Expression of Uroporphyrinogen Decarboxylase or
Coproporphyrinogen Oxidase Antisense RNA in Tobacco Induces Pathogen
Defense Responses Conferring Increased Resistance to Tobacco Mosaic
Virus*
Hans-Peter
Mock
§,
Werner
Heller¶,
Antonio
Molina
**,
Birgit
Neubohn
,
Heinrich
Sandermann Jr.¶, and
Bernhard
Grimm
From the
Institut für Pflanzengenetik und
Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, the
¶ Institut für Biochemische Pflanzenpathologie,
GSF-Forschungszentrum für Umwelt und Gesundheit, D-85764
Oberschleißheim, Germany, and
Biotechnology and Genomics
Center, Novartis Crop Protection, Inc., Research Triangle Park,
North Carolina 27709-2257
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ABSTRACT |
Transgenic tobacco plants with reduced activity
of either uroporphyrinogen decarboxylase or coproporphyrinogen oxidase,
two enzymes of the tetrapyrrole biosynthetic pathway, are characterized by the accumulation of photosensitizing tetrapyrrole intermediates, antioxidative responses, and necrotic leaf lesions. In this study we
report on cellular responses in uroporphyrinogen decarboxylase and
coproporphyrinogen oxidase antisense plants, normally associated with
pathogen defense. These plants accumulate the highly fluorescent coumarin scopolin in their leaves. They also display increased pathogenesis-related protein expression and higher levels of free and
conjugated salicylic acid. Upon tobacco mosaic virus inoculation, the
plants with leaf lesions and high levels of PR-1 mRNA
expression show reduced accumulation of virus RNA relative to wild-type
controls. This result is indicative of an increased resistance to
tobacco mosaic virus. We conclude that porphyrinogenesis as a result of deregulated tetrapyrrole synthesis induces a set of defense responses that resemble the hypersensitive reaction observed after pathogen attack.
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INTRODUCTION |
All organisms contain tetrapyrroles that play important roles in
the transfer of energy, redox sensing, or catalysis. Chlorophylls are
the most abundant tetrapyrroles in plants and are involved in the
harvesting of light and its subsequent conversion to chemical energy.
In response to varying environmental conditions the coordinated synthesis and stoichiometric assembly of the components of the photosynthetic complexes ensure an adjusted electron flow and avoid
exacerbated oxidant production (1). In addition to the regulatory
mechanisms, plants have evolved effective protection systems against
photooxidative stress consisting of enzymic and low molecular weight
antioxidants (2).
Genetic or chemical impairment of chlorophyll or heme biosynthesis
results in the accumulation of photosensitizing tetrapyrroles, e.g. by feeding of the precursor 5-aminolevulinate or by
application of diphenyl ether herbicides acting on protoporphyrinogen
oxidase (3). Formation of reactive oxygen species such as singlet
oxygen is essential for the photodynamic action of tetrapyrroles (3). Expression of antisense or additional sense genes encoding enzymes of
the tetrapyrrole metabolism leads to modified enzymic activities and,
in consequence, to an imbalance of the substrate flow. Transgenic tobacco plants with reduced uroporphyrinogen decarboxylase
(UROD)1 or coproporphyrinogen
oxidase (CPO) were characterized by light-dependent formation of leaf lesions and by stunted growth (4, 5). Reduced
activity of either of the two enzymes may increase the level of
porphyrinogen substrates up to 500-fold in transformants relative to
control plants. These compounds, predominantly uroporphyrinogen or
coproporphyrinogen in UROD or CPO antisense plants, respectively, can
be photooxidized, thus triggering photodynamic cellular destruction. Cellular stress responses observed upon accumulation of these tetrapyrroles included increased steady state levels of antioxidant mRNAs and increased activity of enzymes involved in stress defense. In particular, higher activities of superoxide dismutase in younger leaves and of ascorbate peroxidase in older leaves were observed (6).
In addition to the plastidic superoxide dismutase isoform, mitochondrial and cytosolic isoforms contributed to the increased total
enzyme activity suggesting that tetrapyrroles initially synthesized in
the plastids may spread to other compartments where they induce local
antioxidative defense responses (6). However, limitation of the
detoxifying system was indicated by a decrease in the total content and
a higher percentage of the oxidized form of ascorbate, reduced content
of glutathione, and the loss of tocopherol (4, 6).
In addition to the well established roles of these protective
components, phenolic constituents also contribute to the antioxidative capacity of cells (7, 8). Different phenylpropanoids are formed in
response to various stresses such as wounding, high light, UV
radiation, or pathogen attack. These molecules often exert
antimicrobial and UV protecting or signaling functions (9). We have
continued to examine the protective responses to oxidative stress
following the accumulation of tetrapyrroles in UROD and CPO antisense
plants with the analysis of phenolic constituents and found an
increased content of the coumarin scopolin which is also synthesized in
tobacco upon TMV infection (10, 11). We demonstrate that other pathogen
defense responses are also activated indicating that these cellular
reactions upon porphyrinogenesis resemble a hypersensitive reaction
after pathogen attack.
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EXPERIMENTAL PROCEDURES |
Plant Material--
Tobacco (Nicotiana tabacum var.
Samsun NN) plants expressing antisense RNA for CPO (4) or UROD (5) and
control plants (Samsun NN) were grown under controlled environmental
conditions (24 °C, 300 µmol of photons m
2
s
1, 16/8-h light/dark cycles) that favor development of
necrotic leaf lesions in the transformants (5). For biochemical
analysis plant material from progenies of line 2 of UROD and line 3 of CPO antisense plants was harvested as described previously (6). Additional lines were used for the TMV infection experiment (Fig. 7),
and these have also been characterized previously (4, 5). Necrotic
leaves from individual plants with lesion formation and leaves of the
same developmental stage of transformants without lesion formation as
well as wild-type plants were used for inoculation. For comparative
studies of necrotic and non-necrotic leaf areas of transformants,
tissue was separately harvested from the same leaf, and for each single
experiment, material was pooled from several leaves. The plant material
used for biochemical analysis was frozen in liquid nitrogen, ground to
a fine powder with a mortar and a pestle, and stored at
80 °C
until further processing.
Chemicals--
Acetobromo-
-D-glucose, silver(I)
oxide, and pyridine were purchased from Fluka (Neu-Ulm, Germany).
Drierite and 1,4-diazabicyclo(2,2,2)octane were obtained from Aldrich
(Steinheim, Germany). Scopoletin was purchased from Roth (Karlsruhe,
Germany). HPLC gradient grade methanol was obtained from Riedel-de-Haen
(Seelze, Germany). Other chemicals were obtained from Merck (Darmstadt,
Germany) or Sigma (Deisenhofen, Germany), and all chemicals were of
analytical or the highest purity available. Pre-coated TLC plates SIL
G-200 UV254 were purchased from Macherey und Nagel
(Düren, Germany).
Extraction and Analysis of Phenolic Constituents--
Aliquots
of powdered plant material were extracted repeatedly with methanol in
Eppendorf tubes (final volume 1 ml). Homogenization was accomplished by
a motor-driven pestle which fitted into the Eppendorf tubes. Extracts
were centrifuged at 13,000 × g for 10 min at
4 °C.
Phenolic constituents were analyzed on an HPLC system consisting of a
Waters 600 controller and pump unit and a Waters 996 photodiode array
detector (Waters, Eschborn, Germany) combined with a fluorescence
detector (FP-920, JASCO, Groß-Umstadt, Germany). Data acquisition and
processing were performed with the Millenium software package (Waters,
Eschborn, Germany). Methanolic extracts were diluted with 0.25 volume
of water and centrifuged again. Aliquots of the supernatants were
injected and separated on a RP-18 phase column (LiChrospher, 5 µm,
125-4; Merck, Darmstadt, Germany). The mobile phase used consisted of
0.1% ammonium formate in 2% formic acid (solvent A) and 90% methanol
in solvent A (solvent B) as described previously (12) but with a
slightly modified gradient profile. After 5 min elution with 100%
solvent A, a gradient from 0 to 40% solvent B over 25 min was applied
followed by a gradient from 40% solvent B to 100% B over further 20 min. Spectra from 250 to 400 nm were recorded (1/s) at maximal spectral
resolution of the photodiode array detector (1.2 nm), and chromatograms
of absorbance at 280 nm were extracted from these data sets. The wavelength settings for the fluorescence detector were 300 nm for
excitation and 400 nm for emission.
Chlorogenic acid was identified as the major peak in the UV
chromatograms (280 nm) in methanolic extracts of wild-type leaves or
roots. This peak eluted with the same retention time and displayed an
identical diode array spectrum as authentic chlorogenic acid (CGA) in
the HPLC analysis. For subsequent MS analysis of the major constituent,
appropriate fractions were collected after HPLC and monitoring at 280 nm, evaporated to dryness, and redissolved in 50% methanol, 2% acetic
acid. The purity of the preparation was confirmed by HPLC. ES-MS
spectra were recorded on a Bruker Esquire ion trap system (Bruker
Franzen, Bremen, Germany) equipped with an API interface (Analytica,
Branford, CT). The mass spectrometer was operated in the positive ion
mode with a resolution of 0.4 atomic mass units. Samples were infused
at a flow rate of 80 µl h
1. A temperature of 150 °C
was set for the drying gas (nitrogen). Helium was used as a gas for
collision-induced dissociation. Authentic CGA and purified plant
extracts with tentatively identified CGA both exhibited a peak at
m/z = 355 corresponding to the (M + H+) ion.
For the identification of scopolin, 3 g of powdered plant material
was extracted in 50 ml of methanol (30 min, 4 °C). The extract was
centrifuged (15 min, 6,000 × g). The supernatant was evaporated to dryness, redissolved in 500 µl of methanol, and adjusted to 20% methanol by addition of water. After centrifugation (15 min, 6,000 × g) 200-µl aliquots were injected
for HPLC. To allow better separation of scopolin and chlorogenic acid,
the mobile phase was as follows: 5% methanol (solvent A1) and 80% methanol (solvent B1) and a gradient from 0 to 50% B1 over 30 min.
Elution was monitored by UV absorbance at 280 nm. Scopolin eluting at
19 min was collected from several injections, evaporated to dryness,
and redissolved either in acetate buffer (50 mM, pH 5) for
subsequent digestion with
-glucosidase, or in 50% methanol, 2%
acetic acid for MS analysis. The purity of the scopolin preparation was
confirmed by HPLC using the standard separation system described for
phenolic constituents.
For
-glucosidase digestion, aliquots of isolated scopolin were
redissolved in acetate buffer (50 mM, pH 5) and incubated at 37 °C for 30 min in the presence of 1 unit of almond
-glucosidase (Sigma, Deisenhofen, Germany) dissolved in the same
buffer. Control experiments with buffer alone were run in
parallel. Samples were then transferred to ice, and 4 volumes of
methanol were added. After centrifugation, aliquots of the
supernatant were spiked with either scopoletin or isoscopoletin
dissolved in 80% methanol or 80% methanol alone and analyzed by
HPLC using the standard separation system and fluorescence detection.
The chemical synthesis of scopolin was essentially according to Ref. 13
with the following modifications. A mixture of 38.4 mg of scopoletin
(0.2 mmol), 90.4 mg of acetobromoglucose (0.22 mmol), 46.4 mg of
silver(I) oxide (0.2 mmol), 56 mg of drierite (calcium sulfate, 0.4 mmol), and 1 mg of 1,4-diazabicyclo(2,2,2)octane in 1 ml of dry
pyridine were mixed in an Eppendorf tube and stirred overnight in the
dark. After centrifugation the supernatant was separated and the
solvent removed under a stream of nitrogen. The residue was dissolved
in 0.5 ml of trichloromethane and applied to a preparative silica gel
plate purified twice before use with trichloromethane/methanol 1:1
(v/v) containing 2% (v/v) acetic acid. The plate was developed with
trichloromethane/glacial acetic acid/water, 70:28:2 (v/v/v), and
air-dried. The band at Rf = 0.8-0.9 ahead of the
residual scopoletin was scraped off, the scopolin tetraacetate eluted
with trichloromethane/methanol, 8:2 (v/v), and the solvent removed
in vacuo. The crude scopolin tetraacetate was treated with 5 ml of 0.5 M barium hydroxide at 80 °C for 15 min, and
the reaction mixture was then cooled to room temperature and brought to
pH 7 with 2 M sulfuric acid. The barium sulfate formed was
precipitated by centrifugation, the supernatant separated, and the
solvent removed in vacuo. The residue was chromatographed on
Sephadex LH2O (Pharmacia, Freiburg, Germany) with methanol, and the
scopolin characterized by HPLC combined with diode-array spectroscopy
and by mass spectrometry as described under "Results."
Analysis of conjugated and free SA was performed essentially according
to Ref. 14, with the following minor modifications: 200 mg of powdered
leaf material was used for extraction, and trichloroacetic acid was
replaced by 20% formic acid. The preparations were finally resuspended
in 20% methanol prior to HPLC analysis which was performed using the
HPLC system described above with fluorescence detection. The mobile
phase consisted of solvent A and solvent B2 (80% methanol and 10%
acetonitrile in solvent A), and the column was kept at 30 °C.
Elution was performed at a flow rate of 1 ml min
1 with a
hyperbolic gradient from 10% solvent B to 45% B in 25 min using curve
5 offered by the Millenium software. The column was then washed with
100% solvent B and equilibrated. Salicylic acid eluted at 16.4 min.
Recovery rates for each individual experiment were determined by
spiking parallel samples with appropriate amounts of authentic SA.
Fluorescence Microscopy--
Hand-cut cross-sections of leaves
were mounted in water and viewed under a Zeiss Axiovert microscope with
a Fluar 20 × 0.75 UV. The filter combination used was as follows:
excitation BP 365, DBS 395; emission LP 397. Photos were taken using a
Kodak Elite 400 slide film.
Western Blot Analysis--
Protein extraction,
SDS-polyacrylamide gel electrophoresis, and immunoblotting were
performed as described previously (4). Protein was quantified according
to Ref. 15 using bovine serum albumin as a standard. Antisera raised
against tobacco PR proteins PR-1c, -2, -Q, and -S were a gift of Drs.
P. Geoffroy and B. Fritig (Université Louis Pasteur, Straßbourg,
France). The immunospecificity of the antisera has been described (see
Ref. 16 and references therein).
TMV Inoculation and RNA Analysis--
Samsun NN tobacco plants,
a local lesion host for TMV, UROD, and CPO antisense plants were grown
under controlled conditions favoring development of necrotic lesions
(6). Ten-week-old plants were inoculated with a suspension of the
Flavum strain of TMV (0.5 mg/liter) in a solution of 10 mM
sodium phosphate (pH 7) containing carborundum as described previously
(17). In parallel, other plants were mock-inoculated with buffer and carborundum only. At least two leaves per plant and five plants per
transgenic line or wild-type were used for inoculation. Leaves were
harvested directly before and 5 days after TMV inoculation, and total
RNA was isolated by phenol/chloroform extraction followed by lithium
chloride precipitation (18). Total RNA (7.5 µg) was subjected to
electrophoresis on formaldehyde/agarose gels and blotted to Hybond-N
membranes (Amersham, Braunschweig, Germany) as described (19). Equal
loading of samples was confirmed by including 40 µg/ml ethidium
bromide in the sample loading buffer allowing visualization of RNA
under UV light. 32P-Labeled probes were synthesized using
the random primers DNA-labeling system (Life Technologies, Inc.). The
cDNA probes for tobacco PR-1 and reverse-transcribed
TMV-RNA have been previously described (20). Hybridization and
washing were carried out at 65 °C as described (21). Relative
amounts of TMV transcripts were quantified using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) according to the manufacturer's instructions.
 |
RESULTS |
A Highly Fluorescent Phenolic Compound Accumulates in Leaves of
UROD and CPO Antisense Plants--
Fig.
1 shows chromatograms obtained from
methanolic extracts of leaf 9 of a UROD antisense plant. The main peak
observed by UV detection at 280 nm (24.6 min) (Fig. 1A) was
identified as chlorogenic acid (CGA) by co-chromatography with
authentic CGA, combined with diode array and electrospray mass
spectrometry (ES-MS) as described under "Experimental Procedures."
In leaf extracts of transgenic plants the chromatograms recorded by
using fluorescence detection showed a dominant peak (24.4 min) eluting
very close to CGA (24.9 min) (Fig. 1B). This peak
corresponded to the small signal observed at 24.1 min in the UV
chromatogram (Fig. 1A, see arrow). The insert in
Fig. 1A shows the diode array spectrum of this peak. This
compound was absent in UV chromatograms in extracts of wild-type plants
(data not shown), and only a small signal was found by fluorescence
detection (Fig. 1C). Comparing the integrated peak areas
obtained from control and transformed plants on a fresh weight basis,
the content of this compound was increased (up to 23-fold) in leaves of
the transformants (see Table I below). The content of other constituents remained unchanged or differed only
slightly in leaves of control and transformed plants.

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Fig. 1.
Separation of methanolic leaf extracts by
HPLC. Methanolic extracts were prepared from powdered plant
material and analyzed by HPLC as described under "Experimental
Procedures." UV detection with a diode array detector (250-400 nm)
was immediately followed by fluorescence detection ( EX,
300 nm; EM, 400 nm). The injected volume corresponds to
0.8 mg of leaf material. A, extracts from leaf 9 of UROD
antisense plants monitored by absorbance at 280 nm. Inset
shows the spectrum of the peak eluting at 24.1 min. B, same
injection monitored by fluorescence emission at 400 nm following
excitation at 300 nm. C, fluorescence profile of extracts
from leaf 9 of wild-type tobacco.
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Table I
Accumulation of scopolin in leaves of UROD and CPO antisense plants
relative to wild-type
Methanolic leaf extracts of wild-type (WT), CPO (CPOAS), or UROD
(URODAS) antisense plants were subjected to HPLC analysis on RP-18 as
described for Fig. 1. Chemically synthesized scopolin was used for
calibration. Results from a typical experiment are shown. Each value
represents the mean ± S.D. of three separate extractions. Leaves
were counted from the top to the base. Leaves of transformants were
harvested without separating necrotic and non-necrotic areas.
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Isolation and Identification of the Fluorescent Compound,
Scopolin--
Based on the strong fluorescence (22) and on the UV
spectrum (see inset, Fig. 1A (23)), we
tentatively identified the unknown substance as a coumarin. The
metabolite was isolated from necrotic leaves of UROD antisense plants
by HPLC as described under "Experimental Procedures." It was
identified as scopolin based on HPLC analysis of a
-glucosidase
digest, mass spectrometry, and comparison with authentic standards. The
isolated compound was incubated with or without
-glucosidase and
separated by HPLC (Fig. 2). After
glucosidase digestion the fluorescent peak at 24 min completely
disappeared, and one new peak of approximately the same fluorescence
intensity became apparent. The retention time of this product was
identical to that of scopoletin, the aglycon of scopolin, as
demonstrated by cochromatography with authentic scopoletin (Fig.
2B). The product released by
-glucosidase also displayed
the same diode array spectrum as scopoletin (data not shown).
Isoscopoletin clearly differed in its elution time (28.9 min) under the
same chromatographic conditions and showed a much lower ratio of peak
areas from fluorescence to UV detection (data not shown).
Analysis of the isolated metabolite by electrospray-mass spectrometry
(ES-MS) gave a mono-isotopic mass of 354.1 identical with the value of
354.1 calculated for scopolin,
C16H18O9. The collision-induced
fragmentation of the protonated molecular ion produced one
intense fragment ion at m/z 193.0 (Fig.
3) resulting from the loss of the glucose
moiety. The appearance of this ion and a simultaneous reduction in
intensity of the protonated molecular ion at 355.1 was observed when
the temperature of the drying gas was elevated (data not shown). The
chemical structure was further confirmed by chemical synthesis of
scopolin from scopoletin and acetobromoglucose. The product obtained
after alkaline hydrolysis of the tetra-acetyl intermediate was
identical to the compound isolated from necrotic leaves of UROD
antisense plants when analyzed by the methods described. In addition,
both preparations gave an identical pattern of product ions when the
molecular ions at m/z 193 were further fragmented (data not
shown). The content of scopolin in leaves of transformants and controls
was quantified using the chemically synthesized compound for
calibration (Table I).

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Fig. 2.
HPLC analysis of isolated scopolin incubated
with -glucosidase. Scopolin was isolated
from methanolic leaf extracts of UROD antisense plants as described
under "Experimental Procedures." Incubations in acetate buffer (pH
5) alone or with the addition of -glucosidase were performed at
37 °C for 30 min. Assays were then brought to 80% methanol. HPLC
analysis was performed as described in Fig. 1 with fluorescence
detection. A, buffer control. Inset shows the
structural formula of scopolin. B, -glucosidase digests.
Aliquots of the sample obtained after digestion with -glucosidase
were spiked with buffer (shaded area) and authentic
scopoletin (solid line). Inset shows the
structural formula of scopoletin.
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Fig. 3.
ES-MS/MS spectrum of scopolin isolated from
methanolic leaf extracts of UROD antisense plants. The ES-MS
showed a (M + H+) ion peak at m/z = 355 consistent with the monoisotopic mass of 354.1 as calculated for
scopolin. The protonated molecular ion at m/z 355 was
isolated in the ion trap and was fragmented resulting in the appearance
of one prominent product ion at m/z = 193.0 indicating
the loss of the glucose moiety.
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Scopolin Accumulates in Necrotic Leaf Areas of UROD and CPO
Antisense Plants--
The content of scopolin was quantified in leaf
tissue with or without visible necrotic lesions from UROD (Fig.
4A) and CPO (Fig.
4B) antisense plants. Necrotic and non-necrotic leaf tissues were defined as indicated in Fig. 4C. In both transgenic
lines the amount of scopolin was more than 10-fold higher in the
necrotic tissue compared with non-necrotic areas. The high content of
scopolin and its predominance among the fluorescent compounds found in methanol extracts prompted us to investigate the subcellular
localization of this compound in leaf tissues of transformants. Fig.
5 shows leaf sections of a necrotic (Fig.
5A) and a non-necrotic area (Fig. 5B) from the
same leaf of a UROD antisense plant visualized by fluorescence
microscopy. A white to bright-bluish fluorescence in the sections of
necrotized leaves coincided with dead tissue. Around this tissue, a
strong deep-blue fluorescence surrounded by a layer of red fluorescence
was visible in cells of both the palisade and spongy parenchyma. The
deep-blue fluorescence indicated the accumulation of scopolin in the
vacuoles. Non-necrotic sections of necrotized leaves from UROD
antisense plants (Fig. 5B) displayed similar intensity of
the red fluorescence, whereas the deep-blue fluorescence was
considerably lower, consistent with the quantitative analysis of
scopolin (Fig. 4). Fluorescence analysis of necrotic and non-necrotic
leaf sections of CPO antisense plants gave similar results (data not
shown).

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Fig. 4.
Content of scopolin in necrotic and
non-necrotic leaf areas of transformants. Leaves of UROD antisense
plants or CPO antisense plants were separated into areas with and
without necrotic lesions. Scopolin was quantified in leaf material by
HPLC as described for Fig. 1 using chemically synthesized scopolin as a
standard. A, UROD antisense plants. B, CPO
antisense plants. C, the picture indicates the separation
into necrotic and non-necrotic leaf areas.
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Fig. 5.
Fluorescence microscopy of a necrotic and a
non-necrotic leaf area of a UROD antisense plant. Hand-cut
sections of tobacco leaves from UROD antisense plants were mounted in
water. Excitation with UV light and a long-pass LP 397 filter allowed
monitoring of both blue and chlorophyll-derived
red fluorescence. The bar represents 100 µm.
A, necrotic area; B, non-necrotic area.
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Expression of Pathogenesis-related (PR) Proteins Is Strongly
Induced in UROD and CPO Antisense Plants--
We were next interested
in determining if porphyric plants showed other defense reactions which
are normally observed after pathogen attack. TMV infection of tobacco
induces the expression of PR proteins including
-glucanases (class
two of tobacco PR proteins), chitinases (e.g. PR Q), as well
as other proteins of unknown function (24). Western blot analyses of PR
proteins representing these different classes showed stronger
accumulation of all PR proteins in older leaves of transformants
relative to control plants (Fig.
6A). A similar pattern was
obtained for CPO antisense plants (data not shown). We also compared
the amount of PR proteins in necrotic and non-necrotic areas of the
same leaf from UROD and CPO antisense plants, respectively (Fig.
6B). In both transformants PR proteins mainly accumulated in
the leaf area with necrotic lesions and were found only to a minor
extent in the area devoid of lesions.

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Fig. 6.
Accumulation of PR (pathogenesis-related)
proteins in leaf extracts of wild-type UROD (URODAS),
or CPO (CPOAS) antisense plants. Protein extracts
were separated by SDS-polyacrylamide gel electrophoresis and blotted
onto nitrocellulose. Immunodetection was performed with antibodies
raised against tobacco PR-1, -2, -Q, and -S (Heitz et al.
(16)). A, PR protein expression in leaves of wild-type and
UROD antisense plants at different developmental stages. B,
PR protein expression in necrotic and non-necrotic leaf areas of UROD
and CPO antisense plants. Areas with and without necrotic lesions were
obtained from leaves of UROD or CPO antisense plants (see Fig.
4).
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UROD and CPO Antisense Plants Accumulate Elevated Levels of Free
and Conjugated Salicylic Acid (SA)--
Leaf lesion formation in
tobacco infected with TMV is accompanied by increased levels of SA
(25). We determined the levels of free and conjugated SA in leaves of
UROD and CPO antisense and control plants (Table
II). The contents of free SA was
increased in all leaves of transformants relative to controls but was
more enhanced in UROD antisense plants (up to 5-fold) compared with CPO
antisense plants. The amount of conjugated SA was severalfold higher in
leaves of both transformants, relative to wild-type, and the increase
was more pronounced (up to 25-fold) for CPO antisense plants. We next
determined if SA was uniformly distributed in leaves or accumulated
preferentially in tissue adjacent to the necrotic lesions as was
observed after TMV infection (25). In both UROD and CPO antisense
plants free SA was equally distributed between necrotic and
non-necrotic leaf areas, whereas conjugated SA preferentially
accumulated in leaf tissue with necrotic spots (Table
III). For comparison, in the TMV
inoculation experiment (see Fig. 7) the
amount of free and conjugated SA in Samsun NN controls 5 days after
inoculation was 3.6 µg/g fresh weight and 25 µg/g fresh weight,
respectively.
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Table II
Content of free and conjugated SA in leaves from tobacco control plants
and UROD or CPO antisense transformants
200 mg of leaf material were analyzed for free and conjugated SA by
HPLC as described under "Experimental Procedures." Results from one
of three independent experiments are shown and values are the
means ± S.D. from at least four replicates. Leaves were counted
from the top to the base. WT, wild-type tobacco; URODAS, transformants
expressing UROD antisense RNA; CPOAS, transformants expressing
antisense RNA for CPO.
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Table III
Comparison of SA content in necrotic and non-necrotic leaf areas of
UROD and CPO antisense plants
Necrotic and non-necrotic leaf areas of UROD (URODAS) and CPO (CPOAS)
antisense plants were separated as described for Fig. 4. Content of
free and conjugated SA was analyzed as given in Table II. Values
represent the means ± S.D. from three independent experiments.
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Fig. 7.
PR-1 mRNA accumulation in CPO and UROD
antisense plants correlates with TMV resistance. Several lines of
UROD and CPO antisense plants previously characterized (4, 5) were used
for this experiment. Total RNA was extracted from upper
(a) and lower (b) leaves of wild-type,
UROD, and CPO antisense plants with (L) or without lesions
( ). Total RNA (7.5 µg) was electrophoresed and blotted to nylon
membranes as described under "Experimental Procedures."
A, steady state PR-1 mRNA levels in wild-type
(WT), UROD (URODAS), and CPO (CPOAS) antisense
plants prior to TMV infection. Hybridization was carried out with a
32P-labeled probe derived from a cDNA clone of
PR-1 (20). B, Northern blot analysis of TMV RNA
in leaves of mock-inoculated (M) or TMV-inoculated
(I) plants. Plant material for RNA extraction was harvested
5 days after inoculations. Blots were hybridized with a
32P-labeled probe derived from reverse-transcribed TMV RNA
(20). Two subgenomic TMV RNAs are indicated (RNA1 and
RNA2). C, quantification of the hybridization
signals of TMV RNA shown in B. Amount of TMV in wild-type
was taken as 100%. Quantification was carried out using a
PhosphorImager.
|
|
Tobacco Mosaic Virus (TMV) Infection Is Reduced in UROD and CPO
Antisense Plants--
It has previously been shown that TMV infection
of local lesion hosts leads to a hypersensitive reaction and to
increased resistance against subsequent infections with either TMV as
well as other viruses (26), fungi, and bacteria (27, 28). The development of the hypersensitive reaction and systemic acquired resistance (29) tightly correlates with increased PR protein expression
and SA levels (17). We next investigated if the strong induction of PR
proteins and SA formation would result in resistance to TMV in UROD and
CPO antisense plants. Fig. 7 shows an inoculation experiment with TMV
of wild-type tobacco and several transgenic lines of UROD and CPO
antisense plants (4, 5). Leaves were harvested before TMV inoculation
to analyze PR-1 gene expression levels. As expected from the
Western blot analysis (Fig. 6), necrotic UROD (2L and 15L) and CPO (3L)
antisense plants had high levels of PR-1 mRNA (Fig.
7A). Wild-type controls (WT) and UROD (2, 12, 15)
or CPO (17) antisense plants without visible leaf necrosis showed no
expression of PR-1 (Fig. 7A). Five days after inoculation with TMV total RNA was extracted from leaf tissue, and the
amount of virus RNA was analyzed by Northern blot using a
32P-labeled probe derived from reverse-transcribed TMV RNA
(Fig. 7B). Viral RNA accumulation was reduced in transgenic
plants that were characterized by necrotic lesions and high
PR-1 mRNA expression before inoculation. No significant
differences in the amount of TMV RNA were detected between wild-type
plants and UROD or CPO antisense plants without a necrotic phenotype.
This finding is correlated with the lack of PR-1 protein expression in
these plants (Fig. 7A). Quantification of hybridization
signals corresponding to the two viral RNAs (Fig. 7B)
revealed a 70-90% reduction of TMV in UROD and CPO antisense plants
showing necrotic lesions in comparison to wild type (Fig.
7C).
 |
DISCUSSION |
Accumulation and Localization of Scopolin--
Transgenic plants
with reduced activity of either UROD or CPO accumulated considerable
amounts of a fluorescent compound in close proximity to necrotic leaf
areas (Fig. 4). This compound was identified as scopolin based on its
spectroscopic and fluorescence properties, HPLC analysis, by mass
spectrometry, and by comparative analysis of the aglycone obtained by
-glucosidase digestion. The structure of the product was further
confirmed by chemical synthesis of scopolin which gave identical
results. The data excluded isoscopolin which has been detected in leafy
galls of tobacco (30). Results from fluorescence microscopy suggested
that scopolin was localized in the vacuoles (Fig. 5). Scopoletin and
scopolin accumulation has previously been demonstrated in cell
suspension cultures of tobacco (31). Although scopoletin has mainly
been recovered from the culture filtrate, scopolin accumulated within the cells (31). Vacuolar localization is consistent with the rapid
uptake of the coumarin glucoside esculin into isolated barley leaf
vacuoles (32).
There is substantial evidence that scopolin and scopoletin play an
important role in disease resistance. It has been shown that scopoletin
possesses antimicrobial activity (34-37). In incompatible plant-pathogen interactions a rapid and pronounced synthesis of scopoletin was observed, and a slower and reduced formation was found
in compatible interactions (36-38). Elevated constitutive levels of
scopoletin and scopolin were found in a disease-resistant Nicotiana hybrid (35). Spraying of tobacco plants with
scopoletin but not with scopolin prior to TMV inoculation reduced
lesion formation (35). In potato tubers infected with
Phytophthora infestans a ring of blue fluorescence derived
from accumulated scopolin was observed around the site of infection
(39). Scopolin also accumulated during the hypersensitive reaction of
TMV-infected local lesion host tobacco varieties (10, 11). Its low
toxicity compared with the aglycon scopoletin and its putative vacuolar localization classifies scopolin as a preformed antimicrobial compound
(40). Upon tissue disruption following pathogen attack, scopoletin is
released by the action of
-glucosidases originating from the plant
itself or from the attacking microorganism and exerts its inhibitory
effects. In analogy, the action of a
-glucosidase is required to
exert the antimicrobial effects of the cyanogenic glucoside dhurrin or
the isoflavanoid derivative maackiain, which is stored as a glucoside
in roots of red clover (41, 42).
Lesion Formation in Leaves of UROD and CPO Antisense Plants
Resembles the Hypersensitive Reaction Observed after TMV
Infection--
The strong accumulation of scopolin in leaves of UROD
and CPO antisense plants suggests similarities between the formation of
necrotic lesions upon accumulation of photosensitizing porphyrins and
the hypersensitive reaction following TMV infection. Tobacco leaves
infected with TMV similarly show substantial increase in the levels of
both free and conjugated SA (25) and have stimulated PR protein
expression (17, 43) with a gradual distribution and a maximum near the
center of the lesions (25, 16). It is widely accepted that in infected
plants concentration of SA and induction of PR proteins correlate with
enhanced resistance against pathogens (29, 44-47). It has been shown
that treatment of tobacco with SA or analogs of SA, such as
2,6-dichloroisonicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic
acid (S)-methyl ester, induce PR gene expression
and activate resistance against TMV as well as bacterial and fungal
pathogens (17, 20). TMV inoculation leads to local leaf lesions in
tobacco cultivars bearing the N gene limiting the spread of
the virus (48). In UROD and CPO antisense plants of the cultivar Samsun
NN, the activation of defense responses, e.g. the
accumulation of scopolin (Table I), the local induction of PR proteins
(Fig. 6), and increased content of free and bound SA (Tables II and
III) clearly correlated with a reduction in TMV infection (Fig. 7,
B and C). This result obtained with the
transgenic local lesion host is in analogy to the induced TMV
resistance in the same cultivar by treatment with ethrel (49). Future
experiments will reveal if accumulation of tetrapyrroles in a
susceptible tobacco variety lacking the N gene contributes a
systemic pathogen resistance in uninoculated tissue.
Deregulation of Chlorophyll Biosynthesis Can Initiate Defense
Responses Against Pathogen Attack--
Cellular responses in plants
similar to those observed after pathogen attack can be provoked by a
wide range of other stress situations in plants. UV light, heavy metal,
or ozone treatment, or genetic modification of metabolic pathways can
lead to the induction of PR protein expression (50-53). In addition,
increased resistance to some pathogens has been observed in transgenic
plants expressing cholera toxin (54), yeast invertase (55),
bacterio-opsin (56) as well as in response to reduced catalase activity
(57, 58). Our results are consistent with these and other examples of
altered cell death control by transgene expression (53, 59).
Free porphyrins are potent photosensitizers and have been used in
photodynamic therapy for the treatment of cancer (60). They are also
involved in the mode of action of diphenyl ether type herbicides
(e.g. acifluorfen), which act on protoporphyrinogen oxidase
(61). The phototoxicity of tetrapyrroles is exerted via reactive oxygen
species. Singlet oxygen is generated from molecular oxygen after UV
irradiation of coproporphyrin (62). By using the electron spin
resonance technique a range of free radicals is observed when porphyrin
solutions are illuminated (63). Reduced activities of UROD or CPO led
to the accumulation of tetrapyrroles (4, 5). The limited capacity of
the antioxidative defense mechanisms in UROD and CPO was indicated by
decreased levels of low molecular weight antioxidants (6). Reactive
oxygen species derived from excessive tetrapyrroles could mimic
signaling molecules formed after pathogen attack in triggering defense
responses (64-66). However, other cellular processes resulting from
tetrapyrrole-induced oxidative stress could contribute to the induction
of antimicrobial defense responses.
Evidence was recently provided that lesion formation after TMV
inoculation of tobacco can be attributed to programmed cell death (67).
Programmed cell death was inhibited in plants grown under low oxygen
pressure suggesting that ambient oxygen levels are required for this
response (68). Based on the similarity of antimicrobial defense
responses triggered in UROD or CPO antisense plants and TMV-infected
tobacco, one could speculate that lesion formation in the porphyric
transformants is exerted via programmed cell death (59, 68). In a model
system for the photodynamic therapy of cancer, the application of a
mixture of porphyrins could provoke either apoptotic or necrotic cell
death depending on the incubation protocol (69). It remains a future
challenge to prove concepts of cell death caused by accumulated
tetrapyrroles in plants.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Matthew Terry and Christian
Langebartels for critical reading of the manuscript. Elena Barthel,
Susanne Stich, and Elke Mattes are thanked for their excellent
technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by Grants Gr936/3-1 and
Gr936/4-1 from the Deutsche Forschungsgemeinschaft (to B. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Institut für
Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany. Tel.: 49 039482 5506; Fax: 49 039482 5139; E-mail: mock{at}ipk-gatersleben.de.
**
Present address: Laboratorio de Bioquimica y Biologia Molecular,
Departamento de Biotecnologia, E.T.S.I. Agronomos-UPM, 28040-Madrid, Spain.
The abbreviations used are:
UROD, uroporphyrinogen decarboxylase; CGA, chlorogenic acid; CPO, coproporphyrinogen oxidase; ES-MS, electrospray-mass spectrometry; PR, pathogenesis related protein; SA, salicylic acid; TMV, tobacco mosaic
virus; HPLC, high pressure liquid chromatography.
 |
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