HORTSCIENCE 26(7):910-913. 1991. chromatography (Chicken and Sharom, 1983), to study carbohydrate structure (Chu et al., Detection of Citrus Leaf and Seed 1981) and, when conjugated to ferritin, flu- orescein isothiocyanate, Horseradish perox- Glycoproteins using Biotinylated idase, or biotin derivatives, as histochemical and cytochemical probes (Pena et al., 1981). Each lectin’s characteristic carbohydrate- Lectin Probes binding specificity can distinguish glycopro- Randall P. Niedz1, Michael G. Bausher2, and C. Jack Hearn3 teins separated electrophoretically (Gordon and Pena, 1982). Horticultural Research Laboratory, U.S. Department of Agriculture- The ABC affinity method used is a mod- Agricultural Research Service, 2120 Camden Road, Orlando, FL 32803 ification of an immunohistochemical proce- dure developed by Hsu et al. (1981) for Additional index words. Concanavalin A, SDS-PAGE, electroblotting studying polypeptide hormones. The ABC Abstract. Citrus leaf and seed glycoproteins were detected after sodium dodecyl sulfate method was estimated to be 20 to 40 times polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting onto polyvinyli- more sensitive than the peroxidase-antipe- dene difluoride (PVDF) membrane when probed with biotinylated lectin at 1 µg·ml-1. roxidase (PAP) method. A modified ABC Four lectins representing three carbohydrate-binding groups were used as probes. A method, using biotinylated lectins, was faster preformed avidin-biotin-complex (ABC) was used to detect the glycoprotein-bound and gave better resolution than 125I-labeled lectins and resulted in dark bands and little background staining. Concanavalin A lectins when used to detect electroblotted cell- (ConA) and wheat germ agglutinin (WGA) resulted in the darkest-staining bands. The surface glycoproteins of human skin fibro- four Citrus spp. and one related species studied had unique seed glycoprotein profiles blasts (Gordon and Pena, 1982). It had the when probed with ConA and WGA. This procedure might be useful in clarifying citrus further advantage of being nonradioactive. taxonomy, providing genetic markers, and in physiological studies involving glycopro- Shah and Stegemann (1986), by the same teins. technique, discriminated between two wheat cultivars using wheat germ agglutinin (WGA) Citrus taxonomy is an unresolved, com- and Rhodes, 1976). Barrett believes the fa- and Ulex europaeus agglutinin (UEA). These plex problem. For instance, the number of cultative apomictic reproductive system of two cultivars could not be distinguished by species reported to comprise the genus ranges Citrus is responsible for much of the con- sodium dodecyl sulphate polyacrylamide gel from 162 (Tanaka, 1977) to three (Barrett fusion over what constitutes a true species. electrophoresis (SDS-PAGE) and isoelectric Citrus spp. relationships have been studied focusing. Glycoprotein profiles from four Received for publication 30 Apr. 1990. Mention by numerical taxonomy (Barrett and Rhodes, Citrus spp. and one Citrus relative were ex- of a trademark, warranty, proprietary product, or 1976), isozyme profiles (Torres et al., 1978), amined to determine the usefulness of the vendor does not constitute a guarantee by the U.S. chemical constituents (Rouseff et al., 1987), ABC method in glycoprotein detection in Dept. of Agriculture and does not imply its ap- and Ouchterlony immunoprecipitation pat- Citrus. proval to the exclusion of other products or ven- terns (Morimoto, 1977). Leaf protein extraction. Shoots were in- dors that may also be suitable. We thank Delores This paper presents a sensitive procedure duced on 2-year-old Citrus sinensis L. Os- F. Jordan for her competent assistance and Ran- to detect specific glycoproteins based on the beck cv. Hamlin seedlings under sodium dall C. Smith for preparing the figures. The cost -1 -2 avidin-biotin-complex (ABC) method of Hsu vapor lamps (1200 µmol·s ·m , 16-h pho- of publishing this paper was defrayed in part by toperiod, 25 C). The new, just-elongated flush the payment of page charges. Under postal regu- et al. (1981) by probing electroblotted citrus lations, this paper therefore must be hereby marked leaf and seed proteins with biotinylated lec- of leaves was collected, washed with dis- advertisement solely to indicate this fact. tins. Lectins are carbohydrate-binding pro- tilled water, dried, and the midrib removed. 1Research Geneticist. teins, not immunologically derived and Leaves were frozen in liquid nitrogen and 1Research Plant Physiologist. generally of plant origin. They have been powdered with mortar and pestle. Cold (4C) 2 Research Geneticist. used to isolate glycoproteins by affinity extraction buffer [50 mM Tris·HCl pH 8.4, 910 HORTSCIENCE, VOL. 26(7), JULY 1991 Table 1. Major ConA and WGA-binding gly- or 1.0% crystallized BSA (U.S. Biochemical coproteins in four Citrus spp. and one relative. Corp., Cleveland). Excess blocking agent was removed by washing (3´, 5 min) in HBS. Biotinylated lectins Concanavalin A (Con A), pokeweed agglutinin (PWA), peanut ag- glutinin (PNA), and wheat germ agglutinin (WGA) were obtained from Sigma Chemical Co. (St. Louis). Lectins were dissolved in HBS containing 0.04% sodium azide and stored at – 20C. Blots were then incubated for 60 min in 5 ml HBS containing biotin- Fig. 1. Identification of lectin-binding glycopro- ylated lectin at 1-50 µg·ml. To remove the teins in C. sinensis cv. Hamlin leaf and aced unbound lectin, blots were washed (3´, 5 protein extracts on a 12% SDS-PAGE gel. (A) zSee Fig. 3. min) in HBS. Leaf proteins; (B) seed proteins. Lane 1, WGA; y+, Band present; –, band absent. An avidin-biotinylated alkaline phospha- lane 2, ConA; lane 3, PNA; lane 4, PWA. Blots xSee Fig. 4. tase complex (ABC-AP) was made using the -1 were probed with 1 µg·ml of lectin and gly- Vectastain ABC-alkaline phosphatase kit coproteins detected using the ABC technique of (Vector Laboratories, Burlingame, Calif.) Hsu et al. (1981). Molecular mass markers are and electroblotting. Leaf and seed extracts according to the manufacturer’s instructions. indicated on the left and right. were mixed 1:1 and 1:9, respectively, with To detect biotin-labeled lectins, blots were a solution containing 0.1 M Tris·HCl (pH first incubated in 5 ml ABC-AP solution for kDa 6.8), 2% (v/v) glycerol, 1% (w/v) SDS, 10% 90 min at 37C, washed (3 ´, 5 min) in HBS (v/v) 2-mercaptoethanol and 0.0002% (w/v) to remove unbound ABC-AP, and finally 66 bromophenol blue. The extract then was equilibrated for 15 min before staining in heated for 5 min at 90C and 10 µl was loaded substrate buffer (0.1 M Tris·HCl pH 9.5, 0.1 2+ per lane (»10 µg of protein) for leaf and M NaCl and 50 mM Mg ). Alkaline phos- 42 seed extracts. Electrophoresis was per- phatase was visualized by adding 1.65 mg formed in a Mini-Protean II 7 cm ´ 8 cm nitroblue tetrazolium chloride (NBT) and 0.85 31 ´ 0.75 mm slab cell (Bio-Rad, Richmond, mg 5-bromo-4-chloro-3-indolylphosphate p- Calif.) using the Laemmli buffer system toluidine salt (BCIP) (Bethesda Research (Laemmli, 1970: 0.125 M Tris pH 6.8 stack- Laboratories, Gaithersburg, Md.) to 10 ml 21 ing gel (4% T, 2.67% C), 0.375 M Tris pH substrate buffer. Blots were incubated in 5 8.8 separation gel (8% and 12% T, 2.67% ml of substrate in darkness until bands de- 14 C), and Tris-glycine (0.025 M Tris, 0.192 M veloped. The color reaction was stopped by glycine, 0.1% SDS, pH 8.3) electrode buffer. washing the blots in 20 mM Tris·HCl (pH The proteins were separated by 40 mA con- 7.5), 5 mM ethylene diaminetetraacetate 123 stant current until the dye front reached the (EDTA). Fig. 2. Control blot to determine nonspecific lectin bottom of the gel, i.e., »1 h. Bromophenol In control experiments, the blots were binding and possible endogenous alkaline phos- blue was used as the tracking dye and phos- treated as above but lectins were omitted, or phatase activity. Blot is seed protein extract from phorylase b (97 kDa), BSA (66 kDa), oval- the lectin solution contained 0.5 M of the C sinensis cv. Hamlin run on 12% SDS-PAGE. bumin (42 kDa), carbonic anhydrase (31 inhibiting saccharides for ConA and WGA, Lane 1, ConA; lane 2, ConA incubated with kDa), soybean trypsin inhibitor (21 kDa), a -methyl-D-mannoside and N-acetylglucos- 0.5 M a -methyl-D-mannoside before probing; and lysozyme (14 kDa) were used as molec- amine, respectively. Experiments testing the lane 3, no lectin. Molecular mass markers are ular mass standards. Chemical reagents and four blocking agents were carried out with indicated on the left. standards for SDS-PAGE were purchased ConA only. from Bio-Rad Laboratories. The increased sensitivity of the ABC 150 mM NaCl, 1 mM CaCl2, 1% insoluble After electrophoresis the gels were equil- method is based on the extreme affinity of polyvinylpolypyrrolidone (PVP)] was mixed ibrated for 30 min in transfer buffer: Tris/ the egg white glycoprotein avidin (70,000 with the leaf powder (0.5 ml buffer per 0.1 glycine (14 mM/11 mM), 0.0375% (w/v) SDS, MW) for biotin (dissociation constant g leaf tissue) and stirred for 15 min. This pH 8.0 and 20% (v/v) methanol. Polyvinyl 10-15M). As avidin has four binding sites solution was filtered through cheesecloth and difluoride (PVDF) membrane was prepared for biotin, a “lattice” structure is believed the filtrate centrifuged for 20 min at by a 20-sec soak in methanol, three 5-min to be formed between avidin and biotin-la- 20,000´ g. Protein concentration was de- washes in distilled water, and equilibration beled enzyme with avidin acting as a bridge termined by the Bradford dye-binding method for 15 min in transfer buffer. Proteins were between the biotin-labeled enzyme mole- (Smith, 1988) using bovine serum albumin electroblotted on PVDF (Immobilin-P, Mil- cules (Hsu et al., 1981).