Quality-related endosperm proteins in sulfur-deficient and normal wheat grain

Journal of Cereal Science 5 (1987) 233-245

Quality-related Endosperm Proteins in Sulfur-deficient and Normal Wheat Grain J. G. FULLINGTON*, D. M. MISKELLYt, and D. D. KASARDA*

c. w. WRIGLEYt

* USDA-ARS, Western Regional Research Center, 800 Buchanan St, Albany, CA 94710, U.S.A., t Bread Research Institute of Australia and t CSIRO Wheat Research Unit, PO Box 7, North Ryde, NSW 2113, Australia Received 18 June 1986

Specific groups of proteins associated with the baking properties of flour were identified by extracting flour from normal and sulfur-deficient wheat with a sodium dodecylsulfate (SDS)-Tris buffer either with or without reducing agent. Total proteins (with reducing agent), extract proteins (without reducing agent), and residue proteins (re-extraction of sediment with reducing agent) were fractionated by SDSpolyacrylamide gel electrophoresis (with reducing agent present for all fractions). Significant correlations were obtained between the proportions of certain groups of polypeptides and flour-quality characteristics. The proportion of high molecular weight (HMW) components (Mr 80,000; corresponding to HMW-glutenin subunits) in the residue was strongly positively correlated with resistance to extension and negatively correlated with dough extensibility and breakdown. Of the proteins extracted mainly by SDS without reduction, ro-gliadins in the M r range 51,000 to 80,000 showed the same correlations as the HMW-glutenin subunits, whereas proteins in the M r range 38,000 to 50,000 and 28,000 to 39,000 were positively correlated with dough extensibility. The proteins of these latter ranges correspond mainly to ex-, ~-, and y-gliadins and LMW-glutenin subunits. The proteins of the lowest M r range (M r < 28,000), which corresponded to albumins (and some globulins) and appeared mainly in the unreduced extract, correlated negatively with resistance to extension and positively with dough breakdown and extensibility. Electrophoretic patterns of the residue proteins were similar to those obtained from residues in the SDS and Zeleny tests suggesting that the sedimentation-test residues provide a measure of the proportion of HMW-glutenin subunits present in the flour.

Introduction The sulfhydryl and disulfide groups of wheat endosperm proteins have long been postulated to be important in determining dough properties and the baking quality of wheat flour 1 ,2, but our understanding of their chemical involvement is still vague. It has been suggested that disulfide interchange, catalysed by sulfhydryl groups is necessary for satisfactory dough properties and for a good balance between elasticity and extensibility. Many dough 'conditioners' are reasonably presumed to act by modifying SS-SH Abbreviations used: M r = relative molecular mass; HMW = high molecular weight; LMW = low molecular weight; SDS = sodium dodecylsulfate; S = sulfur; S-S = disulfide; SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis. 0733-5210/87/030233 + 13 $03.00/0

© 1987 Academic Press Inc, (London) Limited

234

J. G. FULLINGTON ET AL.

interchange 3 , e.g. cysteine, glutathione, metabisulfate, and even bromate and iodate. Other compounds that block sulfhydryl groups, such as iodoacetate and Nethylmaleimide, drastically alter dough properties 4 , Further evidence for the importance of sulfur-containing amino acids in gluten function has been provided by the observation that sulfur deficiency, during grain development, drastically alters dough propertiess- 7 • As a result of sulfur deficiency, protein synthesis is redirected in favor of the low-sulfur proteins B, particularly the co-gliadins and the HMW-glutenin subunits, both extractable in the presence of SDS together with an S-S bond breaking agent such as 2-mercaptoethanol 9 (ro-gliadins do not have -SH or S-S groups and can be extracted to a large extent without reducing agent). A loss of dough extensibility and an increase in resistance to extension accompanies these changes in protein compositionS, 7, possibly because a good supply of -SH and S-S groups is required to allow an orderly slipping (extensibility) of molecules or complexes of molecules within the dough as it is stretched to avoid rupture (lack of extensibility). The balance between elasticity and extensibility may be a consequence, however, of the M r distribution of glutenin aggregates, the intrinsic aggregation properties ofsubunits as combined in the glutenin complex, or a combination of these possibilities. For example the average M r ofdisulfide-bonded glutenin aggregates might be decreased by the presence of thiol compounds. Sulfur-deficient flour represents a useful model system with which to study gluten function since it contains' pre-fractionated' proteins with considerable enrichment of sulfur-poor gluten proteins. It would thus also be valuable to know more about the nature of the sulfur groups in sulfur-deficient dough, to determine, for example, how readily S-S groups can be broken, especially in view of reports that certain sulfurcontaining groups of dough are more accessible and reactive 1o . Flour from sulfurdeficient grain was therefore extracted with a dissociating solvent, both in the presence and absence of an S-S breaking agent, and the composition of the extracted proteins was quantitatively analysed by gel electrophoresis, The results were also related to the SDS-sedimentation test, a rapid procedure used to predict grain quality, especially in plant breedingll , 12. Materials and Methods

Flour samples

Twelve grain samples (cultivar Olympic) were selected from a set of field experiments in which nitrogen and sulfur fertilizers were varied between a and 100 kg Njha and a and 50 kg Sjha D, 13. The 12 samples (over 1 kg each) were milled to a flour yield of 72% on a Buhler experimental mill. The resulting flours ranged in protein content from 7·4 to 11·2%, and in sulfur content from 0·088 to 0·181 % (Table I). The results of quality testing of the flours have been previously reported in detai1 8 • Brabender Extensigraph test results were expressed as extensibility, resistance at 5 em, and Resistance Ratio (maximum resistance:extensibility), Dough breakdown was measured with a Brabender Farinograph.

Protein extraction Total proteins were extracted from flour samples as previously described l4 , I. with a buffer comprising 0·062 M Tris-(hydroxymethy1)aminomethane-HCI, pH 6·8, 2% SDS, 5% 2mercaptoethanol, at the rate of 30 mg flour jml. This is referred to as the' total' extract. Flour

PROTEINS IN SULFUR-DEFICIENT AND NORMAL WHEAT

235

protein was also separated into' extract' and' residue' fractions using a similar extraction buffer, but omitting 2-mercaptoethanol, by grinding 30 mg of flour sample in a mortar and pestle with 0·5 ml of this extracting buffer. The mixture was centrifuged in a bench top centrifuge at about 5000 r/min. An additional 0·5 ml of buffer was added to the resulting residue, the mixture was centrifuged again, and the two supernatants were combined to provide the fraction designated as 'extract' protein. 2-Mercaptoethanol was added to this solution for electrophoresis. Extraction buffer containing 5% 2-mercaptoethanol was added in the ratio of 1 ml/30 mg (originally) of flour to the residue remaining after removal of the extract fraction. The mixture was ground in a mortar and pestle, then centrifuged. The solubilized protein from this step is designated the 'residue' protein. The SDS-microsedimentation test protein (SDS test res), which was used for the electrophoregram of Fig. I, was prepared from the 0·181 % S sample of Olympic flour by running the test as described 16 , centrifuging the sediment, and washing it twice with a small volume of the SDS-lactate test solution to remove any remaining soluble proteins. The washed sediment was taken up in SDS-PAGE reducing buffer for electrophoresis. The Zeleny test was carried out according to Pinckney et alP

Electrophoretic analysis SDS extracts of flour, reduced by the addition of 2-mercaptoethanol (1 %) if it were not already present, were fractionated by high-resolution SDS-polyacrylamide gel electrophoresis. A detailed description of the method has already been described previously15, and is an adaptation of the method of Payne and Corfield lH • The gels were stained with Coomassie Brilliant Blue R250 and scanned by means of a Gilford scanning densitometer l ". Absorption curves were divided into regions (areas) Al to AS as follows: AI, polypeptides ofM r > 80,000; A2, 51,000 to 80,000; A3, 40,000-S0,000; A4, 28,000 to 39,000; and A5, 8000 to 27,000. Previous work (Cole et al. le , D. D. Kasarda and N. Fulrath, unpublished results) has indicated that the proteins of these ranges correspond to the foHowing types of endosperm proteins: AI, almost entirely to HMW-glutenin subunits; A2 almost entirely to ro-gliadins; A3, mainly to LMW-glutenin subunits, but with some IX-, 13- or 'Y-gliadins; A4, mainly to ex-, 13- and 'Y-gliadins, but with some LMW-glutenin subunits; A5, mainly to albumins, but with small amounts of globulins. There were small differences between the absolute areas of the total extracts and the sum of the absolute areas of extract and residue fractions for the various samples. Absolute areas corresponding to each extract and residue fraction were multiplied by a factor obtained by dividing the sum of the absolute areas for that total extract by the sum of the absolute areas for both the unreduced extract fraction and the residue fraction of that sample. The resulting corrected areas sum to I00 % and represent the proportions of the total flour protein for each fraction. The resulting unreduced-extract areas are designated EI-E5, the residue areas, RI-R5, and their sum, TI-TS. The areas Tl-TS are approximately equivalent to the total extract areas AI-AS referred to in a previous publicatione. Results and Discussion

Polypeptides of SDS sedimentation test residues Figure 1 shows the SDS-PAGE patterns obtained for total flour proteins and for the residue fraction and sedimentation test residue fractions from a sample of Olympic flour (0,181 % S). The similarity of protein subunit composition between the residue protein fraction and the SDS-sedimentation test residue fraction is apparent. Results for Zeleny test residue fractions were similar, although carried out on a different flour sample. The SDS-PAGE patterns of these residues were notably similar to those of highly-purified glutenin fractions (D. D. Kasarda and N. Fulrath, unpublished results) with the major 10

CER

5

J. G. FULLINGTON ET AL.

236 AI

A2

A3

A4

A5

TOlal prolein

sOS lesl residue

50S re&idue

50S extract Tolal pralein

FIGURE 1. 8DS-PAGE of reduced protein subunits from the SDS residue fraction or from the sediment from the SDS micro-sedimentation test, compared with the total protein, and the SDS extract fractions, all from Olympic flour containing 0·181 % sulfur.

exception being the presence of components in A2 that are absent from the purified glutenin; these components are almost certainly ro-gliadins that seem to be virtually impossible to separate completely from glutenin by solubility methods. In view of the similarity of the sedimentation test residue and our SDS residue, together with the correlation of the SDS sedimentation test and grain quality, analysis of the polypeptide components of the SDS residue appeared warranted to examine possible specific correlations with dough properties. The Olympic flour series used in this study offered a unique opportunity to explore this possibility because in all the flours, the same subunits were present, differing only in their quantity, yet these quantitative differences correlated with widely-different dough properties for the flours of the series. Electrophoretic analyses Typical differences in polypeptide distribution for two flours of the series appear in Fig. 2, which shows densitometric tracings of the solubilized total protein (T) along with the extract (E) and residue (R) fractions for a high-sulfur (0'181 %) and a low-sulfur (0,088 %) sample of Olympic flour. The fractions of the total protein (as represented by areas of the densitometric curves) for each of the M r ranges A I to AS are indicated for E, R, and T (= E + R) for each of the twelve flour samples in Table I and in Fig. 3. Each of the values of Table I is the average of five analyses. Confidence limits at the 95 % level (not shown) were an average of 1·8 percentage points for these values, with the largest range in the T values and the smallest in the R values. The average difference between A (total extract) values and uncorrected E+ R values was 1·6%.

PROTEINS IN SULFUR-DEFICIENT AND NORMAL WHEAT AI

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237

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80

51 40

28 M, (x 10- 3)

80

51 40

28 M, (x 10- 3 )

FIGURE 2. Densitometric scans of the total solubilized protein, the extract fraction, and the residue fraction from samples of Olympic flour containing (a) 0·181 % S or (b) 0·088% S.

Relationships of areas to S: N

The data are arranged in Table I and in Fig. 3(a) in order of increasing S: N in the flour. Our previous study of these samples 9 showed greater correlation between quality parameters and S:N ratio than with simple S content. In considering this approach, the low protein content of the last two flours must be borne in mind. They have high S:N ratios, but the effects of low protein on flour quality must be considered with the effect of high sulfur. Correlations with sulfur, nitrogen and S: N ratios are given in Table II. There was almost no correlation of scan areas with nitrogen. Sulfur limitation for the samples we studied may not have been sufficient to diminish the overall level of protein synthesis (although this should occur at sufficiently low levels of sulfur availability), but mainly may have caused a disproportionation of proteins in favor of those with lower sulfur contents. We did not determine non-protein nitrogen, however, and Shewry et at. 20 noted a significant increase in non-protein nitrogen (presumably mostly amino acids) in barley when sulfur was limited. It may be that actual protein levels for our low-sulfur samples are lower than we report; the difference could result from an accumulation of 10-2

J. G. FULLINGTON ET AL.

238

TABLE 1. Percentages of densitometric scan areas corresponding to twelve flour samples for extracts obtained with SDS in the absence of reducing agents (El-E5), for extracts of the resulting residue with SDS and 2-mercaptoethanol (Rl-R5), and for total protein expressed as the sum of E+R (T1-T5), which was normalized to 100% (see text) Flour S:N (x 100)

Flour sulfur (%)

Flour protein

4·40

0·088

9·7

4'51

0·084

10·5

4·60

0·\06

10·4

4·93

0·095

9·7

5·18

0·103

9·8

5'59

0·093

10·6

6·22

0·139

10·9

Densitometric scan areas

E: R:

T: E:

R: T: E:

R: T: E:

R: T: E:

R: T: E:

R: T: E: R:

T: 7·47

0·154

10·3

E: R: T:

8·00

0·164

10·4

E: R:

T:

8·19

0·181

11'2

8·50

0·116

7·6

8·93

0'128

7·4

E:

R: T: E: R:

T: E: R:

T:

Al

A2

A3

A4

AS

Total

5·1 8'2 13·7 5·5 6·1 12·4 5'5 6·0 12·5 6·3 6'3 12·3 5·3 6·8 12·1 5-8 5·8 11'2 4'7 5·7 10·6 5·3 2·3 8·6 6·2 2·7 7'7 4·1 3·7 7·6 4·6 3'6 7·3 5·2 2·1 7·2

19·3 1·9 17·4 19·1 2·2 16·8 19·0 1·9 15·3 1s.4 2·3 14·9 17·5 1·8 15-8 15·9 1'5 15·2 15·1 2·0 12·2 12·2 1·3 10·3

13·9 5·1 18·7 13-9 4·5 18'7 15-3 5·0 20·4 15·3 5·7 20·4 14-8 5·6 19·7 16·0 5·2 20·1 15·1 5·8 21·1 17·6 4·2 21·8 18·2 3-9 20·7 15·1 6·9 20·0 14·6 6·0 21·0 16·5 4·8 21·8

22·2 3·4 26·8 21'8 3·3 27·6 24'1 3'1 28·1 20·8 3·5 27'3 22-4 3·3 27-6 22'6 3·0 27·7 25·2 3-7 29·5 26·2 2·4 29·6 25·9 2·6 29'7 27·0 4·5 30·1 24·2 3·8 29·5 26·1 3·0 28·9

21·4 0·2 23-3 22-8 1·1 24-6 18·9 1·3 23'7 23·0 1·4 24·9 20·7 1·8 24·0 22-5 1·2 26·1 21-6 1'2 26·5 27·7 1·2 29·7 25'5 1·0 32·0 26·5 0·2 32·8 30·5 0·2 32·2 28·1 1·7 31·7

81·9 18·8 100 83·1 17·2 100 82-8 17·3 100 80·8 19·2 100 80·7 19·3 100 82·8 16·7 100 81·7 18·4 99·9 89·0 11·4 100 88·4 11-6 100 83·5 16·9 100 84·9 15·0 100 86·9 12-9 100

12-6

1·4 9·9 10·8 1-6 9·5 11·0 1·4 9·9 11·0 1·3 10·0

free amino acids (other than cysteine and methionine) or short peptides that resulted from arrested translation of mRNA. Although there was significant correlation of scan areas with sulfur content, the correlation with S: N was generally better. In general, the pronounced decrease in the values ofTl with increasing S: N (an overall

PROTEINS IN

SULFUR·DEFICIENT AND NORMAL WHEAT

239

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FIGURE 3. Distribution of electrophoretic scan areas with respect to (a) the S:N ratios of the flour samples, or (b) Resistance Ratio 9 (the Extensigraph maximum resistance divided by extensibility), with the total protein content of all samples normalized to 100 %. The solid lines are lines-of-best-fit (based on the individual points shown) for the sums (81 to S5) of individual areas for extracts (E) (without 2-mercaptoethanol) and residues (R) (extracted with SDS-2·mercaptoethanol sDlvent). Within the sum areas for each molecular-weight range (see Methods), the residue area is the lower part (below the broken line) and the extract is the upper region.

41% decrease) is accentuated in the residue fractions (RI), which undergo a 74% decrease

from the lowest S: N flour to the highest (Table I). Tl consists almost entirely of HMW-glutenin subunits9 , 14, 15,19, 21. These components generally have about one-third less sulfur than gliadins; this evidently favors their synthesis when sulfur is deficient so that they assume a higher proportion in low-sulfur flours. El does not show such a decrease and the change in Tl can be attributed entirely to the residue proteins (Rl). This raises the possibility that the solubilization procedures we used effected some

240

J. G. FULLINGTON ET AL.

TABLE II. Linear correlation coefficients between individual scan areas (identified in Figures I and 2) and grain/flour attributes. The most significant coefficients (P < 0'001) are underlined Area

%S

%N

S:N

Ext

Res 5

E1 E2 E3 E4 E5 RI R2 R3 R4 R5

-0,36 -0,83 0·61 0·88 0·62 -0,79 -0,65 0·11 0·09 -0,25 -0,83 -0,83 0·81 0-87 0·60

0'07 0·35 0·03 0·00 -0·5::; 0-30 0·36 0·03 0·07 -0,04 0·30 0·36 0·06 0·02 -0,58

-0,37 -0,96 0·55 0·82 0-88 -0-91 -0,80 0·07 0·04 -0·19

-0·25 -0,82 0·71 0·87 0·62 -0,82 -0,69 0·00 -0-02 -0·25 -0·83 -0,83 0·84 0·84 0·60

0·22 0'86 -0,56 -0,75 -0'84 0-90 0·72 0·08 0·01 0·28 0-89 0·87 -0,61 -0,72 -0,83

Tl T2

T3

T4

T5

-.0.:..22 -0,97 0·70 0·80 0·88

Significance of correlation coefficients: 0·58 (P < 0·05); 0·71

Resistance ratio 0·23

!liZ

-0,61 -0'79 -0,85 Q:.2J 0'76 0·12 0-08 0·27 0·92 0·89 -0,64 -0,74 -0,85 (P <

Breakdown

Loaf volume

-0'13 -0,75 0·41 0·51 0·84 -0'76 -0,70 -0,10 -0,09 -0,18 -0,74 -0,77 0·41 0·47 0·84

-0,19 -0,23 0·36 0·49 -0,06 -0,17 -0-14 0·13 0·08 -0,17 -0,22 -0-23 0·53 0·49 -0,09

0'01); 0·82 (P < 0,001).

separation of the lower- and higher-sulfur components of the HMW-glutenin subunits. These subunits have a relatively simple SDS gel electrophoretic pattern (Figs. I and 2) that shows no drastic differences in the proportions of the first three subunits when El and RI are compared, but a fourth subunit with M r near 82,000 has greater intensity in the residue fractions - it may have a tendency to be more extensively crosslinked to other proteins and, hence, less soluble in the unreduced extract than the other HMW-glutenin subunits. This fourth subunit apparently corresponds to one of the ID y subunits (see Thompson et al. 23 for nomenclature), which do have more S-S than most of the other HMW-glutenin subunits (approximately 1·5 mole% vs. 0·5 mole% )21-24. On the other hand, the decrease in T2, which corresponds almost entirely to ro-gliadins, appears to result from the extract proteins (E2) as very little of this fraction is found in the residue, although some components can be noted in Fig. 1. The ro-gliadins contain little sulfur, some containing none at all, and thus they lack the ability to participate in disulfide crosslinking. Their lack of sulfur apparently is responsible for their strong increase in proportion in low-sulfur samples, as for the HMW-glutenin subunits. Both the ro-gliadins and the HMW-glutenin subunits appear to undergo a nearly 100 % increase in their proportions from the highest-sulfur sample to the lowest-sulfur sample even though the HMW-subunits do have a significant amount of sulfur and might be expected to be less favored in their synthesis under low-sulfur conditions. Our data might not be precise enough to show a small difference. The proteins of T3, which correspond mainly to LMW-glutenin subunits, but also include a significant amount of gliadins, were largely found in the unreduced SDS extract (E3). The extracted proteins should include most of the gliadins along with some of the

PROTEINS IN SULFUR-DEFICIENT AND NORMAL WHEAT

241

LMW-glutenin subunits, which are extracted as disulfide-bonded aggregates. A substantial proportion of the proteins (about one-third), however, remained in the residue; these unextracted proteins are most likely incorporated into relatively insoluble glutenin aggregates - perhaps of large size. T3 increased slightly with increase in 8: N showing only a weak correlation (Table II). Our results appear to complement those of Timms et al. 5 , who studied the size distribution of SDS-extractable proteins (without reduction of 8-8) by gel-filtration chromatography. Under conditions of high nitrogen fertilizer supply, but limiting sulfur availability, there was a reduction in the proportion of' higher-molecular-weight glutenin aggregates' in their extracts. Their results might be interpreted as resulting from a decrease in solubility of the highest molecular weight glutenin aggregates, rather than from their absence, in the flour. These HMW aggregates would correspond mainly to our Rl and R3 fractions (disulfide-crosslinked HMW- and LMW-glutenin subunits), and Rl increased dramatically with decrease in S. The proteins ofT4, which correspond mainly to C/.-, p-and y-gliadins, but also include a small amount of LMW-glutenin subunits, were also found almost entirely in the unreduced extract, as were the proteins ofT5, which correspond almost entirely to wheat albumins (although small amounts of globulins are likely to be present in this fraction as well). T4, E4, T5 and E5 all increased with increase in S: N. This was most notable for the albumins of E5, which is to be expected as these proteins have about three times as much sulfur as gliadins and about nine times as much sulfur as the HMW-glutenin subunits. Even so, their increase in proportion is only about 41 % over the series - about equivalent to the decrease found for the HMW-glutenin subunits and ro-gliadins. The changes in proportion we measured for various types of endosperm proteins when sulfur becomes limiting do not appear to be directly related to the amount of sulfur in each type of protein; albumins may be under a different type of control from the gliadin and glutenin proteins. Although it is not especially pertinent to the interpretations of our results, it should be noted that our scan areas for albumins are disproportionately large because these proteins are rich in basic groups as well as sulfur-containing amino acids and tend to bind roughly twice as much dye as the gliadin proteins 1fi • Correlations between scan areas and quality characteristics

Some of the scan areas showed correlations with flour quality measurements for the set of twelve flours. These are shown in Table II in the form of a correlation matrix. The extent of the changes is shown in Table III for selected areas and attributes where significant correlations were found. The percentage of variance (V) accounted for was very high for several of the areas with respect to S: N. This correlation probably indicates the sulfur content of the grain protein, since protein is the main source of nitrogen in the mature grain. The S: N ratio probably indicates the change in properties with change in the proportion of S-rich and S-poor proteins, which has already been shown to vary with the availability of sulfur8 • 9 • Here we show changes relating to solubility fractions. Highly significant correlations with dough quality parameters were obtained for some of the areas (fractions), particularly Brabender Extensigraph parameters (R5, resistance of a dough to extension at an extension of 5 cm; E, maximum extensibility of the dough; and Resistance Ratio (Res Ratio), ratio of maximum resistance to maximum extension).

J. G. FULLINGTON ET AL.

242

TABLE III. Extent of variation of selected scan areas (y) with %8, 8:N, and quality attributes of flour (x) as linear regressions in the form y = mx+b, where m is slope and b is intercept. Percentage (v) of variance accounted for is also shown. Areas with low correlation coefficients have been omitted Flour attribute (x) Area (y) E2 E3 E4 E5 R1 R2

%8 m b v m b v m b v m b v

-100 26 66 30 12 32 65 17 74 78 16 32

m

-57

b

v m

b

v

Tl

m

T2

v m

T3

v m

T4

v m

T5

v m

b b

b b b

v

11

59 -8-1 2·6 36 -65 17 66 -108 28 66 33

17

62 67 20 73 73 17 30

8:N

Ext

Res 5

Res ratio

-185 27 92 43 13 23 97 18 63 178 13 75

-0,61 27 65 0·21 45 0·40 16 74 0·48 15 32

0·04 4·1 72 -0,01 18 25 -0·02 30 52 -0·04 35 68

-105 12 82 -16 2-8 61

-0'36 12 64 -0'05 2·8 42

0'02 -1,7 79 0·003 0·78 47

2·3 0·22 86 0·33 1·0 54

-118 18 89 -201 29 94 46 18 44 98 21 60 173 14 76

-0'40 18 65 -0,66 30 66 0·21 17 68 0·40 20 68 0·45 16 30

0·03 3·0

2·4 5·1 83 3·9 8·5 76 -0·89 23 35 -1,9 31 51 -3·5 32 69

11

77

0·04 4·9 74 -0·01 23 31 -0·02 33 48 -0,04 36 65

3·6 7·5 74 -1'0 18 31 -2,0 28 58 -3,7 32 70

These are shown in Tables II and III. The most significant correlations with quality measurements were found for proteins in higher ranges of M r (> 50,000). The negative correlations between extensibility and Sl and the positive correlation between resistance and TI arise from the residue fraction as they are reflected in the Rl values, but not in El. Significant correlations of the same sign were obtained for T2, but in this case, the

PROTEINS IN SULFUR-DEFICIENT AND NORMAL WHEAT

243

most significant correlations arose from the soluble components of the reduced extract (E2) rather than from the residue. The solubilities are as expected because Al corresponds almost entirely to HMW-glutenin subunits, which are not completely soluble until the glutenin complex (consisting of disulfide-bonded aggregates that include HMW- and LMW-glutenin subunits) is reduced, whereas A2 corresponds almost entirely to ro-gliadins, which contain no sulfur and are largely solubilized in the unreduced SDS extract. The signs of the correlations with extensibility and resistance seem reasonable for the Al proteins (Rl)-it might be expected that an increase in the amount ofHMW-glutenin subunits would decrease dough extensibility while increasing dough resistance. On the other hand, correlations of the same sign for A2 proteins (E2) would not be expected; increase in the amount of co-gliadins might be expected to increase extensibility, as was found for the a-, 13- and ')'-gliadins that predominate in A4 (E4). Either ro-gliadins have a very different action in doughs from other gliadins or these correlations indicate that the effects of the HMW-glutenin subunits predominate strongly over those of ro-gliadins. In the latter case, the correlated synthesis of these two types of low-sulfur (or no-sulfur) storage proteins, which increase in proportion with decrease in available sulfur, would result in both fractions having apparently the same effect on dough properties. Perhaps the relative effects on doughs of co-gliadins as compared with ct-, ~-and ')'-gliadins should be tested. In this regard, Tatham and Shewry 25 concluded that co-gliadins have a conformational structure rather different from those of the other gliadins; ro-gliadins are rich in ~-turns and bear some similarities in conformation to HMW-glutenin subunits 26 • While R3 contributed a significant proportion of the residue fraction, there were no significant correlations of R3 with dough properties. Because R3 includes most of the LMW-glutenin subunits, which are part of disulfide-bonded aggregates, we would have expected these proteins to show some correlation with dough properties. The lack of correlation might be related to the way in which the experiments were carried out and interpreted. The proteins of A3 and A4 (T3 and T4), which have intermediate levels of sulfur, seem to act in a pivotal way - showing little change in their proportions with change in sulfur content. Our result might be thought of in an alternative way: the increase in RI with little change in R3 as S: N decreased indicates that the disulfidebonded complexes of the residue have a much greater proportion of HMW-glutenin subunits. The ratio RI: R3 should show the same correlation as Rl. It may be noted that the higher the molecular weight of a glutenin fraction, the greater the proportion of HMW-glutenin subunits in the complex (Payne and Corfield 18 , D. D. Kasarda and N. Fulrath, unpublished results). The proteins of E3 are probably mainly gliadins, but it is known that substantial amounts of glutenin are extracted from flours by solvents containing SDS without reducing agent 5 and so E3 must include some LMW-glutenin subunits as well. E3 (and T3) correlates significantly with extensibility; this correlation may result from the gliadins, which are known to correlate positively with dough extensibility4, but a contribution from SDS-soluble glutenin complexes (some of which include only LMW-glutenin subunits 27 ) cannot be ruled out. The proteins of E4 (and T4) are almost entirely gliadins and their importance to dough properties can be seen in their strong correlation with extensibility and their negative correlation with resistance and Resistance Ratio (Tables II and III).

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The albumins (and globulins) ofES (and T5) showed strong negative correlations with resistance and Resistance Ratio, along with a strong positive correlation with breakdown (Table II). The correlation with extensibility was weak, however, suggesting that these proteins may not contribute much to the cohesiveness of a dough, instead disrupting the continuity of the proteinaceous matrix - in contrast to gliadins, which show strong correlation with extensibility. Several reports have indicated that poorer loaf volumes are obtained with sulfurdeficient flours 6 , 7,28. Results from other studies 8 , 29, 30 suggest that disulfide bonds of gluten proteins may help to stabilize crumb structure during baking, in addition to the major contribution of starch gelatinization. However, results from the present study (Table II) provided no clear indication of protein fractions that might contribute to such a role in terms of correlation with loaf volume. Relationship to SDS-sedimentation test

The results reported here have the practical significance of helping to provide a chemical explanation of the SDS-sedimentation test l l , which is used as a rapid procedure to predict dough properties. The test depends on determining the settled volume of insoluble material after suspension in an SDS solvent similar to the solvent used in this study. Figure I shows that the polypeptide composition of the residue fractions in the SDS test is virtually the same as the residue material examined in the present study and of glutenin aggregates. Tables II and III highlight the positive role of R I polypeptides in association with dough resistance (thus' strength') and the overall negative relationship of the readily extractable protein with dough resistance. Of course, it must be realized that the results presented here are for environmentally-related changes in only one variety. It is possible that different conclusions might be reached if a range of different genotypes were examined. These results complement the study of Moonen et al. 12 on the basis of the SDS-sedimentation test. We wish to thank Dr P. J. Randall and co-workers (CSIRO, Division of Plant Industry, Canberra) for providing the samples used in this study and Dr Peter Shewry (Rothamsted Experimental Station, Harpenden, U.K.) for helpful discussion of m-gliadin structure. Reference to a company or product by the U.S. Department of Agriculture in this publication is for purposes of information and does not imply approval or recommendation to the exclusion of others that may also be suitable.

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