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Plant Physiol. (1999) 120: 529-538
Enhanced Expression and Activation of the Alternative Oxidase
during Infection of Arabidopsis with
Pseudomonas
syringae pv tomato1
Bert H. Simons,
Frank F. Millenaar,
Lonneke Mulder,
Leendert C. Van
Loon, and
Hans Lambers*
Graduate School of Functional Ecology, Department of Plant Ecology
and Evolutionary Biology, Section of Plant Pathology (B.H.S., L.M.,
L.C.V.L.), and Section of Plant Ecophysiology (B.H.S., F.F.M., L.M.,
H.L.), Utrecht University, P.O. Box 800.84, 3508 TB Utrecht, The
Netherlands; and Utrecht University, P.O. Box 800.84, 3508 TB Utrecht, The
NetherlandsPlant Sciences, Faculty of Agriculture, The
University of Western Australia, Nedlands, WA 6009 Australia (H.L.)
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ABSTRACT |
Cyanide-resistant ("alternative")
respiration was studied in Arabidopsis during incompatible and
compatible infection with Pseudomonas syringae pv tomato
DC3000. Total leaf respiration increased as the leaves became necrotic,
as did the cyanide-resistant component that was sensitive to
salicylhydroxamic acid. Infiltration of leaves with an avirulent strain
rapidly induced alternative oxidase (AOX) mRNA, whereas the increase
was delayed in the compatible combination. The increase in mRNA
correlated with the increase in AOX protein. Increased expression was
confined to the infected leaves, in contrast to the
pathogenesis-related protein-1, which was induced systemically.
Virtually all of the AOX protein was in the reduced (high-activity)
form. Using transgenic NahG and mutant
npr1-1 and etr1-1 plants, we established
that the rapid induction of the AOX was associated with necrosis and
that ethylene, but not salicylic acid, was required for its induction.
Increased pyruvate levels in the infected leaves suggested that
increased substrate levels were respired through the alternative
pathway; however, in the control leaves and the infected leaves,
respiration was not inhibited by salicylhydroxamic acid alone.
Increased respiration appeared to be associated primarily with symptom
expression rather than resistance reactions.
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INTRODUCTION |
Mitochondrial respiration provides the energy necessary to drive
metabolic and transport processes in cells. During electron transport
along the Cyt pathway a proton gradient is generated across the inner
mitochondrial membrane, which allows ATP synthesis. Plant mitochondria
contain an additional alternative electron-transport pathway that
branches off from the Cyt pathway at the level of ubiquinone; it is not
inhibited by cyanide and does not contribute to the formation of a
proton gradient. This pathway is therefore not coupled to ATP synthesis
(Siedow and Umbach, 1995 ). Electron transport along this alternative
pathway involves only a single quinol oxidase, termed the AOX. The
energy of electron flow through the alternative pathway is mainly lost
as heat (Moore and Siedow, 1991; Siedow and Umbach, 1995). This process
seems wasteful and the physiological significance of the alternative
pathway in the metabolism of these plants is still unclear. One example
of alternative pathway activity during an essential part of the life
cycle is in the reproduction of Araceae. The spadix is heated 10°C or
more to volatilize odoriferous compounds that attract pollinators
(Meeuse, 1975; Raskin et al., 1987). The alternative pathway is also
found in most other plant species and plant organs and thus must serve other functions as well.
Cyanide-resistant plant respiration is increased during various stress
conditions, e.g. low temperature, wounding, and plant diseases (Uritani
and Asahi, 1980; Hiser and McIntosh, 1990; Vanlerberghe and McIntosh,
1992; Purvis and Shewfelt, 1993). AOX protein levels are also increased
after wounding, infection, and low temperature conditions (Hiser and
McIntosh, 1990; Vanlerberghe and McIntosh, 1992; Lennon et al., 1997),
suggesting a role for the alternative respiration in stress
alleviation. However, we do not know whether or to what extent the
cyanide-resistant pathway also contributes to respiration under these
conditions. Enhanced operation of the alternative pathway might relieve
the Cyt pathway and prevent overreduction, thus reducing the formation
of harmful radicals (Purvis and Shewfelt, 1993; Wagner and Krab, 1995).
Nevertheless, the significance of the alternative respiratory pathway
during stress remains to be elucidated. It is interesting that
increased expression of both the AOX in Arum lily spadices
during flowering and of PRs during resistance responses in, for
example, tobacco, requires SA as a signal (Raskin et al., 1987; Rhoads
and McIntosh, 1992; Delaney et al., 1994; Lennon et al., 1997). The
latter is associated with the occurrence of SAR against further
infections. Addition of SA to cell suspensions or intact leaves of
tobacco also induces aox gene expression (Rhoads and
McIntosh, 1993; Lennon et al., 1997). Together, these results suggest
that SA acts as a signal in inducing both AOX and resistance responses
in infected plants and that the alternative pathway might be associated
with the resistant state.
The observations described above prompted us to investigate the
possible relationship between the induction of the alternative pathway
and resistance expression in Arabidopsis infection and whether SA is
involved as a signal in both responses. Arabidopsis infected with the
leaf-spotting bacterium Pseudomonas syringae is a
well-described plant-pathogen system in which several features of the
resistance response, as well as the involvement of SA, have been
established (Whalen et al., 1991; Uknes et al., 1993; Cameron et al.,
1994; Delaney et al., 1994). Using an avirulent and a virulent strain
of P. syringae we studied whether the resistance response of
the plant was associated with induction of the alternative pathway. The
expression of the aox gene was compared with the expression
of the gene encoding PR-1, which is a good marker for SA-dependent
expression of SAR in Arabidopsis (Uknes et al., 1993).
To study the significance of SA for the induction of AOX in infected
plants, transgenic and/or mutant Arabidopsis plants were used. Plants
expressing the bacterial nahG gene, encoding the enzyme
salicylate hydroxylase, which converts SA to catechol, are unable to
accumulate SA. These transgenics exhibit an increased susceptibility to
pathogens and are unable to express SAR (Delaney et al., 1994). Mutant
npr1-1 plants that are unable to express PRs or SAR upon
infection (Cao et al., 1994) are defective in the subsequent signaling
pathway. Expression of AOX was monitored in these plants in response to
infection. Because ethylene stimulates cyanide-resistant respiration in
plant organs (Laties, 1982), we also investigated the possible
involvement of ethylene. This hormone plays a role in the responses to
infections in the plant and its production is strongly increased in
infected plant tissues, coinciding with necrosis (Uritani and Asahi,
1980; De Laat and Van Loon, 1982, 1983; Brederode et al., 1991).
Expression of AOX was monitored upon infection of the Arabidopsis
etr1-1 mutant, in which the perception of ethylene was
strongly reduced (Schaller and Bleecker, 1995).
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MATERIALS AND METHODS |
Plant Material, Bacterial Strains, and Growth Conditions
Seeds of Arabidopsis ecotype Columbia (Col-0) wild-type,
nahG transgenic (Delaney et al., 1994), npr1-1
(Cao et al., 1994), etr1-1 (Bleecker et al., 1988), and
rps2-201 (Kunkel et al., 1993) mutants were sown on
quartz sand. After 2 weeks the seedlings were transferred to a mixture
of autoclaved potting soil and sand (12:5). Plants were cultivated in a
growth chamber with 9-h d (200 µmol m 2
s1 at 24°C) and 15-h night (20°C) cycles
and 65% RH. Twice a week plants were supplied with water or modified
one-half-strength Hoagland nutrient solution: 2 mM KNO3, 5 mM
Ca(NO3)2, 1 mM
KH2PO4, 1 mM MgSO4, and trace
elements, pH 7.0 (Hoagland and Arnon, 1938), containing 10 µM Sequestreen (CIBA-Geigy, Basel,
Switzerland).
Pst (Pseudomonas
syringae pv tomato) (Dong
et al., 1991; Whalen et al., 1991) strains DC3000 (pLH12 ; virulent) and DC3000 (pLH12; avirulent) were provided by Dr. A.F. Bent
(University of Illinois, Urbana). These strains were grown at 28°C on
King's medium B (King et al., 1954), containing 40 mg
L1 tetracycline. Nonpathogenic
Pseudomonas fluorescens strains WCS417 and WCS374,
originally isolated from the rhizosphere of wheat (Lamers et al., 1988)
and potato (Geels and Schippers, 1983), respectively, were grown
similarly in the absence of the antibiotic. Escherichia coli
strain DH5 , harboring pGEM-3Z constructs, was grown in Luria-Bertani
medium supplemented with 100 mg L1
ampicillin.
Plants were inoculated for 5 weeks after sowing. For treatment of the
plants, bacterial cultures were washed and resuspended in 10 mM MgCl2. Sterile 10 mM
MgCl2 was used as a control. The bacterial
suspension or the control solution was then pressure infiltrated into
the abaxial side of the leaves using a syringe without a needle
(Swanson et al., 1988). For treatment of leaves with SA, leaves were
infiltrated with neutralized SA solutions. Treatment of leaves with ACC
was carried out by dipping the leaves in a solution of 1 mM ACC and 0.01% Silwet L77 (v/v) in water (Van Meeuwen
Chemicals, Weest, The Netherlands). Controls were performed using
0.01% Silwet (v/v) in water.
Respiration of Infected Leaves
Leaves were detached, cut into four pieces with a razor blade, and
kept in the dark for 15 min before measurement. Pieces were transferred
into an air-tight cuvette containing 20 mM Hepes (pH 7.2)
and 0.2 mM CaCl2 (Atkin et al.,
1993), and O2 uptake was measured as a decrease
of O2 concentration using a Clark-type electrode
(YSI, Yellow Springs, OH). Cyanide-resistant O2
uptake was measured in the presence of 0.5 mM KCN. To
assess whether the cyanide-resistant component was due to the presence
of the alternative pathway, we used an appropriate concentration (2 mM) of the inhibitor SHAM. The effect of SHAM was also
assessed in the absence of KCN.
Quantification of Transcript Levels of AOX and PR-1
The competitive RT-PCR (Siebert and Larrick, 1992) was used to
semiquantitatively determine AOX and PR-1 transcript levels in leaves.
Poly(A+)RNA was isolated from several leaves of
three plants using the QuickPrep Micro mRNA Purification kit (Pharmacia
Biotech, Uppsala, Sweden); 200 ng was used for reverse
transcription with the Ready-To-Go T-Primed First-Strand kit (Pharmacia
Biotech). Competitive RT-PCR was then carried out using two
gene-specific oligonucleotides as primers in the amplification
reaction, 0.8 µL of the first-strand mixture containing the cDNA and
0.8 µL containing 1, 5, 10, 50, 100, and 500 pg of competitor DNA.
The gene-specific oligonucleotides were based on the published sequence
of Arabidopsis AOX (Kumar and Söll, 1992) and yield a fragment of
approximately 350 bp. A 400-bp heterologous competitor DNA fragment,
competing for the same set of primers, was obtained as described by
Siebert and Larrick (1992). After agarose-gel electrophoresis in the
presence of ethidium bromide, the resulting PCR products were
quantified upon UV illumination. The dilution of the competitor DNA,
yielding an approximate equimolar amount of product as the target cDNA, was taken as a measure of target mRNA level. Transcript levels were
expressed as relative values, taking the level in noninfected control
treatments as 1. A similar procedure was carried out to determine PR-1
mRNA levels using the primer set and competitor DNA as described by
Pieterse et al. (1996). Northern blots, probed with the Arabidopsis
AOX-specific PCR product described above, failed to show specific
bands, probably because of low AOX transcript levels. Northern analysis
using total RNA extracts, however, agreed with competitive RT-PCR
results of PR-1 mRNA levels (Pieterse et al., 1996), which confirmed
the validity of the RT-PCR results. All experiments were carried out at
least twice.
Western Blotting
Total leaf extracts were prepared from 100 mg (fresh weight) of
frozen leaf material obtained from several plants. The material was
ground in liquid N2 using a mortar and pestle and
suspended in a total volume of 400 µL of a protein sample mixture
(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and
0.001% bromphenol blue). After the sample was centrifuged for 10 min
at 14,000 rpm in an Eppendorf centrifuge to precipitate cell debris,
the proteins were separated by SDS-PAGE according to the method of
Laemmli (1970) and subsequently electrotransferred to nitrocellulose
filters using a blot-transfer buffer (25 mM Tris, 192 mM Gly, and 20% [v/v] methanol). Immunodetection of the
AOX protein was carried out according to the product protocol of the
AOX monoclonal antibody, aminooxyacetic acid (GT monoclonal
antibodies kindly provided by Dr. T.E. Elthon, University of
Nebraska, Lincoln), which was used as a primary antibody (1:50).
A conjugate of Fab fragments of anti-mouse IgG from sheep and
peroxidase (Boehringer Mannheim) was used as the secondary antibody
(1:25,000). AOX protein was then detected by chemiluminescence using a
substrate (SuperSignal ULTRA, Pierce), according to the protocol
provided by the manufacturer.
Determination of Pyruvate Concentration in Leaves
For the determination of pyruvate concentrations in leaves, about
1 g of fresh leaves was ground as described above. Preparation of
the samples and measurement of the pyruvate concentration was carried
out essentially as described by Wagner and Wagner (1995), with the
addition of an extra purification step in which the final sample
mixture was mixed with active C and subsequently filtered. Conversion
of NADH to NAD+ in the presence of lactate
dehydrogenase (Boehringer Mannheim) was recorded at a wavelength of 340 nm.
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RESULTS |
Total and Cyanide-Resistant, SHAM-Sensitive Respiration in
Leaves Infiltrated with Avirulent or Virulent Bacterial Strains
To determine whether infiltration of Arabidopsis leaves with
either a virulent or an avirulent strain of Pst affected
respiratory activity, we measured rates of O2
uptake of infected leaves and mock-infiltrated leaves. Leaves
infiltrated with the avirulent strain collapsed within 1 d because
of the HR, whereas those infiltrated with the virulent strain started
to necrose 2 d after infiltration. Infiltration of the leaves with
107 CFU mL1 avirulent
pathogen resulted in a rapid increase in total and in
cyanide-resistant, SHAM-sensitive O2 uptake (Fig.
1A). SHAM did not affect leaf respiration
in the absence of KCN (data not shown). The compatible plant-pathogen
combination showed a similar, but delayed, change in the total and
cyanide-resistant, SHAM-sensitive O2 uptake (Fig.
1A). Noninfected leaves of plants that were infiltrated with the
avirulent strain did not show a systemic increase in cyanide-resistant,
SHAM-sensitive respiration (Fig. 1B). The increase of total
O2 uptake in both the compatible and the
incompatible combination coincided with an increase in the
KCN-resistant component that was sensitive to SHAM (i.e. the
alternative pathway). However, the lack of an effect of SHAM in the
absence of KCN provides no information concerning the contribution of
the alternative pathway to respiration in the absence of inhibitors.
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| Figure 1.
Total and cyanide-resistant, SHAM-sensitive
O2-uptake rates of wild-type Arabidopsis leaves infiltrated
with avirulent or virulent strains of Pst, expressed in
units of fresh mass (FM). A, Leaves infiltrated with suspensions
containing 107 CFU of the pathogens per mL. Circles,
Control; triangles, avirulent pathogen; and squares, virulent pathogen.
Open symbols indicate total O2-uptake of the leaves; closed
symbols indicate cyanide-resistant, SHAM-sensitive O2
uptake. B, Systemic, noninfiltrated leaves of mock plants infiltrated
on lower leaves 3 d earlier. Results are mean values ± SE; n = 4.
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Induction of AOX in Response to Infection
AOX transcript levels were determined at intervals in leaves after
infiltration with Pst, with AOX-specific primers yielding a
350-bp DNA fragment. To test whether this fragment was specific for
AOX, the fragment was ligated into a pGEM-3Z vector and transferred into E. coli strain DH5. From the resulting clones, 20 were subjected to a second amplification round, using degenerate
aox-specific nested primers based on highly conserved
sequences of plant AOX, as reported by Whelan et al. (1996). All clones
gave a PCR product of the expected size (about 180 bp). Two of the
clones containing the 350-bp fragment were also subjected to
DNA-sequence analysis. The DNA sequence of the fragment was identical
to the matching part of the published aox sequence from
Arabidopsis, confirming that the amplification product of 350 bp
corresponded to AOX.
In the incompatible combination, AOX transcript levels were enhanced
10-fold within 6 h after infiltration (Fig.
2A). After 22 h, just before total
collapse of the leaves (after which mRNA could no longer be isolated),
AOX transcript levels were increased 100-fold. Leaves infiltrated with
the virulent strain showed a similar increase of AOX transcripts;
however, it was delayed by about 1 d (Fig. 2A). These differential
time-dependent induction patterns were associated with the rate of
necrosis development in the infiltrated leaves. Noninfected leaves of
infected plants did not show any induction after infiltration with
either the avirulent or the virulent Pst strain, indicating
that there was no measurable systemic increase in AOX transcripts (Fig.
2B).
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| Figure 2.
AOX and PR-1 transcript levels in Arabidopsis
treated with avirulent (avir) or virulent (vir) Pst.
Leaves were infiltrated with bacterial suspensions containing
107 CFU, and transcript levels were determined in the
infiltrated leaves and in the unaffected, systemic leaves of the same
plants. Values are given relative to the control level (set at 1) in
mock-infiltrated leaves. ND, Not detectable.
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To compare the induction of AOX transcripts with that of SAR-associated
PR-1 mRNA, we measured PR-1 transcript levels in the same samples. The
results (Fig. 2, C and D) show that, like AOX transcripts, the PR-1
transcript levels increased upon infiltration of the Pst
strains into the leaves. Induction of PR-1 occurred earlier after
infiltration with the avirulent strain than with the virulent strain,
but it reached similar levels by 22 h. In contrast to the AOX
transcript levels, however, PR-1 transcript levels were increased
systemically. This increase developed more rapidly in the incompatible
than in the compatible plant-pathogen combination (Fig.
3, C and D). Identical results were
obtained in independent replicates.
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| Figure 3.
Detection of AOX protein in Arabidopsis leaves
treated with avirulent or virulent Pst. AOX was detected
on western blots, using immunological staining of the protein. Top,
Treated leaves: lanes 1 to 3, at 0, 24, and 48 h, respectively,
after infiltration with the avirulent Pst strain; lanes
4 to 6, at 0, 24, and 48 h, respectively, after infiltration with
the virulent Pst strain; and lanes 7 to 9, at 0, 24, and
48 h, respectively, after mock infiltration. Bottom, Nontreated
leaves (except lane 5): lanes 1 and 2, at 0 and 22 h,
respectively, after infiltration with the avirulent Pst
strain; lanes 3 and 4, at 24 and 48 h, respectively, after
infiltration with a virulent Pst strain; lane 5, control
level in treated leaf immediately after mock infiltration (0 h); and
lanes 6 to 8, at 0, 22, and 48 h. respectively, after mock
infiltration.
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Two Pseudomonas fluorescens strains (WCS374 and WCS417) were
used as nonpathogenic controls to determine whether the induction of
AOX was specific for an interaction with a pathogen. Neither of these
two nonpathogenic strains induced AOX within 2 d after infiltration of the leaves with 107 CFU
mL1 (not shown), nor did they cause any visible
symptoms in the leaves for the duration of the experiment. These
results indicate that AOX induction is associated with infection of
either a susceptible host by a virulent strain or a resistant host by
an avirulent strain of a pathogen rather than with bacterial
infiltration.
It is well established that there is a linear relationship between the
concentration of avirulent bacteria infiltrated into a leaf and the
extent of necrosis (Turner and Novacky, 1974). It has been estimated
that leaves collapse completely when about 25% of the plant cells are
involved in the HR, which occurs upon infiltration with a concentration
of about 107 CFU mL1. To
determine the extent of the correlation between the concentration of
the bacterial suspension and AOX expression, leaves were infiltrated with a range of bacterial concentrations. When 5 × 105 CFU of the avirulent strain per mL was used,
the leaves did not show visible symptoms, whereas a concentration of 2 to 4 × 106 CFU mL1
resulted in a delayed necrosis becoming visible 4 d after
infiltration. Nevertheless, infiltration of no more than 2 × 106 CFU of the avirulent pathogen per mL resulted
in a rapid, substantial increase in AOX transcripts 6 h after
infiltration. No increase in AOX transcripts was detectable in leaves
infiltrated with 5 × 105 CFU of the
virulent pathogens per mL (Table I).
Leaves infiltrated with less than 107 CFU of the
compatible pathogen per mL also showed delayed disease symptoms, which
was associated with a delayed AOX induction (Table I).
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Table I.
Relative AOX transcript levels in leaves of
Arabidopsis ecotype Col-0 infiltrated with different concentrations of
avirulent or virulent Pst
The AOX transcript level in the control treatment (mock) is set at
1.
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Immunodetection of AOX Protein Reveals That It Mainly Occurs in a
Reduced (High-Activity) Form in Infected Plant Leaves
To study whether the induction of the AOX mRNA after bacterial
infiltration is accompanied by a higher concentration of the AOX
protein, AOX was analyzed by western blotting. Leaves infiltrated with
avirulent or virulent Pst showed an increase in AOX protein (Fig. 3A) in accordance with the increase in AOX transcript levels. Two
major bands were visible on western blots, one larger than the 30.6-kD
marker and one with an apparent molecular mass that was smaller than
the 30.6-kD marker. As was found previously (Elthon et al., 1989), a
band that is smaller than the 17.8-kD marker appeared as well; we will
not further discuss this smallest band. As suggested by Kumar and
Söll (1992), the smaller protein might represent a partial
degradation product of the larger protein. However, since AOX in
Arabidopsis is encoded by a multigene family (Saisho et al., 1997), as
it is in soybean (Whelan et al., 1996), the different bands may reflect
the expression of different genes. The intensity of both larger bands
was enhanced in the pathogen-infiltrated leaves, and two smaller bands
became visible. No readily detectable increase of AOX protein was found
in noninfiltrated leaves of treated plants, in agreement with the
finding that AOX transcript levels were not increased systemically.
In vivo the AOX enzyme occurs as a dimer, the subunits of which are
linked by disulfide bridges. The oxidized form, in which the two
monomers are covalently linked, is much less active than the reduced
form of the AOX dimer (Umbach and Siedow, 1993). A large pool of
reduced AOX was detectable in the samples from leaves infiltrated with
either avirulent or virulent Pst (Fig. 3), indicating that a
large portion of the enzyme was in its activated form. As in the intact
roots of Poa annua (Millenaar et al., 1998), the oxidized
form of the protein could be visualized by this method only after a
prolonged exposure of the film.
Induction of AOX in Compatible and Incompatible Plant-Pathogen
Combinations Is Not Dependent on SA but Is Associated with Necrosis
The observation that PR-1 is strongly induced systemically,
whereas AOX is not, indicates that the induction pathways are different
or that the threshold level for the induction of AOX is not reached in
systemic noninfected leaves. To investigate a possible relationship
between systemic effects of SA and the induction of AOX mRNA, leaves of
an Arabidopsis npr1-1 mutant that is unable to express PRs
or SAR upon infection with avirulent Pst were infiltrated
with the pathogen. AOX was normally induced in the infiltrated leaves
(Table II), whereas, as expected, no PR-1
mRNA was detected (not shown). This result clearly demonstrates that
the induction pathways of PR-1 (and SAR) and AOX after infection are
different.
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Table II.
Relative AOX transcript levels in leaves of
different transgenic and/or mutant Arabidopsis plants infiltrated with
avirulent (Avir) or Virulent (Vir) Pst
Leaves were infiltrated with 107 CFU per mL or, as a
control, mock infiltrated with 10 mM MgCl2. For
a description of the different transgenics and/or mutants, see the
text. The AOX transcript level of the control (mock-treated) leaves of
the wild type is set at 1. For wild-type and NahG
plants, PR-1 transcript levels are presented in parentheses, with
control (mock-treated) leaves of the wild type set at 1.
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To investigate whether exogenously applied SA induces AOX in
Arabidopsis, leaves were infiltrated with solutions containing 0.01, 0.1, 0.5, or 1 mM SA. Higher concentrations resulted in toxic effects. At the applied SA concentrations, there was no increase
in AOX transcript levels after 6, 24, or 48 h (not shown). In
contrast, 1 d after infiltration with 1 mM SA, PR-1
transcript levels had increased approximately 10-fold; 0.1 mM was the lowest SA concentration that gave a slight
increase (5-fold) in PR-1 transcripts, which was detected 1 d
after infiltration (not shown).
A possible involvement of SA in AOX induction during pathogenesis was
further investigated in leaves infiltrated with the virulent pathogen
using SA-degrading transgenic nahG plants. No major
differences in disease development were observed compared with those in
wild-type Arabidopsis. Determination of the AOX transcript levels
showed that the AOX induction during the first 22 h was similar in
wild-type and nahG plants (Table II). In contrast, there
were clear differences in symptom development between wild-type and
nahG plants that were infiltrated with the avirulent
pathogen. In the transgenic nahG plants, symptoms appeared
later and necrosis developed as in the compatible combination. Also,
AOX induction in these leaves was delayed and similar to the compatible
combination (Table II). PR-1 transcript levels remained very low in
NahG plants, in contrast to those in the wild type (Table
II), confirming that SA levels in the transgenic plants do not increase
after infection. Thus, the finding that AOX transcript levels were
significantly increased during both the compatible and incompatible
interaction indicates that SA is not essential for AOX induction during
these infections.
On the other hand, the finding that in the NahG plants the
"fast" AOX induction 6 h after infiltration with the avirulent pathogen was abolished (Table II) suggested that SA-dependent processes
must be involved in AOX induction during the incompatible interaction.
Because the HR was also delayed in the transgenic NahG
(Delaney et al., 1994), HR might be associated with the fast AOX
induction. To test this hypothesis, leaves of the Arabidopsis mutant
rps2-201 were infiltrated with the normally avirulent
pathogen Pst DC3000 (avrRpt2). This mutant plant
contains a defect in the rps2 resistance gene (Kunkel et
al., 1993; Yu et al., 1993) and is therefore compatible with
Pst DC3000 (avrRpt2). Upon infiltration of the
leaves with this pathogen, the fast AOX induction observed in the
leaves of wild-type Arabidopsis was absent in the rps2-201 plants. Instead, the induction of AOX in the mutant plant was similarly
timed as that upon infiltration with the virulent pathogen (Table II).
This result implies that the fast AOX induction, as with the
incompatible interaction, is indeed associated with the rapid cell
death that occurs during an HR. The comparatively slow induction of AOX
in the compatible combinations is most likely associated with delayed
necrosis of the plant tissue.
Total Respiration and Cyanide-Resistant, SHAM-Sensitive
O2-Uptake Rates Are Not Affected in Leaves of
etr1-1 Plants Infiltrated with an Avirulent Pst
Strain
To study the possible involvement of ethylene-dependent processes
in AOX induction upon infection, O2 uptake was
measured using wild-type and etr1-1 mutant plants. Leaves
were infiltrated with the avirulent Pst strain, because the
respiratory increase of infected leaves of the wild type was most
predictable with this pathogen. To measure O2
uptake during a prolonged period (2 d), leaves of wild-type plants were
infiltrated with bacterial suspensions, containing from 5 × 105 to 107 CFU. With 5 × 105 CFU, no disease symptoms were visible
until 6 d after infiltration; however, there was a considerable
and rapid increase in cyanide-resistant, SHAM-sensitive
O2 uptake, which remained constant between 20 and 48 h (Fig. 4A). In contrast to
wild-type Arabidopsis, leaves of etr1-1 mutant plants,
infiltrated with the same bacterial suspensions, did not show an
increase in the total or cyanide-resistant, SHAM-sensitive rate of
O2 uptake with time (Fig. 4B). These results
indicate that ethylene-dependent processes are required for the
induction of AOX during infection of Arabidopsis with avirulent
Pst.
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| Figure 4.
Total and cyanide-resistant, SHAM-sensitive
O2-uptake rates of wild-type (A) and etr1-1
(B) Arabidopsis leaves infiltrated with an avirulent strain of
Pst. Leaves were infiltrated with suspensions containing
5 × 105 CFU of the pathogens per mL. Circles,
Control; triangles, avirulent pathogen. Open symbols, Total
O2 uptake of the leaves; closed symbols, cyanide-resistant,
SHAM-sensitive O2 uptake. Results are mean values ± SE; n = 4. FM, Fresh mass.
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Expression of AOX Is Dependent on Ethylene
For a molecular analysis of the possible involvement of ethylene
in the induction of AOX, AOX transcript levels were monitored in leaves
of wild-type and etr1-1 plants infiltrated with the avirulent or virulent Pst strain. In the etr1-1
plants the AOX transcript levels were only slightly increased upon
infection with the avirulent Pst strain, compared with those
in the wild type. The compatible combination did not result in a
detectable increase in AOX transcript levels in etr1-1(data
not shown).
Pyruvate Accumulates in Tissue Infiltrated with the Pathogenic
Bacteria
Infected plant tissues accumulate hexoses (Farrar, 1992), which
can be used in several defense-related biosynthetic pathways and/or
converted into large amounts of respiratory substrate. When the Cyt
pathway is unable to cope with the latter, one might imagine that the
alternative pathway is required. Flooding of the Cyt pathway with a
respiratory substrate is likely to result in the accumulation of
pyruvate (Vanlerberghe and McIntosh, 1996). Pyruvate is also a strong
activator of AOX in vitro (Millar et al., 1993) and thus might be a
feed-forward regulator of the alternative pathway in vivo. Therefore,
levels of pyruvate in the leaf tissues infiltrated with the avirulent
pathogen were determined. Necrotizing leaves showed a 4-fold increase
of the pyruvate concentration 22 h after infiltration (Table
III). It is unlikely that the bacteria contributed significantly to these increased pyruvate levels in the
infected leaves, because their fresh weight was less than 0.01% of the
total fresh weight of the infected leaves. These results suggested that
the mitochondria was flooded with respiratory substrate and that the
alternative electron-transport pathway was fully activated.
View this table:
[in this window]
[in a new window]
|
Table III.
Pyruvate concentration in leaves of
Arabidopsis 22 h after infiltration of the leaves with
avirulent Pst
The data are from two independent experiments. The bacterial suspension
contained 107 CFU mL1.
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| |
DISCUSSION |
An increase in respiration is a widespread phenomenon in
plant-pathogen interactions (Farrar, 1992; Lennon et al., 1997). The
present study suggests that the alternative pathway might be a major
contributor to the increase in respiration in Arabidopsis upon
infection with avirulent and virulent P. syringae strains. This was evidenced by strongly increased AOX transcript levels, increased amounts of reduced (high-activity) AOX protein, and increased
cyanide-resistant, SHAM-sensitive O2-uptake rates
in the infected leaves. Because SHAM did not affect leaf respiration in
the absence of KCN, we have no evidence that the alternative pathway
contributed to the enhanced leaf respiration. Infection with the
tobacco mosaic virus in tobacco also enhances AOX protein levels, but
it has no effect on the contribution of the alternative pathway to leaf
respiration (Lennon et al., 1997).
Because SA acts as a signal in the induction of AOX in Arum
lily flower stalks and in tobacco leaves and as a signal of PRs and SAR
during resistance responses, the following questions have been
addressed: Is AOX induced during plant diseases? Is such induction
associated with the expression of PRs? Do SA and/or ethylene act as a
signal(s) in the induction of AOX during infection in Arabidopsis
leaves? In Arabidopsis AOX was strongly induced in the infected tissue
with both the incompatible and the compatible combination. In treated
leaves the expression of AOX and PR-1 mRNA upon infection was
correlated in time, but in systemic leaves AOX transcript levels were
not increased, in contrast to the transcript levels in PR-1. This is at
variance with the results in tobacco, in which AOX protein levels
increased both in tobacco mosaic virus-infected and in noninfected
systemic leaves (Lennon et al., 1997). Application of SA did not induce
AOX in Arabidopsis, and AOX induction was not abolished in
NahG plants, indicating that SA accumulation is not
essential in the induction of AOX due to bacterial infection in
Arabidopsis. In contrast to AOX transcript levels, PR-1 transcript levels remained low in the NahG plants, even in the
infiltrated leaves. Because PR-1 expression is associated with SA
accumulation, AOX and PR-1 expression in Arabidopsis must be regulated
by different signals. Direct evidence showing the involvement of
different signaling pathways for induction of PR-1 and AOX came from
experiments with the npr1-1 mutant, which is unable to
express PRs and SAR. Immediately upon infiltration of the leaves with
either the avirulent or the virulent Pst strain, this mutant
showed AOX-induction patterns (in time and place) similar to those in
the wild-type plant.
Although SA was clearly not essential for induction of AOX, fast
induction of AOX during the avirulent plant-pathogen combination was
abolished in both nahG and npr1-1 plants,
indicating that SA-dependent processes do play a role. Fast induction
of AOX in the avirulent plant-pathogen combination was associated with
HR. Lower amounts of the avirulent pathogen resulted in lower AOX transcript levels in the leaves (Table II), indicating a direct relationship between the level of induction and the amount of tissue
affected. The delayed induction of AOX in the compatible combination
suggests that HR accelerates but is not essential for the induction of
the AOX. Apparently the AOX induction is associated with the
development of necrosis. It is well known that respiratory changes
occurring as a result of infection in plants are not limited to the
infected cells but also take place in the surrounding tissue (Farrar,
1992). Therefore, the increases in AOX transcript levels, AOX protein,
and total and cyanide-resistant, SHAM-sensitive respiration in
Arabidopsis leaves infected with Pst most likely take place
in the vicinity of necrotizing plant cells. Previous reports have shown
that the increases in total and cyanide-resistant plant respiration can
be stronger in a susceptible plant compared with those in a resistant
one (Farrar and Rayns, 1987). Such increases are caused by the
localization of the pathogen in the avirulent plant-pathogen
combination; consequently, less tissue is affected. The major
difference between the reaction of a resistant and a susceptible plant
appears to be the speed at which respiration increases and AOX
induction occurs.
Ethylene plays a major role in the induction of AOX upon infection.
Based on the transcript levels, AOX induction in the etr1-1 mutant plants was almost completely abolished in leaves infiltrated with either the virulent or avirulent pathogen. Moreover,
O2-uptake rates in the presence of cyanide were
not increased in the leaves of the etr1-1 plant infiltrated
with the avirulent pathogen. Taken together, the results suggest that
strong, local AOX induction during infections is associated with
ethylene production at the site of infection. Ethylene is strongly
increased at an early stage in an HR (De Laat and Van Loon, 1983;
Boller, 1991), indicating a functional role for the rapid action
of AOX in the incompatible combination.
Although local increases of AOX transcripts corresponded well with
increased AOX protein levels and increased cyanide-resistant, SHAM-sensitive respiration, the actual contribution of the alternative pathway to the respiration in infected tissue was not determined. This
would require the use of the O2 discrimination
technique (Day et al., 1996). The AOX pool in the plant tissue
ultimately determines the maximum contribution of the alternative
pathway to total respiration. The actual contribution can be strongly modulated, however, by the state of the disulfide bridge between two
AOX subunits, because the reduced dimer is much more active than the
oxidized, covalently bound form. Our western blotting results show
that, at least during the later stage of infection, the amount of the
reduced (high-activity) AOX dimer was increased. This suggests that AOX
could significantly contribute to respiration in these tissues.
Furthermore, pyruvate levels in the Arabidopsis leaves infiltrated with
the avirulent pathogen were strongly increased. This suggests that the
mitochondria are flooded with respiratory substrate and activator, a
condition that is likely to favor full operation of the alternative
pathway in the affected cells.
How can the resistance response and the energy-wasting alternative
pathway be linked at the physiological level? A schematic representation of the main components and pathways involved in the
physiological response of plant cells to infection and other stresses,
such as wounding and chilling injury, are depicted in Figure
5. Infections and other stresses are
associated with enhanced biosynthesis of aromatic compounds. The
oxidative pentose-phosphate pathway provides erythrose-4-phosphate,
which condenses with PEP into the precursor of numerous
phenylpropanoids that are implicated in resistance reactions. The NADPH
that is produced might act as a reductant in numerous stress-related
reactions. Another source for NADPH is the cytosolic NADP-malic enzyme,
which catalyzes the oxidation of malate to pyruvate and
CO2. This enzyme is induced upon the addition of
elicitors, suggesting that it is involved in primary metabolism changes
after infection (Schaaf et al., 1995). The pentose-phosphate pathway,
which may account for 90% of the breakdown of Glc during infections
(Shaw and Samborski, 1957), bypasses the allosteric adenylate control
of glycolysis. In combination with the enhanced activity of the
cytosolic NADP-malic enzyme, this can lead to an accumulation of
pyruvate, particularly if the Cyt pathway in the affected tissue is
somehow restricted. That the operation of the alternative pathway does
not contribute to the formation of ATP might not be disadvantageous,
because many defense-related reactions require NADPH rather than ATP, as shown in Figure 5.
View larger version (20K):
[in this window]
[in a new window]
| Figure 5.
Major metabolic pathways involved in the
resistance response to pathogens and its association with respiration.
Important intermediates involved in the biosynthesis of several defense
compounds are depicted in boxes. Numbers refer to major control points
for glycolysis due to allosteric inhibition of phosphofructokinase by
ATP (1) and inhibition of oxidative phosphorylation by limiting amounts
of ADP (2). TCA cycle, Tricarboxylic acid cycle; OAA, oxaloacetic
acid.
|
|
It has been proposed that operation of the alternative pathway during
environmental stresses might (partly) relieve the mitochondrial electron-transport pathway (Purvis and Shewfelt, 1993; Wagner and Krab,
1995). This could prevent overreduction that might result in the
formation of harmful radicals. Formation of O2
radicals is instrumental in necrotization, but limited lesion formation during avirulent reactions requires that surrounding tissues be protected. This is clearly illustrated by the occurrence of disease lesion mimics, in which spontaneous necrosis may spread because of
mutations (Dietrich et al., 1994). During infections the formation of
radicals might worsen disease symptoms. For a successful resistance response the production of radicals might be an important feature, because the performance of a plant during disease is determined not
only by its ability to localize the pathogen but also by the its
capacity to minimize tissue damage. Because several other stress
conditions induce AOX, we speculate that cyanide-resistant respiration
is important to the plant for acclimation to adverse conditions. How
much this enables the plant to cope with such stresses awaits
experiments with plants that cannot express AOX.
| |
FOOTNOTES |
1
This work was supported by grants from the
Netherlands Organization for Scientific Research.
*
Corresponding author; e-mail hlambers{at}cyllene.uwa.edu.au; fax
61-8-9380-1108.
Received December 3, 1998;
accepted February 23, 1999.
| |
ABBREVIATIONS |
Abbreviations:
AOX, alternative oxidase.
CFU, colony-forming
unit(s).
HR, hypersensitive reaction.
KCN, potassium cyanide.
PR, pathogenesis-related protein.
RT-PCR, reverse transcriptase-PCR.
SA, salicylic acid.
SAR, systemic acquired resistance.
SHAM, salicylhydroxamic acid.
| |
ACKNOWLEDGMENTS |
The authors wish to thank Dr. Patrick M. Finnigan (Australian
National University, Canberra) for critically reading the
manuscript, Dr. Jim Whelan (University of Western Australia, Nedlands)
for providing advice and the AOX-specific degenerant primers,
Dr. Thomas E. Elthon (University of Nebraska, Lincoln) for
providing the AOX-specific monoclonal antibodies, and Dr. Andrew Bent
(University of Illinois, Urbana) for providing the Pst
strains. We would also like to thank Erik De Vlieger for his help with
obtaining the data concerning the effects of SHAM on leaf respiration.
| |
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S. Akhter, H. C. McDade, J. M. Gorlach, G. Heinrich, G. M. Cox, and J. R. Perfect
Role of Alternative Oxidase Gene in Pathogenesis of Cryptococcus neoformans
Infect. Immun.,
October 1, 2003;
71(10):
5794 - 5802.
[Abstract]
[Full Text]
[PDF]
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A. Gilliland, D. P. Singh, J. M. Hayward, C. A. Moore, A. M. Murphy, C. J. York, J. Slator, and J. P. Carr
Genetic Modification of Alternative Respiration Has Differential Effects on Antimycin A-Induced versus Salicylic Acid-Induced Resistance to Tobacco mosaic virus
Plant Physiology,
July 1, 2003;
132(3):
1518 - 1528.
[Abstract]
[Full Text]
[PDF]
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C. Dutilleul, M. Garmier, G. Noctor, C. Mathieu, P. Chetrit, C. H. Foyer, and R. de Paepe
Leaf Mitochondria Modulate Whole Cell Redox Homeostasis, Set Antioxidant Capacity, and Determine Stress Resistance through Altered Signaling and Diurnal Regulation
PLANT CELL,
May 1, 2003;
15(5):
1212 - 1226.
[Abstract]
[Full Text]
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J. Vahala, R. Ruonala, M. Keinanen, H. Tuominen, and J. Kangasjarvi
Ethylene Insensitivity Modulates Ozone-Induced Cell Death in Birch
Plant Physiology,
May 1, 2003;
132(1):
185 - 195.
[Abstract]
[Full Text]
[PDF]
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S. H. Ordog, V. J. Higgins, and G. C. Vanlerberghe
Mitochondrial Alternative Oxidase Is Not a Critical Component of Plant Viral Resistance But May Play a Role in the Hypersensitive Response
Plant Physiology,
August 1, 2002;
129(4):
1858 - 1865.
[Abstract]
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M. J. Considine, R. C. Holtzapffel, D. A. Day, J. Whelan, and A. H. Millar
Molecular Distinction between Alternative Oxidase from Monocots and Dicots
Plant Physiology,
July 1, 2002;
129(3):
949 - 953.
[Full Text]
[PDF]
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F. F. Millenaar, M. A. Gonzalez-Meler, J. N. Siedow, A. M. Wagner, and H. Lambers
Role of sugars and organic acids in regulating the concentration and activity of the alternative oxidase in Poa annua roots
J. Exp. Bot.,
May 1, 2002;
53(371):
1081 - 1088.
[Abstract]
[Full Text]
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O. W. Nagel, S. Waldron, and H. G. Jones
An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities
Plant Physiology,
November 1, 2001;
127(3):
1279 - 1286.
[Abstract]
[Full Text]
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F. F. Millenaar, M. A. Gonzàlez-Meler, F. Fiorani, R. Welschen, M. Ribas-Carbo, J. N. Siedow, A. M. Wagner, and H. Lambers
Regulation of Alternative Oxidase Activity in Six Wild Monocotyledonous Species. An in Vivo Study at the Whole Root Level
Plant Physiology,
May 1, 2001;
126(1):
376 - 387.
[Abstract]
[Full Text]
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Top
Abstract
Introduction