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

portant for consumer acceptance (Moss, 1971; Miskelly, 1984, 1996), while low L* values indicate undesirable Polyphenol oxidase (PPO) causes darkening and discoloration of discoloration or darkening of noodles. wheat (Triticum aestivum L.) foods such as noodles. Consequently, a simple, nondestructive, quantitative assay for determining PPO on There is a need for a simple, quantitative assay for one to a few wheat seeds could identify superior germplasm and determining the potential noodle color or discoloration eliminate inferior breeding lines, thus greatly assisting the developcharacteristics of wheat germplasm. A test that is nonment of wheat cultivars with superior noodle color. We sought to (i) destructive to the wheat seed, maintains viability, and examine current PPO whole-seed assays and develop an improved which requires as few as one seed would be particularly assay that would facilitate rapid, efficient evaluation of wheat breeding advantageous for permitting plant propagation from lines and cultivars, be amenable to single seeds, and not adversely individual seeds or lines early in breeding programs. affect seed viability; (ii) use the assay to evaluate a large collection Identifying superior germplasm and eliminating inferior of wheat germplasm with the aim of identifying lines with very low breeding lines would greatly assist the development of PPO levels for crossing; and (iii) gain additional information on the wheat cultivars with superior noodle color. location of PPO gene(s). Phenol, L-tyrosine, catechol, methyl catechol, 3,4 dihydroxyphenyalanine (L-DOPA), and caffeic acid were Darkening and discoloration of noodles, especially evaluated as potential substrates. Kinetic studies indicated that raw alkaline, has been associated primarily with the L-DOPA and catechol at pH 6.5 produced the greatest enzyme activactivity of PPO (Kruger et al., 1992, 1994a,b; Baik et ity. L-DOPA did not reduce seed viability, whereas catechol is reportal., 1994, 1995; Crosbie et al., 1996; Miskelly, 1996). The edly toxic to seeds. A standard assay [1.5 mL of 10 mM L-DOPA in presence of PPO has been studied in wheat grain since 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer, pH at least 1907, when tyrosinase was detected in bran (Ber6.5, with 3 to 5 seeds constantly rotated in a 2-mL microcentrifuge trand and Muttermilch, 1907, cited in Lamkin et al., tube for 0.5 or 1 h at room temperature] was used to screen 1953 1981). PPO activity is usually greatest in immature germplasm accessions grown in a common environment. Lines with seeds, and may result from as many as 12 isozymes low levels of PPO (i.e., 10% of the population) were identified; 24 (Taneja et al., 1974; Kruger, 1976). In higher plants, PPO of 66 lines displayed low PPO when evaluated under a second environment. Lastly, chromosome 2D was identified as a location of PPO is a copper-containing metalloprotein that catalyzes the gene(s) based on ‘Langdon’ durum/‘Chinese Spring’ D-genome substihydroxylation of o-monophenols to o-diphenols (E.C. tution lines, and homoeologous group 2 nullisomic/tetrasomic stocks 1.14.18.1; monophenol monooxygenase, tyrosinase, or of Chinese Spring. The L-DOPA standard assay described here procresolase) and the oxidation of o-dihydroxyphenols to vides a robust and efficient method of evaluating germplasm and cultio-quinones (E.C. 1.10.3.2; diphenol oxygen oxidoreducvars for PPO. tase, diphenol oxidase, or catecholase) (see review by Steffens et al., 1994). Because of their electrophillic nature, quinones undergo secondary reactions, such as autoT countries of eastern Asia are major wheat oxidation and polymerization with amino acid groups of users, with as much as 50% of the flour consumed cellular proteins. These secondary reactions of quinones in the form of noodles (Miskelly and Gore, 1991). Two are likely responsible for the brown and black pigmentaof the leading types of noodles in these markets are tion associated with reduced noodle quality. termed white salted and yellow alkaline. Color is a key In addition to the current interest in the relationship quality trait for both of these types of noodles, and can between PPO and Asian noodle color, the reaction of vary dramatically among different wheat cultivars and whole wheat seeds with phenol (Csala, 1972; Fraser and grain lots (Morris et al., 2000). Consequently, any means Gfeller, 1936; Joshi and Banerjee, 1969; Maguire et al., of predicting noodle color from grain is highly desirable. 1975; Walls, 1965; Wrigley, 1976), tyrosine (Mahoney Noodle color is conveniently and routinely measured and Ramsay, 1992), and catechol (Milner and Gould, using the Commission Internationale de l’Eclairage 1951) has been used to characterize cultivars and test 1976 international color measurement system (Wyzecki grain lots for purity. Polyphenol oxidase also plays a and Stiles, 1982) triaxial color space, L*, a*, b*, where role in darkening and discoloration of flat breads L* is the black–white, a* is the red–green, and b* is the (Y. Abrol and co-workers, see Tikoo et al., 1973; Singh yellow–blue axes. Higher L* values, described as greater and Sheoran, 1972; and Faridi, 1988), pan bread (McCalwhiteness or brightness, are considered especially imlum and Walker, 1990), and steamed breads (Dexter et al., 1984). Phenol and tyrosine were used to study the James V. Anderson, USDA/ARS, Biosciences Research Laboratory, genetics of PPO in diploid, tetraploid, hexaploid and 1605 Albrecht Blvd., P.O. Box 5674, State University Station, Fargo, synthetic hexaploid wheats (Bhowal et al., 1969; Fraser ND 58105; Craig F. Morris, 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. Abbreviations: AU, absorbance units; GRIN, Germplasm Resources Received 9/28/2000. *Corresponding author ([email protected]). Information Network; MOPS, 3-(N-morpholino) propane sulfonic acid; L-DOPA, 3,4 dihydroxyphenyalanine; PPO, polyphenol oxidase. Published in Crop Sci. 41:1697–1705 (2001).