THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 267, No. 30, Issue ofOctober 25,pp. 21802-21807,1992
Printed in U.S.A.
0 1992 by The AmericanSociety for Biochemistryand Molecular Biology, Inc
Molecular Cloning and Characterizationof a Gene EncodingPea
Cytosolic AscorbatePeroxidase*
(Received for publication, May 29, 1992)
Ron Mittler and Barbara A. ZilinskasS
From the DeDartment of Biochemistry and Microbiology,
-_ Cook College, Rutgers University,
New Brunswick, New Jersey 08903-0231
A geneencodingcytosolicascorbateperoxidase
(ApxI) from pea (Pisum sativum L.) was isolated and
against the purified enzyme were used to isolate a cDNA clone
encoding the pea cytosolic ascorbate peroxidase (5). Cytosolic
its nucleotidesequencedetermined.Byhomologous
ascorbate peroxidase was found to share littleoverall homolalignment between the ApxI cDNA (Mittler, R., and ogy with classical plant peroxidases. Significant homology
Zilinskas, B. (1901) FEBS Lett. 289, 257-259) and was, however, found in thevicinity of the putative active site
the genomic clone, positions of introns and
exons were with all known peroxidases (5). Although several studies have
determined. The isolatedApxI gene was found to con- demonstrated an increase in ascorbate peroxidase activity in
tain 9 introns, the first of which was located within response to changes in growth conditions and environmental
the 6”untranslated region of mRNA.
the
Southern blot stresses (6-11), little is known about the molecular mechaanalysis ofpeagenomicDNA
suggeststhatinpea
cytosolic ascorbate peroxidase is encoded by a single nism underlying its response to these stresses.
The current report is the first describing the molecular
copy gene. Steady state ApxI transcript levels were
cloning
and characterization of a gene encoding ascorbate
found to increase in response to several stresses imposed by drought, heat, and application of ethephon, peroxidase (ApxP).As a first step toward the understanding
abscisic acid,and the superoxide-generating agent par-of the environmentally controlled expression of the ascorbate
aquat.Increasesinascorbateperoxidase
activity in peroxidase gene, we have also studied the combined changes
in ascorbate peroxidase transcript, protein, and activity levels
response to stresses were less marked than changes
observed in transcript levels; cytosolic ascorbate per- in response to several stresses including drought, heat, and
oxidase proteinlevels measured by immunoblot analy- the chemical stresses imposed by ethephon, abscisic acid
(ABA)? and the superoxide-generating agent paraquat. In
sis remained unchanged.
pea, ApxI was found to occur as a single copy gene which
responds to a relatively broad spectrum of environmental
stresses.
Ascorbate peroxidase (EC1.11.1.11)is a hydrogenperoxidescavenging enzyme, found in higher plants, algae, and some
EXPERIMENTALPROCEDURES
cyanobacteria (1).Functionally and structurally distinct from
Screening of the EMBW Pea Genomic Library-A genomic library,
the typical plant peroxidases superfamily, ascorbate peroxiconstructed in EMBL3 using pea (Pisum satiuum L., cv. Alaska)
dase is unique in having a decided preference toward ascorbate genomic inserts of 8-21 kb in size (Clontech), was used to infect the
as a reductant (2). Ascorbate peroxidase plays a key role in host strain Escherichia coli NM539. Approximately 5 X lo6 plaques
the removal of hydrogen peroxide in the chloroplasts and were screened. Plaques were transferredto nitrocellulose filters
cytosol of higher plants (3). The enzyme exists as two distinct (Schleicher & Schuell), and the DNA was denatured and neutralized
isozymes, chloroplastic and cytosolic, both of which catalyze according to Benton andDavis (12) and UV cross-linked. Filters were
prehybridized at 65 “C for 4 h in 6 X SSC, 0.1% SDS, 0.2 mg/ml
the following reaction.
bovine serum albumin, 0.2 mg/ml Ficoll, and 0.2 mg/ml denatured
calf thymus DNA. Following prehybridization, the filters were incubated with a 32P-labeled pea cytosolic ascorbate peroxidase cDNA
The chloroplastic ascorbate peroxidase isozyme was recently (specific activity of 5-8 X 10’ cpm per fig of DNA and total activity
purified to homogeneity from tea leaves and compared to the of 2-4 X lo6 cpm per ml of hybridization solution) for 16 h under the
same conditions. Random prime labeling of the cDNA insert was
partially purified cytosolic isozyme (2). The two isozymes performed with [w3’P]dCTP as the radiolabeled nucleotide using a
were found to differ in the following aspects: molecular weight, random prime labeling kit (U. S. Biochemical Corp.). The filters were
substrate specificity, pH optimum, and stability. We have washed at 65 “C in0.1 X SSC, 0.2% SDS andexposed to X-Omat AR
recently purified to homogeneity the cytosolic ascorbate per- film (Kodak). Putativepositive clones were rescreened at least twice
oxidase from pea shoots (4). Polyclonal antibodies raised at a lower density using the same procedure.
Southern Hybridization Analysis of Pea Genomic DNA and AscorPeroxidase Genomic Clones-Total genomic DNA isolated from
* This is New Jersey Agricultural Experiment Station Publication bate
pea leaves (13) and EMBLB DNA isolated from recombinant clones
D-01905-2-92, supported in part by state funds and by the United
States Hatch Act. This work is also supported by the Cooperative (14) were digested with BamHI, EcoRV, BamHI+EcoRV, HindIII,
State Research Service and United States Departmentof Agriculture, XbaI and EcoRI, electrophoresed on0.8% (w/v) agarose gels, blotted,
under Agreement 89-3471-4502.The costs of publication of this article
The nucleotide sequence of ApxZ cDNA is found in EMBL nuwere defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in accordance with cleotide sequence database under the accession number X62077. The
nucleotide sequence of the ApxZ is found in the GenBank database
18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be sent: Dept. of Biochemistry under the accession number M93051.
The abbreviations used are: ABA, abscisic acid; p-CMPSA, pand Microbiology, Rutgers University, Cook College, Lipman Hall,
New Brunswick, NJ 08903-0231. Tel.: 908-932-9563; Fax: 908-932- chloromercurisulfonic acid, HSE, heat-shock element; ARE, antiperoxidative element; kb, kilobase; bp, base pair.
8965.
2 ascorbate
+ H202+ 2 monodehydroascorbate + 2 HzO
2 1802
Pea Ascorbate Peroxidase Gene
and hybridized to a radiolabeled full-length cytosolic ascorbate peroxidase cDNA probe. Digested recombinant EMBL3 DNA was also
probed with two fragments of pea ascorbate peroxidase cDNA of 300
bp and 800 bp, corresponding to the 5'- and 3'- ends of the cDNA
clone. Probing duplicate blots of digested recombinant EMBL3 DNA
with the two probes that correspond to the 5'- and 3'-ends of the
cDNA clonerevealed a 5.5-kb BamHI fragment that exclusively
hybridized with both probes and therefore likely contained the entire
gene. Hybridization conditions were identical to those used for the
library screening (referred to as high stringency). In addition, pea
genomic DNA wasalso hybridized at 55 "C and washed with 1X SSC,
0.2% SDS at 55 "C (referred to as moderate stringency).
Isolation of a Genomic
Clone
Encoding ApxI-Recombinant
EMBL3 DNA was isolated and subsequently digested with BamHI
to release the ApxI gene. The restriction fragments were separated
by electrophoresis in 1%Seakem (FMC Corp.) agarose gel and eluted
and purified using Geneclean (BiolOl). The 5.5-kb fragment was
subcloned into pBluescript KS- (Stratagene), which had been linearized with BarnHI and used to transform E. coli XLlB (Stratagene).
DNA Sequence Analysis-Plasmid DNA wasisolated using a magic
miniprep kit (Promega). The ApxI gene, cloned into pBluescript, was
denatured and sequenced according to Sanger et al. (15). Sequencing
reactions were carried out with T-7 Polymerase (U. S. Biochemical
Corp.) with [CP~~SI~ATP
as the radiolabeled nucleotide. Synthetic
17-base primers, complementary to internal sites in the pea ascorbate
peroxidase cDNA (5) and theApxI genomic clone, were synthesized
on a Milligen Biosearch Cyclone instrument and used to prime the
sequencing reactions. Sequencing reactions were analyzed on a denaturing 6% (w/v) polyacrylamide gel. Both strands were sequenced
with no ambiguities.
Primer Extension-An antisense 31-nucleotide primer corresponding to nucleotides 326-356 of the genomic sequence (see Fig. 2) was
synthesized, end-labeled with [Y-~*P]ATP
using T4-polynucleotide
kinase (U.S. Biochemical Corp.; specific activity of 5 X lo5 cpm per
pg of DNA), and used to direct the synthesis of cDNA from total
RNA of 14-day-old leaves using reverse transcriptase (MMLV-RT,
Bethesda Research Laboratories) as described by Dean et al. (16).
The extended product was analyzed on a denaturing 6% (w/v) polyacrylamide gel.
Stress Studies-Pea (P. satiuurn L., cv. Progress 9) seeds were
planted in vermiculite and grown for 14 days in a greenhouse with a
mean air temperature of20 "C and a 14-h light period (provided by
sodium vapor lamps at anintensity of 300 pEm-'s-I). Young seedlings
were provided half-strength Hoagland's solution. Plants were drought
stressed by withholding water for 72 and 96 h (decrease in shoot
relative water content of approximately 10% and 20% respectively)
and compared to control plants of the same developmental stage.
Relative water content was defined as: lOO(actua1shoot weight - dry
weight)/(hydrated weight - dry weight). After the shoots were sampled and weighed (actual shoot weight), they were immersed for 24 h
in distilled water at 4 "C,blotted and weighed again (hydrated weight).
The shoots were then dried for 24 h in a 75 "C oven and weighed (dry
weight). Heat shock was imposed by elevating the temperature to
36 "C for 1 and 5 h under constant illumination (300 pEm-* s-').
Stressed plants were compared to control plants maintained at 20 "C
in the greenhouse and sampled at the same time as were the heatstressed plants. Chemical stresses were imposed by spraying plants
to run-off with paraquat, ABA, and ethephon,each prepared in 0.05%
Tween 20. The pH of the ethephon solution was adjusted to 5.5 with
KOH. Control plants were sprayed with 0.05% Tween 20. Paraquattreated plants were sampled following a 5-h incubation under light
(300 pEm-' s-I). Plants treated with ethephon and ABA weresampled
following a 24-h period under greenhouse conditions. In all cases the
second leafnodewas collected, from approximately 30 plants per
treatment, divided into two portions, flash-frozen in liquid nitrogen,
and stored at -80 "C. One portion served for the analysis of transcript
levels and the other for ascorbate peroxidase immunodetection and
activity measurements.
Northern and WesternBlots Analysis and Ascorbate Peroxidase
Activity Measurements-Total RNA was isolated according to (17)
and quantified spectrophotometrically. Thirty pg of total RNA was
electrophoresed on a 1.2% agarose gel containing 2.2 M formaldehyde,
transferred to a nylon membrane, and probed with a radiolabeled pea
ascorbate peroxidase cDNA insert using high stringency conditions
as described for plaque screening. Protein was extracted in 100 mM
sodium phosphate buffer (pH 7), 1 mM EDTA, and quantified according to Bradford (18).Forty pg of protein was subjected to SDSpolyacrylamide gel electrophoresis and Western blotting as
previously
21803
described (4). Polyclonal antibodies raised against the purified pea
cytosolic ascorbate peroxidase were used for immunodetection. These
antibodies do not cross-react with the pea chloroplastic ascorbate
peroxidase (4)and therefore reflect only changes in cytosolic ascorbate peroxidase abundance. Relative changes in transcript and protein levels analyzed via Northern and Western blots were quantified
using an Ultrascan XL laser densitometer (LKB, Sweden). Ascorbate
peroxidase activity was measured according to Chen and Asada (2)
and Mittler and Zilinskas (4). As previously described (4), the pea
chloroplastic ascorbate peroxidase isvery labile in the absence of
ascorbate; therefore, the extraction of soluble proteins in the absence
of ascorbate results in the immediate inactivation of the pea chloroplastic ascorbate peroxidase. Hence, the ascorbate peroxidase activity
reported herein reflects changes in only the nonchloroplastic ascorbate peroxidase activity. However, in measurements of pea total
ascorbate peroxidase activity, there is also found a heat-stable, nonenzymatic ascorbate peroxidase activity corresponding to approximately 15-20% of the total activity (4); therefore, in order to distinguish between the nonenzymatic activity, as well as residual nonspecific peroxidase activity (1) and
actual
ascorbate peroxidase
enzymatic activity, we have used the inhibitor p-chloromercurisulfonic acid (p-CMPSA) thatinhibits ascorbate peroxidase enzymatic
activity and has no effect on either guaiacol peroxidase activity (2,4)
or nonenzymatic ascorbate peroxidase activity (data not shown).
Protein extracts were therefore incubated with or without 0.5 mM pCMPSA for 10 min at room temperature prior to assaying for ascorbate peroxidase activity. Hence, ascorbate peroxidase activity measured followingextraction in an ascorbate-depleted medium is reported
both as total ascorbate peroxidase activity as well as the amount of
ascorbate peroxidase activity which is inhibited by p-CMPSA, i.e.
cytosolic ascorbate peroxidase.
RESULTS
Isolation of Pea Cytosolic Ascorbate Peroxidase Gene and
Nucleotide SequenceAnalysis-With a pea cytosolic ascorbate
peroxidase cDNA clone (5) as a probe, three genomic clones
were isolated from a pea EMBL3genomic library (Clontech).
The isolated genomic clones were characterized by restriction
mapping and Southern blot analysis. Two of the clones were
identical and contained the entire ApxI nuclear gene, while
the third clone contained a portion of the ApxI gene corresponding to the 3'-end of the gene. A 5.5-kb fragment that
contained the ApxI gene was subcloned into the plasmid
vector pBluescript and sequenced. The sequencing strategy is
shown in Fig. 1. Synthetic primers corresponding to internal
sites in pea cytosolic ascorbate peroxidase cDNA (5) andpea
ApxI gene (as they were determined) were used to prime the
sequencing reactions. The determined nucleotide sequence of
the isolated gene (Fig. 2) was compared with the ascorbate
peroxidase cDNA nucleotide sequence (5). The isolated 5.5kb genomic clone insert was found to contain a full-length,
2.5-kb ApxI gene in addition to 2 and 1 kb, respectively, of
5"upstream and 3'-downstream regions.
Ascorbate Peroxidase Gene Structure-By
homologous
alignment of the ascorbate peroxidase cDNA and gene sequence, positions of introns and exons were determined (Fig.
1). The isolated ApxI gene was found to contain 9 introns,
the first of which was located within the 5"untranslated
region of the mRNA. Introns were found to contain a high
percentage of A + T (average of 72%) and conform to the 5'and 3"splice site consensus sequence (Table I). In contrast,
exons had a lower A T percentage (average of 52%). Primer
extension analysis was performed to map the siteof transcription initiation. A 31-base primer, antisense to position 326356 (Fig. 2), was used to prime a reverse transcriptase reaction
using isolated total pea RNA as template (Fig. 3). Two distinct
products (which did not contain the first 200-bp intron) were
152 and 154 nucleotides in length; these products mapped the
initiation of transcription toa position which is 13 and15 bp
upstream from the 5'-end of the cDNA clone (Fig. 2). The
transcription initiation site which is the closest to the 5'-end
+
21804
Pea Ascorbate Peroxidase Gene
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FIG. 1. Restriction map, exon-intron pattern, and sequencing strategy of the pea cytosolic ascorbate peroxidase gene. Boxes
denote exons, hatched boxes indicate open reading frame, and open boxes represent 5'- and 3"noncoding transcribed regions. Arrows show
the direction and the distance of the nucleotide sequence obtained from each primer.
of the ApxZ gene is referred to as position 1. This initiation
site also conforms to the plant consensus for initiation of
transcription (19). A putative TATAbox was found 25-27 bp
upstream from the transcription start site, and a putative
CCAAT box was found 147 bp upstream from the TATA box.
Several potential regulatory elements were found between
positions -350 and -25; these include two repeats of a putative plant heat-shock element (HSE) (20) and a reversed
antiperoxidative element (ARE) (21) (Fig. 2). In addition, two
repeats of putative
a
xenobiotic responsive element
(CACGCA) (21) were also found in the following positions:
-282 and -302; a G-box core sequence (CACGTG) (22) was
identified at position -137, and a putative GPEI enhancer
(23) was found at position 97, located in the first intron. A
repeat of 11 bases (GTTTTTGCTTC) was found both in the
cDNA 5"untranslated region (position 56, Fig. 2) and in the
first intron (position 199, Fig. 2); however, the significance, if
any, of this sequence is unknown. The nucleotide sequence in
the vicinity of the pea ascorbate peroxidase initiation of
translation ( U G C U U G G ) does not conform to the plant
consensus sequence ( A A C A m G C ) (19); it is not known
whether this difference affects the efficiency of translation.
Deduced Amino AcidSequence and Homology Studies-Pea
cytosolic ascorbate peroxidase was found to share little overall
homology with classical plant peroxidases. However, considerable homology was found with yeast cytochrome c peroxidase (5). In addition, the amino acid sequence is conserved in
the vicinity of the putative active site of all known peroxidases
including ApxZ (5). The partial amino acid sequence of tea
chloroplastic ascorbate peroxidase was recently reported (24).
As shown in Fig. 4, pea ascorbate peroxidase was found to
share little homology with the chloroplastic isozyme from tea;
only 30% identity was found over 197 amino acids (only
homologous tea peptide fragments are shown in Fig. 4). In
contrast, pea ascorbate peroxidase is 40% identical over 250
amino acids with yeast cytochrome c peroxidase (Fig. 4). Note
that a recording error in nucleotides 538-542 and the corresponding deduced amino acids reported in Mittler and Zilinskas (5) has herein been corrected (Fig. 4). The nucleotide
sequence of a cDNA clone encoding ascorbate peroxidase from
Arabidopsis thaliana was also recently reported (25). When
compared with the previously described pea cytosolic ascorbate peroxidase cDNA sequence (5), theA . thaliana ascorbate
peroxidase-deduced amino acid sequence was found to be
highly homologous with that of pea cytosolic ascorbate peroxidase (79% identity over 250 amino acids, Fig. 4).
Pea Cytosolic Ascorbate Peroxidase Is a Single Copy GenePea genomic DNA was isolated and subjected to genomic
Southern blot analysis under high and moderate stringency
(65 "C, 0.1 X SSC, 0.2% SDS, and 55 "C, 1 X SSC, 0.2% SDS,
respectively). In both cases, the same pattern of fragments
hybridized to the pea cytosolic ascorbate peroxidase cDNA
probe (Fig. 5, data shown only for moderate stringency). The
sizeof the hybridizing fragments was consistent with the
restriction map of the obtained genomic clones and the nucleotide sequence data. There was no evidence of cross-hybridization with gene(s) encoding the chloroplastic ascorbate
peroxidase isozyme or with any other peroxidase genes. The
apparent absence of additional cross-hybridizing bands under
the moderate hybridization conditions utilized, which generally allows detection of other conserved members of a gene
family, suggests that cytosolic ascorbate peroxidase is encoded
by a single gene in pea.
Changes in Steady State Ascorbate Peroxidase Transcript
and Protein Levels and Ascorbate Peroxidase Activity in Response to Chemical and
Environmental Stresses-Examination of steady state ApxZ transcript levels and transcriptsize
was performed by Northern blot analysis. The ApxI transcript
was found to be approximately 1050 bases under all treatments and was observed in both roots and leaves of 14-dayold pea plants, aswell as inetiolated leaf tissue of 10-day-old
plants (data not shown). Transcript levels were also found to
increase in the early hours of the day and decrease by night
(data not shown); therefore, when performing the stress experiments,care was takento sample control andtreated
plants at the same time of day. Drought stress, as indicated
by a decrease in relative water content in pea shoot tissue of
approximately 10% and 20%, resulted in a 5- and a IO-fold
increase in steady state transcript levels and in a 1.5- and a
%fold increase, respectively, in ascorbate peroxidase activity
(Fig. 6). Heat-stressed plants (1h, 36 "C) had a 4-fold higher
steady state level of the ApxZ transcript. However, ascorbate
peroxidase activity increased only slightly following the 1-h
heat shock, and steady state ApxZ transcript levels nearly
returned to control levels after 5-h heat shock (Fig. 6). There
were no differences in either activity or transcript abundance
24 h after the5-h heat shock when compared to control plants
(data not shown). The difference between the heat-shock
controltime points (Fig. 6, IC and 5C) demonstrates the
increase in transcript levels between the early ( I C ) and the
later ( 5 C ) time-of-day sampling. As indicated in Fig. 6, paraquat application also resulted inan increase in ApxZ transcript
levels (%fold for both concentrations). However, ascorbate
peroxidase activity increased only following the application
of the lower concentration
M) and decreased following
M). Application
application of the higher concentration
Pea Ascorbate Peroxidase Gene
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G
L
D
I
TARIX1
Splice ,sites, Lngth. and pprcrntapA + T of introns in thr p
cytosolic ascorhatr peroxidawgrnc
Intron
Length
6 0 5 CTTTCGGAACAATTAAGCACCAAGCCGAGCTTGCTCATGCTGCTAACAACGGTC~GATA
A
V
R
L
L
E
P
I
K
E
Q
F
P
I
V
S
Y
A
D
F
6 6 5 TCGCGGTTAGGCTGTTGGAGCCTATTAAGGAGCAATTCCCCTATTGTGAGCTATGCTGATT
Y
Q
7 2 5 TCTACCAGqttqqtaatttttqtqtqtqtttaqtttttaqatttqaatttatqtqqttqt
L A G V
7 8 5 tcaatttttqtqatcatqtqqttqatqqtttattttaatacqtaacqcaqTTGGCTGGTG
V
A
V
E
I
T
G
G
P
E
V
P
F
H
P
G
R
E
8 4 5 TTGTTGCTGTTGACATTACCGGTGGACCTGAAGTTCCTTTCCACCCTGGTAGGGAGqtat
9 0 5 qtttqaccacaactatcqcttttqtcttcaaatctaatttacatqattaqtaaatcaatt
9 6 5 attqqqtatcacttttttcqqttatataatqattqqattcatqttqqtqqqtaccttttt
S'-Splice site
a
A +T
3'.Splice
site
hr,
GAG GTTCG
GGC GTAAG
CAG GTTGG
102
4
442
GAG GTATG
AGG GTCAG
5
87
6
266
ATT GTATG
91
CAC GTAAG
7
8
91
GCG GTAAG
TGC GTAGG
9
93
AG GTAAG
Plant consensus sequence "
1
200
2
3
149
TGTAG
TGTAG
CGCAG
ATCAG
TGCAG
CTTAG
TATAG
TACAG
TGCAG
TGCAG
A
A
T
G
G
G
T
G
A
G
69.0
73.2
70.6
71.9
72.4
69.5
74.7
75.8
73.1
1 0 2 5 ttttaaaqaataqtqtatqtttaatttttatatcatqttcqqacattaqtttqtaaqctt
1 0 8 5 tqatatttqtcactttttqqtqccttctqqttttcaaqaatttccattqqttacataatt
PE A C G T
"
1 1 4 5 qcqqtcaqaatcacaacaattaatctaatqtqatqqaataattqqaaatqcttttcttac
1 2 0 5 atqttttactaaaatqtatqtaaaqtqtqqttatattattttacacaqttqttqatqatq
1 2 6 5 tattctttatctttttttactcaqtttttcaqataqttqaaqctataacaqtccttttqt
D
K
P
E
P
P
P
E
C
R
L
P
D
A
T
1 3 2 5 tttqttttCatatcaqCACAAGCCTGAGCCAcCACCTGAGGGTCGCTTGCCTGATGCCAC
1385
K G
TAAGGqtcaqtqatctqatttqtgatgtgaatqtqaattaatctatatqattqatqtatttatttq
1445
S D H L R D V F G
tctaaqqatttqattcttqattattattqcaqGTTCTGACCATTTGAGCGATGTG~GG
K
A
M
C
L
S
D
Q
D
I
V
A
L
S
C
G
H
T
I
1 5 0 5 AAAGGCTATGGGGCTTAGTGATCAGGACATTGTTGCTCTATCTGGTGGTCACACCA~qt
1565
atqtcataactttaaqctcqctcctacttttattqtaqtattaataaaaccattaatatt
FIG. 2. Nucleotide and deduced amino acid sequencesof the
pea cytosolic ascorbate peroxidase gene. Uppercase lpttprs indicate exons sequences whichwere also found in the corresponding
ascorbate peroxidase cDNA, lowercase letters represent introns, 5 ' nontranscrihed regions, and 3"regions which were not found in ?he
correspondingascorbate peroxidase cDNA.Underlinedsequences
denote putative regulatory regionsreferred to in the text. Shaded
nucleotidesrepresentthe
sequence complementarytotheprimer
extension antisense oligonucleotide.Abbreviations: Trx (uppercase
holded hasps), initiation of transcription; reo. ARE, reversed ARE;
{,'fEI, GPEI enhancer, Poly-A, polyadenylation signal.
FIG. 3. Primerextensionanalysis.
Autoradiogram of the
primerextensionproductseparatedonadenaturingpolvarylamide gel. A 31-base antisense primer (Fig. 2 ) was u s ~ dto prime a
reverse transcriptase reaction using total leaf R S A RS A template. as
described under "Experimental Procedures." I ~ n r n extended
:
products, PI.:; sequencing ladder, A.
(;, and T. Sequencing Inrider was
with thewme3l-hnse
obtained by primingsequencingreactions
antisense primer using the isolated genomic clone as a template.
('.
of lo" M paraquat, which resulted in severe necrosis of plant
tissue, caused a decrease in both ascorbate peroxidase tranN o visible injury was
script and activity (data not shown).
evident in plants treated with either 10". or 10" M paraquat.
Pea Ascorbate Peroxidaqe Gene
2.1806
*
*
VYAEK
WH1F.W
TAPX:
PAPX:
1 MGKSYPTVSPDYQKAIEKAKRKLR...GFIAEKKCAPLILRLAWHSAGTF
47
AApx:
1 MTKNYPTVSEDYKKAVEKCRRKLR. ..GLIAEKNCAPIMVRLAWliSAGTF 4 7
YCCP: 8 0 KGRSY
EDFQKVYNhIALKLREDDEYDNYICYCPVLVRLAWHiSGTW 1 2 5
....
0.0
A. Tnrcrl.C1
HAANAGLVNALKLLET1.K
DERWP. K
CTEEPAAYCEL
4 8 DSKTKTGGPF.GTIKHQAELGANNGLDIAVRLLEPI.KEQFP.IVSYADF
4 0 DCQSRTGGPF.GTMRFDAEQAHGANSGIHIALRLLUPI.REQFP.TISFADF
1 2 6 DKHDNTGGSYGGTYRFKKEFNUPSNncLaNGFKFLEPIHKE.FPWISS.CDL
YGRVDTSS..PPEEGRL.DAA..N
9 7 YQLAGWAVEITGGPEVPFHPCREDKPE..PPPEGRLPDATKGSDHLRDV
9 7 HQLAGWAVEVTGGPDIPFHPCREDKPQ..PPPECRLPDATKCCDHLRDV
1 7 6 F::LGGVTAVQEMQGPKIPWRCGR:'DTPEDTTPDIiGRLPDAnKDAGYVRTF
96
96
175
I).Mal.
"_
"- "_
VSIGGLNPSVNK
145
14 5
226
144
144
225
E1VALSGAHTLGRSRPERSGWGEP.T
FDNSYFKD
FGKAMGLSDQDIVALSGCHTIGAAHKERSGFEGPWTSNPLIFDNSYFTE 1 9 3
FAKQMGLSDKDIVALSGAHTLGRCHKDRSGFEGAWTSNPLIFDNSYFKE 1 9 3
FQI?.I.NMfIDREVVALMGAHALCKTHI.K!l~GYECPWG~A~NVFltlEFYl.t~
273
ELKSA
...............REDIKELLNTK
...............
...............
YAADQ
KDGLLQLPSDKALLTDSVFRPLVEKYAADE
1 9 4 LLTGE
KECLLQLVSDULLDDPVFRPLVEKYAADE
1 9 4 LLSGE
2 7 4 LLNEDWKLEKNDANNEQWDSKSGYY:.!LP~DYSLIQDPK'~LSIVKEYA!:DQ
DEFFK
228
228
323
SNFGWNGT
2 2 9 DVFFADYAEAHLKLSELGFAEA 2 5 0
2 2 9 DAFFADYAEAHMKLSELCFADA 2 5 0
3 2 4 DKFFKDFSYAFEKLLE!IGIT
143
FIG.4. Amino acid sequence homology between ascorbate
peroxidases and yeast cytochrom
c eperoxidase. Comparison of
the deduced amino acid sequence of the pea cytosolic ascorbate
peroxidase (PAI'X) ( 5 ) withhomologous peptide fragments of tea
chloroplastic ascorbate peroxidase ( T A P X ) (24), the deduced amino
acid sequence of A. thaliana ascorbate peroxidase (AAI'X) ( 2 5 ) ,and
the yeast cytochromec peroxidase amino acid sequence ( YCCP) (36).
Amino acids which are identical to the pea ascorbate peroxidase are
shaded. Asterisks indicateproximalanddistalhistidineandthe
conserved arginine found in thevicinity of the putative active siteof
all plant peroxidases.
kb
23.1-
946.5-
8 EBEHX
FIG. 6. C h a n g e s i n s t e a d y state ascorbate peroxidase trans c r i p t a n d p r o t e i nlevels a n d a s c o r b a t e p e r o x i d a s e a c t i v i t y i n
response to chemical and environmental stresses.
Fourteendav-old pea plants were treated. sampled. and analyzed as descrihed
under"Experimental Procedures." Ahhreviations for stresstreatments are as follows: control drought. i . p . , well-watered plants ( ( ' 1 ) ) ;
drought-stressed plants which exhibited decreases of approximately
10 and 20%, respectively, in shoot tissue relative water content ( 1 0 7
and 20%); 1 and -5 h of exposure to heat shock ( I H and 5 H ) and the
corresponding control time points
( I C and iiC); control plants for
paraquat treatment sprayed with 0.05'Y Tween-20 (Tu,);
molar concentrations of paraquatappliedtoplants
(IO-' and lo",); control
plants for ARA and ethephon treatments sprayedwith 0.05'; Tween20 ( T w ) ;application of 1 mM ARA ( A H ) ; application of 14 m%i
ethephon (Eth). Total ascorbate peroxidase activity is indicated by
fillpd bars; p-CMPSA-inhibited ascorbate peroxidase activity is indicated by hatchpd bars.
peroxidase activity (Fig. 6). ARA application caused a 4-fold
increase in ApxI transcript abundance and a 2-fold increase
in ascorbate peroxidase activity(Fig. 6). There were no visible
differences between ARA-treated and untreated plants.
No
significantchanges were observedin ascorhate peroxidase
protein levels as detected withcytosolic ascorbate peroxidase
monospecific antibodies, following each of the stress treatments (Fig. 6).
4.3-
DISCUSSION
As a first stepin the understandingof the environmentally
regulated expression of the pea cytosolic ascorbate peroxidase
gene, we haveisolated,sequenced,and
analyzed the ApxI
2.0gene. Several interesting structural features were identified;
the gene is interrupted by 9 introns, the first of which is
located in the 5"untranslated region of the mature transcript.
Occurrence of an intron in the 5"noncoding region is not a
0 56common trait for plant genes and wasfoundonly
in four
other gene families including phytochrome, sucrose synthase.
actin,andubiquitin
(26-29). I t isnotclearwhetherthe
presence of an intron at this location in the ApxI gene has
any significant effect on transcription; however, the occurrence of an intron in the 5"noncoding region of a reporter
FIG.5. G e n o m i c S o u t h e r n a n a l y s i s of t h e pea ApxI gene.
Total DNA (10 pg) was digested withRarnHl ( R ) ,EcoRV ( E ) , RarnHI gene was found to increase gene expression in two different
+ KcoRV ( R E ) ,Hindlll (If),and XhaI ( X ) ,separated on 0.8% agarose plant transient expression systems (30,31). Nucleotide sequenceanalysis of thepromotor region combinedwith a
gel, and transferred to a nylon membrane. The membrane was hyhridized with the '"P-labeled insert of ascorbate peroxidase cDNA.
primer extension assay revealed a typical promotor structure
Molecular sizes were calibrated hy reference to the migration of X/
that includes a TATA box located 25-27 hp upstream from
Hind111 DNA fragments (indicated in kilobase pairs).
the transcription initiation site and a putative CCAAT hox.
Several potential regulatory sequenceswere identified in the
Application of 14 mM ethephon ((2-chloro-ethy1)phosphonic vicinity of the promotorregion; these include three regulatory
acid), which releasesethylene in treated plant tissues, resulted sequences that were extensively characterized in the regulaxenobiotic
in an epinastic response in pea and in a 6-fold increase in tion of rat glutathione S-transferase (ARE and
ApxI transcript levels and in a 2.4-fold increase in ascorbate responsive element (21) and a GPEI enhancer (23)).Of these
2.3-
Pea Ascorbate Peroxidase Gene
21807
the ARE is the most interesting since it was shown to be ested in studying the effect of a protein synthesis inhibitor
responsible for the hydrogen peroxide-dependent response of (cycloheximide) on the increase in ascorbateperoxidase activthe glutathione S-transferase gene (21). As the role of ascor- ity in response to theapplied stresses. Results obtained from
bate peroxidase is to rid the cell of excess HzOz, it is tantalizing these experiments willallow us to determine whether the
t o suggest that theARE may be involved in regulation of the increase in activity is a result of de nouo ascorbate peroxidase
ApxI gene. An additional possible route for the regulation of synthesis or the outcome of enzymatic activation of preexistthe ApxI gene via HzO, may be suggested by the occurrence ing ascorbate peroxidase polypeptides.
Pea ApxI was found to occur as a single copy gene which
of a heatshock factor binding element
(HSE). Thispossibility
is suggested since hydrogen peroxide was shown to activate displays a relatively low degree of homology to members of
immediate binding of a Drosophila transcriptional factor to the plantperoxidase superfamily. In contrast,ApxI was found
the HSEboth in uiuo and in vitro(32). Although the proximal to share a higher degree of homology with yeast cytochrome
HSE found in the vicinity of the promoter region is not in c peroxidase (36).
A recently published partial amino acid sequence of the tea
perfect agreement with
the HSE consensus (1 base mismatch),
it is well within range of the HSEsfound in other plant heat- chloroplastic ascorbate peroxidase and the recently reported
shock genes, especially cognate heat-shock genes (20). An- deduced amino acid sequence of A. thaliuna ascorbate peroxother common feature of the ApxZ gene with other plant heat- idase (probably a cytosolic ascorbate peroxidase cDNA) were
shock genes is the existence of a region with a very high also found to share the same characteristicpattern of homolpercentage of A T (more then 75% A + T between position ogy when compared to typical plant peroxidases and yeast
-896 and -350) which is also foundupstream from the cytochrome c peroxidase (24, 25). Based on this observation
proximal promotor region of many plant heat-shock genes and othernoted enzymatic and molecular properties common
(20). However, unlike most heat-shock genes, the ApxI gene between ascorbate peroxidases and yeast cytochrome c peroxidase (1,24), it is reasonable to assume that bothascorbate
is interrupted by 9 introns.
We have studied the environmentally regulated expression peroxidases and the yeast cytochrome c peroxidase originate
of pea cytosolic ascorbate peroxidase in response to several from the same ancestral gene.
stresses that were previously suggested to involve oxidative
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Gurley, W. B., and Key, J. L. (1991) Biochemistry 3 0 , 1-12
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-,".I
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1 R. fi75-fiR9
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B. C., Poulos, T. L., and Kraut,J. (1984) J. Biol. Chem. 259,13027script levels and increase in activity. We are currently inter13036
+
""