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
3
.
1
. Cloning of two cDNA fragments of ACS genes from etiolated melon seedlings and
expression of the genes in the seedlings Two cDNA fragments about 1.1 kbp from a
cDNA library of indole acetic acid IAA-treated etiolated seedlings were isolated by PCR amplifi-
cation and their nucleotide sequences determined. The deduced amino acid sequences of both cDNA
fragments contained seven conserved regions found in other ACS isoenzymes from various
plant species data not shown [1], indicating that the genes isolated from melon were CMe-ACS
2
and
3
. RT-PCR analyses were performed to study the expression patterns of CMe-ACS
2
and
3
in etiolated melon seedlings. The seedlings were cut
into six pieces, and the mRNA levels of the ACS genes in each piece were determined Fig. 1. The
mRNA levels of CMe-ACS
1
,
2
and
3
were very low in the tissues not subjected to IAA treatment.
CMe-ACS
2
was strongly expressed in the roots and lower parts of hypocotyls following treatment
with IAA 100 mM for 2 h. The mRNA levels of CMe-ACS
3
were also markedly increased in the hypocotyls and roots following IAA treatment
100 mM. The expressions of CMe-ACS
2
and 3 were low in the cotyledons even following the
treatment of the tissues with IAA 100 mM. Ex- pressions of CMe-ACS
1
could not be detected in etiolated seedlings under the above conditions. A
dose-response experiment of IAA showed that treatment with a low concentration of IAA 1 mM
was sufficient to increase the mRNA levels of CMe-ACS
2
and
3
in the hypocotyls Fig. 2. The expression of CMe-ACS
2
was more sensitive to low levels of IAA than that of CMe-ACS
3
. The effects of plant hormones on the mRNA levels of
the ACS genes in etiolated hypocotyls are shown in Fig. 3. Treatment with naphthalene acetic acid
NAA, 100 mM and 2,4-dichlorophenoxy acetic acid 2.4-D, 100 mM increased the mRNA levels
of CMe-ACS
2
and
3
in etiolated hypocotyls.
Fig. 1. Expression of ACS genes in the etiolated melon seedlings following treatment with indole acetic acid IAA. Melon seedlings were grown for 6 days at 28°C in the dark. Seedlings were cut into six pieces as shown in panel B. The tissue pieces were
incubated in 20 mM phosphate buffer pH 6.8 containing IAA 100 mM for 2 h. To determine the mRNA levels of the ACS genes in the tissues, RT-PCR analysis was performed as described in Section 2. Panel A: lane 1, cotyledon; lane 2, upper
hypocotyl; lane 3, middle hypocotyl; lane 4, lower hypocotyl; lane 5, upper root; lane 6, lower root.
Fig. 2. Effect of indole acetic acid IAA concentration on the mRNA levels of the ACS genes in etiolated hypocotyls. The
hypocotyl tissues were incubated in media with various con- centrations of IAA for 2 h. Lane 1, no IAA; lane 2, IAA 0.1
m
M; lane 3, IAA 0.5 mM; lane 4, IAA 1 mM; lane 5, IAA 10 mM; lane 6, IAA 50 mM.
4A. The mRNA level of CMe-ACS
2
was markedly increased in melon fruit at 3 days after
pollination Fig. 4A. The expression of CMe- ACS
3
was only slightly increased after pollination. Ethylene production in the ripening melon fruit
was increased, and peaked on day 7 after harvest Fig. 4B. The mRNA levels of CMe-ACS
1
,
2
and
3
were very low in the mesocarp tissues at harvest. Those of CMe-ACS
2
and
3
were temporally in- creased in the mesocarp tissues on approximately
the 3rd and 5th days after harvest, although the mRNA level of CMe-ACS
3
was lower than that of CMe-ACS
2
. The increase in the mRNA level of CMe-ACS
1
was noted first in mesocarp tissues at day 5 after harvest Fig. 4B.
3
.
3
. Characterization of a genomic DNA encoding CMe-ACS
2
A genomic DNA encoding CMe-ACS
2
was iso- lated and sequenced accession number D86242.
Southern blot analysis showed that each restric- tion digestion fragment of CMe-ACS
2
by EcoR I, Bgl II, BstX I and Hind III gave a single band,
and the sizes of the fragments were in good agree- ment with those calculated from the nucleotide
sequence Fig. 5. CMe-ACS
2
consisted of five exons and four introns. The number and size of
the exons and the location of introns were identi- cal to those in CMe-ACS
1
of melon [25] and CP-ACS
1
A
1
B of zucchini [27]. The deduced amino acid sequence of CMe-ACS
2
consisted of 490 amino acids 54.09 kDa. The homology of
the deduced amino acid sequence of CMe-ACS
2
to those of other ACS genes is shown in Table 1. The amino acid sequence of CMe-ACS
2
was ho- mologous to those of VR-ACS
1
of mung bean 71 similarity[7], PS-ACS
2
of pea 71 similar- ity [28,29], LE-ACS
6
of tomato 71 similarity [15] and AT-ACS
6
of Arabidopsis 69 similarity [4]. A putative TATA box tatatatat and a poly
A-signal aataa and aattaa were found at 5- and 3-flanking regions, respectively. Nucleotide se-
quences resembling
auxin-responsive elements
AuxREs were found in the 5-flanking region of the CMe-ACS
2
gene Fig. 6.
4. Discussion
