Polygalacturonase Gene Expression in Ripe Melon Fruit Supports a Role for Polygalacturonase in Ripening-Associated Pectin Disassembly

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.Fruit ripening is a genetically programmed event that is characterized by a number of biochemical and physiological processes that alter fruit color, flavor, aroma, and texture (Brady, 1987). Extensive cell wall modifications occur during ripening and are thought to underlie processes such as fruit softening, tissue deterioration, and pathogen susceptibility. These modifications are regulated at least in part by the expression of genes that encode cell wall-modifying enzymes (Fischer and Bennett, 1991). Pectins are a major class of cell wall polysaccharides that are degraded during ripening, undergoing both solubilization and depolymerization. In tomato the majority of ripening-associated pectin degradation is attributable to the cell wall hydrolase PG. Transgenic tomato plants with altered PG gene expression indicated that PG-dependent pectin degradation is neither required nor sufficient for tomato fruit softening to occur (Sheehy et al., 1988; Smith et al., 1988; Giovannoni et al., 1989). However, data from experiments using fruit of the same transgenic lines strongly suggested that PG-mediated pectin degradation is important in the later, deteriorative stages of ripening and in pathogen susceptibility of tomato fruit (Schuch et al., 1991; Kramer et al., 1992).In melon (Cucumis melo) substantial amounts of pectin depolymerization and solubilization take place during ripening (McCollum et al., 1989; Ranwala et al., 1992; Rose et al., 1998), implicating a role for PG in ripening-associated cell wall disassembly in melons. However, melons have been reported to lack PG enzyme activity (Hobson, 1962; Lester and Dunlap, 1985; McCollum et al., 1989; Ranwala et al., 1992). The possibility exists that PG is present in melon but that it does not conform to the expected enzymic properties in terms of abundance and/or lability, a point illustrated by recent reports in apple and strawberry, which were previously reported to lack PG activity but that do in fact accumulate low amounts of protein and/or measurable activity (Nogata et al., 1993; Wu et al., 1993). In light of the unexplained discrepancy between ripening-associated pectin depolymerization and undetectable PG activity in melons, we have undertaken a study to reexamine the status of PG in melon using the rapidly ripening cv Charentais (C. melo cv Reticulatus F1 Alpha).As reported for other cultivars, Charentais melons exhibit substantial solubilization and a downshift in the molecular-mass profile of water-soluble pectins, but this is associated with the later stages of ripening, after softening is initiated (Rose et al., 1998). By utilizing a molecular approach to analyze PG in melon, we have attempted to overcome some of the potential limitations of biochemical methods, such as low abundance of protein, reliance on other cell wall components, and unknown cofactors for activity and/or lability during extraction. In doing so, we have identified and characterized a multigene family encoding putative PGs from Charentais melon, including three PG homologs that are expressed abundantly during fruit ripening. The pattern of PG gene expression correlates temporally with the depolymerization of water-soluble pectins and an increase in pectin-degrading enzyme activity. Three additional PG homologs were also identified and shown to be expressed in mature anthers and fruit-abscission zones, tissues that, similar to ripening fruit, are undergoing cell separation. The most abundant ripening-associated putative PG mRNA, MPG1, was expressed in the filamentous fungus Aspergillus oryzae. The culture filtrate from the transformed A. oryzae strain XMPG1 exhibited endo-PG activity, further supporting a role for endo-PG in ripening-associated pectin disassembly in Charentais melon fruit.