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. 2012;7(11):e50057.
doi: 10.1371/journal.pone.0050057. Epub 2012 Nov 21.

Coexpression of the high molecular weight glutenin subunit 1Ax1 and puroindoline improves dough mixing properties in durum wheat (Triticum turgidum L. ssp. durum)

Yin Li  1 , Qiong Wang, Xiaoyan Li, Xin Xiao, Fusheng Sun, Cheng Wang, Wei Hu, Zhijuan Feng, Junli Chang, Mingjie Chen, Yuesheng Wang, Kexiu Li, Guangxiao Yang, Guangyuan He
Affiliations

Coexpression of the high molecular weight glutenin subunit 1Ax1 and puroindoline improves dough mixing properties in durum wheat (Triticum turgidum L. ssp. durum)

Yin Li et al. PLoS One. 2012.

Abstract

Wheat end-use quality mainly derives from two interrelated characteristics: the compositions of gluten proteins and grain hardness. The composition of gluten proteins determines dough rheological properties and thus confers the unique viscoelastic property on dough. One group of gluten proteins, high molecular weight glutenin subunits (HMW-GS), plays an important role in dough functional properties. On the other hand, grain hardness, which influences the milling process of flour, is controlled by Puroindoline a (Pina) and Puroindoline b (Pinb) genes. However, little is known about the combined effects of HMW-GS and PINs on dough functional properties. In this study, we crossed a Pina-expressing transgenic line with a 1Ax1-expressing line of durum wheat and screened out lines coexpressing 1Ax1 and Pina or lines expressing either 1Ax1 or Pina. Dough mixing analysis of these lines demonstrated that expression of 1Ax1 improved both dough strength and over-mixing tolerance, while expression of PINA detrimentally affected the dough resistance to extension. In lines coexpressing 1Ax1 and Pina, faster hydration of flour during mixing was observed possibly due to the lower water absorption and damaged starch caused by PINA expression. In addition, expression of 1Ax1 appeared to compensate the detrimental effect of PINA on dough resistance to extension. Consequently, coexpression of 1Ax1 and PINA in durum wheat had combined effects on dough mixing behaviors with a better dough strength and resistance to extension than those from lines expressing either 1Ax1 or Pina. The results in our study suggest that simultaneous modulation of dough strength and grain hardness in durum wheat could significantly improve its breadmaking quality and may not even impair its pastamaking potential. Therefore, coexpression of 1Ax1 and PINA in durum wheat has useful implications for breeding durum wheat with dual functionality (for pasta and bread) and may improve the economic values of durum wheat.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Scanning electron microscopy analyses of endosperms from transgenic and control lines.
Seeds from two lines expressing 1Ax1 and Pina (lines HP-19, HP-245), two lines expressing 1Ax1 (lines H-182, H-293), two lines expressing Pina (lines P-121, P-149), one null segregant line (lines N-1) and non-transformed control cv. Luna were subjected to scanning electron microscope analysis to reveal the structure differences of endosperm. Scale bar = 10 µm.
Figure 2
Figure 2. SDS-PAGE and Western blotting analyses of total and starch-bound PINA in transgenic and control lines.
A. SDS-PAGE of TX-114-soluble proteins isolated from flours of transgenic and control lines. B. Western blotting results of total PINA. C. Densitometry quantification of western blotting results of total PINA. D. SDS-PAGE of starch bound puroindolines isolated from flours of transgenic and control lines. E. Western blotting results of starch-bound PINA. F. Densitometry quantification of western blotting results of starch bound PINA. PINA protein is indicated by arrow on both stained SDS-PAGE and Western blots. Data are given as mean ± SEM. *and ** indicates the significant differences with the PINA levels of control variety Chinese Spring at 0.05 or 0.01 probability level, respectively.
Figure 3
Figure 3. Characterization of storage proteins in transgenic and control lines.
A. SDS-PAGE of seed protein extracts from transgenic lines, null segregant line and non-transformed control line. Transgenic 1Ax1 is indicated by arrow on the left side of the gel. B. Characterization of storage proteins from the transgenic and control lines. HMW % glutenin and LMW % glutenin means quantities of HMW-GS and LMW-GS, respectively, expressed related to total quantity of the glutenins (and the same for Ax, Bx and By). x/y: ratio of the x- and y- type HMW-GS. HMW/LMW: ratio of the high and low molecular weight glutenin subunits. Glutenin %: quantity of the glutenins expressed related to total proteins extracted by the sequential extraction methods (and the same for Gliadin %) . Glu/Glia: ratio of the glutenins and gliadins. 1Ax1+Pina = transgenic lines coexpressing 1Ax1 and Pina genes (dark grey); 1Ax1 = transgenic lines expressing only 1Ax1 (light grey); Pina = transgenic lines expressing only Pina (black). Control = both null segregant and non-transformed control lines (white). Data are given as mean ± SEM. Values within the same characteristics of storage proteins followed by the same letter are not significantly different (P<0.05).
Figure 4
Figure 4. Mixograph curves of dough prepared from transgenic and control lines.
Flour samples from two lines coexpressing 1Ax1 and Pina (lines HP-19, HP-245), two lines expressing 1Ax1 (lines H-182, H-293), two lines expressing Pina (lines P-121, P-149), one null segregant line (lines N-1) and non-transformed control cv. Luna were subjected to Mixograph analysis to reveal differences in dough mixing properties.
Figure 5
Figure 5. Combined effects of 1Ax1 and PINA on dough mixing properties.
Eleven mixing parameters were compared by Student’s t test between lines coexpressing 1Ax1and Pina (lines HP-19 and HP-245, represented by dark grey bar), lines expressing only 1Ax1 (lines H-182 and H-293, represented by light grey bar), lines expressing only Pina (lines P-121 and P-149, represented by black bar) and non-transformed control line (cv. Luna, represented by white bar). Data are given as mean ± SEM. *and ** indicates the significant differences with mixing parameters of non-transformed control Luna at 0.05 or 0.01 probability level, respectively.
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Grants and funding

This work was supported by the National Science and Technology Major Project of China (2011ZX08002-004; 2011ZX08010-004) (http://www.nmp.gov.cn/), International S & T Cooperation Key Projects of MoST (2009DFB30340), the National Natural Science Foundation of China (No.30871524 and No.31071403) (http://www.nsfc.gov.cn/Portal0/default152.htm) the National Natural Science Foundation of Hubei, China (2010 CBD 02403) and Wuhan Municipal S & T research project (201120922286). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.