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An Improved Whole-Seed Assay for Screening Wheat Germplasm for Polyphenol Oxidase Activity

^a USDA/ARS, Biosciences Research Laboratory, 1605 Albrecht Blvd., P.O. Box 5674, State University Station, Fargo, ND 58105
^b USDA/ARS Western Wheat Quality Laboratory, E-202 Food Sci. & Human Nutrition Facility East, P.O. Box 646394, Washington State University, Pullman, WA 99164-6394

View larger version (27K): [in a new window]   Fig. 1. pH-Dependent auto-oxidation of monophenolic and dihydroxyphenolic substrate solutions across time. Solutions of 2 g L-1 phenol, and 10 mM L-tyrosine, catechol, methyl catechol, and 3,4 dihydroxyphenylalanine (L-DOPA) were prepared in 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 6.5) or 50 mM Tris buffer (pH 7.5 and 8.5). L-tyrosine was prepared at pH 8.5 only. The solutions were allowed to stand at room temperature, and absorbance at 475 nm was measured at the time points indicated. pH levels are indicated as: 6.5 (), 7.5 (——), and 8.5 (—•—). Absorbance values are the average of two replicates conducted on separate days and measured at 475 nm at the time points indicated. Error bars indicate one standard deviation above and one below the mean value. Where no error bars are apparent, they are less than the size of the symbol, on the order of SD <0.1 absorbance unit.

View larger version (15K): [in a new window]   Fig. 2. Change in absorbance across time for a wheat cultivar with high (Penawawa —•—) or low (ID377s, ——) seed polyphenol oxidase levels. The assay used three seeds incubated with 1.5 mL of 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 6.5) containing 10 mM 3,4 dihydroxyphenylalanine (L-DOPA) in 2-mL microcentrifuge tubes with constant rotation. Absorbance values are the average of three replicate assays measured at 475 nm at the time points indicated. Error bars indicate one standard deviation above and one below the mean value. Where no error bars are apparent, they are less than the size of the symbol, on the order of SD <0.008 absorbance units.

View larger version (33K): [in a new window]   Fig. 3. Relative polyphenol oxidase activity of seeds obtained from four wheat cultivars using the six different substrates indicated. The ordinate shows the change in absorbance at 410 nm (phenol and catechol) or 475 nm [L-tyrosine, methyl catechol, 3,4 dihydroxyphenylalanine (L-DOPA), and caffeic acid]. Catechol, methyl catechol, L-DOPA and caffeic acid reactions were conducted at 10 mM substrate in 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) (pH 6.5); phenol at 2 g L-1 and 50 mM MOPS (pH 6.5); and tyrosine at 10 mM in 50 mM Tris (pH 8.5). Absorbance readings are the average of nine replicate standard assays per cultivar (three seeds per assay). Error bars represent one SD above the mean.

View larger version (26K): [in a new window]   Fig. 4. Frequency histogram of the whole-seed polyphenol oxidase 3,4 dihydroxyphenylalanine (L-DOPA) values of 1,953 wheat germplasm grown under a common environment in Aberdeen, ID, and harvested in 1997.

View larger version (16K): [in a new window]   Fig. 5. Relationship between 3,4 dihydroxyphenylalanine (L-DOPA) values of low polyphenol oxidase (PPO) germplasm lines grown under two environments. The set includes 17 of 20 lines representing the best 1%, and 49 lines randomly drawn from the next 9% of the original population of 1953 lines.

View larger version (23K): [in a new window]   Fig. 6. 3,4 Dihydroxyphenylalanine (L-DOPA) values obtained from chromosome substitution lines of Langdon durum and Chinese Spring. Three replicates of three seeds each from each of the substitution lines indicated were incubated for 2 h in 1.5 mL of 10 mM L-DOPA in 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) (pH 6.5). Solid bars are Chinese Spring D-genome for A-genome substitutions, open bars are Chinese Spring D-genome for B-genome substitutions. Error bars represent one SD above the mean.

View larger version (17K): [in a new window]   Fig. 7. 3,4 Dihydroxyphenylalanine (L-DOPA) values of nullisomic/tetrasomic lines in Chinese Spring wheat. Absorbance values were obtained by incubating four replicates of five seeds each of each line for 1 h in 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) (pH 6.5) containing 5 mM L-DOPA. Error bars represent one SD above the mean.