BLBS102-c12 BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come
240 Part 2: Biotechnology and EnzymologyPG and ExpansinThe influence of the suppression of the expression of two cell-
wall-related proteins in the same transgenic line has been re-
ported (Kalamaki et al. 2003a, Powell et al. 2003). Tomato lines
with simultaneous suppression of the expression of both PG and
the ripening-associated expansin have been produced and their
chemical and physical properties characterized (Kalamaki et al.
2003a, Powell et al. 2003). Expansins are proteins that lack wall
hydrolytic activity and are proposed to act by disrupting the
hydrogen bonding between cellulose microfibrils and the cross-
linking glycan (xyloglucan) matrix (see Fig. 12.1). Expansins
are involved in cell wall disassembly during ripening (Brum-
mell et al. 1999a). Their role in ripening is proposed to be that of
loosening the wall and increasing the accessibility of wall poly-
mers to hydrolytic enzymes (Rose and Bennett 1999). In tomato,
the product of one expansin gene (Exp1) is accumulated exclu-
sively during tomato fruit ripening (Rose et al. 1997, Brummell
et al. 1999b). Juices and pastes prepared from fruit with modified
levels ofExp1exhibit enhanced viscosity attributes (Kalamaki
et al. 2003b).
Double transgenic lines were generated by crossing single
transgenic homozygous lines. Fruit was harvested at the ma-
ture green stage and allowed to ripen at 20◦C, and texture was
determined as the force required to compress the blossom end
of the tomato by 2 mm. Fruit from the double suppressed line
were firmer than controls and single transgenic lines at all ripen-
ing stages. The same results were observed in fruit ripened on
vine. At the red ripe stage, fruit from the double suppressed
line were 20% firmer than controls. The flow properties of juice
prepared from control, single transgenic, and double transgenic
lines at the mature green/breaker, pink, and red ripe stages were
evaluated using a Bostwick consistometer. Average juice vis-
cosity decreased as ripening progressed in all genotypes. Juice
prepared from fruit of the suppressed PG and the double sup-
pressed line at the red ripe stage exhibited higher viscosity than
control (Powell et al. 2003).
In another experiment, an elite processing variety was used
as the wild-type background to suppress the expression of
PG, Exp1 and both PG and Exp1 in the same line (Kalamaki
et al. 2003a). Juice and paste were prepared from control,
single suppressed and double suppressed fruit, and their flow
properties were characterized. In paste diluted to 5 Brix, an
increase in Bostwick consistency of about 18% was observed for
the PG-suppressed and the Exp1-suppressed genotypes. Diluted
paste from the double suppressed genotype exhibited the highest
viscosity; however, this increase in consistency of the double
transgenic line was only by an additional 4% compared to the
single transgenic lines. Analysis of particle size distributions at
5 Brix in juices and pastes of the different genotypes indicated
that suppression of PG or Exp1 results in small increases in the
number of particles below a diameter of 250μm (Fig. 12.5).
Suppression of Exp1 also results in the appearance of some
larger particles, above 1400μm in diameter. The presence of
these larger fragments shows that suppression of Exp1 affects
the way the cells and cell walls rupture during processing.
Biochemical characterization of cell wall polymers did not show133.7176.8234.1
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1660010002000300040005000600070008000Control, pasteSupressed PG/Exp1, pasteFigure 12.5.Particle size determination in juice and paste from
control and suppressed PG/Exp1 fruit. (Based on data presented by
Kalamaki et al. 2003a.)large differences in the amounts of extractable polyuronide or
neutral sugar in sequential cell wall extracts of juices and diluted
concentrates of the various genotypes. Only minor size differ-
ences appear in individual polymer sizes in sequential extracts.
In suppressed PG genotypes, larger pectin sizes were observed
in the CDTA extract. The most dramatic difference was in the
carbonate extracts where the double suppressed-line contained
larger pectin polymers in all paste concentration levels relative
to other genotypes (Kalamaki 2003). Concurrent suppression
of PG and Exp1 in the same transgenic line showed an overall
increase in viscosity compared to control, but only a 4%
additional increase compared with the single transgenic lines.
Particle size distribution in juices and pastes from the double
transgenic line are more poly-disperse (Fig. 12.5), and particles
appear to be more rigid. In this line, an increase in the size of