Measuring polyphenol oxidase activity in a wheat breeding program

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Abstract
High levels of polyphenol oxidase (PPO) have been associated with discoloration of end-use products of wheat, especially certain noodle types. Two whole-seed methods of measuring PPO, one based on 10 mM tyrosine as substrate and the other on 90 mM catechol, were examined and modified to determine their potential as screening tools in large-scale breeding programs. Thirteen spring wheat and two spring triticale genotypes were used to compare the methods. Both methods could measure PPO on individual seeds. All genotypes displayed large seed-to-seed variation for PPO with both substrates. The mean coefficient of variation for the PPO values of individual seeds within genotypes was 39% with tyrosine and 34% with catechol. Furthermore, the PPO values of individual seeds within genotypes were not normally distributed for most genotypes. Identifying genotypes with incremental improvements in PPO would probably require measurement of 70–100 seeds. Approximately 50% of the catecholase activity was associated with the water extract after soaking seeds for 16 h, while all of the tyrosinase activity was still associated with the seed, suggesting that different enzymes are responsible for oxidizing tyrosine and catechol in wheat. While the 10 mM tyrosine assay was nondestructive and allowed plants to be generated from seeds low in PPO, 90 mM catechol reduced germination to less than 20%. Reducing the catechol to 30 mM improved germination to 85%, did not substrate-limit the reaction, and reduced the health risk associated with the assay. Spectral and kinetic differences between the assays were also considered. Key words: Triticum sp., wheat, polyphenol oxidase, catechol, tyrosine

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