Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit

Abstract

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.Tomato fruit ripening is characterized by a number of coordinated biochemical and physiological changes, which collectively alter fruit color, flavor, and texture. Biochemical and genetic analysis (6, 7) has indicated that many of the physiological processes associated with ripening are regulated at the level of gene expression, a view firmly supported by recent analysis ofgene expression during tomato fruit ripening (1,8,11,14,17,28).Among the ripening-associated enzymes studied in tomato, the cell wall-degrading enzyme polygalacturonase has been analyzed in the greatest detail (2,4,5,8,9,12,13,(24)(25)(26). The site of action of polygalacturonase is the cell wall, where it hydrolyzes polyuronides, or pectins. The increase in the level of chelator-soluble polyuronides during ripening and their corresponding decrease in molecular size have been well documented and are attributed to the action of polygalacturonase (15,22,23). Polygalacturonase activity isolated from ripe fruit is comprised of three closely related isozymes, PGl, PG2A, and PG2B (4,5,10,(18)(19)(20)

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

“…The marked downshifts in polyuronide M, were apparent in both Hass and Lula, Mexican and Mexican/Guatamalen strains, respectively (Ryall and Pentzer, 1982). The limited downshift in M, of polyuronides from Sunny tomato fruit is consistent with data for other tomato cultivam, including Rutgers (DellaPenna et al, 1990), an inherently softer cultivar than Sunny (Ahrens and Huber, 1990). Similarly, Smith et al (1990) observed an approximately 50% reduction in weight-average M, of polyuronides during ripening of Ailsa Craig tomato fruit.…”

“…Previous investigators have attempted to explain polyuronide solubility changes in tomato fruit relative to levels of extractable PG. Polyuronide solubilization in AIS derived from the rin tomato mutant (which normally contains very low levels of PG) transformed with the PG structural gene led DellaPenna et al (1990) to conclude that the chelator (EDTA) solubility of tomato polyuronides was PG dependent. Smith et al (1990) observed that AIS derived from tomato fruit transformed with the PG antisense gene exhibited no reduction in chelator-soluble polyuronides, indicating a mechanism for solubilization independent of the enzyme.…”

“…Similarly, Smith et al (1990) observed an approximately 50% reduction in weight-average M, of polyuronides during ripening of Ailsa Craig tomato fruit. Moreover, there is reason to suspect that the data reported by DellaPenna et al (1990) and Smith et al (1990) may overestimate the extent of in vivo polyuronide depolymerization occumng during tomato ripening. In both studies, phenol-acetic acid-water was used to inactivate tissue enzymes before polyuronide extraction and M, analyses, a treatment we have found to be ineffective at completely eliminating polyuronide depolymerization in cell-wall/AIS preparations (Huber, 1992).…”

“…Although recent studies with tomato fruit have deemphasized the relationship between polyuronide metabolism, and in particular PG (EC 3.2.1.15)-mediated polyuronide depolymerization, and fruit ripening and softening (Giovannoni et al, 1989;DellaPenna et al, 1990;Smith et al, 1990), whether a similar situation holds true for other fruits containing PG or other polyuronide depolymerases remains to be established.…”

Ripening-Regulated Susceptibility of Tomato Fruit toBotrytis cinereaRequiresNORBut NotRINor Ethylene

“…Tomato fruit ripening includes the progressive and extensive loss of firmness that is largely a result of metabolism of the cell wall polysaccharides (Brummell, 2006;Cantu et al, 2007Cantu et al, , 2008a. The ripeningassociated disassembly of hemicellulosic and pectic cell wall polysaccharides is known to be severely reduced in rin fruit and apparently is diminished in nor and 1-MCP-treated fruit, since they also have only limited softening (Seymour et al, 1987;Giovannoni et al, 1989;DellaPenna et al, 1990;Maclachlan and Brady, 1994). We have previously shown that fruit susceptibility to B. cinerea depends on LePG and LeExp1, two proteins that participate in the disassembly of the cell wall, which are expressed in ripe fruit even though B. cinerea secretes its own polysaccharidehydrolyzing enzymes (van Kan, 2006;Cantu et al, 2008a).…”