Evaluation of Glycine Germplasm for Nulls of the Immunodominant Allergen P34/Gly m Bd 30k Leina M. Joseph, Theodore Hymowitz,* Monica A. Schmidt, and Eliot M. Herman ABSTRACT Soybean [Glycine max (L.) Merr.] seed contains an immunodomi- nant human allergen P34 or Gly m Bd 30k (mentioned as P34) of the cysteine protease family. Of approximately 16 266 accessions from USDA soybean germplasm screened, 12 P34 null lines were identified among soybean (G. max), wild annual (Glycine soja Sieb. and Zucc.), and wild perennial Glycine spp. Glycine soja were low P34 expressers, while G. max and wild perennial species had nondetectable levels of the allergenic protein. Further investigation of G. max nulls by 2D- IEF/SDS PAGE showed all primary seed proteins present indicating that the loss of P34 was not due to large scale restructuring of protein content. Southern and northern analysis showed no large insertions or deletions to render the gene nonfunctional. The cDNA of both G. max nulls each showed the same six point mutations indicating the two nulls have a single origin. Of these six single nucleotide changes, four are predicted to result in an amino acid alteration. One such alteration re- sults in a serine being replaced by a cysteine residue. The introduction of a cysteine residue might produce a mismatched disulfide bond forma- tion producing an unstable P34 protein in the null soybean accessions. The isolation and introgression of soybean lines with low allergen levels will provide the basis for developing a low allergen line incorporated with other agronomically desirable traits in a breeding program. S OYBEAN is grown primarily for production of seed, which has a multitude of uses in the food and indus- trial sectors. It represents one of the major sources of edible vegetable oil and protein in the food industry. However, soybeans contain allergenic proteins, making it one of the major food sources that cause allergies to sensitive individuals (Herman, 2004). In the USA and Europe, nearly 5 to 8% of babies and 2% of adults are reported to be allergic to soybeans (Heppel et al., 1987); therefore, millions of infants (babies) must avoid soybean- based formula and baby food (Cantani and Lucenti, 1997). Further, the increased use of soy proteins and their de- rivatives in a wide range of processed foods limits soy- sensitive individuals in their choices of soy free processed foods. Currently, the primary treatment for food allergies is to avoid the food source causing the allergy. Although recently enacted labeling laws will assist in identifying foods with soy content, it will still be difficult to avoid soy protein because of its extensive usage in a wide range of processed foods (Herian et al., 1993; Tsuji et al., 1995). Soybean allergic reaction in humans is primarily atopic (skin) reactions and gastric distress which can range from mild to severe and is rarely life-threatening, although there are some reports of soy-induced anaphylaxis (Foucard and Malmheded, 1999). Soybean possesses about 15 proteins recognized by IgEs from soy-sensitive people (Burks et al., 1989; Ogawa et al., 1991, 1993). The dominant soybean allergens consist of P34 and the a-subunit of conglycinin. P34 is a unique member of the papain superfamily of cysteine proteases lacking the catalytic cysteine residue that is replaced by a glycine (Kalinski et al., 1992; Herman, 2005). It is most strongly and frequently recognized by the IgE antibodies in sera of soy-sensitive patients with atopic dermatitis (Ogawa et al., 1991). In several IgE binding studies, more than 65% of soy-sensitive patients react only to P34 pro- tein (Ogawa et al., 1991, 1993; Helm et al., 1998, 2000), suggesting that the 34-kDa protein is the major allergen in soybean and a target allergen for producing low-allergen content hypoallergenic soybean. Epitope mapping of P34 showed at least 12 distinct epitopes on P34 protein through immunological analysis of its allergenicity (Helm et al., 1998 as reported by Yaklich et al., 1999). P34 is a relatively minor seed constituent comprising less than 1% of total seed protein. The lack of P34 in soybean would eliminate a large portion of the allergenicity of soybean seeds. The function of P34 in soybeans is not known with the exception of its role in syringolide elicitor binding activity in disease resistance (Cheng et al., 1998). The P34 protein consists of 379 amino acids having homology to thiol pro- teases (Kalinski et al., 1990). In soybean seed, P34 is post translationally processed from a 46-kDa precursor protein after seed germination. Seed thiol proteases are synthesized only after seed germination, whereas P34 ac- cumulates during seed maturation. Electron microscope immunochemistry with a monoclonal antibody demon- strated that P34 is localized in the protein storage vacuoles but not in the oil bodies (Kalinski et al., 1992). It is mod- erately abundant in soybean seed and cotyledons but is found in low levels in leaves. Using cosuppression gene silencing technique, a trans- genic soybean was produced in which P34 was eliminated (Herman et al., 2003). These soybean plants had ap- parently identical seed composition, development, struc- ture, and phenotypic characters as that of nontransgenic plants, indicating that P34 protein is not required for seed protein processing or maturation. However, regu- latory difficulties and the lack of acceptance of geneti- cally modified soybeans by the baby food and formula industry makes using such an allergen-suppressed soy- bean difficult at the present time. An alternative approach would be to identify low- allergen content soybeans in the soybean germplasm. An initial attempt to find soybeans lacking P34 by screen- L.M. Joseph and T. Hymowitz, Dep. of Crop Sciences, Univ. of Illi- nois, Turner Hall, 1102 S. Goodwin Ave., Urbana, IL 61801; M.A. Schmidt and E.M. Herman, USDA-ARS, Donald Danforth Plant Science Center,975 N. Warson Rd., St. Louis, MO 63132. Drs. Joseph and Schmidt contributed equally to the work. Received 27 Feb. 2006. *Corresponding author (soyui@uiuc.edu). Published in Crop Sci. 46:1755–1763 (2006). Plant Genetic Resources doi:10.2135/cropsci2005.12-0500 ª Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved. 1755 Published online June 20, 2006