Cell-wall Metabolism and Softening during Ripening - Fruit Ripening: Physiology, Signalling and Genomics - page 52

gene transcript for a protein increases
during ripening suggests it plays a role in
ripening, but the true function of the
protein is not always obvious. To ascertain
a function for the cell-wall proteins,
researchers have relied on natural occur-
ring variants (mutants) of a particular fruit
or artifi cially created mutants made by
genetic transformation.

Flavr Savr (Kramer and Redenbaugh,
1994), and Zeneca Seeds in the UK
commercialized their product as a tomato
purée paste (Khachatourians, 2002). Both
groups reported improved shelf-life for the
fruit (Sheehy et al. , 1988; Schuch et al. ,
1991). The fi rmness, as measured by
compressibility, of the freshly picked fruit
was not much different from the non-
transgenic fruit; however, after several days
of storage, the transgenic fruit were
signifi cantly fi rmer (Kramer et al. , 1992;
Brummell and Labavitch, 1997). The fruit
were also more resistant to some common
fruit pathogens, which improved storage
quality. Both groups measured a signifi cant
increase in the viscosity of the processed
fruit, e.g. purées (Schuch et al. , 1991;
Kramer et al. , 1992). A later study of the
Flavr Savr fruit confi rmed and extended
the earlier work by quantifying the
depolymerization of polyuronides in the
transgenic and non-transgenic (wild-type)
fruit (Brummell and Labavitch, 1997).
Depolymerization of polyuronides, as
measured by size fractionation of trans -1,2-
cyclohexanediaminetetraacetic acid (CDTA,
a divalent cation chelator)-extractable cell-
wall carbohydrate, changed markedly
during ripening in both PG-suppressed and
wild-type fruit (Brummell and Labavitch,
1997). However, they observed a small
difference in depolymerization of poly-
uronides in PG-suppressed fruit compared
with wild-type fruit, which correlated with
a small shift in fruit fi rmness (Brummell
and Labavitch, 1997). At least two reasons
can be envisaged for only a slight
difference in depolymerization of poly-
uronides at harvest when the polyuronides
were clearly fragmented in both lines. It is
possible that PG2A, the abundant tran-
script inhibited in these plants, is not
responsible for fragmentation of poly-
uronides during ripening or that there are
additional enzymes that can depolymerize
pectins, such as pectate lyases. Giovannoni
et al. (1989) did an interesting experiment
that sheds light on this problem. They
transformed a ripening inhibited mutant
of tomato, rin , with a construct (gene)
that could express active PG2A at a

4.3.1 Pectin

Let us look at a few examples where the
expression of a protein has been altered to
see how the loss or overexpression of the
protein changed fi rmness, texture and cell-
wall degradation products. PGs are of
interest because they are expressed
abundantly in many fruits during the
softening phase (Crookes and Grierson,
1983). PG can degrade both the middle
lamella between cells and the pectin
matrix within the primary cell wall
(Crookes and Grierson, 1983). Also
important is the fact that it is the pectin
matrix that determines the porosity of the
cell wall and accessibility of enzymes and
proteins to the cellulose microfi brils and
hemicelluloses embedded in it (Carpita
and Gibeaut, 1993). Thus, it seems reason-
able to expect that the pectin fraction must
fi rst be modifi ed in order to provide
protein access to the hemicellulose and
cellulose components of the cell wall.
Tomato is an important model for genetic
manipulation because protocols for genetic
transformation of tomato were established
fairly early and, unlike tree fruits like
apple, pear and avocado, tomato plants
bear fruit within a month or two of
germination. Two separate research groups
used antisense constructs in tomato to
suppress accumulation of PG transcripts,
one in the USA (Sheehy et al. , 1988) and
another in the UK (Schuch et al. , 1991).
These early transformation events are of
particular interest because these transgenic
plants were the fi rst genetically modifi ed
plant products to be commercialized.
Calgene in the USA commercialized the
transgenic fresh fruit using the trade name