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Genome-, Transcriptome- and Proteome-Wide Analyses of the Gliadin Gene Families in Triticum urartu.

Zhang Y, Luo G, Liu D, Wang D, Yang W, Sun J, Zhang A, Zhan K - PLoS ONE (2015)

Bottom Line: An RNA sequencing (RNA-Seq) survey of the dynamic expression patterns of gliadin genes revealed that their synthesis in immature grains began prior to 10 days post-anthesis (DPA), peaked at 15 DPA and gradually decreased at 20 DPA.The phylogenetic analysis demonstrated that the homologs of these α-gliadin genes were present in tetraploid and hexaploid wheat, which was consistent with T. urartu being the A-genome progenitor species.This study presents a systematic investigation of the gliadin gene families in T. urartu that spans the genome, transcriptome and proteome, and it provides new information to better understand the molecular structure, expression profiles and evolution of the gliadin genes in T. urartu and common wheat.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy/The Collaborative Innovation Center of Grain Crops in Henan, Henan Agricultural University, Zhengzhou, China.

ABSTRACT
Gliadins are the major components of storage proteins in wheat grains, and they play an essential role in the dough extensibility and nutritional quality of flour. Because of the large number of the gliadin family members, the high level of sequence identity, and the lack of abundant genomic data for Triticum species, identifying the full complement of gliadin family genes in hexaploid wheat remains challenging. Triticum urartu is a wild diploid wheat species and considered the A-genome donor of polyploid wheat species. The accession PI428198 (G1812) was chosen to determine the complete composition of the gliadin gene families in the wheat A-genome using the available draft genome. Using a PCR-based cloning strategy for genomic DNA and mRNA as well as a bioinformatics analysis of genomic sequence data, 28 gliadin genes were characterized. Of these genes, 23 were α-gliadin genes, three were γ-gliadin genes and two were ω-gliadin genes. An RNA sequencing (RNA-Seq) survey of the dynamic expression patterns of gliadin genes revealed that their synthesis in immature grains began prior to 10 days post-anthesis (DPA), peaked at 15 DPA and gradually decreased at 20 DPA. The accumulation of proteins encoded by 16 of the expressed gliadin genes was further verified and quantified using proteomic methods. The phylogenetic analysis demonstrated that the homologs of these α-gliadin genes were present in tetraploid and hexaploid wheat, which was consistent with T. urartu being the A-genome progenitor species. This study presents a systematic investigation of the gliadin gene families in T. urartu that spans the genome, transcriptome and proteome, and it provides new information to better understand the molecular structure, expression profiles and evolution of the gliadin genes in T. urartu and common wheat.

No MeSH data available.


Related in: MedlinePlus

Identification of the gliadin protein spots from T. urartu after resolution with 2-DE.Gliadins were prepared from mature grains, separated by 2-DE, and further identified via MALDI-TOF/TOF-MS analysis. Shown on the right side is the SDS-PAGE separation of prolamins from T. urartu. The high-molecular-weight glutenin subunit protein spots are not shown because of limited space. The spots in red circles are gliadins, the spots in blue circles are LMW-GSs (spot 14, KM085281, MW: 38.06, pI: 7.91; spot 15, KM085304, MW: 38.56 pI: 8.5; spot 16, KM085275, MW: 37.52, pI: 8.71), and the spot in purple is avenin-3 (TRIUR3_09156, MW: 35.1, pI: 7.66).
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pone.0131559.g003: Identification of the gliadin protein spots from T. urartu after resolution with 2-DE.Gliadins were prepared from mature grains, separated by 2-DE, and further identified via MALDI-TOF/TOF-MS analysis. Shown on the right side is the SDS-PAGE separation of prolamins from T. urartu. The high-molecular-weight glutenin subunit protein spots are not shown because of limited space. The spots in red circles are gliadins, the spots in blue circles are LMW-GSs (spot 14, KM085281, MW: 38.06, pI: 7.91; spot 15, KM085304, MW: 38.56 pI: 8.5; spot 16, KM085275, MW: 37.52, pI: 8.71), and the spot in purple is avenin-3 (TRIUR3_09156, MW: 35.1, pI: 7.66).

Mentions: In the 2-DE gel, 16 major spots in the regions of the gliadins and low-molecular-weight glutenin subunit (LMW-GS) genes were marked (Fig 3), excised, and analyzed. After performing the bioinformatics analysis of the PMFs and MALDI-TOF/TOF-MS spectra, 12 of the spots were found to be gliadin proteins (red circles) (Fig 3, Table 2, S7 and S8 Tables). The MALDI-TOF/TOF-MS spectra obtained from the protein spots were carefully compared with the predicted amino acid sequences of the 16 active gliadin genes isolated in this work. All of the gliadin spots were precisely matched to the genes cloned above (Table 2 and S7 Table). Of the remaining 12 gliadin spots, seven (spots 1, 2, 3, 4, 5, 6, and 7) were α-gliadins and five (spots 8, 9, 10, 11 and 12) were γ-gliadins; none was ω-gliadin (Fig 3 and Table 2). Among the matched α-gliadin spots, spots 2, 4, 5 and 7 were assigned as the products of Gli-α-4, Gli-α-8, Gli-α-9 and Gli-13, respectively, with between 3 and 12 peptides per spot matched to the corresponding gliadin (Fig 3 and S7 Table), whereas the remaining three spots (1, 3 and 6) were found to match the predicted proteins of more than one gliadin gene (Fig 3 and S7 Table). This phenomenon might have been caused by the extremely high similarity between these gliadin proteins, which had similar predicted molecular weights and pIs (Fig 1A and Table 2). Spot 1 contained six peptides that corresponded to both Gli-α-2 and Gli-α-3 and one unique peptide (QPQQLPQFEEIRN) to Gli-α-2 (S7 Table), and all six of the identified peptides in spot 6 could match both Gli-α-10 and Gli-α-11. However, spot 3 could represent a mixture of Gli-α-5, Gli-α-6, Gli-α-7 and Gli-α-12; most of its 11 peptides matched all four of these gliadins, which have similar pIs and molecular weights and share high levels of sequence identity (>98%) (Fig 1A and S7 Table). In addition, spots 8, 9 and 10 were assigned to the predicted Gli-γ-1 protein. This phenomenon has been observed in glutenin subunits previously, but the underlying reasons are still unclear [65]. Furthermore, none of the 16 analyzed spots corresponded to the hypothetical polypeptides encoded by the pseudogenes, which were predicted by ignoring the internal stop codons and frameshift mutations (Tables 1 and 2). Except 12 gliadin protein spots, the remaining four spots in the 2-DE gel were three LMW-GSs (spots 14, 15 and 16; blue circles) and one avenin-3 protein (spot 13; purple) (Fig 3, S6 and S7 Tables). In the avenin-3 protein, presence of even number of cysteine residue lead to the formation of intra-chain disulphide bonds, resulting in the monomers in the 2-DE gels (Fig 3), while only traces of three LMW-GSs were detected as monomers due probably to the presence of six intra-chain disulphide bonds (S7 and S8 Tables) or a cross contamination of gliadin by LMW-GS for the chemincal procedure fractionation [55].


Genome-, Transcriptome- and Proteome-Wide Analyses of the Gliadin Gene Families in Triticum urartu.

Zhang Y, Luo G, Liu D, Wang D, Yang W, Sun J, Zhang A, Zhan K - PLoS ONE (2015)

Identification of the gliadin protein spots from T. urartu after resolution with 2-DE.Gliadins were prepared from mature grains, separated by 2-DE, and further identified via MALDI-TOF/TOF-MS analysis. Shown on the right side is the SDS-PAGE separation of prolamins from T. urartu. The high-molecular-weight glutenin subunit protein spots are not shown because of limited space. The spots in red circles are gliadins, the spots in blue circles are LMW-GSs (spot 14, KM085281, MW: 38.06, pI: 7.91; spot 15, KM085304, MW: 38.56 pI: 8.5; spot 16, KM085275, MW: 37.52, pI: 8.71), and the spot in purple is avenin-3 (TRIUR3_09156, MW: 35.1, pI: 7.66).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489009&req=5

pone.0131559.g003: Identification of the gliadin protein spots from T. urartu after resolution with 2-DE.Gliadins were prepared from mature grains, separated by 2-DE, and further identified via MALDI-TOF/TOF-MS analysis. Shown on the right side is the SDS-PAGE separation of prolamins from T. urartu. The high-molecular-weight glutenin subunit protein spots are not shown because of limited space. The spots in red circles are gliadins, the spots in blue circles are LMW-GSs (spot 14, KM085281, MW: 38.06, pI: 7.91; spot 15, KM085304, MW: 38.56 pI: 8.5; spot 16, KM085275, MW: 37.52, pI: 8.71), and the spot in purple is avenin-3 (TRIUR3_09156, MW: 35.1, pI: 7.66).
Mentions: In the 2-DE gel, 16 major spots in the regions of the gliadins and low-molecular-weight glutenin subunit (LMW-GS) genes were marked (Fig 3), excised, and analyzed. After performing the bioinformatics analysis of the PMFs and MALDI-TOF/TOF-MS spectra, 12 of the spots were found to be gliadin proteins (red circles) (Fig 3, Table 2, S7 and S8 Tables). The MALDI-TOF/TOF-MS spectra obtained from the protein spots were carefully compared with the predicted amino acid sequences of the 16 active gliadin genes isolated in this work. All of the gliadin spots were precisely matched to the genes cloned above (Table 2 and S7 Table). Of the remaining 12 gliadin spots, seven (spots 1, 2, 3, 4, 5, 6, and 7) were α-gliadins and five (spots 8, 9, 10, 11 and 12) were γ-gliadins; none was ω-gliadin (Fig 3 and Table 2). Among the matched α-gliadin spots, spots 2, 4, 5 and 7 were assigned as the products of Gli-α-4, Gli-α-8, Gli-α-9 and Gli-13, respectively, with between 3 and 12 peptides per spot matched to the corresponding gliadin (Fig 3 and S7 Table), whereas the remaining three spots (1, 3 and 6) were found to match the predicted proteins of more than one gliadin gene (Fig 3 and S7 Table). This phenomenon might have been caused by the extremely high similarity between these gliadin proteins, which had similar predicted molecular weights and pIs (Fig 1A and Table 2). Spot 1 contained six peptides that corresponded to both Gli-α-2 and Gli-α-3 and one unique peptide (QPQQLPQFEEIRN) to Gli-α-2 (S7 Table), and all six of the identified peptides in spot 6 could match both Gli-α-10 and Gli-α-11. However, spot 3 could represent a mixture of Gli-α-5, Gli-α-6, Gli-α-7 and Gli-α-12; most of its 11 peptides matched all four of these gliadins, which have similar pIs and molecular weights and share high levels of sequence identity (>98%) (Fig 1A and S7 Table). In addition, spots 8, 9 and 10 were assigned to the predicted Gli-γ-1 protein. This phenomenon has been observed in glutenin subunits previously, but the underlying reasons are still unclear [65]. Furthermore, none of the 16 analyzed spots corresponded to the hypothetical polypeptides encoded by the pseudogenes, which were predicted by ignoring the internal stop codons and frameshift mutations (Tables 1 and 2). Except 12 gliadin protein spots, the remaining four spots in the 2-DE gel were three LMW-GSs (spots 14, 15 and 16; blue circles) and one avenin-3 protein (spot 13; purple) (Fig 3, S6 and S7 Tables). In the avenin-3 protein, presence of even number of cysteine residue lead to the formation of intra-chain disulphide bonds, resulting in the monomers in the 2-DE gels (Fig 3), while only traces of three LMW-GSs were detected as monomers due probably to the presence of six intra-chain disulphide bonds (S7 and S8 Tables) or a cross contamination of gliadin by LMW-GS for the chemincal procedure fractionation [55].

Bottom Line: An RNA sequencing (RNA-Seq) survey of the dynamic expression patterns of gliadin genes revealed that their synthesis in immature grains began prior to 10 days post-anthesis (DPA), peaked at 15 DPA and gradually decreased at 20 DPA.The phylogenetic analysis demonstrated that the homologs of these α-gliadin genes were present in tetraploid and hexaploid wheat, which was consistent with T. urartu being the A-genome progenitor species.This study presents a systematic investigation of the gliadin gene families in T. urartu that spans the genome, transcriptome and proteome, and it provides new information to better understand the molecular structure, expression profiles and evolution of the gliadin genes in T. urartu and common wheat.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy/The Collaborative Innovation Center of Grain Crops in Henan, Henan Agricultural University, Zhengzhou, China.

ABSTRACT
Gliadins are the major components of storage proteins in wheat grains, and they play an essential role in the dough extensibility and nutritional quality of flour. Because of the large number of the gliadin family members, the high level of sequence identity, and the lack of abundant genomic data for Triticum species, identifying the full complement of gliadin family genes in hexaploid wheat remains challenging. Triticum urartu is a wild diploid wheat species and considered the A-genome donor of polyploid wheat species. The accession PI428198 (G1812) was chosen to determine the complete composition of the gliadin gene families in the wheat A-genome using the available draft genome. Using a PCR-based cloning strategy for genomic DNA and mRNA as well as a bioinformatics analysis of genomic sequence data, 28 gliadin genes were characterized. Of these genes, 23 were α-gliadin genes, three were γ-gliadin genes and two were ω-gliadin genes. An RNA sequencing (RNA-Seq) survey of the dynamic expression patterns of gliadin genes revealed that their synthesis in immature grains began prior to 10 days post-anthesis (DPA), peaked at 15 DPA and gradually decreased at 20 DPA. The accumulation of proteins encoded by 16 of the expressed gliadin genes was further verified and quantified using proteomic methods. The phylogenetic analysis demonstrated that the homologs of these α-gliadin genes were present in tetraploid and hexaploid wheat, which was consistent with T. urartu being the A-genome progenitor species. This study presents a systematic investigation of the gliadin gene families in T. urartu that spans the genome, transcriptome and proteome, and it provides new information to better understand the molecular structure, expression profiles and evolution of the gliadin genes in T. urartu and common wheat.

No MeSH data available.


Related in: MedlinePlus