ABSTRACT. Fresh weight and dry weight as well as quantitative and qualitative protein changes in the developing soybean (Glycine max) seed were described ...
Plant Physiol. (1974) 53, 747-751
Proteins of Soybean Seeds II. ACCUMULATION OF THE MAJOR PROTEIN COMPONENTS DURING SEED DEVELOPMEN.T AND MATURATION1 Received for publication October 2, 1973 and in revised form January 10, 1974
J. E. HILL2 AND R. W. BREIDENBACH Department of Agronomy and Range Science, University of California, Davis, California 95616 ABSTRACT Fresh weight and dry weight as well as quantitative and qualitative protein changes in the developing soybean (Glycine max) seed were described from 12 days after flowering until maturity. The seed proteins were separated on sucrose density gradients into three major fractions, having average sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The 2.2S sedimenting proteins predominated at very early stages of development (12 days after flowering) and decreased proportionately throughout maturation. The 7.5S and 11.8S components appeared to be synthesized later in maturity and in larger amounts than the 2.2S proteins. Electrophoretic studies on extracts from whole seeds and on isolated protein fractions confirmed the early abundance of proteins in the 2.2S fraction and revealed temporal differences in the accumulation of three components of the 7.5S fraction. The 11.8S sedimenting fraction appeared throughout seed development as a homogeneous protein which accumulated in the seed with a time course similar to that of the total 7.5S protein fraction.
rates of accumulation of the proteins of maturing soybean have been demonstrated by Kondo et al. (17). The observations of these workers led us to examine further the major protein components of soybean seeds during development and maturation in order to extend the available information on their characterization and accumulation.
MAlERIALS AND METHODS Plant Materials. Seeds of an early maturing soybean, Glycine max var. Portage, obtained from the University of Minnesota seed stocks, were planted in 8-inch clay pots in a sand-peat mix (1:1) containing a full complement of the required nutrients. A nutrient solution (16) was given weekly as a supplemental fertilizer after the third week of growth until flowering. During the period of rapid fruit set (about five weeks from planting), pods 5 to 12 mm long were marked individually, with a tag. Previous correlation of pod length and DAF3 had shown this length is reached 3 to 5 days after the fully expanded flower. Seeds in untagged pods were allowed to develop to maturity. Tagged pods were harvested periodically during seed development, pod and seed weights were obtained immediately, and the seeds of a given age were then pooled, frozen in Dry Ice, and stored at -20 C for further use. Dry weights were determined by placing freshly weighed seeds in a 70 C drying oven, weighing at 24 hr, and reweighing until the weight Large quantities of protein are accumulated over a short was constant. Homogenization and Protein Extraction. Immature seeds period of time during seed development in many legumes (2, 20). These accumulated proteins are few in kind but constitute were cracked and powdered in a Dry Ice-cooled mortar. The a high proportion of the total protein of the mature seed (1). resulting powder was homogenized for 30 sec at full speed in Thus the developing seed has been generally recognized to a VirTis blender in 10 volumes (w/v) of a 0.035 M potassium have potential for studies on the quantitative and qualitative phosphate buffer, pH 7.6, containing 0.4 M NaCl and 0.01 M regulation of protein synthesis (2, 14, 20). Before this potential B3-mercaptoethanol as described by Wolf and Briggs (23). The can be fully realized, however, the temporal pattern of protein brei was filtered through four layers of cheesecloth, and the filtrate centrifuged at 31 ,000g for 20 min. The 31 ,000g accumulation during seed development must be established. The synthesis or accumulation of the seed proteins of several supernatant was used for further studies. A protein balance leguminous species have been studied, including Glycine max sheet was maintained at all stages as a check for recoveries and (3, 17), Pisum sativum (2, 9), Vicia faba (20, 24), and distributions of total protein. Protein was measured by the Phaseolus vulgaris (14). Danielson (9) observed temporal dif- method of Lowry et al. (18). Aliquots of the 31,000g supernaferences in the accumulation of the albumin and globulin tant fractions were precipitated with 5% trichloroacetic acid to fractions of Pisum sativum. The relative amounts of exog- distinguish precipitable high mol wt protein from nonprecipenously supplied amino acids incorporated into these two itable low mol wt Lowry-reactive compounds. Sucrose Density Gradient Centrifugaton. Sedimentation and fractions were recently demonstrated to vary during maturation of these seeds (2). Temporal differences in the relative fractionations of soybean globulins was accomplished as previously described (15). Aliquots of the immature seed supernatants were adjusted to approximately 20 mg of protein per I This study was supported in part by Hatch funds and in part by ml, and 1 ml was layered on the gradients. Peak regions in the E. I. du Pont de Nemours under auspices of a du Pont Young gradient were pooled for further studies. Faculty award to R. W. B. 2 Present address: Department of Environmental Toxicology, 'Abbreviation: DAF: days after flowering. University of California, Davis, Calif. 95616. 747
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Polyacrylamide Discontinuous Gel Electrophoresis. Total protein obtained directly from the 31,000g supernatant preparation and protein fractionated on sucrose density gradients were electrophoresed by the method of Davis (10) as described in the preceding paper (15) except that the protein in some cases was layered over the stacking gel. Scanning and Peak Estimation. The destained gels were scanned on a Gilford linear transport attachment to the Beckman DU monochromator. A computer program using X-Y plots of the gel traces was employed for estimation of the area under overlapping absorption peaks (Criddle and Zaya, personal communication). The protein represented by each peak was calculated as a product of the contribution (%) of its area and of the total protein at that stage in seed development.
RESULTS Figure 1 shows the general pattern of fresh weight and dry weight changes of the maturing seeds. Fresh weight increased rapidly from 12 DAF to about 34 DAF. Thereafter fresh weight declined rapidly as the seeds desiccated. Dry weight (Fig. 1) increased steadily, leveling to a constant value at about 36 DAF. Stages of early embryogenesis prior to the rapid fresh weight increases at 12 DAF were not studied in detail. Figure 1 shows, however, that between 4 and 8 DAF pod weight increased rapidly, suggesting that much of the substrate available for fruit formation is channeled into the pod at this time. The seed proteins were extracted beginning at 12 DAF, and the supernatant of the 31,000g spin was studied. At all stages of maturity, except 12 DAF, at least 90% of the total protein extracted in the crude homogenate was recovered in the supernatant fraction. At 12 DAF only 79% of the extracted protein was recovered in the 31,000g supernatant. This may reflect relatively higher amounts of nonextractable organellar protein in the young cells. Figure 2 shows the changes in protein of the crude homogenates and the 31,000g supernatant fractions as measured by the method of Lowry et al. (18) over the ripening period. The relatively low levels of total protein found at the earliest stages are apparent from Figure 2. In fact, less than 5% of the total protein of the mature seed had been accumulated by 12 DAF. Protein accumulation was very rapid between 12 and 28 DAF and then declined, ceasing at the onset
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Days Affer Flowering FIG. 2. Changes in total protein (-(0-), extractable protein (-*-), precipitable protein (-A-), and soluble Lowry (18) reactive material (-El-), during soybean seed maturation. Extractable protein represents that found in the 3 1,000g supernatant.
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FIG. 3. Protein profiles of whole seed extracts at various stages of seed development prepared on 10 to 30% sucrose density
gradients.
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DAYS AFTER FLOWERING FIG. 1. Fresh weight (-0-), dry weight (-*-) and pod fresh weight (-A&-) accumulation during Portage soybean seed maturation. Each point represents an average value for 30 seed weights. The graph is a composite of several growing periods.
of seed desiccation. Thereafter, actual protein levels decreased slightly. The relative amounts of protein in contrast to low mol wt Lowry-reactive compounds were estimated from precipitation by trichloroacetic acid. The precipitable protein accumulated similarly to total protein (Fig. 2), whereas the amount of low mol wt Lowry-reactive material remained relatively constant during maturation. Figure 3 compares the sucrose density gradient sedimentation profiles at various stages of seed development. At very immature stages in seed development (12 DAF and 17 DAF), much of the protein was widely distributed over the upper portion of the gradient, suggesting that a relatively higher
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PROTEINS OF SOYBEAN SEED.
proportion of the protein was of low to intermediate mol wt. The 1 1.8S component showed a small but definite peak on the gradient as early as 12 DAF. All three major sedimentation fractions found previously in mature seed (15) were readily apparent at 23 DAF, and the gradient profiles thereafter indicated that nearly all of the observable protein was found as one of these three distinctly sedimenting classes. These proteins were further resolved on polyacrylamide gels as reported previously by Hill and Breidenbach (15). Figure 4 illustrates the changes in the relative distribution of these bands during seed development. Electrophoresis of each of the isolated sedimentation fractions from mature seeds revealed that bands 1, 2, and 3 were the major components of the 7.5S fraction whereas bands 4 and 5 were obtained from the 11.8S and 2.2S fractions, respectively (15). At the earliest age, a relatively high amount of protein migrated at the gel front, suggesting, along with the evidence from the sedimentation profile, that much of the protein at 12 DAF was indeed low mol wt. Faint bands in the upper region of the gel were also observed at 12 DAF. One of these bands appears to correspond to the 11.8S (band 4) component, thus correlating with its presence as a small peak on the sedimentation profile at this age. It was not clear whether any of bands 1, 2, or 3 from the 7.5S fraction were present at this age, although there is a trace of at least one such peak. At 17 DAF, two of the three 7.5S bands and the 11.8S band were readily observable (Fig. 4). The third 7.5S band was not detected in the supernatant protein until 28 DAF, and thereafter all of the five major electrophoretic components were observed in varying concentrations for the duration of the ripening period. Semiquantitatively at least, it can be seen from Figure 4 that band 5, reppresenting the smaller protein components, is much more concentrated in young than in mature tissue. The amount relative to the total protein of band 4, (the 11.8S component) seems
39 DAS+
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RELATIVE MOBILITY FIG. 5. Relative mobilities of the isolated 7.5S fraction at various stages of seed development in discontinuous polyacrylamide (5% w/v) gels by the method of Davis (10). are marked changes in the three 7.5S proteins throughout maturation. Figure 5 shows the changes in the three components of the 7.5S fraction after separation on sucrose gradients and analysis by gel electrophoresis at each developmental stage. The isolated 7.5S bands seen in Figure 5 clearly have the same relative mobilities and are present at similar relative concentrations at each age. Band 3 is more apparent at 23 DAF in the isolated 7.5S fraction than in the total protein, where it was masked by the 11.8S band (Fig. 4). Changes in the amounts of each component during the ripening period were estimated by a computer program for determination of the area under overlapping peaks as described in "Materials and Methods." Figure 6a shows the increase in the level of protein from the electrophoretic traces of bands 1, 2, and 3 combined (as an estimate of the total 7.5S protein), band 4 (11.8S), and band 5 (2.2S). Both the total 7.5S and 11.8S proteins begin to increase rapidly 17 DAF, whereas the 2.2S component increases more slowly and constitutes a considerably smaller fraction of the total protein. The electrophoretic profiles were used as the primary criteria for determining temporal changes in protein levels. Figures 6b and 6c, however, compare the peaks from the sucrose gradients as a check on the respective protein levels of the corresponding band(s) from the electrophoretic scans for the 7.5S and 11.8S fractions. Estimates of protein values appeared larger from peaks 1, 2, and 3 combined than from the gradient profile (Fig. 6b), perhaps reflecting the addition of background from the gels. The 11.8S fraction and band 4 gave very similar protein estimates (Figure 6c) throughout seed development. Figure 7 details the changes in the three 7.5S electrophoretic bands over the ripening period. Although these three bands in
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to undergo only slight changes, whereas there
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RELATIVE MOBILffY FIG. 4. Relative mobilities of the whole seed proteins at various stages of seed development in discontinuous polyacrylamide (5% w/v) gels by the method of Davis (10).
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combination yield a pattern of protein accumulation similar to that of the 7.5S sedimenting peak, their individual patterns of accumulation were considerably different. Bands 1 and 2 both began to increase rapidly at 17 DAF. However, band 1 showed very little further increase beyond 23 DAF while band 2 increased almost linearly through 33 DAF. Both of these components decreased slightly between 33 DAF and full
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maturity. Band 3 on the other hand, was not apparent until 23 DAF and increased from this time until maturity.
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Days Affer Flowering FIG. 6. a: Protein changes in the electrophoretic components 1, 2, and 3; 4, and 5 of developing soybean seeds. Peaks 1, 2, and 3 were combined as an estimate of the 7.5S (-E)-) component. Peaks 4 and 5 represent, respectively, the 11.8S (-A-) and 2.3S (-E-) ultracentrifugal components. Relative protein values at different ages were estimated by a computer from the scans of the gels prepared by the Davis (10) procedure. b: Comparison of the protein
changes in the 7.5S fraction as measured from ultracentrifugal (-*-) and electrophoretic (-0-) profiles. The electrophoretic peaks 1, 2, and 3 were combined to estimate the 7.5S component. c: Comparison of the protein changes in the 11.8S fraction as measured from ultracentrifugal (-*-) and electrophoretic (-0D-)
profiles.
28
Days After Flowering FIG. 7. Protein changes in the three individual 7.5S electrophoretic components of development soybean seed. Protein for peak 1 (-(0-), peak 2 (-A-), and peak 3 (--E), were estimated by a computer from the scans of the gels of total protein prepared by the method of Davis (10).
5
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DISCUSSION Satisfactory separation and characterization of the major protein components of soybean seeds has proved difficult to achieve with conventional protein purification techniques. Generally employed procedures (salting out, isoelectric precipitation, cryoprecipitation, etc.) require much time, usually result in considerable cross contamination, and may result in differential denaturation. To avoid these problems, we separated the major protein components present at various stages of development of soybean seeds by direct sucrose density gradient sedimentation of the 31,000g supernatant of seed extracts. This method of extraction and fractionation in dilute salt buffers required a minimum of manipulation and resulted in quantitative recovery of fractions with very little cross contamination as determined by two electrophoretic criteria (15). Millerd et al. (20) has also used sucrose density sedimentation to obtain very clean preparations of the proteins of Vicia faba as verified by immunological criteria. Density gradient sedimentation of the extracts of freshly matured 46-day-old seeds (Fig. 3) revealed a sedimentation profile with three major peaks (2.2S, 7.5S and 11.8S), consistent with the profiles of defatted soybean meals obtained by analytical centrifugation (21, 23) and with previous results from this laboratory (15). A large proportion of the soybean protein found at early stages in development of the seed sedimented at 2.2S. In mature soybean, large quantities of the so-named trypsin inhibitors as well as a number of other proteins in lesser amounts have been reported to have 2S sedimenting properties (6, 7, 11). The function of the 2S proteins is largely unknown, although enzyme activities have been reported for some of them (22). The 7.5S and 11.8S fractions of soybean are the major components of mature seed. Whereas the 11.8S fraction seemed homogeneous by electrophoretic criteria, the 7.5S fraction was heterogeneous. Both temporal and rate differences were apparent in the accumulation of the three electrophoretic components of the 7.5S fraction (Fig. 7). The relationship among the three components of the 7.5S fraction of soybean is
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PROTEINS OF SOYBEAN SEED. II
not known. Although their temporal differences indicated that they were unrelated, all three exhibited a similar sedimentation shift in buffer lacking 0.5 M salt, suggesting some similarity in properties. Electrophoresis of this fraction from mature seeds in an 8 M urea dissociating system yielded only three major components and a few minor bands, indicating that few changes occurred. In contrast, the 11.8S fraction was dissociated into several bands by the same system (15). Preliminary results in this laboratory indicate that the subunits obtained from the three 7.5S bands, under dissociating conditions, show changes parallel to the results reported here in a nondissociating system, whereas the subunit ratios of the 11.8S fraction remain constant throughout development. Changes in the subunit ratios of vicilin during seed development in Vicia faba have similarly led other workers to conclude that this fraction is heterogeneous (24). Studies on soybeans are under way to extend this point and will be reported later. No temporal difference was seen between the early appearance of the 7.5S components as a whole and the appearance of the 11.8S protein. At 12 DAF, Figures 3 and 4 show that both the 11.8S protein and components of the 7.5S fraction were apparently present. More specific criteria are required to determine the identity of these proteins because, at this and earlier stages in seed development, enzymatic proteins exhibiting the same physical properties may be found at concentrations comparable to those of the storage proteins. This raises questions about the reports (13, 24) based on solubility separations, that vicilin precedes legumin in the seed development of Vicia
faba. Additionally, profiles of mature soybean seed extracts on sucrose density gradients were similar to those recently reported for Vicia faba (20) and Pisum aureus (12). Together these studies qualitatively corroborate the interspecies comparisons on the analytical centrifuge by Danielson (8), who reported that a number of leguminous species have similar storage components. Indeed, the temporal accumulation of the protein components of soybean and of Pislim sativum (9), as indicated by sedimentation criteria, substantiates the apparent similarity of these proteins and further suggests that a similar sequence of events leads to their synthesis. Sedimentation criteria alone are not sufficient to completely characterize these proteins. Recent investigations (4, 5, 19, 25) have shown that proteins from related leguminous species seemed similar by solubility or sedimentation criteria but exhibited differences in electrophoretic properties. Comparison of the seed proteins of leguminous species is frequently confusing. They are not well characterized by any single physical parameter. The names vicilin, legumin, glycinin, etc., no longer seem appropriate in view of both the heterogeneity within species and differences between species. However, within a species they are few in number, and the similarities between species that exist indicate an evolutionary conservation that might not be expected if their function is purely storage of amino acids. It is perhaps surprising that they are not more different.
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Acknowledgments-The authors thank J. Zaya and R. S. Criddle for their assistance with the computer program for overlapping absorption peaks and R. Falk for use of the Gilford gel scanner. LITERATURE CITED 1. ALTSCHUL, A. M., L. Y. YATSu, R. L. ORY, AND E. M. ENGLEMANL-. 1966. Seed proteins. Annu. Rev. Plant Physiol. 17: 113-136. 2. BEEVERS, L. AND R. PouLsoN. 1972. Protein synthesis in cotyledons of Pisum sativum L. I. Changes in cell-free amino acid incorporation capacity during seed development and maturation. Plant Physiol. 49: 476-481. 3. BILS, R. F. AND R. W. HOWELL. 1963. Biochemical and cytological changes in developing soybean cotyledons. Crop Sci. 3: 304-308. 4. BOULTER, D., E. DERBYSHIRE, J. A. FRAHM-LELIVELD, A-ND R. 'M. POLHILL. 1970. Observations on the cytology and seed proteins of various African species of Crotalaria L. (Leguminosae). New Phytol. 69: 117-121. 5. BOUJLTER, D., D. A. THrRM5-N, AND E. DERBYSHIRE. 1967. A disc electrophoretic study of globulin proteins of legume seeds with reference to their systematics. New Phytol. 66: 27-36. 6. CATSIMPOOLAS, N., C. EKENSTAM, AN-D E. W. MEYER. 1969. Separation of soybean whey proteins by isoelectric focusing. Cereal Chem. 46: 357-369. 7. CATSIMPOOLAS, N. AND E. LEUTHNER. 1969. The major pH 4.5 soluble proteins of soybean cotyledons. I. Separation by gel filtration, disc electrophoresis and immunoelectrophoresis. Biochim. Biophys. Acta 181: 404-409. 8. DANIELSON, C. E. 1949. Seed globulins of the Gramineae and Leguminosae. Biochem. J. 44: 387-402. 9. DAN'IELSON, C. E. 1952. A contribution to the study of the synthesis of the reserve proteins of ripening pea seeds. Acta Chem. Scand. 6: 149-159. 10. DAVIS, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. 11. ELDRIDGE, A. C., R. L. ANDERSON, AND W. J. WOLF. 1966. Polyacrylamide gel electrophoresis of soybean whey proteins and trypsin inhibitors. Arch. Biochem. Biophys. 115: 495-504. 12. ERICSON, M. AND M. CHRISPEELS. 1973. Isolation and characterization of glucosamine-containing storage glycoproteins from the cotyledons of Phaseolus aureus. Plant Physiol. 52: 98-104. 13. GRAHAM, T. A., AND B. E. S. GuNN;ING. 1970. Localization of legumin and vicilin in bean cotyledon cells using fluorescent antibodies. Nature 228: 8182. 14. HALL, T. C., R. C. MCLEESTER, AND F. A. BLISS. 1972. Electrophoretic analysis of protein changes during the development of the French bean fruit. Phytochemistry 11: 647-4649. 15. HILL, J. E. AND R. W. BREIDENBACH. 1974. Proteins of soybean seeds: I. Isolation and characterization of the major components. Plant Physiol. 53: 742-746. 16. HUFFAKER, R. C., T. RADIN, G. E. KLEINKOPF, AND E. L. Cox. 1970. Effects of mild water stress on enzymes of nitrate assimilation and of the carboxylative phase of photosynthesis in barley. Crop Sci. 10: 471-474. 17. KONDO, K., S. K. MORI, AND M. KAJIMA. 1954. On the components of soybean protein. 2. Formation of the component-protein of the soybean during the ripening duration. Bull. Research Inst. Food Sci., Kyoto Univ. 15: 37-48. 18. LoWRY, 0. H., N. J. RoSEaRoUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 19. McLEESTER, R. C., T. C. HALL, S. M. SUN, AND F. A. BLISS. 1973. Comparison of globulin proteins from Phaseolus vulgaris with those from Vicia faba. Phytochemistry 12: 85-93. 20. MILLERD, A., M. SIMoN, AND H. STERN. 1971. Legumin synthesis in developing cotyledons of Vicia faba L. Plant Physiol. 48: 419-425. 21. NiAsMITH, W. E. F. 1955. Ultracentrifuge studies of soya bean protein. Biochim. Biophys. Acta 16: 203-210. 22. WoLF, W. J. 1970. Soybean proteins: their functional, chemical and physical properties. Agr. Food Chem. 18: 969-976. 23. WOLF, W. J. AND D. R. BRIGGS. 1956. Ultracentrifugal investigations of the effect of neutral salts on the extraction of soybean proteins. Arch. Biochem. Biophys. 63: 40-49. 24. WRIGHT, D. J. AND D. BOULTER. 1972. The characterization of vicilin during seed development in Vzcia faba (L.). Planta 105: 60-65. 25. WRIGHT, D. J. AND D. BOULTER. 1973. A comparison of acid extracted globulin fractions and vicilin and legumin of Vicia faba. Phytochemistry 12: 79-84.
