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

Authors: Beecher, Brian; Skinner, Daniel

Submitted to: Journal of Cereal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2011
Publication Date: 6/1/2011
Citation: Beecher, B.S., Skinner, D.Z. 2011. Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernels. Journal of Cereal Science 53:371-378.

Interpretive Summary: Polyphenol oxidase, or PPO, is a major cause of discoloring in agricultural plant products such as sliced apples, bananas, and wheat dough. In products such as alkaline noodles, PPO activity causes graying and browning of the dough over time. This is considered to be undesirable by consumers, and thus considerable effort is being made to minimize PPO in wheat varieties. In most plants, PPO is produced from many related genes. In a given plant part, some PPO genes are more active (i.e. make a larger contribution and are more influential) than others. Efficiently minimization of PPO levels in wheat products requires an understanding of which PPO genes are involved in the developing kernel. Here we have identified three additional PPO genes in wheat that were not previously known to be produced in wheat kernels. The expression levels these new genes were determined and compared to those of the two previously known wheat PPO genes. We found that the new genes actually appear to account for 72% of the PPO RNA levels in developing wheat kernels, making them major contributors to discoloration of wheat dough products.

Technical Abstract: Polyphenol oxidase (PPO, EC 1.10.31) is a major cause of discoloring in raw dough containing wheat flour. Minimization of PPO activity has proven difficult because bread wheat is genetically complex, composed of the genomes of three grass species. The PPO-A1 and PPO-D1 genes, on chromosomes 2A and 2D, respectively, have been implicated in PPO-mediated discoloration. However, we recently reported that wheat contains multiple paralogous PPO genes. The goal of this study was to identify and quantify expression levels for all PPO genes relevant to wheat grain quality. A novel paralogous gene family distinct from PPO-A1/PPO-D1 family was identified from developing seed cDNA. Full length clones were isolated from genomic DNA of the T. aestivum cultivars ‘Chinese Spring’ and ‘Alpowa’. Three distinct sequences were found in each cultivar. The new sequences were found to be orthologous to one another, and paralogous to the previously described PPO-A1/PPO-D1 group. The new paralogous group was localized to group 2 chromosomes. We propose naming these new genes PPO-A2a, PPO-A2b, PPO-B2a, PPO-B2b, PPO-D2a, and PPO-D2b . Real-time PCR analysis determined that in the wheat cultivar ‘Alpowa’, PPO-A1a, PPO-A2b, PPO-D1b and PPO-D2b were all expressed to substantial levels in developing wheat kernels, while PPO-B2 was not. .Transcript levels varied over the course of grain development, with peak levels observed at 9 to 16 days post-anthesis. On average 90% of PPO transcripts originated from chromosome 2A. These results show that wheat kernel PPO activity is the result of at least two orthologous families of two paralogous genes and that some of these genes are expressed to several-fold greater levels than others. The novel paralogous group 2 genes described here together account for 72% of PPO transcripts in developing wheat kernels of the wheat cultivar ‘Alpowa’.