Journal of Experimental Botany, Vol. 55, No. 401, pp.

13611369, June 2004

DOI: 10.1093/jxb/erh134 Advance Access publication 8 April, 2004

RESEARCH PAPER

Activity and protein level of AO isoforms in pea plants

(Pisum sativum L.) during vegetative development and in
response to stress conditions
Edyta Zdunek-Zastocka2,*, Rustem T. Omarov1, Tomokazu Koshiba3 and Herman S. Lips1
1

Biostress Research Laboratory, J Blaustein Institute for Desert Research and Department of Life Sciences,
Ben-Gurion University of the Negev, Sede-Boqer 84990, Israel
2

Department of Biochemistry, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland

Department of Biological Sciences, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan

Abstract
Among three AO isoforms detected in pea plants, the
activity of PAO-1 was dominant in leaves of seedlings and young leaves of mature plants, while PAO-3
revealed the highest band intensity in old leaves and
roots. PAO-1 and PAO-3 are homodimers consisting
of 145 kDa and 140 kDa subunits, respectively, while
PAO-2 is a heterodimer of one 145 kDa and one
140 kDa subunit. In leaves, the activity of PAO-1 disappeared gradually with leaf ageing, while in roots it
was present only in seedlings but not in mature pea
plants. PAO-3 could oxidize abscisic aldehyde, a precursor of abscisic acid, indicating the possible
involvement of this isoform in ABA synthesis in pea.
The ability of PAO-3 to oxidize abscisic aldehyde was
higher in old leaves than in young ones and
increased signicantly both in roots and leaves of
plants exposed to salinity and ammonium treatments.
A marked increase of the AO protein level was
observed after ammonium application but not under
salinity. Interestingly, the activity of PAO isoforms
may be transcriptionally and post-transcriptionally
regulated during vegetative growth and in response
to stress conditions, and such a regulation might be
particularly important to adjust ABA levels to the
recent requirements of the plant. The observations
suggest that the AO isoforms have different metabolic roles and that the activity and protein level of
each isoform is regulated not only by environmental
conditions but also through plant developmental
stages.

Key words: Aldehyde oxidase, AO, ammonium, Pisum

sativum L., plant development, salinity, stress conditions.

Introduction
Aldehyde oxidase (AO; EC 1.2.3.1) belongs to the family
of molybdenum hydroxylases and catalyses the oxidative
hydroxylation of a number of diverse aldehydes and
aromatic heterocyclic compounds in reactions that necessarily involve the cleavage of a C-H bond (Hille, 1996).
AO has been extensively characterized in animals and
micro-organisms, where it has been implicated in the
detoxication of environmental pollutants and xenobiotics
(Bauer and Howard, 1991). In plants, however, the
information about AO is limited and focused particularly
on an involvement of the enzyme in the nal step of plant
hormone biosynthesis, such as indole-3-acetic acid (IAA)
and abscisic acid (ABA). The enzyme catalyses in the
cytosol the oxidation of indole-3-acetaldehyde (IAAld) to
IAA (Koshiba et al., 1996; Seo et al., 1998) and abscisic
aldehyde (ABAld) to ABA (Taylor et al., 1988; WalkerSimmons et al., 1989; Seo et al., 2000b). The endogenous
ABA level increases upon stress conditions, most notably
under drought and salinity (Munns and Cramer, 1996). The
application of exogenous ABA creates effects similar to
plant-stress responses and increases plant tolerance to
environmental stresses (Fedina et al., 1994; Welbaum
et al., 1997). Elucidation of the regulation mechanisms of
the ABA biosynthetic pathway is, therefore, essential to
understand the adaptive stress-induced mechanisms in
plants.

* To whom correspondence should be addressed. Fax: +48 22 853 09 50. E-mail: zdunek@delta.sggw.waw.pl
Journal of Experimental Botany, Vol. 55, No. 401, Society for Experimental Biology 2004; all rights reserved

Downloaded from http://jxb.oxfordjournals.org/ by guest on March 15, 2014

Received 19 August 2003; Accepted 20 February 2004

1362 Zdunek-Zastocka et al.

Arabidopsis AO genes previously cloned by Sekimoto

et al. (1998). When the Arabidopsis rosette leaves were
detached and exposed to dehydration, the expression of
mRNA encoding AOd increased rapidly, but the activity
was unaffected (Seo et al., 2000a). Drought-induced
increase of abscisic aldehyde oxidase activity was, however, observed in leaves of Arabidopsis thaliana by Bittner
et al. (2001).
The detailed mechanisms of molecular and biochemical
regulation of AO in plants, as well as the physiological
function of AO isoforms are just starting to be unravelled.
In the present study, AO activity changes and the protein
level of each AO isoform following stress application as
well as during plant development have been investigated.
The roles of particular AO isoforms in stress-induced ABA
synthesis and during plant development are discussed.
Materials and methods
Plant material and experimental conditions
Seeds of Pisum sativum L. (cv. Little Marvel) were germinated in
moist vermiculite. After 14 d, the seedlings were transferred to 20 l
containers containing aerated half-strength Hoagland nutrient solution (Hoagland and Arnon, 1938) with an adequate nitrogen supply
(4 mM NO3). The residual cotyledons were removed from the
seedlings after 3 d of adaptation to hydroponics, and the treatments
were initiated. Nitrogen was provided as 4 mM NaNO3 or 2 mM
(NH4)2SO4. Salinity was supplied at a concentration of 100 mM
NaCl. Plants grown with 4 mM NO3 in the absence of salinity were
taken as controls. The pH of the medium (pH 6.5) was adjusted daily
and the nutrient solutions were replaced weekly. Experiments were
conducted in a greenhouse with temperatures ranging between 25
28 C during the daytime and 1518 C during the night, with a 14 h
photoperiod, and a midday photosynthetic photon ux density of
c. 9001000 mmol m2s1.
For the `developmental' experiments, seedlings were harvested 6 d
after sowing while mature plants were collected at the age of 3 and
5 weeks. For the `stress treatments' experiments, plants were
harvested 7 d after application of the treatments. Plants were
separated into roots and young (half expanded), mature (fully
expanded), and old leaves. Fresh samples were used for immediate
enzyme activity assays or frozen in liquid N2 and stored at 80 C for
other analysis.
Native PAGE and AO activity staining
Plant tissues were homogenized immediately after harvesting with
acid-washed sand and an ice-cold extraction medium containing
250 mM TRIS-HCl, pH 8.5, 1 mM EDTA, 10 mM GSH, and 2 mM
DTT. A ratio of 1 g tissue to 3 ml buffer (1:3 w/v) was used for
leaves and 1 g tissue to 2 ml buffer (1:2 w/v) for roots. The
homogenized plant material was centrifuged at 18 000 g and 4 C for
15 min. The resulting supernatant was subjected to native
polyacrylamide gel electrophoresis (PAGE) on 7.5% polyacrylamide gels in a Laemmli buffer system (Laemmli, 1970) in the
absence of SDS at 4 C. Each lane in the gel was loaded with 100 mg
root proteins or 400 mg leaf proteins. After electrophoresis, AO
activity staining was developed at room temperature in a mixture
containing 0.1 M TRIS-HCl, pH 7.5, 0.1 mM phenazine methosulphate, 1 mM MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide), and 1 mM substrate (abscisic aldehyde or
indole-3-aldehyde). AO activity was estimated on the basis of MTT
reduction, which resulted in the development of specic formazan

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Two enzymes, involved in the early steps of ABA

synthesis in chloroplasts, have recently attracted much
attention as the enzymes having a putative key role in the
modulation of ABA levels in the plant. Zeaxanthin
epoxidase (ZEP) converts zeaxanthin to violaxanthin by
a two-step epoxidation reaction and appears to be ratelimiting for ABA biosynthesis in non-photosynthetic
tissues such as roots and developing seeds (Marin et al.,
1996; Audran et al., 1998). The over-expression of ZEP in
transgenic tobacco resulted in enhanced seed dormancy
(Frey et al., 1999). Epoxycarotenoid dioxygenase (NCED)
cleaves the 9-cis-isomers of both violaxanthin and
neoxanthin to cis-xanthoxin and a C25 epoxy apocarotenoid (Cutler and Krochko, 1999). Over-expression
of the LeNCED1 in tomato (Thompson et al., 2000) and
AtNCED3 in Arabidopsis (Iuchi et al., 2001) resulted in
increased ABA levels. It was suggested that the epoxycarotenoid cleavage reaction might be the key regulatory
step of drought-induced ABA biosynthesis (Qin and
Zeevaart, 1999; Iuchi et al., 2000).
The oxidation of abscisic aldehyde to abscisic acid may
constitute another important element in the regulatory
mechanism of stress-induced ABA biosynthesis, most
probably controlled at various steps of the pathway and in a
tissue- and growth condition-specic manner. Plant AO
has only been puried from coleoptiles of maize (Koshiba
et al., 1996). Two AO cDNAs (zmAO-1 and zmAO-2) with
a high homology to each other at both the nucleotide and
the amino acid sequence levels have been subsequently
cloned for maize (Sekimoto et al., 1997). Four cDNAs
(atAO-1, 2, 3, and 4) encoding AO proteins have been
cloned for Arabidopsis (Sekimoto et al., 1998; Hoff et al.,
1998), while three putative AO cDNAs have been isolated
from tomato (Min et al., 2000). Information on AO
regulation in plants is limited and little is known about its
sensitivity to environmental factors. AO activity increased
in ryegrass plants exposed to salinity and/or NH+4, although
the salinity-enhanced activities were more pronounced in
roots than in leaves (Sagi et al., 1998). Root, but not shoot,
AO activity was enhanced by salinity in barley plants,
especially in the presence of ammonium in the nutrient
medium (Omarov et al., 1998). An increase of AO activity,
particularly in old leaves, was observed in pea plants
exposed to salinity, nitrogen deciency as well as to
ammonium treatment (Zdunek and Lips, 2001). Plant
aldehyde oxidase exhibited broad substrate specicity with
respect to different aromatic and aliphatic aldehydes
(Koshiba et al., 1996; Akaba et al., 1998; Koiwai et al.,
2000). However, AO isoforms that can efciently oxidize
abscisic aldehyde to abscisic acid have only been identied
recently in Arabidopsis rosette leaves (Seo et al., 2000a)
and in roots of barley (Omarov et al., 2003). The AOd
isoform, that could efciently oxidize abscisic aldehyde in
rosette leaves of Arabidopsis (Seo et al., 2000a), is
encoded by AAO3 (formerly called atAO-3), one of four

AO isoforms in pea plants 1363

bands. The relative intensity of formazan bands was directly
proportional to enzyme activity (Rothe, 1974). Native PAGE was
carried out with a Protean II xi Cell (Bio-Rad, USA).

Protein determination
Total soluble protein content was estimated by the Bio-Rad Protein
Assay, a modication of the Bradford procedure (Bradford, 1976),
using crystalline bovine serum albumin as a reference.

Results
AO isoforms in pea seedlings
Three bands of aldehyde oxidase activity were detected in
leaves and roots of 6-d-old seedlings using indole-3aldehyde as substrate (PAO-1, PAO-2, PAO-3; Figs 1, 2).
In leaf extracts a faint fourth band with the lowest mobility
in gel was also observed. Mobility of AO isoforms in roots
corresponded to the bands detected in leaves. PAO-1
revealed the highest band intensity in leaf extracts, while

AO isoforms in mature pea plants

Staining of proteins extracted from leaves of 3-week-old

plants with indole-3-aldehyde as the substrate revealed
three AO activity bands (Fig. 1) corresponding to the
location of the AO bands detected in pea seedlings. Only
one band of AO activity was detected in root extracts
(PAO-3, Fig. 2). The ability of leaf AO isoforms to oxidize
indole-3-aldehyde varied between leaves at different
developmental stages. In young leaves, PAO-1 showed
the strongest activity, while in old leaves the highest ability
to produce formazan bands corresponded to PAO-3. In
fully expanded leaves, PAO-1 and PAO-3 were detected at
a similar activity level. When abscisic aldehyde was used
as substrate, two activity bands were detected in leaf
extracts (PAO-2, PAO-3) and one in roots (PAO-3).
In 5-week-old plants, three AO activity bands were
detected with indole-3-aldehyde only in young leaves
(Fig. 1). Two AO activity bands (PAO-2, PAO-3) were
detected in fully expanded leaves, while only one band was
visible in old leaves (PAO-3). When the gel was stained
with abscisic aldehyde, fully expanded and old leaves
revealed only the PAO-3 activity band. The level of PAO-3
activity, developed either with indole-3-aldehyde or
abscisic aldehyde, was signicantly higher in old leaves
when compared with that of young and fully expanded
leaves.
At least three leaf proteins cross-reacted with the antiAO antibodies regardless of leaf developmental stage and
the age of the plant (Fig. 1). However, PAO-1 was
predominantly expressed in young leaves while the PAO-3
protein level was slightly higher in old leaves. In mature
and old leaves the protein content of the PAO-1 and PAO-2
isoforms remained roughly at the same level throughout

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Western blot analysis

Protein extraction was carried out essentially as described above
with the exception that a ratio of 1 g tissue to 1 ml extraction buffer
(1:1 w/v) was used for SDS-PAGE. The extracts were centrifuged at
18 000 g and 4 C for 15 min. Aliquots of the supernatants
containing 300 mg leaf proteins were combined with the same
volume of sample loading buffer (125 mM TRIS-HCl, pH 6.8, 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol
blue), and heated for 5 min at 100 C before being subjected to SDSPAGE.
Native-PAGE and SDS-PAGE were carried out in 7.5%
polyacrylamide gels (Laemmli, 1970). The separated proteins were
electrophoretically blotted onto a nitrocellulose membrane (0.2 mm
pore size; Schleicher and Schull, Dassel, Germany) at 2 mA cm2 for
2 h. Unbound sites on the membranes were blocked with 5% (w/v)
non-fat dry milk (Blotting Grade Blocker, Bio-Rad, USA) in TRISbuffered saline, TBS (20 mM TRIS-HCl, pH 7.5, 150 mM NaCl).
Immunodetection of AO was carried out with polyclonal rabbit
antibodies raised against 30 kDa Arabidopsis AAO1 and AAO2
peptides (Akaba et al., 1999) after a 500-fold dilution in TBS. After
incubation of the blots with the primary antibodies at 4 C overnight,
the membranes were washed three times with TBS-T buffer (TBS
containing 0.05% (v/v) Tween 20) and incubated for 1 h at room
temperature with secondary antibody. As secondary antibodies, the
alkaline phosphataseconjugated goat antirabbit IgG (Sigma, USA)
diluted 1000-fold in TBS were used. The immune-complexes were
visualized by developing the alkaline phosphatase activity with
5-bromo-4-chloro-3-indolyl phosphate (0.36 mM) and nitroblue
tetrazolium (0.32 mM) in 100 mM TRIS-HCl buffer, pH 9.5,
100 mM NaCl, and 5 mM MgCl2.
The subunit composition of AO isoforms was determined as
follows: after separation of leaf AO isoforms by native PAGE and
subsequent staining with indole-3-aldehyde, PAO-1, PAO-2, and
PAO-3 protein bands were extracted from the gel, denatured in SDS
sample buffer, and subjected to SDS-PAGE followed by western
blot analysis.
The molecular weights of AO polypeptides were estimated with
a mixture of highly puried recombinant proteins of specic
molecular weights (Precision unstained protein standards, BioRad, USA): 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa,
25 kDa, 15 kDa, and 10 kDa. SDS-PAGE was carried out with a
Mini-Protean II Cell (Bio-Rad, USA); electrophoretic transfer was
performed with a Mini Trans-Blot Cell (Bio-Rad, USA).

PAO-3 (with the highest mobility in the gel) showed the

strongest ability to oxidize indole-3-aldehyde in root
extracts.
When abscisic aldehyde was used for AO activity
staining, two bands with similar intensities were detected
in leaf extracts (PAO-2 and PAO-3; Fig. 1), while only one
activity band was observed in root extracts (PAO-3; Fig. 2).
Western-blot analysis following protein separation by
native PAGE revealed at least three AO proteins both in
leaves (Fig. 1) and in roots (Fig. 2) of 6-d-old seedlings. In
leaf extracts, a faint fourth protein band with the lowest
mobility in gel was also observed. The location of
immunologically detected proteins corresponded to the
AO activity bands. In leaves, the protein level of PAO-1
was signicantly higher than those of PAO-2 and PAO-3.
Immunodetection of AO proteins was carried out with
polyclonal rabbit antibodies raised against Arabidopsis
30 kDa AAO1 and AAO2 peptides (Akaba et al., 1999). For
extracts of pea plants, both antibodies gave similar results
and the immunoblots obtained with anti-AAO2 antibodies
were chosen for presentation.

1364 Zdunek-Zastocka et al.

plant vegetative development, thus suggesting a posttranslational inactivation leading to a gradual disappearance of activity which was observed in fully expanded and
old leaves of 5-week-old plants. Only one AO protein band
was detected in the roots of 3- and 5-week-old plants
(Fig. 2).
AO isoforms as affected by stress conditions

A signicant increase in abscisic aldehyde (ABAld)oxidase activity was observed both in leaf (Fig. 3) and root
extracts (Fig. 4) after 7 d of exposure to salinity and
ammonium. Within leaves of three different ages the most
pronounced stress-induced enhancement of AO activity
was observed in old leaves. No increase of ABAld-oxidase
activity following stress application was observed in young
leaves. Within two AO isoforms able to oxidize abscisic
aldehyde (PAO-2, PAO-3), the lower one with the highest
mobility in the gel (PAO-3) was particularly induced by
unfavourable growth conditions both in leaves and roots.
The increase in leaf and root ABAld-oxidase activity after
7 d of stress treatments was more pronounced in
ammonium-treated plants than under salinity.
As detected by native western-blot analysis, the level of
AO proteins, particularly of PAO-3, increased signicantly

both in roots and leaves of plants grown under ammonium

treatment (Figs 3, 4). The salt-induced increase of the AO
protein level was much less remarkable.
SDS-PAGE immunoblot analysis revealed, in leaf
extracts, the presence of two polypeptides with molecular
weights of about 145 kDa and 140 kDa each (Fig. 5). A
polypeptide with a molecular weight of about 90 kDa,
which was also observed in the analysed extracts, may be a
proteolytic product of the 145 kDa subunit. Polypeptides
with a similar molecular weight have been previously
reported in maize coleoptiles (Koshiba et al., 1996) and
barley roots (Omarov and Lips, 2000; Omarov et al.,
2003). Stress treatments brought about a signicant
increase in the protein level of the 140 kDa subunit,
especially in ammonium-treated plants (Fig. 5).
Subunit composition of AO isoforms extracted from
pea leaves

Anti-AO antibodies that cross-reacted with the denatured

PAO-1 isoform from pea leaves revealed a polypeptide
with a molecular mass of 145 kDa, while the PAO-3 band
was presented as a single 140 kDa polypeptide. The PAO-2
isoform consisted of one 145 kDa and one 140 kDa
polypeptide (Fig. 6).

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Fig. 1. Activity and protein level of aldehyde oxidase in leaves of seedlings and of mature pea plants. (a) Zymograms of AO activity developed
with abscisic aldehyde and indole-3-aldehyde as substrates. (b) Immunoblot detection of AO proteins carried out with polyclonal rabbit antibodies
raised against Arabidopsis 30 kDa AAO2 peptide. Leaf proteins were extracted from plants grown with 4 mM NaNO3 and were separated (400 mg
lane1) by native PAGE. A, young leaves; B, fully expanded leaves; C, old leaves. The zymograms and immunoblots represent similar results
obtained in ve different experiments.

AO isoforms in pea plants 1365

Discussion
Unlike animal species, where AO is present as a single
gene copy (Li Calzi et al., 1995; Terao et al., 1998), in
plants, the enzyme is represented by a group of several
proteins with different electrophoretic mobility, substrate
specicity, and subunit composition. Studies reporting
organ distribution of aldehyde oxidases showed AO

Fig. 3. Activity and protein level of aldehyde oxidase in old leaves of pea plants exposed for 7 d to salinity and different nitrogen source.
(a) Zymogram of AO activity developed after native PAGE and staining with abscisic aldehyde as substrate. (b) Immunoblot detection of
separated by native PAGE AO proteins carried out with polyclonal rabbit antibodies raised against Arabidopsis 30 kDa AAO2 peptide. Each lane
in the gel was loaded with 400 mg of leaf proteins. A, young leaves; B, fully expanded leaves; C, old leaves. Nitrate was supplied as 4 mM
NaNO3, ammonium as 2 mM (NH4)2SO4, salinity as 100 mM NaCl. Plants grown with 4 mM NO3 in the absence of salinity were the controls.
The zymogram and immunoblot represent similar results obtained in ve different experiments.

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Fig. 2. Activity and protein level of aldehyde oxidase in roots of

seedlings and of mature pea plants. (a) Zymograms of AO activity
developed with abscisic aldehyde and indole-3-aldehyde as substrates.
(b) Immunoblot detection of AO proteins carried out with polyclonal
rabbit antibodies raised against Arabidopsis 30 kDa AAO2 peptides.
Root proteins were extracted from plants grown with 4 mM NaNO3
and were separated (100 mg lane1) by native PAGE. The zymograms
and immunoblots represent similar results obtained in ve different
experiments.

polymorphism of gel electrophoretic bands mainly in

leaves of dicotyledones such as Arabidopsis (Schwartz
et al., 1997), tomato (Sagi et al., 1999), and roots of
monocots such as ryegrass (Sagi et al., 1998), barley
(Omarov et al., 1999), and maize (Barabas et al., 2000). In
Pisum sativum, the polymorphism of aldehyde oxidase
isoforms, observed in both leaves and roots of seedlings,
changes during plant vegetative development. The isoform
with the lowest gel mobility (PAO-1) exhibited the highest
intensity of activity band in leaves of seedlings and young
leaves of mature pea plants, but disappeared gradually with
leaf ageing. The protein content of this isoform in leaves
remained roughly at the same level during vegetative
development, thus indicating a post-translational inactivation taking place, especially in fully expanded and old
leaves of 5-week-old plants (Fig. 1). In roots, the PAO-1
isoform was only detected in seedlings, but not in mature
pea plants (Fig. 2). PAO-1 was unable to oxidize abscisic
aldehyde to abscisic acid (Figs 1, 2, 3, 4). Thus, PAO-1
may play an important role in the development of pea
plants that is unrelated to ABA production, but is linked to
the yet undened events in young organs. A unique
function during plant development was also suggested for
the products of each of the two maize AO cDNAs; zmAO-1
was expressed at a high level in roots, whereas zmAO-2
was expressed in coleoptiles (Sekimoto et al., 1997).
Nothing is known about the involvement of aldehyde
oxidase in any of the developmental processes that occur in
plants and, at the present stage, it is difcult to speculate
about any specic role of the enzyme throughout the life
cycle of pea plants. However, apical meristems of shoots,
young leaves, and developing fruits are considered as the
primary sites of indole-3-acetic acid (IAA) synthesis in
higher plants (Bandurski et al., 1995), from where auxin
moves basipetally toward the root system (Estelle, 1998).
The high activity of PAO-1 in young leaves (Fig. 1) may
therefore suggest the involvement of PAO-1 in IAA
synthesis in these organs. Aldehyde oxidase has been

1366 Zdunek-Zastocka et al.

Fig. 4. Activity and protein level of aldehyde oxidase in roots of pea

plants exposed for 7 d to salinity and different nitrogen source.
(a) Zymogram of AO activity developed after native PAGE and
staining with abscisic aldehyde as substrate. (b) Immunoblot detection
of separated by native PAGE AO proteins carried out with polyclonal
rabbit antibodies raised against Arabidopsis 30 kDa AAO2 peptide.
Each lane in the gel was loaded with 100 mg of root proteins. Nitrate
was supplied as 4 mM NaNO3, ammonium as 2 mM (NH4)2SO4,
salinity as 100 mM NaCl. Plants grown with 4 mM NO3 in the
absence of salinity were the controls. The zymogram and immunoblot
represent similar results obtained in ve different experiments.

assumed to be implicated in IAA biosynthesis from

tryptophan (Koshiba et al., 1996; Seo et al., 1998),
although no direct evidence for this hypothesis has been
reported in any plant.
As determined by immunodetection following SDSPAGE, the three AO isoforms found in leaves of Pisum
sativum were dimers. PAO-1 and PAO-3 are homodimers
consisting of 145 kDa and 140 kDa subunits, respectively,
while PAO-2 is a heterodimer of one 145 kDa and one
140 kDa subunit (Fig. 6). Subunit composition similar to
pea AOs was found for three AO isoforms detected in
seedlings of Arabidopsis thaliana (AOa, AOb, AOg;

Akaba et al., 1999) and in roots of barley (AO2, AO3,

AO4; Omarov et al., 2003). Two different polypeptides,
150 and 145 kDa, forming the three dimeric AO isoforms
in Arabidopsis were products of two cDNAs, AAO1 and
AAO2 (Akaba et al., 1999). Immunodetection of pea AO
proteins was carried out with polyclonal rabbit antibodies
raised against Arabidopsis 30 kDa AAO1 and AAO2
peptides. As it was shown by immunoprecipitation (Akaba
et al., 1999), the anti-AAO1 and anti-AAO2 peptide
antibodies could specically recognize AOa and AOg in
Arabidopsis seedlings, respectively. However, when the
immunoblot analyses were performed after native PAGE,
the anti-AAO1 antibodies reacted with all three
Arabidopsis AOs (AOa, AOb, AOg). In extracts of pea
plants both anti-AAO1 and anti-AAO2 antibodies recognized all three AO isoforms following immunobloting,
although, immunoprecipitation did not remove the AO
proteins from the crude extracts (data not shown). This can
be explained by the fact that the antibodies could recognize
only a small fragment of the AO polypeptide chain
inaccessible in the folded protein, but when the protein
became unfolded during blotting to the nitrocellulose lter
the immunoreactive site was then exposed to the antibody.
There are two AO isoforms that can efciently oxidize
abscisic aldehyde to ABA in leaves and one in roots of pea
plants (Figs 1, 2). So far, the AO isoforms capable of using
abscisic aldehyde as a substrate were found only in the
rosette leaves of Arabidopsis (called AOd; Seo et al.,
2000a) and in roots of barley (called AO2 and AO3;
Omarov et al., 2003). The regulatory function of AO in
abscisic acid biosynthesis, especially synthesis stimulated
by stress conditions, is still debated. When the Arabidopsis
rosette leaves were detached and exposed to dehydration,
the expression of mRNA encoding AOd increased rapidly,
although, the activity was unaffected (Seo et al., 2000a). A
drought-induced increase of abscisic aldehyde oxidase
activity was, however, observed in leaves of Arabidopsis

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Fig. 5. SDS-PAGE immunoblot analysis of AO proteins extracted

from old leaves of pea plants exposed for 7 d to salinity and different
nitrogen sources. Immunoblot detection of AO proteins was carried
out with polyclonal rabbit antibodies raised against Arabidopsis 30 kDa
AAO2 peptides. Each lane in the gel (7.5%) was loaded with 300 mg
of leaf proteins. Nitrate was supplied as 4 mM NaNO3, ammonium as
2 mM (NH4)2SO4, salinity as 100 mM NaCl. Plants grown with 4 mM
NO3 in the absence of salinity were taken as controls. The location of
the SDS-PAGE molecular weight markers is indicated on the left. The
immunoblot represents similar results obtained in three different
experiments.

Fig. 6. Subunit composition of AO isoforms from leaves of pea

plants. AO activity bands (PAO-1, PAO-2, PAO-3) observed after
native PAGE and staining with indole-3-aldehyde as substrate were
extracted from the gel, and subjected to SDS-PAGE. Immunoblot
detection of AO proteins was carried out with polyclonal rabbit
antibodies raised against Arabidopsis 30 kDa AAO2 peptides. Proteins
were extracted from old leaves of plants grown with 4 mM NH4+.

AO isoforms in pea plants 1367

regulation of ABA biosynthesis. The sulphuration step is

reversible in vitro (Wahl and Rajagopalan, 1982; Schwartz
et al., 1997), although, it is not known whether this
reversibility has biological signicance.
In spite of the extensive study of plant AO during recent
years, it is still problematic to ascribe well-dened
physiological roles to this enzyme and to explain the
occurrence of its polymorphism in plant tissue. Apart from
the implication of AO in plant hormones synthesis, the
enzyme may also full other physiological tasks. In fact,
liver AO carries out biochemical detoxication function
(Li Calzi et al., 1995). If AO has a similar detoxication
function in plants, then plant environmental adaptability
may require the maintenance of AO polymorphism to deal
with a wide range of toxic substrates.
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Akaba S, Seo M, Dohmae N, Takio K, Sekimoto H, Kamiya Y,
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thaliana by Bittner et al. (2001). These ndings on stressinduced changes of ABAld-oxidase activity in pea plants
are only the second to be published. In pea plants, there
seems to be only one AO isoform, PAO-3, that might be
responsible for stress-induced ABA production. PAO-3
activity was higher in old than in young leaves and
increased signicantly both in roots and leaves of plants
exposed to salinity or ammonium treatment (Figs 3, 4).
The increase of PAO-3 activity, developed with indole-3aldehyde, but not abscisic aldehyde as the substrate, was
observed previously in roots and leaves of pea plants
exposed to salinity, ammonium, and low nitrogen treatment (Zdunek and Lips, 2001). In the same experiment the
increase of root PAO-3 activity observed under stress was
accompanied by the enhancement in ABA xylem delivery
rate. The ABA accumulated in leaves of stressed pea plants
might, therefore, originate in roots in view of the higher
ABA xylem delivery rate or may be a result of in situ
synthesis in leaves (Zdunek and Lips, 2001). It has been
suggested previously that the old leaves are the main ABA
producers in plant shoots both under control as well as
stress conditions (Zeevaart and Boyer, 1984; Zhang and
Zhang, 1994; Zdunek and Lips, 2001). In pea plants
exposed to salinity and ammonium treatment, the ability of
PAO-3 to oxidize abscisic aldehyde did not change in
young leaves but increased signicantly in old leaves
(Fig. 3). Thus, the observed leaf PAO-3 activity changes
indicate once more that, in pea shoots, the stress-induced
synthesis of ABA takes place in old leaves but not in young
ones.
The level of PAO-3 protein increased signicantly in old
leaves and roots of plants grown with ammonium in the
nutrient solution, but not under salinity (Figs 3, 4). The
changes in relative AO protein abundance observed after
ammonium application may have contributed at least
partially to the changes of PAO activity. Under salt
treatment, however, the post-translational activation of a
latent PAO protein should be taken under consideration.
The activity of AO in the ABA mutants such as aba3 of
Arabidopsis thaliana, aba1 of tobacco, and acca of
tomato could be restored by in vitro anaerobic sulphuration
with dithionite and Na2S (Schwartz et al., 1997; Akaba
et al., 1998; Sagi et al., 1999). Thus, it was assumed that
these mutants are defective in the gene encoding the
sulphurase responsible for the nal step of the AO
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form. The enzyme catalysing this reaction, the Mohydroxylase sulphurase, has been recently cloned in
Arabidopsis (Bittner et al., 2001; Xiong et al., 2001) and
in tomato (Sagi et al., 2002). The gene expression of
Arabidopsis Mo-hydroxylase sulphurase was induced by
drought-stress (Bittner et al., 2001). Therefore, the
sulphuration of the AO-Moco might be an important
regulatory mechanism of AO activity involved in the

1368 Zdunek-Zastocka et al.

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