Biochemical and genetic characterization of wheat (Triticum spp.) kernel polyphenol oxidases

Genetic variability of polyphenol oxidase (PPO) activity was evaluated in hexaploid, tetraploid, and aneuploid wheat (Triticum spp.). A DNA probe to kernel-specific PPO hybridized to PPO clones from an immature wheat kernel cDNA library but not to clones from a seedling cDNA library. Recombinant protein and antibody were developed from the kernel-specific PPO. PPO transcript and antigenic proteins were most abundant during kernel development and decreased rapidly at physiological maturity. PPO proteins were more abundant in immature kernels, but PPO activity was greater in mature kernels. Wheat kernel antigenic protein bands of ∼58, 60, 62, and 75 kD were identified; immunoprecipitated proteins of ∼60 and ∼62 kD were confirmed to be PPOs by LC-MS/MS. The 2D chromosome was implicated as a major locus of PPO activity in cultivar (cv.) 'Chinese Spring' based on antigenic staining intensity of ∼60-62 kD proteins and kernel PPO activity in aneuploid lines. The ∼58 kD protein was not well-correlated with kernel PPO activity, while the ∼75 kD protein was likely a PPO precursor. Tropolone completely inhibited PPO activity extracted from mature kernels but not from immature kernels. Total PPO activity in mature kernels is thus a function of specific PPO isoforms present, their abundance, and activation levels.