Professional Documents
Culture Documents
Euphytica, 1982, Vol.31, 677-690 PDF
Euphytica, 1982, Vol.31, 677-690 PDF
Institute for Cereals Flour and Bread TNO, P.O. Box 15,670O AA Wageningen, the Netherlands
INDEX WORDS
SUMMARY
Gelprotein or SDS-insoluble gel-forming glutenin was isolated from wheat flour by extraction with an
aqueous 1.5% SDS solution. Remarkable intervarietal differences were observed both in amount and subun-
it composition of these proteins.
The amount of gelprotein and the SDS-sedimentation volume both proved to be good parameters for
the bread-making quality of wheat cultivars. A high correlation was observed between amount of gelprotein
and SDS-sedimentation volume. The amount of gelprotein was therefore tentatively assumed to be the
essential basis of the SDS-sedimentation test.
The subunit composition of the gelprotein was studied by SDS-PAGE after reduction of SS bonds by
mercaptoethanol. It was found that the average bread-making quality of wheat cultivars and progeny of
the cross Atlas 66 x Atys which possessedsubunits 3 and 10, coded for by chromosome lD, was significantly
higher than that of wheat samples possessing subunits 2 and 11, their allelic counterparts.
INTRODUCTION
Nowadays it is generally accepted that wheat proteins are responsible for the visco-
elastic and bread-making properties of a dough. However there is still much dispute
as to which protein fraction is involved (WALL, 1979).
Recently, wheat proteins were fractionated in our laboratory by suspending wheat
flour in 1.5% sodiumdodecylsulphate (SDS) solution and ultracentrifugating the sus-
pension (GRAVELAND et al., 1979). A gel layer, mainly consisting of SDS-insoluble
glutenin could be isolated (GRAVELAND et al., to be published). It will be referred to
as gelprotein. Remarkable differences between wheat cultivars both in quantity and
viscosity of the gel layer were observed.
In 1978 AXFORD et al. introduced the SDS-sedimentation test, a relatively simple
method for estimating bread-making quality of wheat cultivars. This led us to study
the relationship between the amount of the SDS-insoluble gelprotein and the SDS-
sedimentation volume and their correlations with bread-making quality.
It has been shown by PAYNE et al. (1979, 1981) and BURNOLJF& BOURIQUET (1980)
that the composition of the high-molecular weight subunits of glutenin can be related
677
J. H. E. MOONEN, A. SCHEEPSTRA AND A. GRAVELAND
MATERIALSANDMETHODS
Wheat samples. Samples of 60 wheat cultivars, grown in 1979, were kindly given by
Koninklijk Kweekbedrijf en Zaadhandel D. J. van der Have at Rilland (NL).
The cultivars Atlas 66 and Atys and Fg progeny from a cross between these were
obtained from the Foundation for Agricultural Plant Breeding in Wageningen; they
had been grown on trial fields in 1979.
Baking test. Standard baking tests and micro-baking tests were performed as described
by SMAK (1972) and MEPPELINK (198 1) respectively.
SDS-sedimentation test. The SDS-sedimentation test was carried out according to Ax-
FORD et al. (1978).
SDS-PAGE. SDS-PAGE was carried out with the buffer system described by laemmli
(1970). Gelprotein (10 mg/ml) or total flour (40 mg/ml) was suspended in a buffer
of 0.062 M Tris-HCl (pH 6.8) 2% SDS and 5% mercaptoethanol. After shaking for
2 h at room temperature the suspensions were boiled for 2 min. After centrifugation
(2000 g) for 5 min at room temperature 10 ul gelprotein solution or 50 ul flour extract
was applied to a vertical polyacrylamide slabgel (180 x 140 x 2.7 mm) which consisted
of a 4% (w/v) acrylamide, 0.107% (w/v) b isacrylamide stacking gel (20 mm) and a
10% (w/v) acrylamide, 0.267% (w/v) bisacrylamide separation gel (120 mm). The elec-
trophoresis was carried in a Pharmacia GE 2/4 system at room temperature, the elec-
trophoresis buffer was cooled with tapwater. Two slabgels were run with 30 mA for
30 min, and 60 mA overnight. Proteins were fixed and stained with 0.2% Coomassie
Brilliant Blue R 250 in HzO/MeOH/HAc (45/45/10; v/v/v) at 60°C for 1 h and des-
tained with HzO/MeOH/HAc (175/10/l 5; v/v/v) at 60 “C overnight.
Statistics. For groups of cultivars or progeny with the same A subunits the average
loaf volume was calculated. Any differences in the average loaf volumes were tested
for significance with Student’s t test. At a probability level of 5% (P < 0.05) the differ-
ence was regarded as ‘probably significant’, at P < 0.01 the difference was ‘significant’
and at P < 0.001 ‘highly significant’.
RESULTSANDDISCUSSION
A I I I I I
v 20 25 30 35 40
V. PROTEIN ( ‘Iour 1 GELPROTEIN ( mg/ g flour )
700 -
:
0
;
600 -
Gl
8
<
E
- QOt
I I I I I
10 11 12 13 14
V. PROTEIN ( flour ) GELPROTEIN ( mg g flour )
I
Fig. 2. Correlation between SDS-sedimentation volume and protein content and amount of gelprotein.
For symbols see legends Fig. 1.
Recently PAYNE et al. (1979, 1981) have suggested that the presence of subunit 1
in wheat cultivars improves bread-making quality. However as shown in Table 2 the
difference in loaf volume between the cultivars with subunit 1 and those without sub-
unit 1 is not significant. Moreover, comparison of group I, 1 and group I, 2 shows that
the addition of subunit 1 did not improve bread-making quality.
However addition of subunit 8 (group I, 1 versus group I, 3) and replacement of
subunit 5 by subunits 4 and 8 (group I, 1 versus group I, 5) improved bread-making
quality significantly (Table 2). The difference between group I, 2 and I, 5 was not
significant; neither were the differences between the subgroups of group II (data not
shown).
Relationship between the A subunits in progeny of the cross Atlas 66 x Atys and bread-
making quality. An elegant method to establish whether the division between wheat
varieties made in the previous section is meaningful in respect to bread-making quality,
is to analyse the progeny of a cross between wheat varieties of which one parent has
the subunits 3 and 10 and the other parent the subunits 2 and 11.
In Fig. 5 SDS-PAGE electropherograms are shown of Fg progeny lines of the cross
Atlas 66 x Atys. Atys contains the subunits 1, 3, 5, 8, 10 and Atlas the subunits
2, 6, 7, 11. Fig. 6 indicates that no relationship exists between protein content of the
flour and loaf volume as determined by the micro-baking test. Besides it is shown
in Fig. 6 which lines possess the subunits 3 and 10, subunits 2 and 11, or the combina-
tion of these subunits. The difference in bread-making quality between group I and
group II proved to be significant (Table 3).
Euphytica 31 (1982) 681
J. H. E. MOONEN, A. SCHEEPSTRA AND A. GRAVELAND
Fig. 3. SDS-PAGE of isolated gelprotein from 10 European wheat cultivars. Lane 1, Sicco; 2, Bastion;
3. Adonis; 4, Okapi; 5, Tundra; 6, Marksman; 7, Carimulti; 8, Kormoran; 9, Hardi; 10, Rivoli; 11, Calibra-
tion proteins, phosphorylase b (97,000), serum albumin (68,000), ovalbumin 43,000) and carbonic anhydrase
(30,000).
SDS-PAGE of single kernels of the F8 lines that possessed in the flour the combina-
tion of A subunits 2,3, 10 and 11 showed that these lines were still impure. The single
kernels possessed either the combination 2, 11 or the combination 3, 10 (results not
shown).
Again the presence of subunit 1 was not accompanied with enhanced bread-making
quality. Neither was there any significant difference in bread-making quality between
Fg lines possessing the subunits 6 and 7 and those possessing the subunits 5 and 8.
In the subgroups of group I and group II no further significant correlations between
subunits and bread-making quality could be found (Table 3).
GENERAL DISCUSSION
Fig. 4. SDS-PAGE of isolated gelprotein and total flour extracts from 4 European wheat cultivars. Lane
1, Marksman flour; 2, Marksman gelprotein; 3, Tundra four; 4, Tundra gelprotein; 5, Bastion flour; 6,
Bastion gelprotein; 7, Sicco flour; 8, Sicco gelprotein.
sedimentation volume (r = 0.95; P < 0.001). Both properties give a rather good indica-
tion of the bread-making quality of wheat cultivars (Fig. 1 and 7) (Fig. 7 is a graphic
representation of Table 1).
However, we should remark that the SDS-sedimentation test was insufficiently ac-
curate in our hands to reveal existing differences in bread-making quality between
the subgroups of group I (data not shown).
It has recently been shown in our laboratory that the SDS-insoluble gelprotein be-
comes SDS-soluble by the action of a redox system present in wheat flour, when a
dough is mixed in the presence of oxygen (GRAVELAND et al., 1980). It is conceivable
that this redox system is also active during the extraction of flour proteins with SDS.
As indicated by GRAVELAND et al. (1979) both mechanical stress and elevated tempera-
tures could activate this redox system, thereby having a negative effect on the amount
The table indicates from left to right variety, type of wheat (summer or winter), country of origin, loaf
volume standard baking test (ml. 100 g flour -I), SDS sedimentation volume (ml. 6 g meal-t). The cultivars
indicated with * are used in the experiments of Fig. 1 and Fig. 2.
Table 3. Correlations between inheritance of A subunits and loaf volumes in Fg progeny of the cross Atlas 66 x Atys.
0 0
A0
00
0
atys
0 o" A
0 02. .
A. . l .
A
8.0°
A oA Oaths 66
0
.
0
I .
In I I I .1 I I
” 12 13 14 15 16 17
Fig. 6. Correlation between loaf volume and protein content of the flour of F6 progeny from the cross
Atlas 66 x Atys.
Open circles : Fg progeny with A subunits 3 and 10
Closed circles : Fg progeny with A subunits 2 and 11
Triangles : Fg progeny with A subunits 2,3, 10 and 11
800
1
;2,700-
8
<
-i
lr!
2
?a00 -
%
2
500-
400 -
I I 1 1 I 1 I I
40 50 60 70 60 90 100
Fig. 7. Correlation between loaf volume and SDS-sedimentation volume of 60 wheat cultivars.
Open circles : cultivars with A subunits 3 and 10
Closed circles : cultivars with A subunits 2 and 11
The dotted circles indicate that the average SDS-sedimentation volume of cultivars with A subunits 3 and
10 is significant higher than the average SDS-sedimentation volume of cultivars with A subunits 2 and
11.
in Fig. 6 that the higher bread-making quality of the Fg progeny from Atlas 66 x
Atys possessing subunits 3 and 10, was definitely not caused by higher protein contents
of their flours.
The higher bread-making quality of cultivars with subunits 3 and 10 has to be ex-
plained by the fact that the cultivars of group I contained more SDS-insoluble glutenin
and higher SDS-sedimentation volumes than did the cultivars of group II. The mean
SDS-sedimentation volume of group I was 80.2 f 13.0 ml and that of group II 66.9
f 14.2 ml (see also Fig. 7). From Fig. 2 we can deduce that these values correspond
with 32.1 f 3.5 and 28.5 + 3.8 mg gelprotein/g flour respectively. The difference
in average amount of gelprotein between group I and group II was highly significant
(P < 0.001). To explain this we could assume that the A subunits 3 and 10 form more
stable and/or more numerous disultide bonds with the other glutenin subunits than
do the A subunits 2 and 11.
688 Euphyiica 31 (1982)
SCREENING FOR BREAD-MAKING QUALITY IN WHEAT
ACKNOWLEDGEMENT
This investigation was carried out with financial support of the Stichting Nederlands
Graan-Centrum, Wageningen.
NOTE
In recent work, the presence of a further subunit 2* was established in some wheat
cultivars, in accordance with the studies of G. J. LAWRENCE & K. W. SHEPHERD (Austr.
J. Biol. Sci 33 (1980) 221-233) and P. I. PAYNE, L. M. HOLT & C. N. LAW (Theor.
Appl. Genet. 60 (1981) 229-236). This subunit bears a positive effect on bread-making
quality. In a further paper, the identification of this subunit and its use in screening
breeder’s samples for baking quality will be described.
REFERENCES
ARAKAWA, T., M. YOSHIDA,H. MORSHITA,J. HONDA&D. YOHEZAWA, 1977. Relation between aggregation
behavior of glutenin and its polypeptide composition. Agric. Biol. Chem. 41: 995-1001.
AXFORD, D. W. E., E. E. MCDERMOTT& D. G. REDMAN, 1978. Small-scale test of bread-making quality.
Milling Feed and Fertilizer 66: 18-20.
AXFORD, D. W. E., E. E. MCDERMOTT & D. G. REDMAN, 1979. Note on the sodium dodecyl sulphate
test of bread-making quality: Comparison with Pelshenke and Zeleny tests. Cereal Chem. 56: 582-584.
BIETZ,J. A., K. W. SHEPHERD& J. S. WALL, 1975. Single-kernel analyses of glutenin: use in wheat genetics
and breeding. Cereal Chem. 52: 513-532.
BOLLING, H. & K. M~~NZING,1980. Die Verwendung der SDS-Sedimentation zur Beurteilung von Backwei-
zen. Getreide, Mehl und Brot 34: 611.
BURNOUF,T. & R. BOURIQUET,1980. Glutenin subunits of genetically related European hexaploid wheat
cultivars: their relation to bread-making quality. Theor. Appl. Genet. 58: 107-I 11.
GRAVELAND, A., P. BONGERS& P. BOSVELD,1979. Extraction and fractionation of wheat flour proteins.
J. Sci. Food Agric. 30: 71-84.
GRAVELAND, A., P. BOSVELD, W. J. LICHTENDONK &J. H. E. MOONEN, 1980. Superoxide involvement in
the reduction of disullide bonds of wheat gelprofeins. Biochem. Biophys. Res. Commun. 93: 1189-l 195.
GRAVELAND, A., P. BOSVELD,W. J. LICHTENDONK,J. H. E. MCXNEN & A. SCHEEPSTRA,Extraction and
fractionation of wheat flour proteins. II. J. Sci. Food Agric. to be published.
LAEMMLI, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage
T4. Nature 227: 68&685.
LAWRENCE,G. J. & K. W. SHEPERD,1980. Variation in glutenin subunits of wheat. Aust. J. Biol. Sci. 33:
221-233.
MEPPELINK, E. K., 1981. EinsatzmGglichkeit des Microbackversuches in der Weizenziichtung. Getreide,
Mehl und Brot. 35: 107-109.
ORTH, R. A. & W. BUSHUK, 1974. Studies on glutenin VI. Chromosomal location of genes coding for
subunits of glutenin of common wheat. Cereal Chem. 51: 118-126.
PAYNE, P. I. & K. G. CORFIELD, 1979. Subunit composition of wheat glutenin proteins, isolated by gel
filtration in a dissociating medium. Planta 145: 83-88.
PAYNE, P. I., K. G. CORFIELD &J. A. BLACKMAN, 1979. Identification of a high-molecular-weight subunit
of glutenin whose presence correlates with bread-making quality in wheats of related pedigree. Theor.
Appl. Genet. 55: 153-159.
PAYNE, P. I., L. N. LAW & E. E. MUDD, 1980. Control by homoeologous group I chromosomes of the
high-molecular-weight subunits of glutenin, a major protein of wheat endosperm. Theor. Appl. Genet.
58: 113-120.
PAYNE, P. I., K. G. CORFIELD, L. M. HOLT & J. A. BLACKMAN, 1981. Correlations between the inheritance
of certain high-molecular-weight subunits of glutenin and bread-making quality in progenies of six crosses
of bread wheat. J. Sci. Food. Agric. 32: 51-60.
SMAK, C. 1972. New approach to determine the browness of bread crust. Correlation between crust colour
and protein content. Cer. Chem. 49: 554-560.
WALL, J. S., 1979. The role of wheat proteins in determining baking quality. In: D. L. LAIDMAN & R.
G. WYN JONES (Eds), Recent advances in the biochemistry of cereals. Academic Press, pp 275-311.
ZEHATSCHEK, W., 1980. Das Elektrophoresemuster von Weizenglutenin bei Sorten mit unterschiedlichem
Qualitltsniveau. Getreide, Mehl und Brot 34: 239-243.