Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernels

Authors: Beecher, Brian; Skinner, Daniel

Submitted to: American Association of Cereal Chemists Meetings
Publication Type: Abstract Only
Publication Acceptance Date: 6/14/2010
Publication Date: 10/25/2010
Citation: Beecher, B.S., Skinner, D.Z. 2010. Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernels. American Association of Cereal Chemists Meetings.

Interpretive Summary: Abstract Only -- Summary not required.

Technical Abstract: Polypheol oxidase (PPO, Ec 1.10.31) is a major cause of discoloring in raw dough containing wheat flour. PPO is a ubiquitous enzyme that occurs in the outer layers of wheat kernels. High levels of flour PPO have been associated with dimished end-product color and brightness in a variety of products, particularly Asian noodles. PPO catalyzes the oxidation of endogenous phenolic substrates in flour, resulting in the formation of dark pigmented products that diminish product quality. Mimimization of PPO activity is therefore desirable. This has proven difficult because bread wheat is genetically complex, composed of the genomes of three grass species. The PPOA1 and PPOD1 genes, on chromosones 2A and 2D, respectively, have been implicated in PPO activity. Recently we found that in addition to PPOA1 and PPOD1, wheat contains multiple paralogous PPO genes. The goal of this study was to determine which if any of these genes were expressed in developing wheat kernels. We also wished to quantify expression levels of all the expressed PPO sequences in wheat kernel development. RNA was isolated from developing wheat kernels, reversed transcribed, and amplified using degenerate oligonucleotide primers designed against conserved PPO regions. Four distinct PPO sequences were detected. Full length clones were isolated from cDNA and genomic DNA. An additional related sequence was detected in genomic DNA. Of the five sequences, two were the previously described PPOA1 and PPOD1 genes. The three new sequences were found to be homeologus to one another, and paologous to teh PPOA1/PPOD1 group. We propose nameing these new genes PPOA2, PPOB2, and PPOD2. Real-time PCR analysis determined that PPOA1, PPOA2, PPOD1 and PPOD2 were all expressed in substantial amounts in developing wheat kernels.