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Plant Physiol. (1998) 117: 1351-1361
Developmental and Light Regulation of Desacetoxyvindoline
4-Hydroxylase in Catharanthus roseus (L.) G. Don.1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The expression of desacetoxyvindoline 4-hydroxylase (D4H), which catalyzes the second to the last reaction in vindoline biosynthesis in Catharanthus roseus, appears to be under complex, multilevel developmental and light regulation. Developmental studies with etiolated and light-treated seedlings suggested that although light had variable effects on the levels of d4h transcripts, those of D4H protein and enzyme activity could be increased, depending on seedling development, up to 9- and 8-fold, respectively, compared with etiolated seedlings. However, light treatment of etiolated seedlings could stop and reverse the decline of d4h transcripts at later stages of seedling development. Repeated exposure of seedlings to light was also required to maintain the full spectrum of enzyme activity observed during seedling development. Further studies showed that a photoreversible phytochrome appeared to be involved in the activation of D4H, since red-light treatment of etiolated seedlings increased the detectable levels of d4h transcripts, D4H protein, and D4H enzyme activity, whereas far-red-light treatment completely reversed this process. Additional studies also confirmed that different major isoforms of D4H protein exist in etiolated (isoelectric point, 4.7) and light-grown (isoelectric point, 4.6) seedlings, suggesting that a component of the light-mediated activation of D4H may involve an undetermined posttranslational modification. The biological reasons for this complex control of vindoline biosynthesis may be related to the need to produce structures that could sequester away from cellular activities the cytotoxic vinblastine and vincristine dimers that are derived partially from vindoline.
Alkaloids are physiologically active secondary metabolites
containing heterocyclic nitrogen in their structures (Pelletier, 1970 The molecular mechanisms mediating the effects of other environmental
factors on alkaloid biosynthesis are less well documented. Light, which
plays a critical role in plant growth and development, may also affect
alkaloid biosynthesis. For example, during the early stages of tobacco
seedling development, the rate of nicotine biosynthesis is associated
with radicle elongation. A brief pulse of light interfered with radicle
growth and reduced nicotine accumulation (Weeks and Bush, 1974 The effects of light on alkaloid accumulation have also been studied in
C. roseus. This plant, which belongs to the Apocynaceae family, produces more than 100 monoterpenoid indole alkaloids, including the powerful cytotoxic drugs vinblastine and vincristine. These alkaloids are dimers formed from the condensation of
catharanthine and vindoline (Svodoba and Blake, 1975 The transformation of tabersonine to vindoline involves six strictly
ordered enzyme reactions (Fig. 1):
aromatic hydroxylation, O-methylation, hydration of the
2,3-double bond, N(1)-methylation, hydroxylation at position
4, and 4-O-acetylation (Balsevich et al., 1986
We recently isolated cDNA and genomic clones of D4H that display a high
degree of homology with a well-characterized family of plant and fungal
dioxygenases (Vazquez-Flota et al., 1997 The present study describes in greater detail the relationship between
seedling development and the role played by light in the activation of
D4H. The results indicate that expression of D4H may be regulated by
transcriptional, translational, and posttranslational controls.
Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
).
These complex molecules are widespread in the plant kingdom, and it is
estimated that about 30% of all plants contain alkaloids (Robinson,
1981). Most theories propose a role for alkaloids in the interaction of
plants with their environment, either by providing a chemical defense
against pathogens or by participating in different plant-insect
interactions (Bennet and Wallsgrove, 1994; Grayer and Harborne,
1994; Rhodes, 1994). The contributions of alkaloids to plant fitness to
their surroundings may be modulated by the rate and type of alkaloids
produced in response to biotic and abiotic factors (Robinson, 1981;
Bennet and Wallsgrove, 1994; Kutchan, 1995). Some aspects of the
molecular basis for pathogen-induced alkaloid synthesis have been
studied in Papaver somniferum (Facchini et al., 1996),
Eschscholtzia californica (Dittrich and Kutchan, 1991;
Kutchan, 1993), and Catharanthus roseus (Eilert et al., 1987; Pasquali et al., 1992; Roewer et al., 1992). Cell suspensions from these species responded to the addition of fungal elicitors by
activating the transcription of key alkaloid pathway genes, which was
followed by the appearance of corresponding enzyme activities and the
accumulation of indole alkaloids.
).
However, after cotyledons were open, a 10-h photoperiod triggered a
70% increase in nicotine content over untreated etiolated seedlings
(Weeks and Bush, 1974). Light-dependent enhancement of nicotine
biosynthesis was also observed in 6-week-old plants, in which a
correlation between photoperiod length and nicotine accumulation was
found (Tso et al., 1970). Phytochrome seems to be involved in this
process, since a red-light pulse given at the end of the day promoted a
further nicotine accumulation, whereas a similar far-red-light
treatment reversed these effects (Tso et al., 1970).
). Early studies
have shown that the pattern of alkaloids extracted from C. roseus seedlings was greatly affected by development and light
(Mothes et al., 1965; Scott, 1970). Etiolated seedlings contained high
levels of the late vindoline precursor tabersonine, which upon
illumination was transformed stoichiometrically into vindoline
(Balsevich et al., 1986; De Luca et al., 1986). In contrast,
catharanthine, which accumulated to high levels in etiolated seedlings,
was hardly affected by the light regime (Scott, 1970; Balsevich et al.,
1986). These studies suggested that light is a major limiting factor in
the conversion of tabersonine to vindoline and in the formation of
dimeric indole alkaloids (Balsevich et al., 1986; De Luca et al., 1986,
1988).
; De Luca et
al., 1986). The first of these reactions is catalyzed by tabersonine
16-hydroxylase, a Cyt P450-dependent monooxygenase associated with
microsomal cell fractions, whereas the next reaction is catalyzed by a
cytosolic S-adenosyl-L-Met, 16 hydroxytabersonine O-methyltransferase (St. Pierre and De
Luca, 1995). The enzyme involved in the hydration of the double bond of
the 16-methoxy compound has yet to be characterized, but the product
from this hydroxylase is N-methylated by a
thylakoid-associated S-adenosyl-L-Met,
S-adenosyl-L-Met:2,3-dihydro-3-hydroxytabersonine-N-methyltransferase, which forms desacetoxyvindoline (De Luca et al., 1985; Dethier and De
Luca, 1993). The second-to-the-last reaction involves the 4-hydroxylation of desacetoxyvindoline and is catalyzed by a
cytosolic 2-oxoglutarate-dependent dioxygenase known as D4H (De Carolis et al., 1990; De Carolis and De Luca, 1993). Final
O-acetylation of deacetylvindoline to yield vindoline is
catalyzed by a cytosolic DAT (De Luca et al., 1986; Powers et al.,
1990; B. St. Pierre, P. Laflamme, A.-M. Alarcoj, and V. De Luca,
unpublished data). In addition, these studies revealed that expression
of tabersonine 16-hydroxylase, D4H, and DAT in developing C. roseus seedlings is light regulated. However, although D4H and DAT
activities are detected exclusively under conditions resulting in
vindoline biosynthesis, expression of tabersonine 16-hydroxylase occurs
at low levels in C. roseus cell cultures that do not
accumulate vindoline (St. Pierre and De Luca, 1995).
View larger version (21K):
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Figure 1.
The pathway for vindoline biosynthesis. SS,
Strictosidine synthase; TDC, tryptophan decarboxylase.
). Expression of D4H appears to
be regulated by cell-, tissue-, development-, and
environment-specific controls. Enzyme assays and RNA-blot hybridization studies showed that hydroxylase activity followed closely
the levels of d4h transcripts, occurring predominantly in
young leaves and in much lower levels in stems and fruits. In contrast,
etiolated seedlings containing considerable levels of d4h
transcripts had almost undetectable hydroxylase activity. Exposure of
seedlings to light resulted in a rapid increase in enzyme activity
without any further increase in transcript levels, and continued
exposure to light was necessary to maintain transcript levels later in
seedling development (Vazquez-Flota et al., 1997).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Seedling Treatments
Light Treatments
Etiolated seedlings were exposed to white light from 60-W cool-white fluorescent tubes (General Electric-Sylvania) and 60-W incandescent bulbs (Phillips Royale, Scarborough, Ontario, Canada) at the times indicated in the figures. Photon fluence rate was calibrated at 20 µmol m
2 s1 with
a photometer (model Li-189, Li-Cor, Lincoln, NE). Unless specified
otherwise, light-exposed seedlings were grown under an 18-h
photoperiod. For red-light treatments, white light was provided as
described above and filtered (no. 19 filter, Roscolux, Port
Chester, NY). This filter transmitted only wavelengths longer than 575 nm, and greater than 90% of the irradiance at wavelengths longer than
650 nm. These characteristics produced red light with approximately the
same photon fluence rate as the red component of the original
white-light source (Aerts and De Luca, 1992). Far-red-light irradiation
was obtained by filtering white light (filter nos. 19, 83, and 89, Roscolux). Such a filter combination transmitted only wavelengths
longer than 710 nm (Aerts and De Luca, 1992). The spectral quality of
light transmitted through the filters was verified using a
spectrophotometer (model DU-65, Beckman).
External Carbon Source Application
Etiolated seedlings received 1 mL of filter-sterilized (Millex-GP, Millipore) Suc stocks to give a final concentration of 100 or 300 mM per Petri dish, and were treated as described in Figure 4.
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Enzyme Analysis
Batches of 100 seedlings submitted to various treatments were collected under a dim-green (25 W) safelight (DecoColor, General Electric), frozen in liquid nitrogen, and kept at
80°C until analysis. D4H was extracted and assayed by the direct method described by De Carolis et al. (1990), and enzyme activity was expressed on a
per-seedling basis.
Immunological Studies
Protein Purification and Antibody Production
Anti-D4H antibodies were raised against recombinant D4H protein in New Zealand White female rabbits. The expression construct pQD4H-19 was engineered from the cDNA clone cD4H-3 into the expression vector pQE30 (Qiagen, Chatsworth, CA), as described previously (Vazquez-Flota et al., 1997). This vector
provides an affinity tag containing six His residues at the N terminus
of the recombinant protein, which allows the specific purification of
D4H. Escherichia coli BB4 cells (Stratagene) containing the
expression construct pQD4H-19 were grown at 37°C and 300 rpm in 250 mL of Luria-Bertani medium (Sambrook et al., 1989) to an
A600 of 0.6, and bacterial cultures were
induced and extracted as described previously (Vazquez-Flota et al.,
1997). The sonicated bacterial extract (Brandon, Danbury, CT)
was loaded onto a 5-cm Ni-NTA agarose-affinity resin (Qiagen) packed in a 1- × 10-cm chromatographic column (C-10, Pharmacia). The
buffers and procedures used for purification of recombinant D4H were
those recommended by the manufacturer.
Immunological Analysis
Desalted seedling extracts (PD-10 columns, Pharmacia) were diluted with Laemmli buffer (Laemmli, 1970), and the equivalent of one-half of
each seedling (protein content, 8-12 µg; Bradford, 1976) was
submitted to 12% SDS-PAGE. After electrophoretic transfer (Towbin et
al., 1979) to nitrocellulose membranes (Protran, Schleicher & Schuell),
the extracts were blocked with 9% skim milk powder diluted in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween; Sambrook et al., 1989), washed twice with TBST, and then incubated for
1 h at room temperature with the primary antibody diluted 10,000-fold in 3% skim milk powder suspended in TBST (total antiserum protein approximately 70 µg). Membranes were washed twice with TBST
and probed with horseradish peroxidase conjugated to donkey anti-rabbit
IgG diluted 1:4000 in TBST (Amersham). The immunoblots were revealed
with a mixture of oxidizing and luminescence reagents (Renaissance
chemiluminescence reagent, New England Nuclear) according to the
instructions of the manufacturer. D4H antigens were detected by
autoradiography on Kodak X-Omat AR film. To distinguish variations in
the amounts of immunoreacted protein, films were exposed to probed
membranes from 30 to 120 s.
Two-Dimensional IEF-SDS-PAGE and Immunoblotting
Seedlings were grown for 8.5 d in continuous darkness or 7-d-old etiolated seedlings were exposed for a further 36 h to white light before harvesting. Seedlings were extracted and fractionated with 30% to 70% ammonium sulfate as described previously (DeCarolis et al., 1990) and were desalted on PD-10 columns. Protein was mixed to yield a final concentration of 9 M urea, 1% Triton X-100, 5%
-mercaptoethanol, and 2% ampholytes (1.6% in the
pH range 5.0-7.0 and 0.4% in the pH range 3.0-10.0; BioLytes,
Bio-Rad). Forty micrograms of total protein from dark- or light-induced seedlings was submitted to IEF (O'Farrel, 1975), followed by 12% SDS-PAGE in the second dimension (Laemmli, 1970). An identical IEF-SDS-PAGE gel was also run with a mixture of 25 µg of protein each
from extracts of dark- and light-induced seedlings. The pH gradient
(range 3.0-10.0) was calibrated with IEF standards (Bio-Rad) that had
been applied simultaneously and detected with Ponceau red reversible
staining (Sambrook et al., 1989). The IEF-SDS-PAGE gels were then
processed for immunoblotting as described in the previous section for
SDS-PAGE gels.
Nucleic Acid Extraction and Analysis
Total seedling RNA was extracted, submitted to electrophoresis on agarose gels, and transferred onto nitrocellulose membranes as described previously (Vazquez-Flota et al., 1997). Transcripts were
detected by hybridization to the radioactive cDNA clone
cD4H-3 under conditions reported previously (Vazquez-Flota
et al., 1997). Equal loading of RNA was ensured by inspection of gels
stained with ethidium bromide.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Developmental and Light Regulation of D4H
The appearance of D4H enzyme activity in developing etiolated seedlings was detected at low levels, with the highest activities observed between d 5 and 7 (Fig. 2A). Despite the low levels of enzyme activity observed, D4H protein was easily detected during the early stages of growth, and it appeared to decrease continuously after 7.5 d until it could no longer be detected by d 11 (Fig. 2A). The levels of D4H transcript also increased from the beginning of the experiment (d 4), reached a maximum 24 to 48 h later, and decreased thereafter (Fig. 2A) (Vazquez-Flota et al., 1997).
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Appearance of Different Isoforms of D4H Protein in Etiolated and Light-Grown Seedlings
The expression of d4h transcripts and the presence of significant amounts of D4H protein in etiolated seedlings (Fig. 2), which contain little or no measurable enzyme activity, suggested that some modification of D4H protein was required for enzyme activity. It was previously shown that D4H exists as three charged isoforms (De Carolis and De Luca, 1993), and it was speculated that light treatment
might also trigger a posttranslational modification, leading to the
generation of one of these isoforms and to the activation of D4H
activity. When desalted protein extracts from etiolated and light-grown
seedlings were submitted to two-dimensional protein electrophoresis and
to immunoblotting with anti-D4H antibody, different isoforms of D4H
protein could be observed (Fig. 3).
|
).
The Light Stimulation of D4H Is Not Caused by an Increase in Carbon Availability
To determine if light activated D4H as a result of increased carbon availability due to the activation of photosynthesis, 7-d-old etiolated seedlings were fed from an external carbon source. Neither etiolated seedlings nor light-treated etiolated seedlings grown in the presence of Suc (Fig. 4) or Glc (data not shown) displayed enhanced levels of d4h transcripts, D4H protein, or enzyme activity compared with the light treatment alone (Fig. 4). These initial experiments suggested that light may exert a more direct effect on D4H and that induction may not be caused by the activation of photosynthesis. These results were not unexpected, because etiolated seedlings accumulate equivalent concentrations of vindoline-pathway intermediates, which are quantitatively converted into vindoline by light treatment (Balsevich et al., 1986; De Luca et
al., 1986).
D4H Requires Light to Remain Fully Active
Studies were also conducted to determine if the continuous presence of light is necessary to maintain high levels of enzyme activity. Seven-day-old etiolated seedlings exposed to light for 24 h were returned to dark conditions for 24 or 48 h, and each dark-treated plant was subsequently reexposed to light for another 24-h period. The results indicated that d4h transcripts, D4H protein, and D4H activity decrease as a result of the light/dark transition (Fig. 5). The maximum decreases in these three parameters were observed after 48 h in the dark, and the levels were comparable with those detected at the beginning of the experiment (Fig. 5). Reexposure of 24- and 48-h dark-treated seedlings to light caused an increase in d4h transcripts, D4H protein, and D4H activity (Fig. 5), indicating that regular exposure to light is necessary to maintain the levels of D4H activity during seedling development.
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Phytochrome Is Involved in D4H Light Activation
Previous studies have suggested that phytochrome may be involved in the light activation of D4H (De Carolis, 1994) and DAT (Aerts and De
Luca, 1992). Five- and seven-day-old etiolated seedlings were treated
with red light for various lengths of time at a continuous photon
fluence of 20 µmol m2
s1 and were kept in the dark for another
24 h before processing. As noticed in earlier figures, 5-d-old
seedlings showed a more pronounced response to red-light treatment than
did older seedlings (Fig. 6). When
5-d-old seedlings were exposed to red light for a minimum of 15 min,
both d4h transcripts and D4H protein levels increased, but
D4H activity remained at background levels. A minimum of 30 min of red
light in 5-d-old etiolated seedlings was required to obtain maximum D4H
activity (Fig. 6A), whereas longer exposures to red light did not
result in any further increase in enzyme activity. Red-light treatment
of 7-d-old etiolated seedlings resulted in a slight but continuous
increase in enzyme activity with 15 to 120 min of exposure (Fig. 6B).
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The availability of a rapid and sensitive assay for D4H enzyme
activity (De Carolis et al., 1990 Light Participates in Processes Controlled by Seedling
Development
View larger version (52K):
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Figure 7.
Red-light kinetics of D4H activation and its
photoreversibility by far-red light in 5-d-old (A) and 7-d-old (B)
etiolated seedlings. For the kinetic analysis, 5- and 7-d-old etiolated
seedlings were either harvested immediately (5 d and 7 d) or
exposed to red light (R) for 30 min and harvested after 8 h (R/8
h), 16 h (R/16 h), or 24 h (R/24 h) of further dark growth.
Five- and seven-day-old etiolated seedlings were also exposed to
repetitive 30-min red-light and far-red-light (FR) treatments (R/FR and
R/FR/R), and samples were harvested after another 24 h of dark
growth. The top, middle, and bottom panels show the levels of
d4h transcripts, D4H antigens, and D4H enzyme
activities, respectively. The data in the bottom panels are the
averages ± SE of three separate experiments.
), and the control of terpenoid
biosynthesis (Tanaka et al., 1989; Yamamura et al., 1991). In C. roseus seedlings, red-light activation of D4H (Fig. 6A) (De
Carolis, 1994) and DAT (Aerts and De Luca, 1992) appears to be under
this type of photoreversible control. The inducing effects of a 30-min
red-light pulse on the accumulation of d4h transcripts, D4H
protein, and enzyme activity were reversed by a 30-min far-red-light
treatment, and the reversion was prevented by a subsequent 30-min
red-light pulse (Fig. 7).
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; De Carolis and De Luca, 1993), D4H
cDNA clones (Vazquez-Flota et al., 1997), and a highly specific
anti-D4H antibody has made it possible to study the expression of D4H
at multiple levels.
). The appearance of D4H protein followed closely the levels
of hydroxylase transcripts in etiolated seedlings but these produced
only low-D4H enzyme activities throughout the time course. Treatment of
etiolated seedlings with light did activate D4H enzyme activity, but
this depended on the age at which seedlings were exposed (Fig. 2).
Five-day-old seedlings appeared to be optimally primed to respond to
light treatment, producing the highest D4H activities (Fig. 2B), which
also correlated with the most appropriate developmental stage for
vindoline accumulation (De Luca et al., 1986; Aerts et al., 1994). In
contrast, younger seedlings did not respond well to light treatment
(data not shown), and older seedlings (Fig. 2C) were only capable of a
more limited response, producing maximal D4H activities directly
related to their developmental stage of growth (Figure 2, compare B and
C). The importance of seedling development in the light response was
corroborated when 9- and 11-d-old etiolated seedlings were treated with
light, and the D4H activities reached only those of later stages of
development of continuously illuminated seedlings (Fig. 2C, bottom, and
data not shown). The light treatment, therefore, appears to activate processes already triggered and controlled by seedling development.
Appearance of D4H Enzyme Activity Is under Complex Regulatory Control
The differential effects of light on the expression of D4H transcripts, protein, and enzyme activity (Fig. 2) at various stages of seedling development suggest that multiple levels of control may be involved in the regulation of D4H. The results shown in Figure 2, A and B, show that even though D4H transcripts and protein appear in dark-grown seedlings, light is required for the appearance of significant hydroxylase activity. The modulation by light of these three parameters appears to vary with seedling development and decreases progressively with the age of etiolated seedlings. A possible explanation of these results may involve several levels of control in which light modulates development-related transcription, translation, and undetermined posttranslational modifications (Fig. 3) that would activate or inactivate the enzyme.) and by the fact that D4H exists as a single-copy gene
(Vazquez-Flota et al., 1997). The purified protein could be resolved by
IEF and SDS-PAGE into three 45-kD isoforms with pI values of
4.6, 4.7, and 4.8. The results presented in this paper suggest that the
pI-4.7 isoform, which also occurs in dark-grown seedlings (Fig. 3A),
may be inactive, and that light treatment may convert this isoform into
an active, more acidic isoform by an undetermined posttranslational
modification (Fig. 3B). In this context, it is interesting to note that
DAT, which is involved in the last step of vindoline biosynthesis, also
appears to exist as isoforms with various specific activities (Fahn et
al., 1985; Powers et al., 1990).
A Photoreversible Phytochrome Is Involved in the Activation of D4H
The red-light activation of D4H could be reversed by a subsequent far-red-light treatment, strongly suggesting the involvement of phytochrome in the light regulation of D4H (Figs. 6 and 7). A minimum 30-min red-light pulse was necessary to saturate the D4H response (Fig. 7A), resulting in increased production or accumulation of d4h transcripts and D4H protein, whereas far-red-light treatment completely reversed this process (Fig. 7). The significant increase of D4H protein appearing within 8 h of red-light treatment of 5-d-old etiolated seedlings (Fig. 7A) suggests that the signal transduction pathway between photoreception of the light stimulus and activation of D4H may be shorter than previously anticipated (Aerts et al., 1992; De Carolis, 1994). It is interesting
to note that the kinetics of D4H activation by red light was time
dependent (Fig. 7). The minimal dose of red light required for the
induction of D4H does not allow its classification as a low-fluence
response. However, the relatively short time span between reception of
the light stimulus during the induction of D4H activity and its
photoreversibility strongly suggests that vindoline biosynthesis is
closely associated with the photomorphogenetic process in C. roseus. These results, together with those showing that regular
intervals of light treatment were required to maintain enzyme activity
(Fig. 5), suggest the involvement of light-requiring factors in the
transduction of the light stimulus that results in the activation of
D4H.
), the small subunit of Rubisco
(Keller et al., 1991), and starch phosphorylase (St. Pierre et al.,
1996). This report suggests that these mechanisms may regulate alkaloid
biosynthesis for an undetermined but important reason. Developmental
studies have shown that the complete pathway leading to catharanthine
biosynthesis occurs in etiolated seedlings, whereas several of the
terminal steps in vindoline biosynthesis appear only upon light
stimulation. Chemical inducers of vindoline biosynthesis such as methyl
jasmonate (Aerts et al., 1994) appear to be effective only if light is
applied and only within a specific developmental time frame (F.A.
Vazquez-Flota and V. De Luca, unpublished data), suggesting an intimate
association between the light activation of vindoline biosynthesis and
light-dependent developmental processes.
). It is reasonable, therefore, to
suggest that the combined presence of catharanthine and vindoline in
the cell would lead to the production of the antimitotic dimers
vinblastine and vincristine. In this way, light activation of the
terminal steps in vindoline biosynthesis may be coupled with essential
and undetermined ontogenetic processes required to sequester cytotoxic
vinblastine and vincristine dimers, which would otherwise kill the
plant. Specialized laticifers and idioblasts have been shown to exist
in C. roseus (Yoder and Mahlberg, 1976; Eilert et al., 1985;
Mersey and Cutler, 1986), but their potential roles in alkaloid
biosynthesis and accumulation remain to be shown.
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FOOTNOTES |
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Received February 19, 1998;
accepted May 9, 1998.
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ABBREVIATIONS |
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Abbreviations: D4H, desacetoxyvindoline 4-hydroxylase. DAT, acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase.
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ACKNOWLEDGMENTS |
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We thank Benoit St. Pierre, Pierre LaFlamme, and Gabriel Guillet for reading the manuscript and for helpful discussions. Sylvain Lebeurier is gratefully acknowledged for maintenance of plants in the greenhouse and in growth chambers.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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De Luca V
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) and the
molecular-mass range (
) were calibrated with commercial standards
(two-dimensional SDS-PAGE standards and SDS-PAGE low-range
molecular-mass standards, respectively, from Bio-Rad).
