Aberrant Processing of Polyphenol Oxidase

Bruce's Sport is a mutant grapevine (Vitis vinifera L.) with green and white variegated fruit derived from the Sultana variety. The white regions of tissue have decreased polyphenol oxidase (PPO) activity resulting in a reduced capacity for browning. Active PPO from Sultana grapes was purified and had an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots indicated that mature Sultana grapes contained a single 40-kilodalton PPO, and young Sultana berries also had small quantities of a 60-kilodalton protein. Bruce's Sport grapes had much less of the 40-kilodalton PPO and greater amounts of the 60-kilodalton band. Protease digestion of Bruce's Sport extracts decreased the proportion of the 60-kilodalton protein and increased the 40-kilodalton band. A cDNA clone of grape PPO was used to probe a northern blot of Sultana and Bruce's Sport RNA and hybridized to a 2.2-kilobase transcript in both grapevines. The level of PPO mRNA was high in the early stages of berry development but then declined. The results suggest that in grapevine the active 40-kilodalton form of PPO is synthesized as a precursor protein of at least 60 kilodaltons, and normal processing is interrupted in Bruce's Sport resulting in the accumulation of the 60-kilodalton inactive preform of PPO. ppO2 (EC 1.10.3.1.), also known as catechol oxidase, is a copper-containing enzyme catalyzing the oxidation of o-diphenols to o-diquinones. These quinones polymerize to form the familiar brown pigments associated with browning in plants. The physiological function of PPO is as yet unknown, although it has been associated with disease resistance (27). Other functions such as mediation of the Mehler reaction or involvement in pseudocyclic phosphorylation (22) have also been suggested. PPO is encoded in the nucleus and thought to be transported to the chloroplast in an inactive form (25). It is located on thylakoid membranes in healthy green leaves (24) and immature green olive fruit (21). In Vicia faba leaves, PPO is colocalized with PSII proteins on the thylakoid membranes (9). The chloroplastic location of this enzyme ensures that it is normally separated from its phenolic substrates within the 'This work was supported by a grant from the Australian Dried Fruits Research Council. 2Abbreviations: PPO, polyphenol oxidase; kb, kilobase pair(s); BTP, Bis Tris Propane; PVDF, polyvinylidene difluoride; CNBr, cyanogen bromide. vacuole and thus browning only occurs when plant cells are damaged. The molecular mass of PPO has not yet been clearly established. Gel filtration of the PPO of Mucuna pruriens determined its molecular mass to be 90 kD (28). The protein was found to be a dimer, denaturation revealing two subunits of 42 kD. Partially denaturing gels stained for PPO activity revealed a 40-kD PPO in Vicia faba, the same size as the denatured purified protein (26). The PPOs purified from olive, sago palm, and spinach also consisted of a single subunit of 40 to 42 kD (2, 14, 23). Western blots probed with a polyclonal antibody raised against purified broad bean PPO indicated that a 43to 45-kD band was present in broad bean, bush bean, lettuce, mung bean, soybean, spinach, and tobacco (8). In vitro translation of leaf mRNA isolated from each of these species produced a protein of approximately 45 kD (4), resulting in the suggestion that in a range of plant tissues PPO is synthesized as a 45-kD protein without a transit sequence. Although PPO has been extensively studied in grapes (Vitis vinifera L.), much of the work has been concerned with browning during juice or wine production (29). The existence of multiple forms of PPO in grapes has been reported by a number of authors. Wolfe (31) noted at least three forms of PPO in 55 grapevine varieties, whereas as many as eight bands staining for PPO activity were described by Wissemann and Lee (30) and Sanchez-Ferrer et al. (19). There is also little consensus in the literature concerning the mol wt of grape PPO, with values ranging from 15,000 in Noble grapes (7) to 85,000 in DeChaunac (10). Few reports, however, have described PPO purified to homogeneity, and most estimates of mol wt have been determined under nondenaturing conditions. Nakamura et al. (13) purified PPO from Koshu grapes and estimated the mol wt of the single PPO to be 39,000 by gel filtration and 41,000 by SDS-PAGE. In Australia, the grape variety Sultana (Syn. Thompson Seedless, Kishmish, Sultanina) is used extensively for the production of dried fruit. Bruce's Sport is a mutant of Sultana, first described by Antcliff and Webster (1). It produces variegated leaves, although this is not constitutively expressed and first appears after one-third of the growing season. The berries, however, are always variegated and have green and white stripes running along the length of the fruit. Bruce's Sport has an inherently low capacity for browning because it lacks PPO activity in the white regions of berry tissue. The green sections, however, have the same activity as berries of Sultana (16). The aim of this work was to determine the basis