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Journal of Experimental Botany, Vol. 50, No. 330, pp. 63–69, January 1999
Aldehyde oxidase in roots, leaves and seeds of barley
(Hordeum vulgare L.)
Rustem T. Omarov1,3, Shuichi Akaba2, Tomokazu Koshiba2 and S. Herman Lips1
1 Biostress Research Laboratory (J. Blaustein Institute for Desert Researches) and Department of Life Sciences
(Faculty of Natural Sciences), Ben-Gurion University of the Negev, Sede Boqer 84990, Israel
2 Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192–0397, Japan
Received 8 April 1998; Accepted 19 August 1998
Abstract
Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves,
roots and seeds of barley (Hordeum vulgare L.) plants
were studied. Differences in substrate specificity and
mobility in native PAGE between AO proteins
extracted from roots, leaves and seeds have been
observed. Four clear bands of AO reacting proteins
were detected in barley plants capable of oxidizing a
number of aliphatic and aromatic aldehydes such as
indole-3-aldehyde, acetaldehyde, heptaldehyde, and
benzaldehyde. Mouse polyclonal antibodies raised
against purified maize AO cross-reacted with barley
AO proteins extracted from roots, leaves and seeds.
At least three different AO proteins were detected in
roots on the basis of their mobility during PAGE after
native Western blot analysis while in leaves and seeds
only one polypeptide cross-reacted with the antibody.
SDS-immunoblot analysis showed marked differences
in molecular weight between subunits of the AO
bands extracted from roots, leaves and seeds. Two
distinct subunit bands were observed in roots, with
relatively close molecular weights (160 kDa and
145 kDa), while a single subunit with a molecular
weight of 150 kDa was observed in leaf and seed
extracts.
Menadione, a specific and potent inhibitor of animal
AO did not affect barley AO proteins. Root and leaf
AO differed in their thermostability and susceptibility
to exogenous tungstate. The AO proteins in plants
may be a group of enzymes with different substrate
specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.
Key words: Aldehyde oxidase, barley, roots, leaves, seeds,
molybdenum cofactor (MoCo).
Introduction
AO (EC 1.2.3.1) is a member of the family of molybdenum hydroxylases, iron sulphur flavoproteins involved
in the metabolism of a broad range of natural and
xenobiotic compounds, most of which have been studied
in animals and microorganisms. Early observations demonstrated the presence of AO activities in oat coleoptiles
(Rajagopal, 1971), potato tubers (Rothe, 1974), cucumber seedlings (Bower et al., 1978), and pea seedlings
(Miyata et al., 1981). Much of the interest in AO in
plants stems from its involvement in the biosynthesis of
abscisic acid (ABA) and indole acetic acid (IAA). The
enzyme seems to catalyse the last step of the biosynthesis
of these hormones, through the oxidation of indole3-acetaldehyde (IAAld ) to IAA ( Koshiba et al., 1996)
and abscisic aldehyde (ABAld) to ABA ( Taylor et al.,
1988). Molybdenum cofactor (MoCo)-deficient mutants
of barley and tobacco lacked AO and XDH activities and
were almost totally impaired in their capacity to produce
ABA ( Walker-Simmons et al., 1989; Leydecker et al.,
1995). The flacca mutant of tomato lacks aldehyde oxidase and xanthine dehydrogenase, but not nitrate reductase
(Marin and Marion-Poll, 1997) suggesting that the
expression of the genes responsible for the synthesis of
MoCo is unaffected. The aba3 mutant of Arabidopsis
(Arabidopsis thaliana) may be affected in its capacity to
introduce a third sulphur atom into the Mo-centre of AO
and XDH (Schwartz et al., 1997). Similarly, the mutants
3 To whom correspondence should be addressed. Fax: +972 7 6596752. E-mail: [email protected]
© Oxford University Press 1999
64
Omarov et al.
aba1 in tobacco and flacca in tomato may not be affected
in their capacity to produce MoCo but rather on the
sulphurylation of the Mo hydroxylases.
Maize (Zea mays L.) coleoptile AO has been reported
to be a flavin- and molybdenum-containing enzyme with
a molecular mass of 300 kDa composed of two identical
subunits of 150 kDa ( Koshiba et al., 1996). A leaf AO
of citrus (Citrus sinensis L.) catalysed the oxidation of
indole-3-aldehyde to indole-3-carboxylic acid ( Winer
et al., 1993).
Maize AO has been found to have biochemical properties similar to the animal enzyme ( Koshiba et al., 1996).
The tomato AO protein exhibited a considerable amount
of amino acid sequence homology to AO and xanthine
dehydrogenase of various organisms (Ori et al., 1997).
Two cDNAs for maize AO (zmAO-1 and zmAO-2) have
been cloned from maize coleoptiles (Sekimoto et al.,
1997). On the basis of their very high homology it was
suggested that both AO proteins were related but may
play different roles in the plant. Recently four cDNAs
(atAO-1, 2, 3 and 4) encoding MoCo-containing AOs
have been cloned in Arabidopsis (Sekimoto et al., 1998).
Limited information is available on AO distribution in
different plant organs and little is known about its physiological roles. It has been reported recently that activity
staining of Arabidopsis seedlings AO after native PAGE
revealed localization of AO in an organ-specific manner
(Seo et al., 1998). In Arabidopsis at AO-1 and at AO-2
genes were expressed at higher levels in lower hypocotyls
and roots of seedlings, while at AO-3 was slightly higher
in cotyledons and upper hypocotyls (Sekimoto et al.,
1998). Root AO but not the leaf enzyme was enhanced
by salinity in barley plants, especially in the presence of
ammonium in the nutrient medium (Omarov et al., 1998).
AO activity increased remarkably in roots and shoots of
ryegrass plants following applications of salinity and
ammonium to nutrient medium (Sagi et al., 1998).
In the present paper, some biochemical properties and
differences of AO proteins in roots and leaves of barley
are described using native PAGE, Western blot analysis
and other assays. The main goal of the present study is
the characterization of different AO proteins observed in
plant organs and, eventually, to define their principal
specific functions in each part of the plant.
Materials and methods
Plant material
Barley (Hordeum vulgare L.) cv. Steptoe seedlings were grown
in 20 l containers with nutrient solutions as previously described
(Savidov et al., 1997a). The seeds were germinated in 0.2 mM
CaSO in the dark at room temperature over 3 d. Uniform
4
plants, 23 pot−1, were grown in 4–8 replications for each
treatment in a completely randomized block design. The
experiments were conducted in a greenhouse, with average day
temperatures between 20 °C and 25 °C during the day, and
between 8 °C and 12 °C during the night. Midday PPFD was
900–1000 mmol m−2 s−1. NH Cl at 4 mM was supplied as the
4
nitrogen source in half-strength modified Hoagland nutrient
solution (Hoagland and Arnon, 1938) in order to enhance AO
activity in roots (Omarov et al, 1998). Plants grown in
vermiculite were irrigated twice daily with identical volumes of
nutrient solution to prevent an increase of more than 10% in
the EC of the leachate as compared with that of the
input solution.
Extraction and assay of AO
Barley roots, leaves and seeds were frozen in liquid nitrogen
immediately after harvesting and ground in an ice-cold mortar
with acid-washed sand and ice-cold extraction medium. The
extraction medium contained 250 mM TRIS-HCl (pH 7.5),
1 mM EDTA, 1 mM dithiothreitol, 5 mM reduced glutathione
(GSH ), 5 mM FAD, and 50 mM leupeptin. The tissue/buffer
ratios were 152 (w/v) for roots and leaves and 156 (w/v) for
seed material. The homogenized plant material was centrifuged
in a Centrikon T-124 refrigerated centrifuge at 27 000 g at 4 °C
for 15 min. The resulting supernatant was used in subsequent
assays.
AO activity was detected in polyacrylamide gels by staining
after native electrophoresis. Native gels were prepared with
7.5% acrylamide gel (Laemmli, 1970) in the absence of SDS at
4 °C. The gel was immersed after electrophoresis in 0.2 M
phosphate buffer, pH 7.5, for 10 min followed by gentle shaking
at room temperature in a reaction mixture containing 0.1 M
TRIS-HCl, pH 8.0, 0.1 mM phenazine methosulphate, 1 mM
MTT
(3[4,5-dimethylthiazol-2-yl ]-2,5-diphenyltetrazoliumbromide) and 1 mM substrate (heptaldehyde, indole-3-aldehyde,
benzaldehyde or acetaldehyde). Native PAGE was carried out
with a Protean II xi Cell (Bio-Rad, USA).
Western blot analysis and protein determination
The AO proteins extracted from the plant material were
subjected to Western blotting. Ground tissue (1 g FW ) was
extracted with 3 ml TRIS-HCl buffer, pH 7.5, 3 mM DTT,
1 mM EDTA, 5 mM GSH, and 50 mM leupeptin. After
centrifugation of part of the crude extract, the resulting
supernatant was added to SDS-buffer at a ratio of 154 (v/v).
Native-PAGE was carried out as described above. SDSPAGE was performed in a 10% polyacrylamide gel (Laemmli,
1970). The resulting gel with the separated proteins was then
electrophoretically transferred on to a nitrocellulose membrane
(0.2 mm pore size; Schleicher and Schüll, Dassel, Germany).
Blotting time was 1 h at 2 mA cm−2. Blots were blocked for
90 min in 5% (w/v) bovine serum albumin in TBS.
Immunodetection of AO was carried out with polyclonal mouse
antibodies raised against the purified maize AO ( Koshiba et al.,
1996) after a 500-fold dilution in TBS and secondary antibodies
(anti-mouse IgG, Sigma) diluted 1000-fold in TBS. The antigen/
primary antibody complex was detected by binding of alkalinephosphatase-linked goat anti-mouse IgG (Sigma, USA).
Phosphatase activity was developed by staining with 5-bromo4-chloro-3-indolyl phosphate and nitroblue tetrazolium
(BCIP/NBT, Sigma Fast@ tablets). Molecular weight of proteins
was estimated with a mixture of protein standards: myosin
(202 kDa), b-galactosidase (109 kDa), bovine serum albumin
(78 kDa), and ovalbumin (46.7 kDa).
Binding of the antibodies to barley AO proteins was detected
by immunoprecipitation using protein-A-Sepharose CL-4B
(Pharmacia). All of the activity (detected after native-PAGE)
in the samples was removed when the antibodies and Sepharose
Aldehyde oxidases in barley 65
were added, while all of the activity remained in the supernatant
when control mouse serum was used.
Soluble proteins were estimated by the Bio-Rad micro assay
modification of the Bradford procedure (1976) using crystalline
bovine serum albumin as a reference.
MoCo determination
Molybdenum cofactor activity in plant tissue was estimated
using a pleiotropic nit-1 mutant of Neurospora crassa according
to Nason et al. (1970). The original procedure (Mendel et al.,
1985) was carried out with recent modifications (Sagi et al.,
1997; Savidov et al., 1997b).
Results
Differences between AO proteins extracted from plant
organs
AO proteins were detected following gel electrophoresis
of extracts of roots, leaves and seeds of barley. At least
four bands were observed, three of them (AO-2, AO-3,
AO-4) in roots, with marked differences in mobility
during gel electrophoresis and subsequent staining with
indole-3-aldehyde as substrate (Fig. 1). Additional aldehydes used were benzaldehyde, acetaldehyde and heptaldehyde. The AO proteins showed different affinity for the
substrates used. Enzyme activity was most prominent
with indole-3-aldehyde as substrate and differences in
intensity of AO bands were not significant. Two bands
(AO-3 and AO-4) were detected in gels with benzaldehyde, acetaldehyde and heptaldehyde as substrates
( Fig. 1B). The AO-4 band, with the highest mobility in
the gel, showed its strongest affinity for heptaldehyde,
acetaldehyde and benzaldehyde. The AO-1 band in roots
could be observed with benzaldehyde, although its activity
was very low (Fig. 1B). A single AO band (AO-1) was
determined in leaves using each of the substrates although
it reacted best with indole-3-aldehyde. High AO activity
in seeds was detected with heptaldehyde, indole3-aldehyde and benzaldehyde. Acetaldehyde was a poor
substrate for seed AO (not shown).
Immunological analysis
Polyclonal mouse antibodies raised against maize AO
( Koshiba et al., 1996) cross-reacted with at least three
proteins in roots and a fourth band in leaves and seeds
after native PAGE (Fig. 2A). These proteins corresponded to the location of AO proteins as revealed by
their staining with aldehydes in gels after native PAGE
( Fig. 1A). The differences in molecular weight between
the AO proteins extracted from roots, leaves and seeds
persisted following exposure to SDS-PAGE and immunoblotting. Two polypeptides with molecular weights of
about 160 kDa and 145 kDa each were detected in roots
( Fig. 2B). Only one protein band appeared after immunoblotting of leaf AO, with a molecular weight of 150 kDa.
Immunoblotting of seed extracts yielded two polypeptides
with molecular masses of 150 kDa and 85 kDa each after
SDS-PAGE. The 85 kDa polypeptide may be either a
degradation piece of the 150 kDa subunit produced by
the extraction procedure or a latent unused precursor. A
polypeptide with similar molecular weight has been
observed previously in maize coleoptiles ( Koshiba et al.,
1996) and tomato leaves (Ori et al., 1997).
Other properties of AO in barley roots, leaves and seeds
Root AO lost almost all activity when heated at 80 °C
for 2.5 min prior to native PAGE (Fig. 3). Leaf and seed
AO remained active after heat pretreatment of the extract
at 85 °C for 2.5 min before native PAGE ( Fig. 3). The
highest activity of leaf AO was detected with samples
preheated between 65 and 80 °C.
Menadione, a potent inhibitor of animal AO did not
affect enzyme activity of roots and leaves of plants (data
not shown) confirming the lack of inhibition previously
reported for AO of maize coleoptiles ( Koshiba et al.,
1996).
Effect of tungstate on root and leaf AO activity
Fig. 1. Native PAGE of AO proteins in barley. (A), AO isoforms in
barley roots, leaves and seeds detected after native PAGE and developed
with indole-3-aldehyde as substrate. (B) AO protein bands of barley
roots developed with: indole-3-aldehyde (1), benzaldehyde (2), acetaldehyde (3), and heptaldehyde (4). Amount of proteins loaded per each
lane: 230 mg for leaves, 230 mg for roots and 259 mg for seeds.
Addition of 250 mM Na WO to the growth medium
2
4
inhibited AO activity in roots and leaves of 6-d-old
seedlings. Tungstate inhibition of AO in roots was more
prominent than in leaves ( Figs 4, 5). Na MoO (250 mM )
2
4
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Omarov et al.
Fig. 2. Western blot analysis of AO proteins in barley organs. (A) Immunoblot made after native PAGE of extracts of leaves, roots and seeds of
barley. (B) Immunoblot following SDS-PAGE of extracts of leaves, roots and seeds of barley. Arrows indicate molecular weight of AO polypeptides
and standard protein markers.
Fig. 3. Native PAGE of AO proteins of barley roots, leaves and seeds
following heat pre-treatment prior to electrophoresis. Extract samples
were heated in a water bath at different temperatures during 2.5 min
followed by immediate cooling in ice. AO activity was determined with
indole-3-aldehyde as substrate.
Fig. 5. AO activity in roots of barley seedlings as affected by tungstate
and molybdate. AO protein bands were developed with acetaldehyde
and indole-3-aldehyde as substrates. 110 mg of proteins were loaded per
each lane.
Discussion
Fig. 4. AO activity in leaves of barley seedlings as affected by tungstate
and molybdate. AO activity was developed with indole-3-aldehyde as
substrate. 230 mg of proteins were loaded per each lane.
did not increase AO activity very much either in the
leaves or the roots of seedlings. Tungstate also decreased
MoCo activity in leaves (Fig. 6) and roots of seedlings
(data not shown), although to a lower extent than AO.
Several isoforms of AO in potato tubers (Rothe, 1974)
and in maize coleoptiles ( Koshiba et al., 1996) have been
observed. Recent observations have shown that staining
of AO after native PAGE of Arabidopsis seedling extracts
revealed the distribution of AO in an organ-specific
manner. One AO-1 (with higher substrate preference for
indole-3-aldehyde) was highly expressed in roots of both
wild-type and sur 1 auxin-overproducing mutant plants
and also specifically in the hypocotyl of the mutant
seedlings, whereas AO-3 was detected in cotyledons exhibiting a marked substrate preference for naphthaldehyde
(Seo et al., 1998).
Four AO proteins were observed in barley plants, of
Aldehyde oxidases in barley 67
Fig. 6. MoCo activity in leaves of barley seedlings as affected by
tungstate. Bars indicate standard error of the mean.
which at least three were detected in plant roots while
only one major band was observed in leaves and seeds,
capable of oxidizing to different degrees a number of
aliphatic and aromatic aldehydes (Fig. 1). Western
blotting revealed three of the AO proteins in roots
( Fig. 2A). Plant AO catalyses the oxidation of abscisic
aldehyde (ABAld ) to ABA ( Walker-Simmons et al., 1989;
Sindhu et al., 1990; Leydecker et al., 1995) and indole3-acetaldehyde (IAAld ) to IAA ( Koshiba et al., 1996).
Are these multiple functions of a single enzyme protein?
In view of the differences observed between leaf and root
AO, the feasibility that similar but not identical proteins
catalyse the oxidation of ABAld and IAAld should not
be ignored. The main site of ABA synthesis is the root
from where the hormone is transported to the shoot,
especially under drought or saline conditions ( Wolf et al.,
1990; Bano et al., 1993). ABA synthesis in roots was
enhanced under salinity conditions (Schnapp et al., 1990),
complementing recent observations on the enhancement
of AO activity in barley roots by salinity and ammonium
and the depression of AO activity by nitrate in the
nutrient medium (Omarov et al., 1998). It is not completely clear yet how much of the increased levels of AO
in roots of salt-stressed plants may be the result of
enhanced expression of genes coding for the enzyme
components (MoCo and AO apoprotein) or by posttranslational modulation of existing enzyme molecules as
shown for the Mo-enzyme nitrate reductase ( Kaiser and
Huber, 1996; Bachmann et al., 1996; Moorhead et al.,
1996).
AO proteins extracted from roots and leaves exhibited
different mobility during native gel electrophoresis
( Fig. 1). Differences between AO proteins extracted from
roots, leaves and seeds were also detected after SDSPAGE followed by Western blots. Two polypeptides with
molecular weights of 160 kDa and 145 kDa were detected
in roots ( Fig. 3), combinations (dimerization) of which
may generate the four isomers observed in native PAGE
of barley tissue extracts.
Root, leaf and seed AO proteins differed in their
susceptibility to high temperature pretreatments. Root
AO was almost completely inactivated in extracts subjected to heating at 80 °C for 2.5 min while leaf AO was
far more stable and showed even increased activity when
pretreated at temperatures of 65–80 °C (Fig. 3). AO of
maize coleoptiles lost all its activity after boiling for 5 min
but was relatively stable at 60–70 °C ( Koshiba and
Matsuyama, 1993). AO activity of cucumber seedlings
was enhanced by heating enzyme preparations at 70 °C
(Bower et al., 1978).
The presence of AO in seeds (Figs 1A, 2) may be
related to ABA production in the dormant embryo. AO
in dry seeds of barley was localized almost exclusively in
the embryo while the endosperm exhibited mere traces
(not shown). Seed dormancy and ABA content of several
plant species were very low in acid soils due to insufficient
uptake of Mo, but it increased following Mo applications
to the soil or the leaves (Modi and Cairns, 1994). In dry
pea (Pisum sativum L.) seeds, exogenous sodium molybdate was absolutely required for MoCo determination
( Kildibekov et al., 1996). Therefore, the increase in ABA
level in seeds following the application of exogenous Mo
suggests the involvement of a MoCo-containing enzyme
(presumably AO) in ABA synthesis and seed dormancy.
Tungsten substituted for molybdenum in the MoCocontaining nitrate reductase inactivating the enzyme
(Deng et al., 1989; Huber et al., 1994; Mendel, 1997).
Xanthoxal, presumably the first free C molecule in the
15
ABA biosynthesis pathway (Milborrow et al., 1997; Lee
and Milborrow, 1997), increased by 2.6-fold when AO
activity was blocked by tungstate in avocado (Persea
americana) fruits. The effect of tungstate on root and leaf
barley AO proteins (Figs 4, 5) provides additional evidence of Mo involvement in the catalytic centres of the
AO proteins observed in roots and leaves. AO inhibition
by tungstate in roots of barley seedlings was more prominent than in leaves ( Figs 4, 5). This may indicate (a) differences in the biochemical properties of root and leaf AO
proteins; (b) different rates of enzyme turnover which is
required for the insertion of tungsten into pterin instead
of Mo or (c) a limited transport of tungstate from roots
to shoots.
AO in plants consists of a group of several proteins
with different electrophoretic mobility, substrate specificity and subunit composition. These subunits, which in
different combinations give rise to the isozymes observed,
may be the products of the cDNAs isolated from maize
(Sekimoto et al., 1997), tomato (Ori et al., 1997) and
68
Omarov et al.
Arabidopsis (Sekimoto et al., 1998). Present evidence does
not allow at this stage to pinpoint which and how much
each of the resulting dimers is involved in specific metabolic tasks in the plant organs studied. This diversity of
AO proteins may be related to special metabolic roles in
the plant organs and/or different locations within a
given organ.
Acknowledgements
This work has been possible thanks to the financial support of
AID/CDR to project CA15–124, the Fohs Foundation ( USA)
and ICA (Israel ). Our gratitude to RR Mendel ( Technical
University of Braunschweig, Germany) for valuable discussions.
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