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Plant Physiol. (1999) 119: 755-764
Differential Expression of Three Members of the
1-Aminocyclopropane-1-Carboxylate Synthase Gene
Family in
Carnation1
Michelle L. Jones2 and
William R. Woodson*
Department of Horticulture and Landscape Architecture, Purdue
University, West Lafayette, Indiana 47907-1165
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ABSTRACT |
We
investigated the expression patterns of three
1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation
(Dianthus caryophyllus cv White Sim) under conditions
previously shown to induce ethylene biosynthesis. These included
treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene,
LiCl, cycloheximide, and natural and pollination-induced flower
senescence. Accumulation of ACC synthase transcripts in leaves
following mechanical wounding and treatment with
2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA
gel-blot analysis. As in other species, the carnation ACC synthase
genes were found to be differentially regulated in a tissue-specific
manner. DCACS2 and DCACS3 were preferentially expressed in styles,
whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not
induce increased accumulation of ACC synthase transcripts in carnation
flowers, whereas the expression of ACC synthase was up-regulated by
auxin, ethylene, LiCl, pollination, and senescence in a
floral-organ-specific manner. Expression of the three ACC synthases
identified in carnation did not correspond to elevated ethylene
biosynthesis from wounded or auxin-treated leaves, and there are likely
additional members of the carnation ACC synthase gene family
responsible for ACC synthase expression in vegetative tissues.
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INTRODUCTION |
The gaseous plant hormone ethylene plays an important regulatory
role in growth and development. In plant tissues ethylene production
typically is low, but, increases at developmental stages such as
ripening and senescence and in response to mechanical and environmental
stresses (Yang and Hoffman, 1984 ). In higher plants ethylene is
synthesized from Met via the following route: Met S-adenosylmethionineACCethylene (Adams and Yang,
1979). The conversion of S-adenosylmethionine to ACC, which
is catalyzed by the enzyme ACC synthase, represents one of the
rate-limiting reactions in the biosynthesis of ethylene, and the
induction of ethylene biosynthesis has been shown to require de novo
synthesis of ACC synthase (Yang and Hoffman, 1984; Kende, 1989).
Recently, the cloning of ACC synthase genes from a number of different
species has demonstrated that the enzyme is encoded by a multigene
family, the members of which are differentially regulated in a
tissue-specific manner by a variety of signals, including auxin
treatment, wounding, anaerobiosis, ripening, senescence, and
Li+ (Kende, 1993). Although there is evidence for
the regulation of ACC synthase at the posttranscriptional level
(Chappell et al., 1984; Felix et al., 1991, 1994; Spanu et al., 1994),
expression studies indicate that the induction of ACC synthase activity
is most often the result of the increased accumulation of ACC synthase mRNAs (Kende, 1993; Zarembinski and Theologis, 1994).
Highly divergent ACC synthase multigene families have been identified
and characterized in Arabidopsis (Liang et al., 1992, 1996; Van der
Straeten et al., 1992), tomato (Olson et al., 1991; Rottmann et
al., 1991; Yip et al., 1992; Lincoln et al., 1993; Oetiker et al.,
1997), mung bean (Botella et al., 1992a, 1992b, 1993; 1995; Kim et al.,
1992, 1997), zucchini (Huang et al., 1991), rice (Zarembinski and
Theologis, 1993), potato (Destefano-Beltran et al., 1995; Schlagnhaufer
et al., 1995), and orchid (Bui and O'Neill, 1998). The Arabidopsis
gene family includes at least five members whose expression is
differentially induced by hormones, developmental cues,
Li+, and the protein-synthesis inhibitor CHX
(Liang et al., 1996). Four of the ACC synthase genes in tomato have
been shown to be differentially regulated in fruit and hypocotyls
during ripening by wounding and auxin treatment (Yip et al., 1992). ACC
synthase mRNAs are also induced during flower senescence and following pollination in carnation and orchid (Park et al., 1992; Woodson et al.,
1992; O'Neill et al., 1993; Jones and Woodson, 1997; Bui and O'Neill,
1998). Few studies of other species have included the investigation of
ACC synthase expression in floral tissue. We were interested in the
differential regulation of ethylene biosynthesis in carnation floral
organs during senescence. We show that three members of the carnation
ACC synthase gene family are differentially expressed in floral organs
in response to various chemical stimuli, pollination, and
senescence.
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MATERIALS AND METHODS |
Plant Material
Greenhouse-grown carnation (Dianthus caryophyllus L. cv
White Sim) plants were used in all experiments. Flowers were harvested at anthesis, when the styles had fully elongated. Stems were recut to
10 cm, placed in deionized water, and held in the laboratory.
Flower Treatments
For treatment with ethylene, flowers were sealed in a 24-L chamber
and ethylene was injected to yield a final concentration of 10 µL
L 1. For NBD treatments, liquid NBD was injected
onto filter paper in a 24-L chamber to yield a concentration of 2500 µL L1 after volatilization. Intact carnation
flowers and leaves were treated with a number of known inducers of ACC
synthase. These inducers included 50 mM LiCl, the synthetic
auxin 2,4-D (100 µM), and 25 µM CHX, an
inhibitor of protein synthesis. Intact flowers were held in a solution
containing the various treatments for 24 h. Ethylene production
from individual flower organs was then determined. Treatments also
included pollinated and naturally senescing flowers. Flowers were
pollinated by brushing cv White Sim stigmas with freshly dehisced cv
Starlight anthers (Larsen et al., 1995). Flower organs were collected
12 and 24 h after pollination.
Harvested flowers were also left in a solution of water on the
laboratory bench, and flower organs were assayed 6 d after harvest, when the petals were inrolling. A more detailed study of
pollinated flowers included styles collected from flowers at various
times from 1 to 48 h after pollination. Leaves were treated with
LiCl and 2,4-D by placing the cut bases in the respective solutions.
After the leaves were treated for 24 h, ethylene production was
determined. Leaves also were wounded with a steel brush, and ethylene
production was measured after 2 h.
Ethylene Measurements
Individual styles, ovaries, receptacles, and petals isolated from
intact flowers after treatment were enclosed in 6-mL vials with a
rubber septum. Following a 15-min incubation period, 1-mL gas samples
were withdrawn from the vials and analyzed for ethylene using a gas
chromatograph (Varian, Sugarland, TX) equipped with an activated
alumina column and a flame-ionization detector. Leaves were sealed in
25-mL vials for 30 min for ethylene determination. Each experiment
utilized a replication of at least six flowers or leaves per treatment,
and the graphed values represent the mean ± SE
ethylene production for the replications. All experiments were conducted a minimum of three times with similar results.
RNA Extraction and Gel-Blot Analysis
Treated carnation tissue was frozen in liquid
N2 and stored at  80°C until being used for
RNA extraction. Total RNA from carnation tissue was extracted as
described by Lawton et al. (1990) and quantified
spectrophotometrically. Ten micrograms of total RNA was separated by
electrophoresis through a 1% (w/v) agarose gel containing 2.2 M formaldehyde. The separated RNAs were transferred to
membranes (Nytran, Schleicher & Schuell) and cross-linked with a
controlled UV light source (Stratalinker, Stratagene). Membranes were
prehybridized and hybridized as previously described (Jones et al.,
1995). Membranes were hybridized for 20 h at 42°C with 5 × 105 cpm mL1 32P-labeled
cDNA. Membranes were washed in 2× SSC (1× SSC is 0.15 M
NaCl and 15 mM sodium citrate, pH 7.0) and 0.1% SDS for 15 min at room temperature, followed by 15 min at 55°C, and then 15 min in 0.2× SSC and 0.1% SDS at 55°C. Blots were exposed to Kodak XAR-5
film at 80°C for 5 d using a single intensifying screen. Blots
were used for multiple hybridizations by stripping in boiling 0.1%
SDS.
Probes used for the detection of ACC synthase were either partial
cDNAs, including the coding region, or gene-specific probes containing
the 3 untranslated regions of the three ACC synthase cDNAs. Probes
containing the coding regions included a 1250-bp fragment of DCACS1
(Park et al., 1992), a 1175-bp PCR clone, DCACS2 (Henskens et al.,
1994), and a 1515-bp PCR clone, DCACS3. ACC synthase gene-specific
probes were constructed utilizing restriction sites at the periphery of
the 3 end of the coding region to give inserts approximately 250 to
300 bp in length that represented the 3 untranslated regions of these
ACC synthase cDNAs. The DCACS1-3 probe corresponds to bp 1662 to 1950 from DCACS1 (previously called caracc3, accession no. M66619) and
DCACS3-3 corresponds to bp 1268 to 1515 from DCACS3 (accession no.
AF049137). The PCR clone DCACS2 isolated by Henskens et al. (1994, accession no. X66605) does not include the 3 untranslated region of
the cDNA.
To construct the gene-specific probe for DCACS2 the 3 end of this ACC
synthase cDNA was amplified by reverse-transcriptase PCR. A sense
primer specific to the DCACS2 sequence and a nonspecific oligo(dT)-antisense primer were utilized by reverse-transcriptase PCR
of pollinated style total RNA to clone the 3 end of DCACS2 (data not
shown). The PCR clone was identified based on sequence identity to the
original DCACS2 clone. The nucleotide sequence data for the
PCR-amplified 3 end of DCACS2 will appear in the database under the
accession no. AF049138. The gene-specific probe DCACS2-3 corresponds
to bp 429 to 709. To demonstrate equal loading of RNA samples,
membranes were reprobed with rRNA (Goldsbrough and Cullis, 1981).
Specificity of Gene-Specific Probes
Approximately 10 ng of cDNA representing the coding regions
and 3 untranslated regions of DCACS1, DCACS2, and DCACS3 were electrophoresed through a 1% agarose gel. The gel was depurinated for
10 min in 0.25 M HCl, denatured for 30 min in 0.5 M NaOH and 1.5 M NaCl, and neutralized for 30 min in 1 M Tris (pH 8.0) and 1.5 M NaCl. The
DNA was transferred to membranes and cross-linked with a controlled UV
light source, as described above. Membranes were probed with the
DCACS1, DCACS2, and DCACS3 cDNA clones and the gene-specific probes
DCACS1-3, DCACS2-3, and DCACS3-3. Prehybridization,
hybridization, and washing were carried out under the conditions
described above for the RNA gels, with the addition of a 15-min wash in
0.2× SSC and 0.1% SDS at 65°C.
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RESULTS |
Homology among the Carnation ACC Synthases
DCACS3 shares the highest amino acid identity (83.7%) with the
carnation ACC synthase DCACS2, whereas amino acid identity between
DCACS3 and DCACS1 is only 66.7% (Table
I). The high homology between DCACS2 and
DCACS3 made it difficult to distinguish between the transcripts of
DCACS2 and DCACS3 when using the full-length clones as probes for RNA
gel-blot analysis under high-stringency conditions (Fig.
1). When probes were constructed that
included only the 3 untranslated regions of the cDNAs, each probe
detected only its corresponding cDNA. Whereas nucleotide identity of
the coding regions of DCACS2 and DCACS3 was 79.2%, the homology
between the 3 untranslated regions of DCACS2 and DCACS3 was only
42.7% (Table I).
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Table I.
Comparison of the percentage of nucleotide identity
and percentage of amino acid identity (in parentheses) between the
coding regions (cds) and the percentage of nucleotide identity between
the 3 untranslated regions (3 UTR) of the carnation ACC synthases
DCACS1, DCACS2, and DCACS3
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| Figure 1.
DNA gel-blot analysis showing the specificity of
the ACC synthase gene-specific probes. Each lane contained 10 ng
of DNA corresponding to the full-length cDNA of DCACS1, DCACS2, and
DCACS3. The blot was hybridized with the full-length
32P-labeled cDNA probes DCACS1, DCACS2, and DCACS3 and
probes consisting of the 3 untranslated region of the cDNA of
DCACS1-3, DCACS2-3, and DCACS3-3.
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Differential Expression of ACC Synthase in Carnation Floral Organs
Intact carnation flowers were treated with a number of known
inducers of ACC synthase to characterize the differential regulation of
the three members of the ACC synthase gene family that have been
identified in carnation. Intact flowers were placed in aqueous solutions of 25 µM CHX, 50 mM LiCl, 100 µM 2,4-D, or water. After 24 h of treatment floral
organs were removed from the treated flowers for ethylene analysis.
Floral organs also were assayed from intact flowers treated with 10 µL L1 ethylene for 24 h, from pollinated
flowers 12 or 24 h after pollination, and from naturally senescing
flowers 6 d after harvest.
The patterns of expression of the three ACC synthase genes could be
determined using gene-specific probes in the styles, ovaries, receptacles, and petals in response to the various treatments. RNA
gel-blot analysis revealed that the carnation ACC synthases were
differentially regulated in a tissue-specific manner. All three ACC
synthase transcripts were detected in styles in response to various
treatments, but DCACS2 and DCACS3 were preferentially expressed (Fig.
2). Only DCACS3 mRNAs could be detected
at low levels in the control/untreated styles, and expression of DCACS3 was enhanced by all of the treatments except CHX. Pollination, flower
senescence, and treatment of carnation flowers with exogenous ethylene
and LiCl resulted in increased levels of stylar ethylene production.
Expression of DCACS2 was induced in styles by Li, ethylene,
pollination, and senescence. DCACS1 mRNAs exhibited an increase in
senescing styles that was equivalent to levels of DCACS2 and DCACS3
transcripts, but lesser increases in DCACS1 mRNAs were observed
following treatment with LiCl or ethylene or after 24 h of
pollination.
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| Figure 2.
Ethylene production and expression of ACC synthase
in carnation styles in response to various inducers of ACC synthase
activity. Flowers were treated with water as a control (C) or with 25 µM CHX, 50 mM LiCl, 100 µM
2,4-D, or 10 µL L1 ethylene for 24 h, at which
time styles were collected for analysis. Styles were also collected
from pollinated flowers 12 and 24 h after pollination (P12 and
P24, respectively) and from senescing flowers 6 d after harvest
(6D). A, Ethylene production by the styles. Each bar consisted
of the average ± SE of styles from six flowers. B,
Accumulation of ACC synthase mRNAs in styles following treatment. Each
lane contained 10 µg of total RNA. Blots were hybridized with the
gene-specific ACC synthase probes DCACS1-3, DCACS2-3, and
DCACS3-3 and with an rRNA probe. ACC synthase blots were exposed to
film for 5 d.
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Auxin treatment of flowers induced elevated ethylene production in
ovaries that was greater than all other treatments except 24 h of
pollination (Fig. 3). In ovaries ACC
synthase transcripts were primarily those of DCACS3. DCACS1 transcripts
were detected in ovaries by the gene-specific probes only following
treatment with ethylene. Very low levels of DCACS2 transcripts were
detectable in all samples except control and CHX. Basal levels of ACC
synthase mRNAs corresponding to DCACS3 were detected in untreated
ovaries but did not appear to be enhanced by any of the treatments,
despite elevated ethylene production from these ovaries. DCACS3 mRNA
levels decreased below the constitutive level detected in untreated
ovaries in ethylene-treated, 12-h-pollinated, and senescing
ovaries.
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| Figure 3.
Ethylene production and expression of ACC synthase
in carnation ovaries in response to various inducers of ACC synthase
activity. Flowers were treated with water as a control (C) or with 25 µM CHX, 50 mM LiCl, 100 µM
2,4-D, or 10 µL L1 ethylene for 24 h, at which
time ovaries were collected for analysis. Ovaries were also collected
from pollinated flowers 12 and 24 h after pollination (P12 and
P24, respectively) and from senescing flowers 6 d after harvest
(6D). A, Ethylene production by the ovaries. Each bar consisted
of the average ± SE of six ovaries. B, Accumulation
of ACC synthase mRNAs in ovaries following treatment. Each lane
contained 10 µg of total RNA. Blots were hybridized with the
gene-specific ACC synthase probes DCACS1-3, DCACS2-3, and
DCACS3-3 and with an rRNA probe. ACC synthase blots were exposed to
film for 5 d.
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In receptacles, despite the induction of ethylene biosynthesis by
2,4-D, ethylene, pollination, and senescence, only treatment with 2,4-D
resulted in a substantial increase in ACC synthase mRNAs (Fig.
4). These mRNAs corresponded to all three
ACC synthases. Very low levels of ACC synthase transcripts were also
detected in all samples by the DCACS2 and DCACS3 gene-specific probes.
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| Figure 4.
Ethylene production and expression of ACC synthase
in carnation receptacles in response to various inducers of ACC
synthase activity. Flowers were treated with water as a control (C) or
with 25 µM CHX, 50 mM LiCl, 100 µM 2,4-D, or 10 µL L1 ethylene for
24 h, at which time receptacles were collected for analysis.
Receptacles were also collected from pollinated flowers 12 and 24 h after pollination (P12 and P24, respectively) and from senescing
flowers 6 d after harvest (6D). A, Ethylene production by the
receptacles. Each bar consisted of the average ± SE of six receptacles. B, Accumulation of ACC synthase
mRNAs in receptacles following treatment. Each lane contained 10 µg
of total RNA. Blots were hybridized with the gene-specific ACC synthase
probes DCACS1-3, DCACS2-3, and DCACS3-3 and with an rRNA probe.
ACC synthase blots were exposed to film for 5 d.
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In contrast to styles, petals showed preferential expression of DCACS1
(Fig. 5). Ethylene-treated,
2,4-D-treated, 24-h-pollinated, and senescing petals all produced
elevated levels of ethylene, and both DCACS1 and DCACS2 mRNAs
accumulated in response to these treatments. In all of these
treatments, DCACS1 transcript abundance was greater than DCACS2. An
increase in DCACS3 mRNAs was detected in petals only in response to
2,4-D.
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| Figure 5.
Ethylene production and expression of ACC synthase
in carnation petals in response to various inducers of ACC synthase
activity. Flowers were treated with water as a control (C) or with 25 µM CHX, 50 mM LiCl, 100 µM
2,4-D, or 10 µL L1 ethylene for 24 h, at which
time petals were collected for analysis. Petals were also collected
from pollinated flowers 12 and 24 h after pollination (P12 and
P24, respectively) and from senescing flowers 6 d after harvest
(6D). A, Ethylene production by the petals. Each bar consisted
of the average ± SE of petals from six flowers. B,
Accumulation of ACC synthase mRNAs in petals following treatment. Each
lane contained 10 µg of total RNA. Blots were hybridized with the
gene-specific ACC synthase probes DCACS1-3, DCACS2-3, and
DCACS3-3 and with an rRNA probe. ACC synthase blots were exposed to
film for 5 d.
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Pollination-Induced Expression of ACC Synthase in Carnation Styles
Inhibition of pollination-induced ethylene production by
pretreating carnation styles with aminoethoxyvinylglycine, an inhibitor of ACC synthase, suggests that early-pollination-induced ethylene requires ACC synthase activity in the style (Woltering et al., 1993).
To identify the ACC synthase gene responsible for
early-pollination-induced ethylene in carnation styles, RNA gel-blot
analysis was performed on styles from 1 to 48 h after pollination
using the gene-specific probes. RNA gel blots showed enhanced
accumulation of DCACS3 transcript as early as 1 h after
pollination (Fig. 6). Induction of
DCACS2, the other ACC synthase previously shown to be preferentially
expressed in styles, was delayed to 6 h after pollination. DCACS1
transcripts were detected at much lower levels and were not induced
until 24 h after pollination. To identify which genes were
directly induced by pollination and which were induced by subsequent
ethylene production, flowers were placed in an atmosphere of 2500 µL
L1 NBD, an inhibitor of ethylene action,
immediately after pollination. NBD treatment did not block the enhanced
accumulation of DCACS3 transcripts but completely inhibited the
induction of both DCACS1 and DCACS2 in pollinated styles. In
NBD-treated flowers, DCACS3 mRNAs continued to increase in styles until
12 h after pollination, after which time the transcript abundance
decreased.
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| Figure 6.
Expression of ACC synthase in styles from
pollinated flowers treated with NBD or air. Each lane contained 10 µg
of total RNA. Blots were hybridized with the gene-specific ACC synthase
probes DCACS1-3, DCACS2-3, and DCACS3-3 and with an rRNA probe.
ACC synthase blots were exposed to film for 5 d. HAP, Hours after
pollination.
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Expression of ACC Synthase in Vegetative Tissue
To determine whether the three ACC synthases identified from
carnation were flower specific, RNA gel-blot analysis was conducted to
determine expression of the ACC synthase mRNAs in leaves. Leaves were
treated with LiCl or 2,4-D or wounded to induce ethylene biosynthesis.
Although all treatments induced ethylene, treatment of isolated leaves
with 2,4-D for 24 h resulted in the greatest increase in ethylene
production (Fig. 7). No ACC synthase
transcripts were detected in control leaves by the three probes, and,
despite induction of ethylene biosynthesis by all of the treatments,
only LiCl treatment resulted in the induction of DCACS2 mRNAs in
leaves.
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| Figure 7.
Ethylene production and ACC synthase expression in
carnation leaves. Leaves were wounded with a wire brush or treated with
50 mM LiCl or 100 µM 2,4-D. A, Ethylene
production by control leaves, wounded leaves 2 h after wounding,
and LiCl- or 2,4-D-treated leaves 24 h after treatment. Bars
represent the averages ± SE of six leaves. B,
Accumulation of ACC synthase mRNAs in control, wounded, and treated
leaves. Each lane contained 10 µg of total RNA. Blots were hybridized
with the gene-specific ACC synthase probes DCACS1-3, DCACS2-3, and
DCACS3-3 and with an rRNA probe. ACC synthase blots were exposed to
film for 5 d.
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DISCUSSION |
As has been discovered in many species, members of the ACC
synthase multigene family in carnation are differentially regulated in
a tissue-specific manner. The carnation ACC synthases were found to be
induced by auxin, LiCl, ethylene, senescence, and pollination. No
induction or enhancement of ACC synthase genes was detected when
protein synthesis was inhibited by CHX, although induction of ACC
synthase by CHX has been demonstrated in many other species
(Zarembinski and Theologis, 1994). Among treatments, expression of
DCACS2 and DCACS3 was primarily localized to the gynoecium, whereas
DCACS1 was more abundantly expressed in petals. In styles and petals
transcript levels of one or more of the ACC synthase genes were
up-regulated by treatments that increased ethylene production, although
there was not always a good correlation between ethylene production
rates and transcript levels. Despite high levels of ethylene evolution
following most treatments in ovaries, receptacles, and leaves, these
tissues had little up-regulation of ACC synthase mRNAs, as detected by
the three gene-specific probes.
Auxin is a well-documented inducer of ethylene biosynthesis, and at
least one member of each ACC synthase gene family identified to date
has been shown to be induced or enhanced in response to treatment with
auxin (Huang et al., 1991; Nakagawa et al., 1991; Botella et al.,
1992b; Kim et al., 1992; Yip et al., 1992; Zarembinski and Theologis,
1993; Abel et al., 1995; Destefano-Beltran et al., 1995). Some of these
genes, including ACS4 from Arabidopsis (Abel et al., 1995), LEACS3 from
tomato (Yip et al., 1992), and VRACS6 from mung bean (Yoon et al.,
1997), are specifically induced by auxin, whereas other
auxin-responsive ACC synthase genes are induced by other stimuli as
well (Huang et al., 1991; Lincoln et al., 1993; Botella et al., 1995).
This study failed to identify a single auxin-regulated member of the
carnation ACC synthase gene family, because all three genes were
induced by auxin in various organs. DCACS3 was the only ACC synthase to
be up-regulated in styles, receptacles, and petals in response to
treatment with 2,4-D. The up-regulation of DCACS3 mRNAs by 2,4-D in the
petals when there was little regulation by ethylene suggests that
DCACS3 is an auxin-responsive gene.
In 1984, Boller discovered that Li+ greatly
enhanced ACC synthase activity in tomato fruit, and it was
Li+- and IAA-enhanced accumulation of ACC
synthase in zucchini fruits that made it possible to isolate the first
ACC synthase cDNA (Sato and Theologis, 1989). Recent expression studies
identified ACC synthase genes that are induced by
Li+. These Li-inducible genes include ACS5 in
etiolated Arabidopsis seedlings (Liang et al., 1992), CPACS1 in
zucchini fruit (Huang et al., 1991), and OSACS1 and OSACS3 in rice
(Zarembinski and Theologis, 1993). Plants grown under normal conditions
do not contain Li+, but it is one of the
strongest inducers of ACC synthase activity in plants (Liang et
al., 1996). Li+ inhibits the activity of
inositol-phosphate phosphatases, but the molecular mechanism of action
of ACC synthase induction in plants is not well understood (Liang et
al., 1996). In carnation flowers the induction of ACC synthase by
Li+ was very tissue specific, with induction of
all three ACC synthase genes only in the styles.
DCACS1 is a senescence-related ACC synthase that is regulated by
ethylene (Park et al., 1992; Woodson et al., 1992). Studies by ten Have
and Woltering (1997) found tissue-specific expression of the ACC
synthase genes in carnation, with DCACS1 predominantly expressed in
senescing petals and DCACS2 preferentially expressed in the gynoecium.
We have shown that the recently identified ACC synthase DCACS3
cross-hybridizes with DCACS2. Using gene-specific probes, we confirmed
that DCACS1 is predominantly expressed in petals, whereas both DCACS2
and DCACS3 are preferentially expressed in the gynoecium during
ethylene-induced and natural flower senescence. Although similar
regulation of DCACS2 and DCACS3 by ethylene and senescence were
observed in the style, only DCACS2 transcripts were detected in
senescing petals. The absence of DCACS3 transcripts in senescing petals
and the abundance of constitutive levels of DCACS3 in ovaries suggests
that the expression of DCACS3 may be more specific to the gynoecium
than that of DCACS2.
In styles DCACS2 and DCACS3 transcripts correlated well with ethylene
biosynthesis. This is consistent with the transcriptional regulation of
ACC synthase and de novo synthesis of the ACC synthase enzyme leading
to increased ethylene production. In petals the patterns of expression
and ethylene production were not so easy to interpret. Whereas 24 h after pollination petals were producing the most ethylene,
ethylene-treated and senescing petals had the largest accumulation of
DCACS1 transcripts. Although ACC synthase is generally considered to be
regulated at the level of transcription, regulatory mechanisms at the
posttranscriptional and -translational levels may be equally important
means of regulating the production of ACC and ethylene in many systems.
Evidence for this type of regulation has been demonstrated at the level
of mRNA splicing (Olson et al., 1995), processing of the C terminus of
the enzyme (Li and Mattoo, 1995), and posttranslational modification of
the enzyme by phosphorylation (Felix et al., 1994; Spanu et al., 1994). Further experiments are necessary to determine whether
posttranscriptional processing of ACC synthase is involved in the
regulation of ethylene biosynthesis in carnation flowers. Considering
that five or more ACC synthase genes have been identified in
Arabidopsis, tomato, and mung bean (Liang et al., 1996; Kim et al.,
1997; Oetiker et al., 1997; Yoon et al., 1997), it is likely that the
three ACC synthase genes described in this paper represent only a few
of the members of the carnation ACC synthase gene family. When gel blots of pollinated and senescing petal RNA are probed with the full-length DCACS1 probe (cDNA clone no. 403; Park et al., 1992), the
patterns of expression are more closely correlated with ethylene levels
(M.L. Jones, unpublished data). These data support the existence of
additional ACC synthase genes with sequence homology to the
senescence-related ACC synthase DCACS1.
Pollination accelerates ethylene biosynthesis and coordinates
developmental changes that occur during the natural senescence of
flowers (Stead, 1992). Like senescence in unpollinated flowers, pollination also induces preferential accumulation of DCACS1 in petals
and DCACS2 and DCACS3 in the gynoecium. In styles pollination-induced ethylene can be defined temporally by three distinct peaks (Larsen et
al., 1995; Jones and Woodson, 1997). The time points 12 and 24 h correspond to the second and third peaks of ethylene
production from the style, respectively. We previously reported that
ACC synthase mRNAs detected by the full-length DCACS2 probe increased in styles by 1 h after pollination (Jones and Woodson, 1997).
Using gene-specific probes, we differentiated between DCACS2 and DCACS3
transcripts and have shown in Figure 6 that early increases in ethylene
biosynthesis following pollination were the result of DCACS3 gene
expression, whereas the expression of DCACS2 and DCACS1 corresponded to
the second and third peaks of ethylene production, respectively.
Pollination-induced expression of DCACS1 and DCACS2 in styles was
prevented by NBD, indicating that these genes were transcriptionally
regulated by ethylene. We previously showed that the stylar ethylene
produced within the third peak and a portion of the second peak is
autocatalytic and can be prevented by treatment of the flower with NBD
(Jones and Woodson, 1997). Pollination-induced expression of DCACS3 was independent of ethylene action; therefore, DCACS3 represents a pollination-responsive gene.
Recently, Bui and O'Neill (1998) showed similar regulation of ACC
synthase in the gynoecium of orchid. They showed that
early-pollination-induced ethylene biosynthesis from the gynoecium was
the result of pollination-responsive ACC synthases (Phal-ACS2 and
Phal-ACS3), and autocatalytic ethylene production from the flower
resulted from the expression of an additional ACC synthase gene
(Phal-ACS1). In orchids auxin contained in the pollinia has been
proposed as the primary pollination signal, and the application of
auxin to the stigma duplicates postpollination events, including
ethylene production, flower fading (Curtis, 1943; Burg and Dijkman,
1967), ovary growth, and ovule differentiation (O'Neill et al., 1993;
Zhang and O'Neill, 1993). Consequently, both pollination-responsive
ACC synthase genes identified in orchid were induced by application of
auxin to the stigma. The ethylene-independent regulation of DCACS3 by
pollination and its responsiveness to 2,4-D suggest that auxins may
also play a role in postpollination development and signaling in
carnation flowers.
In the ovary the induction of ethylene biosynthesis did not correlate
well with ACC synthase gene expression. Basal levels of DCACS3 mRNAs
were detected in ovaries but were not enhanced by any of the
treatments, whereas DCACS2 mRNAs were only slightly up-regulated by
various treatments including pollination and senescence. In contrast,
ACC oxidase mRNAs have been shown to be highly up-regulated by
pollination and senescence in the carnation ovary (Woodson et al.,
1992; Jones and Woodson, 1997). Large increases in DCACO1 mRNAs can be
detected in pollinated and senescing ovaries by exposing blots for only
18 h, whereas 5-d exposures are required for detection of ACC
synthase transcripts (Jones and Woodson, 1997). The comparatively low
levels of ACC synthase transcripts in carnation ovaries have led us to
propose that ACC transport to the ovary is necessary for the sustained
increases in ethylene biosynthesis induced by pollination and
senescence (Jones and Woodson, 1997).
Similar to the basal level of DCACS3 detected in carnation gynoecia,
low steady-state levels of LEACS2 have been shown in green tomato
fruits (Yip et al., 1992). Yip et al. (1992) suggested that this low
level of LEACS2 expression in green tomato fruits is responsible for
the system I ethylene (Yang and Hoffman, 1984) produced in
preclimacteric fruits. Although the basal levels of DCACS3
transcripts detected in carnation gynoecia may be responsible for the
low levels of ethylene produced by these organs, the constitutive levels of DCACS3 mRNAs detected in control ovaries seem relatively high
compared with the low levels of ethylene production measured. Untreated
ovaries from preclimacteric flowers have very low levels of ACC oxidase
activity compared with the levels of ACC synthase activity, and ACC
oxidase mRNAs have not been detected in these ovaries by RNA gel-blot
analysis (Woodson et al., 1992; Jones and Woodson, 1997; ten Have and
Woltering, 1997). This evidence suggests that ACC oxidase limits
ethylene production in preclimacteric ovaries. Although ACC synthase is
often considered to be the rate-limiting step in ethylene biosynthesis,
activity of the enzyme ACC oxidase has also been shown to limit
ethylene biosynthesis in flowers and fruits (Yang and Hoffman, 1984).
DCACS3 mRNA abundance in ovaries from ethylene-treated,
12-h-pollinated, and 6-d-senescing flowers was actually lower than basal levels in control ovaries, indicating the presence of negative regulation or repression of ACC synthase transcripts. ten Have and
Woltering (1997) showed that treating carnation flowers with exogenous
ethylene resulted in increases in ACC synthase mRNAs in the ovary by
3 h, but this accumulation then decreased after 18 h of
exposure. These experiments also suggested that there is a negative
regulation or inhibition of ACC synthase by ethylene in the ovary. A
detailed study involving multiple exposure times and ethylene
concentrations will be needed to more accurately assess the regulation
of DCACS3 by ethylene in carnation ovaries.
As was observed with carnation ovaries, the induction of ethylene
biosynthesis by various treatments did not correlate well with ACC
synthase transcripts in leaves and receptacles. In these tissues the
three members of the carnation ACC synthase gene family described in
this paper were insufficient to catalyze the observed ethylene
biosynthesis. Although ethylene biosynthesis in the absence of ACC
synthase transcripts could be the result of ACC transport from other
parts of the flower, as has been suggested for ovaries, reports of
increased ACC synthase activity in senescing receptacles provide
evidence for de novo synthesis of ACC in the receptacle and suggest
that an ACC synthase gene responsible for ethylene biosynthesis in the
receptacle tissue remains to be identified (Woodson et al., 1992).
The phytohormone ethylene is associated with carnation flower petal
senescence, and auxin treatment of flowers has also been shown to
induce ethylene biosynthesis and petal senescence (Sacalis, 1989). In
contrast, both ethylene and auxin cause increases in the fresh weight
of the ovary similar to those following pollination (Nichols, 1971).
Following pollination, ethylene biosynthesis is induced in styles,
ovaries, and petals, but the results of ethylene action in these floral
organs is dramatically different. Whereas ethylene induces senescence
of the petals and styles, organs that have now completed their function
in pollen reception, the ovary is now induced to grow and develop into
a fruit. Considering their differing roles in reproduction, it is not
unexpected that ethylene biosynthesis would be differentially regulated
within the individual organs of a flower.
Using gene-specific probes we have identified differential patterns of
ACC synthase gene expression in carnation flowers and leaves. The
differential regulation of the members of the carnation ACC synthase
gene family following pollination and senescence will be useful in
determining the role of ACC and ethylene in interorgan communication
within the flower. In addition, the expression of the
pollination-responsive DCACS3 can be used to help identify the primary
pollination signal or pollen factor responsible for inducing ACC
synthase immediately after pollination in the style and setting into
motion the subsequent postpollination events. Except for moderate
accumulation of DCACS2 mRNAs in response to LiCl treatment of leaves,
the three carnation ACC synthases appeared to be flower specific.
Experiments that resulted in increased ethylene biosynthesis but no
detectable increases in ACC synthase mRNAs suggested that the carnation
ACC synthase gene family has additional unidentified members that are
not detected by the gene-specific probes. To gain a complete
understanding of the regulation of ethylene biosynthesis during flower
senescence, it will be necessary to identify these members and compare
their expression patterns with ACC synthase and ACC oxidase enzyme
activity, as well as expression of the ACC oxidase genes.
| |
FOOTNOTES |
1
This research was supported by grants from the
U.S. Department of Agriculture/National Research Initiative Competitive
Grants Program (no. 92-37304-7867) and the American Floral Endowment. This is publication no. 15,897 of the Purdue University Office of
Agricultural Research Programs.
2
Present address: Department of Horticulture and
Landscape Architecture, Colorado State University, Fort Collins, CO
80523-1173.
*
Corresponding author; e-mail woodson{at}purdue.edu; fax
1-765-494-0808.
Received August 20, 1998;
accepted November 6, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CHX, cycloheximide.
NBD, 2,5-norbornadiene.
| |
ACKNOWLEDGMENTS |
We would like to acknowledge Dr. Ernst Woltering for supplying
us with the DCACS2 clone and to thank Drs. Ed Ashworth and David Rhodes
for reading the manuscript.
| |
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Top
Abstract
Introduction