D. G Lindsay, CEBAS-CSIC, Murcia
8.2.2 Fruit texture during ripening
Fruit ripening is accompanied by disassembly of several cell wall polymers, including pectin and hemicellulose, which are primarily responsible for ripening-associated changes in fruit texture. Extensive studies on ripening- associated pectin disassembly and the expression of the endo-polygalacturonase (PG) gene family, suggested that tomato fruit texture could be modified by transgenic modification of PG gene expression. The expression of both antisense and sense constructs of the tomato fruit PG catalytic subunit (PG2) gene resulted in greater than 95% reduction in PG activity (Sheehyet al., 1988; Smithet al., 1998; 1990a). Fruit with reduced expression of PG were analyzed for alterations in the expression of other cell wall hydrolases and none were detected (Smithet al., 1990b). Fruit with reduced PG activity provided the basis for testing the significance of this PG during softening and ripening as well as the basis for the commercial introduction of fresh and processed tomato fruit whose texture was modified by this genetic modification. Analysis of cell wall polymers of these fruit demonstrated that diminished PG expression contributed to reduced depolymerization of the chelator-solubilized pectins and increased viscosity of processed tomato products but did not reduce fruit softening (Tayloret al., 1991;
Caringtonet al., 1993; Fenwicket al., 1996; Brummell and Labavitch, 1997;
Porretta and Poli, 1997; Porretta et al., 1998). The effect of antisense suppression of a single fruit PG on fruit softening may be partially offset by expression of other tomato PG genes in ripening fruit (Sitrit and Bennett, 1998).
Interestingly, transgenic plants with reduced expression of fruit PG did not exhibit changes in leaf abscission, suggesting that PGs involved in abscission are distinct from those that participate in fruit ripening (Taylor et al., 1991). The tomato fruit PG gene has also been inactivated by transposition and stabilization of a maize transposon,DS, within the PG gene (Cooley and Yoder, 1998).
Suppression of the non-catalytic subunit of the PG1 isozyme complex in transgenic tomato plants by expression of an antisense gene construct also reduced pectin metabolism during fruit ripening (Watson et al., 1994).
Specifically, the reduced expression of the PG1 subunit, a regulatory subunit, reduced cell wall pectin solubilization and depolymerization, suggesting that the dynamics of pectin associations and structure in the cell wall are determined by several factors, perhaps some acting cooperatively (Watsonet al., 1994).
Several other fruit characteristrics have been measured in tomato fruit with suppressed PG gene expression. Transgenic tomato fruit were evaluated for sensory characteristics and their color and flavor outperformed a similar variety that was heterozygous for therin (ripening inhibited) locus, a variety that had been bred for long shelf life (Sozzi Quiroga and Fraschina, 1997). The tomatine content of transgenic fruit was unaffected by antisense suppression of PG (Furui et al., 1998). Furthermore, PG antisense fruit generally had improved integrity and were less susceptible to cracking and pathogen attack specifically at the over-ripe stage (Krameret al., 1992; Hadfield and Bennett, 1998). However, the susceptibility of PG suppressed transgenic tomato fruit to Colletotrichum gloeosporioideswas not measurably different than in wild-type fruit (Cooperet al., 1998).
Pectin methylesterases
Because methylation of pectins affects their structural properties in the cell wall as well as their susceptibility to pectinases, expression of pectin methylesterases (PMEs) has been altered to modify pectin metabolism of tomato fruit (Tiemanet al., 1992; Tieman and Handa, 1994; Tieman et al., 1995). Suppression of the expression of a single PME in tomato by the transgenic introduction of a truncated sense tomato PME gene resulted in significantly higher molecular weight pectins isolated from the fruit cell walls (Thakuret al., 1996a). Processed tomato products made from the PME suppressed transgenic lines also exhibited increased serum viscosity and reduced serum separation (Thakuret al., 1996b;
Erringtonet al., 1998). Analysis of the PME mRNA in the transgenic plants suggested that the reduction in the endogenous PME mRNA resulted from interference by the transgenic mRNA with post-transcriptional processing of the endogenous PME mRNA (Mishra and Handa, 1998). Transgenic expression of the sequence encoding 71 amino acids of tomato fruit PG linked to the tomato PME sequence under control of the 35S CaMV promoter resulted in suppression of both PG and PE simultaneously (Seymouret al., 1993).
Expansins
Expansins are cell wall proteins that have been proposed to participate in disruption of hydrogen bonding between hemicellulose and cellulose polymers at the surface of the cellulose microfibril. These polymeric associations are particularly important for cell expansion and other developmental events in which cell wall disassembly occurs, such as tissue softening during fruit ripening (Roseet al., 1997). Several expansin genes are expressed during tomato fruit
development and ripening (Brummell et al., 1999b). The over-expression and suppression of one expansin gene, LeExp1, was examined in tomato plants expressing a sense full-length or truncated LeExp1 gene (Brummell et al., 1999c). Transgenic fruit from plants over-expressingLeExp1were significantly less firm at the mature green and breaker stages and analysis of the cell wall material demonstrated that precocious depolymerization of the hemicellulose structure of the wall correlated with constitutive over-expression of LeExp1.
However, expression in tomato of a cucumber hypocotyl expansin,CsExp1, did not result in phenotypic alterations of transgenic tomato fruit, suggesting that divergent expansin proteins may have distinct functions or substrates in vivo (Rochange and McQueen-Mason, 2000). Suppression ofLeExp1 in transgenic tomato resulted in increased fruit firmness, especially at the early (e.g. Breaker) stage of ripening (Brummell et al., 1999c). Surprisingly, in the LeExp1 suppressed fruit, polyuronide depolymerization but not hemicellulose depolymerization was reduced in the later stages of ripening in these fruit.
1, 4 endo-glucanases
Because hemicellulose, and xyloglucans specifically, are disassembled in ripening fruit substantial research has focused on endo--1, 4-glucanases as a class of enzymes because they have the capacity to cleave the -1,4-glucan linkages of xyloglucan. At least two endo--1,4-glucanases are expressed in ripening tomato fruit, and one of them,Cel1, is also expressed in fruit abscission zones (Lashbrook et al., 1994). Analysis of expression patterns demonstrated that the mRNA corresponding to a second endo--1,4-glucanase, Cel2, is more abundant in ripening fruit. However, the expression of both Cel1 and Cel2 during ripening suggested that the two endo--1,4-glucanases could act synergistically on their substrates in the softening cell wall. Transgenic plants in which Cel1 expression was suppressed by an antisense gene construct produced fruit that softened normally, and abscission was partially reduced (Lashbrook et al., 1998). Transgenic plants engineered for suppression of the Cel2gene also exhibited no changes in fruit ripening or softening but also were altered in abscission zones, requiring greater force for the abscission zone breakage (Brummellet al., 1999a).
Galactosidases
A recent report indicated that antisense suppression of galactosidase gene expression in ripening tomato fruit reduced softening by approximately 40%
(Gross and Smith, USDA report). At least four of seven tomato fruit - galactosidases are expressed during ripening (Smith and Gross, 2000), and the release of galactosyl residues is the most dynamic cell wall change during ripening. As breakdown of galactose-containing polymers in the fruit cell wall is abundant during ripening, reduction of galactosidases, perhaps in concert with other cell wall hydrolases and expansin proteins may provide the basis to regulate the softening process of ripening tomato fruit.