Journal of Cereal Science 48 (2008) 457e463 www.elsevier.com/locate/jcs
Introgression of transgenes into a commercial cultivar confirms differential effects of HMW subunits 1Ax1 and 1Dx5 on gluten properties J. Michael Field a, Dhan Bhandari b, Arturo Bonet b, Claudia Underwood c, Helen Darlington c, Peter Shewry c,* a
b
Advanta Seeds UK Ltd., Docking, King’s Lynn, Norfolk PE31 8LS, UK Campden & Chorleywood Food Research Association, Chipping Campden, Gloucestershire GL55 6LD, UK c Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Received 7 August 2007; received in revised form 15 October 2007; accepted 17 October 2007
Abstract Transgenes encoding the HMW subunits 1Ax1 and 1Dx5 have been transferred from ‘‘model’’ wheat lines into the commercial French bread wheat cultivar Soissons, using three backcrosses. Five pairs of BC3 expressing and null lines were isolated from each cross and multiplied to provide grain for functionality studies. Analysis of white flour samples confirmed the expression of the transgenes. SE-HPLC and Reomixer studies showed that the two transgenes had differential effects on dough functional properties. Thus, subunit 1Dx5 resulted in detrimental effects on dough development which were associated with decreased extractability of large glutenin polymers. In contrast, lines expressing subunit 1Ax1 contained increased proportions of extractable large glutenin polymers with three lines showing higher torque at similar mixing times (i.e. increased dough strength). This confirms the results obtained with the model wheat lines and shows that the 1Ax1 transgene can be used to increase dough strength in commercial cultivars. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Transgenic wheat; Gluten proteins; Mixing properties
1. Introduction Transgenesis is a powerful tool to determine fundamental aspects of gene function and structure/function relationships in wheat and other cereals and also has the potential to develop novel variation for use in plant breeding programmes (Shewry and Jones, 2006). Early work on transgenic wheat focused on the manipulation of grain quality, and in particular on the expression of additional copies of genes encoding the high molecular weight (HMW) subunits of wheat glutenin (Altpeter et al., 1996; Alvarez et al., 2000; Barro et al., 1997; Blechl and Anderson,
* Corresponding author. Tel.: þ44 1582 763133. E-mail address:
[email protected] (P. Shewry). 0733-5210/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.10.010
1996; Blechl et al., 2007) because of their role in determining dough strength (Payne, 1987; Shewry et al., 2003). However, the procedure for wheat transformation is still not routine and the efficiency still varies widely between genotypes. Hence, most transgenic lines are generated in ‘‘model’’ genotypes selected for their ease of transformation rather than in high yielding modern cultivars which may provide a more effective assessment of the effects of the transgenes. In particular, the cultivar Bobwhite has high embryogenic capacity and has been used in a number of studies (Altpeter et al., 1996; Anderson and Blechl, 2000; Blechl and Anderson, 1996; Blechl et al., 2007, 1998; Bregitzer et al., 2006). Ideally, it will eventually be possible to transform all wheat genotypes with a similar efficiency but this is not likely without considerably more investment. An alternative strategy is to introgress the transgenes from ‘‘model’’ genotypes into advanced cultivars, using classical plant breeding.
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We have reported detailed studies of a series of transgenic wheat lines produced in two model backgrounds selected to compare the impacts of two HMW subunit transgenes (Barro et al.,1997, 2002, 2003; Darlington et al., 2003; Rooke et al.,1999, 2003; Shewry et al., 2006). However, these lines were not selected to determine the potential for improving the quality of advanced commercial wheats by transgenesis and to evaluate this we have transferred the transgenes to a current commercial cultivar, Soissons, using conventional crossing. Analysis of these lines demonstrates that studies in model lines can be translated to commercial cultivars, including the ability to increase dough strength. 2. Materials and methods The production and characterisation of the transgenic wheat lines B102-1-2 (expressing HMW subunit 1Ax1), B72-8-12 and B73-6-1 (both expressing HMW subunit 1Dx5) has been reported previously (Barro et al., 1997; Rooke et al., 2003). These lines were grown in a glasshouse under containment conditions and crossed with cv Soissons. The F1 lines were then backcrossed three times to cv Soissons and five pairs of null and expressing lines selected from each cross. These lines were multiplied with cv Soissons and B102-1-2 in a containment glasshouse, using a randomised block design with three 20 cm pots of each line, each containing five plants. Grain from the blocks (i.e. from 15 plants) was milled with a Brabender Quadrumat Junior laboratory mill. Grain nitrogen was determined by Dumas analysis using a Leco FP428 combustion analyser and multiplied by 5.7 to give grain protein. SDSePAGE was carried out as described by Fido et al. (1995) and quantified by gel scanning using phoretixTM software (Nonlinear Dynamics, Newcastle, UK), as described by Shewry et al. (2006). SE-HPLC was as described by Millar (2003) and Morel et al. (2000). Dough mixing was measured using a Reomixer with data analysis as described by Anderson (2003). 3. Results and discussion Soissons is a winter wheat cultivar bred in France but also grown in the UK, where its early maturity can be an advantage in marketing. It is also favoured by millers and bakers because it mills well with a good extraction rate and gives strong and stable dough suitable for breadmaking and blending with weaker wheats. The high quality is consistent with the HMW subunit composition of Soissons, which contains ‘‘quality associated’’ subunits encoded by all three genomes (1Ax2*, 1Dx5 þ 1Dy10, 1Bx7 þ 1By8). This gives a quality score of 10 according to the system of Payne et al. (1987). We therefore selected Soissons as a parent for introgression of transgenes from three lines. The 1Dx5 transgene is expressed in lines B73-6-1 and B72-8-12 but at higher levels in the former (as discussed below) while the 1Ax1 transgene is expressed in B102-1-2. These lines were crossed with Soissons and the F1 seeds backcrossed three times to Soissons, selecting at each generation for lines with similar gluten
Fig. 1. SDSePAGE of transgenic and control lines of cv Soissons. (A) From crosses with B72-8-12; (B) from crosses with B73-6-1; (C) from crosses with B102-1-2. G Indicates pairs of sister lines expressing the transgene/not expressing the transgene (null). HMW subunits are labelled 1, 2*, 5, 7, 8, 10 with 5* indicating a larger form of subunit 5 present in lines derived from B73-6-1. The arrow in (C) indicates a faint band corresponding to subunit 1Ax1 in a putative control line, showing the presence of contamination. The brackets in (B) indicate a group of u-gliadins whose pattern differs in lines 184/185 and 188/189 from that in the other pairs of lines.
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protein composition by SDSePAGE to that of Soissons except for the additional HMW subunit encoded by the transgene. At this stage, single homozygous null and homozygous expressing seeds were selected from five individual plants from each cross and multiplied without further selection for functionality testing. SDSePAGE analyses of total grain protein extracts from these 15 pairs of lines are shown in Fig. 1, together with Soissons and B102-1-2 as controls. This showed that one of the putative control lines from the B102-1-2 cross (line 192) was contaminated with transgenic grain (see faint band indicated by the arrow in Fig. 1C), and this line and its corresponding null control (line 193) were therefore omitted from further studies. The transgenic lines derived from B736-1 also contain a larger form of subunit 1Dx5 (labelled 5* in Fig. 1B) which may be encoded by a rearranged gene. This has been discussed by Rooke et al. (1999). Fig 1B also shows that two patterns of u-gliadins are present in the lines derived from B73-6-1, being different in lines 180, 181, 182, 183, 186 and 187 and in lines 184, 185, 188 and 189, respectively (see brackets in Fig. 1B). However, gel scanning showed that this group of bands accounted for similar proportions of the total proteins irrespective of the pattern: 7.69 G 0.99% of the total in the first group of lines and 7.80 G 1.29% in the second. Previous studies showed that transgenes encoding subunit 1Ax1 are integrated at two unlinked loci in B102-1-2 (Rooke et al., 2003). This was confirmed in the present study, analyses of F3 grain from 140 F2 plants showing that 68 were homozygous positive, 12 homozygous null and 60 heterozygous. This corresponds to a 9:(3 þ 3):1 ratio (c2 ¼ 3.746 on 2df; p > 0.05) showing the presence of two unlinked loci. Segregation in the staining intensity of subunit 1Ax1 was apparent in the crosses made with this line, and selection was therefore made for lines in which the band stained less intensely than in B102-1-2, assuming that these contained only single transgene loci. The resulting lines did not segregate when selfed and were therefore assumed to be homozygous and contain only single transgene loci. The flour protein content was determined as ranging between 13.00 and 14.04% dry wt (Table 1). No significant differences were observed between the protein contents of the transgenic lines expressing the 1Dx5 transgene and the
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corresponding null control lines. In the case of the lines derived from B102-1-2, the mean protein content of the null lines was about 0.6% less than that of the transgenic lines but this difference was not significant statistically (13.38 G 0.42% compared with 14.04 G 0.42%). Quantitative gel scanning of SDSePAGE separations (Table 1) showed that the subunit 1Ax1 encoded by the transgenes accounted for approximately 4% of the total gluten proteins in the lines derived from B102-1-2. Subunit 1Dx5 accounted for an average of 8.6% of the total gluten proteins in the transgenic lines derived from B73-6-1 and 4.3% of the total gluten proteins in the lines derived from B72-8-12, but this protein represented the combined products of the endogenous gene and transgenes. The endogenous 1Dx5 protein accounted for about 3.3% of the total gluten proteins in the null segregant lines from both crosses. It can be concluded that the relative expression level of the 1Dx5 transgene(s) was greater in the lines derived from B73-6-1 than in the lines derived from B72-8-12. However, it is not possible to calculate precise expression levels of the transgenes in these lines. This is because compensatory effects may occur in the expression of other storage protein genes, including the endogenous form of the 1Dx5 gene. The proportions of monomeric, oligomeric and polymeric gluten proteins in the flours were determined by SE-HPLC essentially as described by Morel et al. (2000). This method uses sonication in 1% SDS in 0.1 M phosphate buffer, pH 6.9 to extract the total grain proteins, which are then separated into five fractions corresponding broadly to high molecular mass glutenin polymers (F1), lower molecular mass glutenin polymers (F2), u-gliadins (F3), a-type and g-type gliadins (F4) and albumins and globulins (F5). The total area (F1e F5) therefore provides an estimate of the total extractable proteins. The sonication procedure results in shearing of glutenin polymers and hence the sizes of the polymers separated by SE-HPLC do not accurately reflect the sizes of those present in vivo. However, the relative amounts of the peaks do relate to dough strength with %F1/%F2 and (%F3 þ %F4)/%F1 showing particularly strong correlations (Millar, 2003). The SE-HPLC results for the 30 lines and for the control samples of Soissons and B102-1-2 are summarised in Table 2.
Table 1 Proportions of subunits 1Ax1 and 1Dx5 in the lines expressing the transgenes and in control lines, expressed as % total HMW subunits and % total gluten protein Lines
170, 172, 171, 173, 180, 182, 181, 183, 190, 194, 191, 195, Soissons B102-1-2
174, 175, 184, 185, 196, 197,
176, 177, 186, 187, 198 199
178 179 188 189
Origin of lines
Transgenes expressed
Total protein (N 5.7)
Cross with B72-8-12 Cross with B72-8-12 Cross with B73-6-1 Cross with B73-6-1 Cross with B102-1-2 Cross with B102-1-2 Parent Transgenic parent
1Dx5 Null 1Dx5 Null 1Ax1 Null e 1Ax1
13.28 13.00 13.24 13.00 14.04 13.38 13.50 12.86
(0.64) (0.67) (0.85) (0.54) (0.42) (0.42)
HMW subunit as % total HMW subunits
HMW subunit as % gluten protein
1Ax1
1Dx1
1Ax1
1Dx1
e e e e 18.2 (1.79) e 20.2 (1.95) e
24.3 19.7 40.9 19.0 e e e 32.0
e e e e 3.65 (0.37) e 3.40 (0.23) e
4.3 3.3 8.6 3.3 e e e 3.68
(2.21) (2.09) (2.37) (1.89)
(2.30)
(0.39) (0.26) (0.96) (0.28)
(0.13)
Values are means of the individual lines with standard deviations in parentheses. Values for Soissons and B102-1-2 are for single samples. Note: the values for subunit 1Dx5 include the protein encoded by the endogenous gene in lines derived from B72-8-12 and B73-6-1, and include a minor band corresponding to a rearranged form of subunit 1Dx5 (indicated as 5* in Fig. 1B) in the lines derived from B73-6-1.
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Table 2 The total area (AT) and ratios of peak areas of total grain protein fractions separated by SE-HPLC Lines 170, 172, 171, 173, 180, 182, 181, 183, 190, 194, 191, 195, Soissons B102-1-2
174, 175, 184, 185, 196, 197,
176, 177, 186, 187, 198 199
178 179 188 189
Origin of lines
Transgenes expressed
AT (AU)
%F1/%F2
(%F3 þ %F4)/%F1
Cross with B72-8-12 Cross with B72-8-12 Cross with B73-6-1 Cross with B73-6-1 Cross with B102-1-2 Cross with B102-1-2 Parent Transgenic parent
1Dx5 Null 1Dx5 Null 1Ax1 Null e 1Ax1
20.30 26.50 18.84 26.90 28.47 27.7 27.0 28.8
0.586 0.634 0.594 0.642 0.645 0.622 0.64 0.54
4.08 (0.30) 3.05 (0.09) 4.41 (0.44) 2.98 (0.09) 2.85 (0.1) 3.06 (0.16) 2.99 3.75
(1.50) (1.09) (1.07) (1.06) (0.85) (0.77)
(0.015) (0.005) (0.015) (0.019) (0.006) (0.015)
The values for the lines expressing the transgenes and the corresponding null control lines are the means of the individual lines with the standard deviations in parentheses. The values for Soissons and B102-1-2 are for single samples.
The differences between the control lines are consistent with their known difference in quality. Although B102-1-2 expresses the ‘‘quality associated’’ 1Ax1 transgene the
A
background line (L88-31) expresses only two subunits encoded by chromosome 1B (1Bx17 þ 1By18) (Lawrence et al., 1988) and hence the derived transgenic also shows lower
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Time (min) Fig. 2. Reomixer traces for the transgenic, null and control lines. (A) Lines derived from B73-6-1; (B) lines derived from B72-8-12; (C) lines derived from B102-12 (omitting lines 192/193 due to contamination of line 192); (D) mean traces of the series of transgenic/null lines shown in (A), (B), (D), omitting line 196. In (A) and (B) the null lines are shown as small dashed lines, expressing lines as large dashed lines and cv Soissons as unbroken lines. The same notation is used in (C) but with line 196 in red and B102-1-2 in blue.
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10 9 8 190 191 194 195 196 197 198 199 Soissons B102-1-2
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Time (min) Fig. 2 (continued)
quality than commercial cultivars (Popineau et al., 2001). Thus, although B102-1-2 had slightly higher extractable protein (AT) the ratio of %F1/%F2 was substantially lower and that of (%F3 þ %F4)/%F1 higher than in Soissons reflecting a lower content of HMW glutenin polymers. The introgressed lines expressing the 1Ax1 transgene also gave slightly higher values for AT but in this case %F1/%F2 was higher and (%F3 þ %F4)/%F1 lower, indicating increases in the proportions of HWM polymers. In contrast, the reverse effect was observed in the transgenic lines expressing the 1Dx5 protein from B72-8-12 or from B73-6-1, with the AT and %F1/%F2 being lower and the (%F3 þ %F4)/%F1 being higher in the transgenic compared with the control lines. Previous studies of B73-6-1 and B72-8-11b (which expresses the 1Dx5 transgene in the same L88-31 background as B72-8-12) showed similar effects. These appeared to result from the fact that the additional
subunit led to the formation of highly cross-linked polymers which could only be efficiently extracted by sonication when dithiothreitol was used to reduce disulphide bonds (Popineau et al., 2001). The dough mixing properties of the lines were determined using a Reomixer which measures the torque during dough development (Fig. 2). Soissons showed a typical mixing curve for a strong breadmaking wheat, showing maximum torque at about 8 min. In contrast, B102-1-2 showed weaker mixing properties with the torque reaching a maximum at about 3 min and then decreasing. Some variation was observed between the individual null and expressing lines derived from the cross with B73-6-1 (Fig. 2A) but this was not related to the differences in the patterns of u-gliadins in these lines. Variation was also observed between the individual null and expressing lines derived from the cross with B72-8-12 (Fig. 2B). Nevertheless, the effects
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Fig. 3. Principle components analysis of mixing characteristics determined using the Reomixer. The group in the lower left part of the figure comprises all null lines and transgenic lines derived from B102-1-2 (expressing the 1Ax1 transgene) while that in the lower right part comprises lines derived from B72-8-12 and B73-6-1 (expressing the 1Dx5 transgene). Numbers correspond to those used in Tables 1 and 2 with Soissons being 200 and B102-1-2 being 201.
were essentially the same in both of these series of lines derived from crosses to incorporate the transgene encoding subunit 1Dx5. In both series the transgenic lines failed to show normal dough development with the curves reaching a low plateau after only about 1.5 min. A similar failure to develop was reported for B73-6-1 and B72-8-11b by Popineau et al. (2001) using Mixograph analyses. They also reported that the viscoelastic plateau of gluten fractions isolated from these lines was similar to that of gluten fractions which had been enzymically cross-linked using transglutaminase, providing support for the proposed role of the subunit 1Dx5 in promoting cross-linking. The Reomixer curves obtained with the lines derived from crosses with B102-1-2 were more similar to that obtained with Soissons (Fig. 2C), but three of the lines expressing the transgene (190, 194, 198) clearly showed higher torques at similar mixing times. In contrast, line 196 gave a different curve with slow development over a long mixing time. We cannot account for this difference, as line 196 had a similar protein content (14.02%) and showed a similar level of subunit expression level to other lines in the series: 3.8 G 0.23% of the total gluten proteins in line 196 compared with a mean of 3.65 G 0.28% total gluten proteins for the series. Similarly, the values for %F1/%F2 and (%F3 þ %F4)/%F1 were within the range of the series: 0.65 and 2.75 compared with means for the whole series of 0.645 G 0.006 and 2.845 G 0.1, respectively. The mean Reomixer traces for the six series of lines (but excluding line 196 and lines 192 and 193 which showed evidence of contamination) are shown in Fig. 2D. The differences between the Reomixer traces of the lines are also illustrated by principle components analysis (PCA),
with PC1 and PC2 together accounting for about 88% of the total variation in the analysis (C. Anderson, personal communication) (Fig. 3). In this analysis, increasing peak torque (i.e. peak dough development and strength) is associated with negative values for PC1 while higher stability (i.e. decreased breakdown) is associated with positive values for PC2. Hence, strong breadmaking varieties are usually positioned in the bottom left hand quadrant (and occasionally in the top left hand quadrant) while weaker biscuit wheats are usually positioned in the bottom right hand quadrant. In Fig. 3, the lines expressing the 1Dx5 transgene form a discrete cluster with high scores for PC1 and low scores for PC2, while B102-1-2 is isolated in the top left hand quadrant. The remaining lines form a group in the bottom left hand quadrant having similar overall properties to Soissons. However, lines 190, 194, 196 and 198 are all at lower positions than their null counterparts, which indicates greater dough stability. Lines 190, 194 and 198 are to the right of the two controls (Soissons, B102-1-2) which indicates increased strength and longer peak development times. Although these increases are modest, it should be noted that Soissons is a relatively strong cultivar and hence only small increases can be expected. In conclusion, we have shown that introgression of transgenes from ‘‘model’’ into commercial genotypes is a valid strategy for improving grain quality, with the differential effects of the expressed proteins being retained in the advance background. Furthermore, we have shown that expression of additional genes encoding subunit 1Ax1 results in increased dough strength in a cultivar of good breadmaking quality, validating the strategy for future work.
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