Characterization and comparative analysis of HMW glutenin 1Ay alleles with differential expressions

Abstract

Background: High-molecular-weight glutenin subunits (HMW-GSs) have been considered as most important seed storage proteins for wheat flour quality. 1Ay subunits are of great interest because they are always silent in common wheat. The presence of expressed 1Ay subunits in diploid and tetraploid wheat genotypes makes it possible to investigate molecular information of active 1Ay genes.

Results: We identified 1Ay subunits with different electrophoretic mobility from 141 accessions of diploid and tetraploid wheats, and obtained the complete ORFs and 5' flanking sequences of 1Ay genes including 6 active and 3 inactive ones. Furthermore, the 5' flanking sequences were characterized from 23 wild diploid species of Triticeae. All 6 active 1Ay possess a typical HMW-GS primary structure and some novel characteristics. The conserved cysteine residue within the repetitive domain of y-type subunits was replaced by phenylalanine residue in subunits of 1Ay (Tu-e1), 1Ay (Tu-e2), 1Ay (Ta-e2) and 1Ay (Td-e). Particularly, 1Ay (Ta-e3) has an unusual large molecular weight of 2202 bp and was one of the known largest y-type HMW-GSs. The translations of 1Ay (Tu-s), 1Ay (Ta-s) and 1Ay (Td-s) were disrupted by premature stop codons in their coding regions. The 5' flanking sequences of active and inactive 1Ay genes differ in a few base substitutions and insertions or deletions. The 85 bp deletions have been found in promoter regions of all 1Ay genes and the corresponding positions of 6 species from Aegilops and Hordeum.

Conclusion: The possession of larger molecular weight and fewer conserved cysteine residues are unique structural features of 1Ay genes; it would be interested to express them in bread wheat and further to examine their impact to processing quality of wheat. The 1Ay genes from T. urartu are closer to the genes from T. turgidum dicoccon and T. aestivum, than those from T. monococcum aegilopoides. The 85 bp deletion and some variations in the 5'flanking region, have not interrupted expression of 1Ay genes, whereas the defects in the coding regions could be responsible to the silence of the 1Ay genes. Some mutational events in more distant distal promoter regions are also possible causes for the inactivation of 1Ay genes.

Figure 1

SDS-PAGE analysis of high-molecular-weight glutenin subunits (HMW-GSs) of diploid and tetraploid wheat species. a Diploid accessions of T. urartu: (Tu1) PI428309, (Tu2) PI 428308, (Tu3) PI 428318, (Tu4) PI 428310; b, c: Diploid accessions of T. monococcum aegilopoides: (Ta1) PI 427928, (Ta2) PI 427759, (Ta3) PI 428007, (Ta4) PI 427622, (Ta5–6) Citr 17665, (Ta7–8) PI 277123, (Ta9–10) PI 306526; d: Tetraploid wheat accessions of T. turgidum dicoccon: (Td1–2) PI 355475, (Td3–4) PI 355477; CS: Chinese spring. The SDS-PAGE profiles of HWM-GSs showed 1Ay subunits were differentially expressed in some accessions of T. urartu, T. monococcum aegilopoides and T. turgidum dicoccon while 1Ax subunits were expressed in all accessions (marked by tailed-arrows). The expressed 1Ay subunits were marked by solid and the hollow arrows indicated the area where the absent subunit band might have been.

Figure 2

PCR amplification of HMW-GS ORFs. Lane1–3: PI 428309, PI 428318, PI 428308 (T. urartu); lane 4–7: PI 428007, PI 277123, PI 306526, PI 427928 (T. monococcum aegilopoides) and lane 8 and 9: PI355475, PI355477 (T. turgidum dicoccon); M is 1 Kb DNA ladder.

Figure 3

Comparison of the primary structure of 1Ay subunits from different wheat species. Signal peptide was underlined; N-terminal and C-terminal regions were boxed, respectively. Conserved cysteine residues were indicated by solid arrows while the substitutions of cysteine residues with phenylalanine residue (F) were marked by hollow arrows. The in-frame stop codons were represented by asterisks and boxed.

Figure 4

Comparison of the 5' flanking sequences of 9 Glu-A1-2 alleles characterized in this study with those of Glu-A1-2, Glu-B1-2 and Glu-D1-2, represented by 1Ay (Cheyenne), 1By9 and 1Dy10. The regulatory elements E motif, N motif, partial HMW enhancer and complete HMW enhancer were boxed and labelled, respectively. TATA box was indicated by asterisks; and translational start codon was underlined. This comparison showed that the 85-bp fragment (marked by shadow) was deleted at the 5' flanking sequences of all alleles of Glu-A1-2. The 5' flanking sequences of Glu-A1-2 alleles from wild diploid, tetraploid and hexaploid wheat species shared high degree of homology.

Figure 5

Comparative analysis of partial 5' flanking region sequences of y-type HMW-GSs from 23 wild diploid relative species of wheat. The deletion of 85 bp fragment (marked by shadow) was also observed in six diploid species of Ae. umbellulata (U), Ae. uniaristata (N), H. bogdanii (H), H. brevisubulatum (H), H. bulbosum (I) and H. spontaneaum (H).

Figure 6

Phylogenetic relationships of 1Ay alleles from diploid, tetraploid and hexaploid wheat species with previously published HMW-GS genes encoded by Glu-B1-2 and Glu-D1-2 loci (represented by 1By9 and 1Dy10, respectively). The phylogenetic tree was created based on the multiple alignment of the 5' flanking sequences plus the sequences encoding the signal peptides and N-terminal regions. The corresponding sequence of 1Dx5 was used as outgroup, and bootstrap analysis was conducted with 1000 replicates.