response to different signals, such as wounding, ripening, senescence and the presence of auxin, as
well as in different tissue types [1]. Ethylene production was increased in a climac-
teric fruit during ripening, and ethylene induced the expression of ripening-related genes in the fruit
and accelerated ripening. The low level of ethylene produced in fruit during the preclimacteric stage
the system 1 ethylene production was suggested to act as a stimulus for the induction of genes for
ethylene production at the ripening stage [22]. Melon is a climacteric fruit, in which ethylene
production is known to increase markedly during ripening. It was previously found that a wound-re-
sponsive ACS gene CMe-ACS
1
was mainly ex- pressed in mesocarp tissues in the ripening fruit,
which played a key role in the marked increased in the production of ethylene at the ripening stage
[23]. The mRNA of CMe-ACS
2
was also detected in a ripening melon fruit at a preclimacteric stage
[23]. CMe-ACS
2
was isolated from auxin-treated etiolated seedlings, but the expression patterns and
the stimuli for gene induction were not clarified. The mRNA levels of ACS genes were usually very
low in ripening melon fruit, and the nucleotide sequences of some ACS genes were similar to each
other. It is not easy to detect the mRNA levels of each ACS gene by Northern hybridization. If one
is to understand the expression patterns of ACS genes in plant tissues, RT-PCR analysis could be
employed as a useful tool to identify low levels of mRNA in the tissues.
In this experiment, two cDNA fragments encod- ing the auxin-responsive ACS gene CMe-ACS
2
and
3
were isolated from etiolated melon seedlings, and their expression patterns in the etio-
lated seedlings and developing melon fruit were studied. A genomic DNA encoding CMe-ACS
2
was isolated and its structure determined.
2. Material and methods
2
.
1
. Plant materials Melon seedlings Cucumis melo L. cv. AMS
were grown for 6 days at 28°C in the dark. The tissues about 0.5 g were incubated in petri dishes
90 mm in diameter with 2 ml of 20 mM phos- phate buffer pH 6.8 containing various chemicals
for 2 h at 28°C. The tissue in the petri dish was placed in a gas-tight box 3 l for treatment with
ethylene or 2,5-norbornadiene NBD. The tissue was frozen by liquid nitrogen, and stored at
− 80°C. Melon plants cv. Takami were grown in
a greenhouse. Fruits were harvested on day 55 after pollination and incubated at 25°C for prede-
termined durations.
2
.
2
. Estimation of ethylene concentration A melon fruit was placed in a gas-tight box 12
l at 25°C for 1 h. The ethylene concentration in the box was measured by gas chromatography
Hitachi 3000 using an alumina column f2.5 mm × 1 m [23].
2
.
3
. Cloning of auxin-responsi6e ACS genes from melon
Total RNA was isolated from auxin-treated 100 mM etiolated melon seedlings and a cDNA
library from the RNA was constructed in pBlue- script SK − using a vector primer [24]. Two
ACC synthase-related gene fragments about 1.1 kbp were obtained by PCR amplification of the
cDNA.
A genomic library of melon was constructed into the lGEM11 vector Promega, Madison,
USA and screened with a
32
P-labeled cDNA probe of pCMe-ACS2 [25]. The inserted DNA in
the lGEM11 vector was subcloned in pBluescript SK − .
2
.
4
. Analysis of RNA RT-PCR analysis was performed to determine
the levels of mRNA of the ACS genes in the tissues. The first strand of cDNA was synthesized
with 10 mg of the total RNA and the Rv primer which was specific for a conserved sequence of the
ACS genes box 7 [1]. A cDNA fragment of ACS mRNA was amplified up to 20 cycles with the Rv
primer and one of the Fw primers Fw1, 2 and 3 specific for each ACS gene, and was detected by
Southern hybridization with a
32
P-labeled probe specific for each ACS mRNA.
Fw primers: Fw1 CMe- GACCATTGCAAGAAGGAACTC
ATTC, ACS
1
GTTGAATGCGATAGAAATCTT Fw2 CMe-
ATCC, ACS
2
CGAAGAGGAGGAAGTTTGGA Fw3 CMe-
AGAGA, ACS
3
Rv primer AARCAAACYCGRAACCAWCC
TGGYTC. To determine the levels of mRNA in the total
RNA fraction, the level of mRNA from the actin gene was measured by RT-PCR analysis with
actin-specific primers a forward primer CAY- ACWGTYCCNATHTAYGARGGNTA
and a
reverse primer CTGATRTCNACRTCRCAYT- TCAT.
2
.
5
. General methods The general methods adopted for the manipula-
tions of DNA and RNA have been described by Sambrook et al. [26].
3. Results
