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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
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.)
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.
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 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
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 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 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.
Plant Material
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).
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
min 1; 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.
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.
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.
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.
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.
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.
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.
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.
Received March 12, 1998;
accepted August 21, 1998.
Abbreviations:
Cad, cadaverine.
HR, hypersensitive reaction.
PA, polyamine.
PR, pathogenesis-related.
Put, putrescine.
SA, salicylic
acid.
SAG, salicylic acid 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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Abstract
Introduction
-), 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.
) or
distilled water (
). Data are the mean values of triplicate
samples. Error bars indicate SD. The experiment was
repeated twice with similar results. FW, Fresh weight.
), and basal (
