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Wheat germ agglutinin evidence for a genetic basis of multiple forms
175
Biochimica et Biophysica A cta, 444 ( 176 ) 1 7 5 " 1 8 0 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27973
WHEAT GERM AGGLUTININ EVIDENCE FOR A GENETIC BASIS OF MULTIPLE FORMS
R O B E R T H. R I C E *
Department o f Biochemistry al~ Biophysics, University o f California, Davis, Calif. 95616 (U.S.A.) (Received December 22nd, 1975)
Summary Germ from hexaploid wheat (Triticum aestivum L.) contained three forms of agglutinin separable by ion-exchange column chromatography at pH 3.8, while germ from tetraploid wheat (Triticum turgidum L. {durum group)) contained only two such forms. The different number of forms, not due to protein modification occurring during the purification process, was demonstrable in commercial germ and in bran fractions containing germ from single wheat varieties. This evidence for a genetic basis of lectin multiple forms in wheat indicates the advisability of using genetically identified plant varieties in lectin research.
Introduction Plant-derived hemagglutinating proteins and glycoproteins (lectins) have been isolated as closely related multiple forms from many plant sources [1--3]. In some cases, the forms from a single plant species have exhibited differences in carbohydrate- or celt-binding specificity [4--6], an important consideration for their use in complex carbohydrate and cell surface studies. By analogy to isozymes, multiple lectin forms have been suggested to arise from multiple genes coding for polypeptides differing in primary structure or from protein modification prior to or during purification [7]. Although it appears likely from structural considerations that the multiple forms of some lectins are products of different genes [3,6], a genetic basis for multiplicity has not been demonstrated. Multiple forms of the widely used what germ agglutinin have been detected * P r e s e n t address: D e p a r t m e n t o f B i o l o g y , M a s s a c h u s e t t s Institute of T e c h n o l o g y , C a m b r i d g e , Ma. 02139 (U.S.A.)
176 in commercial germ by several laboratories [8--10]. In one study, these forms had the same molecular weight and dimeric subunit structure, had similar but distinct amino acid compositions, and underwent subunit interchange (except for a minor disulfide-bonded form) upon treatment with denaturants, high salt, or pH extremes [10]. Since bread wheat (Triticurn aestivum L., often called 7" vulgare in lectin literature) has multiple forms of enzymes and steerage proteins attributable to its hexaploid nature [11], comparison of wheat germ agglutinins from hexaploid (aestivum group) and tetraploid (durum group) varieties might allow a more straight-forward demonstration of multiple lectin structural genes than would be possible using the diploid plants from which m a n y other lectins are obtained. Methods Agglutinin was purified from commercial wheat germ or from bran fractions containing germ obtained by small scale grinding of whole wheat in a Brabender Quadramat mill for removal of flour. Germ or bran fractions were defatted in light petroleum (b.p. 38--52°C), air dried, extracted as a 10~ suspension in 0.05 M HC1 [12] (final pH, 2.4--3.2), and centrifuged at low speed (6000 × g for 15 min). The supernatant was adjusted to pH 6 with solid Tris(hydroxymethyl)aminomethane prior to addition of (NH4LSO4 (0.35 g/ml). The precipitated protein was recovered by low speed centrifugation, resuspended in H20 at one-tenth the original volume, and dialyzed overnight against 0.01 M acetic acid. The supernatant obtained from the dialyzed material by low speed centrifugation was adjusted to neutrality with solid Tris(hydroxymethyl)aminomethane and to 0.1 M in NaC1, clarified by low speed centrifugation, and applied to a column of ovomucoid-Sepharose [13]. Typically, an extract from 50 g of commercial germ was applied to a 1.4 × 20 cm column of the affinity resin (cleaned in 8 M urea between uses). The column was washed with 0.15 M NaC1 made 0.01 M in sodium phosphate buffer (pH 7.2) until m280nmA 1 .... of the effluent was less than 0.05. The agglutinin was eluted with 0.1 M acetic acid, dialyzed against 0.1 M NaC1 made 0.1 M in sodium acetate buffer (pH 3.8), and an aliquot (generally 20--60 A280n m units) was applied to a 1 × 47 cm column of sulfopropyl-Sephadex C-50. The column was rinsed with 0.1 M NaC1 in 0.1 M sodium acetate buffer (pH 3.8) and eluted with a linear gradient employing 200 ml each of 0.1 M NaC1 and 0.7 M NaCl in 0.1 M sodium acetate buffer (pH 3.8). The agglutinin peaks were distinguished by salt concentrations at their elution positions as indicated by conductivity measurements. In repurification experiments designed to test protein modification during this extraction process, the starting material consisted of 2 g of commercial germ (equivalent to 2 A280nm units of agglutinin) suspended in 20 ml of 0.05 M HC1 containing 30--37 A2s0nm units of agglutinin purified through the ovomucoid-Sepharose affinity ehromatolttaphy step from commercial germ of a different type. Results
The purified wheat germ agglutinin multiple forms were separated according to charge at pH 3.8 by ion-exchange column chromatography [8] as illustrated
177 in Fig. 1. Panel (a) shows the three forms separable in this way using commercial germ from a mixture of soft white varieties of T. aestivum. These forms, labeled I, lI, and III in order of elution, correspond to those observed in previ-
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Fig. 1. Ion-exchange c o l u m n chromatographic separation of wheat germ agglutinin multiple forms derived from hexaploid and tetraploid wheats. The samples examined were: (a), commercial germ from a mixture of soft white 7'. a e s t i v u m varieties; (b), bran fraction containing germ from T. a c s t i v u m , var. Scout 66; (c), commercial germ from a m i x t u r e of 7'. turglidum varieties, (d), bran fraction containing germ from T. turg i d u m , var. Leeds; (e), multiple agglutinin f o r m s from commercial T. t u r z i d u r n germ after repurification in the presence of commercial 7'. a c s t i v u r n germ; (f), multiple agglutinin forms from commercial "1". aestiuurn germ after repurification in the presence of commercial T. t u r g i d u m germ. Absorbanee, • -~; NaCI concentration, - . . . . . .
178 ous work [10]. Panel (b) shows the three peaks of agglutinin obtained from Scout 66, a hard red winter hexaploid wheat. Though form lIl was obtained in slightly different relative amounts, the chromatographic patterns of agglutinin forms were quite similar in panels (a) and (b), three peaks eluting between about 0.25 M and 0.35 M NaC1. This result supports the suggestion that different wheat varieties may yield different proportions of the agglutinin multiple forms [14], though multiple forms appear characteristic of hexaploid wheat. In contrast to the patterns obtained with hexaploid wheat, samples from tetraploid wheat gave only two peaks of agglutinin activity separable by this chromatographic method. A commercial germ mixture from Triticum turgidum varieties (panel (c)) gave peaks corresponding in elution positions to forms I and III obtained in (a) and (b). The durum variety Leeds gave essentially the same pattern (panel (d)) as shown in (c), where the agglutinin peak labeled l contained about twice the material present in peak III, as judged by optical absorption. Mixing experiments indicated that the difference in number of agglutinin forms exhibited by hexaploid and tetraploid wheats was not due to proteolytic or other modifications occurring during the purification process. Agglutinin derived from commercial durum germ upon repurification in the presence of commercial 7'. aestivum germ (panel (e)) or, similarly, agglutinin derived from T. aestivum germ upon repurification in the presence of durum germ (panel (f)) showed minimal alteration of chromatographic behavior on the ion-exchange column. In both cases, the small changes in elution patterns, such as the low a m o u n t of form I! in panel (e), were attributable to the contribution of agglutinin from the small aliquots of germ added for purposes of repurification. The possibility of lectin modification prior to purification has not been excluded but appears remote. In these experiments bran fractions containing germ, obtained by grinding whole wheat for removal of flour, were found satisfactory for purification of the lectin. Bran fractions from single wheat strains exhibited 5--10% of the agglutinin content of purified commercial germ on a weight basis, roughly in proportion to the germ content, while agglutinating activity in the flour fraction (not detected) was at least an order of magnitude lower in Scout 66. Agglutinin purified through the affinity chromatography step had more residual contaminating material from bran fractions than from purified germ, judging by the ion-exchange chromatograms, though no agglutinating activity (tested as previously described [10]) was detected in neutralized column fractions except in the numbered lectin peaks *
* A p e a k o f m a t e r i a l f r o m S c o u t t 6 6 b r a n f r a c t i o n w a s o b s e r v e d e l u t i n g f r o m t h e i o n - e x c h a n g e colu m n a t 0 . 6 M N a t 1 . T h e m a t e r i a l w a s s u b s t a n t i a l l y h o m o g e n e o u s u p o n s o d i u m d o d e c y l s u l f a t e gel e l e c t r o p h o r e s i s [ 1 5 ] ( w i t h m o b i l i t y c o r r e s p o n d i n g to a b o u t 1 2 0 0 0 d a l t o n s ) a n d u p o n a l u m i n u m lactate gel elcctrophoresis [16] (with mobility about half that of wheat purothionin at pH 3.1). The nature of its interaction with ovomucoid-Sepharose, to which it adhered upon reapplication, a n d its p o s s i b l e c o r r e s p o n d e n c e to t h e a l b u m i n f r a c t i o n o f t h e s a m e m o l e c u l a r w e i g h t o b t a i n e d b y E w a r t [ 9 ] w e r e n o t s t u d i e d f u r t h e r d u e t o its l a c k o f a g g l u t i n a t i n g a c t i v i t y a n d t h e l o w a m o u n t s available.
179 Discussion These results indicate a genetic basis for multiple forms o f wheat germ agglutinin. The presence of three chromatographically separable forms suggests the existence of diverged triplicate genes for the lectin in hexaploid wheat. As found for isozymes of wheat alcohol dehydrogenase [11], the A, B, and D genomes present in bread wheat {derived from three diploid ancestors [17 ] ) may each contribute one form. By this rationale, agglutinin forms I and III purified from tetraploid durum germ could correspond to products of genes on chromosomes of the A or B genomes, while form II corresponds to a product of the D genome which durum lacks. A fourth agglutinin form, obtained from commercial hexaploid germ as a minor c o m p o n e n t of form II by column chromatography at pH 3.8 [10], has not been detected in germ from tetraploid wheat, suggesting it is also a product of the D genome. A more detailed genetic analysis will be necessary, however, to show that the presence of an agglutinin form is due to a single gene and to find its chromosomal location or to identify unambiguously the contributing genome. In addition, it remains to be shown whether diploid wheat genomes of divergent ancestry code for wheat germ agglutinin since the lectin, of uncertain function in vivo, is not known to be required for the survival of the species. In view of recent advances in wheat cytogenetics [17], further genetic analysis of the agglutinin appears feasible and may prove valuable for study of lectin regulation and function in plants. Wheat has provided an advantageous system for the present work due to its polyploid nature. While their applicability to diploid plants from which many other agglutinins are derived is not direct, the results obtained emphasize the general possibility of a genetic basis of lectin multiple forms and indicate the advisability of using genetically identified plant varieties in lectin research. Acknowledgements I thank Dr. M.E. Etzler for encouragement and support of these experiments and for valuable advice; Dr. D.D. Kasarda for supplying Scout 66 bran fraction, performing aluminum lactate gel electrophoresis, and providing helpful suggestions; Dr. C.O. Qualset for valuable discussion and for supplying Leeds durum; and General Mills, Inc., Vallejo, CA for generous donations of commercial wheat germ. This work was supported by United States Public Health Service postdoctoral fellowship CA-01605 from the National Cancer Institute and by United States Public Health Service grant GM-21882 held b y Dr. M.E. Etzler. References 1 2 3 4 5 6 7 8
Lis, H. and Sharon, N. ( 1 9 7 3 ) A n n u . Rev. B i o c h e m . 4 2 , 5 4 1 - - 5 7 4 C a r t e r , W.G. a n d E t z l e r , M.E. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 2 7 5 6 - - 2 7 6 2 W a x d a l , M.J. ( 1 9 7 4 ) B i o c h e m i s t r y 13, 3 6 7 1 - - 3 6 7 7 A l l e n , L.W., S v e n s o n , R . H . a n d Y a c h n i n , S. ( 1 9 6 9 ) P r o c . Natl. A c a d . Sci. U.S. 63, 3 3 4 - - 3 4 1 M a t s u m o t o , I. a n d O s a w a , T, ( 1 9 7 0 ) A r c h . B i o c h e m . B i o p h y s . 1 4 0 , 4 8 4 - - 4 9 1 N i c o l s o n , G . L . , B l a u s t e i n , J. a n d E t z l e r , M.E. ( 1 9 7 4 ) B i o c h e r n s i t r y 1 3 , 1 9 6 - - 2 0 4 S h a r o n , N. a n d Lis, H. ( 1 9 7 2 ) S c i e n c e 1 7 7 , 9 4 7 - - 9 5 9 Allen, A . K . , N e u b e r g e r , A. and Sharon, N. ( 1 9 7 3 ) B i o c h e m . J. 1 3 1 , 1 5 5 - - 1 6 2
180
9 10 ll 12 13 14 15 16 17
E w a r t , J . A . D , ( 1 9 7 5 ) J. Sci. Fd. Agric. 26, 5 - - 2 2 R i c e , R . H . a n d Etzler, M.E. ( 1 9 7 5 ) B i o c h e m i s t r y 1 4 , 4 0 9 3 - - 4 0 9 9 H a r t , G.E. ( 1 9 7 0 ) Proe. Natl. A c a d . Sci. U.S. 66, 1 1 3 6 - - 1 1 4 1 B l o c h , R. a n d B u r g e r , M.M. ( 1 9 7 4 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 58, 1 3 - - 1 9 Marchesi, V.T. ( 1 9 7 2 ) in M e t h o d s in E n z y m o l o g y ( G i n s b e r g , V., ed.), Vol. 28, p p . 3 5 4 - - 3 5 6 , A c a d e m ic Press, N e w Y o r k L o t a n , R., G u s s i n , A.E.S., Lis, H. a n d S h a r o n , N. ( 1 9 7 3 ) B i o c h e m , B i o p h y s . Res. C o m m u n . 52, 656--662 W e b e r , K. a n d O s b o r n , M. ( 1 9 6 9 ) J . Biol. C h e m . 2 4 4 , 4 4 0 6 - - 4 4 1 2 N i m m o , C.C., O ' S u l l i v a n , M.T. a n d B e r n a r d i n , J . E . ( 1 9 6 8 ) Cereal C h e m . 45, 2 8 - - 3 6 Sears, E.R. ( 1 9 7 4 ) in H a n d b o o k o f G e n e t i c s ( K i n g , R . C . , ed.), Vol. 2, pp. 5 9 - - 9 1 , P l e n u m Press, N e w York
Biochimica et Biophysica A cta, 444 ( 176 ) 1 7 5 " 1 8 0 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27973
WHEAT GERM AGGLUTININ EVIDENCE FOR A GENETIC BASIS OF MULTIPLE FORMS
R O B E R T H. R I C E *
Department o f Biochemistry al~ Biophysics, University o f California, Davis, Calif. 95616 (U.S.A.) (Received December 22nd, 1975)
Summary Germ from hexaploid wheat (Triticum aestivum L.) contained three forms of agglutinin separable by ion-exchange column chromatography at pH 3.8, while germ from tetraploid wheat (Triticum turgidum L. {durum group)) contained only two such forms. The different number of forms, not due to protein modification occurring during the purification process, was demonstrable in commercial germ and in bran fractions containing germ from single wheat varieties. This evidence for a genetic basis of lectin multiple forms in wheat indicates the advisability of using genetically identified plant varieties in lectin research.
Introduction Plant-derived hemagglutinating proteins and glycoproteins (lectins) have been isolated as closely related multiple forms from many plant sources [1--3]. In some cases, the forms from a single plant species have exhibited differences in carbohydrate- or celt-binding specificity [4--6], an important consideration for their use in complex carbohydrate and cell surface studies. By analogy to isozymes, multiple lectin forms have been suggested to arise from multiple genes coding for polypeptides differing in primary structure or from protein modification prior to or during purification [7]. Although it appears likely from structural considerations that the multiple forms of some lectins are products of different genes [3,6], a genetic basis for multiplicity has not been demonstrated. Multiple forms of the widely used what germ agglutinin have been detected * P r e s e n t address: D e p a r t m e n t o f B i o l o g y , M a s s a c h u s e t t s Institute of T e c h n o l o g y , C a m b r i d g e , Ma. 02139 (U.S.A.)
176 in commercial germ by several laboratories [8--10]. In one study, these forms had the same molecular weight and dimeric subunit structure, had similar but distinct amino acid compositions, and underwent subunit interchange (except for a minor disulfide-bonded form) upon treatment with denaturants, high salt, or pH extremes [10]. Since bread wheat (Triticurn aestivum L., often called 7" vulgare in lectin literature) has multiple forms of enzymes and steerage proteins attributable to its hexaploid nature [11], comparison of wheat germ agglutinins from hexaploid (aestivum group) and tetraploid (durum group) varieties might allow a more straight-forward demonstration of multiple lectin structural genes than would be possible using the diploid plants from which m a n y other lectins are obtained. Methods Agglutinin was purified from commercial wheat germ or from bran fractions containing germ obtained by small scale grinding of whole wheat in a Brabender Quadramat mill for removal of flour. Germ or bran fractions were defatted in light petroleum (b.p. 38--52°C), air dried, extracted as a 10~ suspension in 0.05 M HC1 [12] (final pH, 2.4--3.2), and centrifuged at low speed (6000 × g for 15 min). The supernatant was adjusted to pH 6 with solid Tris(hydroxymethyl)aminomethane prior to addition of (NH4LSO4 (0.35 g/ml). The precipitated protein was recovered by low speed centrifugation, resuspended in H20 at one-tenth the original volume, and dialyzed overnight against 0.01 M acetic acid. The supernatant obtained from the dialyzed material by low speed centrifugation was adjusted to neutrality with solid Tris(hydroxymethyl)aminomethane and to 0.1 M in NaC1, clarified by low speed centrifugation, and applied to a column of ovomucoid-Sepharose [13]. Typically, an extract from 50 g of commercial germ was applied to a 1.4 × 20 cm column of the affinity resin (cleaned in 8 M urea between uses). The column was washed with 0.15 M NaC1 made 0.01 M in sodium phosphate buffer (pH 7.2) until m280nmA 1 .... of the effluent was less than 0.05. The agglutinin was eluted with 0.1 M acetic acid, dialyzed against 0.1 M NaC1 made 0.1 M in sodium acetate buffer (pH 3.8), and an aliquot (generally 20--60 A280n m units) was applied to a 1 × 47 cm column of sulfopropyl-Sephadex C-50. The column was rinsed with 0.1 M NaC1 in 0.1 M sodium acetate buffer (pH 3.8) and eluted with a linear gradient employing 200 ml each of 0.1 M NaC1 and 0.7 M NaCl in 0.1 M sodium acetate buffer (pH 3.8). The agglutinin peaks were distinguished by salt concentrations at their elution positions as indicated by conductivity measurements. In repurification experiments designed to test protein modification during this extraction process, the starting material consisted of 2 g of commercial germ (equivalent to 2 A280nm units of agglutinin) suspended in 20 ml of 0.05 M HC1 containing 30--37 A2s0nm units of agglutinin purified through the ovomucoid-Sepharose affinity ehromatolttaphy step from commercial germ of a different type. Results
The purified wheat germ agglutinin multiple forms were separated according to charge at pH 3.8 by ion-exchange column chromatography [8] as illustrated
177 in Fig. 1. Panel (a) shows the three forms separable in this way using commercial germ from a mixture of soft white varieties of T. aestivum. These forms, labeled I, lI, and III in order of elution, correspond to those observed in previ-
TI',LL
" 0 4
04 "'~-~ ..... " ~t~ l I TTr (b)
0.2 0.1
]X
0.4. ~
0,4
0.2
0.3 0.2 0.1
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~
0,4 0,3 0.2
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Z,
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i
~rrr
,0.4 0.3 0.2 -0.1
-
0
0.2
40 80 120 Fraction (2 ml)
Fig. 1. Ion-exchange c o l u m n chromatographic separation of wheat germ agglutinin multiple forms derived from hexaploid and tetraploid wheats. The samples examined were: (a), commercial germ from a mixture of soft white 7'. a e s t i v u m varieties; (b), bran fraction containing germ from T. a c s t i v u m , var. Scout 66; (c), commercial germ from a m i x t u r e of 7'. turglidum varieties, (d), bran fraction containing germ from T. turg i d u m , var. Leeds; (e), multiple agglutinin f o r m s from commercial T. t u r z i d u r n germ after repurification in the presence of commercial 7'. a c s t i v u r n germ; (f), multiple agglutinin forms from commercial "1". aestiuurn germ after repurification in the presence of commercial T. t u r g i d u m germ. Absorbanee, • -~; NaCI concentration, - . . . . . .
178 ous work [10]. Panel (b) shows the three peaks of agglutinin obtained from Scout 66, a hard red winter hexaploid wheat. Though form lIl was obtained in slightly different relative amounts, the chromatographic patterns of agglutinin forms were quite similar in panels (a) and (b), three peaks eluting between about 0.25 M and 0.35 M NaC1. This result supports the suggestion that different wheat varieties may yield different proportions of the agglutinin multiple forms [14], though multiple forms appear characteristic of hexaploid wheat. In contrast to the patterns obtained with hexaploid wheat, samples from tetraploid wheat gave only two peaks of agglutinin activity separable by this chromatographic method. A commercial germ mixture from Triticum turgidum varieties (panel (c)) gave peaks corresponding in elution positions to forms I and III obtained in (a) and (b). The durum variety Leeds gave essentially the same pattern (panel (d)) as shown in (c), where the agglutinin peak labeled l contained about twice the material present in peak III, as judged by optical absorption. Mixing experiments indicated that the difference in number of agglutinin forms exhibited by hexaploid and tetraploid wheats was not due to proteolytic or other modifications occurring during the purification process. Agglutinin derived from commercial durum germ upon repurification in the presence of commercial 7'. aestivum germ (panel (e)) or, similarly, agglutinin derived from T. aestivum germ upon repurification in the presence of durum germ (panel (f)) showed minimal alteration of chromatographic behavior on the ion-exchange column. In both cases, the small changes in elution patterns, such as the low a m o u n t of form I! in panel (e), were attributable to the contribution of agglutinin from the small aliquots of germ added for purposes of repurification. The possibility of lectin modification prior to purification has not been excluded but appears remote. In these experiments bran fractions containing germ, obtained by grinding whole wheat for removal of flour, were found satisfactory for purification of the lectin. Bran fractions from single wheat strains exhibited 5--10% of the agglutinin content of purified commercial germ on a weight basis, roughly in proportion to the germ content, while agglutinating activity in the flour fraction (not detected) was at least an order of magnitude lower in Scout 66. Agglutinin purified through the affinity chromatography step had more residual contaminating material from bran fractions than from purified germ, judging by the ion-exchange chromatograms, though no agglutinating activity (tested as previously described [10]) was detected in neutralized column fractions except in the numbered lectin peaks *
* A p e a k o f m a t e r i a l f r o m S c o u t t 6 6 b r a n f r a c t i o n w a s o b s e r v e d e l u t i n g f r o m t h e i o n - e x c h a n g e colu m n a t 0 . 6 M N a t 1 . T h e m a t e r i a l w a s s u b s t a n t i a l l y h o m o g e n e o u s u p o n s o d i u m d o d e c y l s u l f a t e gel e l e c t r o p h o r e s i s [ 1 5 ] ( w i t h m o b i l i t y c o r r e s p o n d i n g to a b o u t 1 2 0 0 0 d a l t o n s ) a n d u p o n a l u m i n u m lactate gel elcctrophoresis [16] (with mobility about half that of wheat purothionin at pH 3.1). The nature of its interaction with ovomucoid-Sepharose, to which it adhered upon reapplication, a n d its p o s s i b l e c o r r e s p o n d e n c e to t h e a l b u m i n f r a c t i o n o f t h e s a m e m o l e c u l a r w e i g h t o b t a i n e d b y E w a r t [ 9 ] w e r e n o t s t u d i e d f u r t h e r d u e t o its l a c k o f a g g l u t i n a t i n g a c t i v i t y a n d t h e l o w a m o u n t s available.
179 Discussion These results indicate a genetic basis for multiple forms o f wheat germ agglutinin. The presence of three chromatographically separable forms suggests the existence of diverged triplicate genes for the lectin in hexaploid wheat. As found for isozymes of wheat alcohol dehydrogenase [11], the A, B, and D genomes present in bread wheat {derived from three diploid ancestors [17 ] ) may each contribute one form. By this rationale, agglutinin forms I and III purified from tetraploid durum germ could correspond to products of genes on chromosomes of the A or B genomes, while form II corresponds to a product of the D genome which durum lacks. A fourth agglutinin form, obtained from commercial hexaploid germ as a minor c o m p o n e n t of form II by column chromatography at pH 3.8 [10], has not been detected in germ from tetraploid wheat, suggesting it is also a product of the D genome. A more detailed genetic analysis will be necessary, however, to show that the presence of an agglutinin form is due to a single gene and to find its chromosomal location or to identify unambiguously the contributing genome. In addition, it remains to be shown whether diploid wheat genomes of divergent ancestry code for wheat germ agglutinin since the lectin, of uncertain function in vivo, is not known to be required for the survival of the species. In view of recent advances in wheat cytogenetics [17], further genetic analysis of the agglutinin appears feasible and may prove valuable for study of lectin regulation and function in plants. Wheat has provided an advantageous system for the present work due to its polyploid nature. While their applicability to diploid plants from which many other agglutinins are derived is not direct, the results obtained emphasize the general possibility of a genetic basis of lectin multiple forms and indicate the advisability of using genetically identified plant varieties in lectin research. Acknowledgements I thank Dr. M.E. Etzler for encouragement and support of these experiments and for valuable advice; Dr. D.D. Kasarda for supplying Scout 66 bran fraction, performing aluminum lactate gel electrophoresis, and providing helpful suggestions; Dr. C.O. Qualset for valuable discussion and for supplying Leeds durum; and General Mills, Inc., Vallejo, CA for generous donations of commercial wheat germ. This work was supported by United States Public Health Service postdoctoral fellowship CA-01605 from the National Cancer Institute and by United States Public Health Service grant GM-21882 held b y Dr. M.E. Etzler. References 1 2 3 4 5 6 7 8
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