Plant Physiology 132:1973-1981 (2003)
© 2003 American Society of Plant Biologists
PLANTS INTERACTING WITH OTHER ORGANISMS
Induction of Hypersensitive Cell Death by Hydrogen Peroxide Produced through Polyamine Degradation in Tobacco Plants1
Hiroshi Yoda,
Yube Yamaguchi and
Hiroshi Sano*
Research and Education Center for Genetic Information, Nara Institute of
Science and Technology, Nara 630–0192, Japan
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ABSTRACT
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Screening immediate-early responding genes during the hypersensitive
response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana
tabacum) plants, we identified a gene encoding ornithine decarboxylase.
Subsequent analyses showed that other genes involved in polyamine biosynthesis
were also up-regulated, resulting in the accumulation of polyamines in
apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine
biosynthesis,  -difluoromethyl-ornithine, however, suppressed
accumulation of polyamines, and the rate of HR was reduced. In contrast,
polyamine infiltration into a healthy leaf induced the generation of hydrogen
peroxide and simultaneously caused HR-like cell death. Polyamine oxidase
activity in the apoplast increased up to 3-fold that of the basal level during
the HR, and its suppression with a specific inhibitor, guazatine, resulted in
reduced HR. Because it is established that hydrogen peroxide is one of the
degradation products of polyamines, these results indicate that one of the
biochemical events in the HR is production of polyamines, whose degradation
induces hydrogen peroxide, eventually resulting in hypersensitive cell
death.
One of the early events during the hypersensitive response (HR) in
pathogen-attacked plants is the production of reactive oxygen intermediates,
including superoxide (O2–), hydrogen peroxide
(H2O2), hydroxyl radical (OHÿ), and others.
Reactive oxygen intermediates restrict pathogens by strengthening cell walls
through oxidative cross-linking (Bradley
et al., 1992 ), by directly attacking pathogens
(Levine et al., 1994), and by
acting as signal molecules to induce defense responses such as rapid
hypersensitive cell death (Hammond-Kosack
and Jones, 1996; Alvarez et
al., 1998). This latter event prevents pathogens from spreading
from the site of entry. To examine the molecular mechanisms underlying HR in
tobacco (Nicotiana tabacum) plants, an experimental system with the
tobacco mosaic virus (TMV) and intact leaves of a tobacco cultivar carrying
the resistant (N) gene has often been used. When tobacco plants
carrying the N gene are inoculated with TMV and incubated at
30°C, at which temperature the N gene dose not function, viral
particles multiply. When transferred to 20°C (temperature shift), the
N gene is activated, resulting in lethal HR
(Gianinazzi, 1970). The system
is simple and synchronizable, making it suitable to examine signal
transduction pathways active in the HR.
Polyamines are small, positively charged aliphatic amines at cellular pH
values and therefore bind to negatively charged molecules, including nucleic
acids, acidic phospholipids, and proteins
(Cohen, 1998). Consequently,
they modulate DNA-protein (Shah et al.,
1999), and protein-protein interactions
(Thomas et al., 1999). Common
natural polyamines include putrescine, spermidine, and spermine, along with
related minor compounds and conjugated forms. Their pathway of biosynthesis is
well established (Kumar et al.,
1997). The first rate-limiting step is catalyzed by Orn
decarboxylase (ODC), which converts Orn into putrescine. Putrescine is then
successively converted to spermidine and spermine by spermidine synthase and
spermine synthase, respectively, with addition of propylamino groups derived
from decarboxylated S-adenosyl-Met, which was generated through a
reaction involving S-adenosyl-Met decarboxylase. In addition to this
major pathway, another pathway to putrescine from Arg has been proposed,
catalyzed by the Arg decarboxylase-producing intermediates, agmatine and
N-carbamoyl putrescine. Genes encoding these enzymes have been cloned
from various organisms (Walden et al.,
1997).
In animal cells, polyamines are considered to have specific roles in
embryonic development (Kusunoki and
Yasumasu, 1978), in control of the cell cycle
(Alm et al., 2000), in
carcinogenesis (Seiler et al.,
1998), and in immune system functions
(Seiler and Atanassov, 1994).
A recent study also suggested polyamines to be linked with cell proliferation
and likely with apoptosis as well (Thomas
and Thomas, 2001). This is based on the observation that the gene
encoding ODC is activated during apoptosis induced by overexpression of c-Myc,
a transcription factor belonging to the Myc oncogene family
(Bello-Fernandez et al., 1993),
which is important for both cell proliferation and apoptosis
(Packham and Cleveland, 1994).
A possible role of polyamines in apoptosis was proposed involving the
generation of hydrogen peroxide through their degradation by flavin-containing
polyamine oxidase (Ha et al.,
1997; Bonneau and Poulin,
2000).
In plants, polyamines are also thought to play important roles in growth,
development, and stress responses (Walden
et al., 1997). For example, the level of polyamines is reported to
fluctuate during plant-microbe interactions
(Walters, 2000). In tobacco
plants infected with TMV, enzymatic activity of ODC and Arg decarboxylase
increases during HR, resulting in elevated concentrations of their products
and conjugates, mainly in necrotic regions
(Martin-Tanguy et al., 1973;
Negrel et al., 1984). Also,
spermine accumulates in intercellular spaces and induces pathogenesis-related
proteins during HR (Yamakawa et al.,
1998). Accumulation of polyamines has been observed in tobacco
cultivars resistant to TMV, but not in TMV-susceptible counterparts
(Marini et al., 2001).
However, investigations of polyamine function have been mainly focused on
changes in their levels and spectrum, leaving the biological significance to
be determined. Although genetic analyses have been conducted at the gene level
(DeScenzo and Minocha, 1993;
Masgrau et al., 1997), the
available information on their impact on the HR is limited.
In this paper, we provide evidence that ODC and therefore polyamines are
critical components in induction of hypersensitive cell death during pathogen
attack in tobacco plants, and that this is largely based on production of
hydrogen peroxide through their degradation by polyamine oxidase.
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RESULTS
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Accumulation of Transcripts for Polyamine Biosynthetic Enzymes
Using the intact tobacco-TMV system, we compared the mRNA populations in
TMV-infected wild-type tobacco plants (Nicotiana tabacum cv Xanthi
nc) carrying the N gene, whose product confers resistance against
TMV, before and after temperature shift, by fluorescence differential display
(Yoda et al., 2002). Screening
identified a particular gene encoding ODC
(Yoda et al., 2002), which
converts Orn to putrescine in polyamine biosynthesis
(Walters, 2000). To determine
whether other genes involved in polyamine biosynthesis might also be activated
during the HR, RNA gel-blot analysis was performed with cDNA probes for
sperimidine synthase and S-adenosyl-Met decarboxylase together with
one for ODC. The results clearly showed that transcripts for all of them
accumulated after temperature shift, reaching maximum levels after about 12 h
(Fig. 1A). Subsequent RNA
gel-blot analysis using tobacco cv Samsun nn plant, which does not carry the
N gene, and therefore is susceptible to TMV, showed that transcripts
for ODC were not induced after temperature shift
(Fig. 1B). These results
indicated that expression of ODC is under the control of the
N gene and suggested de novo production of polyamines during the
HR.
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Figure 1. Transcript accumulation of polyamine biosynthesis-related genes. Healthy
leaves of wild-type plants with (tobacco cv Xanthi nc; A and B) or without
(tobacco cv Samsun nn) N gene (B) were harvested, inoculated with TMV
or mock, and maintained in an incubator at 30°C for 48 h and then at
20°C for appropriate time intervals as indicated in terms of post
temperature shift (h). Total RNAs were isolated, and RNA gel-blot analysis was
conducted with 32P-labeled probes for ODC, sperimidine synthase
(SPDS), and S-adenosyl-Met decarboxylase (SAMDC). Experiments were
repeated twice to confirm similar results. As an internal standard for RNA
loading, the transcript level for the actin gene was estimated. Accession
numbers: ODC, AF233849; SAMDC, AF033100; and SPDS, AB006692.
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Inhibition Effect of Polyamine Synthesis
The relationship between polyamines and the HR was then examined using two
inhibitors of polyamine biosynthesis; -difluoromethyl-Orn (DFMO) as an
irreversible inhibitor of ODC and methylglyoxalbis(guanylhydrazone) (MGBG) as
a competitive inhibitor of S-adenosyl-Met decarboxylase
(Fig. 2A). When detached leaves
were inoculated with TMV in the presence of DFMO, necrotic lesions became much
smaller in comparison with those of the controls
(Fig. 2B). Similarly, when
detached leaves were inoculated with TMV in the presence of MGBG, the size and
number of lesions also became smaller and less than those of the controls,
respectively (Fig. 2B). Because
TMV was found to proliferate almost equally in infected leaves in the presence
or absence of either DFMO or MGBG by RNA gel-blot analyses (data not shown),
small lesions were not due to variation in the quantity of TMV particles.
Thus, results from two independent pharmacological experiments strongly
indicated that polyamines contribute to hypersensitive cell death.
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Figure 2. Effects of DFMO and MGBG on lesion formation during HR. A, Polyamine
biosynthesis pathway in plants and inhibition sites by DFMO and MGBG. ADC, Arg
decarboxylase; dSAM, decarboxylated S-adenosyl-Met; SAM,
S-adenosyl-Met. B, Size of necrotic lesions. Detached leaves were
treated with water, 10 mM DFMO, or 10 mM MGBG at the
time of TMV infection (48 h before temperature shift). Lesions developed 48 h
after temperature shift in the presence of DFMO, and those developed 24 h
after temperature shift in the presence of MGBG. Controls inoculated in the
absence of drugs are shown in the left panels.
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Polyamine Accumulation in Apoplasts
Because the above-described experiments indicated polyamines to accumulate
during the HR, the amounts of polyamines were directly measured in
TMV-infected leaves. Preliminary estimation of polyamine levels showed no
change in either mock-treated or TMV-inoculated leaves after temperature shift
(data not shown). However, because spermine was reported to accumulate in the
intercellular spaces during HR against TMV infection
(Yamakawa et al., 1998), the
amounts of polyamines in apoplast were then examined. No polyamines were
detectable in apoplast of healthy uninoculated leaves and mock-treated leaves
at any time point (data not shown), indicating that temperature shift (low
temperature) did not induce accumulation of polyamines. In contrast,
putrescine and spermidine began to increase in apoplasts after temperature
shift in TMV-infected leaves (Fig.
3), reaching approximately 20 and 12 nmol, respectively, per gram
fresh weight after 50 h (Fig. 3, A and
B). Their accumulation was suppressed to less than one-half of
these values by DFMO treatment (Fig. 3, A
and B). Spermine was not detectable under the experimental
conditions employed (Fig. 3C).
These observations indicated that polyamines are one of the causative or
triggering elements for HR onset.
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Figure 3. Accumulation of polyamines in apoplast. Time course of polyamine
accumulation in apoplast of TMV-infected leaves with (black circle) or without
DFMO (white circle) treatment. At the indicated time points after temperature
shift, apoplastic fluid was extracted and benzoylated with benzoyl chloride.
The benzoylated samples were separated and quantified for putrescine (A),
spermidine (B), and spermine (C) by HPLC. Average values and SDs
are from three independent experiments each with one individual sample.
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Induction of Hydrogen Peroxide and Cell Death by Polyamines
In animal cells, it is known that hydrogen peroxide, which is produced
through oxidation of polyamines, plays a critical role in apoptosis
(Parchment, 1993;
Lindsay and Wallace, 1999). To
determine whether this is also the case for the HR in tobacco plants, each
polyamine was infiltrated into apoplast of a healthy leaf
(Fig. 4A). Two days after
infiltration, infiltrated areas with spermidine and spermine were collapsed
due to cell death, whereas water treatment and putrescine showed no effects
(Fig. 4B). When duplicated
samples were then stained with diaminobenzidine (DAB), the generation of large
amounts of hydrogen peroxide was visually observed to have accumulated in
collapsed areas (Fig. 4C).
These results suggested that polyamines are catabolized in the apoplast during
the HR, resulting in the generation of hydrogen peroxide.
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Figure 4. Induction of cell death and generation of hydrogen peroxide by polyamines.
A, Infiltration of chemicals. Indicated polyamines at the concentration of 10
mM were infiltrated into a healthy tobacco leaf. As the control,
water was also infiltrated (Water). Tested samples were putrescine (Put),
spermidine (Spd), and spermine (Spm). B, Cell collapse at infiltration sites.
Photograph was taken 48 h after infiltration. C, Detection of hydrogen
peroxide. 3,3'-Diaminobenzidine (DAB) solution was infiltrated 6 h after
the first infiltration and incubated for further 6 h. Sample leaf was briefly
boiled in ethanol and observed for hydrogen peroxide, which is seen as
reddish-brown color.
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Polyamine Degradation by Polyamine Oxidase
Putrescine (diamine), spermidine, and spermine (polyamines) are oxidized by
diamine oxidase and polyamine oxidase, respectively, yielding hydrogen
peroxide (Sebela et al.,
2001). Hence, an apoplast fluid from healthy leaves was assayed
for these enzymatic activities. The results indicated that, whereas putrescine
remained intact, spermidine and spermine were efficiently degraded in the
presence of the crude extract (Fig.
5A). The enzymatic activity was subsequently examined in the
presence of a polyamine oxidase-specific competitive inhibitor, guazatine.
Results showed that when spermidine was used as the substrate, guazatine
reduced the enzymatic activity up to 50% and 10% of the control by 10- and
100-fold excess concentrations to the substrate, respectively
(Fig. 5B). When spermine was
used as the substrate, however, guazatine was less effective, showing little
inhibition at 10-fold excess and a 40% reduction at 100-fold excess
concentrations to the substrate, respectively
(Fig. 5B). The difference might
result from spermine being the most preferable substrate for the enzyme in
vitro (Fig. 5A). Further
analyses by fractionating the apoplastic fluid through a molecular sieve
revealed the molecular size of protein having the activity to be approximately
50 kD (Fig. 5C). This size
agrees with those reported for polyamine oxidase from barley (Hordeum
vulgare; Radova et al.,
2001) and maize (Zea mays;
Tavladoraki et al., 1998). On
the basis of these observations, it was concluded that polyamine oxidase was
in fact responsible for release of hydrogen peroxide.
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Figure 5. Identification of polyamine oxidase. A, Time course analysis of polyamine
oxidase activity. Apoplastic fluid from wild-type healthy leaves was extracted
and subjected to assay at the indicated time (h). Concentrations of hydrogen
peroxide generated in the reactions containing 10 µL of apoplastic fluid
and 5 mM of the indicated substrate were estimated. Average values
and SDs are from two independent experiments, each with duplicated
samples. B, Inhibition of enzymatic activity by guazatine. Apoplast fluid from
wild-type healthy leaves was treated with the indicated concentrations of
guazatine for 30 min before polyamine oxidase activity was assayed. The
numbers in brackets represent ratios of inhibitor to substrate. Average values
and SDs are from two independent experiments, each with duplicated
samples. C, Fractionation of polyamine oxidase. Apoplastic fluid from healthy
leaves was fractionated by gel filtration. Polyamine oxidase activity of each
fraction was measured. The relative molecular mass (Mr)
was estimated from the standard curve obtained from marker proteins of albumin
(67 kD), ovalbumin (43 kD), chymotrypsinogen (25 kD), and ribonuclease A (13.7
kD).
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Polyamine Oxidase Activity during HR
The activity profile of polyamine oxidase in apoplasts during HR was then
examined. When spermidine was employed as the substrate, a constant level of
polyamine oxidase activity was found to be present in mock-treated leaves
(Fig. 6A). In TMV-inoculated
leaves, however, the level began to increase, reaching up to 3-fold that of
the control 12 h after temperature shift
(Fig. 6A). In contrast, the
activity was apparently constant during HR when spermine was used as the
substrate (Fig. 6A). These
results suggested that polyamines are readily degraded whenever they are
supplied to apoplast, and that, even so, degradation activity is enhanced by
further induction of polyamine oxidase on recognition of TMV. The catalytic
activity, however, was dependent on the substrate: approximately 10-fold
higher with spermine than with spermidine
(Fig. 6A). Putrescine was not
degraded (data not shown), consistent with the finding that it was not
catabolized by apoplastic fluid (Fig.
5A). To confirm the function of polyamine oxidase in planta,
effects of its inhibition during HR were examined. Healthy leaf cuttings were
inoculated with TMV and pretreated with guazatine 12 h before temperature
shift. The formation of necrotic lesions was weak and incomplete
(Fig. 6B), supporting the idea
that polyamine oxidase plays a critical role during hypersensitive cell
death.
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Figure 6. Polyamine oxidase activity in planta and effects of its suppression. A,
Polyamine oxidase activity in apoplast. Apoplastic fluids from leaves
inoculated with TMV (black bar) or mock (white bar) were extracted at the
indicated time period after temperature shift (h) and were subjected to
polyamine oxidase activity assay. The concentration of hydrogen peroxide
generated in the reaction containing 10 µL of apoplastic fluid and either 5
mM spermidine (left panel) or 5 mM spermine (right
panel) was estimated. Average values and SDs are from three
independent experiments, each with one individual sample. Experimentally
measured values were statistically tested and confirmed to be P 
0.02. B, Inhibition of necrotic lesion formation by polyamine oxidase
inhibition. Detached leaves were treated with 10 mM guazatine 12 h
before temperature shift. Lesions developing 24 h after temperature shift in
the absence (water) and presence (guazatine) of the inhibitor are shown in the
panels.
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DISCUSSION
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The present paper documents that polyamines produced during the HR can
serve as direct substrates for hydrogen peroxide production, which could
contribute to induction of hypersensitive cell death.
Polyamines in Hypersensitive Cell Death
Recent studies with animal cells revealed that polyamines possess dual
apparently conflicting functions, both preventing and inducing apoptosis
(Thomas and Thomas, 2001). The
prevention is based on the fact that polyamines stimulate growth and promote
passage through the cell cycle. The induction of apoptosis is considered to be
mediated by c-Myc, a potent transactivator of the ODC gene, whose product then
up-regulates polyamine synthesis. For the latter case, polyamines are
considered to be enzymatically catabolized yielding hydrogen peroxide, which
is biologically responsive. Polyamines are degraded by a variety of oxidases,
among which flavin-containing polyamine oxidases and copper-containing diamine
oxidases play major roles (Morgan,
1998,
1999). Polyamine oxidase in
mammalian cells can convert spermine and spermidine, by oxidative cleavage,
back to spermidine and putrescine, respectively, resulting in the formation of
hydrogen peroxide through the following pathway.
In fact, catabolic products of polyamine analogs by polyamine oxidase were
reported to induce programmed cell death in animal cells
(Ha et al., 1997). It has thus
been suggested that, during apoptosis of animal cells, oxidative degradation
of polyamines results in production of hydrogen peroxide, which directly
induces cell death or indirectly transmits signals leading to this outcome
(Parchment and Pierce, 1989;
Ha et al., 1997;
Bonneau and Poulin, 2000).
In plants, however, the relationship between polyamines and hydrogen
peroxide has so far not been examined in detail. During the HR against
pathogens, ODC activity and polyamines are reported to increase around and/or
in necrotic lesions (Torrigiani et al.,
1997), with accumulation in apoplast of pathogen-infected plants
(Yamakawa et al., 1998;
Walters, 2000). Hydrogen
peroxide was shown to be produced through the degradation of polyamines by
polyamine oxidase through the following pathway
(Sebela et al., 2001), which
differs from that of mammalian cells.
Hydrogen peroxide has also been considered to be a causative element for
hypersensitive cell death (Levine et al.,
1994). Thus, we speculated that the two phenomena might be closely
related each other. However, no experimental work has hitherto been performed
to support this idea, leaving open questions as to the physiological functions
of polyamines during HR and as to the source(s) of hydrogen peroxide in
planta. Our current study revealed that a set of genes involved in polyamine
biosynthesis is simultaneously up-regulated upon pathogen infection, resulting
in rapid production of polyamines. Accumulated polyamines are degraded by
polyamine oxidase in apoplast, yielding hydrogen peroxide, which efficiently
induces hypersensitive cell death. These findings clearly provide a possible
missing link between polyamines and hydrogen peroxide during HR, and shed
light on the biochemical events that constitute HR.
Degradation of Polyamines by Polyamine Oxidase
In plant cells, both polyamine oxidase and diamine oxidase are reported to
be located in apoplast (Slocum and Furey,
1991; Laurenzi et al.,
2001). In the present study, we also found polyamine oxidase
activity in apoplast fluid, and we consequently speculated that accumulated
polyamines were degraded by this enzyme in apoplast during HR, as in the case
with animal cells. The present experimental results clearly substantiate this
hypothesis by showing direct associations among polyamine production, their
degradation catalyzed by polyamine oxidase, and hydrogen peroxide formation.
The time-course analyses suggested that within several hours after
elicitation, newly synthesized polyamines are efficiently degraded by
polyamine oxidase, releasing hydrogen peroxides in apoplast. A question then
arises as to which polyamine serves as the main substrate for polyamine
oxidase in apoplasts during HR. Although spermine appeared to be the better
substrate than spermidine in vitro, the former was shown to be hardly detected
in apoplast, being less than one-twentieth that of the latter under the normal
condition (Masgrau et al.,
1997). This is consistent with our current results, showing no
detectable accumulation regardless of HR, and suggests spermine to be less
probable as the main substrate for the enzyme.
In contrast, spermidine was induced, but the level was maintained low
during the initial 30 h of HR. Such a profile change is consistent with that
of polyamine oxidase activity, showing a constant basal level regardless of
the infection and a 3-fold increase by TMV recognition. These results can be
best explained by assuming that, upon onset of HR, spermidine is newly
produced and transported into apoplast, where it is readily degraded by
polyamine oxidase. Putrescine also accumulated during the HR, but the
infiltration assay showed that it apparently induced neither hydrogen peroxide
nor hypersensitive cell death, suggesting that it has some other function.
Taken together, it is conceivable that spermidine may be the main substrate
for polyamine oxidase and therefore that the rate limiting step of this system
is the production of this polyamine. It is worthy of note that a simple
infiltration experiment here facilitated the detection of polyamine oxidase
activity in planta. This method is useful for identification of in vivo
polyamine and/or diamine oxidase activities and might be generally applicable
to other plants.
Alternative Pathways for Hydrogen Peroxide Production
One of the earliest events that occurs after pathogen recognition is
activation of NAD(P)H oxidase in plasma membranes
(Keller et al., 1998). This
results in synthesis of superoxide radicals in apoplast, which spontaneously
dismutate to give other active oxygen intermediates, including hydrogen
peroxide and hydroxyl radical (Bestwick et
al., 1997). The mechanism by which NAD(P)H oxidase contributes to
production of hydrogen peroxide temporally and spatially during HR is
currently not clear, but such a rapid NAD(P)H oxidase-dependent production of
hydrogen peroxide may be necessary to directly dispatch pathogens, to induce
cell death of infected and adjacent cells, and to enhance production of
defense signal molecules, including salicylic acid
(Leon et al., 1995). One such
signal molecule, most probably jasmonic acid
(Walters et al., 2002), might
induce expression of genes involved in polyamine biosynthesis, resulting in
accumulation and subsequent hydrogen peroxide generation through degradation
in apoplast.
It is well known that the oxidative burst after temperature shift in
tobacco plants consists of two phases; phase I is rapid and transient, and
phase II, arising a few hours after infection, persists for a longer period,
up to several hours (Allan et al.,
2001). Because of the late expression of genes involved in
polyamine biosynthesis and persistent accumulation of their products, we
speculate that hydrogen peroxide produced by polyamine degradation may
contribute to phase II. However, onset of the HR was not completely suppressed
in the presence of guazatine, which substantially reduced polyamine
degradation. This implies that some other mechanism(s) might simultaneously
operate in generation of hydrogen peroxide at the time of polyamine
catabolism. If this is the case, a question arises as to how these sources of
hydrogen peroxide, NAD(P)H oxidase, polyamine oxidase, and other(s), are
coordinately regulated, and which is more critical for production of hydrogen
peroxide during HR. Furthermore, it should be also determined which reactive
oxygen intermediates (O2–,
H2O2, OHÿ, and others) contribute most to HR.
Further investigations are required to address these questions and to
substantiate our hypothesis.
It should be mentioned that plants might be capable of using both Orn and
Arg for polyamine biosynthesis (Evans and
Malmberg, 1989). Transcripts for Arg decarboxylase are also
induced during HR (H. Yoda, Y. Yamaguchi, and H. Sano, unpublished data).
However, little evidence has so far been presented for an active involvement.
Arg decarboxylase from oats (Avena sativa) has been shown to be
localized at thylakoid membranes of chloroplasts
(Borrell et al., 1995), whereas
ODC is generally found in the cytoplasm
(Tiburcio et al., 1990). One
possibility is that plants are equipped with two alternative pathways for
polyamine biosynthesis that function independently in the plant defense
network. Another possibility is that Arg decarboxylase regulates polyamine
levels in a similar manner as in mammalian cells, in which agmatine, a product
of Arg decarboxylase, has been shown to exert effects by inducing an antizyme
for ODC (Babal et al.,
2001).
Concluding Remarks
It has thus far been a matter of speculation if hydrogen peroxide generated
through polyamine degradation contributes to the HR, including hypersensitive
cell death. The current study strongly supports this hypothesis by identifying
ODC and polyamine oxidase activities and simultaneous generation of hydrogen
peroxide during the HR in tobacco plants. To our knowledge, this is the first
concrete observation as to the biochemical role of polyamines in the HR.
However, the conclusion must be qualified because polyamines and their
conjugates have been reported to be multifunctional, raising the possibility
that they play some other role(s) even during the HR. Further analyses
including their distribution and quantification during the HR will be of help
to better understand the mechanism for plant disease resistance.
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MATERIALS AND METHODS
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Plant Materials and Treatments
Leaves of 2-month-old tobacco plants (Nicotiana tabacum cvs Xhanti
nc and Samsum nn) were inoculated with TMV (10 µg
mL–1) as previously described
(Yoda et al., 2002) and
incubated at 30°C under continuous light for 48 h and then at 20°C
(temperature shift). For treatment with polyamine biosynthesis inhibitors, 10
mM DFMO (Sigma-Aldrich, St. Louis) and 10 mM MGBG
(Sigma-Aldrich) solution, and with the polyamine oxidase inhibitor, 10
mM guazatine, these agents were absorbed at cutting sites of
detached leaves immediately after TMV infection (48 h before temperature
shift) and 12 h before temperature shift, respectively. Polyamines (10
mM each) were infiltrated into detached leaves (tobacco cv Xhanti
nc) with a 1-mL syringe without a needle. Incubation was at 25°C under
continuous light. For DAB (Sigma-Aldrich) staining, DAB-HCl solution (1 mg
mL–1, pH 3.8) was infiltrated 6 h after the first
exposure to polyamine, and incubation was performed for a further 6 h. DAB
deposits were visualized after washing leaves in boiled 100% (v/v) ethanol for
15 min to remove chlorophyll.
RNA Isolation and Gel-Blot Analysis
Total RNA was isolated by the acid guanidinium thiocyante-phenolchloroform
(AGPC) method (Chomczynski and Sacchi,
1987), and gel-blot analyses were performed as previously
described (Yoda et al., 2002).
The probe for ODC was synthesized with a pair of specific primers:
forward, 5'-GGATGGCCGGCCAAACAATC-3', and reverse,
5'-TCAGCTTGGATAAGAATAAGCG-3'. Probes for cDNAs encoding spermidine
synthase and S-adenosyl-Met decarboxylase were prepared by digesting
the plasmid containing each gene with appropriate restriction enzymes.
Polyamine Quantification
To extract polyamines from apoplast of tobacco leaves, 15 leaf-discs (15 mm
in diameter) were cut out, weighed, and submerged in water in vacuo.
Subsequently, they were subjected to centrifugation to recover apoplastic
fluid by placing them in a 10-mL syringe, which was set in a 50-mL Falcon
tube. Extracted fluid-containing polyamines were derivatized with benzoyl
chloride as described (Flores and Galston,
1982). Subsequent benzoylated polyamines were separated and
quantified by HPLC (Waters, Milford, MA) with a reverse-phase (C18) column and
an UV detector (254 nm) at room temperature. The solvent system was run
isocratically at 64% (v/v) methanol, at a flow rate of 1 mL
min–1. Standard curves for estimation were
obtained by measuring different known amounts of each polyamine.
Assay for Polyamine Oxidase
Apoplastic proteins from 500 leaf-discs (15 mm in diameter) of healthy
tobacco leaves were recovered by centrifugation as described above. This
recovery procedure was repeated twice, and second fluid was employed for
enzymatic activity. A 10-µL aliquot of recovered fluid was reacted with 5
mM putrescine, spermidine, or spermine for indicated minute(s), and
measurement of hydrogen peroxide was performed for 30 s with a luminometer
(Lumat LB 9507, EG & G Berthold, Wildbad, Germany) in a 40 µL total
volume of 62.5 mM Tris-HCl (pH 8.0) containing 125 µM
luminal (Bolwell et al., 1995).
For inhibition of polyamine oxidase activity, guazatine was added to 10-µL
aliquots of apoplast solution, and mixtures were incubated at room temperature
for 30 min before performance of chemiluminescence reactions as described
above. Produced hydrogen peroxide was measured using luminol. For assays for
TMV or mock-inoculated leaves, 18 leaf discs (15 mm in diameter) were cut out
at indicated times after temperature shift and submerged in 10 mM
Tris-HCl (pH 8.0) buffer in vacuo. The recovered second fluid was measured in
the reaction with each 5 mM polyamines for 1 min as described
above.
Gel Filtration
Apoplast fluid containing polyamine oxidase activity was collected from 500
leaf discs (15 mm in diameter) of healthy tobacco leaves as described above.
The collected fluid (about 5 mL) was concentrated to about 0.2 mL by
centrifugation with filter devices (CentricutminiU-10, Kurabou, Tokyo), and
then subjected to gel filtration on a column of 16/60 Superdex 75 pregrade
(Amersham Biosciences, Uppsala) equilibrated with 10 mM Tris-HCl
(pH 8.0) containing 0.2 M NaCl. Each 1-mL fraction was collected at
a flow rate of 1 mL min–1, polyamine oxidase
activity was measured by chemiluminescence using luminol as described above,
and fractions containing the highest activity were determined. The relative
molecular mass (Mr) was estimated from the standard curve
obtained from marker proteins.
|
ACKNOWLEDGMENTS
|
|---|
We are grateful to Drs. Takashi Hashimoto (Nara Institute of Science and
Technology) and Kenzo Nakamura (Nagoya University, Japan) for generous gifts
of cDNA clones of spermidine synthase and S-adenosyl-Met
decarboxylase, respectively. We also thank Drs. Nozomu Koizumi (Nara Institute
of Science and Technology) and Malcolm A. Moore (Intermal, Nagoya, Japan) for
valuable suggestions and critical reading of the manuscript, respectively.
Received April 2, 2003;
returned for revision April 24, 2003;
accepted May 9, 2003.
|
FOOTNOTES
|
|---|
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.024737.
1 This work was supported by the Research for the Future Program of the Japan
Society for the Promotion of Science (grant no. JSPS-RFTF00L01604). 
*
Corresponding author; e-mail
sano{at}gtc.aist-nara.ac.jp;
fax 81–743–72–5659.
|
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ABSTRACT
RESULTS