Progress 10/01/03 to 09/30/04
Outputs
Some wheat varieties produce dark Asian noodle products, which are completely unacceptable to consumers. Polyphenol oxidase (PPO) is implicated as the leading cause of such discoloration. We are developing predictive tests for PPO and noodle discoloration in wheat (Bettge, 2004). We are also characterizing wheat PPO at the molecular and biochemical levels and correlating these data with noodle quality in wheat cultivars that differ in noodle discoloration. We tested the role of PPO in Asian noodles by adding tropolone (PPO inhibitor) to noodles. We observed that tropolone inhibited up to 50% of discoloration in the high PPO line Klasic but did not significantly inhibit discoloration in the low PPO line ID377s. Therefore, PPO activity, noodle discoloration, and tropolone inhibition of discoloration were all positively correlated. PPO was extracted from wheat bran in a MOPS-pefabloc-KCl buffer, pH 6.5. Extracting with the detergent NP-40 (0.2%) activated PPO, but 0.1% SDS
did not activate PPO. Approximately 80% of the total PPO enzyme was insoluble in our extraction procedures. Extracted wheat PPO had negligible activity when tyrosine, a monophenol, was used as substrate. We evaluated several diphenols as potential tyrosine co-substrates. Chlorogenic acid proved the best candidate. PPO was purified and characterized from a high PPO wheat cultivar (Anderson & Morris, 2003A). N-terminal sequence analysis confirmed the purified protein as a PPO. Primers were used to clone three partial genomic wheat PPOs, two of which were distinct. Antibodies were raised against two conserved taPPO1 peptide sequences and were affinity-purified. Using these antibodies, we observed up to 6 bands of varying intensity on Western blots of a partially purified FPLC PPO fraction. This implies the possibility of several isoforms of PPO in wheat kernels. We identified a full length cDNA to a wheat bread clone (BT009357) located at DuPont. We isolated two clones from developing
seed cDNA library of cv. Butte 86 that show strong homology to the DuPont wheat clone and other plant PPOs. One of the Butte 86 clones (AY515506) is nearly full-length. The DuPont clone was also used to probe RNA isolated from developing seeds of two high PPO lines (Penawawa and Klasic) and two low PPO lines (ID377s and Langdon). All four show varying degrees of hybridization but Penawawa and Klasic clearly showed much higher levels of PPO transcript that the other two varieties. PPO transcript levels were greatest during early seed development (0-14 dpa) and declined after that. PPOs similar to the DuPont clone appear to be seed-specific, whereas PPOs similar to the Tigst & Morris (2002) PPO clone appear to be expressed in anthers and ovaries.
Impacts
Some wheat varieties from the Pacific Northwest produce dark Asian noodle products, which are unacceptable to consumers. This problem has hurt U.S. market share in Asia. Polyphenol oxidase (PPO) causes much of this discoloration. We are developing rapid predictive tests for discoloration and characterizing wheat ppO at the biochemical and molecular levels. This research will accelerate testing of germplasm and accelerate development of new wheat varieties with improved Asian noodle color and improved export potential to the Pacific Rim countries.
Publications
- J.V., and C.F. Morris. 2003A. Purification and Analysis of Wheat Grain Polyphenol Oxidase (PPO) Protein. Cereal Chem. 80(2):135-143.
- Anderson, J.V., and C.F. Morris. 2003B. Characterization and Expression of Polyphenol Oxidase in Developing Wheat Grain. Abstr., Am. Soc. Plant Biologists.
- Bettge, A.D. 2004. Collaborative study on L-DOPA, wheat polyphenol oxidase assay (AACC Method 22-85. Cereal Foods World 49(6): 338-342.
- Fuerst, E.P., C.F. Morris, and J.V. Anderson. 2003. Correlation of wheat polyphenol oxidase characteristics and Asian noodle discoloration. Program Book, 2003 American Association of Cereal Chemists Annual Meeting, p.115 (Abstract).
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Progress 10/01/02 to 09/30/03
Outputs
Some wheat varieties produce dark Asian noodle products, which are completely unacceptable to consumers. Understanding the biochemical basis for such discoloration may lead to the development of improved procedures for testing wheat germplasm. Polyphenol oxidase (PPO) is implicated as the leading cause of such discoloration. We are characterizing the genetic and biochemical characteristics of wheat polyphenol oxidases and correlating these data to noodle quality in wheat cultivars that differ in noodle discoloration. We have purified and characterized a wheat kernel PPO (Anderson & Morris, 2003A). This protein migrated at 67 kDa in denaturing SDS-PAGE and at 45 kDa in non-denaturing SDS-PAGE. N-terminal sequence analysis confirmed the purified protein as a PPO. We have cloned three partial genomic wheat PPOs; TaPPO1 and TaPPO2 were were 98% similar (over 1040 bp) (Anderson & Morris, 2003B). However, TaPPO3 showed less than 50% amino acid sequence identity. In studies
comparing the cultivars, 'Klasic' and 'ID377s', noodle color was significantly darker in 'Klasic' than 'ID377s', and PPO enzyme activity was higher in 'Klasic' than 'ID377s', when measured in whole kernels, enzyme extracts, and in insoluble enzyme activity (Fuerst et al., 2003).
Impacts
The results of this research will enhance testing of germplasm and allow development of new varieties with improved noodle color and improved export potential to the Pacific Rim countries.
Publications
- Anderson, J.V., and C.F. Morris. 2003A. Purification and Analysis of Wheat Grain Polyphenol Oxidase (PPO) Protein. Cereal Chem. 80(2):135-143.
- Anderson, J.V., and C.F. Morris. 2003B. Characterization and Expression of Polyphenol Oxidase in Developing Wheat Grain. Abstr., Am. Soc. Plant Biologists.
- Fuerst, E.P., C.F. Morris, and J.V. Anderson. 2003. Correlation of wheat polyphenol oxidase characteristics and Asian noodle discoloration. Program Book, 2003 American Association of Cereal Chemists Annual Meeting, p.115 (Abstract).
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