... in a glasshouse at. 20 C day/ 15 C night with supplementary incandescent lighting to .... developing at 20 Ctook place between 25 and 40 days after flowering ...
Plant Physiol. (1981) 67, 958-961
0032-0889/81/67/0958/04/$00.50/0
f?-Conglycinins in Developing Soybean Seeds' Received for publication March 27, 1980 and in revised form October 28, 1980
KENWYN R. GAYLER AND GEOFFREY E. SYKEs Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, 3052, Australia ABSTRACT The temporal sequence of development of the major proteins of seeds of soybean (Meff.) has been studied during development of cotyledons from flowering to maturity. A well-defrned difference occurred in the tmes of appearance and the periods of maximum accumulation of a-, a'-, and /8subunits of betaconglycinin. Whereas a- and a'-subunits appeared 15 to 17 days after flowering, accumulation of ,8-subunit did not commence until 22 days after flowering. Such alterations in subunit composition infer that changes also occurred in the amino acid composition of betaconglycinin during maturation, particularly in the content of methionine which is low in the l8-subunit.
Marked changes occur in the proteins accumulating in cotyledons during seed development in many legume species (4, 5, 10, 18). The yield and quality of the proteins eventually stored in mature seeds depends both on the relative rates of synthesis of these proteins and on the length of time for which such synthesis occurs. Variations both in the rates of synthesis of the major proteins, legumin and vicilin, and in the times at which their syDtheses commence have been demonstrated in developing cotyledons of Pisum sativum (9, 10). An ordered sequence of appearance also occurs among the group of proteins antigenically related to the mature vicilin fraction (10). Differences have also been shown between legumin and vicilin fractions in Viciafaba both in the time at which accumulation commences and also in the rates of synthesis (18). In soybean the major equivalent proteins are glycinin (11 S) and /?-conglycinin (7 S) (1, 6). Previous studies on developing soybean pods have shown that although proteins of both the 11 S and 7 S types appear early in cotyledonary development, there are major differences between the 7 S proteins in immature and mature cotyledons (5). During development a complex series of changes has been shown to occur in the proportions of three fractions obtained from the 7 S proteins by electrophoresis on polyacrylamide gels (5). Since it is now known that fi-conglycinin is composed of at least six isomers in mature tissue (13, 17) the significance of these developmental changes is unclear. All the isomers of fl-conglycinin are combinations of only three major polypeptides, the a-, a'-, and ,8-subunits combined as trimers (14, 15). In this paper, therefore, the 7 S proteins in developing soybean seeds have been reexamined under conditions which dissociate the secondary complexes. It is expected that the component polypeptides studied in this way should be related more directly to the primary products of protein synthesis. 'This research was supported by the Australian Research Grants Committee.
MATERIALS AND METHODS Plant Materials. Glycine max L. Merr. cv. Wayne was grown in 8-cm pots in sandy loam and fertilized every 2 weeks with a complete nutrient solution. Plants were grown in a glasshouse at 20 C day/ 15 C night with supplementary incandescent lighting to maintain a photoperiod of 16 h. During flowering, which commenced after 6 weeks and continued for 4 weeks, individual flowers were tagged for subsequent harvest at intervals from 10 days after flowering to maturity. Seeds were removed from pods, weighed, and stored frozen at -75 C. Protein Extraction. Both seed coat and embryo were removed from mature seeds. Cotyledons were finely ground, suspended in 8 volumes (w/v) of distilled H20 and lyophilized to improve the subsequent extraction (10). Seeds from developing pods were similarly treated while still frozen. Protein was extracted from the lyophilized powder by homogenization in 30 volumes (w/v) of 60 mM Tris-HCl buffer, pH 7.5, containing 10 mms 8-mercaptoethanol. The mixture was stirred at room temperature for I h and centrifuged at 15,000g for 40 min. The supernatant was decanted through a plug of glass wool to remove lipid and stored at -15 C. This modification of the method of Thanh and Shibasaki (12) extracted a total of 0.51 g protein/g lyophilized powder in three sequential extractions. The first extract contained 89%o of this extractable protein, and there were no qualitative differences between sequential extracts as revealed by electrophoresis. Protein was estimated by the method of Lowry et al. (8) after initial precipitation in 9% (w/v) trichloroacetic acid. Electrophoresis on Slabs of Polyacrylamide Gel. To dissociate subunit complexes, undenatured protein solutions were heated to 80 C for 2.5 min in 60 mm Tris-HCl buffer, pH 7.5, containing 2% (w/v) SDS and 0.15 M f.i-mercaptoethanol prior to electrophoresis on slabs of polyacrylamide gel I mm thick. Electrophoresis was carried out in the discontinuous buffer system of Laemmli (7) using a stacking gel of 6% (w/v) acrylamide in 60 mM Tris-HCI buffer, pH 7.5, a 15% (w/v) separating gel in 0.38 M Tris-HCl buffer, pH 8.9, and an electrophoretic buffer containing 25 mM Tris and 0.19 M glycine. All three buffers contained 0.1% (w/v) SDS. Nondissociating gels containing 5% (w/v) acrylamide were run in the discontinuous system of Davis (3) using a 3% (w/v) acrylamide stacking gel. Detection of Proteins on Gels. Before staining, gels were first fixed in 50%o (w/v) trichloroacetic acid for 5 h at room temperature, and washed twice with distilled H20. Gels were stained by incubation in 200 ml of 0.1% (w/v) Coomassie Blue R (Sigma) in ethanol:acetic acid:water (5:1:5, v/v/v) at 45 C for 45 min and destained by incubation in 600 ml of 7.5% (w/v) acetic acid at 60 C for 7 h with one change. Under these conditions, dye-binding to particular proteins was linearly related to protein content up to 5 ag, the upper limit of protein in the bands scanned in this paper. Dye-binding on stained gels was measured with a Gelman DCD- 16 Digital Computing Densitometer at a wavelength of 660 nm. The protein represented by each peak was calculated as the product of the contribution (%) of its area and of the total protein
958
DEVELOPMENT OF SOYBEAN fi-CONGLYCININS
Plant Physiol. Vol. 67, 1981
content in the sample. Corrections were made for differences in dye-binding ofthe different proteins from scans of purified protein samples. In all cases, comparisons were made only for proteins separated and stained on the same gel. Identification of Bands. Identification of the components of the major storage proteins among the many minor proteins present in the total extract was by the combination of electrophoretic mobilities and amino acid profiles. Glycinin and /1-conglycinin markers were purified from mature seeds essentially by the method of Thanh and Shibasaki (12, 17) using isoelectric precipitation and chromatography on Biogel A-5M. Purified f-conglycinin and glycinin produced single peaks s20,w = 7.3 S and S20,w = 11.5 S, respectively, at ionic strength ,u = 0.5. ,8-Conglycinin also produced a single peak s20w = 9.8 S at it = 0.1. Mobilities of the subunits of these purified proteins during electrophoresis on polyacrylamide gels in the presence of SDS were compared with those of markers of known molecular weight and with previously published relative mobilities in similar electrophoretic systems (2). The purified a-, a'-, and fl-subunits used for calibration of dyebinding were purified from 1.5 mg of purified ,B-conglycinin by electrophoresis in the presence of SDS on 3-mm thick slabs of 12.5% (w/v) acrylamide. Bands were excised from unstained sections of gels, ground in 1% (w/v) SDS, and extracted ovemight at room temperature. Acrylamide was removed by centrifugation through Miracloth and filtration, the filtrate dialyzed against 70% (w/v) isopropanol for 36 h and dried under vacuum. The amino acid profiles of each of the subunits of fi-conglycinin prepared in this way were in agreement with previously published data (14). fl-Subunit was also prepared independently from a 7 S species composed entirely of f8-subunits of f-conglycinin (Sykes and Gayler, unpublished results).
RESULTS
Temporal Sequence of Protein Accumulation. Although the most rapid rate of accumulation of total protein in cotyledons developing at 20 C took place between 25 and 40 days after flowering, increase in the protein content per cotyledon had commenced as early as 15 days and continued through to maturity (Fig. 1). Changes in individual proteins were, therefore, examined from the earliest stage of development where cotyledons could be harvested. Proteins from cotyledons ranging in age from 15 to 70 days after flowering were separated into their component polypeptides
by electrophoresis on SDS-containing polyacrylamide gels (Fig. 2). Major changes in the proportions of the dissociated polypeptides occurred over this period. Whereas those polypeptides which 80 002 60 ". D
0 10 20 30 40 50 60 70
C
E
40
Age IDA.F) -
a_ 20
0
0
20 Age
60 40 (D.A.F)
80
FIG. 1. Development of soybean seeds. Changes in fresh weight (0), dry weight (0) and protein content (E) of seeds measured from 15 DAF to maturity.
959 - cc
Oc
60K-
5OK-
-p
40K-
Acidic Ai A2 A3 A4 F2 (1)
30K-
Basic
20K-
JB
B2
Bs3 B4
-F2(2)
1S
17
20
22
25
30
35
40
M
FIG. 2. Polypeptides from seeds developing on soybeans. The total protein was extracted in 60 mm Tris-HCl, pH 7.5, 10 mm ,B-mercaptoethanol from cotyledons ranging in age from 15 days after flowering to maturity (M) and 36 i.g protein per track subjected to electrophoresis on polyacrylamide gels in the presence of SDS after reduction in fl-mercaptoethanol. The nomenclature of Moreira et al. (11) has been used for the acidic (F2(1), F2(2) Al, A2, A3, A4) and Basic (B1, B2, B3, B4) subunits of glycinin and the nomenclature of Thanh and Shibasaki (14) for a-, a'-, and fl-subunits of fi-conglycinin. Molecular weights are shown in kdaltons (K).
had been assembled into the fi-conglycinins and into glycinin increased rapidly throughout development of the cotyledon, the proportion of most other proteins decreased. In general, the polypeptides corresponding to ,f-conglycinin and those detectable from glycinin, appeared at essentially the same time, between 15 and 17 DAF2. There was one clear exception. The polypeptide which ran with an apparent mol wt of 48 kdaltons in this system appeared up to 5 days after all other components of the storage proteins and was subsequently identified as the f8subunit of fi-conglycinin. In general, the polypeptides which accumulated from 15 days onwards also had the same electrophoretic mobilities as the equivalent polypeptides in mature seeds. One of the basic subunits of glycinin however showed a change in apparent mol wt from 21 to 20 kdaltons between 40 days and maturation (Fig. 2). Inasmuch as at least two of the basic subunits of glycinin were not resolved from each other in this system, however, the possibility remains that this also was the result of a change in proportions of subunits. Estimation of Rates of Accumulation of Proteins During Development. Measurements of the levels of the three major subunits of ,B-conglycinin by densitometry showed that not only was the initial appearance of fl-subunit delayed until 22 DAF, but its maximum rate of accumulation was also reached only 25 to 30 DAF (Fig. 3). By contrast, a and a'-subunits had appeared by 17 DAF and went through most rapid accumulation 15 to 20 DAF. Despite its late appearance, however, synthesis of fl-subunit continued for at least 10 days after the other subunits reached their maximum levels, and by maturity had reached a level equivalent to them. During maturation, therefore, a dramatic change occurred in the proportions of the subunits of f8-conglycinin present. That this change was the underlying cause of the changes in the proportions of the 7 S fractions previously observed (5), was confirmed by separation
2Abbreviation: DAF, days after flowering.
Plant Physiol. Vol. 67, 1981
GAYLER AND SYKES
960 0-04 a~~~~~~~~~~~~~~~a
0-03
-
0-02
-
E
study on developing soybean have also been observed in other legumes, including Vicia (18) and Pisum. The appearance of a polypeptide of mol wt 30 kdaltons among the vicilin subunits in peas 8 days after the 50 kdalton polypeptide (10), closely parallels the development of fl-subunit in soybean. It too persists to maturity. Since such alterations in subunits appear to be widespread among the vicilin-like proteins, it is unlikely that thewererelatively grown
low temperature at which the soybeans in this study Low temperature induced the late appearance of the may, however, have slowed development and thereby assisted in detection of this subunit. *0 X 40 30 35 15 20 25 At least for soybean, the model proposed for the tertiary strucAge ture of its vicilin-like protein would allow a varying complement of in developing seeds. Levels of the of subunits to be assembled into a complete storage protein. In FIG. 3. Subunits ,B-conglycinin subunitsof,B-conglycinin in soybean at different ages (DAF) as determined the model proposed for there are no covalent bonds by gel scanning at 660 nm after staining in Coomassie Brilliant Blue. between the subunits (15). In fact, it has been suggested that at Corrections were made for differences in dye binding of a'- (0), a- (0), physiological pH and low ionic strength the subunits of fi-congly,B (A)-subunits using relative staining intensities of 0.8, 1.0 and 1.2, cinin can exist as and organized monomers in equilibrium with the respectively. trimer (7S) form and with a hexamer (10S) form (16). Such would reversible binding between the subunits from the to the 7 trimers of S complete assembly appear permit Day 17 of subunits available during development varying complement and possibly also allow the rearrangement of subunits among the 7S trimers. Alterations in the proportions of subunits of the kind observed E in this work would also result in changes in the amino acid Day 20 fraction. Because the mecomposition of the total I. thionine content in particular of the fl-subunit of ,B-conglycinin is less than (14), its preferone-third that of the aand a'-subunits co ential accumulation in the latter stages of pod development (Fig. Day 3) seems to be one factor contributing to the overall nutritional deficiency in methionine characteristic of mature soybean protein. Go 2 ' 0101
,B-subunit.
0
I
J
M
(OAF)
,B-conglycinin
of,8-conglycinin
,B-conglycinin
D
40
a
22
is extended to Mr. J. Shearer for excellent technical assistance, and to Mr. G. Halloran, School of Agriculture, for the provision of plant growth facilities.
Acknowledgment-Appreciation
,li,
Day
25
LITERATURE CITED !,
B5, B3.1
S6s
B34
02
FIG. 4. f8-Conglycinin isomers in developing seeds separated on nondissociating polyacrylamide gels. Gels loaded'with constant total extractable protein (48 jig) were run in the system of Davis (3), then stained and scanned as in Figure 3. Ages shown are DAF. B2 to B6 refer to the isomers of 3-conglycinin (13). The apparent drop in B5,6 late in maturation is due to the
use
of
a
constant
loading of total protein throughout but only
showing the sections of the gels containing ,8-conglycinins.
of the protein extracts from developing cotyledons on polyacrylamide gels under nondissociating conditions (Fig. 4). On such gels the isomers of fi-conglycinin, B5 and B6, which contain only aand a'-subunits separate as a group from isomers B2, B3, and B4 which contain a-, a'-, and f-subunits (13). When extracts of the sections of gels labeled B5, B6 in Figure 4, were subjected to a second electrophoresis in the presence of SDS, only a- and a'subunits were detected; from those labeled B2, B3, and B4, a-, a', and ,f-subunits were obtained. As shown in Figure 4, only isomers of f8-conglycinin containing a- and a'-subunits were detected in extracts of soybean cotyledons for the first 20 DAF, but between 22 and 40 days increasing proportions of the fl-containing isomers also accumulated. DISCUSSION
The changes in proportions of the major subunits of the 7 S or vicilin-like protein, fi-conglycinin, which have been shown in this
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DAvIs
McLEESTER,
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RW KOSHIYAMA I,
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T4. Nature 217: 680-685
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DEVELOPMENT OF SOYBEAN f-CONGLYCININS
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