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Effect of rye chromosome arm 2RS on flour proteins and physical dough properties in bread wheat
Journal of Cereal Science 10 (1989) 169-173
RAPID COMMUNICATION Effect of Rye Chromosome Arm 2RS on Flour Proteins and Physical Dough Properties in Bread Wheat R. B. GUPTA*t, K. W. SHEPHERD* and F. MACRITCHIEt
* University of Adelaide. Waite Agricultural Research Institute, Department of Agronomy, Glen Osmond, SA, 5064, Australia and t CSIRO Division of Plant Industry, Wheat Research Unit, P.O. Box 7, North Ryde. NSW, 2113, Australia Received 20 July 1989
The interchain disulphide-linked protein of wheat, glutenin, has been shown to have positive effects on physical dough properties l-3. Cereal rye also contains interchain disulphide-linked proteins, namely high molecular weight (HMW) and 75 k y-secalins4, but their effects on dough quality are not known. The 75 k y-secalins constitute approximately half of the mature rye endosperm proteins 4 and are controlled by the Sec-2 locus located on chromosome arm 2RS li • This paper describes the effects of chromosome arm 2RS, which codes for these 75 k y-secalins, on the amount and composition of flour proteins and physical dough properties. This chromosome arm had been transferred from rye cultivar Imperial into bread wheat cultivar Timgalen by producing a 2BL-2RS translocation line, but this stock was found to be poorly fertile 6 • Its fertility has been improved, however, by altering the genetic background?, and in the present study, this stock was further backcrossed into Gabo, and fertile lines with and without rye chromosome arm 2RS (chromotypes), were isolated. The backcross 1 F2 (BCl F2) seeds were analysed by one-dimensional SDS-PAGE 3 [Fig. leA)] and the 75 k y-secalins were used as genetic markers to score for the presence or absence of this chromosome arm. Twenty seed (10 for each chromotype) were selected and multiplied individually in the glass house, and the progeny were screened on gels to confirm their homozygosity. Finally, 14 lines (seven lines for each chromotype) were chosen that were homogeneous and morphologically similar to each other. Since Timgalen [Fig. leA), slot a], the original parent hybridized with this translocation, has Gabo in its parentage and has the same gliadin 8 and low molecular weight (LMW) glutenin subunits 9 band patterns as Gabo [Fig. leA), slot b] and showed only a few differences in HMW subunits of glutenin lO [Fig. leA)], uniformity for protein composition was quickly attained among these backcross lines. Moreover, selection for similar plant morphology increased the overall genetic similarity of these lines. Nevertheless, 7 lines of each class were selected to accommodate the effects of background gene segregation. These lines were grown in field-plots (dimension 0·6 x 4·0 m) at Roseworthy Agricultural College in South Australia in a completely randomized block, with two replications. 0733-5210/89/060169+05 $03.00/0
© 1989 Academic Press Limited CER 10
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FIGURE 1. A. One-dimensional SDS-PAGE patterns, under reducing conditions, of total flour proteins from (a) Timgalen, (b) Gabo, (c) chromotypes 2BL-2BS and (d) 2BL-2RS and ofSE-HPLC fractions from a 2BL-2RS line that correspond to areas I (e, f), II (g) and III (h) in Fig. IB. B. SE-HPLC elution profiles of sonicated proteins from a 2BL-2RS line (------) and a normal
RYE CHROMOSOME ARM 2RS AND WHEAT QUALITY
171
The physical dough properties and protein concentration in the flour from each plot were determined using the Brabender Extensograph and a micro-Kjeldahl method, respectively, as described earlier:l. To determine the relative and absolute amounts of protein fractions of different molecular sizes in flours, proteins were completely extracted by sonicating flour samples in 2 % (wIv) SDS solution (pH 6,9) and were separated by Size Exclusion-HPLC ll using a Protein-Pak 300 column and acetonitrile/ water (1/1) with 0·1 % of trifluoroacetic acid as eluting solvent. The total SE-HPLC chromatogram was divided into three areas I, II and III [Fig. I(B)], and SDS-PAGE of the fractions corresponding to these areas revealed that they contained mainly glutenin (polymers), gliadins (monomers) and albumins/globulins (monomers), respectively [Fig. l(A), slots e-h]. Area I also contained some polymeric albumins 9 and globulins ll . The unreduced gel patterns of the fractions from area I (results not shown) gave only streakiness indicating that this contained only polymeric proteins. The data obtained were subjected to one-way analysis of variance, and, since there were no significant differences between the replicates (P> 0'08) for the parameters measured, the data from these replicates have been pooled (Table I). The 2BL-2RS translocation lines gave significantly higher dough resistance (RmaJ than the 2BL-2BS lines (normal lines). The increase in dough extensibility (Ext) was not significant. The doughs did not exhibit any stickiness in the mixing bowl as found with wheat-rye IR substitution and translocation lines 12 . The SE-HPLC elution profiles of total protein from these lines revealed that the 2BL-2RS lines had significantly higher amounts of polymeric proteins than the normal lines (absolute area I, Table I). However, differences in the amounts of monomeric proteins (absolute area II, III) between these lines were insignificant. This suggested that the higher protein levels in 2BL-2RS lines are largely due to increased amount of polymers (glutenins). Moreover, the increase in the polymers is associated with the 75 k y-secalins because they are additional polymeric proteins in the 2BL-2RS lines to those present in the normal sibs [Fig. leA), slots c, d]. The proportion of polymeric proteins was also significantly greater in the 2BL-2RS lines (% area I, Table I). Hence, the increase in Extensograph resistance for these lines can be attributed to the above changes in its glutenin quantity. However, it is important to note that the 75 k y-secalins form ~-turns that confer elasticity (Dr P. R. Shewry, pers. comm.), and this may also contribute to the greater dough elasticity in the 2BL-2RS lines. Contrary to these findings, Field and Shewry'\ suggested that incorporation of 7S k ysecalins in bread wheat might bring deleterious effects on bread-making and other technological properties. They argued that these secalins, due to the formation of alcohol-soluble oligomers and polymers, might decrease the proportion of insoluble glutenins (aggregates of larger sizes) correlated with good bread-making quality. In our study, however, the 2BL-2RS lines showed a greater proportion of large interchain disulphide-linked aggregates (see the leading end of peak 1), and these aggregates contained the secalin subunits [Fig. leA), slot e]. The data presented here thus provide evidence that incorporation of the 75 k y-secalins in bread wheat increases glutenin level, which leads to greater dough elasticity. The problem of the reduced grain yield of the 2BL-2RS line (Table I) needs to be overcome, however, to make it a useful line for breeding purposes. 9-2
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TABLE 1. Dough physical properties and grain yield of 2RS chromotypes (mean of 14 per chromotype) SE-HPLC area
Extensograph Chromotypes 2BL-2RS 2BL-2BS Significance (P)
Relative area (%)
?' p:l
Absolute area
R max (BU)
Ext (em)
I
II
III
I
II
III
298 271
17-4 17-0
48-8 45-8
39-6 40-5
11-7 13-6
76 65
62 56
0-006
0-432
< 0-001
0·103
0-635
<0-001
0·15
Protein
Grain wt
(%)
(g)
19 19
13-8 11-8
330 372
0-96
0-001
0-0~9
0
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RYE CHROMOSOME ARM 2RS AND WHEAT QUALITY
173
We are grateful to Mr P. Ellis, Mr G. McCormack and Dr 1. L. Batey for their technical assistance. R. B. G. acknowledges financial support from the University of Adelaide and the Australian Wheat Research Council.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12.
MacRitchie, F. J. Cereal Sci. 6 (1987) 259-268. Payne, P. I. Ann. Rev. Plant Physiol. 38 (\987) 141-153. Gupta, R. B., Singh, N. K. and Shepherd, K. W. Theor. Appl. Genet. 77 (1989) 57-64. Field, J. M. and Shewry, P. R. J. Cereal Sci. 6 (\987) 199-210. Shewry, P. R., Parmar, S., Fulrath, N., Kasarda, D. D. and Miller, T. E. Can. J. Genet. Cytol. 28 (1986) 76-83. May, C. E. and Appels, R. Cereal Res. Cornrnun. 6 (1978) 231-234. May, C. E. and Appels, R. TheOl·. Appl. Genet. 68 (1984) 163-168. Wrigley, C. W., Lawrence, G. J. and Shepherd, K. W. Aust. J. Plant Physiol. 9 (1982) 15-30. Gupta, R. B. (1989) Ph. D. Thesis, University of Adelaide. Lawrence, G. J. Aust. J. Agric. Res. 37 (1986) 125-133. Singh, N. K., Donovan, G. R., Batey, I. L. and MacRitchie, F. Cereal Chern. (in press). Dhaliwal, A. S., Mares, D. J., Marshall, D. R. and Skerritt, 1. H. Cereal Chern. 65 (1988) 143--149.
RAPID COMMUNICATION Effect of Rye Chromosome Arm 2RS on Flour Proteins and Physical Dough Properties in Bread Wheat R. B. GUPTA*t, K. W. SHEPHERD* and F. MACRITCHIEt
* University of Adelaide. Waite Agricultural Research Institute, Department of Agronomy, Glen Osmond, SA, 5064, Australia and t CSIRO Division of Plant Industry, Wheat Research Unit, P.O. Box 7, North Ryde. NSW, 2113, Australia Received 20 July 1989
The interchain disulphide-linked protein of wheat, glutenin, has been shown to have positive effects on physical dough properties l-3. Cereal rye also contains interchain disulphide-linked proteins, namely high molecular weight (HMW) and 75 k y-secalins4, but their effects on dough quality are not known. The 75 k y-secalins constitute approximately half of the mature rye endosperm proteins 4 and are controlled by the Sec-2 locus located on chromosome arm 2RS li • This paper describes the effects of chromosome arm 2RS, which codes for these 75 k y-secalins, on the amount and composition of flour proteins and physical dough properties. This chromosome arm had been transferred from rye cultivar Imperial into bread wheat cultivar Timgalen by producing a 2BL-2RS translocation line, but this stock was found to be poorly fertile 6 • Its fertility has been improved, however, by altering the genetic background?, and in the present study, this stock was further backcrossed into Gabo, and fertile lines with and without rye chromosome arm 2RS (chromotypes), were isolated. The backcross 1 F2 (BCl F2) seeds were analysed by one-dimensional SDS-PAGE 3 [Fig. leA)] and the 75 k y-secalins were used as genetic markers to score for the presence or absence of this chromosome arm. Twenty seed (10 for each chromotype) were selected and multiplied individually in the glass house, and the progeny were screened on gels to confirm their homozygosity. Finally, 14 lines (seven lines for each chromotype) were chosen that were homogeneous and morphologically similar to each other. Since Timgalen [Fig. leA), slot a], the original parent hybridized with this translocation, has Gabo in its parentage and has the same gliadin 8 and low molecular weight (LMW) glutenin subunits 9 band patterns as Gabo [Fig. leA), slot b] and showed only a few differences in HMW subunits of glutenin lO [Fig. leA)], uniformity for protein composition was quickly attained among these backcross lines. Moreover, selection for similar plant morphology increased the overall genetic similarity of these lines. Nevertheless, 7 lines of each class were selected to accommodate the effects of background gene segregation. These lines were grown in field-plots (dimension 0·6 x 4·0 m) at Roseworthy Agricultural College in South Australia in a completely randomized block, with two replications. 0733-5210/89/060169+05 $03.00/0
© 1989 Academic Press Limited CER 10
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1
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o iii "5
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FIGURE 1. A. One-dimensional SDS-PAGE patterns, under reducing conditions, of total flour proteins from (a) Timgalen, (b) Gabo, (c) chromotypes 2BL-2BS and (d) 2BL-2RS and ofSE-HPLC fractions from a 2BL-2RS line that correspond to areas I (e, f), II (g) and III (h) in Fig. IB. B. SE-HPLC elution profiles of sonicated proteins from a 2BL-2RS line (------) and a normal
RYE CHROMOSOME ARM 2RS AND WHEAT QUALITY
171
The physical dough properties and protein concentration in the flour from each plot were determined using the Brabender Extensograph and a micro-Kjeldahl method, respectively, as described earlier:l. To determine the relative and absolute amounts of protein fractions of different molecular sizes in flours, proteins were completely extracted by sonicating flour samples in 2 % (wIv) SDS solution (pH 6,9) and were separated by Size Exclusion-HPLC ll using a Protein-Pak 300 column and acetonitrile/ water (1/1) with 0·1 % of trifluoroacetic acid as eluting solvent. The total SE-HPLC chromatogram was divided into three areas I, II and III [Fig. I(B)], and SDS-PAGE of the fractions corresponding to these areas revealed that they contained mainly glutenin (polymers), gliadins (monomers) and albumins/globulins (monomers), respectively [Fig. l(A), slots e-h]. Area I also contained some polymeric albumins 9 and globulins ll . The unreduced gel patterns of the fractions from area I (results not shown) gave only streakiness indicating that this contained only polymeric proteins. The data obtained were subjected to one-way analysis of variance, and, since there were no significant differences between the replicates (P> 0'08) for the parameters measured, the data from these replicates have been pooled (Table I). The 2BL-2RS translocation lines gave significantly higher dough resistance (RmaJ than the 2BL-2BS lines (normal lines). The increase in dough extensibility (Ext) was not significant. The doughs did not exhibit any stickiness in the mixing bowl as found with wheat-rye IR substitution and translocation lines 12 . The SE-HPLC elution profiles of total protein from these lines revealed that the 2BL-2RS lines had significantly higher amounts of polymeric proteins than the normal lines (absolute area I, Table I). However, differences in the amounts of monomeric proteins (absolute area II, III) between these lines were insignificant. This suggested that the higher protein levels in 2BL-2RS lines are largely due to increased amount of polymers (glutenins). Moreover, the increase in the polymers is associated with the 75 k y-secalins because they are additional polymeric proteins in the 2BL-2RS lines to those present in the normal sibs [Fig. leA), slots c, d]. The proportion of polymeric proteins was also significantly greater in the 2BL-2RS lines (% area I, Table I). Hence, the increase in Extensograph resistance for these lines can be attributed to the above changes in its glutenin quantity. However, it is important to note that the 75 k y-secalins form ~-turns that confer elasticity (Dr P. R. Shewry, pers. comm.), and this may also contribute to the greater dough elasticity in the 2BL-2RS lines. Contrary to these findings, Field and Shewry'\ suggested that incorporation of 7S k ysecalins in bread wheat might bring deleterious effects on bread-making and other technological properties. They argued that these secalins, due to the formation of alcohol-soluble oligomers and polymers, might decrease the proportion of insoluble glutenins (aggregates of larger sizes) correlated with good bread-making quality. In our study, however, the 2BL-2RS lines showed a greater proportion of large interchain disulphide-linked aggregates (see the leading end of peak 1), and these aggregates contained the secalin subunits [Fig. leA), slot e]. The data presented here thus provide evidence that incorporation of the 75 k y-secalins in bread wheat increases glutenin level, which leads to greater dough elasticity. The problem of the reduced grain yield of the 2BL-2RS line (Table I) needs to be overcome, however, to make it a useful line for breeding purposes. 9-2
-..l
tv
TABLE 1. Dough physical properties and grain yield of 2RS chromotypes (mean of 14 per chromotype) SE-HPLC area
Extensograph Chromotypes 2BL-2RS 2BL-2BS Significance (P)
Relative area (%)
?' p:l
Absolute area
R max (BU)
Ext (em)
I
II
III
I
II
III
298 271
17-4 17-0
48-8 45-8
39-6 40-5
11-7 13-6
76 65
62 56
0-006
0-432
< 0-001
0·103
0-635
<0-001
0·15
Protein
Grain wt
(%)
(g)
19 19
13-8 11-8
330 372
0-96
0-001
0-0~9
0
C
...,
'"t:I
>-
..., r-. t"
RYE CHROMOSOME ARM 2RS AND WHEAT QUALITY
173
We are grateful to Mr P. Ellis, Mr G. McCormack and Dr 1. L. Batey for their technical assistance. R. B. G. acknowledges financial support from the University of Adelaide and the Australian Wheat Research Council.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12.
MacRitchie, F. J. Cereal Sci. 6 (1987) 259-268. Payne, P. I. Ann. Rev. Plant Physiol. 38 (\987) 141-153. Gupta, R. B., Singh, N. K. and Shepherd, K. W. Theor. Appl. Genet. 77 (1989) 57-64. Field, J. M. and Shewry, P. R. J. Cereal Sci. 6 (\987) 199-210. Shewry, P. R., Parmar, S., Fulrath, N., Kasarda, D. D. and Miller, T. E. Can. J. Genet. Cytol. 28 (1986) 76-83. May, C. E. and Appels, R. Cereal Res. Cornrnun. 6 (1978) 231-234. May, C. E. and Appels, R. TheOl·. Appl. Genet. 68 (1984) 163-168. Wrigley, C. W., Lawrence, G. J. and Shepherd, K. W. Aust. J. Plant Physiol. 9 (1982) 15-30. Gupta, R. B. (1989) Ph. D. Thesis, University of Adelaide. Lawrence, G. J. Aust. J. Agric. Res. 37 (1986) 125-133. Singh, N. K., Donovan, G. R., Batey, I. L. and MacRitchie, F. Cereal Chern. (in press). Dhaliwal, A. S., Mares, D. J., Marshall, D. R. and Skerritt, 1. H. Cereal Chern. 65 (1988) 143--149.