Molecular cloning and characterisation of grape berry polyphenol oxidase

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Abstract
Polyphenol oxidase (PPO) was purified to homogeneity from Sultana grapes yielding a single protein with an apparent molecular mass of 40 kDa as determined by SDS-PAGE. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified 40 kDa grape PPO protein was used to amplify a 1650 bp cDNA clone (GPO1M) by 3′ rapid amplification of cDNA ends (3′-RACE). GPO1M hybridized to a single 2.2 kb transcript from grape berry mRNA indicating the presence of further upstream sequence which was cloned using 5′-RACE PCR. The complete 1990 bp cDNA (GPO1) encodes a 67 kDa protein consisting of a 10.6 kDa chloroplast transit peptide, a 40.5 kDa catalytic unit containing two copper-binding regions and a 16.2 kDa C-terminal extension. Southern analysis suggested the presence of only one PPO gene in grapevine. High levels of gene expression were found in young developing berries, leaves and roots, but there was little expression in mature tissues. Biogenesis of PPO in grapevine tissues, appears to involve synthesis of a 67 kDa precursor protein which is imported into the chloroplast and processed to remove a 10.6 kDa chloroplast transit peptide from the N-terminus and a 16.2 kDa peptide of unknown function from the C-terminus.