAAAATA CTTTAAAAAT TAAAT~G ACATTGTATT 1958 ATACGTAT~ CTTGCGTTTT TTGT~TTGTT~TT~TGA ~T~GAGCACTT~TT~TGAAACTAAAA 2028 ~CTCTTACA AC~TTCATT ACCTTCATAT AGTTTG~GG CTAGT~TCT CAC~TGATA ~CTATATTG 2098 AAAGTTGTGT TATCGTTG~ AAATTAAAAT ~TTTTTATT TCTGTATCTT C~TGAT~T AAACAAAT~ 2168 ~GCTTCAAA G~TATTTTT GTAAATTT~ AAAACG~TG ATATTATTGT CTAC~TTTC TTCAAATCAT 2238 CATTAT~TT GTTTTGCTCT TTTGAAAAAA TAAAATGGAG ATATT~TTT GCTTG~CTC AAAACGAC~ 2308 CGTAGCAC~ CTCCTTTCCA CTCG~T~T CCATTTGGAG GCTTTTTTTC ATTACAG~C ACACACTCTC 2378 TCTCC~T~ ACCTAAAATG ~CATTCTCC GACCTeCGAC GTCATCATCA TCTTCGTCGT TTCCTCCATA 2448 CCCAAAGCCC GTTTCATT~ CCCCTCCGGT ATCTTTCACT CTCATCCACA ACCCCATAAA CCTCTGCTCT 2518 ATAAACCCAC CATTCACC~ CGCTGGTC~ C

67

Fig. 3. Complete sequence of the EcoRV-SalI fragment shown in Fig. 2. Coordinates are numbered from the first base of the putative methione translation initiator codon (ATG) which was identified by aligmnent with the sequences of known cytosolic APX polypeptides (Table 1). The nearest consensus TATA box to the coordinate ATG and putative polyadenylation sequences are underlined. Putative exon and intron sequences are shown in upper and lower case, respectively

Using the 5'- and 3'-splice site consensus sequences (Senapathy et al. 1990) it was possible to deduce a spliced m R N A sequence (Fig. 2). Putative introns were found to contain a high A + T content (average of 63%) which is typical of dicotyledonous species (Chen and Asada 1989). A possible polyadenylation signal (Joshi 1987) was found 269 bases downstream of the stop codon and a potential TATA box was located 176 bases upstream of the ATG and 42 bases upstream of the predicted transcription start (Joshi 1987). Such a location would give the A P X l b m R N A a 122-base leader sequence, with a 68% A + T content. Two of the potential regulatory elements described for the cAPX gene in pea, a G-box core sequence (CACGTG) and the palindromic element of heat-shock- protein gene promoters (TTCNNGAA) (Mittler and Zilinskas 1992b) were found at positions - 3 4 9 and

- 293, respectively. Unlike the other cAPX genes (Kubo et al. 1993; Mittler and Zilinskas 1992b) we could find no sequences typical of an intron donor/acceptor consensus in the 5' untranslated region.

The derived amino-acid sequence of A P X l b was compared with all the known cAPXs by searching the EMBL database (Table 1). The obtained high homology levels were a further indication that A P X l b is a typical cAPX. It should be noted that the cAPX encoded by A P X l b is no more homologous to that encoded by A P X l a than to those found in other plant species. Interestingly, an un- known carrot c D N A was identified as a cAPX (EMBL accession number Z17398).

A computer comparison of the structure of the polypeptides encoded by A P X l a and A P X l b revealed no significant differences between the two predicted proteins

68

Table 1. Similarities and identities between APX deduced protein sequences. Pairwise similarities and identities are shown below and above the diagonal, respectively. References or database accession numbers appear in the text


APXla APXlb Pea Soybean Spinach Radish Carrot

APXla 77.2 78.4 78.8 83.2 91.2 79.6 APXlb 88.4 - 78.8 79.2 77.2 77.2 79.2 Pea 90.0 87.6 90.8 84.8 79.6 82.8 Soybean 89.2 88.0 95.6 84.8 79.6 81.2 Spinach 90.4 86.8 90.8 91.2 - 85.2 80.8 Radish 94.8 88.0 89.6 90.0 92.0 - 78.4 Carrot 88.4 87.6 88.8 88.4 88.0 88.0 -

(data not shown). At the nucleotide level, while exons shared 72% homology, the non-coding regions of the two genes revealed no significant homologies.

Mappin9 of two c A P X loci. The above data strongly indicated that there are at least two cAPX genes in the Arabidopsis genome. We therefore investigated if the two genes were from two separate loci. Restriction fragment length polymorphisms (RFLPs) were obtained for A P X l a and A P X l b (Fig. 4) by digesting genomic DNA from Arabidopsis ecotypes Columbia and Landesberg erecta with DraI and SacI, respectively and hybridizing with the probes indicated in Figs. 2 and 4. These polymorphisms were used to map both genes onto the R F L P linkage map of the Arabidopsis nuclear genome (Lister and Dean 1993). The A P X l b gene maps on linkage group 3 at 3.1 cM from the marker m583, while A P X l a maps on linkage group 1 to one of two possible positions either side of the NCC1 marker. With the number of recombinant inbred (RI) lines scored for the NCC1 marker (25; Lister and Dean 1993) it was not possible to determine the precise location of A P X l a , but it maps within 1 cM of NCC1.

Until now cAPX was considered to be the product of a single gene; however, we now provide unequivocal evidence that cAPX in Arabidopsis is encoded by a small gene family. This contrasts with previous observations in this species (Kubo et al. 1993) and in pea (Mittler and Zilin- skas 1992b), although the conditions used to identify the cAPX genes in the latter cases may not have been optimal for detection of a second gene. The wide range of habitats exploited by Arabidopsis in Europe, Asia and Africa (Anderson and Mulligan 1992) might explain the re- quirement and presence of multiple genes coding for cAPX in a small genome with almost no repetitive D N A (Meyerowitz 1992).

Screening for expression of A P X l a and APXlb under non- stress conditions. In order to check if the two genes were differentially transcribed, 3 'RACE-PCR was performed using primers specifically designed for detecting A P X l a and A P X l b transcripts (see Materials and methods). Un- der the conditions used the A P X l a transcript was identified in all the organs analyzed (leaf, root, stem + flower) but no A P X l b transcripts could be detected. However, since A P X l b had all the characteristics of an active gene (TATA box, transcription-start consensus sequence, some sequence-specific binding domains), and the gene immedi- ately adjacent, a r ibosomal protein gene, was active (data not shown), we propose that it is only expressed either in particular tissues or under particular environmental

Fig. 4A, B. Restriction fragment length polymorphisms (RFLPs) for APX1 genes. Southern blots of genomic DNA (5 lag) of Arabidopsis ecotypes Columbia (C) and Landsberg erecta (L) cut with SacI or DraI and probed with sequences specific for APXla (A; DraI) or APXlb (B; SacI). The probe for APXlb is shown in Fig. 2. The probe for APXla RFLP analysis was an ApaI-BglII fragment (coordinates 1-202; Kubo et al. 1993)

conditions. However, an initial screening with Paraquat did not reveal any induction of expression, suggesting that the enzyme was not induced by H202 produced by chloroplast reactions (data not shown). In contrast, an A P X l b promoter- [3-glucuronidase (GUS) gene fusion was active in the leaves of plants bombarded using biolis- tic techniques, suggesting that the promoter of A P X l b is functional (data not shown). Although it cannot be ex- cluded that the sequence reported here may be a pseudogene copy of a functional APX gene, we consider this gene to be active since none of the reported features of a typical pseudogene (in-frame termination codons, absence of obvious acceptor/donor consensus sequences for introns, unusual codon usage; (Vanin 1985) could be detected.


All our at tempts to isolate plastidial APX genes failed. To date these genes have not been isolated, despite inten- sive studies by several groups including our own. Never- theless, the notable absence of pAPX genes (encoding the soluble or m e m b r a n e - b o u n d enzymes) from the databases must p rompt a re-appraisal of our unders tand- ing of the relat ionships between the APX forms at a molecular level.

M.S. gratefully acknowledges the support from the Junta Nacional de Investigaq~to Cientifica e Tecnolbgia, Portugal (grant number BD/394/90-IE). This work was supported by the Biotechnological and Biological Sciences Research Council through a grant-in-aid to the John Innes Centre.

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Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

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