Ethylene Biosynthesis - Fruit Ripening: Physiology, Signalling and Genomics - page 189

the key to understanding the regulation of
ethylene production during plant growth
and development. In bacteria, however,
there is clear evidence that ethylene
biosynthesis occurs via the intermediate
2-oxo-4-methylthiobutyric acid and not
ACC (Primrose, 1979).
Frequently, the ethylene literature states
that ACS is the regulatory step in ethylene
biosynthesis. This is partly true, but
transcriptional regulation of ACO is also an
important control point for ethylene
synthesis, as explained below. The
demonstration that ACO1 mRNA appears
rapidly after wounding indicates that early
conclusions that ACO is always con-
stitutive, and that control is only exerted at
the level of ACS, are not entirely correct
(discussed below). In addition, there are
multiple ACO and ACS genes controlled by
multiple transcription factors (TFs). These
gene families can generate multiple
isoforms of ACO and ACS and some have
different half-lives and regulation. In
principle, the pathway could be controlled
at the level of transcription, translation or
enzyme modifi cation/protein degradation
of either ACS or ACO. In fact, both
enzymatic steps are transcriptionally
regulated, and ACS activity is also con-
trolled post-translationally. It was neces-
sary to purify and study the enzymes,
clone and sequence their genes, and
investigate mutants with altered ethylene
evolution in order to reveal and under-
stand these complex control mechanisms.

and provided probes for gene identifi cation
(Sato et al. , 1991; Van der Straeten et al. ,
1992). Cloning of the genes showed that
ACS is a member of a multigene family,
related to the aminotransferase class of
proteins, which form dimers in vivo . In
Arabidopsis , ACS genes encode a range of
active homo- and heterodimer isoforms of
ACS with different kinetic properties
(Yamagami et al. , 2003; Tsuchisaka and
Theologis, 2004). These isoforms can have
different affi nities for pyridoxal phosphate
and show structural and regulatory
differences. It is believed that this range of
ACS forms can function in different
cellular environments and substrate con-
centrations. The crystal structure of ACS
from apple (Capitani et al. , 1999) showed
that the amino acids in the active site are
identical to those of chicken mitochondrial
aminotransferase. ACS binds the pyridoxal
phosphate cofactor at a critical lysine
residue (Lys278 in tomato ACS), which is
conserved in other ACS isoforms (Yip et
al. , 1990).
There was a widespread belief that the
fi nal step in the biosynthesis of ethylene
by EFE required membrane integrity (Yang
and Hoffman 1984; see also John, 1991)
because, when attempts were made to
solubilize the activity, it disappeared.
Yang had studied the activity in tissue
slices and used analogues to study the
stereospecifi city of the reaction (Hoffman
et al. , 1982), but there was a lack of
appreciation of the requirements of EFE
(now called ACO). The problem was
resolved when the author's group, after a
systematic search, identifi ed a cDNA clone
from a tomato fruit ripening cDNA library,
TOM13, encoding a mRNA that was
expressed both in ripening fruit and
rapidly in wounded leaves, situations
where large amounts of ethylene are
produced (Smith et al. , 1986; Holdsworth
et al. , 1987) (Figs 10.3 and 10.4). Later, it
was shown that this mRNA is also
expressed in senescing leaves (Davies and
Grierson, 1989) where again there is a
requirement for ethylene synthesis.
Sequencing of the mRNA showed that it
appeared to encode a soluble protein, and

10.2 Identifi cation of Genes for ACO
and ACS

ACS is a pyridoxal phosphate-requiring
enzyme. It was fi rst studied in homo-
genates of tissue that synthesized large
amounts of ethylene and was purifi ed by
several groups using conventional protein
purifi cation procedures (Bleecker et al. ,
1986; Nakajima and Imaseki, 1986;
Nakajima et al. , 1988). An antibody raised
against ACS was used to identify the
mRNA translation product, which enabled
the amino acid sequence to be predicted