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Plant Physiol. (1998) 116: 979-989
Reduction of Light-Induced Anthocyanin Accumulation in Inoculated
Sorghum Mesocotyls1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Sorghum (Sorghum bicolor L. Moench) accumulates the anthocyanin cyanidin 3-dimalonyl glucoside in etiolated mesocotyls in response to light. Inoculation with the nonpathogenic fungus Cochliobolus heterostrophus drastically reduced the light-induced accumulation of anthocyanin by repressing the transcription of the anthocyanin biosynthesis genes encoding flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. In contrast to these repression effects, fungal inoculation resulted in the synthesis of the four known 3-deoxyanthocyanidin phytoalexins and a corresponding activation of genes encoding the key branch-point enzymes in the phenylpropanoid pathway, phenylalanine ammonia-lyase and chalcone synthase. In addition, a gene encoding the pathogenesis-related protein PR-10 was strongly induced in response to inoculation. The accumulation of phytoalexins leveled off by 48 h after inoculation and was accompanied by a more rapid increase in the rate of anthocyanin accumulation. The results suggest that the plant represses less essential metabolic activities such as anthocyanin synthesis as a means of compensating for the immediate biochemical and physiological needs for the defense response.
Plants are able to make different physiological adjustments in
response to a wide range of stimuli in their environment. In response
to light, etiolated seedlings of some sorghum (Sorghum bicolor) cultivars accumulate anthocyanin pigments in epidermal tissue of the mesocotyl (Orczyk et al., 1996
The biosynthesis of anthocyanidins and 3-deoxyanthocyanidins
represents two partially overlapping, competing pathways in
sorghum. Both compounds are derived from the phenylpropanoid and
flavonoid pathways. Activities of the key phenylpropanoid branch-point
enzymes PAL and CHS, and expression of their respective genes, are
induced by light as well as by fungal infection (Lue et al., 1989
The attempted infection of plants by pathogens often evokes extensive
and multicomponent defense mechanisms that might include activation of
phenylpropanoid biosynthesis (Dixon and Paiva, 1995 Plant Material and Fungal Inoculation
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Weiergang et al., 1996).
Anthocyanins constitute a large class of flavonoids that account for
pigmentation, serve to attract animals for pollination and seed
dispersal, and are also believed to be important as protectants against
UV irradiation (Holton and Cornish, 1995). In response to attempted
fungal infection, sorghum synthesizes a group of structurally related
compounds, the 3-deoxyanthocyanidins, which serve as phytoalexins.
Phytoalexins are low-molecular-weight antimicrobial compounds produced
by plants in response to infection or stress (Nicholson and
Hammerschmidt, 1992; Smith, 1996). The 3-deoxyanthocyanidins differ
from other anthocyanidins in that they are not hydroxylated at the
number 3 carbon of the flavonoid oxygen heterocycle (Fig. 1). They were shown to accumulate
within inclusions in epidermal cells of sorghum leaves under pathogen
attack (Snyder and Nicholson, 1990; Synder et al., 1991). The
inclusions become pigmented, indicating the presence of the
phytoalexins, and then move within the cell toward the site of
attempted penetration. The inclusions eventually burst and release
their contents, which kills both the fungus and the cell that
synthesized them.
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Figure 1.
Chemical structures of the 3-deoxyanthocyanidin
phytoalexins. MW, Molecular weight.
;
Orczyk et al., 1996). The compounds originate from the condensation of p-coumaroyl CoA and malonyl CoA to form naringenin chalcone,
which is converted to the flavanone naringenin. It is from naringenin that both the anthocyanidins and the 3-deoxyanthocyanidins are derived
(Dixon and Paiva, 1995; Holton and Cornish, 1995; Hipskind et al.,
1996b) (Fig. 2).
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Figure 2.
Biosynthetic pathways for
3-deoxyanthocyanidins and anthocyanidins. The proposed route of
3-deoxyanthocyanidin synthesis originates with the first committed step
in phenylpropanoid biosynthesis. Phe is deaminated by PAL to form
cinnamic acid, which is hydroxylated by C4H to form
p-coumaric acid. Subsequently, p-coumaric
acid is activated to a high-energy CoA thiol-ester by 4CL, which then serves as one of two substrates for flavonoid biosynthesis mediated by
CHS. The biosynthetic routes for normal anthocyanidins and for
3-deoxyanthocyanidins are proposed to diverge from one another after the synthesis of the flavanone naringenin. C4H, Cinnamic acid
4-hydroxylase; 4CL, 4-hydroxycinnamic acid:CoA ligase; CHI, chalcone
isomerase; F3 
H, flavonoid 3-hydroxylase. Solid arrows indicate
established pathways; dashed arrows indicate proposed metabolic steps
in 3-deoxyanthocyanidin synthesis.
), modification of
cell wall structures by deposition of Hyp-rich proteins (Brisson et
al., 1994) and lignin (Bruce and West, 1988), production of PR proteins
(van Loon et al., 1994), production of hydrolytic enzymes such as
chitinase and glucanases (Kombrink et al., 1991), generation of active
oxygen species (Mehdy, 1994), and synthesis of inhibitors of cell
wall-degrading enzymes (Degra et al., 1988). Thus, induction of defense
mechanisms would inevitably consume considerable amounts of available
cellular resources, including substrates and energy. It has been
suggested that repression of some cellular functions is likely to occur
to ensure a metabolic balance during the host's responses to the
stress of disease (Kombrink and Hahlbrock, 1990). Consistent with this,
we have observed that anthocyanin accumulation in sorghum is altered
after inoculation with fungal pathogens. This observation suggests that
infected plants produce the 3-deoxyanthocyanidin phytoalexins at the
expense of the light-induced accumulation of anthocyanins. In the
present study we demonstrate that inoculation with the fungus
Cochliobolus heterostrophus, a nonpathogen of sorghum,
results in the synthesis of the deoxyanthocyanidin phytoalexins and the
activation of genes encoding for PAL, CHS, and a PR protein, PR-10. In
contrast to these stimulatory effects, the accumulation of anthocyanins
and the expression of structural genes for anthocyanidin biosynthesis were repressed.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
1) of
C. heterostrophus. Tween 20 served as a wetting agent (100 µL 100 mL1) in the inoculum suspension. The
resulting suspension was misted onto seedlings with an atomizer, and
the plants were incubated at 100% RH under constant light (60 µE
m2 s1) at room
temperature.
Extraction of Anthocyanins and Phytoalexins
Triplicate samples of mesocotyl tissue (approximately 200 mg each) were collected at different time intervals after inoculation. The mesocotyls were excised from the seedlings, and 1-cm segments were removed from the base and the apex of each mesocotyl. The remaining tissue was cut into segments, weighed, and placed in 1 mL of HPLC-grade methanol. The anthocyanins and phytoalexins that had accumulated were allowed to leach from the tissue at 4°C for 24 h, after which time the extracts were analyzed.HPLC Analysis
The composition of plant extracts was determined by HPLC. Separation was carried out on two reverse-phase C-18 Ultrasphere columns (Beckman) connected in tandem. The dimensions of the two columns were 250 × 4.6 mm and 150 × 4.6 mm. Solvent A was 0.6% perchloric acid and solvent B was 100% HPLC-grade methanol. Samples (20 µL) were injected and eluted isocratically with 40% solvent B at a flow rate of 0.8 mL min1.
Compounds were detected at 480 and 535 nm for the presence of phytoalexins and anthocyanins, respectively, with an absorbance detector (model 190, Beckman) at a sensitivity of 0.001 absorbance units full scale. The anthocyanin concentrations were expressed in
terms of cyanidin equivalents with a commercially available cyanidin
chloride standard (ICN). Phytoalexin standards included luteolinidin,
apigeninidin, and the apigeninidin acyl ester, each of which had been
isolated previously in this laboratory (Hipskind et al., 1990). The
phytoalexin concentrations were determined based on extinction
coefficients of 13,800 m1
cm1 for luteolinidin, 18,000 m1 cm1 for
apigeninidin (Stafford, 1966), and 12,700 m1 cm1 for
the apigeninidin acyl ester (Hipskind et al., 1990). The concentration
of 5-methoxyluteolinidin was estimated from the extinction coefficient
for luteolinidin (Lo et al., 1996). The standards were chromatographed
and their retention times and peak areas were determined with an
integrator (model 3396A, Hewlett-Packard). Retention times were used to
identify the flavonoid compounds in the test samples, and the amount of
each compound in a sample was determined by comparing the compound's
peak area with that of a known concentration for an individual
standard.
MALDI-TOF MS Analysis
Plant extracts were analyzed at the Purdue Mass Spectrometry Center with a PerSeptive Biosystems (Framingham, MA) Voyager MALDI-TOF instrument operating in a positive-ion linear mode with a nitrogen laser (387 nm) at an accelerating voltage of 28 kV. The matrix used was
-cyano-4-hydroxycinnamic acid (10 mg mL1)
dissolved in H2O:1% trifluoroacetic
acid:acetonitrile (4:1:5). Samples were diluted 10-fold in methanol and
then mixed with the matrix solution in a ratio of 1:12.
Northern-Blot Analysis
Total RNA was isolated from liquid nitrogen-frozen mesocotyl tissues by the procedure of Hipskind et al. (1996b). Aliquots of 10 µg were denatured and fractionated on a formaldehyde-1.0% agarose
gel in 0.2 M Mops buffer, 0.5 m
sodium acetate, 0.1 m EDTA, pH 7.0. That the amount of RNA
was approximately the same in each lane was determined with ethidium
bromide-stained gels. After electrophoresis, RNA was transferred by
capillary action to a ZetaProbe Membrane (Bio-Rad) for at least 16 h, then covalently cross-linked to the membrane with a UV cross-linker
(Fisher Scientific). Individual membranes were prehybridized for 15 min
at 65°C in 0.5 m sodium phosphate buffer (pH 7.0)
containing 7% (w/v) SDS. Membranes were then hybridized for 18 h
in the same buffer with the addition of denatured
32P-labeled DNA probes. The hybridized membranes
were washed briefly in 40 mm sodium phosphate buffer
containing 5% SDS and then washed for an additional 45 min at 65°C
with a fresh buffer solution. This wash was replaced with the same
buffer containing 1% SDS and the membranes were washed twice for 45 minutes at 65°C. The membranes were then exposed to x-ray film
(X-Omat AR, Kodak) with intensifying screens at 
70°C.
DNA Probes
The cDNA probes were A1 and A2 from maize encoding DFR and ANS, respectively, F3H from barley, and CHS and PAL from sorghum. A1 and A2 were obtained from U. Wienand and A. Gierl (Max Planck Institute, Köln, Germany). F3H was obtained from M. Meldgaard (Carlsberg Laboratory, Copenhagen, Denmark). PAL and CHS were obtained from C. Magill (Texas A&M University, College Station). A full-length sorghum cDNA clone isolated in this laboratory and corresponding to a member of the PR-10 gene family was also used as a probe. Radioactive DNA probes were prepared by random-primer labeling with a labeling kit (Deca-Prime, Ambion, Austin, TX).| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Symptoms of Infection in Mesocotyls of DK46 and DK18 Plants
Sorghum cvs DK46 and DK18 differ in their responses to light (Hipskind et al., 1996b). When exposed to light, etiolated mesocotyls of cv DK46 gradually turned pink, indicating the presence of
anthocyanin pigments (Fig. 3, A-C). In
contrast, etiolated mesocotyls of cv DK18 did not accumulate
anthocyanin pigments in response to light (Fig. 3, G-I). Seedlings of
both cultivars were inoculated with C. heterostrophus
conidia and placed under constant light, as were uninoculated control
plants. At 24 h after inoculation, lesions with reddish-brown
pigmentation characteristic of the phytoalexins (Nicholson et al.,
1987) were found along the mesocotyl surface of infected cv DK46 plants
(Fig. 3D). At this time, anthocyanin pigmentation was often absent from
the mesocotyl surface. At 48 h after inoculation anthocyanin
pigments started to accumulate; however, tissue lacking anthocyanin
pigments could still be found surrounding lesions where the
phytoalexins were produced in response to attempted fungal infection
(Fig. 3E). At 72 h after inoculation anthocyanin pigments had
accumulated in all of the epidermal tissue in which the phytoalexins
had not been synthesized (Fig. 3F). The anthocyanin pigmentation was
still less intense than that of control plants kept under light for the
same period of time (Fig. 3C). Inoculated mesocotyls of cv DK18
exhibited lesions with intense phytoalexin accumulation but did not
accumulate anthocyanin pigments (Fig. 3, J-L). By 48 h after
inoculation, lesions had coalesced (Fig. 3K), and by 72 h lesions
with phytoalexins covered much of the mesocotyl surface (Fig. 3L). The
extent of lesion development and phytoalexin accumulation in cv DK18
mesocotyls appeared to be much greater than that which occurred in cv
DK46 plants at each time interval. It should be noted that lesion
development does not reflect the extent of fungal growth; rather, it
reflects the extent of deoxyanthocyanidin phytoalexin synthesis around the sites of attempted infection. We have shown previously that neither
pathogens nor nonpathogens of sorghum grow in the plant tissue that is
responding by the production of phytoalexins (Nicholson et al.,
1987, 1988; Snyder and Nicholson, 1990).
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HPLC Analysis of Anthocyanin and Phytoalexin Accumulation
HPLC was used to follow the accumulation of individual 3-deoxyanthocyanidin and anthocyanin pigments in sorghum mesocotyls. The HPLC profile of the pigments extracted from uninoculated control mesocotyls of cv DK46 revealed a major peak with a retention time of 21.5 min (Fig. 4A). Acid hydrolysis (boiling in 2% HCl for 2 h) of the extract resulted in a single peak that co-chromatographed with a cyanidin chloride standard (24.0 min) when separated by HPLC (data not shown). Thus, the anthocyanin that accumulated was a derivative of cyanidin. In addition to the accumulation of the anthocyanin pigment (21.5 min), inoculated mesocotyls of cv DK46 also accumulated each of the known 3-deoxyanthocyanidin phytoalexins (Fig. 4B). The phytoalexin components were luteolinidin (16.2 min), 5-methoxyluteolinidin (19.5 min), apigeninidin (22.0 min), and the caffeic acid ester of arabinosyl 5-O-apigeninidin (24.8 min). Each of these compounds was also produced by inoculated mesocotyls of cv DK18 (Lo et al., 1996).
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MALDI-TOF MS Analysis of Anthocyanin and Phytoalexin Compounds
MALDI coupled with TOF has been used for routine mass-spectrometric analysis of a wide range of biomolecules, including peptides and proteins, oligonucleotides, oligosaccharides, and glycoproteins (Edmondson and Russell, 1996). It is a soft
desorption-ionization technique that determines the
Mr of both fragile and nonvolatile high-mass
molecules. In the past we used plasma desorption MS to identify
anthocyanin and 3-deoxyanthocyanidin compounds in plant extracts (Wood
et al., 1994). Recently, we have demonstrated that MALDI-TOF is more
efficient for the analysis of these flavonoids, especially for the
anthocyanins that have higher Mrs because of the
presence of sugar residues in the anthocyanidin glycoside. (J.A. Sugui,
C. Bonham, S.-C. Lo, K. Wood, and R.L. Nicholson, unpublished data).
Quantification of Anthocyanin and 3-Deoxyanthocyanidins in Plant
Extracts
Expression of Anthocyanin Biosynthesis Genes F3H, DFR,
and ANS
Expression of PAL and CHS Genes
Expression of PR-10 Gene
) and in members
of the Asteraceae (Takeda et al., 1986). The structure of cyanidin 3-dimalonyl glucoside is shown in Figure
6. The aglycone cyanidin is structurally
similar to luteolinidin except for the hydroxylation at the number 3 carbon of the flavonoid oxygen heterocycle where glycosylation occurs.
The 535 m/z ion probably represents the fragmentation of a
malonyl group from the 621 m/z ion during MS analysis.
MALDI-TOF data for pigment extracts from inoculated cv DK46
mesocotyls revealed the anthocyanin ion of 621 m/z, and the
3-deoxyanthocyanidin ions of 255 m/z (apigeninidin), 271 m/z (luteolinidin), 285 m/z
(5-methoxyluteolinidin), as expected (Fig. 5B). The ion at 269 m/z has also been reported to occur in the inoculated cv
DK18 plants (Hipskind et al., 1996b). This Mr is consistent with that of a methyl ether of apigeninidin, a compound that
was recently found in grain of Sorghum caudatum and
identified as 7-O-methylapigeninidin,
Mr 269 (Pale et al., 1997). In our HPLC
system this compound probably co-chromatographed with apigeninidin (Fig. 4) and was not detected. Although HPLC analysis revealed the
presence of the acyl ester of apigeninidin, it was not detected by
MALDI-TOF analysis.
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Figure 5.
MALDI-TOF analysis of anthocyanin and
3-deoxyanthocyanidin phytoalexins in cv DK46 mesocotyls. A, Anthocyanin
extracted from uninoculated plants 48 h after exposure to light. A
major ion at 621 m/z and a minor ion at 535 m/z were detected; 621 and 535 m/z
correspond to the Mr of dimalonyl and
monomalonyl derivatives of cyanidin 3-glucoside, respectively. B,
Pigment extracted from plants 48 h after inoculation and
exposure to light. Major ions corresponding to the
molecular masses of luteolinidin (271 m/z), 5-methoxyluteolinidin (285 m/z), and apigeninidin (255 m/z) were detected in addition to the anthocyanin ion
(621 m/z). The 269 m/z ion is believed to
be a methylated derivative of apigeninidin. M, Matrix peak.
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Figure 6.
Chemical structure of cyanidin-3-dimalonyl
glucoside.
). Because the presence of pigmented 3-deoxyanthocyanidin compounds in extracts from infected tissue would
have interfered with the spectrophotometric measurement of
anthocyanins, we quantified anthocyanin contents by HPLC and expressed
them in terms of cyanidin equivalents. The time course of accumulation
of the anthocyanin cyanidin 3-dimalonyl glucoside in uninoculated and
inoculated cv DK46 mesocotyls is shown in Figure
7. The anthocyanin was first detected
in uninoculated tissues 12 h after exposure to light and continued
to accumulate through 60 h. Anthocyanin production was appreciably
lower in inoculated tissues and was first detected at 24 h after
inoculation (Fig. 7). The total anthocyanin that accumulated was 2.5 times less than that in uninoculated control plants by 72 h after
inoculation (Fig. 7).
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Figure 7.
Accumulation of the anthocyanin cyanidin
3-dimalonyl glucoside in uninoculated ( 
) and inoculated (
) cv
DK46 mesocotyls after exposure to light. Anthocyanin concentration is
expressed as cyanidin equivalents with cyanidin chloride as a
standard.
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Figure 8.
Accumulation of 3-deoxyanthocyanidin phytoalexins
in inoculated cvs DK46 and DK18 mesocotyls. A, Luteolinidin; B,
5-methoxyluteolinidin; C, apigeninidin; and D, caffeic acid ester of
arabinosyl-5-O-apigeninidin. The concentration of
each phytoalexin was determined based on known extinction coefficients.
The concentration of the 5-methoxyluteolinidin was estimated using the
extinction coefficient (13,800 m 1
cm1) for luteolinidin.
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Figure 9.
Temporal expression of different genes in
mesocotyls of uninoculated (U) and inoculated (I) cv DK46 plants.
Northern blots are of total RNA (10 µg) from mesocotyls taken at
different time intervals, probed with cDNA clones for F3H, DFR, ANS,
PAL, CHS, and PR-10. Numbers indicate hours after inoculation of plants with C. heterostrophus conidia.
; Heller and Forkman, 1988). Unlike the anthocyanin-biosynthesis genes F3H, DFR, and ANS,
accumulation of the PAL and CHS transcripts was
stimulated when the plants were simultaneously inoculated and exposed
to light.
Temporal Relationship between the Production of Anthocyanins and Phytoalexins
A close temporal relationship was demonstrated between the accumulation of anthocyanin pigments and the synthesis of 3-deoxyanthocyanidin phytoalexins in inoculated cv DK46 plants (Fig. 10). Anthocyanin accumulated at a slow rate after its first detection at 24 h after illumination/inoculation and accumulated more rapidly at 48 h after illumination/inoculation. In contrast, the rate of accumulation of the total 3-deoxyanthocyanidin phytoalexins was observed to decrease over the same time period, indicating a shift back to the synthesis of the light-induced anthocyanin pigment. It is also important to note that elevated accumulation of transcripts of the anthocyanin biosynthesis genes F3H, DFR, and ANS preceded these changes in inoculated tissues (Fig. 9).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Accumulation of anthocyanins is induced by various environmental
stimuli, including UV and red irradiation (Shichijo et al., 1993; Reddy
et al., 1994), low temperature (Christie et al., 1994), pathogen attack
(Harrison and Strictland, 1980; Heim et al., 1983; Hipskind et al.,
1996a), and several plant-growth regulators, such as cytokinins
(Deikman and Hammer, 1995), GA (Mealem-Beno et al., 1997), and ethylene
(Woltering and Somhorst, 1990). Stimulation of anthocyanin accumulation
has often been correlated with the activation of genes involved in the
anthocyanin biosynthetic pathway. Using MALDI-TOF in conjunction with
HPLC techniques, we showed that sorghum cv DK46 accumulates cyanidin
3-dimalonyl glucoside as the major anthocyanin. Synthesis of
red-light-induced anthocyanin in sorghum has been shown to be enhanced
by low temperature during the preirradiation period (Shichijo et al.,
1993). In contrast to the inductive effects of most other environmental
stresses, we demonstrated that fungal inoculation drastically reduced
anthocyanin accumulation in sorghum mesocotyls. Northern-blot analysis
of the expression of the genes encoding F3H, DFR, and ANS in inoculated mesocotyls indicated that anthocyanin synthesis was inhibited at the
transcriptional level and could thus be correlated with a reduced
expression of these genes.
) and directs the flow of carbon to the
synthesis of cyanidin and pelargonidin-based anthocyanins. Subsequent
reactions involve an NADPH-dependent reduction of the carbonyl group at
the number 4 carbon of the C-ring by DFR, which is encoded in maize by
the A1 gene (Reddy et al., 1987; Holton and Cornish, 1995).
It is believed that the removal of the subsequent hydroxyl group occurs
via the enzyme ANS, which is the putative product of the maize
A2 gene (Weiss et al., 1993). A coordinate repression of the
F3H, DFR, and ANS genes after inoculation
suggests a decrease in the activities of the enzymes they encode,
thereby reducing the flow of carbon to the anthocyanin-synthesis
pathway. Disease-induced reduction of anthocyanin accumulation in plant tissues has been investigated only to a limited extent. Inoculation with tungro virus reduced the contents of both anthocyanins and flavanols in rice plants, and the reduction was markedly greater in the
susceptible cultivar than in either a cultivar with intermediate resistance or a tolerant cultivar (Mohanty and Sridhar, 1989). Similarly, susceptible cultivars of maize inoculated with C. heterostrophus (B. maydis) accumulated significantly
lower levels of anthocyanins before lesion development (Heim et al.,
1983). It is not clear, however, if these effects are also the result
of transcriptional repression.
; Smith, 1996). Sorghum is one of the few
monocotyledonous plants known to synthesize phytoalexins. An important
feature of the sorghum phytoalexins is that they are produced in a
site-specific manner (Snyder and Nicholson, 1990). These compounds are
localized at the site of attempted fungal penetration in both
mesocotyls and juvenile leaves (Nicholson et al., 1987, 1988; Snyder
and Nicholson, 1990). Our results demonstrate that each of the
previously identified 3-deoxyanthocyanidin phytoalexins was synthesized
in inoculated cv DK46 plants. These plants also synthesized the
anthocyanin cyanidin 3-dimalonyl glucoside in response to light,
but that synthesis was significantly repressed as a result of
inoculation (Fig. 10).
suggested
that if the gene activation and mRNA and protein synthesis required for
defense are to be attained, then it is likely that genes for relatively
less-important metabolic activities are simultaneously repressed.
Anthocyanin accumulation could be considered one of the less-essential
metabolic activities that are sacrificed for the extensive and
multicomponent defense response. Mutations in anthocyanin genes
generally have no deleterious effect on plant growth and development
(Holton and Cornish, 1995). Repression of anthocyanin accumulation and
the associated expression of anthocyanin genes is likely to play a
compensatory role in the defense response of sorghum cv DK46 plants.
). The carbonyl group at the number 4 carbon of naringenin and/or eriodictyol is reduced to form apiferol and/or luteoferol, respectively. The newly
formed hydroxyl group is thought to be subsequently removed by a
dehydroxylation and oxidation reaction, resulting in the formation of
apigeninidin and luteolinidin from apiferol and luteoferol, respectively. In spite of the apparent structural similarities between
the end products and intermediates, synthesis of the
3-deoxyanthocyanidins does not involve the expression of genes similar
to those encoding DFR and ANS in the anthocyanin branch pathway
(Hipskind et al., 1996b). The repression of these genes in inoculated
sorghum mesocotyls would increase the availability of naringenin, which
is the last common precursor of the two types of flavonoids for the
biosynthesis of the 3-deoxyanthocyanidin phytoalexins.
).
). We demonstrated here that, upon simultaneous treatment of mesocotyls with light and fungal inoculation,
PAL and CHS genes were even more strongly induced
than by light alone and, in the case of PAL, gene expression
was enhanced over an extended period of time (Fig. 9). In many plant
species, key branch enzymes of the phenylpropanoid pathway, such as PAL
and CHS, are encoded by multiple genes (Dixon and Paiva, 1995).
PAL and CHS genes are always among the
anthocyanin biosynthesis genes that are transcriptionally activated by
external stimuli (Reddy et al., 1994; Deikman and Hammer, 1995; Boss et
al., 1996; Mealem-Beno et al., 1997). The concomitant but opposite
effects of fungal inoculation on the PAL and CHS
genes and the other anthocyanin biosynthesis genes (F3H, DFR,
and ANS) suggest the presence of PAL and CHS multigene
families in sorghum, the members of which may be regulated by different
environmental stimuli and have specific roles in plant defense. This
suggestion is further substantiated by our previous findings that the
accumulation of phytoalexin-associated PAL and CHS transcripts occurred
in a light-independent manner (Weiergang et al., 1996). Southern-blot
analysis with a sorghum CHS partial cDNA fragment (approximately 600 bp) revealed completely different hybridization patterns in cv DK46
total DNA digested with different restriction enzymes (S.-C. Lo,
unpublished data). These data were further evidence for a CHS gene
family.
-glucanase was systemically
activated. In contrast, a localized induction of genes for
phenylpropanoid synthesis occurred specifically at the infection site
(Kombrink and Hahlbrock, 1990). It is possible that in the sorghum
mesocotyl, defense genes other than those related to the phytoalexin
response are induced in the regions of the tissue that fail to produce
anthocyanin pigment. For example, we demonstrated an increase in
abundance of mRNA of a gene encoding a PR protein in inoculated plants
(Fig. 9). This work was carried out with a probe made from a previously isolated full-length cDNA encoding a member of the PR-10
gene family in sorghum. Production of PR proteins represents another common defense response in addition to the accumulation of phytoalexins (van Loon et al., 1994; Smith, 1996). Several PR proteins in potato are
1,3--glucanases and chitinases (Kombrink et al., 1988).
). In cultured parsley cells, fungal
elicitor exhibited dual activity by blocking the light-induced
accumulation of flavonoids through the repression of the transcription
of the CHS gene and by stimulating the secretion of furanocoumarin
phytoalexins (Lozoya et al., 1991). Transient repression of
cell-cycle-related genes was observed in suspension-cultured parsley
cells simultaneously with the activation of PAL and CHS genes in
response to treatment with fungal elicitor (Logemann et al., 1995).
Therefore, a consequential shutdown of comparatively less-important
metabolic activities might represent a general phenomenon in plant
defense to achieve "balanced cellular economy" (Kombrink and
Hahlbrock, 1990).
).
Anthocyanin and 3-deoxyanthocyanidin are two classes of structurally
related, but functionally distinct, flavonoid compounds. The metabolic
shift from the light-induced accumulation of anthocyanin pigments to
the pathogen-stimulated synthesis of 3-deoxyanthcyanidin phytoalexins
in sorghum also represents a novel model for the study of metabolic
regulation in response to light and pathogen attack. A unique feature
of this system is that the consequences of the metabolic changes are
clearly observable (Fig. 3). An understanding of the regulation in the
diversion of metabolism leading to resistance expression would provide
insights for the development of innovative strategies to enhance
disease resistance in sorghum and related crop species.
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FOOTNOTES |
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Received September 2, 1997;
accepted November 28, 1997.
The accession number for the nucleotide sequence for sorghum PR-10
described in this article is U60764.
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ABBREVIATIONS |
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Abbreviations: ANS, anthocyanidin synthase. CHS, chalcone synthase. DFR, dihydroflavanone 4-reductase. F3H, flavanone 3-hydroxylase. MALDI-TOF, matrix-assisted laser desorption ionization-time of flight. PAL, Phe ammonia-lyase. PR, pathogenesis-related.
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ACKNOWLEDGMENTS |
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The authors acknowledge Janyce A. Sugui for her capable assistance in MALDI-TOF analysis. We also thank the following people for providing cDNAs used in this study: C. Magill (PAL and CHS), M. Meldgaard (F3H), and U. Wienand (A1 and A2).
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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