Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

Abstract

BackgroundThe gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.ResultsThe complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST) data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained nine cysteine residues. Only one of the encoded proteins was a perfect match with a sequence reported in NCBI. Contigs from four different publicly available EST assemblies encoded proteins that were perfect matches with some, but not all, of the Butte 86 gamma gliadins and the complement of identical proteins was different for each assembly. A specialized database that included the sequences of Butte 86 gamma gliadins was constructed for identification of flour proteins by tandem mass spectrometry (MS/MS). In a pilot experiment, proteins corresponding to six Butte 86 gamma gliadin contigs were distinguished by MS/MS, including one containing the extra cysteine residue. Two other proteins were identified as one of two closely related Butte 86 proteins but could not be distinguished unequivocally. Unique peptide tags specific for Butte 86 gamma gliadins are reported.ConclusionsInclusion of cultivar-specific gamma gliadin sequences in databases maximizes the number and quality of peptide identifications and increases sequence coverage of these gamma gliadins by MS/MS. This approach makes it possible to distinguish closely related proteins, to associate individual proteins with sequences of specific genes, and to evaluate proteomic data in a biological context to better address questions about wheat flour quality.

New insights into the effects of high temperature, drought and post-anthesis fertilizer on wheat grain development

“…Hajheidari et al (2007) detected changes in 121 spots with drought, but only 57 were identified. In addition, 36 minor spots that changed with fertilizer were not identified in the Altenbach et al (2011) study. Some proteins are difficult to identify by MS because they are of very low abundance.…”

“…Yang et al (2011) applied a water deficit from anthesis and Hajheidari et al (2007) limited the amount of water in field grown wheat from the booting stage throughout grain development. Grove et al (2009) examined proteins that changed in response to fertilization with sulfur (S) while Dupont et al (2006a,b) and Altenbach et al (2011) focused on proteins that changed in response to nitrogen (N) fertilization. Most studies assessed protein levels either late in development or at maturity.…”

“…Both are involved in glucose degradation. Glyceraldehyde-3-phosphate dehydrogenase (GADPH), involved in glycolysis, and granule-bound starch synthase responded to both high temperatures and drought, and triosephosphate isomerase, also Majoul et al, 2003 Thesee 18 C/10 C 3 4 C/10 C d Majoul et al, 2004 Thesee 18 C/10 C 3 4 C/10 C d Dupont et al, 2006a,b Butte 86 24 C/17 C plus NPK fertilization d,e 37 C/28 C plus NPK fertilization d,e Dupont et al, 2006a,b Butte 86 24 C/17 C minus NPK 37 C/28 C d minus NPK Sancho et al, 2008 Spark, Rialto, Soissons, Beaver 19.5 C daily mean, wet 23.2 C daily mean, dry (10% capacity) f Hurkman et al, 2009 Butte 86 24 C/17 C plus NPK fertilization e 37 C/28 C g plus NPK fertilization e Laino et al, 2010 T. durum 'Svevo' 20 C/17 C h 37 C/17 C h Yang et al, 2011 Vinjett 20 C/12 C i , then 24 C/16 C j (cA-cB) 20 C/12 C i , then 32 C/24 C j (cA-htB) Yang et al, 2011 Vinjett 20 C/12 C i , then 24 C/16 C j (cA-cB) 32 C/24 C i , then 32 C/24 C j (htA-htB) Yang et al, 2011 Vinjett 20 C/12 C i , then 24 C/16 C j (cA-cB) 20 C/12 C i , then 24 C/16 C þ water deficit j (cA-wdB) Hajheidari et al, 2007 Arvand Field grown, irrigated k Field grown, water deficit l Hajheidari et al, 2007 Khazar-1 Field grown, irrigated k Field grown, water deficit l Hajheidari et al, 2007 Kelk Afghani Field grown, irrigated k Field grown, water deficit l Grove et al, 2009 Bastian Minus S fertilization Plus S fertilization Altenbach et al, 2011 Butte 86 Table 2 Summary of non-gluten proteins identified in various 2-DE/MS studies that showed significant changes in relative abundance in response to temperature, drought or fertilizer treatments. þ indicates that spot increased, -indicates that spot decreased and þ/À indicates that one spot in a single study increased while another decreased.…”

Isolation and phylogenetic analysis of novel γ-gliadin genes in genus Dasypyrum

“…The molecular cloning and sequencing of these genes are helpful for better understanding the relationships of their structures and functions, and will provide vital information on gene evolution (Guo et al, 2010). In the Triticeae, γ-gliadin genes have been cloned from the genus Triticum (Anderson et al, 2001;Qi et al, 2009;Altenbach et al, 2010), Aegilops (Qi et al, 2009;Huang et al, 2010), Thinopyrum , and Crithopsis (Guo et al, 2010). However, no γ-gliadin genes have been cloned from the genus Dasypyrum.…”

Molecular characterization of gliadins of Chinese Spring wheat in relation to celiac disease elicitors

“…A total of 13 unique γ-gliadin encoding genes have been reported to be present in the Chinese Spring wheat genome, and 11 of them are active according to the ESTs and NGS analysis (Anderson et al 2013). In the cultivar Butte 86, nine γ-gliadin genes 5 encoding full length proteins were identified by EST analysis (Altenbach et al 2010).…”