Antisense technology has shown that neither polygalacturonase nor pectin methylesterase alone are responsible for tomato fruit softening, leading to the likelihood that other enzymes or factors are important. Our laboratory recently found that α and β-galactosidase from avocado fruit solubilized tomato fruit pectin in vitro. Previously, Pressey (Plant Physiol. 1983,71:132) found that the activity of one of three α-galactosidase isozymes from tomato fruit increased during ripening and was capable of degrading cell wall galactan, suggesting a role for the enzyme in fruit softening. Increased β-galactosidase activity was observed in a number of other fruit during ripening. In the present study, NaCl extraction of tomato pericarp yielded relatively high levels of cc- and β-galactosidase activity. At least two isozymes of each were resolved during Mono-Q HPLC α-Galactosidase was further purified by additional Mono Q and Superose 12 gel filtration HPLC. Gel filtration and SDS-PAGE yielded an apparent molecular weight of 44 kD. The partially pure α-galactosidase had a specific activity of 294 μmol product/min per mg protein, a Km of 317 μm, a pl of 5.0, and a pH optimum of 5.5. Activity was inhibited 67% by α-d-galactose. Preliminary results show that β-galactosidase can also be purified by the same techniques. Following further purification, the isozymes will be sequenced and cloned. A second approach being used in an attempt to identify cDNA clones for the α- and β-galactosidase genes from tomato fruit involves using heterologous cDNA clones from guar (Overbeeke et al., 1989; Plant Molecular Biology 13:541-550) and carnation (Raghothama et al., 1991; Plant Molecular Biology 17:61-71), respectively, to screen a ripening tomato fruit cDNA library. Basic molecularbiological techniques will be used to elucidate the role of these enzymes in tomato fruit ripening.
THE IMPORTANCE OF α AND β-GALACTOSIDASE IN TOMATO FRUIT RIPENING