Ethylene is synthesized in plant tissues through the following pathway:
L
-methionine AdoMet ACC ethylene. ACC synthase and ACC oxidase
catalyze the last two reactions [13,14]. The increase in ethylene production in carnation petals is ac-
companied by the expression of genes for both ACC synthase DC-ACS
1
[15] and ACC oxidase DC-ACO
1
corresponding to pSR120 cDNA [16]. Petal wilting in flower senescence is caused by
the decomposition of cell constituents by hy- drolytic enzymes such as protease and nuclease
[17,18]. CPase is probably one of the enzymes responsible for hydrolytic degradation of cell com-
ponents leading to cell death during senescence of petals [17,18]. A CPase gene is up-regulated during
natural, pollination-induced and exogenous ethyl- ene-induced senescence of carnation petals [19]. So
far, it has been believed that autocatalytic ethylene production and petal wilting are regulated in con-
cert and can not be separated, since they occur closely in parallel.
Transgenic plants with suppressed ethylene pro- duction have been used as a powerful tool for
understanding the regulation of the biosynthesis and action of ethylene in plant tissues. For in-
stance, Theologies et al. [20], using a transgenic tomato with suppressed ethylene production, re-
vealed that both ethylene-independent and ethyl- ene-dependent signal transduction pathways were
responsible for ripening of tomato fruit.
In the present work, we generated a transgenic carnation plant harboring a sense ACC oxidase
transgene, and examined the response of their petals to exogenous ethylene with regard to the
induction of in-rolling, ethylene production and expression of genes involved in these events. Fur-
thermore, we examined the changes in the levels of the transcripts for ACC synthase, ACC oxidase
and CPase in petals of non-transformed carnation flowers after treatment with DPSS. We report that
the expression of ACC synthase and ACC oxidase genes, which leads to ethylene production and that
of CPase, which leads to wilting of petals, are differentially regulated in carnation petals.
2. Materials and methods
2
.
1
. Plant materials Carnation Dianthus caryophyllus L. cvs. Nora
or Reiko flowers at the full-opening stage their outermost petals were at right angles to the stem
of the flower were obtained from a local grower. Ethylene production from flowers was monitored
daily as described previously [21].
The shoots of carnation cv. Nora were cultured and maintained according to the method of
Firoozabady et al. [22]. Shoot clusters were sub- cultured monthly for more than one year and used
for transformation.
2
.
2
. Cloning of cDNAs coding for ACC oxidase, ACC synthase and CPase
cDNAs encoding ACC oxidase corresponding to pSR120 [23], ACC synthase corresponding to
DC-ACS
1
, formerly CARACC3 [16] and CPase [19] were obtained by RT-PCR cloning from total
RNAs obtained from senescing carnation petals. RT-PCR was performed according to an ordinal
procedure with necessary optimization. The up- stream and downstream primers for RT-PCR
were: 5-CCCCTCTAGAATGGCAAACATTGT- CAACTT-3
and 5-GGGGTCTAGATCAAG-
CAGTTGGAATGGGAC-3, respectively, for the ACC oxidase cDNA; 5-CCCCACTAGTATGG-
GTTC TTATAAGGGTGT-3 and 5-GGGGAC- TAGTTTATGTTCTTGCTTTAACAA-3 for the
ACC synthase cDNA; and 5-GCAAGCTTAT- CATCTTCAGTCGTGGTC-CGT-3 and 5-TT-
GAATGAAAACCTTCACGATGATGTCTTC-3 for the CPase cDNA. cDNA fragments were lig-
ated into pBluescript II SK + Stratagene, and the resultant plasmids were amplified in Es-
cherichia coli XL-1 Blue Stratagene. The cloned cDNAs were sequenced for both strands using the
PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit with AmpliTaq DNA Poly-
merase, FS and a 373A DNA sequencer Applied Biosystems. Sequence data were analyzed using
DNASIS software Hitachi Software Engineering.
The cloned cDNA for ACC oxidase was 1250 bp in size and contained an open reading frame of
966 bp. The nucleotide sequence of the coding region was identical to that of pSR120 [6] except
for one nucleotide. The cDNA had G instead of C at position 479 in the nucleotide sequence of the
coding region of pSR120 cDNA, which would change Ala to Gly at position 147 in the deduced
amino acid sequence. The cDNA for ACC syn- thase was 1566 bp in size and contained an open
reading frame 1554-bp that shares 99.6 simi- larity with DC-ACS
1
cDNA [16]. On the other hand, the cDNA for CPase was 1306 bp in size
and contained an open reading frame that shared 99.2 similarity with the sequence re-
ported previously [19].
2
.
3
. Construction of a sense ACC oxidase transgene
For the transformation of carnation plant, we prepared a binary vector, pMLH2113-DCACO
OR + , by inserting the coding sequence of ACC oxidase cDNA in sense orientation into
pMLH2113-GUS [24] at the BamHISacI site created
by removing
a GUS
region. The
pMLH2113-GUS vector
was constructed
by adding a HPT gene to an original vector
pBE2113-GUS [25] that drives the GUS gene under the control of CaMV35S promoter with
enhancer sequences El2 and V [24]. Correct ori- entation of the insert was confirmed by PCR.
The plasmids, pMLH2113-GUS and pMLH2113- DCACO OR + , were introduced into Agrobac-
terium tumefaciens EHA101 by the freeze-thaw method [26].
2
.
4
. Transformation and regeneration of transgenic carnation plants
Transformation by
Agrobacterium-mediated gene transfer and regeneration of carnation
plants were performed by the method of Firooz- abady et al. [22] with necessary modification.
Briefly, leaf explants, pulled off from shoot clus- ters of the shoot culture, were mixed with bacte-
ria in Minimal A medium [27] for 15 min, and cocultivated on BD medium [22] supplemented
with 100 mM acetosyringone for a week at 24°C in the dark. For selection of transformants, we
used hygromycin as a selection agent. Survived explants were subcultured repeatedly on Gerlite-
based solid media supplemented with necessary nutrients and plant hormones until they formed
normal shoots with distinct internodes. The shoots were rooted and they were transplanted
to vermiculite in pots, then to soil. The plants were grown in a containment greenhouse until
they flowered.
Carnation plants of a non-transformed control line cv. Nora, not through tissue culture, were
also grown and flowered in the same greenhouse.
2
.
5
. Treatment with exogenous ethylene of petals of the transgenic carnation flowers
Carnation flowers were harvested at their full opening stage day 0. Calyxes were removed,
and petals at the outermost and adjacent whorls were detached from receptacles. The petals de-
tached from each flower were divided into two groups. Each petal was placed vertically in a 30-
ml glass vial with the basal portion to the bot- tom, and 2-ml distilled water was added to the
vial to avoid desiccation. Half of the vials with petals were enclosed in a 1-l glass jar and ethyl-
ene was injected to a final concentration of 10 ml l
− 1
through a rubber septum on a lid. The other half were enclosed in the same way but fresh air
was injected instead of ethylene. The jars con- taining the vials with petals were left under
white fluorescent light 12 mmol s
− 1
m
− 2
at 25°C for 18 h. Then, the vials were taken out
from the jars and placed in open air for 1 h. Ethylene production from the petals was mon-
itored by enclosing the vials with petals in 450- ml glass containers for 1 h. A 1-ml gas sample
was taken into a hypodermic syringe from the container through a rubber septum of a sam-
pling port, and analyzed for ethylene with a gas- chromatograph 263-30, Hitachi equipped with
an alumina column and a flame ionization detec- tor.
Petals were detached from flowers after the as- say for ethylene production, immediately frozen
in liquid N
2
and stored at − 80°C until isolation of RNA.
2
.
6
. Treatment with DPSS of flowers of non-transgenic carnation
Flowers of carnation cv. Reiko were used at the
full-opening stage
day 0.
Stems were
trimmed to 5 cm. DPSS was administered to the flowers by immersing their cut ends in 0.1 mM
DPSS solution for 24 h from day 0. Then, the flowers were left with their cut end placing in
water under white fluorescent light 40 mmol m
− 2
s
− 1
at 23°C. Control flowers were treated similarly without DPSS application. At 1-day in-
tervals, petals were collected from the outermost
and adjacent whorls of the 3 randomly chosen flowers, combined and stored at − 80°C until use.
2
.
7
. RNA gel blot analysis Total RNA was isolated by the SDS-phenol
method [28] from carnation petals. mRNA was obtained from the total RNA using Oligotex-dT30
Takara according
to the
manufacturer’s instruction.
In the experiment with petals of the transgenic carnation treated with exogenous ethylene Fig. 1,
10 mg of total RNA was denatured at 65°C for 15 min in 10 mM MOPS buffer at pH 7.0, containing
2.5 mM Na – acetate, 0.5 mM EDTA, 2.2 M form- aldehyde, and 50 wv formamide. The dena-
tured RNA was separated on a 1.0 agarose gel containing 2.2 M formaldehyde and transferred to
a membrane filter Hybond N + , Amersham Pharmacia Biotech. Blots were prehybridized at
50°C for 3 h in 10 mM Na – phosphate buffer at pH 6.5, containing 5 × SSC 1 × SSC is 0.15 M
NaCl, 15 mM Na – citrate, pH 7.0, 10 × Den- hardt’s solution, 0.5 SDS, 50 formamide and
0.1 mg l
− 1
denatured salmon sperm DNA, fol- lowed by hybridization at 50°C for 16 h in the
solution of the same composition but containing 600 ng ml
− 1
of denatured DIG-labeled probes see below. Blots were washed twice, for 10 min each,
at room temperature in the solution containing 2 × SSC and 0.1 SDS, followed by two washes
for 15 min each at 68°C in the solution containing 0.2 × SSC and 0.1 SDS. Hybridization signals
were detected with a DIG Luminescent Detection Kit Hoffman-La Roche and by exposure to X-
ray film RX-U, Fuji Photo Film. The duration of exposure was 4, 10 and 4 h for transcripts of ACC
oxidase, ACC synthase and CPase, respectively. For DNA probes, we used the cDNAs coding for
ACC oxidase, ACC synthase and CPase obtained from senescing carnation petals. The DNA probes
were labeled with a PCR DIG Labeling Mix Hoffmann-La Roche.
On the other hand, in the experiment with petals treated with DPSS Fig. 2, two mg of mRNA was
similarly treated and blots were hybridized in the solution containing 5 × 10
5
cpm ml
− 1
of
32
P-la- beled probes. The cDNA probes were labeled with
32
P-dCTP by random priming using Multiprime DNA labeling systems Amersham Pharmacia
Biotech according to the manufacturer’s instruc-
Fig. 1. Effects of exogenous ethylene treatment on in-rolling wilting, ethylene production and levels of transcripts for
ACC oxidase, ACC synthase and CPase in petals of the control and the transgenic carnations. Petals detached from
flowers at full opening stage day 0 were treated with or without 10 ml l
− 1
ethylene for 18 h. NT, the control line; sACO, the transgenic line. A In-rolling symptoms: photo-
graphs were taken 1 h after the end of the ethylene treatment. B Ethylene production; ethylene production was measured
at 1 h through 2 h after the end of the ethylene treatment. Data are means 9 S.E. of four samples, each with five petals.
C RNA gel blot analysis of transcripts for ACC oxidase, ACC synthase and CPase: 10 mg of total RNAs isolated from
petals was separated on an agarose gel and hybridized with DIG-labeled DC-ACO
1
, DC-ACS
1
or CPase probes. The analysis was performed with petals obtained from two indi-
vidual flowers 1 and 2 for the control line and three individ- ual flowers 1, 2 and 3 for the transgenic line. Equal loading
of total RNAs was confirmed by ribosomal RNAs visualized by ethidium bromide staining of the agarose gel.
Fig. 2. Changes in the levels of transcripts for ACC oxidase, ACC synthase and CPase in petals of carnation flowers
treated with DPSS. Carnation flowers at day 0 were treated with 0.1 mM DPSS for 24 h, then left for sampling of the
petals at given time. Two micrograms of mRNAs isolated from petals was separated on an agarose gel and hybridized
with
32
P-labeled DC-ACO
1
, DC-ACS
1
or CPase probes. For reference, the mRNAs isolated from petals of flowers, left to
senesce naturally for 5 days, were treated similarly.
3
.
2
. Two distinct responses to exogenous ethylene of the petals from the transgenic carnation
To investigate whether the petals of the trans- genic line with the sense ACC oxidase transgene
respond to ethylene, we treated the petals ob- tained from the transgenic and control lines with
10 ml l
− 1
ethylene for 18 h at the full opening stage day 0, and compared their senescence be-
havior. The petals of both lines exhibited in- rolling symptoms after the ethylene treatment
Fig. 1A. On the other hand, ethylene production from the petals after the ethylene treatment was
quite different between the two lines Fig. 1B. Petals of the transgenic line produced ethylene at
0.5 9 0.1 nmol h
− 1
g
− 1
, whereas those of the control line did so at 10.3 9 1.4 nmol h
− 1
g
− 1
. Ethylene production was negligible in the petals
that had not been treated with ethylene in both lines.
Fig. 1C shows the levels of the transcripts for ACC oxidase, ACC synthase and CPase in the
petals of the transgenic and control lines after the treatment with or without ethylene. A large
amount of transcripts for both ACC oxidase and ACC synthase accumulated in the petals of the
control line after ethylene treatment, whereas did only a little in the petals of the transgenic line.
The large difference in the levels of the transcripts between the control and the transgenic lines corre-
sponded with the difference in ethylene produc- tion between the two lines. Also, the difference in
the transcript levels between the two lines after ethylene treatment was probably a reflection of
the difference found in the flowers of the two lines, which underwent natural senescence as men-
tioned above. On the other hand, the transcript for CPase accumulated at almost the same level in
the petals of both the transgenic and control lines.
The present finding that neither ACC synthase nor ACC oxidase transcripts accumulated in the
petals after the ethylene treatment were different from those reported by Savin et al. [29] who
showed a substantial accumulation of these tran- scripts in the petals of a carnation plant trans-
formed with an antisense ACC oxidase transgene after ethylene treatment. However, their results
might be caused by the use of a high concentra- tion of ethylene, 150 ml l
− 1
, which might cause an excess accumulation of transcripts.
tions. Hybridization signals were detected with an image analyzer FLA2000, Fuji Photo Film.
3. Results
