Project Number: 5348-43440-004-03
Project Type: Trust

Start Date: Aug 15, 2002
End Date: Aug 14, 2005

Objective:
Our long-term goal remains the same: to genetically reduce or eliminate PPO and other discoloration systems in wheat, thereby greatly enhancing the consumer appeal of wheat foods such as Asian noodles and steam breads. The resultant genetically-superior wheat varieties will enhance sustainable, profitable wheat production by making U.S. wheat more valuable and competitive. Food processors will also benefit. Component 1. Measure the alkaline and white salted noodle color brightness of a genetically-defined set of contrasting wheat varieties over multiple locations and years; use these data (and the wheat materials themselves) to resolve biochemical differences among lines. Component 2. Evaluate association of PPO enzyme characteristics with results of noodle discoloration and response to different exogenous phenolic substrates using different contrasting wheat genotypes. Component 3. Purify at least one major protein showing PPO activity, confirm its identity via peptide sequencing, and possibly generate monoclonal antibodies as a future PPO detection tool (time permitting).

Approach:
Component 1. Measure the alkaline and white salted noodle color brightness of a genetically-defined set of contrasting wheat varieties over multiple locations and years; use these data (and the wheat materials themselves) to resolve biochemical differences among lines. Component 2. Evaluate association of Polyphenol Oxidase (PPO) enzyme characteristics and exogenous phenolic substrates with noodle discoloration. We will evaluate directly the association of noodle discoloration from these samples with the biochemical characteristics, discussed below. In these assays we emphasize L-tyrosine and L-DOPA as substrates. These two substrates are used because of their widespread use in characterizing monophenolase and diphenolase activities (respectively), and because of their wide-spread use in screening germplasm. PPO enzyme will be extracted using standard biochemical protocols and assayed against exogenous phenolic substrates. Enzyme activation will be conducted using SDS and urea. Peroxidase and Laccase activity will be assayed since these are also capable of oxidizing phenols. Activity gels will be stained for PPO activity as the simplest approach to observing possible isoforms in extracts of the 12 wheat lines. When antibodies are available from Component 3, we will use western blotting to determine which bands observed in activity gels (above) correspond to the purified PPO. Component 3. Purify at least one major protein showing PPO activity, confirm its identity via peptide sequencing, and generate monoclonal antibodies as a future PPO detection tool. PPO will be purified sequentially from bran. Purified protein bands showing PPO activity will be sent to the Mayo Clinic's Protein Core Facility for custom tryptic-digest peptide sequence analysis. Final purified PPO protein will also be used to generate mAb in mice. WSU RSO BL-1, 01/01/03. Documents Trust with CSREES. Log 21276. Formerly 5348-43440-003-10T (10/04).

Project Team

Morris, Craig

Project Annual Reports

FY 2003

Related National Programs

Plant, Microbial & Insect Genetic Res., Genomics, & Genetic Improv. I (301)   Quality and Utilization of Agricultural Products (306)