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Apoplastic Polyamine Oxidation Plays Different Roles

in Local Responses of Tobacco to Infection by the

Necrotrophic Fungus Sclerotinia sclerotiorum and the
Biotrophic Bacterium Pseudomonas viridiflava1[W]
Marı́a Marina, Santiago Javier Maiale, Franco Rubén Rossi, Matı́as Fernando Romero, Elisa Isabel Rivas,
Andrés Gárriz2, Oscar Adolfo Ruiz, and Fernando Luis Pieckenstain*
Unidad de Biotecnologı́a 1, Instituto Tecnológico de Chascomús/Universidad Nacional de General San

Martı́n-Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (IIB-INTECH/UNSAM-CONICET),

½AQ1 B7130IWA Chascomus, Argentina

The role of polyamine (PA) metabolism in tobacco (Nicotiana tabacum) defense against pathogens with contrasting pathogenic
strategies was evaluated. Infection by the necrotrophic fungus Sclerotinia sclerotiorum resulted in increased arginine decar-

boxylase expression and activity in host tissues, as well as putrescine and spermine accumulation in leaf apoplast.
Enhancement of leaf PA levels, either by using transgenic plants or infiltration with exogenous PAs, led to increased necrosis
due to infection by S. sclerotiorum. Specific inhibition of diamine and PA oxidases attenuated the PA-induced enhancement of
leaf necrosis during fungal infection. When tobacco responses to infection by the biotrophic bacterium Pseudomonas viridiflava
were investigated, an increase of apoplastic spermine levels was detected. Enhancement of host PA levels by the above-
described experimental approaches strongly decreased in planta bacterial growth, an effect that was blocked by a PA oxidase
inhibitor. It can be concluded that accumulation and further oxidation of free PAs in the leaf apoplast of tobacco plants occurs
in a similar, although not identical way during tobacco defense against infection by microorganisms with contrasting
pathogenesis strategies. This response affects the pathogen’s ability to colonize host tissues and results are detrimental for
plant defense against necrotrophic pathogens that feed on necrotic tissue; on the contrary, this response plays a beneficial role
in defense against biotrophic pathogens that depend on living tissue for successful host colonization. Thus, apoplastic PAs play
important roles in plant-pathogen interactions, and modulation of host PA levels, particularly in the leaf apoplast, may lead to
significant changes in host susceptibility to different kinds of pathogens.

Polyamines (PAs) are natural aliphatic polycations RNA structure (Igarashi and Kashiwagi, 2000). In
ubiquitous in prokaryotic and eukaryotic cells and are addition to their free forms, PAs also exist as conju-

essential for cell growth, proliferation, and differenti- gates, covalently bound to small molecules and pro-
ation (Cohen, 1998). Although the mechanism of PA teins. The most abundant PAs, namely the diamine
action in many of these processes is not completely putrescine, the triamine spermidine, and the tetra-
clear, it is well known that they are positively charged amine spermine are synthesized from the amino acids
at cellular pH, and therefore bind to negatively charged Orn and Arg by two alternative pathways. One of

molecules such as nucleic acids, acidic phospholipids, these pathways is shared by almost all organisms and
½AQ3 and proteins (Igarashi et al., 1982). In this way, PAs involves the decarboxylation of Orn by the enzyme
modulate DNA-protein (Shah et al., 1999) interactions, Orn decarboxylase (ODC; EC to form putres-

protein-protein (Thomas et al., 1999) interactions, and cine. This diamine is then successively aminopropylated
to produce spermidine and spermine by spermidine
synthase (EC and spermine synthase (EC
This work was supported by grants from Consejo Nacional de, respectively. The aminopropyl groups are
Investigaciones Cientı́ficas y Técnicas (PIP 5740–CONICET, Argen- donated by decarboxylated S-adenosyl-Met, a com-
tina) and Agencia Nacional de Promoción Cientı́fica y Tecnológica pound synthesized by S-adenosyl-Met decarboxylase
(PICT 04–26517, ANPCYT, Argentina). (AdoMetDC; EC from S-adenosyl-Met. An
Present address: Laboratory of Gene Regulation and Develop- alterative pathway occurs in plants and bacteria, where
ment, National Institutes of Health, Building 6A, Room B1A13, Arg is decarboxylated to agmatine by Arg decarboxyl-
Bethesda, MD 20892–2716 USA. ase (ADC; EC, and agmatine is then trans-
* Corresponding author; e-mail:
formed into putrescine via N-carbamoyl-putrescine.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
In addition to the above-mentioned biosynthetic
described in the Instructions for Authors ( is: pathways, oxidative deamination mediated by amine
½AQ2 Fernando Luis Pieckenstain ( oxidases plays an important role in the regulation of
The online version of this article contains Web-only data. cellular PA levels (Tiburcio et al., 1997). Amine oxi- dases include the copper-containing diamine oxidases
Plant Physiology, August 2008, Vol. 147, pp. 1–15, Ó 2008 American Society of Plant Biologists 1
Marina et al.

(DAOs) and the FAD-containing PA oxidases (PAOs). increase their concentration in apoplastic spaces dur-
DAOs (EC from plants oxidize the diamines ing plant-pathogen interaction (Beckers and Spoel,
putrescine and cadaverine at the primary amino 2006; Grant and Lamb, 2006). Thus, it is of great
groups. PAOs (EC use FAD as a cofactor interest that Yamakawa et al. (1998) found TMV infec-
and catalyze the oxidation of spermidine, spermine, tion to induce spermine accumulation in apoplastic
and/or their acetylated derivatives at the secondary fluids of tobacco plants, this tetraamine acting as an
amino groups (Cohen, 1998). The action of DAO on inducer of acidic pathogenesis-related gene expression
putrescine yields pyrroline, hydrogen peroxide, and in a salicylic acid-independent manner. In addition,
ammonia, while PAO action on spermidine and these authors demonstrated that changes in PA levels
spermine yields pyrroline and 1-(3-aminopropyl) pyr- elicited by TMV infection at the whole leaf level were
rollinium, respectively, as well as 1,3-diaminopropane not correlated with those occurring specifically in
and hydrogen peroxide. 1-(3-Aminopropyl) pyrrolli- intercellular spaces, thus suggesting that apoplastic
nium, which exists as the iminium cation, is converted PA metabolism deserves further attention in relation
to the bicyclic compound 1,5-diazabicyclononane with plant responses to pathogen attack. Later works

(Cohen, 1998). There is evidence that DAO and PAO demonstrated that spermine causes mitochondrial

are predominantly located in the cell wall (Angelini dysfunction via a signaling pathway that stimulates
et al., 1993; Šebela et al., 2001), and the production of the activity of mitogen-activated protein kinases
hydrogen peroxide resulting from PA has been corre- (Takahashi et al., 2003), which in turn activates a
lated with developmental cell wall maturation and subset of HR-specific genes (Takahashi et al., 2004).
lignification in several plant species (Cona et al., 2003; This observation stresses the relevance of spermine

Paschalidis and Roubelakis Angelakis, 2005; Cona accumulation in the intercellular space of pathogen-
et al., 2006a). infected leaves as a potential mechanism of plant
PA metabolism undergoes significant modifications defense activation.
during plant responses to various abiotic and biotic To our knowledge, information about the role of
stresses (for review, see Flores, 1990; Walters, 2003). apoplastic PAs in plant defense against pathogens is
The levels of free and conjugated PAs, as well the restricted to the tobacco-TMV pathosystem, the con-
activities of PA biosynthetic and catabolic enzymes sequences of fungal or bacterial infection on apoplastic
have been shown to increase during the hypersensi- PA metabolism having not been studied so far. More-
½AQ4 tive response (HR) of barley (Hordeum vulgare) to the over, a comparison of the changes in PA metabolism
powdery mildew fungus Blumeria graminis f. sp. hordei elicited in a given plant host by pathogenic organisms
(Cowley and Walters, 2002). A similar picture was with contrasting (necrotrophic and biotrophic) patho-
found during the HR of a tobacco (Nicotiana tabacum) genic strategies was not performed so far. Therefore,

cultivar resistant to the Tobacco mosaic virus (TMV). this work aimed to determine the role of plant PAs as
In this case, ODC, ADC, and AdoMetDC activities part of local defense responses to plant infection by
were found to increase, thus leading to elevated con- pathogenic microorganisms with contrasting patho-
centrations of free and conjugated PAs, mainly in genesis strategies, with emphasis on the consequences

necrotic lesions (Torrigiani et al., 1997). Moreover, it of apoplastic PA oxidation on the ability of plant
was later demonstrated that both PA biosynthesis and pathogens to colonize host tissues. For this purpose,
catabolism are up-regulated during the HR of tobacco experiments were carried out using tobacco as a plant
plants to TMV (Marini et al., 2001). Thus, the above- host, and two different pathogens. One of them was
mentioned works indicate that PA oxidation plays the cosmopolitan and polifagous ascomycete Scleroti-

a role in cell death programs associated to the HR. nia sclerotiorum, which infects more than 400 plant ½AQ5
Moreover, hydrogen peroxide derived from PA oxida- species. This fungus is considered a necrotroph be-
tion has been shown to contribute to wound healing cause it kills host cells during the infection process

(Angelini et al., 2008) and cell wall reinforcement in and causes extensive tissue damage (Bolton et al.,
response to invasion of chickpea (Cicer arietinum) 2006). The other pathogen employed in this study
tissues by the fungal pathogen Ascochyta rabiei (Rea was Pseudomonas viridiflava, a bacterium that causes
et al., 2002). tomato (Solanum lycopersicum) pith and basal stem rot
The bulk of the work performed in relation with (Malathrakis and Goumas, 1987), and is also pathogenic
plant PAs during the response to pathogen attack has on other hosts, including tobacco (Alippi et al., 2003).
pointed to metabolic changes occurring at the organ or Similarly to other Pseudomonas species (Glazebrook,
tissue level, without focusing on intercellular spaces. 2005), P. viridiflava multiplies asymptomatically in the
Most phytopathogenic bacteria multiply in the inter- intercellular spaces of the host early during the infec-
cellular space, also known as the apoplast, and fre- tion process, chlorosis and necrosis being evident only
quently, this is the first site of pathogenic fungi at later stages. Therefore, this bacterium can be consid-
invasion (Glazebrook, 2005). Moreover, pathogenesis- ered as a biotroph, at least during the initial phase of
related proteins are accumulated in the intercellular infection. This work contributes to the understanding
space (Van Loon, 1997), and it has been shown that of the relevance of apoplastic PA accumulation and
some hormones such as salicylic acid or its glucoside, oxidation in relation with plant tolerance to necrotro-
which inhibit the multiplication of certain pathogens, phic and biotrophic pathogens.
2 Plant Physiol. Vol. 147, 2008
Apoplastic Polyamine Oxidation in Plant Defense

RESULTS with noninoculated controls. Apoplastic spermine level

was below the detection limit in control leaves, and
Accumulation of Free PAs in Leaf Extracts and
reached 0.013 nmol mL21 in infected ones (Fig. 1B).
Apoplastic Fluids of Tobacco Plants
Infected by S. sclerotiorum
Leaf Infection by S. sclerotiorum Increases ADC
Tobacco ‘Xanthi-nc’ leaves developed typical brownish Activity and Induces ADC Transcript Accumulation
necrotic lesions 24 h after inoculation (HAI) with S.
sclerotiorum mycelium, and lesion size further increased To determine the biochemical basis of the above-
as a function of time. Samples for free PA determination described changes in PA levels elicited by S. sclerotiorum
in leaf extracts and apoplastic fluids were taken from infection, the activities of several enzymes involved in
noninfected tissues adjacent to the lesions 48 HAI, and PA biosynthesis were determined. For this purpose,
were found to be free of fungal mycelium by observation leaf samples were taken from noninfected tissues
under a binocular microscope. In leaf extracts, putres- adjacent to the lesions 48 HAI, as described in the
cine and spermidine were the most abundant free PAs, preceding section. ADC activity was 4-fold higher in

their levels being not affected by fungal infection (Fig. extracts obtained from leaves infected by S. sclerotiorum

1A). A small but significant decrease of spermine level than in controls (mock-inoculated) 48 HAI. (Fig. 2A).
was detected in leaf extracts of inoculated plants, as On the contrary, ODC and AdoMetDc activities were
compared with controls (Fig. 1A). When apoplastic PA not affected by fungal infection (Fig. 2A). ADC, ODC,
levels were evaluated, an increase of free putrescine, and AdoMetDc activities were also evaluated in IWFs
spermidine, and spermine was detected in leaves infected and found to be extremely low, being ,0.5% of the

by S. sclerotiorum, although the increase in spermidine activities detected in leaf extracts (data not shown), thus
levels lacked statistical significance (Fig. 1B). Thus, the suggesting that the apoplastic compartment makes no
levels of free putrescine in intercellular washing fluids significant contribution to the activity of these enzymes
(IWFs) of infected leaves increased by 7-fold, as compared detected in leaf extracts.
Given that ADC was the sole enzymatic activity
found to be modified as a consequence of fungal
infection, the steady-state levels of ADC mRNA were
analyzed by semiquantitative reverse transcription
(RT)-PCR and found to be higher in infected than in ½AQ6
mock-inoculated leaves (Fig. 2B).

PA Accumulation Increases Disease Severity in Leaves

Infected by S. sclerotiorum
As demonstrated in previous sections, infection of
tobacco leaves by S. sclerotiorum resulted in the induc-

tion of changes in free PA levels, both in leaf extracts

and apoplastic fluids. To understand the consequences
of changes in PA levels on the local response to fungal
infection and further disease development, tobacco
leaf discs with enhanced PA levels were used as a

target for inoculation with S. sclerotiorum and subse-

quent determination of the percentage of necrotic
tissue, as an indicator of disease severity. For this

purpose, two alternative approaches were employed.

The first approach aimed to modify cellular PA levels,
and was based in the use of transgenic tobacco
‘Wisconsin W38 TetR/Oat’ ADC plants that express ½AQ7
Figure 1. Free PA levels in tobacco leaves infected by S. sclerotiorum. an oat (Avena sativa) ADC gene under the control of a
Leaves were inoculated on the adaxial surface by placing discs of tetracycline-inducible promoter (Masgrau et al., 1997).
S. sclerotiorum mycelium cut from the edge of an actively growing In this way, ADC activity and putrescine and spermi-
colony. A and B, Samples for free PA determination in leaf extracts (A) dine levels were, respectively, 3-, 2-, and 3-fold higher
and IWFs (B) were taken from noninfected tissues adjacent to the in extracts obtained from discs induced by floating in a
lesions 48 HAI. Putrescine (Put), spermidine (Spd), and spermine (Spm)
tetracycline solution during 24 h than in extracts
concentrations were determined in mock-inoculated (white bars) and
obtained from noninduced discs to which no tetracy-
S. sclerotiorum-inoculated (black bars) plants using HPLC after deriv-
atization with dansyl chloride. The values are reported in nanomoles cline was added (data not shown). No changes in
relative to leaf fresh weight (FW) in A and to the volume of IWF in spermine levels were detected in tetracycline-induced
B. Statistical differences in the level of each PA between mock- discs. Inoculation of W38 TetR/Oat ADC leaf discs
inoculated and infected plants according to Student’s t test are shown with S. sclerotiorum led to the development of necrotic
as: *, P # 0.05; ***, P # 0.001. lesions, the proportion of infected discs being similar
Plant Physiol. Vol. 147, 2008 3
Marina et al.

The second approach for the modification of PA

levels prior to inoculation with S. sclerotiorum aimed
toward the modification of apoplastic PAs, and con-
sisted of vacuum infiltration of leaf discs of wild-type
Xanthi-nc tobacco plants with 5 mM solutions of pu-
trescine, spermidine, and spermine in water. Infiltra-
tion with these PAs increased lesion size, respectively,
by 12-, 18-, and 15-fold, as compared with control discs
infiltrated with water (Fig. 4).

Activity and Inhibition of Amine-Oxidizing Enzymes

in Tobacco Leaf Apoplast

DAO and PAO activities were detected in vitro

using IWFs as a source of enzyme and PAs as sub-

strates (Fig. 5, A and B), these activities being not
affected by S. sclerotiorum infection (Supplemental Fig.
S1A). A temporal analysis of DAO and PAO activities
throughout the life cycle of tobacco plants was also
performed, both activities being found to decline as

plant age increased (Supplemental Fig. S2). To evalu-
ate the participation of DAO and PAO in lesion de-
velopment as a consequence of PA accumulation
Figure 2. A, ADC, ODC, and AdoMetDC activities in tobacco leaves triggered by S. sclerotiorum infection, the ability of
infected by S. sclerotiorum. Leaves were inoculated with S. sclerotio- specific inhibitors to interfere with the activity of these
rum and samples for the determination of enzyme activities were taken enzymes was tested. Two of the inhibitors used were
from noninfected tissues adjacent to the lesions 48 HAI. ADC, ODC, N,N#-diaminoguanidine, a compound reported to act
and AdoMetDC activities were determined in mock-inoculated (white as a competitive inhibitor of soybean (Glycine max)
bars) and S. sclerotiorum-inoculated plants (black bars) as described in DAO (Nikolov et al., 1990; Xing et al., 2007) and
‘‘Materials and Methods.’’ Statistical differences in each enzymatic guazatine, which competitively inhibits maize (Zea
activity between mock-inoculated and infected plants according to mays; Cona et al., 2004) and tobacco (Yoda et al., 2006)
Student’s t test are shown as **, P # 0.01. B, Steady-state levels of ADC
PAOs. In addition, the PA analog 1,19-bis(etilamino)-

mRNA in tobacco leaves infected by S. sclerotiorum. Samples were

taken as described above. Semiquantitative RT-PCR analysis of ADC
5,10,15-triazanonadecane (Bacchi et al., 2002), which
mRNA was performed with RNA extracted from mock-inoculated and has been found by our group to inhibit oat and maize
S. sclerotiorum-inoculated plants. ADC and actin cDNA bands were
visualized by ethidium bromide staining in agarose gels.

(approximately 100%) in those induced with tetracycline

and in controls (data not shown). However, percent-
age of leaf necrosis was 4-fold higher in tetracycline-

induced discs of the W38 TetR/Oat ADC line than in

noninduced controls (Fig. 3). Thus, induction of the
above-mentioned modifications of PA levels increased

leaf necrosis caused by S. sclerotiorum. Similar treat-

ments were applied to leaf discs obtained from a
transgenic tobacco line (W38 TetR), which harbors the
tetracycline inducible promoter but no heterologous
ADC gene. In this case, percentage of leaf necrosis in
tetracycline-induced discs was similar to that of non- Figure 3. Necrosis caused by S. sclerotiorum infection on leaf discs of
induced controls (Fig. 3). In addition, no changes in PA transgenic tobacco plants that overexpress an oat ADC gene. Leaf discs
levels were detected in leaf discs of the transgenic W38 of transgenic tobacco W38 TetR/Oat ADC and W38 TetR plants were
TetR line treated with tetracycline (data not shown). In incubated in 5 mM, pH 6.5, MES buffer amended with tetracycline
this way, results obtained with the W38 TetR line served (2 mg mL21; 1tet) or without amendments (2tet) for 24 h, prior to
inoculation with S. sclerotiorum. The size of the necrotic area around
to verify that tetracycline itself exerts no effect on PA
the inoculation site was determined 48 HAI. using the Image-ProPlus V
levels and leaf necrosis in leaves infected by S. sclero- 4.1 software (Media Cybernetics) and was expressed as a percentage of
tiorum, thus confirming that the previously mentioned the whole disc area. Statistical differences in the percentage of leaf
effects of this antibiotic on lesion development in W38 necrosis between discs induced with tetracycline and noninduced
TetR/Oat ADC plants were actually due to the induc- controls of the same transgenic line according to Student’s t test are
tion of the ADC transgene. shown as ***, P # 0.001.

4 Plant Physiol. Vol. 147, 2008

Apoplastic Polyamine Oxidation in Plant Defense

to undetectable levels (data not shown), while the level

of 1,3-diaminopropane raised to 0.1 nmol mL21 in
control discs (Fig. 6B). At this time after infiltration, the
level of apoplastic of 1,3-diaminopropane was 2-fold
higher in discs infiltrated with spermine than in
controls (Fig. 6B). Coinfiltration of spermine along
with 1,19-bis(etilamino)-5,10,15-triazanonadecane de-
creased apoplastic 1,3-diaminopropane concentration to
levels similar to those of controls (Fig. 6 B). In discs
infiltrated only with 1,19-bis(etilamino)-5,10,15-triazano-
nadecane, apoplastic 1,3-diaminopropane levels were
similar to those of controls (Fig. 6B). In summary, results
obtained in this section demonstrated that N,N#-
Figure 4. Necrosis caused by S. sclerotiorum infection of tobacco leaf diaminoguanidine, 1,19-bis(etilamino)-5,10,15-triazano-

discs infiltrated with PAs. Tobacco W38 leaf discs were vacuum infiltrated nadecane, and guazatine are able to inhibit apoplastic

with 5 mM putrescine (Put), spermidine (Spd), and spermine (Spm), and PA oxidation in tobacco leaves.
were then inoculated with S. sclerotiorum mycelium. Discs infiltrated
with water were used as controls. The size of the necrotic area around the
inoculation site was determined 48 HAI. using the Image-ProPlus V 4.1
Severity of Tobacco Leaf Rot Caused by S. sclerotiorum
software (Media Cybernetics) and was expressed as a percentage of the Is Reduced by Inhibition of Apoplastic PA Oxidation

whole disc area. Statistical differences in the percentage of leaf necrosis
Results presented in the previous section suggested
between discs infiltrated with PAs and controls according to one-way
ANOVA and Dunnett’s test are shown as **, P # 0.01.
that the use of amino-oxidase inhibitors could contrib-

½AQ8 PAOs in vitro (J. Maiale, unpublished data) was also

employed. The ability of the above-mentioned com-
pounds to inhibit amine-oxidizing enzymes under the
experimental conditions employed in this work was
tested in vitro, using putrescine and spermine as sub-
strates. In this way, N,N#-diaminoguanidine was found
to inhibit DAO activity by 58%, while the inhibition
caused by 1,19-bis(etilamino)-5,10,15-triazanonadecane

and guazatine reached 17% and 30%, respectively (Fig.

5A). 1,19-Bis(etilamino)-5,10,15-triazanonadecane and
guazatine also inhibited PAO activity by 64% and 75%,
respectively (Fig. 5B).

Given that no information was available regarding the

ability of 1,19-bis(etilamino)-5,10,15-triazanonadecane
to inhibit PA oxidation in vivo, the effect of this com-
pound on spermine oxidation was also evaluated in
vivo, by infiltration of leaf discs and subsequent de-

termination of apoplastic spermine levels, as well as

those of 1,3-diaminopropane, one of the products of
spermine oxidation. As expected, infiltration of leaf

discs with spermine led to a significant increase in the

apoplastic concentration of this tetraamine 4 h after
infiltration, as compared with controls, in which
spermine was undetectable (Fig. 6A). Disc infiltration
with 1,19-bis(etilamino)-5,10,15-triazanonadecane in
combination with spermine resulted in higher apo- Figure 5. In vitro determination of tobacco apoplastic DAO and PAO
plastic levels of this tetraamine, its concentration being activities and the effect of amine oxidase inhibitors. Leaf discs were
3-fold higher than in discs infiltrated only with sperm- infiltrated with 100 mM, pH 8.0, Tris-HCl buffer and IWFs were
ine (Fig. 6A). Apoplastic spermine was undetectable in collected as described in ‘‘Materials and Methods.’’ DAO (A) and
discs infiltrated only with 1,19-bis(etilamino)-5,10,15- PAO (B) activities in IWFs were determined using [1,4-14C]putrescine
and [1,4-14C]spermine as substrates, respectively. DPyrroline and
triazanonadecane, as occurred with controls (Fig. 6A).
1[3-aminopropyl]pyrroline (1[3-AP]P) are products of DAO and PAO
At this time point, 1,3-diaminopropane was not de- activities, respectively. The inhibitors N,N#-diaminoguanidine (AG),
tected in IWFs obtained from leaf discs subjected to 1,19-bis[etilamino]-5,10,15-triazanonadecane (SL-11061), and guaza-
any infiltration treatment (data not shown). tine (G) were added to the reaction mixture at a final concentration of
When apoplastic PAs were evaluated 24 h after 0.5 mM. Different letters indicate statistically significant differences
infiltration with spermine, this tetraamine decreased (P # 0.05) according to one-way ANOVA and Bonferroni’s test.

Plant Physiol. Vol. 147, 2008 5

Marina et al.

effect of this compound (Mackintosh and Walters,

2003; Dreassi et al., 2007). As a consequence, guazatine
was not included in the experiments aimed to evaluate
the effect of amino oxidase inhibitors on leaf rot sever-
ity; in that lesion development could be restricted
because of a direct effect of this compound on fungal
growth. Thus, lesion size was determined in leaf
discs inoculated with S. sclerotiorum after infiltration
with N,N#-diaminoguanidine and 1,19-bis(etilamino)-
5,10,15-triazanonadecane, either separately or in combi-
nation with PAs.
As demonstrated in previous sections, infiltration
with putrescine, spermidine, and spermine led to an
increase in the percentage of leaf necrosis, as com-

pared with controls infiltrated with no PAs (Fig. 7, A

and B). In this way, infiltration with putrescine in-
creased leaf necrosis by 2-fold. However, when
putrescine was coinfiltrated with the DAO inhibitor
N,N#-diaminoguanidine, the percentage of leaf necro-

Figure 6. Inhibition of spermine oxidation by 1,19-bis (etilamino)-
5,10,15-triazanonadecane (SL-11061) in tobacco leaf discs. Discs were
infiltrated with 5 mM spermine (Spm) and 1,19-bis(etilamino)-5,10,15-

triazanonadecane (SL-11061), either separately or in combination.

Discs infiltrated with water were used as controls. Intercellular washing
fluids were collected 4 (A) and 24 (B) h after incubation. Apoplastic
spermine levels, as well as those of the product of spermine oxidation
1,3-diaminopropane (Dap) were determined using HPLC after deriva-

tization with dansyl chloride. 1,3-Diaminopropane and spermine were

undetectable 4 and 24 h after infiltration, respectively. Different letters
indicate statistically significant differences (P # 0.01) according to one-
way ANOVA and Bonferroni’s test.

ute to determine whether apoplastic PA oxidation

plays a role during plant defense against S. sclerotiorum
infection. Taking into account that PA accumulation in

the leaf apoplast led to enhanced severity of leaf rot

caused by S. sclerotiorum, it was decided to evaluate
whether this effect is mediated by PA oxidation. For
this purpose, the compounds demonstrated in previ- Figure 7. A and B, Effects of DAO (A) and PAO (B) inhibition on necrosis
ous sections to act as tobacco amine oxidase inhibitors of tobacco leaf discs infected by S. sclerotiorum. A, Leaf discs were
were used to evaluate their effect on leaf rot severity. vacuum infiltrated with 5 mM putrescine (Put) and N,N#-diaminoguani-
Previously, the effect of these compounds on mycelial dine monohydrochloride (AG), either separately or in combination. B,
growth of S. sclerotiorum was tested in vitro. Con- Discs were vacuum infiltrated with 5 mM spermidine (Spd), 5 mM
centrations up to 100 mM of N,N#-diaminoguanidine spermine (Spm), and 5 mM 1,19-bis(etilamino)-5,10,15-triazanonadecane
(data not shown) and 1,19-bis(etilamino)-5,10,15- (SL-11061), according to the combinations indicated in the figure. In
both panels, leaf discs infiltrated with water were used as controls. After
triazanonadecane (J. Maiale, unpublished data) proved
infiltration, discs were inoculated with S. sclerotiorum and incubated
to exert no effect on mycelial growth when added to the for 48 h. The size of the necrotic area around the inoculation site was
growth medium. Guazatine, on the contrary, strongly determined using the Image-ProPlus V 4.1 software (Media Cybernet-
inhibited mycelial growth in concentrations as low as ics) and was expressed as a percentage of the whole disc area. Different
5 mM (J. Maiale, unpublished data), which is in good letters indicate statistically significant differences (P # 0.05), according
agreement with the previously reported fungicidal to one-way ANOVA and Bonferroni’s test.

6 Plant Physiol. Vol. 147, 2008

Apoplastic Polyamine Oxidation in Plant Defense

sis was similar to that of control discs. Moreover, lesion

size was reduced by 69% in discs infiltrated only with
N,N#-diaminoguanidine, as compared with controls
infiltrated with water (Fig. 7A). Infiltration with sper-
midine and spermine increased lesion size by 2- and
3-fold, respectively, as compared with controls. Sper-
midine and spermine-mediated enhancement of leaf
necrosis was blocked by coinfiltration of these PAs along
with the PAO inhibitor 1,19-bis(etilamino)-5,10,15-
triazanonadecane, being reduced to values as low as
45% and 35% of controls, respectively (Fig. 7B). When
discs were infiltrated only with 1,19-bis(etilamino)-
5,10,15-triazanonadecane, necrosis decreased to 41%
of controls (Fig. 7B).

Oxidation of Apoplastic PAs and Its Relation with the
Generation of ROS Figure 8. Superoxide production in leaf discs of tobacco infiltrated
with PAs and amine oxidase inhibitors. Leaf discs were infiltrated with
Besides 1,3-diaminopropane, PA oxidation also leads 5 mM putrescine (Put) and 5 mM diamineguanidine (AG), either separately
to hydrogen peroxide formation. Taking into account or in combination. Another set of leaf discs was infiltrated with 5 mM

the diversity and importance of the roles played by spermine (Spm), either separately or in combination with 5 mM each of
hydrogen peroxide and other reactive oxygen species the amine oxidase inhibitors 1,19-bis(etilamino)-5,10,15-triazanona-
(ROS) in plant defense against pathogen attack, it was decane (SL-11061) and 5 mM guazatine (G), as indicated in the figure.
Discs infiltrated with water were used as controls. NBT was added to all
decided to evaluate whether oxidation of apoplastic
the infiltration solutions at a final concentration of 0.01% w/v. After
PAs contributes to ROS accumulation in the apoplastic infiltration, discs were maintained in the infiltration solution and
compartment of tobacco leaves. In this way, hydrogen
incubated for 3 h in the dark at 30°C. Stained leaf discs were mounted
peroxide production by leaf discs infiltrated with 5 mM on glass slides and scanned. Images were inverted to obtain a negative
putrescine, spermidine, and spermine was evaluated prior to transformation into black and white 8-bit images. Formazan
using 3,3#-diaminobenzidine, no accumulation of this color intensity (in a negative corresponding to the lighter tones of gray)
active oxygen species being detected (data not shown). was determined by using the Image-ProPlus V 4.1 software (Media
In addition, superoxide generation in leaf discs Cybernetics). Different letters indicate statistically significant differ-
infiltrated with putrescine and spermine was evalu- ences (P # 0.05), according to one-way ANOVA and Bonferroni’s test.

ated, using nitro blue tetrazolium (NBT). Infiltration

with either 5 mM putrescine or spermine led to super- ples for the determination of free PAs and PAO and
oxide accumulation in the intercellular spaces, as DAO activities in leaf extracts and apoplastic fluids
evidenced by the formation of a blue formazan pre- were taken from tissues adjacent to the infiltration

cipitate. In this way, superoxide levels in discs in- zone, 20 and 48 HAI. Prior to using IWFs for the above-
filtrated with these PAs were 2-fold higher than in mentioned determinations, bacteria were eliminated
water-infiltrated controls, in which superoxide levels by centrifugation to minimize contamination of this
were very low (Fig. 8). Coinfiltration of discs with 5 mM fraction with metabolites from bacterial origin. The
putrescine and 20 mM N,N#-diaminoguanidine reduced IWFs obtained in this way were free of bacterial cells,

superoxide formation to levels similar to those of con- as demonstrated by the absence of bacterial colonies
trols (Fig. 8). Similarly, coinfiltration of 5 mM spermine after inoculation of 20 mL aliquots onto solid King’s B
with 20 mM 1,19-bis(etilamino)-5,10,15-triazanonadecane medium. No changes in PA levels were evident in leaf

or 20 mM guazatine also reduced superoxide generation extracts of inoculated plants at both HAI times, as
to levels similar to those of controls (Fig. 8). compared with controls (data not shown). On the
contrary, changes in apoplastic spermine levels were
Changes in Apoplastic PA Levels and Amine-Oxidizing
evident in leaves infected by P. viridiflava, the levels of
Enzymes in Response to Infection by the Biotrophic
this tetraamine being 4- and 10-fold higher in leaves
½AQ9 Bacterial Pathogen P. viridiflava
infected by P. viridiflava than in controls, when evalu-
ated 20 (Fig. 9A) and 48 (Fig. 9B) HAI, respectively. As
As a whole, results obtained in previous sections occurred after leaf infection by S. sclerotiorum, DAO
demonstrated that infection of tobacco leaves by the and PAO activities were not affected by P. viridiflava
necrotrophic fungus S. sclerotiorum induces PA accu- infection (Supplemental Fig. S1B).
mulation in the apoplastic compartment, which results
in PA oxidation and thus increases leaf rot severity. PA Accumulation and Oxidation Decreases Leaf
This prompted us to evaluate whether similar re- Colonization by P. viridiflava
sponses operate during infection of tobacco leaves by
biotrophic pathogens. For this purpose, tobacco plants The effect of increased PA levels on P. viridiflava
were infiltrated with the bacterium P. viridiflava. Sam- multiplication in planta was evaluated by two alter-
Plant Physiol. Vol. 147, 2008 7
Marina et al.

based on vacuum infiltration of leaf discs obtained

from wild-type Xanthi-nc plants with a 5 mM solution
of spermine in water. Infiltration with this tetraamine
strongly decreased bacterial growth in inoculated
discs, as compared with controls infiltrated with water
(Fig. 11). Inclusion of both 1,19-bis(etilamino)-5,10,15-
triazanonadecane and spermine in the infiltration so-
lution partly reverted the inhibitory effect of spermine
on bacterial growth in planta, this parameter reaching
an intermediate value between that of discs infiltrated
only with spermine and controls (Fig. 11). Moreover,
infiltration of discs with 1,19-bis(etilamino)-5,10,15-
triazanonadecane and no spermine resulted in higher
bacterial growth than in controls infiltrated with water,

this treatment leading to the highest bacterial growth

in planta, as compared to all the treatments evaluated
(Fig. 11).

Infection by pathogenic microorganisms has been
reported to induce modifications in PA metabolism in
different plant hosts (for review, see Walters, 2003),
which in many cases consisted of PA accumulation,
Figure 9. Changes in apoplastic free PA levels of tobacco leaves either in their free or conjugated forms. Although the
infected by P. viridiflava. Plants were inoculated by leaf infiltration of a
understanding of the physiological significance of
bacterial suspension (5 3 108 CFU mL21) in a sterile solution of 10 mM
MgCl2, pH 7.0, by means of a hypodermic syringe without a needle
pathogen-induced changes in PA metabolism of plants
(black bars). IWFs were obtained from tissues adjacent to the inocu- is far from being complete, PAs have been shown to
lation zone 20 (A) and 48 (B) HAI. Mock-inoculated plants (white bars) play several roles in plant defense against pathogen
were infiltrated with 10 mM MgCl2, pH 7.0. Putrescine (Put), spermi- attack. Conjugated PAs exert antimicrobial effects

dine (Spd), and spermine (Spm) concentrations in IWFs were deter- (Peng and Kuc, 1992; Walters et al., 2001), and the
mined using HPLC after derivatization with dansyl chloride. Statistical
differences in the level of each PA between mock-inoculated and
infected plants according to Student’s t test are shown as *, P # 0.05; **,
P # 0.01.

native approaches similar to those previously de-

scribed for the determination of leaf rot severity
caused by S. sclerotiorum. The first approach consisted

of tetracycline-mediated induction of heterologous

ADC gene expression in leaf discs of transgenic to-
bacco W38 Tet/Oat ADC plants, which results in

2- and 3-fold increases in putrescine and spermidine

levels, respectively, as described in previous sections.
Bacterial growth was strongly reduced in tetracycline-
induced discs of W38 Tet/Oat ADC plants, as com-
pared with noninduced discs of the same transgenic
½AQ10 line, in which 3.3 3 109 colony-forming units (CFU)
mL21 were detected (Fig. 10). Incubation in tetracy- Figure 10. Growth of P. viridiflava in leaf discs of tobacco plants that
cline of leaf discs obtained from the transgenic W38 overexpress an oat ADC gene. Discs of transgenic tobacco W38 TetR/
TetR line, which expresses no heterologous ADC gene, Oat ADC and W38 TetR plants were incubated for 24 h in 5 mM MES,
had no effect on bacterial growth, as compared with pH 6.5, buffer amended with tetracycline (2 mg mL21; 1tet) or without
amendments (2tet). Discs were then inoculated by infiltration of a
discs of the same line incubated in the absence of the
bacterial suspension (5 3 108 CFU mL21) in sterile 10 mM MgCl2, pH
antibiotic (Fig. 10). In this way, the transgenic W38 7.0, and were incubated for 48 h. Bacteria were recovered from the discs
TetR line served to verify that tetracycline itself does and the number of CFUs was determined by plating on King’s B medium.
not inhibit bacterial growth in planta. Statistical differences in the percentage of leaf necrosis between discs
The second approach for the modification of tobacco induced with tetracycline and noninduced controls of the same trans-
PA levels prior to inoculation with P. viridiflava was genic line according to Student’s t test are shown as *, P # 0.05.

8 Plant Physiol. Vol. 147, 2008

Apoplastic Polyamine Oxidation in Plant Defense

enzymes. When PA levels were evaluated in tissue

extracts, a slight decrease in free spermine levels was
the sole change induced by fungal infection. A very
different picture was found when apoplastic PA levels
were evaluated, free putrescine and spermine levels
being significantly higher in infected plants than in
noninfected controls. Similarly, Yamakawa et al. (1998)
found that apoplastic spermine levels of tobacco
plants undergo distinctive changes in response to
infection by the TMV, which are not evidenced by
the evaluation of PA levels in leaf extracts. Thus, it is of
interest that pathogens as different as S. sclerotiorum
and TMV both induce accumulation of apoplastic
spermine. PA accumulation in apoplastic fluids of

tobacco leaves infected by S. sclerotiorum could be due

Figure 11. Effect of PAO inhibition on growth of P. viridiflava in
to an increase in the activity of enzymes involved in
tobacco leaf discs. Leaf discs were vacuum infiltrated with 5 mM spermine PA biosynthesis. In support of this view, both ADC
(Spm) and 5 mM 1,19-bis(etilamino)-5,10,15-triazanonadecane (SL- activity and mRNA levels were found to be increased
11061), either separately or in combination. Discs infiltrated with water in leaves infected by S. sclerotiorum. As opposed to
were used as controls. Discs were inoculated by infiltration of a what was reported for the interaction between tobacco

bacterial suspension (5 3 108 CFU mL21) in 10 mM MgCl2, pH 7.0, and and TMV, PA biosynthetic enzymes other than
incubated for 48 h. Bacteria were recovered from the discs and the ADC were not induced by S. sclerotiorum infection.
number of CFUs was determined by plating on King’s B medium. However, it should be kept in mind that Arg decar-
Different letters indicate statistically significant differences (P # 0.05), boxylation mediated by ADC is considered to be a
according to one-way ANOVA and Bonferroni’s test.
rate-limiting step in PA biosynthesis, an increase in the
activity of this enzyme being enough to enhance the
oxidation of free PAs has been proposed to contribute levels of all the PAs in the biosynthetic pathway. Thus,
to cell-death processes occurring during the HR of the increase in host ADC activity probably contributed
tobacco plants to TMV (Marini et al., 2001). In addi- in a significant degree to apoplastic PA accumulation
tion, the tetraamine spermine acts as an inducer of in infected tobacco plants. In addition, PAs released
pathogenesis-related proteins (Yamakawa et al., 1998) from conjugated and bound pools could also contrib-
and also participates in signaling cascades that acti- ute to the raise in apoplastic free PA levels. The

vate HR-specific genes in tobacco (Takahashi et al., intracellular location of ADC and other PA biosyn-
2003, 2004; Kusano et al., 2007). In this work, the thetic enzymes has been demonstrated by other au-
consequences of apoplastic PA oxidation on host tissue thors (Kaur-Sawhney et al., 2003), and in this work, the
colonization by microorganisms with contrasting activities of these enzymes were found to be extremely

pathogenic strategies, and its relation with plant tol- low in IWFs. Therefore, mechanisms of PA export to
erance to these kinds of pathogens, were investigated. the apoplastic compartment are expected to partici-
pate in the raise of apoplastic PA leaves in response to
Changes in PA Metabolism of Tobacco Plants Infected fungal infection, a subject that deserves further inves-
by the Necrotrophic Fungus S. sclerotiorum tigation. It is also worth pointing out that S. sclerotio-

rum, like the majority of fungi studied so far, lacks

Studies conducted so far in relation with the role of ADC activity (Pieckenstain et al., 2001). In addition,
PAs in plant-pathogen interactions have focused spermine levels in S. sclerotiorum cells are very low,

mainly on viruses (Negrel et al., 1984; Bellés et al., both in mycelium and ascospores (Gárriz et al., 2003,
1991; Torrigiani et al., 1997; Yamakawa et al., 1998; 2004). Thus, the increase in ADC activity and apo-
Hiraga et al., 2000; Marini et al., 2001; Yoda et al., 2003; plastic spermine occurring in leaves infected by S.
Yoo et al., 2004) and biotrophic fungi causing rusts and sclerotiorum is expected to be derived from the host
mildews on several different hosts (Walters et al., 1985; plant and not to result from contamination with fungal
Walters and Wylie, 1986; Cowley and Walters, 2002). metabolites during sample preparation. To our knowl-
However, the attention previously devoted to the edge, this is the first report about changes in PA levels
effects of necrotrophic plant pathogens on host PA and biosynthetic enzymes in response to plant infec-
metabolism has been very scant (Angelini et al., 1993; tion by a necrotrophic pathogen.
Rea et al., 2002). In this work, tobacco plants infected
by the ascomycete S. sclerotiorum were used as a model The Consequences of Alterations in PA Metabolism and
for the study of perturbations induced by necrotrophic Inhibition of Amine Oxidase Activities on Severity of
pathogens on host PA metabolism. Healthy tissues S. sclerotiorum Infection on Tobacco Plants
adjacent to necrotic lesions, which were proved to be
free of fungal mycelium, were used as a target for the To evaluate the consequences of changes in host PA
study of changes in free PA levels and PA biosynthesis levels on tolerance to pathogen infection, tobacco leaf
Plant Physiol. Vol. 147, 2008 9
Marina et al.

discs with enhanced PA levels were infected with S. S. sclerotiorum. Therefore, the utility of the synthetic
sclerotiorum. Accumulation of free putrescine and PA analog 1,19-bis(etilamino)-5,10,15-triazanonadecane
spermidine in leaf discs of transgenic tobacco plants (Bacchi et al., 2002), which was found in our laboratory
as a consequence of tetracycline-induced overexpres- to inhibit oat and maize PAOs in vitro (J. Maiale,
sion of an oat ADC gene, led to a strong increment in unpublished data), was evaluated. Thus, this com-
the necrosis caused by S. sclerotiorum infection. Simi- pound was found to inhibit tobacco PAO activity in
larly, enhancement of apoplastic putrescine, spermi- vitro in a similar degree to guazatine. Taking into
dine, or spermine levels by means of infiltration also account the scarcity of information about the effects of
caused a strong increase of leaf disc necrosis caused by 1,19-bis(etilamino)-5,10,15-triazanonadecane on plant PA
this fungus. Thus, the two above-mentioned alterna- metabolism, its ability to inhibit PAO activity in vivo
tive approaches demonstrated that enhanced PA levels was also evaluated. In this way, 1,19-bis(etilamino)-
increase the severity of S. sclerotiorum infection. The 5,10,15-triazanonadecane was found to inhibit tobacco
question then rose about the mechanism by which PA PAO activity in vivo. Even though endogenous sperm-
accumulation in plant tissues increases disease sever- ine levels in control (noninfiltrated) plants were not

ity. Yoda et al. (2003) demonstrated that PA infiltration high enough to be used as an indicator of PAO inhibi-

into tobacco tissues induces hypersensitive cell death tion by 1,19-bis(etilamino)-5,10,15-triazanonadecane,
as the result of hydrogen peroxide formation due to this compound led to an increase in apoplastic sperm-
PA oxidation, and proposed this mechanism to be ine levels when coinfiltrated with this tetraamine into
involved in the HR of tobacco plants to TMV. Al- leaf discs, as detected shortly (4 h) after infiltration.
though effective against plant pathogens that depend Moreover, 1,19-bis(etilamino)-5,10,15-triazanonadecane

on living host tissues, such as viruses and biotrophic decreased the accumulation of the product of sperm-
fungi and bacteria, the HR is far from being beneficial ine oxidation 1,3-diaminopropane in discs infiltrated
as a plant response to infection by necrotrophic mi- with spermine, as evaluated 24 h after infiltration. Thus,
croorganisms, in that host-cell death favors tissue these results demonstrate that 1,19-bis(etilamino)-
colonization by this kind of pathogens (Govrin and 5,10,15-triazanonadecane acts as a PAO inhibitor in
Levine, 2000; Mayer et al., 2001; Glazebrook, 2005). tobacco leaves. Interestingly, and as opposed to gua-
Therefore, the contribution of PA oxidation to the zatine, 1,19-bis(etilamino)-5,10,15-triazanonadecane
development of necrotic lesions in tobacco leaves exerted no inhibition of mycelial growth of S. sclero-
infected by S. sclerotiorum was investigated. As an tiorum in vitro. On the basis of the results obtained,
initial approach, the activity of amine oxidases in- N,N#-diaminoguanidine and 1,19-bis(etilamino)-5,10,15-
volved in PA catabolism was evaluated in IWFs triazanonadecane were selected for further experi-
obtained from leaf discs. Taking into account that ments aimed to inhibit tobacco DAO and PAO activities

amine oxidases involved in PA catabolism are pre- during the response to S. sclerotiorum infection. More-
dominantly located in cell walls (Angelini et al., 1993; over, results obtained in this work demonstrate that the
Šebela et al., 2001), IWFs were used as a source for the PA analog 1,19-bis(etilamino)-5,10,15-triazanonadecane
evaluation of these enzyme activities in vitro. Both is a powerful tool for the study of the physiological

DAO and PAO activities were detected and, although roles of PAOs, similarly to other inhibitors such as
they were not affected by S. sclerotiorum infection, N-prenylagmatine (Cona et al., 2006b).
basal DAO and PAO activities could contribute to the The DAO inhibitor N,N#-diaminoguanidine was
development of necrotic lesions as a consequence of found to attenuate the development of necrotic lesions
PA accumulation in the apoplast after infection. There- caused by S. sclerotiorum infection, both in leaf discs

fore, it was decided to study the consequences of in infiltrated with putrescine and in control discs infil-
vivo tobacco amine oxidase inhibition on the devel- trated with water. Similarly, the PAO inhibitor 1,19-
opment of necrotic lesions during leaf infection by bis(etilamino)-5,10,15-triazanonadecane diminished S.

S. sclerotiorum. For this purpose, compounds capable sclerotiorum rot in leaf discs infiltrated with spermidine
of inhibiting amine oxidase activities of the plant host and spermine, as well as in control discs infiltrated
without exerting fungicidal effects would be useful. with water. As a whole, these results demonstrate that
In this regard, N,N#-diaminoguanidine, a compound DAO and PAO-mediated PA oxidation occurs during
reported to inhibit soybean DAO (Nikolov et al., 1990; infection of tobacco tissues by S. sclerotiorum and, far
Xing et al., 2007), was found in this work to inhibit from being beneficial to the host, contributes to disease
tobacco DAO, and exerted no effect on mycelial growth development. This observation is in good agreement
of S. sclerotiorum in vitro. Guazatine has been demon- with previous works that found oxidative responses to
strated to inhibit tobacco PAO activity (Yoda et al., increase the severity of infections caused by necrotro-
2006), but is a powerful fungicidal agent (Mackintosh phic pathogens in plant hosts (Govrin and Levine,
and Walters, 2003; Dreassi et al., 2007), which was also 2000; Mayer et al., 2001). On the other hand, results of
found to cause a strong inhibition of mycelial growth of this work are in apparent contradiction with those of
S. sclerotiorum in vitro (J. Maiale, unpublished data). As Rea et al. (2002), who found DAO to participate in cell
a consequence, a PAO inhibitor other than guazatine wall strengthening as part of chickpea (Cicer arietri-
was necessary for in vivo studies about the role of num) local responses to Ascochyta rabiei, and proposed
PAO in the response of tobacco leaves to infection by that this enzyme contributes to plant tolerance to
10 Plant Physiol. Vol. 147, 2008
Apoplastic Polyamine Oxidation in Plant Defense

infection by this necrotrophic fungus. However, these The Role of PAs and PA Oxidation in the Response
authors based their conclusions on experiments per- of Tobacco to Infection by the Biotrophic
formed by infiltrating a DAO inhibitor into chickpea Bacterium P. viridiflava
tissues once infection was already established, as
opposed to this work, in which infiltrations with the Results obtained in this work demonstrate that
inhibitors were done prior to infection. In this way, tobacco plants respond to S. sclerotiorum infection by
differences in the methodological approaches em- accumulating PAs in the leaf apoplast and also that PA
ployed in both works, as well as in the pathogenic oxidation leads to enhanced cell death. Thus, apo-
strategies of both organisms and defense mechanisms plastic PA accumulation and oxidation, far from being
of both hosts probably explain these seemingly con- beneficial to the host, contributed to increased disease
tradictory findings. severity. It is not surprising that such a response
enhances the development of lesions caused by a
necrotrophic fungus, given that these kinds of patho-
gens feed on necrotic tissue. To our knowledge, this is

PA Oxidation and the Generation of Active the first report about PA accumulation and oxidation
Oxygen Species playing a detrimental role in plant responses to path-

Hydrogen peroxide is known to play several roles in ogen infection.
plant defense against pathogen attack. In addition to The question then arose whether responses of to-
the proposed direct antimicrobial effect (Mehdy et al., bacco PA metabolism to infection by a biotrophic
1996; Rojkind et al., 2002), hydrogen peroxide acts via pathogen are similar to those developed in response

signal transduction pathways that lead to the expres- to a necrotrophic one. Thus, to address this question,
sion of defense genes (Orozco-Cárdenas et al., 2001; experiments similar to those performed with S. scler-
Torres et al., 2006). Moreover, hydrogen peroxide otiorum were done using P. viridiflava as a pathogen.
formed through PA oxidation significantly contributes No previous information was available regarding the
to hypersensitive cell death in tobacco (Yoda et al., role of host PA metabolism in response to biotrophic
2003). In this work, infiltration of leaf discs with PA bacteria. As do many other biotrophic bacteria, P.
concentrations capable of leading to an increase in leaf viridiflava propagates in the apoplastic space after
necrosis caused by S. sclerotiorum infection, were not penetration into the host. In this work, this bacterium
found to result in the accumulation of detectable was found to induce spermine accumulation in apo-
hydrogen peroxide levels. In this regard, it should be plastic fluids, as evaluated 20 and 48 HAI. It is worth
noticed that PA concentrations used for infiltration pointing out that spermine was not detected in P.
experiments were in the range of those concentrations viridiflava extracts (data not shown), which suggests

detected in IWFs after leaf infection by S. sclerotiorum. that the accumulation of this tetraamine in infected
In this way, hydrogen peroxide formed through oxi- tissues resulted from the host’s metabolic machinery,
dation of PA quantities used in this work is expected to not from bacterial origin. Moreover, IWFs were freed
½AQ11 reach very low levels, which probably are not detected from bacteria by centrifugation prior to the determi-

by the DAB method employed. nation of PA levels, thus minimizing the risk of sample
As opposed to hydrogen peroxide, superoxide rad- contamination with bacterial metabolites. Deliberate
ical was found to be accumulated when leaf discs were modification of leaf PA levels prior to infection either
infiltrated with PA concentrations capable of increas- by using leaf discs from the transgenic plants previ-
ing leaf necrosis caused by S. sclerotiorum. PA-induced ously described or by PA infiltration, demonstrated
that enhanced host PA levels result in a significant

accumulation of superoxide was decreased by amine

oxidase inhibitors, thus demonstrating that produc- decrease of bacterial growth in planta. More specifi-
tion of this active oxygen species was the result of cally, it was demonstrated that spermine infiltration

PA oxidation. In this way, superoxide produced after inhibited bacterial growth, this effect being due to
PA accumulation and oxidation in the apoplast prob- PAO-mediated oxidation of this tetraamine. This hy-
ably contributes to the development of leaf necrosis pothesis is supported by the observation that the PAO
during infection by S. sclerotiorum, as a consequence of inhibitor 1,19-bis(etilamino)-5,10,15-triazanonadecane
the toxicity of this active oxygen species on host reverted the inhibition of bacterial growth caused by
tissues. spermine infiltration. Moreover, 1,19-bis(etilamino)-
The way in which superoxide was produced after 5,10,15-triazanonadecane decreased bacterial growth
PA oxidation remains to be explained. A possible in discs not infiltrated with exogenous spermine. As a
explanation is that hydrogen peroxide is converted whole, these results demonstrate that PA accumula-
into superoxide by the nonenzymatic reactions (Fenton, tion and further oxidation contribute to restrict the
½AQ12 1894; Haber-Weiss, 1934). The fact that hydrogen per- propagation of P. viridiflava in tobacco tissues.
oxide was not detected in this work does not rule out Taking into account the previously discussed roles
this hypothesis, given that this active oxygen species of PA oxidation in the generation of hydrogen perox-
could be produced in low amounts and be converted ide and superoxide radical, it is tempting to speculate
rapidly into other active oxygen species by the above- that active oxygen species derived from spermine
proposed mechanism. oxidation are involved in the reduction of P. viridiflava
Plant Physiol. Vol. 147, 2008 11
Marina et al.

growth in tobacco leaves. However, it cannot be ruled Plant Material and Growth Conditions
out that spermine plays additional roles during the Tobacco (Wisconsin W38 and Xanthi-nc) seeds were disinfected with 70%
response of tobacco plants to P. viridiflava infection, as (v/v) ethanol for 2 min, 5% (w/v) sodium hypochlorite, and 0.1% (w/v) SDS
well as in response to S. sclerotiorum. Spermine has for 15 min and thoroughly rinsed with sterile distilled water. Disinfected seeds
been shown to act as a systemic inducer of PR gene were dispensed in petri dishes containing Murashigue and Skoog agar medium
(Murashigue and Skoog, 1962) and incubated in a plant growth chamber. After
expression (Yamakawa et al., 1998) and participates in
2 weeks, plantlets were transferred to pots filled with a mixture of soil, sand,
signaling cascades that activate HR-specific genes in and perlite (1:1:1), and were irrigated with half-strength Hoagland’s nutrient
tobacco (Takahashi et al., 2004). Therefore, additional solution (Hoagland and Arnon, 1950). Plants were grown for 6 to 8 weeks in a
work will be necessary to determine whether sperm- growth chamber with a 16-h/8-h photoperiod at 24/21 6 2°C and 55/75 6 5%
ine also plays such roles in tobacco plants infected by relative humidity (day/night) and a photon flux density of 200 mmol m22s21
provided by cool-white fluorescent and incandescent lamps. Transgenic tobacco
the two pathogens studied in this work. Wisconsin W38 TetR/Oat ADC and tobacco Wisconsin W38 TetR (Masgrau et al.,
1997) were cultivated under similar conditions.

CONCLUSION Plant Inoculation and Disease Analysis

PA metabolism of tobacco plants undergoes similar, Inoculation with S. sclerotiorum

although not identical, changes in response to infec- Tobacco plants were inoculated with S. sclerotiorum by placing discs of
tion by two pathogens with opposing pathogenic mycelium on the adaxial face of leaves of the third or fourth pair. Mycelium
strategies. These changes lead to the accumulation of discs were cut from the edge of a colony actively growing on Czapek-Dox
apoplastic free putrescine and spermine in response to medium. Two discs of mycelium were placed per leaf, each one on different
sides of the mid-vein, and leaves were covered with a translucent film to

infection by the necrotrophic fungus S. sclerotiorum,
maintain high relative humidity. A similar procedure was used for the inoc-
and free spermine in response to the biotrophic bac- ulation of leaf discs. In this case, a disc of mycelium was placed in the center of
terium P. viridiflava, these PAs acting as substrates of 18-mm-diameter leaf discs, which were subsequently dispensed on petri dishes
amine oxidases. PA accumulation and oxidation play a containing water-agar (0.8% w/v). Inoculated plants and leaf discs were
½AQ13 role that is beneficial for the host during local re- incubated in the plant growth chamber for 48 to 72 h, until development of
necrotic lesions around the inoculation site. Leaves and leaf discs inoculated
sponses to infection by the biotrophic bacterium
with a disc of Czapek-Dox medium not inoculated with S. sclerotiorum were
P. viridiflava, by restricting bacterial growth in planta. used as controls (mock-inoculated). The size of the necrotic area around the
½AQ14 On the contrary these responses are detrimental in inoculation site was determined using the Image-ProPlus V 4.1 software (Media
local defense to infection by the necrotrophic fungus S. Cybernetics) and was expressed as a percentage of the whole disc area.
sclerotiorum, and lead to increased disease severity. It
still remains to be determined whether reactions sim- Inoculation with P. viridiflava
ilar to those found in this work are of general occur-

rence in other plant hosts infected by necrotrophic and Inoculums of P. viridiflava were prepared by streaking bacteria from 280°C
onto King’s B agar-medium plates, incubating plates at 28°C for 24 h and
biotrophic pathogens. However, the findings of this scraping bacterial cells off plates in 10 mM MgCl2, pH 7.0, to yield 5 3 108 CFU
work are in good agreement with the fact that host per milliliter. Bacterial suspensions (0.5 mL) were infiltrated into tobacco
responses leading to cell death contribute to restricting leaves using a 1-mL hypodermic syringe without a needle as described by Wei

infection by biotrophic pathogens, but play a negative et al. (1992). Leaves infiltrated with 10 mM MgCl2, pH 7.0, were used as
controls. A similar procedure was used for the inoculation of leaf discs, which
role in local responses to necrotrophic organisms. As a
were immersed for 30 min in a bacterial suspension obtained as described
summary, it can be concluded that apoplastic free PAs above, subsequently covered with a translucent film and incubated under
play an important role in local responses to pathogen continuous light. After 2 h, discs were rinsed with sterile 10 mM MgCl2, pH 7.0,
infection by affecting the ability of pathogenic micro- dispensed onto petri dishes containing water-agar, and incubated for 48 h in a
organisms to colonize host tissues. This work also plant growth chamber. For the evaluation of bacterial growth, leaf discs were

ground in 250 mL of 10 mM MgCl2, pH 7.0, and the extracts thus obtained were
demonstrates that modification of plant PA metabo- plated on King’s B agar medium. The number of CFU mL21 was determined
lism by means of a transgenic approach based on the after 24-h incubation at 28°C. Leaf discs infiltrated with 10 mM MgCl2, pH 7.0,

use of PA biosynthetic genes under the control of an were used as controls.

inducible promoter can contribute to modifying plant
responses to pathogen infection. Chemicals
Standard chemicals and guazatine acetate of the highest purity avail-
able were purchased from Sigma Chemical. 1,19-Bis(etilamino)-5,10,15-triazano-
MATERIALS AND METHODS nadecane was kindly gifted by Dr. Benjamin Frydman (SLIL Biomedical
Corporation). N,N#-Diaminoguanidine monohydrochloride was purchased
Fungal and Bacterial Strains from ICN Biomedicals. L-[1-14C]Orn was purchased from NEN Life Sciences
An isolate of Sclerotinia sclerotiorum from the IIB-INTECH Fungal Culture Products. L-[1-14C]Arg and S-adenosyl-L-[carboxyl-14C]Met were purchased
Collection (IFCC 458/02), originally isolated from sunflower (Helianthus from Amersham Pharmacia Biotech. Inhibitors and PAs were dissolved in
annuus) capitula with head rot symptoms, was used for all the experiments. distilled water and stored at 220°C until used.
The fungus was routinely maintained in potato-dextrose agar slants at 4°C.
Prior to inoculation on tobacco (Nicotiana tabacum), mycelium was grown in In Vivo O22 Production Assays
solid Czapek-Dox medium (50 g L21 Glc, 2 g L21 NaNO3, 1 g L21 KH2PO4, 0.5 g
L21 MgSO4.7H2O, 0.5 g L21 KCl, 0.05 g L21 FeSO4.7H2O, 20 g L21 agar, pH 5.5– Superoxide (O22) accumulation in tobacco tissues was determined with
6.0) at 24°C. Pseudomonas viridiflava strain Pvalb8 (Alippi et al., 2003) was NBT, which reacts with O22 producing a blue formazan precipitate. Leaf discs
maintained at 280°C in King’s B medium (King et al., 1954) amended with were infiltrated with 5 mM putrescine and 5 mM N,N#-diaminoguanidine
20% glycerol. monohydrochloride, either separately or in combination. Another set of leaf

12 Plant Physiol. Vol. 147, 2008

Apoplastic Polyamine Oxidation in Plant Defense

discs was incubated with 5 mM spermine, either separately or in combination For the determination of DAO activity, 190 mL aliquots of IWFs obtained as
with 5 mM each of the amine oxidase inhibitors 1,19-bis(etilamino)-5,10,15- described previously were mixed with 10 mL of a substrate solution containing
triazanonadecane and 5 mM guazatine. Discs infiltrated with water were used 10 mM nonradioactive putrescine and 5 nCi mL21 [1,4-14C]putrescine. A similar
as controls of O22 accumulation in the absence of chemical treatments. NBT procedure was used for the determination of PAO activity, by using a
was added to all the infiltration solutions at a final concentration of 0.01% substrate solution consisting of 10 mM nonradioactive spermine and 5 nCi
(w/v). After infiltration, discs were incubated in the infiltration solution for mL21 [1,4-14C]spermine. For inhibition of amine oxidases, guazatine acetate,
3 h in the dark at 30°C with gentle shaking. Stained leaf discs were mounted 1,19-bis(etilamino)-5,10,15-triazanonadecane, and N,N#-diaminoguanidine
on glass slides and scanned. Images thus obtained were inverted to render a monohydrochloride were added to the reaction mixture in a final concentra-
negative prior to transformation into black and white 8-bit images. Formazan tion of 0.5 mM.
color intensity (in a negative corresponding to the lighter tones of gray) was
determined by using the Image-ProPlus V 4.1 software (Media Cybernetics).
Analysis of ADC Gene Expression by RT-PCR
Discs infiltrated with 10 mM MnCl2, a highly effective O22 dismutating
catalyst agent, developed no blue formazan precipitate, thus confirming the Total RNA was extracted from frozen leaf tissue using TRI reagent (Sigma)
specificity of the method for the detection of O22. according to the manufacturer’s instructions. First strand of cDNA was
obtained by using the following mixture: 2 mg of total RNA, 0.5 mM dNTPs,
Preparation of IWFs 1 mL of moloney murine leukemia virus RT (200 units mL21; Promega), 5 mL 53 ½AQ17
reaction buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, 50 mM DTT, pH

IWFs were collected from tobacco leaves as described by De Wit and 8.3), 10 pmol of oligo(dT) primers (Biodynamics SRL), and distilled water up to

½AQ15 Spikman (1982) with some modifications. For the determination of apoplastic a total volume of 25 mL. The reaction mixture was incubated at 37°C for 1 h.
PA concentrations, leaf discs (;50) were vacuum infiltrated (two cycles, 667 PCR amplification was done with 2 mL of RT reaction as template, 4 mL of
mbar, 20 min) with distilled water, while 0.1 M, pH 8.0, Tris-HCl buffer with 103 Taq polymerase buffer (500 mM KCl, 100 mM Tris-HCl, 15 mM MgCl2, pH
½AQ16 0.5 mM phenylmethanesulfonyl fluoride was used for the infiltration of 9.0), 0.05 mM dNTPs, 0.2 mL Taq polymerase (5 units mL21; Promega), 12.5
discs to be used for the determination of DAO and PAO. After gently drying pmol of primers 5#-TGGCCTTCAGGAGTATGCATC-3# (sense), and 5#-CCA-
on filter paper, leaf discs were introduced into a 10-mL syringe and IWFs CGAATAGCAGCAGCAGA-3# (antisense) corresponding to NtADC1 (Gen-

were recovered by centrifugation for 20 min at 2,000g (4°C) in a 50-mL Bank accession no. AF127240), or 5#-GGATTCTGGTGATGGTGTTAG-3#
Nalgene centrifuge tube. Chlorophyll and cytosolic nonphosphorylating (sense) and 5#-ACTTCCTCTCAGGTGGAGCTAC-3# (antisense) correspond-
glyceraldehyde-3-phosphate dehydrogenase activity were determined in ing to tobacco actin (GenBank accession no. X63603), in a total volume of
IWFs as described by Lichtenthaler (1987) and Bustos and Iglesias (2002), 40 mL. Amplification conditions for both genes were 20, 24, 27, and 30 cycles
respectively, to verify that the infiltration procedure caused no significant (94°C, 1 min; 58°C, 1 min; and 72°C, 1 min) and a final elongation at 72°C for
cellular disruption. The activity of these enzymes and chlorophyll levels were 7 min. Amplification products were visualized by staining gels with ethidium
only 1.0% to 2.0% of those obtained by grinding the whole leaf discs, thus bromide and equal loading of RNAs was confirmed by monitoring the levels
demonstrating that the degree of cellular disruption was extremely low, and of actin gene. Bands were analyzed by densitometry using the Gel Pro
the IWFs obtained are not expected to be contaminated with cellular contents. Analyzer 3.0 software (Media Cybernetics).

Determination of Free PA Concentration in Leaf Pharmacological Treatments

Extracts and IWFs PAs (putrescine, spermidine, and spermine), guazatine acetate, 1,19-
bis(etilamino)-5,10,15-triazanonadecane, and N,N#-diaminoguanidine mono-
To determine free PA levels in leaf extracts, plant material (300 mg) was

hydrochloride were infiltrated into the apoplastic space of leaf discs (18-mm
ground in liquid nitrogen, extracted in 600 mL 5% (v/v) perchloric acid, and
diameter) using a vacuum pump (2 cycles, 667 mbar, 20 min). All these
incubated overnight at 4°C. After centrifugation at 10,000g for 15 min, 10 mL of
chemicals were employed at a concentration of 5 mM. After infiltration, discs
100 mM 1,7-heptanediamine (ICN Biomedicals) was added as internal stan-
were placed in petri dishes containing water-agar and incubated in the plant
dard to 200 mL aliquots of leaf extracts. For PA determinations in the
growth chamber during the time periods indicated for each experiment.
apoplastic compartment, IWFs obtained as described in the previous section

were mixed with perchloric acid to a final concentration of 5% (v/v). Then,

200 mL of saturated Na2CO3 and 400 mL of dansyl chloride (10 mg mL21 Induction of ADC Expression in Transgenic Plants
acetone) were added and the mixture was incubated overnight in the dark at
room temperature. Reaction was stopped by adding 100 mL of Pro (100 mg Leaf discs obtained from transgenic tobacco Wisconsin W38 TetR/Oat ADC
mL21) and dansylated PAs were extracted in 500 mL of toluene. Organic phase plants, which express an oat (Avena sativa) ADC gene under the control of the
was vacuum evaporated and dansylated PAs were dissolved in 200 mL of tetracycline-inducible promoter TetR (Masgrau et al., 1997), were incubated in
acetonitrile and analyzed by reversed phase HPLC as described previously a tetracycline solution (2 mg L21 in 5 mM MES, pH 6.5) for 24 h. Discs obtained

(Marcé et al., 1995). from transgenic tobacco Wisconsin W38 TetR plants, which harbor the
tetracycline-inducible promoter but no heterologous ADC gene, were treated

Enzyme Activity Assays
ADC, ODC, and AdoMetDC were extracted by homogenization of leaf Statistics
tissue (500 mg fresh weight) in 2 volumes of 100 mM, pH 7.5, phosphate buffer
containing 0.5 mM EDTA, 10 mM dithiothreitol, 1 mM pyridoxal phosphate, Treatments consisted of three to five replicates, and each experiment was
and 20 mM sodium ascorbate. Crude extracts thus obtained were clarified by conducted at least two times with similar results. Results from representative
centrifugation at 10,000g for 10 min. All the above-described procedures were experiments are shown as means 6 SD. Data were analyzed by appropriate
carried out at 4°C. Protein concentration in the supernatants was determined Student’s t test or ANOVA followed by posthoc comparisons by Bonferroni or
as described by Bradford (1976), using bovine serum albumin as the standard. Dunnet’s test.
Enzyme activities were determined by mixing 190 mL of the extract with 10 mL
of the substrate solution in a glass tube fitted with a rubber stopper and a filter Sequence data from this article can be found in the GenBank/EMBL data
paper disc soaked in 2 N KOH. Substrate solutions for the determination of libraries under accession numbers nnn. ½AQ18
ADC, ODC, and AdoMetDC activities contained 1 mM nonradioactive sub-
strate amended with 5 nCi mL21 L-[1-14C]Arg, 5 nCi mL21 L-[1-14C]Orn, and
2.5 nCi mL21 S-adenosyl-L-[carboxyl-14C]Met, respectively. After incubation Supplemental Data
for 1 h at 37°C, the reaction was stopped and 14CO2 was released by adding
Supplemental Figure S1. In vitro determination of apoplastic DAO and
200 mL of 10% (v/v) PCA. After allowing 1 h at 37°C for distillation of 14CO2,
PAO in tobacco plants infected by S. sclerotiorum and P. viridiflava.
the paper was immersed in 200 mL of scintillation cocktail (4 g Omnifluor L21
toluene), and radioactivity was determined in a Beckman LS 5000 scintillation Supplemental Figure S2. In vitro DAO and PAO activities of tobacco as a
counter. function of plant age.

Plant Physiol. Vol. 147, 2008 13

Marina et al.

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