Colaprico G

Lafiandra D

Margiotta B

Urbano M

English

Sydney eScholarship

 1B- and C-type low molecular weight glutenin subunits in tetraploid wheat germplasm  Margiotta B1, Colaprico G1, Lafiandra D2, Urbano M1 1 CNR Institute of Plant Genetics, Via G. Amendola 165/A, 70126 Bari, Italy 2DABAC, University of Tuscia, Via SC De Lellis, 01100 Viterbo, Italy  ABSTRACT Electrophoretic analyses of B and C-type low molecular weight glutenin subunits (LMW-GSs) in several tetraploid wheat accessions and in durum wheat lines, carrying a 1BL.1RS translocation and null at Gli-2 locus, have allowed the identification of new variants at the Gli-1/Glu-3 loci.  INTRODUCTION The B and C-type low molecular weight glutenin subunits (LMW-GSs) are structural components of the glutenin polymer affecting dough viscoelastic properties. They are encoded by genes at Glu-3, Gli-1 and Gli-2 loci present on the short arms of the homoeologous group 1 and 6 chromosomes in tetraploid and hexaploid wheats1. The B-type, considered typical LMW-GS, have been also subdivided into LMW-s, LMW-m, LMW-i, according to their first amino acid residue of the mature proteins. In durum wheat allelic variation at the linked Gli-B1/Glu-B3 loci has also been associated with pasta-making quality. A consistent relationship between the presence of a gliadin protein, termed γ-45 and gluten strength and the presence of a different gliadin protein, γ-42 and gluten weakness2 has been demonstrated. This was later shown to be due to genetic linkage of genes coding for these allelic gliadin components and those encoding for two groups of low-molecular-weight glutenin subunits, designated LMW-2 and LMW-1, the former group of subunits being the functionally active polypeptides determining gluten strength3. The C group of LMW-GS is quantitatively present in a lower amount, with respect to the B group, and has been studied to a limited extent4. N-terminal amino-acid sequencing has shown that they are made up of α/β and γ−gliadin-like components. This led to the hypothesis that they are encoded by genes present at the Glu-3 locus and possibly at the Gli-1 and Gli-2 loci. It has been suggested that the presence of gliadin-like subunits in glutenin preparations is very likely due to mutations that affect the number and/or the distribution of cysteine residues5. It is likely that mutated genes have acquired one extra or less cysteine residue, so that the encoded polypeptides frequently have odd numbers of cysteine residues. An odd number of cysteines makes such subunits act as chain terminators of growing glutenin polymer chains, which would presumably have a negative effect on flour quality6.  In order to increase knowledge about the role played by LMW-GS on quality characteristics of durum wheat and detect novel variation, B and C-type subunits have been extracted from different varieties and lines of tetraploid wheats and analysed by one- and two-dimensional electrophoresis and chromatographic techniques (RP-HPLC).  MATERIALS AND METHODS Plant material. A collection of 237 accessions of tetraploid wheats T. turgidum ssp. turanicum, ssp.  polonicum, ssp. carthlicum, aneuploid lines of the cv. Langdon, a durum wheat line carrying a 1BL.1RS translocation and a durum line null at Gli-A2 locus, have been analysed along with different cultivars and lines of durum and bread wheats used as references.  Electrophoretical analyses. Total proteins have been extracted from crushed endosperm halves following fractionated precipitation with hydro-alcoholic solvent and gliadin analysed by A-PAGE (pH 3.1). In order to obtain glutenin fractions enriched in B or C subunits, we used different precipitation steps involving increasing concentrations of propan-1-ol, based mainly on the modified methods described by Verbruggen et al. 7 and Masci et al.4.  Total proteins and the above fractions enriched of B+C, B- and C-type LMW-GSs have been separated by SDS-PAGE (1DE) and subsequently by IEFxSDS-PAGE (2DE) following the modified procedure reported by Ikeda et al.8.  Chromatographical analyses. Fractions containing LMW-GSs have been prepared for reversed-phase high performance liquid chromatography (RP-HPLC). Analyses have been performed essentially according to the procedure of Marchylo et al. 9. RESULTS AND DISCUSSION Electrophoretic separation of different groups of LMW-GS present in the durum wheat cultivar Svevo is reported in Fig.1. Though some overlap is evident between the B- and C- fractions, the results can be used in further studies.  A similar separation of the B and C-type LMW-GSs present in the durum wheat cultivar Langdon and derived D-genome substitution lines, have permitted complete chromosomal assignment of components of the two group of subunits after their electrophoretic and chromatographic separations indicating association of the majority of C-type subunits to the Gli-1 and Gli-2  2 loci. The procedure adopted to obtain glutenin fractions enriched in B- or C-type subunits, has been applied to identify novel LMW-GS alleles in the T. turgidum ssp. turanicum, polonicum, carthlicum collection showing, however, the difficulties in resolving the electrophoretic and chromatographic patterns even if carried out by different precipitation steps.   Figure 1. SDS-PAGE separation of total proteins (T), B+C, B- and C-type LMW-GSs present in the durum wheat cv. Svevo.  Additional information on the C-type subunits were deduced by the 2DE of durum wheat cultivar Svevo and a derived near isogenic line in which a deletion on the short arm of the chromosome 6A is responsible of the absence of the entire Gli-A2 associated gliadin components. In this case, electrophoretic separation showed that absence of 6A encoded gliadin components was associated to the absence of some C-type subunits compared to Svevo (Fig. 2).    Figure 2. Comparison of enriched B- and C-type LMW-GS fractions  from cv. Svevo (a) and Svevo null at Gli-2 locus, separated by 2D gel electrophoresis.   RP-HPLC of the glutenin fractions extracted from different tetraploid wheats indicated the existence of variation in the B-type subunits, as reported in T. polonicum by other authors10, with most of the glutenin alleles similar to that of cultivated wheat. Analysis of C-type subunits showed extensive allelic variation compared to the one present in durum wheat cultivars (Fig. 3 and 4). Out of 237 accessions of domesticated tetraploid wheats analysed, 26 of T. turanicum, 19 of T. carthlicum and 20 of T. polonicum LMW-GS alleles have been detected. The new alleles provide the basis for further studies, at the molecular level, on these glutenin fractions to assess their structural organization in the gluten complex.   Figure 3. RP-HPLC separations of C-type LMW-GSs from durum wheat cv. Svevo and accessions of T. turanicum (16), T. polonicum (5), T. carthlicum (28).    Figure 4. RP-HPLC separations of C-type LMW-GSs from accessions  6, 1, 16, 53 of  T. turanicum.   Comparison of  electrophoretic and chromatographic analyses of B- and C-type LMW-GSs from tetraploid wheat T. turgidum ssp. durum and ssp. carthlicum, turanicum and polonicum have indicated the existence of a large variation in this material providing additional information about the association to Glu-3, Gli-1 and Gli-2 loci. The role of structural and quantitative variation associated to different allelic subunits will be assessed in order to shed light on this complex protein mixture strongly affecting wheat technological properties. REFERENCES 1. Jackson, E.A., Holt, L.M., Payne, P.I. 1983.  Characterisation of high molecular-weight gliadin and low-molecular-weight glutenin subunits of wheat endosperm by two-dimensional electrophoresis and the chromosomal localisation  3of their controlling genes. Theor. Appl. Genet., 66: 29–37. 2. Damidaux, R., Autran, J.C., Grignac, P., Feillet, P., 1978. Mise en e´vidence de relations applicables en sélection entre l’électrophorégramme des gliadines et les propriétés viscoélastiques du gluten de Triticum durum Desf.  associés à la qualité culinarie intrinséque des variétés. C.R. Acad. Sci. Paris, Ser. D 278: 701-/704. 3. Pogna, N.E., Autran, J.C., Mellini, F., Lafiandra, D., Feillet, P., 1990. Chromosome 1B-encoded gliadin and glutenin subunits in durum wheat: genetics and relationship to gluten strength. J. Cereal Sci. 11: 15-34. 4. Masci, S., Rovelli, L., Kasarda, D.D., Vensel, W.H.,  Lafiandra, D., 2002. Characterisation and chromosomal localisation of C-type low-molecular-weight glutenin subunits in the bread wheat cultivar Chinese Spring. Theor. Appl. Genet., 104: 422–428. 5. Masci, S., Lafiandra, D., Porceddu, E., Lew, E.J-L., Tao, H.P., Kasarda, D.D.  1993. D Glutenin subunits: N-terminal sequences and evidence for the presence of cysteine. Cereal Chem. 70: 581–585. 6. Tao, H.P., Kasarda, D.D. 1989. Two-Dimensional gel mapping and N-terminal sequencing of LMW-glutenin subunits. J. Exp. Bot. 40: 1015–1020. 7. Verbruggen, I.M., Veraverbeke, W.S., Vandamme, A., Delcour, J.A., 1998. Simultaneous isolation of wheat high-molecular-weight and low-molecular-weight glutenin subunits. J. Cereal Sci. 28: 25–32. 8. Ikeda, T.M., Araki, E., Fujita, Y.,  Yano, H. , 2006. Characterization of low-molecular-weight glutenin subunit genes and their protein products in common wheats. Theor. Appl. Genet., 112: 327–334. 9. Marchylo, B.A., Hatcher, D.W., Kruger, J.E. 1988. Identification of wheat cultivars by reversed-phase high-performance liquid chromatography of storage proteins. Cereal Chem. 65(1): 28-40. 10. Liu, C.Y., Shepherd, K.W. 1996. Variation of B subunits of glutenin in durum, wild and less-widely cultivated tetraploid wheats. Plant Breed. 115: 172-178. ACKNOWLEDGEMENT This work is supported by AGRO-GEN public/private laboratory for Plant Genomics (Lab.6  n. DM 18092). We also thank Dr. E. De Angelis and M. Lestingi for technical assistance.   

B- and C-type low molecular weight glutenin subunits in tetraploid wheat germplasm

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B- and C-type low molecular weight glutenin subunits in tetraploid wheat germplasm

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