Plant Science 159 2000 173 – 181
Cloning of an auxin-responsive 1-aminocyclopropane-1-carboxylate synthase gene CMe-ACS
2
from melon and the expression of ACS genes in etiolated melon seedlings and melon fruits
Yasushi Ishiki
a
, Akiko Oda
a
, Yuka Yaegashi
a
, Yoshikazu Orihara
a
, Tomoe Arai
a
, Tetsuo Hirabayashi
c
, Hiroki Nakagawa
a
, Takahide Sato
a,b,
a
Faculty of Horticulture, Chiba Uni6ersity,
648
Matsudo, Chiba
271
, Japan
b
Graduate School of Science and Technology, Chiba Uni6ersity,
1
Yayoi-cyo, Inage, Chiba
263
, Japan
c
The Japan Horticultural Producti6ity Institute,
207
Shinbashidai, Kamishiki, Matsudo, Chiba
271
, Japan Received 7 February 2000; received in revised form 18 May 2000; accepted 18 May 2000
Abstract
Two cDNA fragments pCMe-ACS2 and 3 encoding auxin-responsive 1-aminocyclopropane-1-carboxylate synthase ACS; EC.4.4.1.14 have been isolated from melon, and the expression patterns of the genes in etiolated melon seedlings and melon fruit
have been determined by RT-PCR analysis. The deduced amino acid sequences of pCMe-ACS2 and 3 were homologous to those of AT-ACS
6
and
4
, which were auxin-responsive ACS genes of Arabidopsis. Both CMe-ACS
2
and
3
were auxin-responsive ACS genes and their expressions in roots and hypocotyls were induced by treatment with indole acetic acid IAA, 100 mM. The mRNA
level of CMe-ACS
2
in the fruit increased after pollination. Those of both CMe-ACS
2
and
3
temporarily increased in the mesocarp tissues at the preclimacteric stage from day 3 to day 5 after harvest during ripening, while that of CMe-ACS
3
was lower than that of CMe-ACS
2
. The increase in the mRNA level of CMe-ACS
1
wound- and ripening-induced gene, T. Miki, M. Yamamoto, N. Nakagawa, O. Ogura, H. Mori, H. Imaseki, T. Sato, Nucleotide sequence of a cDNA for 1-aminocyclopropane-1-
carboxylate synthase from melon fruits, Plant Physiol. 107 1995 297 – 298. in the mesocarp tissue was not observed until 5 days after harvest. A genomic DNA encoding CMe-ACS
2
was isolated and its nucleotide sequence was determined. Nucleotide sequences resembling the auxin-responsive elements AuxRE D1 and D4 the TGTCTC element in the GH
3
gene from soybean, and the auxin-responsive domain AuxRD B in PS-IAA
4 5
from pea were found in the 5-flanking region of the CMe-ACS
2
gene. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
ACC synthase; Auxin; Ethylene; Melon Cucumis melo L.; Ripening fruit www.elsevier.comlocateplantsci
1. Introduction
Ethylene is a gaseous plant hormone, and is produced in plants during seed germination, leaf
abscission, organ senescence and fruit ripening, and its production is also greatly increased by stress and
the presence of auxin [1]. 1-Aminocyclopropane-1- carboxylate ACC synthase ACS, S-adeno-
syl-
L
-methionine methylethioadenosine-lyase, EC 4.4.1.14, which catalyzes the conversion of S-
adenosylmethionine to ACC, is a rate-limiting en- zyme in the ethylene production pathway of higher
plants [1,2], and is encoded by a multigene family. The amino acid sequences encoded by all ACS genes
contain seven conserved regions [1]. Using appro- priate primers specific for the conserved sequences,
several ACS genes have been isolated from Ara- bidopsis [3 – 6], mung bean [7 – 12], rice [13], tomato
[14 – 18] and winter squash [19 – 21]. Each member of this gene family is differentially expressed in
The sequences of pCMe-ACS
3
and CMe-ACS
2
reported herein have been deposited in the DNA Data Bank of Japan under the
accession numbers D86241 and D86242, respectively. Corresponding author. Tel.fax: + 81-47-3088863.
E-mail address
:
satomidori.h.chiba-u.ac.jp T. Sato. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 9 8 - 3
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
