Physiological and Molecular Plant Pathology (2000) 56, 95–106
doi : 10.1006\pmpp.1999.0255, available online at http:\\www.idealibrary.com on
β-Aminobutyric acid-mediated enhancement of resistance in tobacco to
tobacco mosaic virus depends on the accumulation of salicylic acid
J. S I E G R I S T*, M. O R O B ER and H. B U C H E N A U E R
Institute of Phytomedicine, UniŠersity of Hohenheim, D-70593 Stuttgart, Germany
(Accepted for publication December 1999)
A number of chemical and biological agents are known as inducers of systemic acquired resistance (SAR)
to tobacco mosaic virus (TMV) in tobacco plants. In the present study, a local spray application of the
non-protein amino acid DL-β-aminobutyric acid (BABA) was effective in enhancing resistance to TMV
in tobacco plants containing the N gene. In contrast, the isomer α-aminobutyric acid (AABA) showed a
much lower activity whereas γ-aminobutyric acid (GABA) was completely inactive, indicating a strong
isomer specificity of aminobutyric acid in triggering enhanced virus resistance.
Rapid cell death was detected in tobacco leaf tissues after foliar application of BABA, subsequently
resulting in the development of macroscopically visible, necrotic lesions. BABA-induced cell death was
associated with the rapid generation of superoxide and hydrogen peroxide. As further consequences, the
occurrence of lipid peroxidation in treated tissues, a local and systemic increase of salicylic acid (SA) levels
and the expression of PR-1a, a molecular marker of SAR in tobacco, could be observed. None of
these responses was detectable after treatment with GABA.
Enhancement of virus resistance by BABA was found to be strictly dependent on SA-mediated signal
transduction since it could not be detected in salicylate hydroxylase (nahG) expressing transgenic tobacco
plants. These findings suggest that in tobacco, primary processes triggered by foliar application of BABA,
resemble those initiated by microbes during a hypersensitive response (HR) that result in SAR activation.
# 2000 Academic Press
Keywords : β-Aminobutyric acid ; BTH ; hypersensitive response ; reactive oxygen species ; systemic
acquired resistance ; tobacco ; tobacco mosaic virus.
INTRODUCTION
Natural disease resistance of plants is based on preformed
and inducible mechanisms. The latter are activated in the
case of pathogen contact as an active response of plant
cells after recognition of the invader. Defense mechanisms
can be expressed locally, at the site of pathogen attacked
cells as well as systemically, in uninfected tissues. They
include changes like papilla formation, cell wall lignification, production of reactive oxygen species (ROS),
initiation of a hypersensitive reaction (HR), expression of
pathogenesis-related proteins (PRs) and accumulation of
antimicrobial metabolites [28 ]. In recent years, the battery
of the plants’ own defense reactions has attracted a lot of
attention both in applied agriculture and basic research.
* To whom all correspondence should be adressed. E-mail :
jsieg!uni-hohenheim.de
Abbreviations used in the text : AABA, DL-α-aminobutyric
acid ;
BABA,
DL-β-aminobutyric
acid ;
BTH,
benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester ;
DAB, diaminobenzidine ; GABA, γ-aminobutyric acid ; HR,
hypersensitive response ; NBT, nitroblue tetrazolium ; ROS, reactive oxygen species ; SA, salicylic acid ; SAR, systemic
acquired resistance ; TBARS, thiobarbituric acid reactive substances ; TMV, tobacco mosaic virus.
0885–5765\00\030095j12 $35.00\0
In this relation, scientific activities were mainly focused
on the phenomenon of induced disease resistance. In
general, induced resistance can be defined as an activation
of mechanisms that allow plants to defend themselves
against a broad spectrum of pathogens without any
changes of the plant genome [51 ]. Depending on the
activating stimulus, induced resistance can be devided in
pathogen-induced systemic acquired resistance (SAR)
and rhizobacteria-mediated induced systemic resistance
(ISR). In the case of SAR, SA-dependent signal transduction was found to be imperative for triggering defense
responses [27, 34 ]. In contrast, ISR seems to operate in an
SA-independent manner [39 ]. Essential elements of the
SAR and the ISR pathway have already been characterized [15, 52 ].
Several natural and synthetic agents have been decribed
as activators of defense-related processes when applied to
plants and some of these may have potential application
in agriculture [26, 58 ]. However, in only a few cases have
the criteria definded by Kessmann et al. [26 ] been clearly
demonstrated. Thus, besides an effective disease control,
chemical agents should be shown to induce plant specific
defense responses. At the moment, the best characterized
compounds within the group of chemical activators of
# 2000 Academic Press
96
J. Siegrist et al.
disease resistance are 2,6-dichloroisonicotinic acid (INA)
and benzo[1, 2, 3]thiadiazole-7-carbothioic acid-S-methyl
ester (BTH) which are both synthetic substances. These
inducers were used in a number of recently published
studies dealing with mechanisms involved in SAR [45 ].
With the help of transgenic tobacco and Arabidopsis
plants that constitutively express the bacterial nahG gene
encoding a salicylate hydroxylase, it was demonstrated
that both chemicals enter the SAR signalling pathway
downstream of SA [17, 53 ]. Meanwhile, BTH was
introduced as a commercial product (Bion2, Novartis
Ltd., Basel, Switzerland) in Europe for the protection of
wheat against powdery mildew by means of SAR [19 ].
Other inducing agents like probenazole [55 ] or Milsana4,
an extract from the giant knotweed Reynoutria sachalinensis
[12 ], were exclusively active in only a few selected
pathosystems and can therefore not be considered as
effective inducers in general.
Besides BTH and INA, the non-protein amino acid
DL-β-aminobutyric acid (BABA) was described as SAR
inducer in several plant species belonging to the Solanaceae.
BABA treatment was effective in activating plants for a
sucessful defense against foliar diseases [7–9, 20, 47, 50 ]
and also against soilborne pathogens [4, 30 ]. In addition,
the inducing efficiency of BABA was recently demonstrated in cultured parsley cells, a model system for the
testing of SAR-inducing agents [44 ]. In contrast to
BABA, the isomers DL-α-aminobutyric acid (AABA) and
γ-aminobutyric acid (GABA) showed only weak or no
activity in parsley cell cultures [44 ] and in all plant–
pathogen interactions tested so far [7–9, 20, 47, 50 ]. This
indicates a high isomer specificity of aminobutyric acid in
defense activation.
In tomato, Cohen and coworkers were able to correlate
BABA-induced resistance to Phytophthora infestans with the
accumulation of several PR proteins in treated and in
non-treated parts of the plants [8 ]. When tested in Šitro,
BABA exhibited no direct activity against various
pathogens [7–9, 20, 47, 50 ] which is a further important
prerequisite for abiotic inducers of disease resistance [26 ].
However, except for these biochemically based studies on
BABA-activated mechanisms, the mode of action of this
resistance-inducing chemical was not further examined in
detail. In the present study the classical SAR test system
tobacco-tobacco mosaic virus (TMV) was used to gain an
insight into primary BABA-induced processes in plants.
MATERIALS AND METHODS
Materials
BTH was supplied by K.-L. Nau (Novartis Ltd.,
Frankfurt, Germany). BABA was purchased from Fluka
(Buchs, Switzerland), all other chemicals came from
Sigma (Deisenhofen, Germany). Tobacco PR-1a c-DNA
was provided by L. Friedrich (Novartis Ltd., Research
Triangle Park, U.S.A.).
Plants
Seeds of transgenic PR-1a : uidA tobacco plants [10 ] were
provided by D. F. Klessig (Rutgers University, NJ,
U.S.A.), seeds of transgenic NahG-10 tobacco [18 ] came
from K. Lawton (Novartis Ltd., Research Triangle Park,
U.S.A.). Non-transgenic tobacco (Nicotiana tabacum cv.
Xanthi-nc), bearing the N gene derived from Nicotiana
glutinosa for hypersensistive resistance to TMV, was used
for inducer verification. Tobacco plants were grown
under controlled conditions in a 14 h\10 h day\night
cycle at 24mC and 18mC, respectively.
Inducer treatment
Chemical treatment was performed by spraying aqueous
solutions of the aminobutyric acids (10 m) on leaves 3
and 4 of tobacco plants with six fully developed leaves.
BTH (0.5 m) was applied in the form of the commercial
plant activator Bion2 (WP 50, Novartis Ltd., Frankfurt,
Germany). For biological induction of resistance, corresponding leaves of tobacco plants were inoculated with
TMV (10 µg ml−" in 25 m sodium phosphate buffer pH
7.0) using celite (0.1 mg ml−") as abrasive.
Challenge inoculation with TMV
Control plants and inducer-treated tobacco plants were
challenge inoculated on leaf 5 with TMV (10 µg ml−" in
25 m phosphate buffer pH 7.0). Since N gene tobacco
is genetically resistant to TMV by local lesion formation,
evaluation of inducer effects on these plants was performed
7 days after inoculation by counting the number of local
lesions as well as by measuring the diameter of 20 lesions
on each leaf. In this system, effective inducers mediate an
enhancement of resistance by reducing the symptom
severity [17 ].
Quantification of SA
Total contents of SA (the sum of free and conjugated SA)
in tobacco leaves were determined according to Mo$ lders
et al. [37 ] with some modifications. For SA extraction,
tobacco leaf material (1 g) was grinded in liquid nitrogen
and subsequently homogenized in a mortar after addition
of 5 ml MeOH (90 % v\v). The homogenate was
centrifuged for 15 min at 4mC with 15 000 rpm and the
resulting supernatant carefully removed. After resuspenion of the pellet in 5 ml MeOH (90 % v\v) and a
further centrifugation, both methanolic supernatants were
combined and vacuum-reduced to 1\10 of the original
β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus
volume. Extracts were directly hydrolysed for 2 h at 80mC
in the presence of 10  HCl to release glucosidically
bound SA. After acidic hydrolysis, pH of the extract was
adjusted to 6.5 with 1  NaOH. Total SA-contents were
analysed by HPLC (Pharmacia, Uppsala, Sweden) using
a fluorescence detector (LC 304, Linear Instruments,
Reno, U.S.A.). Recovery rates were determined by adding
SA standards to blank samples. SA levels of test samples
were respectively corrected for recovery.
Analysis of PR-1a gene expression
Expression of PR-1a was checked in wild-type tobacco
(Nicotiana tabacum cv. Xanthi-nc) by Northern gel blot
analysis. Total RNA was extracted from tobacco leaf discs
(100 mg FW) which were collected from inducer-treated
and non-treated leaves according to Ka$ stner et al. [23 ].
RNA (10 µg) was separated on 1.1 % (w\v) agarose gels
which contained 6 % (v\v) formaldehyde as described
previously [43 ]. Equal loading of samples was verified by
adding ethidium bromide (0.01 mg ml−") in the sample
buffer. RNA was blotted overnight onto a positively
charged nylon membrane (Hybond-Nj, Amersham Life
Science, Buckinghamshire, U.K.) by capillary transfer
using 10iSSC (1iSSC l 0.15 m sodium chloride,
0.015  sodium citrate pH 7). After transfer, RNA was
covalently bound to the membrane by using an u.v. crosslinker (Amersham Life Science).
Prehybridisation was carried out for 4 h at 47mC in
2iSSC, 5iDenhard’s solution [1iDenhard’s l 1 m
EDTA (pH 8.0)], 0.02 % (v\v) each of polyvinylpyrrolidone, Ficoll and bovine serum albumin, 1 % (v\v)
formamide, sonicated salmon sperm DNA (100 µg ml−")
and 1 % (w\v) SDS. $#P-labelled cDNA probes were
synthesized by PCR amplification of insert DNA present
in a pBluecript vector (Stratagene, La Jolla, U.S.A.) by
using the primer OL 7 (5h-GGT GGC GGC TCT AG-3h)
and OL 53 (5h-GTA AAA CGA CGG CCA GTG AGC3h). A highly labelled probe was obtained by running
PCR with dNTP concentration of 65 µ except dATP
which was used at 6.5 µ in each sample. In addition 1 µl
[α-$#P] dATP (10 mCi ml−" ; Amersham Pharmacia
Biotec, Freiburg, Germany) was included in each PCR
sample. Hybridization to labelled PR-1a cDNA was
carried out for 16 h at 47mC. The blots were then washed
3i for 15 min at 65mC with 1iSSC containing 1 %
(w\v) SDS . After air-drying, blots were exposed to X-ray
film at k80mC.
As a second approach to demonstrate PR-1a gene
activation after inducer treatment, the expression of a
chimeric PR-1a : uidA reporter gene in transgenic tobacco
plants was tested. Activity of β-glucuronidase (GUS) was
determined according to Conrath et al. [10 ]. The
fluorescence of 4-methylumbelliferone (MU) was
measured with a spectrofluorometer (Hitachi F 2000).
97
GUS activity is given in nanomoles MU mg−" protein h−"
at 37mC. Protein content of samples was determined
according to Bradford [3 ].
EŠaluation of cell death, callose formation and lipid
peroxidation
Histochemical monitoring of cell death was carried out by
infiltrating Evans blue in tobacco leaf discs according to
Schraudner et al. [42 ]. As a second method, leaf discs were
stained with trypan blue to visualize cell death as described
previously [25 ]. For further microscopical evaluation of
BABA- and TMV-mediated lesion formation, leaf discs
were heated for 3 min in a mixture of lactic acid, phenol
and water (1 : 1 : 1, v\v\v). After cooling, the discs were
destained for 2 days in the same mixture [42 ].
Formation of callose was determined by staining leaf
discs with aniline blue according to Dietrich et al. [14 ].
Lipid peroxidation was estimated by measuring the
accumulation of thiobarbituric acid reactive substances
(TBARS) spectrophotometrically as described by
Rusterucci et al. [40 ]. The content of lipid peroxides was
determined by using the molar extinction coefficient for
malondialdehyde (1.56i10& −" cm−").
Histochemical detection of ROS
Generation of superoxide anions (O −) was verified by
#
staining leaf discs with nitroblue tetrazolium (NBT) as
described by Schraudner et al. [42 ]. Histochemical
detection of hydrogen peroxide (H O ) was performed by
# #
infiltrating 3,3h-diaminobenzidine 4 HCl (DAB) according to Thordal-Christensen et al. [49 ]. Infiltrated leaf discs
were destained in ethanol : chloroform (4 : 1) containing
0.15 % (w\v) TCA according to Hu$ ckelhoven and Kogel
[21 ].
RESULTS
BABA enhances resistance to TMV in tobacco
The aminobutyric acid isomers AABA, BABA and GABA
were tested for their potential to enhance resistance to
TMV in tobacco plants that contain the N gene. As a
positive control, the chemical activator BTH was included
which was already demonstrated as a highly effective
inducer in tobacco [17 ]. Compared to the untreated
control, plants which were sprayed on lower leaves either
with 10 m BABA or 0.5 m BTH, exhibited significantly
smaller lesions on upper leaves after challenge inoculation
with TMV (Table 1). Furthermore, the number of lesions
was considerably reduced in treated plants (data not
shown). Foliar application of BABA at concentrations
below 5 m resulted in a markedly reduced efficacy, at
0.5 m BABA was completely inactive (data not shown).
In contrast to BABA, a treatment of tobacco plants with
10 m GABA did not enhance resistance to TMV (Table
J. Siegrist et al.
T      T    1.EffectofatreatmentwithBTH,AABA,BABAor
GABA on the expression of resistance in tobacco plants (Xanthinc) to TMV. Resistance was assessed by inoculating an upper leaf
(leaf 5) with TMV (10 µg mlk"), 7 days after application of the
inducers to leaf 3 and 4.
Treatment
None (control)
BTH (0.5 m)
AABA (10 m)
BABA (10 m)
GABA (10 m)
TMV lesion size (mm)a
1.55p0.24
0.25p0.11
1.10p0.16
0.53p0.30
1.47p0.18
aValues are the meanspSD of five experiments with five
plants per treatment
F. 1. Northern Blot analysis of total RNA extracted from
tobacco (Xanthi-nc) leaves sprayed with 10 m BABA, 10 m
GABA or 0.5 m BTH. Seven days after inducer application,
RNA from treated leaves (lanes 1, 3, 5 and 7) as well as from
non-treated leaves (lanes 2, 4, 6 and 8) was separated by
electrophoresis and probed with $#P-labelled tobacco PR-1a
cDNA.
1). GABA was ineffective up to 50 m (data not shown).
The application of AABA (10 m) resulted in slightly
reduced TMV lesion sizes after challenge inoculation
(Table 1). Due to its weak activity, AABA was not
applied in the further experiments.
BABA induces local and systemic PR-1a gene expression
Induction and maintenance of resistance in tobacco is
closely associated with the coordinated activation of SAR
genes [54 ]. Genes coding for proteins of the PR-1 group
are highly expressed after treatments with chemical and
biological inducers [54 ]. In tobacco plants, foliar application of 10 m BABA resulted in the induction of PR1a mRNA in the treated as well as in non-treated
(systemic) leaves (Fig. 1). As expected, BTH (0.5 m)
also induced local and systemic PR-1a expression. No
accumulation of PR-1a mRNA was detectable after
application of 10 m GABA (Fig. 1).
As an additional experimental approach, transgenic
tobacco plants containing a chimeric PR-1a : uidA reporter
gene construct were used to demonstrate the activation of
the PR-1a promotor by aminobutyric acid derivatives. As
GUS activity (nmol MU h–1 × mg protein)
98
120
Treated
Non-treated
100
*
80
60
*
40
*
20
*
0
Control
BTH
BABA
GABA
F. 2. Expression of a chimeric PR-1a : uidA reporter gene in
treated (leaf 4) and non-treated (leaf 5) parts of transgenic
tobacco plants. GUS activity was determined 7 days after
application of 0.5 m BTH, 10 m BABA or 10 m GABA to
leaf 3 and 4. *Means from four experiments (five plants per
treatment) significantly different from control (P 0.05).
shown in Fig. 2, maximal GUS activity was detected in
leaves sprayed with BTH which was used as a positive
control. Lower but significantly induced activities were
determined in leaves treated with BABA. In contrast,
almost no GUS activity was measured in control plants
and in plants sprayed with GABA. However, increased
GUS activities were also evident in non-treated leaves of
tobacco plants induced by a local application of BTH or
BABA, indicating a systemic activation of the PR-1a
promotor by both chemicals (Fig. 2).
BABA mediates local cell death
Foliar application of BABA to tobacco plants resulted in
the appearance of HR-like lesions on the treated leaves.
Lesions were macroscopically visible 24 h after inducer
treatment, especially on the adaxial leaf surface. Lesions
subsequently increased in size within the next 24 h (not
shown). Compared to the formation of local lesions after
TMV inoculation of tobacco plants bearing the N gene, a
greater number of smaller lesions appeared in BABAtreated tissues (Fig. 3). In contrast to BABA, no visible
signs of cell death were detectable after GABA application
(not shown).
Staining of tobacco leaf material with trypan bue and
Evans blue indicated a rapid initiation of cell death by
BABA. Starting 14–16 h after inducer application,
localized blue spots, indicating irreversible uptake of
Evans blue and therefore cell death, were detected in
BABA-treated tissues [Fig. 4(a)]. Blue coloured regions
expanded up to 48 h after inducer treatment [Fig. 4(b)].
Microscopical evaluation of leaf discs stained with trypan
blue, revealed clusters of dead cells surrounded by
apparently unaffected cells [Fig. 4(c)]. Besides cell death,
β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus
99
(a)
(b)
F. 3. Lesion phenotype on tobacco leaves provoked by various inducer treatments. (a) Phenotype of necrotic lesions, 6 days after
application of BABA (10 m) or TMV (10 µg ml−") inoculation. (b) Microscopical appearance of lesions in destained tobacco leaf
tissue, 6 days after application of BABA (10 m) or TMV (10 µg ml−") inoculation.
100
J. Siegrist et al.
a staining of leaf discs with aniline blue showed a rapid
deposition of callose around dying cells [Fig. 4(d)].
Histochemical localization of BABA-mediated generation of
ROS
Occurrence of cell death is expected to be closely related
to the generation of ROS [35 ]. For this reason, histochemical staining methods for the detection of O − and
#
H O were performed. Infiltration of NBT revealed a
# #
short period of O − generation at 6–8 h after BABA
#
application [Fig. 5(a)]. Subsequently, production of H O
# #
was detectable by DAB staining by 12–14 h after inducer
treatment. Staining continued increasing up to 48 h after
BABA treatment [Fig. 5(b) and (c)]. The spatial
distribution of O − production was more or less diffuse
#
[Fig. 5(a)], whereas H O accumulation was distinctively
# #
localized in regions where cell death could be detected by
staining with Evans blue [Figs 4(b) and 5(c)].
BABA induces lipid peroxidation
Since cell death resembling an HR was detected as a
consequence of foliar BABA application, lipid peroxidation in tobacco leaf tissues was verified as a further
typical marker related to cell death. The extent of
membrane damage in control leaves and BABA-treated
leaves was estimated with the thiobarbituric acid assay.
BABA-induced lipid peroxidation was evident by
increased TBARS contents, measurable 48 h after inducer
application. Compared to control plants, a three-fold
higher TBARS level was determined as maximal response
at 96 h after BABA treatment (Fig. 6).
SA accumulation as prerequisite for BABA-mediated
enhancement of Širus resistance
Activation of SAR by HR-inducing microbes depends on
the accumulation of SA [18 ] whereas chemical activators
like BTH and INA induce SAR in an SA-independent
manner [17, 53 ]. Transgenic tobacco that constitutively
expresses a salicylate hydroxylase gene (nahG) from
Pseudomonas putida cannot accumulate significant amounts
of SA [18 ]. This transgene is a helpful tool to demonstrate
the involvement of SA in SAR expression. Since BABA
was found to induce SA accumulation in wild type
tobacco (Fig. 7), transgenic NahG plants were used to test
the relationship between BABA-induced enhancement of
TMV resistance and SA accumulation. For comparison,
non-transgenic tobacco plants and the well characterized
inducers BTH and TMV were included in this set of
experiments. As shown in Fig. 8 and already known from
the literature [17 ], BTH enhanced resistance in wild type
Xanthi-nc as well as in the transgenic line NahG-10,
whereas TMV was exclusively active as inducer in Xanthinc and not in NahG-10. Interestingly, BABA treatment
resulted in the same phenotype of resistance as seen in the
case of the biotic induction by TMV. BABA as an
effective inducer in non-trangenic tobacco, exhibited no
activity in transgenic NahG plants (Fig. 8). A quantitative
evaluation of the plants revealed a significant reduction of
disease symptoms in wild type and transgenic tobacco by
BTH compared to the control, but no enhanced resistance
in TMV-preinoculated or BABA-treated NahG tobacco
plants (Fig. 9). These results indicate that BABA-mediated
enhanced resistance to TMV in tobacco is SA-dependent.
In addition, it is interesting to note that BABA-mediated
lesion formation was still evident in NahG tobacco (not
shown).
DISCUSSION
BABA mediates local and systemic accumulation of SA
SA is known to play an important role in the process of
SAR activation by HR-inducing microbes [27 ]. To
estimate whether the occurrence of BABA-mediated cell
death could be correlated with an accumulation of SA,
the content of total SA in tobacco plants was determined.
Increased SA levels were found in the leaves 7 days after
spraying with BABA [Fig. 7(a)]. As previously described
in the literature [17, 33 ], an inoculation with TMV
caused a massive accumulation of SA whereas SA contents
did not increase in tobacco plants treated with the plant
activator BTH [Fig. 7(a)]. Similarly, SA accumulated in
the upper (systemic) leaves of BABA-treated tobacco
plants but at a lower level than in directly treated leaves.
Again, SA contents measured in systemic leaves of BABAtreated and TMV-preinoculated plants were much higher
than in control and BTH-treated plants [Fig. 7(b)].
It is expected that chemical inducers of disease resistance
will play the key role for the integration of the SAR
concept in modern plant protection strategies [31 ]. For
this reason, the search for new inducing agents as well as
the elucidation of the mode of action of already available
inducers has intensified in recent years [58 ]. Indeed, only
a limited number of inorganic, natural organic or synthetic
compounds with a generally accepted resistance-inducing
activity are known so far [26, 45 ]. However, even in case
of the best studied inducers, INA and BTH, little
knowledge exists about their mode of action and the
cellular receptors or target molecules with which they
interact [41 ]. The resistance inducing potency of BABA
has also been documented in a number of studies
[7–9, 20, 47, 50 ]. The highest efficacy of the compound
was demonstrated in several members of the Solanaceae.
Therefore, tobacco was used in the present study to
β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus
CON
(a)
101
BABA
(a)
(b)
(b)
(c)
(c)
(d)
F. 4. Histochemical detection of cell death by Evans
blue\trypan blue staining and of callose formation by anilin blue
staining in BABA-treated tobacco leaves. Trypan blue-stained
leaf discs were examined by light microscopy, anilin blue-stained
discs by fluorescence microscopy. (a) Control (left) ; Evans bluestained leaf disc, 16 h after application of 10 m BABA (right).
(b) Control (left) ; Evans blue-stained leaf disc, 48 h after
application of 10 m BABA (right). (c) Trypan blue staining,
48 h after application of 10 m BABA. (d) Anilin Blue staining,
24 h after application of 10 m BABA.
F. 5. Histochemical localization of ROS in tobacco leaf tissue
following application of 10 m BABA. For the detection of O −,
#
leaf discs were vaccum-infiltrated with NBT. H O accumulation
# #
was demonstrated by DAB infiltation. (a) NBT staining, 8 h
after BABA application. (b) DAB staining, 12 h after BABA
application. (c) DAB staining, 48 h after BABA application.
examine biochemical and molecular mechanisms involved
in BABA-induced resistance. In the pathosystem tobaccoTMV, BABA-mediated enhancement of virus resistance
could be demonstrated. In contrast, the aminobutyric
acid derivative GABA was completely inactive, the isomer
AABA had a much lower activity indicating a high isomer
specificity of aminobutyric acid in triggering enhanced
virus resistance. Similar results have been obtained in
tomato, pepper and sunflower [8, 20, 47, 50 ]. In a more
related study, using the system tobacco-Peronospora
tabacina, only BABA but not AABA and GABA was found
to induce resistance against blue mold [7 ]. Thus, data
available at the moment suggests a very specific interaction
of BABA with unknown cellular components in treated
plant tissues. As a further characteristic of plant activation
by BABA, relatively high concentrations of the chemical
were needed to trigger resistance in almost all systems
102
J. Siegrist et al.
20
12
(a)
*
10
15
*
*
8
10
SA (µg g–1 FW)
TBARS (nmol g–1 FW)
Control
BABA
5
6
4
0
0
24
48
72
96
Time after application (h)
2
F. 6. Effect of a treatment with BABA (10 m) on the content
of TBARS in tobacco leaves, indicating lipid peroxidation.
*Means from three experiments (five plants per treatment),
significantly different from control (P 0.05).
0
800
BTH
TMV
BABA
BTH
TMV
BABA
(b)
700
600
SA (ng g–1 FW)
tested so far. Foliar applied concentrations of BABA in the
millimolar range (1–20 m) were usually utilized to
achieve plant protection [8, 20, 47 ]. In addition, effective
BABA concentrations used for inducing resistance in
plants by root-drenching were 5–40 m [4, 20, 30, 50 ].
Compared to BABA, a much lower concentration
(0.5 m) of the plant activator BTH was sufficient for the
enhancement of resistance in tobacco to TMV, indicating
differences in the uptake and\or in the mode of action of
both inducers.
Activation of certain marker genes encoding PR
proteins is a common feature associated with resistance
induction. Thus, in tobacco, PR 1-a gene expression could
be demonstrated locally and systemically after foliar
BABA application. This is in agreement with the results
obtained by immunoblot analysis of extracts from tobacco
plants sprayed with BABA, where PR-1 protein accumulation was already shown [7 ]. In addition, BABAinduced resistance in tomato and pepper was also found to
be correlated with the induction of PR-proteins [8, 22 ].
However, no PR protein accumulation could be detected
in tobacco after stem injection of BABA although the
plants were strongly protected against Peronospora tabacina
[7 ]. This indicates that different processes may be
activated by BABA in tobacco depending on the location
of inducer application. A foliar spray treatment with
BABA triggers PR gene expression and virus resistance,
whereas a stem application seems to activate yet unknown
mechanisms which mediate resistance against the blue
mold pathogen but were not related to PR protein
accumulation. This view is further supported by the
observation that a stem application of BABA did not
Control
500
400
300
200
100
0
Control
F. 7. Local and systemic accumulation of SA in tobacco plants
treated with BABA (10 m), BTH (0.5 m) or preinoculated
with TMV (10 µg ml−"). Total SA contents were determined in
leaf 4 (a) and leaf 5 (b) of tobacco plants, 7 days after
application of the inducing agents to leaf 3 and 4. Values are
meanspSD from six experiments with five plants per treatment.
enhance virus resistance in tobacco (Siegrist et al.,
unpublished results).
The occurrence of cell death as a rapid reaction
following application of BABA was an unexpected result
β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus
103
F. 8. Phenotype of TMV lesions in wild type (Xanthi-nc) tobacco (upper row) and in transgenic NahG tobacco (lower row), 1
week after challenge inoculation with TMV (10 µg ml−") on leaf 5. Inducers [0.5 m BTH, TMV (10 µg ml−"), 10 m BABA] were
applied to lower leaves (leaf 3 and 4), 7 days before challenge inoculation.
Lesion size (mm)
3
Xanthi-nc
NahG-10
2
1
*
*
0
Control
BTH
*
TMV
*
BABA
F. 9. Effect of various treatments [0.5 m BTH, TMV (10 µg
ml−"), 10 m BABA] on the expression of resistance in wild-type
tobacco plants (Xanthi-nc) and in transgenic NahG tobacco
against TMV. Resistance was assessed by inoculating an upper
leaf (leaf 5) with TMV (10 µg ml−"), 8 days after application of
the inducers to leaf 3 and 4. *Means from three experiments
(5 plants per treatment) significantly different from control
(P 0.05).
obtained in the present study. Indeed, a critical review of
the literature dealing with BABA-induced resistance
revealed that this HR-resembling reaction of plant tissues
to foliar application of the chemical was already noticed
by other authors, but was not discussed in relation to
plant defense activation [7, 8, 47 ]. Moreover, it was also
observed that AABA, which exhibited a slight but
reproducible activity in the tobacco\TMV system (Table
1), induced cell death when applied to tobacco leaves.
However, compared to BABA, lesion formation occurring
after AABA treatment was markedly less pronounced
(Siegrist et al., unpublished results).
Further studies from our lab provide additional
evidence for BABA-mediated cell death in plants. In
tomato, foliar application of the inducer resulted in SAR
expression against Phytophthora infestans [29 ]. In this system,
the plant phenotype observed after BABA treatment was
nearly identical to that appearing in the case of a biotic
SAR induction mediated by local lesion forming
pathogens like tobacco necrosis virus [2, 29 ]. Furthermore,
it was recently demonstrated that BABA also causes cell
death in the roots of tomato plants after a soil drench
application leading to resistance against Fusarium oxysporum
f.sp. lycopersici [4 ]. This kind of plant response prompted
us to examine how BABA-induced cell death could be
linked to the enhancement of virus resistance in tobacco
and the processes subsequently observed. As the first
detectable reaction, a generation of ROS was evident,
indicating an initial interaction of BABA with a cellular
target which directly or indirectly provokes an oxidative
burst. The nature of this interacting component is still
unknown and awaits further studies. It could be localized
104
J. Siegrist et al.
in the apoplast or in cell wall-associated regions, since a
small portion of radiolabelled BABA remained in the cell
wall of treated tomato leaves whereas no binding to
cytoplasmatic proteins could be detected [6 ].
Besides ROS generation, lipid peroxidation was
detected in tobacco tissues, providing further evidence for
typical cell death-aligned responses initiated by BABA.
However, increased TBARS contents were not obvious
until 2 days after inducer treatment when necrotic lesion
formation was already macroscopically visible. The reason
could be the low sensitivity of the thiobarbituric acid
assay that does not allow detection of small changes in
already damaged membranes at earlier stages.
In the case of a biotic induction of disease resistance,
localized cell death and the formation of HR-lesions
resulting in defense gene activation are characteristically
triggered by viruses and microbes [45 ]. For this reason,
we compared typical responses that appeared after
treatment of tobacco plants with the biotic inducer TMV
and the chemical inducer BABA. In agreement with
already published data [33 ], local and systemic accumulation of SA was evident in tobacco after TMV
inoculation. Interestingly, we could also find increased SA
levels in treated and in non-treated parts of tobacco plants
sprayed with BABA, indicating an involvement of SA in
BABA-mediated enhancement of TMV resistance. This
assumption was further supported in experiments with
NahG plants. As expected, BTH was found to operate
independently of SA. Whereas, enhancement of resistance
was blocked in plants induced by TMV and BABA. From
these results, it can be concluded that BABA triggers
mechanisms in tobacco leaf tissues which are typical for
biotic inducers of resistance acting by local lesion
formation. Therefore, under our experimental conditions,
BABA seems to imitate a pathogen attack. However, the
formation of necrotic lesions in BABA-treated NahG
tobacco indicates that primary processes initated by this
inducer are in no way affected by the low SA level of the
transgenic plants.
Apart from microbial initiation of cell death, known
lesion-mimic mutants of certain plants display necrotic
lesions that resemble those of an HR, and exhibit activated
defense mechanisms as well as SAR. Intensively studied
examples are cell death mutants from Arabidopsis thaliana
which express spontaneous lesion formation in the absence
of pathogens [14 ]. These mutants showed elevated
concentrations of SA, constitutive expression of PR
proteins and enhanced resistance against virulent
pathogens [14, 56 ]. More recently, lesion-mimic mutants
of rice have been described which were resistant against
the blast fungus Magnaporthe grisea [48 ]. A further line of
evidence for cell death-associated activation of SAR
without any microbial stimulus can be deduced from
studies with lesion-mimic transgenic plants. For example,
tobacco and potato plants that constitutively express a
bacterio-opsin proton pump displayed necrotic lesions,
elevated SA levels, increased PR gene expression and
enhanced resistance against pathogens [1, 36 ]. Further
transgenic, lesion-forming plants with similar properties
have been described [13 ].
Other chemicals and natural compounds, besides
BABA, are known to induce resistance in plants by
triggering HR-like responses. In potato, a spray treatment
with arachidonic acid generated massive local lesion
formation and subsequently SAR against Phytophthora
infestans [5, 11 ] and Alternaria solani [11 ]. In this system, a
local but not systemic accumulation of SA was demonstrated [11 ]. Nevertheless, by using NahG potato plants,
SA was found to play an essential role in arachidonic acidmediated SAR [59 ].
Other examples of cell death-inducing agents are
chemicals like the herbicide Paraquat or the dye Rose
Bengal, that act by producing ROS when applied to
plants as foliar sprays. Both chemicals have been
demonstrated as effective SAR inducers in tobacco
[16, 46 ]. Furthermore, inorganic phosphate salts which
induce SAR in different plants were recently described to
operate by causing ROS generation, cell death and SA
accumulation [38 ]. Similar results have been obtained
after application of elicitins, proteins excreted by Phytophthora spp. [24 ]. Cell death-associated activation of
defense genes and SA accumulation is also known from
tobacco plants treated with ozone or exposed to u.v.-light
[57 ].
In the case of BABA-mediated lesion formation, as well
as in most of the above mentioned examples, a complex
regulation seems to be involved that link cell death
initiation and the succeeding processes leading to disease
resistance. In contrast, cell death caused by unspecific
damage of plant tissues, e.g. dry ice treatment, did not
activate SA synthesis and plant defense against pathogens
[32 ; Siegrist et al., unpublished results].
In conclusion, our data provide evidence that foliar
application of the chemical inducer BABA to tobacco
plants results in the activation of mechanisms resembling
those initiated by necrotizing microbes and viruses that
trigger SAR.
We thank Margit Schmidtke, Carmen Kolle and Gisela
Moll for excellent technical assistance. We are grateful to
Prof. Dan Klessig (Rutgers University, NJ, U.S.A.) and
Dr Kay Lawton (Novartis Ltd., Research Triangle Park,
U.S.A.) for providing seeds of transgenic tobacco. We also
thank Dr Leslie Friedrich (Novartis Ltd., Research
Triangle Park, U.S.A.) for providing tobacco PR 1a cDNA and Dr Jan Hinrichs-Berger for reviewing the
manuscript. The financial support of our studies by the
Deutsche Forschungsgemeinschaft is acknowledged.
β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus
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