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Plant Physiol. (1998) 116: 419-428
Three 1-Aminocyclopropane-1-Carboxylate Synthase Genes Regulated
by Primary and Secondary Pollination Signals in Orchid
Flowers1
Anhthu Q. Bui2 and
Sharman D. O' Neill*
Section of Plant Biology, Division of Biological Sciences,
University of California, Davis, California 95616
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ABSTRACT |
The
temporal and spatial expression patterns of three
1-aminocyclopropane-1-carboxylate (ACC) synthase genes were
investigated in pollinated orchid (Phalaenopsis spp.)
flowers. Pollination signals initiate a cascade of development events
in multiple floral organs, including the induction of ethylene
biosynthesis, which coordinates several postpollination developmental
responses. The initiation and propagation of ethylene biosynthesis is
regulated by the coordinated expression of three distinct ACC synthase
genes in orchid flowers. One ACC synthase gene
(Phal-ACS1) is regulated by ethylene and participates in
amplification and interorgan transmission of the pollination signal, as
we have previously described in a related orchid genus. Two additional
ACC synthase genes (Phal-ACS2 and
Phal-ACS3) are expressed primarily in the stigma and
ovary of pollinated orchid flowers. Phal-ACS2 mRNA
accumulated in the stigma within 1 h after pollination, whereas
Phal-ACS1 mRNA was not detected until 6 h after
pollination. Similar to the expression of Phal-ACS2, the
Phal-ACS3 gene was expressed within 2 h after pollination in the ovary. Exogenous application of auxin, but not ACC,
mimicked pollination by stimulating a rapid increase in ACC synthase
activity in the stigma and ovary and inducing Phal-ACS2
and Phal-ACS3 mRNA accumulation in the stigma and ovary, respectively. These results provide the basis for an expanded model of
interorgan regulation of three ACC synthase genes that respond to both
primary (Phal-ACS2 and Phal-ACS3) and
secondary (Phal-ACS1) pollination signals.
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INTRODUCTION |
Pollination of flowers is a key regulatory event in plant
reproduction. In many flowers pollination causes an initial, dramatic increase in ethylene production in the stigma and style and a subsequent increase in ethylene production by other floral organs. Moreover, ethylene has been shown to play a regulatory role in postpollination developmental events (Larsen et al., 1993 ; O'Neill et
al., 1993; Zhang and O'Neill, 1993; O'Neill, 1997; O'Neill and
Nadeau, 1997). In higher plants ethylene is formed via a biosynthetic pathway involving the conversion of AdoMet to ACC and of ACC to ethylene (Adams and Yang, 1979). ACC synthase (EC 4.4.1.14) catalyzes the conversion of AdoMet to ACC, whereas ACC oxidase converts ACC to
ethylene. ACC synthase is generally considered to be the rate-limiting
step in the ethylene biosynthetic pathway (Yang and Hoffman, 1984). ACC
synthase has been purified from several plant tissues, and more than 20 genes and cDNAs encoding ACC synthase have been cloned from numerous
plant species (for review, see Zarembinski and Theologis, 1994). It has
been shown in several systems that ACC synthase is encoded by a
divergent gene family, the members of which are differentially
regulated in a tissue-specific manner during plant growth and
development, and its expression is regulated at the transcriptional
level (Kende, 1993).
In spite of substantial investigation, the precise nature of
pollen-borne signals that initiate postpollination development is still
uncertain (O'Neill, 1997; O'Neill and Nadeau, 1997). Pollen-borne
ACC, which is present at high levels in petunia and tobacco pollen, has
been proposed to be the primary pollination signal because it
contributes to the early phase of ethylene production in the stigma
upon pollination (Whitehead et al., 1983, 1984; Hill et al., 1987), but
this conclusion has been contested (Hoekstra and Weges, 1986; Pech et
al., 1987; Woltering et al., 1993, 1995).
Alternatively, pollen-borne auxin or the physical stress of pollen-tube
growth through the transmitting tract are candidates for the triggering
of postpollination developmental events (Abeles and Rubenstein, 1964;
Burg and Burg, 1966; Hoekstra and Weges, 1986; Singh et al., 1992;
Larsen et al., 1993; Nadeau et al., 1993; Zhang and O'Neill, 1993). In
particular, results from several studies demonstrate that auxin can
duplicate the postpollination effects of induction of ethylene
production and fading of flowers in Phalaenopsis spp. and
Vanda spp. orchids (Curtis, 1943; Burg and Dijkman, 1967),
as it can induce ovary growth and ovule differentiation in a manner
comparable to that triggered by pollination (O'Neill et al., 1993;
Zhang and O'Neill, 1993). A substantial quantity of auxin has also
been reported to be present in the pollinia of several orchid species
(Stead, 1992).
Regardless of the identity of the primary pollination signal, one of
its initial effects is the induction of ethylene biosynthesis, which
serves to coordinate developmental events in multiple floral organs. We
previously described the mechanism of interorgan regulation of ethylene
biosynthesis in pollinated flowers of a Phalaenopsis sp.
hybrid (cv Doritaenopsis), and identified an ACC synthase gene (Ds-ACS1, formerly called OAS1) that is
expressed in the gynoecium and labellum in response to ethylene
(O'Neill et al., 1993). The Ds-ACS1 gene was proposed to
play a role in amplification and propagation of the pollination signal
to distal floral organs but did not account for the initial induction
of ethylene biosynthesis that was triggered by the primary pollination
signal.
The objectives of the current study were to determine whether
additional ACC synthase genes were expressed in the stigma of the
pollinated Phalaenopsis spp. flower and whether these were regulated by the primary pollination signal. Two additional ACC synthase cDNAs were isolated from the stigma and ovary of pollinated orchid flowers: a stigma-specific cDNA (Phal-ACS2) and an
ovary-specific cDNA (Phal-ACS3) encoding a highly divergent
ACC synthase isozyme. The expression of Phal-ACS2 was
regulated by auxin and the cDNA accumulated primarily in the stigma,
whereas Phal-ACS3 was induced by unknown pollination
factor(s) and partially by auxin, and was closely correlated with the
pattern of ACC synthase activity found in the ovary of pollinated
Phalaenopsis spp. These results indicate a pattern of
sequential expression of ACC synthase genes regulated by primary
pollination signals (Phal-ACS2 and Phal-ACS3) and
by ethylene (Ds-ACS1), which acts as a secondary pollination
signal involved in amplification and propagation of the pollination
signal.
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MATERIALS AND METHODS |
Orchid plants of the genus Phalaenopsis (cv SM9108 Silk
Moth Wedding Gown; Stewart Orchids, Carpinteria, CA) were maintained under optimal growth conditions, as described by Gordon (1990), in a
greenhouse at the University of California, Davis. Flowers were divided
into the following floral organ units: stigma (column), ovary (ovary
and adjacent pedicel), labellum, and perianth (three sepals and two
petals) as previously described by O'Neill et al. (1993).
Experimental Treatments
For the pollination time-course experiments, flowers were
pollinated in planta by removing the anther cap and pollinia with blunt
forceps and then placing the pollinia on the stigma. At the indicated
intervals after pollination, floral parts were collected into liquid
N2, pulverized into a fine powder, and stored at
 80°C until later use in RNA isolation and enzyme assays. For the
0-h time point, flowers were pollinated and then immediately collected into liquid N2. Six flowers were used for each
time point, with the floral parts pooled at each sampling time.
For experiments examining the effect of pollination-associated factors,
flowers were treated in planta as follows. ACC treatment was achieved
by applying 10 nmol of ACC (Sigma) in a sample volume of 15 µL
directly to each stigma. Unless otherwise stated, auxin treatment was
carried out by applying 1 µg of NAA (5.4 nmol) in a 20-µL volume at
pH 6.5 to 6.8 directly to each stigma. For the treatment with different
amounts of NAA solution, 0.1, 1, 5, or 10 µg of NAA was applied in a
20-µL volume directly to each stigma. Control flowers were treated in
a similar manner with 20 µL of KPO4 buffer (pH
6.5-6.8).
For experiments determining the effects of NBD on mRNA abundance,
control and experimental groups of flowers were treated in parallel as
follows. Flowers were harvested by excision at the pedicel/floral stalk
junction, placed in tubes containing distilled
H2O, and transported to the laboratory for
immediate treatments. For NBD treatment, six flowers were incubated in
an atmosphere of 2000 µL/L NBD (Aldrich) in a sealed 35-L tank for 26 or 48 h, as previously described (O'Neill et al., 1993). For ACC-plus-NBD treatment, six flowers were pretreated with NBD for 2 h and then removed from the tank. Ten nanomoles of ACC was applied in a
sample volume of 15 µL directly to each stigma. ACC-treated flowers
were then returned to the sealed tank for 24 h of additional NBD
treatment. Six control flowers were treated in a similar manner except
that 15 µL of sterile double-distilled H2O was
applied to the stigma of each flower.
For NBD-plus-ethylene treatment, six flowers were pretreated with NBD
for 24 h and then transferred to another sealed tank supplied with
10 µL/L ethylene for 24 h. For NBD-plus-NAA and NBD-plus-pollination treatments, 12 flowers were pretreated with NBD
for 4 h and then pollinated or treated with 1 µg of NAA (pH 6.5-6.8) in a sample volume of 20 µL. These flowers were returned to
the sealed tank for 6 h of additional NBD treatment. As controls for the latter treatments, 12 flowers were incubated in a growth chamber at high RH for 4 h. Sterile, double-distilled
H2O (20 µL) was applied to each stigma of six
flowers designated as AIR. Meanwhile, the identical amount of NAA
solution was applied to each stigma of six flowers designated as NAA.
These control flowers were returned to the growth chamber for an
additional 6 h. After treatments the floral parts were collected
into liquid N2, pulverized into a fine powder,
and stored at 80°C until later use in RNA isolation and enzyme
assays.
Assay of ACC Synthase Activity in Orchid Floral Tissues
ACC synthase activity was assayed in extractions of floral organs
at various times after pollination, as described by Yu et al. (1979)
with modifications. Two grams of frozen, pulverized tissue was
homogenized in 3 mL of 100 mm Hepes-KOH (pH 8.5), 4 mm DTT, 10 µm PLP, and 20% (v/v) glycerol.
The homogenate was centrifuged at 25,000g for 20 min at
4°C in a Sorvall SM-24 rotor (DuPont). The supernatant was then
passed through a layer of sterile Miracloth (Calbiochem), and the
extract was dialyzed at 4°C for 36 h with one change of 10 mm Hepes-KOH (pH 8.5), 10% (v/v) glycerol, and 10 µm PLP. ACC synthase activity was measured by incubating 200 µg of the enzyme extract with 200 µm AdoMet, 10 µm PLP, and 200 mm Hepes-KOH (pH 8.5) in a
total sample volume of 600 µL for 60 min at 30°C. The amount of ACC
formed was assayed according to the method of Lizada and Yang (1979).
To ensure that there was no contamination of enzyme with extracted ACC,
a duplicate reaction containing 200 µg of enzyme extract was measured
without the substrate AdoMet. The ethylene released to the gas phase
was collected and determined by GC using a Carle Analytical GC 211 equipped with a flame-ionization detector and a SP4270 integrator (Spectra-Physics, San Jose, CA). To determine the efficiency of the
conversion of ACC to ethylene, 2 nmol of ACC was added as an internal
standard to a duplicate reaction mixture and degraded as described
above. Protein concentration was determined according to Bradford
(1976) using commercially available reagents (Bio-Rad).
For the experiments with ACC, NAA, and pollination, flowers were
treated in planta with 10 nmol of ACC, 0.1 or 1 µg of NAA solution,
or pollinated for 2 h. For the pollination experiment, flowers
were hand pollinated in planta. The stigma tissue was collected at 1, 1.5, 2, 2.5, 4, and 8 h after pollination for RT-PCR analysis and
ACC synthase activity assays.
RT-PCR Analysis
Total RNA was isolated from the stigma and ovary tissues of orchid
flowers from 1 to 4 h after pollination as previously described. To ensure that the RNA was free of DNA, total RNA (10 µg) was digested with 10 units of RNase-free DNase I (Promega) in a solution containing 40 mm Tris-HCl (pH 7.9), 10 mm NaCl,
6 mm MgCl2, and 40 units of RNasin
(Promega) at 37°C for 15 min. The reaction mixture was extracted with
RNase-free buffer-saturated phenol and chloroform:isoamyl alcohol
(24:1, v/v), followed by ethanol precipitation using standard
procedures (Sambrook et al., 1989).
RT-PCR analysis was performed as described by Froh-man et al.
(1988) with the following modifications. One microgram of DNA-free RNA
was reverse transcribed in a total volume of 20 µL containing 0.5 µg of oligo(dT)17 primer (Pharmacia), 2.5 mm deoxynucleotide triphosphates, and 200 units of
SUPERSCRIPT RNase H reverse transcriptase
(GIBCO-BRL) in a RT buffer supplied by the company. RNA and the primer
were denatured at 70°C for 10 min in the reaction buffer, then cooled
on ice before addition of nucleotides and the enzyme. First-strand cDNA
synthesis was carried out at room temperature for 10 min and then at
42°C for 1 h. After incubation, the enzyme was denatured by
heating to 90°C for 5 min and then cooled on ice for 10 min. The
reaction mixture was treated with 2.4 units of RNase H (Pharmacia) at
37°C for 20 min to degrade RNA. Ten microliters of the cDNA reaction mixture was used for PCR amplification.
PCR amplification was performed using Taq DNA polymerase
(Promega) in a reaction volume of 50 µL containing PCR buffer
(Promega), 1.25 mm deoxynucleotide triphosphates, and 20 pmol of each PCR primer. A thermocycler (model 480, Perkin-Elmer Cetus)
was used to perform the first cycle (94°C, 4 min; 50°C, 2 min; and
72°C, 2 min), followed by 30 cycles (94°C, 1 min; 55°C, 2 min;
and 72°C, 1.5 min). The sequences of the oligonucleotide primers
corresponding to the conserved regions RDLKW and DEIYS are as follows:
upstream, 5 -CAGAATTCAG(A/G)GA(C/T)CT(A/C/G/T)AA(A/G)TGG-3;
downstream, 5-CAGGATCC(A/C/G/T)GA(A/G)TA(A/G/T)AT(C/T)TC (A/G)TC-3
(EcoRI and BamHI recognition sites are
underlined; these sites were introduced for cloning). The PCR fragments
were purified as described below, followed by PCR reamplification using
the same set of primers.
For cloning and sequencing of PCR products, 45 µL of the PCR mixtures
was digested with EcoRI and BamHI (Promega) and
then fractionated on a 1.5% low-melting agarose gel. DNA bands of
approximately 300 bp were individually excised from the gel and
purified using a standard procedure (Sambrook et al., 1989). The
purified PCR products were ligated to pBluescript II
(KS+) (Stratagene), and the ligation products
were transformed into Escherichia coli DH5 cells.
Recombinant bacteria were selected, and plasmid DNA was isolated and
subsequently analyzed by restriction digestion for the presence of
300-bp inserts. The complete nucleotide sequence of the cDNA clones was
determined for both strands by the dideoxy chain-termination method
(Sanger et al., 1977) using a DNA sequencing kit (Sequenase version
2.0, United States Biochemical). Clones encoding ACC synthase isozymes
were identified by analyzing DNA sequence data using the BLAST program
(Altschul et al., 1990; Gish and States, 1993). Analysis of nucleic
acids and amino acid sequences was carried out using the software
package by the University of Wisconsin Genetics Computer Group
(Devereux et al., 1984).
RNA Isolation and Blot Hybridization Analysis
Total RNA isolation and RNA blot-hybridization analysis were
carried out as previously described (O'Neill et al., 1993). RNA blots
were hybridized with Ds-ACS1, Phal-ACS2, and
Phal-ACS3 cDNA insert probes. Blots were washed twice in a
2× SSC, 0.1% SDS solution at room temperature for 15 min each, and
then twice in a 0.2× SSC, 0.1% SDS solution at 55°C for 30 min
each. The blots were exposed to Kodak XAR 5 film with one intensifying
screen (Cronex Light, DuPont) at 80°C for 4 to 7 d. Genomic
DNA gel-blot hybridization analysis with the Ds-ACS1,
Phal-ACS2, and Phal-ACS3 cDNA insert probes
indicated that each hybridized to a distinct and unique restriction
fragment, indicating that each probe hybridized to a unique sequence
under the hybridization conditions used in these studies.
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RESULTS |
Pollination Induces ACC Synthase Activity in Orchid Floral Organs
Pollination causes an increase in ethylene production in all
floral organs of orchid flowers (O'Neill et al., 1993). To determine if this increase was the result of de novo synthesis of ACC in the four
organs or of the translocation of ACC from one organ to the others,
crude extracts from all organs were assayed for ACC synthase activity.
Figure 1 shows that levels of enzyme
activity in the stigma (column), labellum, and ovary were initially
very low but increased to peak levels at 24 h after pollination.
ACC synthase levels increased first in the stigma, with elevated levels of ACC synthase activity apparent by 12 h after pollination and increasing further until 24 h, at which point the enzyme level was
significantly greater than at 12 and 48 h (Student's t
test analysis, P < 0.03). In contrast, the perianth had an
extremely low level of ACC synthase enzyme activity for a 72-h period
after pollination (Fig. 1), consistent with our previous results
indicating that ACC synthase mRNA did not accumulate in this floral
organ (O'Neill et al., 1993).
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| Figure 1.
ACC synthase activity in floral organs of
pollinated orchid flowers. Enzyme activity was determined in the
stigma, ovary, labellum, and perianth. Mean values in nanomoles of ACC
per milligrams of protein per hour were obtained from five independent
reactions for each floral organ.
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To examine the early induction of ACC synthase in the stigma, a more
detailed series of time points after pollination was examined (Fig.
2). The level of enzyme activity in the
stigma increased within 1.5 h after pollination. ACC synthase
activity in the stigma at 1.5 h after pollination was higher than
that observed in the ovary and labellum at 12 h after pollination
(see Fig. 1), indicating that ACC synthase activity is induced first in
the stigma of pollinated flowers. Exogenous application of auxin (NAA)
also mimicked pollination by rapidly inducing ACC synthase activity in
the stigma (Fig. 2B). Overall, the above data show that the most
rapidly responding tissue with regard to pollination-induced ACC
synthase activity is the stigma, and that this response may be mediated
by pollen-borne auxin.
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| Figure 2.
ACC synthase activity in the stigma of orchid
flowers. A, Enzyme activity after pollination. B, Enzyme activity in
the stigma at 2 h after pollination (as the control), or after
2 h of treatment with 10 nmol of ACC and 0.1 or 1.0 µg of NAA.
Mean values were determined from five independent reactions.
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Three ACC Synthase cDNAs from the Gynoecium of Pollinated Orchid
Flowers
To isolate ACC synthase cDNAs that may contribute to the early
induction of ACC synthase enzyme activity, total RNA isolated from
stigmas and ovaries of orchid flowers at 2 or 4 h after
pollination was subjected to RT-PCR analysis. Several cDNA clones were
isolated that corresponded to Phal-ACS2 and
Phal-ACS3, from the stigma and ovary, respectively. Figure
3 shows that these cDNA fragments share
63 and 54.1% identity at the nucleotide and amino acid levels, respectively. Additionally, Phal-ACS2 and
Phal-ACS3 share 74 and 62% nucleotide identity, and 72.9 and 48.2% amino acid identity, respectively, with the corresponding
region of Ds-ACS1, respectively, indicating that these new
cDNA fragments represent genes encoding distinct ACC synthase isozymes
(Fig. 3). Genomic DNA gel-blot hybridization analysis with the
Ds-ACS1, Phal-ACS2, and Phal-ACS3 cDNA
insert probes indicated that each hybridized to a distinct and unique
restriction fragment (A.Q. Bui and S.D. O'Neill, unpublished data),
indicating that each probe hybridized to a unique sequence under the
hybridization conditions used in these studies. By analogy to other
higher plants, it is likely that ACC synthases in orchids are encoded
by a relatively large gene family, but only ACS1, ACS2, and ACS3 have been detected at the level of
mRNA in orchid flowers (O'Neill et al., 1993; Zarembinski and
Theologis, 1994).
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| Figure 3.
Comparisons of nucleotide and deduced amino acid
sequences of Ds-ACS1, Phal-ACS2, and
Phal-ACS3 cDNAs. A, Alignment of nucleotide sequences of
ACC synthase cDNAs. B, Clustal analysis of predicted amino acid
sequences of cDNAs. Sequences denoting upstream and downstream primers
for PCR amplification are underlined. Asterisks represent identical
nucleotides and amino acids to counterparts of the
Ds-ACS1 cDNA. The five boxed amino acid residues are the invariant amino acids conserved in various aminotransferases (Rottmann et al., 1991).
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Ds-ACS1 Encodes Ethylene-Regulated ACC Synthase
We previously isolated two nearly identical ACC synthase cDNA
clones, Ds-ACS1 and Ds-ACS2 (formerly called
OAS1 and OAS2; GenBank accession nos. L07882 and
L07883; O'Neill et al., 1993). Because of the nearly identical
sequences of Ds-ACS1 and Ds-ACS2, we have
concluded that they represent transcripts from a single gene
(Ds-ACS1). Because Ds-ACS1 mRNA
accumulation in the stigma and ovary of pollinated flowers was
abolished by pretreatment with aminoethoxyvinylglycine, an inhibitor of
ACC synthase, we concluded that the Ds-ACS1 gene was
ethylene regulated (O'Neill et al., 1993). To verify this hypothesis
in flowers of Phalaenopsis spp., in this study we treated
flowers with ACC or ethylene in the presence of NBD, an inhibitor of
ethylene action. Figure 4 shows that
stigma-applied ACC induced the accumulation of Phal-ACS1
mRNA (homologous to Ds-ACS1) in the stigma, ovary, and
labellum; however, this accumulation was inhibited by NBD. In addition,
exogenous ethylene overcame the inhibitory effect of NBD on
Ds-ACS1 mRNA accumulation in the stigma, ovary, and labellum
(Fig. 4). These data confirm that ethylene induces the accumulation of
Phal-ACS1 mRNA in orchid flowers.
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| Figure 4.
Effects of ACC and ethylene on the induction of
Ds-ACS1 mRNA in orchid flowers. A, mRNA abundance in the
stigma (S) and ovary (O). B, mRNA abundance in the perianth (P) and
labellum (L). Treatments were performed on detached flowers as
described in ``Materials and Methods''. For the stigma and ovary,
each lane contained 1 µg of poly(A+) RNA. For the
perianth and labellum, each lane contained 2 µg of
poly(A+) RNA. Numbers at right indicate the approximate
sizes of the detected mRNAs. ETH, Ethylene induced.
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Two Distinct ACC Synthase Genes Are Expressed in the Stigma and
Ovary of Pollinated Orchids
To examine whether expression patterns of Phal-ACS2 and
Phal-ACS3 were correlated with patterns of ACC synthase
activity in the stigma and ovary of pollinated flowers, we carried out
RNA blot-hybridization analysis. Figure
5A shows that the Phal-ACS2 cDNA insert hybridized to a 1.95-kb mRNA in the stigma of pollinated orchid flowers, and that the level of this transcript was detectable at
2 h and increased to a peak at 24 h. In sharp contrast,
Phal-ACS3 mRNA was only barely detectable in the stigma of
pollinated orchid flowers (Fig. 5A). Phal-ACS2 mRNA was only
weakly detected in the ovary at 24 h after pollination (Fig. 5B)
and was not detected in the labellum and perianth of pollinated flowers
(data not shown). These data indicate that the Phal-ACS2
gene is predominantly expressed in the stigma of the pollinated orchid
flowers, and its expression closely parallels the early increase of ACC
synthase activity in this tissue after pollination.
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| Figure 5.
Accumulation of three distinct ACC synthase mRNAs
in the orchid gynoecium after pollination. A, Accumulation of
Ds-ACS1 and Phal-ACS2 mRNAs in the
stigma. B, Accumulation of Phal-ACS3 mRNA in the ovary.
Each lane contains 30 µg of total RNA. An actin cDNA probe was used
as a control for equal loading of RNA. Numbers at right indicate the
approximate sizes of the detected mRNAs. HAP, Hours after
pollination.
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Figure 5B shows that the Phal-ACS3 cDNA insert hybridized to
a 2.2-kb mRNA in the ovary of the pollinated orchid flowers, and that
the mRNA accumulation level was relatively low at 6 h but
increased to a peak at approximately 24 h. An extremely low level
of this mRNA was also detected at 36 h in the stigma (Fig. 5A),
but it was not detected in the labellum and perianth of pollinated flowers (data not shown). Overall, these data indicate that the Phal-ACS2 gene is expressed predominantly in the stigma of
pollinated orchid flowers and the Phal-ACS3 gene is
predominantly expressed in the ovary. Furthermore, the expression of
the Phal-ACS3 gene correlates with the pattern of ACC
synthase activity in the ovary of pollinated orchids, as shown in
Figure 1.
Auxin Induces Phal-ACS2 and Phal-ACS3
Gene Expression in the Orchid Gynoecium
Figure 2B shows that, as in the case of pollination, NAA induced
the increase in ACC synthase activity in the stigma within 2 h. By
contrast, ACC did not stimulate induced ACC synthase activity in this
tissue over a short time period. To determine whether auxin directly
induced expression of the Phal-ACS2 gene in the stigma of
orchid flowers, we treated the stigma with NAA solution and collected
tissue for RNA-blot-hybridization analysis. Figure 6A shows that during 24 h of NAA
treatment, Phal-ACS2 mRNA was induced to accumulate in the
stigma by 2 h and increased to its peak after 8 h of NAA
treatment. By contrast, Phal-ACS1 mRNA was not detected in
the stigma until 8 h after NAA treatment. Comparison of the
expression patterns of Phal-ACS2 mRNA between NAA treatment and pollination revealed a decline in the level of mRNA after 8 h
of NAA treatment; this suggested that a continuous supply of auxin or
other pollination signals may be required for the continuous induction
of this transcript. As shown in Figure 6B, levels of
Phal-ACS2 mRNA increased with increased concentration of the
applied NAA solution. This induction was not mediated by ethylene
because the level of Phal-ACS2 mRNA accumulation induced by
NAA treatment was relatively the same in the presence or absence of NBD
(Fig. 6C). Additionally, neither the application of ACC nor
emasculation stimulated the accumulation of Phal-ACS2 mRNA in the stigma (data not shown). Thus, all of the above data strongly indicate that auxin induces the expression of the Phal-ACS2
gene, as well as the increase in ACC synthase activity in the stigma tissue. Several other auxin-regulated ACC synthase genes have been
identified in other plants (Nakagawa et al., 1991; Botella et al.,
1992; Abel et al., 1995).
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| Figure 6.
Accumulation of Ds-ACS1 and
Phal-ACS2 mRNAs induced by auxin (NAA) in the stigma. A,
Time course (in hours) of ACC synthase mRNA accumulation after the
application of 1 µg of NAA. B, Effects of different levels of NAA on
mRNA accumulation in orchid flowers in planta 6 h after the
application. C, Induction of ACC synthase mRNA accumulation after the
application of 1 µg of NAA for 6 h in the presence or absence of
NBD. Each lane contained 30 µg of total RNA. Numbers at right
indicate the approximate sizes of mRNAs. POLL., Pollination.
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Previously, Zhang and O'Neill (1993) demonstrated that auxin played a
crucial role in ovary and ovule development in orchid plants.
Therefore, we investigated whether auxin (NAA) induced the accumulation
of Phal-ACS3 mRNA in the ovary tissue. Figure 7A shows that levels of
Phal-ACS3 mRNA accumulation in the ovary increased during a
24-h period after the application of NAA solution to the stigma tissue.
Furthermore, as shown in Figure 7C, this mRNA accumulation was also
induced by NAA in the presence of NBD, indicating that auxin and not
ethylene induces the expression of the Phal-ACS3 gene.
However, the levels of mRNA induced by NAA were much lower and
induction occurred later than in the ovary of pollinated flowers,
compared at appropriate time points (Figs. 5B and 7A). These results
might be attributable to either too small an amount of exogenous NAA
being applied or the additional effects of pollination factor(s) other
than auxin. Figure 7B demonstrates that the amount of exogenous NAA was
not the factor influencing the level of Phal-ACS3 mRNA
accumulation, because as much as 10 µg of NAA solution did not result
in a detectable level of Phal-ACS3 mRNA at 6 h after
the treatment. Thus, it is likely that pollination factor(s) other than
auxin also induce the Phal-ACS3 mRNA.
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| Figure 7.
Accumulation of Ds-ACS1 and
Phal-ACS2 mRNAs induced by auxin (NAA) in the ovary. A,
Time course (in hours) of ACC synthase mRNA accumulation after the
application of 1 µg of NAA. B, Effects of different levels of NAA on
mRNA accumulation in flowers in planta 6 h after the application.
C, Induction of ACC synthase mRNA accumulation after the application of
1 µg of NAA for 6 h in the presence or absence of NBD. Each lane
contained 30 µg of total RNA. Numbers at right indicate the
approximate sizes of mRNAs. POLL., Pollination.
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DISCUSSION |
In many flowers pollination leads to increased production of
ethylene, which is involved in developmental changes in floral organs
such as floral color change, perianth senescence, ovary growth, and
ovule development (Stead, 1992). In carnation, orchid, and petunia
flowers, such pollination-induced ethylene production is associated
with the expression of ACC synthase and ACC oxidase genes (Woodson et
al., 1992; O'Neill et al., 1993; Tang and Woodson, 1996; O'Neill,
1997; O'Neill and Nadeau, 1997). However, the nature of pollen-pistil
interactions leading to the expression of ACC synthase genes in floral
organs is poorly understood. In this study we further investigated a
role of ACC synthase in the postpollination syndrome by cloning
additional ACC synthase cDNAs and examining expression patterns of the
corresponding genes in pollinated Phalaenopsis spp. orchids.
To our knowledge, Phal-ACS2 is the first ACC synthase gene
to be induced in the stigma of pollinated flowers. Thus, the expression
pattern of Phal-ACS2 may be useful in searching for primary
pollination signals that trigger the initial production of ethylene in
the stigma of pollinated orchid flowers. The identification of auxin as
a regulator of Phal-ACS2 gene expression suggests that auxin
may be one such pollination signal.
Unlike Phal-ACS2, the induction of Phal-ACS1 by
auxin is mediated by ethylene, because this expression is inhibited
when flowers are pretreated with aminoethoxyvinylglycine (O'Neill et
al., 1993). In this study we confirmed that, like Ds-ACS1
expression in Doritaenopsis (a Phalaenopsis
hybrid cultivar), Phal-ACS1 gene expression is regulated by
ethylene in that its expression is inhibited by NBD but can be restored
by ethylene treatment. Furthermore, the accumulation of
Phal-ACS1 mRNA in the stigma of pollinated flowers was
initially detected at 6 h, a time when increased levels of
ethylene are first detected in this organ (J.A. Nadeau and S.D.
O'Neill, unpublished data). Taken together, the Phal-ACS1
and Phal-ACS2 genes are differentially expressed in the
stigma of pollinated orchids, and it is likely that their combined
expression is responsible for the de novo synthesis of ACC required for
sustained ethylene production in this organ.
In carnation pollination results in a high level of ACC in the ovary,
suggesting the activation of ACC synthase gene expression in this organ
(Nichols et al., 1983). Two previously isolated ACC synthase cDNAs did
not hybridize strongly to an mRNA in the ovary of pollinated carnation,
suggesting that a divergent ACC synthase gene may be expressed in this
organ (Woodson et al., 1992; Henskens et al., 1994). In contrast to
what is seen in carnation, the 2.2-kb Phal-ACS3 mRNA
accumulated concomitantly with the increase in ACC synthase activity in
the ovary of pollinated orchid flowers, suggesting that the expression
of the Phal-ACS3 gene results in ACC synthase enzyme
activity in this organ. This mRNA accumulation was also induced by
auxin in the ovary; however, its level was severalfold less than that
induced by pollination, indicating that auxin is probably not the sole
pollination factor stimulating the accumulation of Phal-ACS3
mRNA. We hypothesize that an unknown pollination factor has a
synergistic effect with auxin in stimulating Phal-ACS3 gene
expression in the ovary.
In this study high levels of ACC synthase activity were detected in the
ovary of pollinated orchid flowers. However, very low levels of
ethylene and ACC oxidase activity are measured in this organ (Nadeau et
al., 1993; O'Neill et al., 1993). These data suggest that only a small
amount of ACC synthesized in the ovary is converted into ethylene,
which is consistent with the previous report that a small amount of
ethylene, together with auxin, regulates ovary growth and ovule
differentiation, but that high levels of ethylene result in cell death
in the ovary (Zhang and O'Neill, 1993). We hypothesize that a portion
of the ACC synthesized in the ovary is available for translocation to
other floral organs, especially the perianth. The translocation of ACC
to the perianth is plausible because this organ senesces several days
after pollination, even though it does not contain ACC synthase mRNA or
enzyme activity (Nadeau et al., 1993; O'Neill et al., 1993).
It is now widely appreciated that both ethylene biosynthesis and
ethylene perception contribute to the regulation of ethylene responses
in plants (Wilkinson et al., 1995). In this study we have focused only
on the coordinated regulation of ethylene biosynthesis after
pollination, and Figure 8 summarizes the
expression patterns of ethylene biosynthetic genes in floral organs of
pollinated orchid flowers based on data from studies carried out in our
laboratory.
View larger version (55K):
[in this window]
[in a new window]
| Figure 8.
A model of interorgan regulation of ACC synthase
and oxidase gene expression in pollinated orchid flowers. ACO, ACC
oxidase; ACS, ACC synthase; AUX, auxin induced; ETH, ethylene
induced; and PS, Phalaenopsis species.
|
|
Pollen-borne signals, including auxin, induce the expression of
Phal-ACS2 in the stigma, leading to increased ACC synthase activity. Newly formed ACC is oxidized by basal constitutive ACC oxidase to ethylene, which then stimulates the expression of
Phal-ACS1 and Phal-ACO1 genes for the
autocatalytic production of ethylene (Nadeau et al., 1993). ACC or
ethylene itself is translocated from the stigma to the labellum and
perianth. In the labellum ethylene induces Phal-ACS1 and
Phal-ACO1 mRNA accumulation, leading to the autocatalytic
production of ethylene, which is responsible for hyponasty and
senescence of the labellum. In the perianth ACC translocated from the
stigma, ovary, or labellum is oxidized to ethylene by ACC oxidase,
which triggers wilting and senescence. In the ovary pollen-borne auxin
and unknown pollination factor(s) induce Phal-ACS3 gene
expression, resulting in increased ACC synthase activity and ACC
synthesis. Based on the low level of ACC oxidase and ethylene
production in the ovary, a limited amount of ACC is likely to be
converted to ethylene, and this organ may also be a source of ACC for
translocation to the perianth. The biochemical tools are now available
to extend this model to include the regulation of ethylene perception
and ethylene signal transduction that may also participate in
regulating postpollination responses in flowers.
| |
FOOTNOTES |
1
This research was supported by grants from the
U.S. Department of Agriculture National Research Initiative Competitive
Grant Program-Plant Growth and Development Program (USDA 91-37304-6464 and 93-37304-6464) and the Binational Agriculture Research and Development Fund (US 1867-90R) to S.D.O.
2
Present address: Department of Molecular, Cell,
and Developmental Biology, 405 Hilgard Avenue, University of
California, Los Angeles, CA 90095-1606.
*
Corresponding author; e-mail sdoneill{at}ucdavis.edu; fax
1-916-752-5410.
Received June 18, 1997;
accepted September 30, 1997.
| |
ABBREVIATIONS |
Abbreviations:
AdoMet, S-adenosylmethionine.
NBD, 2,5-norbornadiene.
PLP, pyridoxal 5-phosphate.
RT, reverse transcription.
| |
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Abstract
Introduction