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Plant Physiol. (1998) 118: 1213-1222
Spermine Is a Salicylate-Independent Endogenous Inducer for Both
Tobacco Acidic Pathogenesis-Related Proteins and Resistance against
Tobacco Mosaic Virus Infection1
Hiromoto Yamakawa,
Hiroshi Kamada,
Masaya Satoh2, and
Yuko Ohashi*
Institute of Biological Sciences, University of Tsukuba, Tsukuba,
Ibaraki 305, Japan (H.Y., H.K.); and Department of Molecular Genetics,
National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan (M.S., Y.O.)
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ABSTRACT |
Intercellular
spaces are often the first sites invaded by pathogens. In the spaces of
tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco
(Nicotiana tabacum L.) leaves, we found that an inducer
for acidic pathogenesis-related (PR) proteins was accumulated. The
induction activity was recovered in gel-filtrated fractions of low
molecular mass with a basic nature, into which authentic spermine (Spm)
was eluted. We quantified polyamines in the intercellular spaces of the
necrotic lesion-forming leaves and found 20-fold higher levels of free
Spm than in healthy leaves. Among several polyamines tested,
exogenously supplied Spm induced acidic PR-1 gene
expression. Immunoblot analysis showed that Spm treatment increased not
only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein
accumulation. Treatment of healthy tobacco leaves with salicylic acid
(SA) caused no significant increase in the level of endogenous Spm, and
Spm did not increase the level of endogenous SA, suggesting that
induction of acidic PR proteins by Spm is independent of SA. The size
of TMV-induced local lesions was reduced by Spm treatment. These
results indicate that Spm accumulates outside of cells after lesion
formation and induces both acidic PR proteins and resistance against
TMV via a SA-independent signaling pathway.
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INTRODUCTION |
PAs are found in a wide range of organisms from bacteria to plants
and animals, especially in proliferating cells. They are basic, small
molecules believed to promote plant growth and development by
activating synthesis of nucleic acids (Bertossi et al., 1965 ; Walden et
al., 1997). Increases in endogenous PAs caused by environmental stresses such as high osmotic pressure, low temperature, and low pH
have been reported (Young and Galston, 1983; Flores and Galston, 1984;
McDonald and Kushad, 1986). Roles for PAs in plant-microbe interactions
have also been proposed. Infection of the brown rust Puccinia
hordei, a fungal pathogen, caused a remarkable increase in Spd
levels in barley leaves (Greenland and Lewis, 1984). Infection of
Phytophthora infestans raised both Spd and Spm levels in
susceptible and resistant cultivars of potato (Stroinski and Szczotka,
1989). In resistant wheat cultivars, both fungal and bacterial
pathogens elicit the production of amide conjugates of phenolic acid
and PA, which may function as phytoalexins (Samborski and Rohringer, 1970). In barley seedlings synthesis of antifungal compounds, hordatines, which contain PAs, increases upon fungal infection (Smith
and Best, 1978). These findings indicate a role for PAs in the
resistance against bacterial or fungal attack in plants.
Among various host plant-pathogen combinations, an experimental system
using TMV and tobacco (Nicotiana tabacum
L.) cultivars resistant to TMV offers advantages to the study of
certain defense mechanisms, because molecular markers of the defense
response, such as SA, jasmonic acid, ethylene, and PR proteins, have
been characterized and their analytical systems established. To our knowledge, no studies on the production and function of endogenous PAs
in the TMV-tobacco system have been previously published. In this paper
we describe the results of qualitative and quantitative analyses of PAs
in TMV-infected and local lesion-forming tobacco leaves, and predict
the roles of Spm in the induction of acidic PR proteins and resistance
to TMV.
Systemic infection of tobacco plants by TMV causes severe mosaic
symptoms on young leaves with a dwarf phenotype. However, in tobacco
cultivars carrying the "N" resistance gene against TMV, infected
cells die, thus preventing further viral multiplication and
translocation to the neighboring cells. Consequently, visible necrotic
lesions are formed at the infection sites. This
phenomenon is called the HR (Goodman and Novacky, 1994) and is thought
to be a typical response to pathogen attack in plants. Lesion formation is accompanied by production of low-molecular-signaling compounds such
as SA (Malamy et al., 1990) and ethylene (De Laat and Van Loon, 1983),
with the associated induction of a number of PR proteins (Van Loon and
Van Kammen, 1970). Tobacco PR proteins consist of at least five
families, each of which contains both acidic and basic isoforms (Van
Loon et al., 1994). PR-2 and PR-3 proteins have  -1,3-glucanase and
chitinase activities, respectively (Kauffmann et al., 1987; Legrand et
al., 1987). They could inhibit the growth of pathogens in vitro (Vigers
et al., 1992; Niderman et al., 1995).
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| Figure 1.
Inducer activity for PR gene
expression in gel-filtrated fractions of the intercellular fluid from
TMV-infected tobacco leaves. Intercellular fluid of TMV-infected and
necrotic lesion-forming tobacco leaves was concentrated and applied to
a Sephadex G-15 column. The gel-filtrated fractions were tested for
their ability to induce acidic PR-1a gene expression in
leaf discs of PR1a-GUS transgenic tobacco plants. GUS
activity in the leaf discs was determined 3 d after treatment with
each fraction. The underlining bar shows the fractions to which
authentic Spm was eluted. The experiment was repeated twice with
similar results. FW, Fresh weight.
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Transgenic plants overexpressing cDNAs such as those for
PR-1, PR-3, and PR-5 were found to
have enhanced resistance to fungal pathogen infection (Alexander et
al., 1993; Vierheilig et al., 1993; Liu et al., 1994), suggesting their
antifungal role in plants. Exogenously supplied SA induces accumulation
of acidic PR proteins in healthy tobacco leaves (White, 1979; Ohshima
et al., 1990). In addition, SA acts as a natural signal for PR proteins
in local lesion-forming tobacco leaves (Klessig and Malamy, 1994).
However, with the exception of SA and its derivatives, such as methyl
SA (Shulaev et al., 1997), little is known about the natural inducers of acidic PR proteins. Plant hormones such as auxin, cytokinin, and
GA3 (Ohashi and Matsuoka, 1987a; Ohashi and
Ohshima, 1992), sugars (Herbers et al., 1996), and thiamine (Malamy et
al., 1996) have all been reported as natural occurring inducers;
however, there is little evidence for an increase in their
concentration in response to the HR. Although synthetic compounds such
as polyacrylic acid (Gianinazzi and Kassanis, 1974), eosin
yellowish (Ohashi and Matsuoka, 1985), 2,6-dichloroisonicotinic acid
and its methyl ester (Ward et al., 1991), and a benzothiadiazole
derivative (Friedrich et al., 1996) have been shown to effectively
induce acidic PR proteins, they are not natural compounds that are
generally found in plants.
Acidic PR proteins that are induced by SA treatment or local lesion
formation accumulate in the intercellular spaces to levels up to
several percent of total soluble proteins (Parent and Asselin, 1984;
Ohashi and Matsuoka, 1987b; Hosokawa and Ohashi, 1988). Because many
phytopathogenic fungi invade intercellular spaces of host plants at
early stages of infection, and since most phytopathogenic bacteria
multiply in the intercellular spaces, PR proteins that accumulate in
the spaces could directly inhibit pathogen growth. SA and its
glucoside, known as inhibitors of the multiplication of certain
pathogens, including TMV, were found in the intercellular spaces after
local lesion formation (Seo et al., 1995), indicating that this area is
an important battlefield in the fight against pathogens.
Here we describe the accumulation of Spm outside of cells in
TMV-infected tobacco leaves, and discuss its possible role as an
inducer of acidic PR gene expression in the defense of
plants against pathogen infection.
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MATERIALS AND METHODS |
Plant Material
Tobacco (Nicotiana tabacum L. cv Samsun NN) plants,
both wild-type and transformants harboring a PR1a-GUS
chimeric gene, were grown in a greenhouse under natural light and
controlled temperature at 20°C to 30°C. Well-expanded upper leaves
of 6-week-old plants were detached and used for the following analyses:
For TMV infection, a whole leaf was inoculated with purified TMV
suspension (TMV-OM, an ordinary strain of TMV in Japan, 25 µg
mL 1, unless otherwise described) using
carborundum (mesh no. 600) and incubated at 22°C under continuous
illumination at 100 µE m2
s1 for appropriate time intervals; for mock
inoculation, only mechanical wounding was conducted by rubbing the leaf
surface with water and carborundum. For chemical treatments, pieces or
discs from healthy leaves were floated in a solution containing an
appropriate concentration of sodium salicylate (pH 7.0) or PAs.
Recovery of PR-1 Inducers from the Intercellular
Fluid of TMV-Infected Tobacco Leaves
Eight-hundred grams of necrotic lesion-forming tobacco leaves was
submerged 4 d after TMV infection in 1 mM DTT solution
in vacuo. The intercellular fluid of the leaves was recovered into the
solution and then it was concentrated by freeze-drying. The resulting
dried material from the intercellular spaces was solubilized in 1 mM DTT and subjected to gel filtration using a Sephadex
G-15 column (10 × 300 mm, Pharmacia). The eluted solution (1 mL)
was fractionated and evaluated for biological activity to induce GUS activity, as described below. Authentic Spm in free basic form was
applied to the same column as a standard, and the eluted fractions were
monitored by pH.
Analysis of PR-1 Gene Expression
Analysis of PR-1a gene expression was performed with
transgenic tobacco plants harboring a PR1a-GUS gene.
Previously, we used transgenic tobacco plants containing a
GUS chimeric gene with 2.4 kb of the 5 flanking region and
+29 bp from the transcription start site of the PR-1a gene
in the pTRA415 plasmid (Ohshima et al., 1990), in which SA treatment
increased GUS activity 10- to 20-fold over a considerable level of
basal constitutive GUS activity. In the current study we used new,
improved plants containing the GUS coding region driven by
2.4 kb of the 5 flanking region and +77 bp of the tobacco
PR-1a gene. For generation of the PR1a-GUS plants, the 35S promoter region in the pBI121 vector
(Jefferson et al., 1987) was replaced by the PR-1a promoter.
In mature leaves of the adult transgenic plants, basal levels of GUS
activity were almost null, and treatment with SA at 2 mM
induced the basal activity by 1000-fold after 2 d of incubation
(data not shown). We selected one representative plant for the analysis
of PR-1a gene expression. In the transgenic plant the
time-course analysis of GUS induction by SA showed similar kinetics to
that of immunologically detected PR-1 proteins (Ohshima et al., 1990).
GUS activity was determined as described by Jefferson et al. (1987)
with modifications by Kosugi et al. (1990). Leaf discs were cut out
from fully expanded upper leaves of PR1a-GUS transgenic tobacco plants and used for GUS assay before and after chemical treatment. The fluorescence of the reactant was measured with a
spectrofluorometer (model FP-777, Jasco, Easton, MD). GUS activity is
given as nanomoles 4-methyl-umbelliferone per gram leaf fresh weight
produced in 1 min at 37°C.
Immunoblot Analysis
Analysis of PR proteins was performed by immunoblotting. Leaf
material was ground in 2 volumes of 84 mM citric acid-32
mM sodium phosphate buffer, pH 2.8. After centrifugation at
15,000g for 30 min, the resultant supernatant fluid was
subjected to precipitation by 80% saturation with ammonium sulfate.
The pellet was dialyzed against 50 mM sodium phosphate
buffer, pH 7.0, containing 2 mM DTT. PR proteins were
separated by two types of PAGE. For 15% SDS-PAGE, a protein solution
from 3 mg leaf fresh weight (corresponding to 15 µg of protein) was
used per lane, basically according to the standard procedure
(Gallagher, 1996). For 15% native-PAGE separation, a protein solution
from 3 mg leaf fresh weight was used per lane according to the method
of Davis (1964). The separated proteins were blotted onto an
Immobilon-P transfer membrane (PVDF, pore size 0.45 µm, no. IPVH 000 10, Millipore) using a semidry electroblotting system. Immunoblotting
was performed basically according to the standard method of Gallagher
et al. (1996). For immunodetection, rabbit polyclonal antibody against
PR-1a protein (Ohashi and Matsuoka, 1985) and new antibodies prepared
as described by Ohashi and Matsuoka (1985) using purified tobacco PR-N,
PR-P, and PR-S proteins were used to detect PR-1, PR-2, PR-3, and PR-5 proteins, respectively. An alkaline phosphatase-conjugated anti-rabbit IgG was used as the secondary antibody. Each PR protein was identified in gel by mobility in comparison with known
Mr markers for SDS-PAGE, or purified
standard PR proteins for native-PAGE.
Quantification of PAs
Free PAs in both whole tobacco leaves and the intercellular spaces
were quantified. For extraction of PAs in whole leaves, fresh leaf
tissue (1.5 g) was homogenized and PAs were extracted with 5 mL of 0.5 M perchloric acid. For extraction of PAs in the intercellular fluid, 35 leaf discs (18 mm in diameter) were cut out
from leaves, immediately weighed, washed with distilled water, and
submerged in water in vacuo. Subsequently, the water-infiltrated leaf
discs were subjected to centrifugation at 2000g for 20 min to recover the intercellular fluid from the discs that were placed in a
25-mL disposable Terumo syringe sitting inside a 50-mL disposable Falcon tube. PAs in these two extracts were derivatized with benzoyl chloride using diaminohexane as an internal standard basically according to the method described by Flores and Galston (1982). Separation and quantification of PA derivatives were carried out using
a HLPC system (model LC-10A, Shimadzu, Tokyo, Japan) equipped with a UV detector under the following conditions: column, Shimadzu Shim-pack CLC-ODS (6 × 150 mm); column temperature, 45°C;
mobile phase, 64% (v/v) methanol; flow rate, 0.8 mL
min1; and detection, 254 nm.
Quantification of SA
SA and its conjugate SAG were extracted from 2 g of leaf
material and quantified essentially as described by Malamy et al. (1992). SAG was quantitated following enzymatic hydrolysis with -glucosidase (EC 3.2.1.21; from almonds; Sigma). Separation and quantification were performed using a HPLC system equipped with a
spectrofluorescence detector (model RF-550A, Shimadzu). Analysis conditions were as follows: column, µBondasphere 300 (Waters), 5-µm
C-18 (3.9 × 150 mm); column temperature, 25°C; mobile phase, 23% (v/v) methanol in 20 mM sodium acetate, pH 5.0, isocratic; flow rate, 1 mL min1; excitation
wavelength, 313 nm; and emission wavelength, 405 nm. All data were
corrected for losses.
Evaluation of Spm-Induced Resistance against TMV Infection
To elucidate acquired resistance to TMV induced by Spm treatment,
the PA was fed through petioles of detached leaves at a final average
concentration in the leaf tissue of 0, 150, 300, or 500 µM, and the leaves were incubated at 22°C under 100 µE m2 s1 of
continuous light for 0 or 2 d. Then, the leaves were inoculated with TMV (10 µg mL1). After an additional
4 d of incubation, the diameters of the necrotic lesions were
measured separately in three areas: apical, middle, and basal, using
enlarged photocopies of the leaves. At least 100 local lesions from
four leaves were measured for each area. The mean values of the
diameter and SD were calculated. The significance of
differences in lesion size between Spm-treated sections and controls
was assessed with a Student's t test.
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RESULTS |
Inducer Activity for PR Gene Expression in the
Intercellular Fluid of TMV-Infected Leaves
By vacuum-infiltration of 800 g of TMV-infected and local
lesion-forming tobacco leaves in 1 mM DTT, we recovered the
solution into which the intercellular fluid of the leaves was
solubilized. The concentrate resulting from freeze-drying of the
solution was subjected to gel filtration to identify compounds with
inducer function for acidic PR-1 gene expression. When leaf
discs from PR1a-GUS transgenic tobacco plants were treated
with 500 µM SA, GUS activity was induced to 1038 nmol
4-methyl-umbelliferone g1 fresh weight
min1 2 d after the treatment, which
corresponds to 1000 times the activity in the water-treated control.
Using this system, gel-filtrated fractions were subjected to induction
of GUS activity. Two major peaks were found positive for induced
expression of the acidic PR-1 gene, as shown in Figure
1. The fractions of numbers 15 to 18 in
the first peak contained compounds with a molecular mass of less than
300 D, and without exception exhibited an alkaline pH. We thought that
the fractions may contain PAs, which are representative organic
compounds with similar characteristics, and tested whether authentic
Spm molecules are present in the same fractions. As expected, the
fractions for Spm completely overlapped the active fractions (Fig. 1).
Therefore, we examined the occurrence and role of PAs in local
lesion-forming tobacco plants.
The presence of the second major peak of GUS activity in the
gel-filtrated fractions (Fig. 1) suggests the presence of other inducers of PR gene expression.
Increase in Free Spm in the Intercellular Fluid of TMV-Infected
Leaves
We extracted total endogenous free PAs after homogenization of
necrotic lesion-developing tobacco leaves 4 d after TMV
inoculation, and determined quantitatively each PA by HPLC.
Unexpectedly, free Put was increased by about 60% by mechanical
wounding resulting from mock inoculation and TMV infection. However,
although Cad was increased slightly by TMV infection, Spd and Spm were
decreased after wounding and TMV infection (Table
I). Next, we extracted free PAs in the
intercellular fluid of tobacco leaves 3 and 5 d after TMV
inoculation and found the Spm content to be 18- and 29-fold higher than
in healthy leaves, respectively (Fig. 2). Mock inoculation did not induce an increase in Spm. Whether there was
an increase in Put, Cad, and Spd in the spaces was not clear because
their contents were relatively low and separation from other substances
was unsuccessful under our analysis conditions.
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Table I.
Content of free PAs in TMV-infected and local
lesion-forming tobacco leaves
Free PAs in leaves of healthy plants (Healthy), 4-d mock-inoculated
plants (Mock), and TMV-inoculated plants were quantified and expressed
as nanomoles in 1 g of leaf fresh weight. The experiment was
repeated twice with similar results.
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| Figure 2.
Induced accumulation of Spm in the intercellular
spaces of local lesion-forming tobacco leaves. Free Spm in the
intercellular fluid was quantified after TMV or mock inoculation. Data
are the mean values of triplicate samples. Error bars indicate
SD. The experiment was repeated twice with similar results.
FW, Fresh weight.
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Spm Induces Expression of the PR-1a Gene in
Tobacco Leaves
The effect of PAs on acidic PR-1 gene expression
was studied in the PR1a-GUS transgenic tobacco system 3 d after treatment with various PAs. As shown in Figure
3, only very low levels of GUS activity
were found in healthy and water-treated leaf discs. Although free Put
or Cad treatment could not increase the activity, free Spd at a final
concentration of 150 and 500 µM raised it 2.3- and
5.8-fold compared with the water-treated control, respectively. Free
Spm at 50 µM (pH 8.5), 150 µM (pH 8.9), and
500 µM (pH 9.4) increased GUS activity 4-, 33-, and
48-fold, respectively. To confirm that the induction was specific for
Spm rather than the alkaline pH, Spm was applied after neutralization
with HCl to pH 7.0, or in Mes buffer at pH 5.5. The increase in GUS
activity was lowered a minimal amount by alterations in pH. However,
the extent of the increase was basically unchanged by Spm-HCl (Fig. 4B) and Spm in Mes buffer (Fig. 4C).
Thus, the inducing effect of Spm was detected at all pH levels tested.
Time-course analysis after treatment with 300 µM Spm
showed that the kinetics of GUS induction by Spm was similar to that by
50 µM SA (Fig. 5). On the
1st d of treatment, only slight GUS activity was detected, but this
activity increased linearly, reaching 910-fold and 1060-fold the
control level 3 d after Spm and SA treatment, respectively.
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| Figure 3.
Induction of PR1a-GUS gene
expression by PAs. GUS activity in the leaf discs of
PR1a-GUS transgenic tobacco plants was determined 3 d after the treatment with various PAs. For a positive control, leaf
discs were treated with 50 µM SA (pH 7.0). Data are the
mean values of triplicate samples. Error bars indicate SD.
The experiment was repeated twice with similar results. FW, Fresh
weight.
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| Figure 4.
Induction of GUS activity by Spm at various pH
levels. Leaf discs of PR1a-GUS plants were treated with
free Spm in water (pH 8.5, 8.9, and 9.4 at 50, 150, and 500 µM, respectively) (A), with Spm neutralized with HCl (pH
7.0) (B), or with Spm in the presence of 10 mM Mes buffer
(pH 5.5) (C). GUS activity in the leaf discs was determined 3 d
after the treatment. For a positive control, leaf discs treated with 50 µM SA (pH 7.0) were used. Data are the mean values of
triplicate samples. Error bars indicate SD. The experiment
was repeated twice with similar results. FW, Fresh weight.
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| Figure 5.
Time-course analysis of PR1a-GUS
gene expression induced by Spm and SA. Leaf discs of
PR1a-GUS plants were treated with distilled water (DW,
- -), 300 µM Spm (pH 9.2) (- -), or 50 µM SA (- -), and GUS activity in the leaf discs was
determined after 0 to 3 d. Data are the mean values of triplicate
samples. Error bars indicate SD. The experiment was
repeated twice with similar results. FW, Fresh weight.
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In this experimental system, exogenously supplied Spd or Spm at 500 µM often induced small necrotic lesions in the edges of leaf discs. Treatment with 300 µM of these PAs sometimes
caused faint necrotic lesions on young leaves. However, this necrosis barely affected the Spm-induced GUS activity in the leaf discs of
PR1a-GUS transgenic tobacco plants. Also, 150 µM Spm, which did not cause visible necrosis during the
experiment, clearly induced both GUS activity and expression of diverse
PR proteins, as described below, suggesting that possible chemical
injury by high levels of Spm is not the cause of PR gene
activation.
Spm Induces Accumulation of a Set of Acidic PR Proteins
Induction of PR proteins by Spm was also confirmed at the protein
level. Some acidic PR proteins were immunologically determined using
specific antibodies. The results in Figure
6A show that acidic PR-1 proteins with
similar Mrs, PR-1a, PR-1b, and PR-1c, migrated as one band in 15% SDS-polyacrylamide gels and that the signal was clearly increased by Spm treatment in a
concentration-dependent manner. One of the acidic PR-5 proteins, PR-S,
showed a more sensitive response to Spm treatment than acidic PR-1
proteins. Acidic PR-2 proteins carrying -1,3-glucanase activity
contain three isoforms with slightly different
Mrs, PR-2, PR-N, and PR-O. These three isoforms were also increased by Spm treatment, although small amounts
of PR-N and PR-O proteins were found in healthy leaves. Two acidic PR-3
proteins, PR-P and PR-Q, and basic chitinases were found in healthy
leaves, resulting in faint bands upon immunodetection, and they were
further increased by exogenously supplied Spm. To separate the acidic
PR-2 and PR-3 proteins from the basic ones, native-PAGE was performed
in a basic gel. In this gel system only the acidic proteins that have
high mobilities can be separated from the basic proteins with low
mobilities. As shown in Figure 6B, all acidic PR-2 and PR-3 proteins
were shown to be induced by Spm treatment, as confirmed by mobility
equal to that of standard acidic PR proteins, PR-2, PR-N, PR-O, PR-P,
and PR-Q, respectively.
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| Figure 6.
Immunoblot analysis of acidic PR-1, PR-2, PR-3,
and PR-5 proteins. Leaf pieces were treated with 150, 300, or 500 µM Spm for 3 d. After tissue homogenization, soluble
protein was extracted. To evaluate acidic PR protein induction, 50 µM SA-treated leaves and leaves 3 d after TMV
infection were also extracted as positive controls. Fifteen micrograms
of protein (equivalent to 3 mg of fresh leaf material) was subjected to
SDS-PAGE (A) or native-PAGE (B). Immunodetection was performed with
specific antibodies against individual purified PR proteins (see
``Materials and Methods''). Each PR protein was identified by
comparison of its mobility with that of Mr
markers or standard samples of purified PR proteins. BCs, Basic
chitinases. The experiment was repeated twice with similar results.
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Exogenously Supplied SA Does Not Induce Accumulation of Spm and Spm
Fails to Induce SA
Time-course analysis showed that the kinetics of PR-1
gene induction by Spm was similar to that by SA (Fig. 5). To assess whether PR gene activation by Spm occurs through SA, the
relationship between Spm and SA as signaling compounds for
PR gene expression was analyzed. First, the Spm in the
intercellular fluid was quantified in leaves floated in a 500 µM SA solution. The basal amount of Spm in the
intercellular fluid decreased gradually with or without SA treatment,
suggesting that SA had no significant effect on synthesis, secretion,
or degradation of Spm at least within 4 d after treatment (Fig.
7). Second, SA and SAG were quantified in
the leaves treated with 300 µM Spm. As shown in Figure
8, Spm failed to raise the levels of
endogenous SA and SAG within 4 d after the treatment, whereas the
levels increased to 61.4 and 181 nmol g fresh
weight1 in TMV-infected leaves, corresponding
to 370 and 140 times the level in healthy leaves, respectively.
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| Figure 7.
Effect of SA treatment on the amount of Spm
recovered from the intercellular fluid. Free Spm was quantified in the
intercellular fluid of tobacco leaves that were incubated for 2 or
4 d in 500 µM SA solution or in distilled water
(DW). Data are the mean values of triplicate samples. Error bars
indicate SD. The experiment was repeated twice with similar
results. FW, Fresh weight.
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| Figure 8.
Effect of exogenously supplied Spm on the
accumulation of SA and SAG. SA (A) and SAG (B) were quantified 2 or
4 d after incubation with 300 µM Spm ( ) or
distilled water (). As a positive control, leaves were used 4 d
after TMV inoculation ( ). Data are the mean values of triplicate
samples. Error bars indicate SD. The experiment was
repeated twice with similar results. FW, Fresh weight.
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Spm Induces TMV Resistance in Tobacco Leaves
Spm was induced in the intercellular spaces of TMV-infected and
local lesion-forming tobacco leaves. Because local acquired resistance
against secondary infection by pathogens is established in the tissues
around local lesions (Ross, 1961), we studied whether Spm induces
resistance against TMV infection. Spm solution was fed through petioles
of detached tobacco leaves at 300 µM. The leaves were
inoculated with TMV immediately or 2 d after the treatment and
incubated for an additional 4 d. The diameters of the lesions on
the apical, middle, and basal parts of the leaves were measured separately. When TMV inoculation was performed immediately after Spm
treatment, the size of the developed lesions showed only a slight
reduction to 98%, 92%, and 85%, respectively, of that in water-treated control leaves. However, when leaves were inoculated 2 d after the treatment, Spm application reduced lesion size to 80%, 70%, and 56% of the respective controls (Fig.
9). Evaluation by a Student's
t test resulted in a P value less than 0.001 for each part
between Spm- and water-treated leaves. In another experiment, detached
leaves were treated with 150, 300, and 500 µM Spm, and after 2 d of preincubation, TMV was inoculated. Four days later, the diameters of necrotic lesions formed on the leaves were measured. The result in Figure 10 shows that Spm treatment significantly reduced
lesion size in all leaf parts (P < 0.001 in Student's t test). The reduction was dose dependent.
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| Figure 9.
Induced resistance against TMV infection by Spm.
Detached tobacco leaves were inoculated with TMV 2 d after the
treatment with 300 µM Spm solution or distilled water and
incubated for a further 4 d at 22°C. Then, the lesion size was
measured in three areas: apical (), middle ( ), and basal ()
sections were evaluated using enlarged photocopies. At least 100 lesions from four leaves were measured for each section. Data are mean
values. Error bars indicate SD. Evaluation by a Student's
t test resulted in P < 0.001 for each section between
Spm- and water-treated leaves. Lesions that developed in basal parts of
preincubated leaves are shown in B. The experiment was repeated twice
with similar results.
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| Figure 10.
Induction of resistance against TMV by Spm.
Detached tobacco leaves were treated with various concentrations of Spm
for 2 d and inoculated with TMV. The diameter of developed local
lesions was measured after 4 additional d of incubation. The
measurement was performed separately in three areas: apical (),
middle (), and basal (). At least 100 lesions from 4 leaves were
measured for each section. Data are mean values. Error bars indicate
SD.
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DISCUSSION |
There is limited evidence that PAs play a role in plant
self-defense. Here we report the quantitative analysis of PAs in both whole-leaf tissues and the intercellular spaces, and propose a new Spm
signaling pathway for PR protein gene expression that differs from that
of SA. The Spm accumulated in the intercellular spaces of TMV-infected
leaves may function as a natural signal molecule to induce PR proteins
and confer resistance against further TMV infection.
Low-molecular-mass substances exhibiting basic pH were found in
the intercellular extract of TMV-infected tobacco leaves to have
inducing activity for acidic PR-1 genes. The results of
quantitative analysis of free PAs showed a specific increase in free
Spm in the intercellular spaces after local lesion formation. However, we could not detect such an increase when whole-leaf tissues were used,
suggesting the following possibilities. First, synthesis of Spm is
highly activated in necrotic tissue, yet the total amount of Spm
decreases because consumption is enhanced. This possibility is
supported by the finding that Put and Spd, precursors of Spm, are
synthesized and highly accumulated around necrotic lesions on
TMV-infected tobacco (Torrigiani et al., 1997). We in the present study
and Scalet et al. (1991) found that the amount of Put was significantly
increased by mechanical wounding in both TMV-infected and
mock-inoculated tobacco leaves and in wounded chickpea leaves, respectively. The second possibility is that the increase in Spm in the
intercellular fluid is the result of leakage from damaged cells in
necrotic lesions. In healthy tobacco leaves, only 1% of total Spm (0.3 nmol g1 fresh weight) is found in the intercellular
spaces. However, 60% of Spm (7.5 nmol g1 fresh weight)
is detected in these spaces in local lesion-forming leaves 4 d
after TMV inoculation (Fig. 2). The area occupied by necrotic lesions
in the TMV-infected leaves was less than 30% of total leaf area, and
the other 70% was free from cell disruption. Even if all of the Spm in
the disrupted cells in local lesions leaked out to the intercellular
fluid, 7.5 nmol g1 fresh weight, which is the value for
Spm found in the intercellular spaces 4 d after TMV infection,
could not be calculated from 12.7 nmol g1 fresh weight,
the value for total Spm of TMV-infected leaves (Table I), except by
contribution of positive Spm transportation to the intercellular fluid
in adjacent healthy cells. These results suggest enhanced production
and secretion of Spm to the spaces in lesion-formed leaves but not
leakage from disrupted cells. For the last possibility, it is also
likely that increased Spm could be converted to conjugated forms such
as hydroxycinnamic acid amides (for review, see Tiburcio et al., 1990).
In the analysis of PR1a-GUS plants, Spm, among various PAs,
could increase GUS activity. Although alkaline pH enhanced the GUS
induction by Spm, the induction was always observed at different pHs of
the solution, suggesting that expression of the PR-1a gene is specifically induced by Spm rather than by alkaline pH. It is
interesting that the kinetics of induction of GUS activity by Spm were
almost the same as that by SA. Immunoblot analysis showed that Spm
induces a diverse range, not only of acidic PR proteins such as PR-1a,
PR-1b, PR-1c, PR-2, PR-N, PR-O, PR-P, PR-Q, and PR-S, but also of basic
PR-3 proteins. These findings strongly suggest that Spm is an
endogenous signal for accumulation of acidic and probably also basic PR
proteins.
Because exogenously applied Spm induced acidic PR proteins, which
are widely considered to be molecular markers of HR, we also expected
Spm to induce acquired resistance against TMV. In fact, exogenously
supplied Spm reduced the lesion size in a concentration-dependent manner, suggesting that Spm enhances the resistance of tobacco against
TMV infection. This phenomenon is well correlated with dose-dependent
induction of PR proteins by Spm (Fig. 6).
We showed that TMV spread was considerably inhibited by Spm, which also
induced acidic PR proteins in a concentration-dependent manner in
tobacco leaves. It is known that overexpression of acidic PR proteins
results in acquisition of certain antifungal activities in tobacco
plants (Alexander et al., 1993). However, as there is no direct
evidence that PR proteins inhibit TMV multiplication, we speculate that
Spm would induce PR protein accumulation and TMV resistance separately.
PR protein induction appears to be one of the self-defense mechanisms
induced by Spm, and other molecules contributing to virus resistance
could be induced by Spm.
In the signaling pathway leading to induction of acidic PR proteins,
the relationship between Spm and SA as the signals was evaluated.
Exogenously supplied SA caused no significant change in the amount of
Spm for at least 2 to 4 d after the treatment. Conversely,
exogenously supplied Spm did not raise levels of endogenous SA and SAG.
These results suggest two independent pathways for acidic PR
protein induction: SA mediated and Spm mediated (Fig. 11).
View larger version (24K):
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| Figure 11.
Proposed model for the pathway signaling acidic
PR protein induction and acquisition of resistance. Spm accumulated in
the intercellular spaces of TMV-infected tobacco leaves induced a high
level of acidic PR protein expression and conferred resistance to
further infection by TMV. This induction was not affected by SA. See
text for details.
|
|
Based on the evidence described in this paper, we propose a critical
role for the intercellular spaces in terms of Spm accumulation. The
leaf tissues infected with TMV and the neighboring tissues gradually
dehydrate with completion of local lesion formation, so the fluid in
the intercellular spaces becomes concentrated. Therefore, the level of
Spm accumulated in the intercellular spaces after lesion formation
would be high enough to induce expression of PR proteins and to confer
acquired resistance. It should be noted that induction of GUS activity
by Spm in the PR1a-GUS transgenic plants is relatively weak
when compared with that by SA. Spm reduces lesion size to a lesser
extent than SA, which reduces it by about 80% (Conrath et al., 1995);
however, this response may serve as a back-up system of SA signaling to
ensure HR at leaves infected by TMV. To our knowledge, this is the
first evidence directly linking PA to the plant-defense response
against viral infection.
| |
FOOTNOTES |
1
This work was supported by grants from the
Center of Excellence and Core Research for Evolutional Science and
Technology.
2
Present address: Faculty of Integrated Arts and
Sciences, University of Tokushima, Tokushima 770, Japan.
*
Corresponding author; e-mail yohashi{at}ss.abr.affrc.go.jp; fax
81-298-38-7044.
Received March 12, 1998;
accepted August 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Cad, cadaverine.
HR, hypersensitive reaction.
PA, polyamine.
PR, pathogenesis-related.
Put, putrescine.
SA, salicylic
acid.
SAG, salicylic acid -glucoside.
Spd, spermidine.
Spm, spermine.
TMV, tobacco mosaic virus.
| |
ACKNOWLEDGMENTS |
We are grateful to Shinsuke Fujihara (Shikoku National
Agricultural Experiment Station, Kagawa, Japan) for valuable advice regarding PA quantification. We also thank Hiroki Matsufuru, Shigemi Seo, Norihiro Ohtsubo, Ichiro Mitsuhara, Kamal A. Malik, Shunichi Kosugi, Tomoya Niki, Susumu Hiraga, and Taka Murakami for helpful discussions, and Y. Gotoh, H. Ochiai, Y. Naitoh, and Y. Matsuda for
maintenance of the plants.
| |
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Top
Abstract
Introduction