Agriculture Reference
In-Depth Information
6
Control
5
4
3
2
TOM13 antisense
No. of antisense genes
1
1
2
0
0
2
4 6
Time (days from start of colour change)
8
10
Fig. 10.5. Reduction in ethylene synthesis by inhibiting TOM13 ( ACO1 ) expression in ripening tomato, by
expressing antisense genes in transgenic plants. The control value for ethylene production (compare with
Fig. 10.3) is reduced by over 85% with one antisense TOM13 ( ACO1 ) gene (heterozygote) and over 95%
with two antisense genes (homozygote). Redrawn from Hamilton et al. (1990).
related cDNA in Xenopus oocytes (Spanu
et al. , 1991). The similarity between the
TOM13 ( ACO ) predicted amino acid
sequence (Holdsworth et al. , 1987) and
fl avanone-3-hydroxylase suggested that the
enzyme would require, among other things,
anaerobic extraction (Ververidis and John,
1991) and Fe(II) and ascorbate (see
Hamilton et al. , 1991). Armed with this
information, it was relatively easy to purify
soluble EFE (ACO) and study its properties
in detail.
ACO is a member of the non-haem iron
oxygenase/oxidase superfamily of enzymes
that utilizes Fe(II) and ascorbate rather
than oxoglutarate as cofactors (Schofi eld
and Zhang, 1999). It requires O, and also
CO 2 (from bicarbonate), which activates the
enzyme (Zhang et al. , 2004). Without CO 2 ,
ACO is rapidly inactivated. Early work by
Holdsworth et al. (1988) identifi ed at least
three genes in tomato, and it is now known
that there are six related tomato sequences,
although the catalytic activity of only three
has been confi rmed in vitro (Bidonde et al. ,
1998). From the crystal structure of Petunia
ACO, it has been deduced that it forms a
complex with Fe(II), coordinated by
His177, Asp179 and His234 (Zhang et al. ,
2004). The oxidation of ACC occurs
concomitantly with the reduction of O 2 ,
presumed to be by ascorbate, to generate
CO 2 , cyanide and two molecules of H 2 O
(Bassan et al. , 2006).
The importance of ethylene in con-
trolling fruit ripening and leaf senescence
was demonstrated by directly inhibiting
the expression of tomato ACO1 and ACS2
in transgenic tomato plants (Hamilton et
al. , 1990; Oeller et al. , 1991; Picton et al. ,
1993; John et al. , 1995). Lack of ethylene
synthesis in these plants and fruit led to
partial or complete inhibition of ripening
and could be reversed by supplying
ethylene externally (Oeller et al. , 1991).
Other attempts were also made to inhibit
ethylene in transgenic plants. The DNAP
company developed a transgenic variety
(called Endless Summer) in which a trun-
cated ACS gene caused silencing of the
endogenous gene, thus limiting ACC, and
therefore ethylene, production during fruit
ripening. Monsanto produced a transgenic
tomato expressing a bacterial ACC deamin-
ase gene that reduced the amount of ACC
available for ethylene synthesis, whereas
Agritope used an S -adenosylmethionine
hydrolase from bacteriophage T3 to reduce
the level of a precursor to ACC. None of
these ventures has been commercially
successful so far, partly for patent reasons,
partly due to consumer resistance and also
 
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