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Plant Physiol. (1999) 120: 571-578
Aldehyde Oxidase and Xanthine Dehydrogenase in a
flacca Tomato Mutant with Deficient Abscisic Acid and
Wilty Phenotype1
Moshe Sagi*,
Robert Fluhr, and
S. Herman Lips
Biostress Research Laboratory, Department of Life Science, Faculty
of Natural Sciences, Ben-Gurion University of the Negev, Sede Boqer
84990, Israel (M.S., S.H.L.); and Department of Plant Science,
Weizman Institute of Science, Rechovot, Israel (R.F.)
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ABSTRACT |
The flacca tomato
(Lycopersicon esculentum) mutant displays a wilty
phenotype as a result of abscisic acid (ABA) deficiency. The Mo
cofactor (MoCo)-containing aldehyde oxidases (AO; EC 1.2.3.1) are
thought to play a role in the final oxidation step required for ABA
biosynthesis. AO and related MoCo-containing enzymes xanthine dehydrogenase (XDH; EC 1.2.1.37) and nitrate reductase (EC 1.6.6.1) were examined in extracts of the flacca tomato genotype
and of wild-type (WT) roots and shoots. The levels of MoCo were found to be similar in both genotypes. No significant XDH or AO
(MoCo-containing hydroxylases) activities were detected in
flacca leaves; however, the mutant exhibited
considerable MoCo-containing hydroxylase activity in the roots, which
contained notable amounts of ABA. Native western blots probed with an
antibody to MoCo-containing hydroxylases revealed substantial, albeit
reduced, levels of cross-reactive protein in the flacca
mutant shoots and roots. The ABA xylem-loading rate was significantly
lower than that in the WT, indicating that the flacca is
also defective in ABA transport to the shoot. Significantly, in vitro
sulfurylation with Na2S reactivated preexisting XDH and AO
proteins in extracts from flacca, particularly
from the shoots, and superinduced the basal-level activity in
the WT extracts. The results indicate that in flacca,
MoCo-sulfurylase activity is impaired in a tissue-dependent manner.
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INTRODUCTION |
ABA is a plant growth regulator involved in various processes,
including the reactions of plants to environmental stress and seed
maturation (Zeevaart and Creelman, 1988 ). In higher plants ABA is
derived from an epoxy-carotenoid precursor that is oxidatively cleaved
to produce xanthoxin (Parry et al., 1988). After the cleavage reaction,
xanthoxin is converted to ABA by a series of ring modifications to
yield abscisic aldehyde, which is oxidized to ABA by AO (EC 1.2.3.1), a
MoCo-containing enzyme (Walker-Simmons et al., 1989; Leydecker et al.,
1995). In addition to AO, plant MoCo-containing enzymes include NR (EC
1.6.6.1) and XDH (EC 1.2.1.37).
XDH and AO (MoCo-containing hydroxylases) from various organisms have
been characterized as homodimers of 150-kD subunits. They have a high
degree of homology in their amino acid sequence and contain binding
sites for two Fe-S centers and a MoCo-binding region (Ori et al., 1997;
Sekimoto et al., 1997). Whereas NR requires a dioxo-Mo center
(Rajagopalan and Johnson, 1992), XDH and AO incorporate mono-oxo-MoCo
in which the second oxygen is replaced by an S ligand. AO belongs to a
multigene family (Ori et al., 1997) and appears to display a broad
range of substrate specificities (Koshiba et al., 1996; Ori et al.,
1997; Sekimoto et al., 1997; Omarov et al., 1999), among them the
oxidation of indole-3-acetaldehyde to IAA (Bandurski et al., 1995;
Koshiba et al., 1996).
The characterization of ABA-deficient mutants has been valuable in
elucidating the function of ABA and the pathway of ABA biosynthesis. A
number of mutants with reduced capacity to synthesize ABA have been
described. These include flacca, notabilis, and sit in tomato (Lycopersicon esculentum),
dr in potato, aba1 in wild tobacco,
nar2a in barley, and aba3 in Arabidopsis
(Walker-Simmons et al., 1989; Taylor, 1991; Schwartz et al., 1997). The
nar2a mutant in barley was shown to lack AO, XDH, and NR
activities, suggesting a lesion in the synthesis of the MoCo, which all
three enzyme activities require (Walker-Simmons et al., 1989). In
contrast, shoots of aba1, aba3, and flacca
apparently lack AO and XDH activities but not NR, suggesting an
additional step in MoCo biosynthesis (Leydecker et al., 1995; Marin and
Marion-Poll, 1997; Schwartz et al., 1997). The Arabidopsis
aba3 mutant may have lost its ability to replace one of the
oxygens of dioxo-MoCo by an S ligand required for AO and XDH activity
(Schwartz et al., 1997).
To our knowledge, the molecular basis for ABA mutations in tomato has
yet to be described. ABA content in flacca leaves was about
20% to 26% of that in the WT (Neill and Horgan, 1985; Linforth et
al., 1987; Taylor et al., 1988; Rock et al., 1991). Plants of
flacca display a marked tendency to wilt, apparently because of excessive transpiration resulting from a lack of control of stomata
closure (Tal, 1966). It has been suggested that the primary reason for
the wilty phenotype was the lower endogenous ABA content in the leaves
(Imber and Tal, 1970). Previous studies of flacca mutants
focused on changes in enzyme activity (Marin and Marion-Poll, 1997) or
the determinations of ABA concentration and synthesis in shoots (Imber
and Tal, 1970; Taylor et al., 1988) but not roots, although roots are
the main site of ABA synthesis (Bano et al., 1993). Thus,
characterization of the Mo-enzymes involved in hormone biosynthesis in
roots and shoots of flacca and the WT are required. The main
goal of this study was to examine the simultaneous analysis of
enzymatic and immunological characteristics of the MoCo-containing hydroxylases in the whole plant, as well as to estimate the capacity of
roots to produce and transport ABA to the shoots.
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MATERIALS AND METHODS |
Plant Material
WT and flacca seeds of tomato (Lycopersicon
esculentum Mill. cv Rheinlands Ruhm) were germinated and allowed
to establish for 14 d on wet filter paper. Uniform plants were
transplanted to pots with dune sand (96% sand, 2% silt, and 2% clay,
pH 8.25 and electrical conductivity 0.7 decisiemens
m 1) irrigated with 2.5 mM
(NH4)2SO4
as the N component of a modified one-half-strength Hoagland solution
(Hoagland and Arnon, 1938). In the greenhouse average day temperatures
during the growth period fluctuated from 20°C to 25°C, and average
night temperatures fluctuated from 8°C to 12°C. Midday PPFD in the
greenhouse was 900 to 1000 µmol m2
s1.
Tissue Extraction
Shoot and root samples were obtained from 6- to 8-week-old plants
and extracted immediately. Crude extracts for assays of NR and MoCo
were prepared as previously described (Gao et al., 1996). Crude
extracts for assays of XDH and AO in native-gel electrophoresis and
western analysis were prepared in a modified version of the method
described previously by Sagi et al. (1998). Tissue was macerated with
acid-washed sand in an ice-cold extraction medium containing 250 mM Tris-HCl (pH 8.5), 1 mM EDTA, 1 mM DTT, 5 mM L-Cys, 80 µM Na2MoO4,
10 µM antipain, 0.1 mM PMSF, 10 mM GSH, and 0.03 mM FAD. Samples of 1 g of
shoot or root were extracted in 2 and 1 mL of buffer (1:2 and 1:1,
w/v), respectively. The homogenized plant material was centrifuged at
30,000g in a refrigerated centrifuge (model RC-5, Sorvall)
at 3°C to 5°C for 15 min. The resulting supernatant was used for
subsequent assays.
Crude extracts for AO assays in vitro were prepared according to the
method of Triplett et al. (1982). Samples of 1 g fresh weight of
tissue were ground in liquid N2, and the
resulting powder was mixed with 0.25 g of polyvinylpolypyrrolidone
and then extracted in 1 mL of 50 mM potassium-phosphate
buffer, pH 7.5. The homogenized plant material was centrifuged as
described above. The supernatants were brought up to 60% saturation
with solid ammonium sulfate. After stirring for 30 min, the mixture was
centrifuged at 40,000g for 20 min. The pellet was suspended
in 1 to 2 mL of 50 mM potassium-phosphate buffer,
pH 7.5, and desalted on a 1.5- × 30-cm Sephadex G-25 column (Pharmacia) equilibrated with 50 mM
potassium-phosphate buffer, pH 7.5. Crude extracts used for XDH assays
in vitro were prepared as described for AO, using 50 mM Tris-HCl buffer, pH 8.48. All of the preceding
steps were carried out at 4°C.
Enzyme Activity and Protein Analysis
NR (EC 1.6.6.1) activity was measured in crude extracts as
described previously (Gao et al., 1996). MoCo activity in plant tissue
was estimated using the MoCo-deficient nit-1 mutant of Neurospora crassa, complemented with tomato MoCo released by
heating extracts at 80°C for 90 s, following the original
procedure of Mendel et al. (1985), as recently modified by Sagi et al.
(1997).
AO and XDH activities in vitro, before or after in vitro sulfurylation,
were assayed, monitoring the decrease of
A600 of the electron donor DCIP
(Courtright, 1967; Rajagopalan and Handler, 1967; Perez-Vicente et al.,
1988) in a spectrophotometer (Genesis-2, Milton Roy, Rochester, NY).
The 1.5-mL AO assay reaction mixture contained 500 to 1000 µg of
protein of the desalted extract, 0.002% DCIP, 0.1 mM phenazine methosulfate, and 2 mM indole-3-aldehyde in 50 mM potassium-phosphate buffer, pH 7.4. The 1.5-mL
XDH reaction mixture contained 1000 µg of protein of the desalted
extract, 0.002% DCIP, and 0.6 mM hypoxanthine in
50 mM Tris-HCl buffer, pH 8.48. AO and XDH
activities were expressed as nanomoles DCIP reduced per milligram
protein per minute. Soluble proteins in the assays were measured
(Bradford, 1976) using crystalline BSA as a reference.
Gel Electrophoresis and Analysis of Enzyme Activity
Enzyme electrophoresis and staining were carried out using
1.5-mm-thick slabs of 7.5% native-polyacrylamide gels loaded with 300 mg of shoot proteins or 100 mg of root proteins. Enzyme activities of
AO and XDH were estimated in gels by staining, after native electrophoresis, using
3(4,5-dimethylthiazolyl-2)2,5-diphenyltetrazolium-bromide, which resulted in the development of specific formazan bands. The
quantity of formazan was directly proportional to enzyme activity during a given incubation time and in the presence of excess substrate and tetrazolium salt (Rothe, 1974). Quantitative analyses were made by
scanning the formazan bands with a computing laser densitometer using
Image Quant version 3.19.4 software (Molecular Dynamics, Sunnyvale,
CA). XDH activity was determined using hypoxanthine as a specific
substrate (Mendel and Muller, 1976), and specificity was
confirmed with allopurinol, an XDH inhibitor (Leydecker et al., 1995).
AO activity was detected after immersing gels in 0.2 M
phosphate buffer, pH 8.0, for 10 min followed by gentle shaking in a
reaction mixture containing 0.1 mM phenazine
methosulfate and 1 mM
3(4,5-dimethylthiazolyl-2)2,5diphenyltetrazolium-bromide in the presence of 1 mM indole-3-aldehyde or 1 mM acetaldehyde in 0.1 M Tris-HCl buffer, pH
8.48, at 25°C.
Western Analysis of MoCo-Containing Hydroxylases
The MoCo-containing hydroxylase proteins in shoots and roots were
detected by western blotting. Native-PAGE loaded with flacca and WT crude extracts, with and without treatments of sulfurylation or
heating to 80°C for 90 s, were carried out as described above. SDS-PAGE was performed in 10% polyacrylamide gels (Laemmli, 1970). The
resulting gels with the separated proteins were then blotted onto a
nitrocellulose membrane (0.2 µm pore size; Schleicher & Schüell). Blotting time was 1.5 h at 2 mA
cm2. Immunodetection of MoCo-containing
hydroxylases was carried out with polyclonal guinea pig antibodies
raised against recombinant TAO1 (tomato
aldehyde oxidase 1)
polypeptides (Ori et al., 1997). The TAO1 sequence contains
binding sites for two Fe-S centers and the Mo-binding regions of XDH
and AO of various organisms (Ori et al., 1997). Primary antibodies were
diluted 500-fold in TBS and secondary antibodies (anti-guinea pig IgG;
Sigma) were diluted 1000-fold in TBS. Phosphatase activity was
developed by staining with 5-bromo-4-chloro-3-indolyl phosphate and
nitroblue tetrazolium.
In Vitro Sulfurylation of Crude Extracts
This procedure was carried out following a modification of the
method described previously by Wahl and Rajagopalan (1982). Detection
of AO and XDH in vitro or on polyacrylamide gels was carried out with
desalted WT and flacca extracts. One-half milliliter of root
or shoot extracts was desalted on G-25 Sephadex and incubated at 32°C
for 40 min with 10 µL of 0.1 M dithionite and 4 µL of 0.5 M Na2S; 5 µL
of 1.25 mM methyl viologen was used as an
indicator of reducing conditions, while gently flushing
N2 through the mixture to maintain anaerobic
conditions. After sulfurylation, the extracts were desalted through
Sephadex G-25 (Sigma). XDH and AO activities were determined in gels
after electrophoresis and also by in vitro assays.
Collection of Xylem Sap and ABA Determination
Xylem exudate was collected 1 h after sunrise for a period of
2 h. Shoots were removed with a smooth horizontal cut with a razor
blade 1 cm below the first leaves. Exudate was collected at 15-min
intervals followed immediately by ABA determination. ABA in shoots,
roots, and exudate of xylem sap was analyzed by ELISA with monoclonal
antibodies as described by Mertens et al. (1985).
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RESULTS |
Activities of MoCo-Containing Enzymes
MoCo-containing enzymes and MoCo levels were measured as described
in ``Materials and Methods''. The WT and flacca exhibited
significant NR activity and MoCo levels in shoots and roots. Root NR
activity in WT was 3-fold higher than in flacca. The MoCo
levels in the roots and shoots of both genotypes was similar, with
higher levels in roots than in shoots (Table
I).
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Table I.
NR and MoCo activities in WT and flacca plants
Letters following values indicate statistical significance of
separation between the varieties Duncan test; (P < 0.05, n = 3 different experiments with eight replications
each).
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Qualitative analysis of the MoCo-containing hydroxylases was carried
out in nondenaturating-PAGE. The substrates indole-3-aldehyde, acetaldehyde, and hypoxanthine (a substrate for XDH) were used as
indicators of AO activity. WT extracts exhibited at least three XDH and
AO activity bands in roots and two bands in shoots with hypoxanthine,
indole-3-aldehyde, or acetaldehyde (Fig.
1). Shoot extracts of flacca
did not exhibit detectable XDH or AO in activity gels. However, root
extracts of flacca revealed one band of MoCo-containing hydroxylase activity that appeared to comigrate with the upper band
detected in WT extracts.
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| Figure 1.
Native-PAGE of shoot and root extracts of WT and
flacca (flc) tomato genotypes showing AO
and XDH activities. Activity gels were loaded with 300 µg of soluble
protein of the crude extract of shoots or 100 µg of soluble protein
of roots and stained with the appropriate substrate as described in
``Materials and Methods''.
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Immunoblot Analysis of MoCo-Containing Hydroxylases
Native-PAGE fractionation of WT and flacca root and
shoot extracts followed by immunoblot analysis with antibodies raised against recombinant TAO1 was carried out to detect MoCo-containing hydroxylase cross-reacting proteins. The blots revealed a few slowly
migrating major bands in the WT and flacca extracts. Their mobility was similar to the activity bands detected in MoCo-containing hydroxylase substrate staining gels (compare Figs. 1 and
2). The relative amounts (density) of
protein detected in flacca were approximately 40% to 46%
and 10% to 17% of that in the WT in roots and shoots, respectively
(Fig. 2). The activity of MoCo-containing hydroxylase proteins in crude
extracts of the WT and flacca was relatively stable during
heat treatment at 80°C for 90 s (data not shown). Heat stability
of MoCo-containing hydroxylases in plant shoots was reported earlier
(Bower et al., 1978, Koshiba and Matsuyama, 1993; Montalbini, 1998).
Rapidly migrating MoCo-containing hydroxylase cross-reacting proteins,
which did not correlate with positions of substrate activity staining
in the gel, exhibited lower thermostability than the active proteins in
the roots and shoots of both genotypes (Fig. 2, lower arrows).
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| Figure 2.
Immunoblot analysis of MoCo-containing
hydroxylases. Approximately 200 µg of total soluble protein from root
and shoot extracts were fractionated on native-PAGE and examined with
anti-TAO1 antibodies. Untreated (Control) and preheated (80°C for
90 s) treatments were used. flc,
flacca tomato genotype.
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SDS-PAGE of plant extracts followed by immunoblotting analysis revealed
in the WT shoots the expected polypeptide with a molecular mass of 150 kD and three polypeptides with molecular masses of 78, 76, and 72 kD.
It is interesting that the 150-and 78-kD bands were conspicuously
absent from the flacca shoot extracts (Fig. 3).
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| Figure 3.
Immunoblot analysis of MoCo-containing
hydroxylases. Crude extracts of roots and shoots of WT and
flacca (flc) tomato plants were
fractionated on SDS-PAGE. The gel was loaded with approximately 50 µg
of total soluble protein and examined with anti-TAO1 antibodies.
Relative density measurements of the bands are indicated at the bottom
of the panels.
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Polypeptides with molecular masses of 150, 102, 78, 76, and 72 kD were
detected in root extracts of both genotypes. The relative amounts of
protein detected in roots and shoots of flacca were significantly lower than in the WT (Fig. 3, relative densities of bands
a-e). The multiplicity of bands detected may represent proteolytic
products resulting from physiological processes in the plant or
degradation during the extraction process (Ichida et al., 1993; Koshiba
et al., 1996; Ori et al., 1997).
Sulfurylation of MoCo-Containing Hydroxylases
Na2S and dithionite can directly sulfurylate
the dioxo-MoCo moiety (Wahl et al., 1982; Schwarz et al., 1997). This
procedure was applied to extracts of the WT and flacca. In
vitro sulfurylation of WT extracts under anaerobic conditions increased
enzyme activities in shoot and root extracts by nearly 2-fold, as
measured by the DCIP reduction assay (Fig.
4). The effect of in vitro sulfurylation in flacca shoot extracts was an enhancement of 10- to
100-fold for XDH and AO activities, whereas in flacca roots
it was proportional to the changes observed in the WT root extracts.
Qualitative analysis of XDH and AO levels using activity gels revealed
a similar pattern of increased activity after sulfurylation, as
observed in in vitro assays (Fig. 5). A
modest increase in XDH and AO bands was observed in the WT and
flacca roots, whereas a larger increase was observed in
flacca shoots. In vitro sulfurylation under anaerobic
conditions of flacca and WT desalted shoot crude extracts
preheated to 80°C for 90 s revealed a similar pattern of
activity as that for the unheated extract (data not shown).
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| Figure 4.
AO and XDH activities of shoot and root extracts
of the WT and flacca (flc) genotypes.
Extracts were assayed in vitro with hypoxanthine and indole-3-aldehyde
as the substrates. The assays were carried out with desalted extracts,
with or without preincubation with Na2S and dithionite.
Enzyme activities are expressed as nmol DCIP mg1
min1. Lowercase letters indicate statistical significance
of the separation between treatments carried out by the multiple range
test (Duncan test; P < 0.05, n = 5). The data
represent one of three different experiments that yielded essentially
identical results.
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| Figure 5.
Native-PAGE of XDH and AO from shoot and root
extracts of the WT and flacca (flc)
genotypes. XDH and AO activities were detected with hypoxanthine and
indole-3-aldehyde as the substrates. The assays were carried out with
desalted extracts, with or without preincubation with Na2S
and dithionite, under anaerobic conditions. The zymogram is one of
three different experiments that yielded essentially identical
results.
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ABA Determination
The considerable AO activity measured in the roots, as determined
by aldehyde-containing substrates, suggests that flacca roots may have the ability to synthesize ABA via ABA-aldehyde. The
levels of ABA measured in the shoots, roots, and xylem sap of
flacca were 23%, 67%, and 67%, respectively, of those of
the WT (Table II). The ABA xylem-loading
rate was estimated on the basis of [ABA] in sap exudate and the
exudate flow rate. A significantly lower exudate flow rate was detected
in flacca than in WT plants, implying a significantly
reduced ABA xylem loading rate (Table II).
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Table II.
ABA accumulation in plant organs and measured
exudate [ABA] and flow rate of WT and flacca
The letters following values in a column indicate statistical
significance of separation between the varieties (Duncan test; P < 0.05, n = 4). The data represent one of two
experiments. ABA was extracted, collected, and analyzed as described in
``Materials and Methods'' and by Walker-Simmons et al. (1988) using
monoclonal antibodies (Mertens et al., 1985).
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DISCUSSION |
In an attempt to elucidate the molecular lesion that causes
reduced ABA content in flacca mutants, we measured
parameters that are relevant to MoCo production, and we measured
the activity of MoCo-dependent hydroxylases. The levels of MoCo
were found to be similar in flacca and WT plants and cannot
be responsible for the flacca phenotype. flacca
lacked significant XDH and AO activities in its leaves, although
considerable MoCo levels and NR activity were observed in the tissue
(Table I). However, in the root, XDH and AO activities were readily
detected by native-PAGE activity gels and by DCIP reduction assays.
Sulfurylation of flacca leaf extracts resulted in
recovery of XDH and AO activities. The results suggest that
flacca is defective in the sulfurylation of MoCo in the
MoCo-containing hydroxylases and that the expression of this mutation
has a tissue-specific determinant. The flacca mutation
appears to be most similar to the Arabidopsis aba3 mutant (Schwartz et al., 1997). Tissue differences in the activity of MoCo
have been observed in the Cnx2 and Cnx3 genes.
These genes, responsible for defined steps in MoCo biosynthesis, were
expressed mainly in the roots of Arabidopsis and are related to MoCo
enzymes other than NR (Hoff et al., 1995).
Our observations show that the dioxo type of MoCo should not be rate
limiting in flacca. Unexpectedly, however, in
flacca roots the NR activity that is dependent on the
dioxo-type of MoCo was only 33% of the corresponding level in WT
roots. The lower NR activity may reflect modifications of NR in the
root because of the lower water potential in flacca plants
(Bradford, 1983). Reduced water potential impairs the driving force for
flow of phloem sap, which in turn compromises the level of total
measured soluble sugar in flacca roots (Johnson et al.,
1992; Guerrier and Bourgeais-Chaillou, 1994). Under conditions of
carbohydrate restriction, nitrate reduction is reduced (Radin et al.,
1978; Oaks, 1986).
Despite the lack of AO activity in shoots, considerable [ABA]s were
measured in flacca organs. They were 23%, 67%, and 67% of
the concentration found in WT shoot, root, and xylem exudate, respectively (Table II). ABA detected in flacca leaves may
have originated in the roots or may be the product of a minor shunt pathway converting ABA-aldehyde via ABA-alcohol to ABA in the shoots
(Taylor et al., 1988; Rock et al., 1991). Impaired stomatal closing of
flacca was corrected by foliar applications of ABA (Imber
and Tal, 1970) or by dipping leaf petioles into solutions containing
ABA (Neill and Horgan, 1985). This suggests that the reduced levels of
ABA and/or its reduced mobilization rate were not enough to initiate
correct stomatal closure. Our results indicate that the
flacca mutation affects ABA transport from the root to the
shoot. The reduced root pressure, resulting in a low ABA xylem-loading rate in addition to lower levels of root ABA synthesis, probably further exacerbates the ability of the plant to transport ABA to the
shoot (Table II).
Sulfurylation of shoot and root extracts in vitro using dithionite and
Na2S under anaerobic conditions activated
preexisting inactive XDH and AO proteins in flacca mutant
extracts (Figs. 4 and 5). The sulfurylation assay used desalted
extracts that probably removed free MoCo. This suggests that dioxo-MoCo
may be bound to the inactive MoCo-containing hydroxylase apoproteins and are rendered active by in vitro sulfurylation under reducing conditions.
The activity of the AO and XDH enzymes that were recovered after in
vitro sulfurylation was lower in flacca than in the WT extracts, presumably because flacca contains fewer total
(active and inactive) AO and XDH proteins. The lower number of these
MoCo-containing hydroxylase proteins in flacca may be due to
the decreased stability of the inactive enzyme molecules. Only small
amounts of the expected full-sized 150-kD monomer were observed in
flacca extracts after SDS-PAGE, although lower molecular
mass cross-reacting polypeptides were evident. Alternatively,
the biosynthesis of MoCo-containing hydroxylase proteins in
flacca may be restricted under conditions in which enzymatic
sulfurylation is either limited or absent. We note that the amount of
cross-reactive protein detected by immunoblot procedures does not
always correlate with the activity recovered. For example, in shoots,
flacca AO and XDH activity after sulfurylation was
approximately 70% of that in the WT, whereas the amount of
cross-reacting protein was only approximately 17% of that in the WT.
The antibodies used in the present work were prepared from areas
conserved among the multigene AO and XDH families. Therefore,
cross-reacting material should be taken as a global indication of
protein amounts that do not necessarily correlate with enzymatic
activity measured with a specific substrate.
Significant increases in MoCo-containing hydroxylase activities were
detected after the in vitro sulfurylation of the WT extracts, indicating that a considerable amount of inactive enzyme was present in
the flacca and WT extracts. The inactive state may be a
normal feature of MoCo-containing hydroxylase metabolism or may be a result of isolation procedures. Thus, MoCo-containing hydroxylase sulfurylase may play a regulatory role in plants, modulating the levels
of active AO and XDH during stress or development. One can speculate
that sulfurylation of MoCo-containing hydroxylases may constitute the
site stimulated by stress and the addition of
NH4+ in barley (Omarov et al., 1998) and ryegrass
(Sagi et al., 1998). In this respect, we note that the sulfurylation
step is reversible in vitro (Wahl and Rajagopalan, 1982; Schwartz et
al., 1997). The question remains, however, whether this reversibility
has biological significance.
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FOOTNOTES |
1
This work was supported by the U.S. Agency for
International Development/Cooperative Development Research
(project nos. C12-157 and CA15-024), by the Fohs Foundation, Israel
Charitable Association, and by the Jewish National Foundation
(the Ramat Negev Research and Development Project).
*
Corresponding author; e-mail gizi{at}bgumail.bgu.ac.il; fax
972-7-6596752.
Received January 29, 1999;
accepted March 9, 1999.
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ABBREVIATIONS |
Abbreviations:
AO, aldehyde oxidase.
DCIP, 2,6-dichloroindophenol.
MoCo, Mo cofactor(s).
NR, nitrate reductase.
XDH, xanthine dehydrogenase.
WT, wild type.
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ACKNOWLEDGMENTS |
The authors are grateful to Rustem Omarov for suggestions and
helpful discussions and to Genia Shichman for excellent technical assistance.
| |
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Abstract
Introduction