ARCELIN SEED PROTEINS reported earlier for SARCI ( 18 ) except the wild bean lines

Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L. , were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and Mrs were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M,s, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-ld and -2) could be distinguished by M,s and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross-reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-It and arcelin-4 also could be distinguished from each other based on Mrs and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean a-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family. In screening 210 wild accessions from the common bean (Phaseolus vulgaris L.) germplasm collection, Schoonhoven and Cardona (22) found several accessions that had natural resistance to two important bruchid pests, the Mexican bean weevil (Zabrotes subfasciatus Boheman.) and the common bean weevil (Acanthoscelides obtectus Say.). It was later shown that most of these resistant wild accessions contained arcelin seed proteins that had never been observed in cultivated beans (19, 21). The wild bean genotypes containing arcelin could be grouped by electrophoretic pattern and genetic analysis into four allelic forms, Arl1, Arl-2, Arl-3, and Arl-4 (19; gene designations, ref. 1), that were codominantly inherited, dominant to the absence of arcelin, and tightly linked to the PHA2 locus ( 1 9). Support provided by the Graduate School and the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by the Plant Cell Research Institute, Inc., Dublin, CA. 2 Abbreviations: PHA, phytohemagglutinin; PHA-E, the erythrocyte-agglutinating subunit of phytohemagglutinin; PHA-L, the leucocyte-agglutinating subunit of phytohemagglutinin; SARC 1 . 4, P. vuilgaris Sanilac-derived lines containing arcelin (18); ddH,O, distilled deionized water; Caps, 3-cyclohexylamino1 -propanesulfonic acid; SSC, 0.15 M NaCl, 15 mm sodium citrate (pH 7.0); IEF, isoelectric focusing; kb, kilobases. 204 Arcelin1 is the most thoroughly characterized of the protein variants. Using artificial seeds reconstituted from bean flour with the addition of purified arcelin protein, arcelin1 was shown to be the component of the wild lines that confers resistance to Z. subfasciatus (18). Arcelin and PHA isolated from an arcelin1-containing line had similar solubility properties, subunit Mrs, deglycosylated Mrs, and amino acid compositions; however, they had very different isoelectric points, and PHA agglutinated native erythrocytes but arcelin agglutinated only Pronase-treated cells (20). These similarities and differences might be explained by the observation that the cDNA nucleotide-derived amino acid sequence of arcelin1 is approximately 60% identical to PHA-E and PHA-L (18). Arcelin has one methionine (PHA-E and PHA-L have no methionine) and additional attached oligosaccharide residues (18, 20) that may affect the chemistry and conformation of the mature protein. Another difference between arcelin1 and PHA concerns their effects on seed-feeding insects. Janzen et al. (11) proposed that phytohemagglutinins are involved in the specificity of host-insect interactions between Phaseolus species and seed-feeding predators. Although phytohemagglutinins from P. vulgaris did not provide protection to P. vulgaris predators, they did provide protection against seed-feeding pests of other Phaseolus species. Osborn et al. (18) showed that arcelin-1 protected seeds against P. vulgaris pests and recently the four arcelin variants were found to be associated with different levels and types of resistance to the Mexican bean weevil or the common bean weevil (3, 8). Characterization of arcelin variants and comparisons to each other as well as to PHA may aid in understanding specific host-pest interactions, thereby enabling plant breeders to plan an effective strategy to control bruchid pests. In this paper, the solubilities and mol wt of the four arcelin variants are reported. The isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences of arcelin1, -2, and -4 also have been reported. These properties are compared among the arcelin variants and to those ofPHA proteins. Lines containing these proteins also are compared for genomic DNA restriction fragment patterns of arcelin and PHA genes. MATERIALS AND METHODS