at concentrations as low as 400 ng/ml, was developed and used to follow the distribution of this lectin in the plant during its life cycle. The lectin was first detected ...
Plant Physiol. (1978) 61, 847-850
Development and Distribution of Dolichos biflorus Lectin as Measured by Radioimmunoassay' Received for publication June 28, 1977 and in revised form December 19, 1977
CRAIG F. TALBOT AND MARILYNN E. ETZLER Department of Biochemistry and Biophysics, University of California, Davis, California 95616 ABSTRACT A radioimmunoassay, capable of detecting the Dolichos biflorus lectin at concentrations as low as 400 ng/ml, was developed and used to follow the distribution of this lectin in the plant during its life cycle. The lectin was first detected in the seeds of the plant 27 days after flowering and rapidly attained the high level of lectin present in the mature seed. The lectin content of the plant is highest in the seeds and cotyledons and decreases as the storage materials of the cotyledons decrease. A low but measurable amount of material that reacts with antibodies to the seed lectin was detected in the leaves, stems, and pods of the plant. This material gives a precipitin band of only partial identity to the seed lectin when tested in immunodiffusion against antiserum to the seed lectin. No lectin was detected by the radioimmunoassay in the roots of the plant at any stage of development.
Many plants contain agglutinins, called lectins, that can combine specifically with particular carbohydrate residues (16, 17). Although these lectins have become useful tools for the characterization of complex carbohydrates in solution and on cell surfaces, very little is known about their natural role in the plants. Over the last 20 years, a variety of functions have been postulated for the lectins, including such roles as storage proteins, transport of sugars, protective agents, growth regulators, and specific attractants for Rhizobia (1, 4-6, 11, 13-15, 21, 25, 29); however, at present there is not sufficient evidence to confirm any of these roles as the true physiological function of the lectins. Such a confirmation must await further information on the distribution and development of the lectins during the life cycle of the plant. The lectins are major components of the seeds of a number of plants, particularly leguminous plants, where they have been found to constitute as high as 9% of the total N of the seed extract (10, 16). Although there have been reports that lectins are confined primarily to the seeds of plants, several investigators have detected lectins in other parts of the plant by testing the abilities of plant extracts to agglutinate erythrocytes (11, 13, 19, 29) and to precipitate with antibodies to the lectin in immunodiffusion assays (13, 20, 24, 25). In the present study we report the development of a sensitive radioimmunoassay for the Dolichos biflorus lectin and its use in following the development and distribution of this lectin throughout the life cycle of the plant. The specificity and structure of the D. biflorus lectin have been extensively characterized (8-10), and the present study was conducted in an attempt to gain some insight into the role of this lectin in the plant. 'This research was supported by Grant GM 21882 from the United States Public Health Service to M. E. E. 847
MATERIALS AND METHODS D. biflorus seeds were obtained from F. W. Schumacher, Sandwich, Massachusetts. ['4C]Formaldehyde (40-60 mCi/mmol) was from New England Nuclear. Polyclar AT was purchased from GAF Corp., New York. The D. biflorus lectin was isolated from seed extracts as previously described (10) by adsorption onto insoluble polyleucyl hog blood group A+H substance and specific elution from this immunoadsorbent with 10 mM N-acetyl-D-galactosamine. After removal of the hapten by chromatography on Bio-Gel P- 10, the lectin was concentrated in a Diaflo ultrafiltration apparatus and stored in PBS.2 Lectin concentration was determined either by A at 280 nm (E,1?1 = 1.38) or by N determination. The lectin contains 15% N. The isolated lectin was labeled with ['4Cjformaldehyde by the reductive alkylation method of Rice and Means (23). Different preparations of labeled lectin had specific radioactivities ranging from 2.8 to 7.9 x 108 cpm/,umol; the labeled lectin was identical to native lectin in its ability to react in quantitative precipitin analyses with antiserum to the lectin. Antiserum to the lectin was produced in a rabbit by injecting it subcutaneously over the lymph nodes in the shoulder area with a mixture of 1 mg of lectin with Freund's complete adjuvant. Three weekly injections were made, and the rabbit was bled I week after the final injection. Prior to the first injection preimmunization serum was obtained from the rabbit. The sera were preserved by adding merthiolate and phenol to final concentrations of 0.01 and 0.25%, respectively. Immunodiffusion was performed by the Ouchterlony (22) method in 1% ionagar in PBS. Nitrogen was determined by a modified ninhydrin method (26). Quantitative precipitin assays were done by a microprecipitin technique in a final volume of 250 ,l. The tubes were incubated at 37 C for 1 hr, stored at 4 C for I to 4 days, and then centrifuged 15 min at 9,500g. Nitrogen determinations were done on the washed precipitates. In the case of the radioimmunoassays, 200 Al of supernatant was mixed with 0.8 ml of H20 and combined with 10 ml of a Triton scintillation cocktail and counted 10 min in a Packard Tri-Carb scintillation counter. D. biflorus seeds were planted in UC Mix 1, Fertilizer II(c) (2) and allowed to germinate and grow in a greenhouse. Under these conditions no nodulation of the roots was detected. RESULTS Radioimmunoassay. The radioimmunoassay developed for measuring D. biflorus lectin is a competitive assay based on the ability of the lectin to inhibit the precipitation of D. biflorus 14Clectin by antibodies to the lectin. The concentrations of 14C-lectin
2Abbreviation: PBS: 0.01 M phosphate buffer (pH 7.2) containing 0.15 M NaCl and 0.02% NaN3.
TALBOT AND ETZLER
848
and antiserum used in the radioimmunoassay were selected from the antigen excess side of the equivalence zone of a quantitative precipitin curve and gave a bound to free ratio of ' C-lectin of approximately 4.5. A standard curve was obtained for the radioimmunoassay by combining various amounts of unlabeled D. biflorus lectin with a standard amount of "4C-lectin in 0.15 M NaCl and adding the antiserum. The curve is quite reproducible and fairly linear in the region from 0 to 60% inhibition (Fig. 1). Most concentrations of lectin assayed were adjusted so that they were in the range between 15 and 60% inhibition. Several standards containing known amounts of lectin were run in every set of radioimmunoassays. Under the conditions used, 150 ,l was the maximum volume of sample that could be assayed; the radioimmunoassay could thus detect lectin concentrations of as little as 400 ng/ml. Lectin Distribution i Gerninating Seeds and Plants. During the first 4.5 days of germination, four germinating seeds were removed at successive 12-hr intervals and dissected into cotyledons, seed coats, and where applicable hypocotyls, leaves, and roots. The samples were homogenized in ice-cold PBS with a Potter Elvehjem homogenizer using a l-ml volume for the cotyledons and 0.2- to 0.3-ml volumes for the other tissues. The homogenates were centrifuged at 27,000g for 10 min, and the supernatants were assayed for lectin by radioimmunoassay and for N by the ninhydrin method (26). Under these conditions, only the cotyledons contained levels of lectin that could be detected by the radioimmunoassay. The amount of lectin relative to N content of the cotyledon extracts remained constant up to 96 hr (Fig. 2), thus indicating that during the absorption of the cotyledons, the amount of lectin diminishes at about the same rate as the other cotyledon reserves. As shown below, by day 7 the total amount of lectin in the entire plant is less than 1% of the original amount of lectin in the cotyledons; the loss of lectin from the cotyledons must therefore be due to degradation of lectin and not to transport of lectin to other parts of the plant. Lectin Distrbution during Development of Plant. After 7 days, the leaves, stems, and roots of the growing plants were assayed at weekly intervals until the plants flowered and produced mature seeds. In order to reduce the possible interference of plant phenolic compounds with the assays, the plant tissues were treated as described by Loomis (18). The tissue was frozen in liquid N2, ground to a powder, and mixed with an equal amount (w/w) of purified Polyclar AT and a 5-fold (w/v) excess of 0.1 M Kphosphate buffer (pH 7.2) containing 0.15 M isoascorbic acid and 2 mm thioglycolate. The frozen plant powder was thawed in this mixture, filtered through cheesecloth, and centrifuged at 27,000g
Plant Physiol. Vol. 61, 1978
for 15 min. The supernatant was dialyzed against PBS, concentrated by ultrafiltration on a PM-10 membrane, and assayed for lectin and N. Low levels of lectin were detected in the stems and leaves of the plants throughout the life cycle (Fig. 3); at all times the stems contained more lectin/,tg of N than the leaves. The amount of lectin in the stems and leaves began to increase gradually about 9 to 10 weeks after germination of the plant; this increase coincided with the time at which buds appeared on the plants. The amount of lectin in stems and leaves continued to increase until the 13th and 16th weeks, respectively, when the levels reached approximately twice the amount of lectin/pg of N that had been present in these tissues up through the 8th week (Fig. 3). No lectin was detected in the roots of these plants throughout the entire study. In an effort to determine the relationship of the leaf and stem lectin to the seed lectin, the extracts of these tissues were tested in immunodiffusion against antiserum to the seed lectin. Both the leaf and stem extracts gave a precipitin band of only partial identity to the seed lectin (Fig. 4). Lectin Content of Developng Pods and Seeds. Under the greenhouse conditions used in this study, the D. biflorus plants developed buds 9 to 10 weeks after germination, and the flowers opened I to 2 weeks later. The flowers remained open for less than 24 hr, thus allowing the determination of an accurate reference point for pod and seed development. 800700-
-1000
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800
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400-
-600
300-
-400
c
Z 0
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>O 0
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t z
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-200 100
5 3 4 2 DAYS AFTER GERMINATION FIG. 2. Lectin content of cotyledons during germination of seeds. At 12-hr intervals from the time of planting, cotyledons were homogenized and assayed for N and lectin. (0): ng of lectin/ug of N; (U): total amount of N in cotyledon. Each point is the average of 12 assays. 0
6
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Z
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9I.g LECTIN FIG. 1. Standard curve for radioimmunoassay of D. biflorus lectin. Antiserum (1.5 1A) to the lectin was added to D. biflorus '4C-lectin (0.68 ,ug) in the presence of various amounts of unlabeled lectin; the final volume was 250 ,ul. After I hr at 37 C, the solutions were stored at 4 C for I to 4 days, centrifuged, and the supernatants were counted as described in the text.
FIG. 3. Lectin content of stems and leaves of D.
biflorus plant during
life cycle. (B): ng of lectin/,ug of N of stems; (0): ng of lectin/Ag of N of leaves. Each point is the average of three or four separate preparations using tissue from three or four plants/preparation. The points at 17 and 18 weeks are each from one preparation. Buds appeared on the plants during the 9th to 10th week and flowering occured in the I th to 12th week. Mature seeds were formed by the 16th to 18th week.
Plant Physiol. Vol. 61, 1978
DISTRIBUTION OF DOLICHOS BIFLORUS LECTIN
FIG. 4. Immunodiffusion of stem extract and seed lectin against antiserum to the seed lectin. Well 1: rabbit antiserum to purified seed lectin, well 2: purified seed lectin; well 3: extract of stems from 6-week-old plants. The stem extract shows a band of partial identity with the seed lectin. Similar results (not shown) are obtained with leaf extracts. 1200z
z
1000. 800.
t600400-
2006
18 12 24 30 36 DAYS AFTER FLOWERING
42
FIG. 5. Lectin content of developing seeds of D. biflorus plant. At various times after flowering, the two to six seeds in a pod were pooled,
homogenized, and assayed for lectin and N. Each point is the average of data from seeds of two to six pods except for days 29, 35, 36, and 37 when the seeds from only one pod were assayed.
Lectin was not detected in the seeds during the first 26 days after flowering, but appeared abruptly in large quantities at day 27 and reached a maximum level by day 28 (Fig. 5). The rapid increase in lectin during the 27th day is equivalent to the accumulation of approximately 210 jig of lectin/seed. Only very small amounts of lectin (less than 1% of the seed lectin) were detected in the pods during this same period of development.
DISCUSSION The seeds of the D. biflorus plant contain a lectin that can selectively agglutinate blood group A erythrocytes (3) and precipitate blood group A substance (7) due to its specificity for terminal nonreducing a-N-acetyl-D-galactosamine residues (10). The radioimmunoassay for this lectin developed in this study provides a sensitive way of quantitating this lectin independent of methods based on its activity. Using this radioimmunoassay, we determined that the lectin accounts for approximately 10%,o of the total N of mature seed extracts. This value agrees closely with the 9% value obtained in a previous study in which the lectin was estimated by its ability to precipitate with blood group A substance (10). Using this sensitive radioimmunoassay, no lectin was detected in the developing seeds until 27 days after the plant flowered at which time the amount of lectin increased rapidly to the level
849
found in mature seeds. Although no data are presently available on the rate of protein synthesis in D. biflorus seeds, the rapid increase in lectin during the 27th day of development falls well within the range of values reported for rate of protein increase in soybean seeds (12). Regardless of the compatibility of rate of lectin increase with possible rate of protein accumulation, the possibility that the rapid increase in lectin may be due to a conversion of a precursor rather than de novo protein synthesis cannot be excluded. The lectin in the seeds is confined primarily to the cotyledons, and it appears to be degraded at about the same rate as the other reserve substances as the cotyledons diminish in size. Similar observations have been reported for the lectins from Lens culinaris and Pisum sativum (24, 25). Low levels of a material that cross-reacts with antibodies against the seed lectin were found in the stems and leaves of the plant at all stages of its development. This cross-reactive material in these tissues as measured by the present radioimmunoassay never accounted for more than 0.08% of the total N of these tissues. These amounts are based on the assumption that the material in stems and leaves is identical to the seed lectin and can thus react with antibodies to all of the determinants of the seed lectin. The immunodiffusion data presented above show that this assumption is not correct and that the cross-reactive material from stems and leaves gives a precipitin band of only partial identity to the seed lectin. Mialonier et al. (20) have made a similar observation in extracts of leaves from Phaseolus vulgaris. Thus, the values reported for the cross-reactive material in the leaves and stems must be considered as representing the minimal amount of this substance present in these tissues. In a recent study (27, 28) we have isolated and characterized the cross-reactive material from the leaves and stems of the D. biflorus plant and found that it contains one subunit electrophoretically identical to subunit I of the seed lectin and a second subunit which is larger than the seed lectin subunits but identical at the NH2-terminal end. Unlike the seed lectin, the isolated crossreactive material does not agglutinate blood type A erythrocytes or bind to insoluble blood group A substance. These findings raise the possibility that the cross-reactive material may be a precursor to the lectin and in combination with the data presented above, establish a framework for further studies on the development of
the lectin in D. biflorus plants.
Acknowledgment-The authors are grateful to J. E. DeVay for his advice and help in providing greenhouse facilities for this study. LITERATURE CITED 1. ALBERSHEIM P, AJ ANDERSON 1971 Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proc Nat Acad Sci USA 68: 1815-1819 2. BAKER KF, ed 1957 The UC System for Producing Healthy Container-grown Plants. Univ Calif Exp Station Ext Service Manual 28, 332 pages. Univ Calif, Div Agric Serv, Ag Exp Stat-Exp Serv 3. BIRD GWG 1952 Relationship of the blood sub-groups Al, A2 and A,B, A2B to haemagglutinins present in the seeds of Dolichos bflorus. Nature 170: 674 4. BoHnLOOL BB, EL SCHMIDT 1974 Lectins: a possible basis for specificity in the rhizobiumlegume root nodule symbiosis. Science 185: 269-271 5. BOHLOOL BB, EL SCHMIDT 1976 Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment or lectin binding. J Bacteriol 125: 1188-1194 6. BOYD WC 1%3 The lectins: their present status. Vox Sang 8: 1-32 7. BOYD WC, E SHAPLEIGH 1954 Specific precipitating activity of plant agglutinins (lectins). Science 119: 419 8. CARTER WG, ME ETZLER 1975 Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin. Biochemistry 14: 5118-5122 9. CARTER WG, ME ETZLER 1975 Isolation, characterization, and subunit structures of multiple forms of Dolichos bflorus lectin. J Biol Chem 250: 2756-2762 10. ETZLER ME, EA KABAT 1970 Purification and characterization of a lectin (plant hemagglutinin) with blood group A specificity from Dolichos biflonus. Biochemistry 9: 869-877 11. HAMBLIN J, SP KENT 1973 Possible role of phytohaemagglutinin in Phaseolus vulgaris L. Nature New Biol 245: 28-30 12. HILL JE, RW BREIDENBACH 1974 Proteins of soybean seeds. I. Accumulation of the major protein components during seed development and maturation. Plant Physiol 53: 747-751 13. HOWARD IK, HJ SAGE, CB HORTON 1972 Studies on the appearance and location of hemagglutinins from a common lentil during the life cycle of the plant. Arch Biochem Biophys 149: 323-326
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14. JANZEN DH, HB JuISTER. IE LIENER 1976 Insecticidal action of the phytohemagglutinin in black beans on a bruchid beetle. Science 192: 795-796 IS. KAtISS H, C GLASER 1974 Carbohydrate-binding proteins from plant cell walls and their possible involvement in extension growth. FEBS Lett 45: 304-307 16. LIENER IE 1976 Phytohemagglutinins (phytolectins). Annu Rev Plant Physiol 27: 291-319 17. Lts H, N ShARON 1977 In M Sela, ed, The Antigens, Vol 4, pp 429-529. Academic Press, New York 18. LooMis WD 1969 Removal of phenolic compounds during the isolation of plant enzymes. Methods Enzymol, 13: 555-563 19. MAKELA 0 1957 Studies in hemagglutinins of leguminosae seeds. Ann Med Exp Biol Fenn 35: Suppi 11, 1-133 20. MIALONIER G, J-P PRIVAT, M MONSiuNy, G KAHILEM, R DtJRANI) 1973 Isolement, proprietes physico-chimiques et localisation in vivo d'une phytohemagglutine (lectine) de Phaseolus vulgaris L. (var. rouge). Physiol Veg II: 519-537 21. MIRELMAN D, E GALUN, N SHIARON, R LOTAN 1975 Inhibition of fungal growth by wheat germ agglutinin. Nature 256: 414-416 22. 01iuhiTERLONY, 0 1948 In vitro method for testing the toxin-producing capability of diphtheria
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bacteria. Acta Pathol Microbiol Scand 25: 186-191 23. RICE RH, G MEANS 1971 Radioactive labeling of protein in vilro. J Biol Chem 246: 831-832 24. Rou(;E P 1974 Etude de la phytohemagglutinene des graines de Lentille au cours de la germination et des premiers stades du developpement de la plante. Evolution dans les cotyledons. CR Acad Sci Paris 278: 449-452 25. RouC;E P 1976 Biosynthese des hemagglutinines au cours de la maturation des graines de Pois. CR Acad Sci Paris 282: 621-623 26. SU-IfFFMAN G, EA KABAT, W ThioMPSON 1964 Immunochemical studies on blood groups XXX. Cleavage of A, B, and H blood-group substances by alkali. Biochemistry 3: 113-120 27. TALBOT CF, ME ETZLER 1977 Isolation and characterization of inactive form of lectin from stems and leaves of Dolichos biflorus. Fed Proc 36: 795 28. TALBOT CF, ME ETZLER 1978 Isolation and characterization of a protein from leaves and stems of Dolichos biflorus that cross reacts with antibodies to the seed lectin. Biochemistry. In press 29. ToMs GC, A WESTERN 1971 In J Harborne, D Boulter, BL Turner, eds, Chemotaxonomy of the Leguminosae. Academic Press, New York
