Plant material and genetic transformation
The LMW-m and LMW-s type genes and their mutated versions used for wheat transformation were those reported by Ferrante et al.. One gene, named B1133-WT, corresponds to a native gene that was presumed to code for a LMW-m protein, although it is reported in GenBank as a γ-gliadin [GenBank:M11077] ; another one, named B1133-T23N, corresponds to the same gene mutated in position 23 to replace a threonine with an arginine. The third gene, named 42K-N23T, derives from a LMW-s type gene [EMBL:HG529977X], mutated in position 23 to replace an arginine with a threonine. This gene was isolated from genomic DNA of the bread wheat cultivar Yecora Rojo with primers reported in . These three genes were cloned separately into the SalI-XbaI restriction sites of pLRPT vector under control of the endosperm-specific HMW-GS Dx5 promoter . The cloning of each gene was achieved by PCR, amplifying the B1133-WT or B1133-T23N with primers SalHB1133F 5′-acagtcgacatgaagaccttcctcgtcttt-3′ and XbaHisFlagR 5′-tctagatcacttgtcatcgtcatccttgtagtcgtgatggtgatggtgatggt-3′, containing the sequences for the His- and FLAG-tags (see also below), whereas the 42K-N23T gene was amplified with the primers LMW42KSalF 5′-acagtcgacatgaagaccttcctcatcttt-3′ and LMW42KXbaIR 5′-tctagatcagtaggca ccaactccggt-3′. Since in the past we experienced multiple problems in LMW-42K gene [EMBL:Y17845] isolation, mostly due to rearrangements occurring during the cloning procedure because of the particular organization of the repeated domain , we deliberately decided not to add tags to this gene construct, which, although helping in protein identification and purification, might contribute to cloning difficulties and/or rearrangements.
PCR reactions were prepared in 50 μl containing 5 μl of 10X FastStart High Fidelity Reaction Buffer (Roche Diagnostics, Monza, Italy), 100 ng of genomic DNA, dNTP Mix 10 mM, 200 ng of each primer, 2.5 units of Fast Start High Fidelity DNA polymerase (Roche Diagnostics, Monza, Italy). The PCR program was: 95°C 2 min, 1 cycle, 95°C 1 min, 62°C 2 min, 72°C 2 min, 30 cycles; 72°C, 5 min. The amplification products were recovered from a 1.2% agarose gel, digested with SalI-XbaI and ligated into pLRPT. Constructs were verified by nucleotide sequencing and the B1133-WT showed a single substitution which caused the replacement of a serine with a phenylalanine in seventh position of the deduced mature sequence. Despite this substitution, this gene was used because we reasoned it would not interfere with our hypothesis.
The plasmid pAHC20  carrying the bar gene that confers resistance to the bialaphos herbicide, was co-bombarded in immature embryos of the durum wheat cultivar Svevo with each of the above LMW-GS genes in a 1:3 molar ratio by following the procedure reported by Volpi et al..
Construct schemes are reported in Additional file 1.
Genomic DNA extraction and PCR analysis of transformed wheat plants
Genomic DNA was extracted from 0.2 g of green tissue as reported in .
Transformed T0 plants were identified by PCR by using primers specific for the HMW-GS DX5 promoter and the terminator (PRDX5F 5′-catgcaggctaccttccac, PRDX5R 5′-cggtggactatcagtgaattg . The PCR conditions were those reported in the previous paragraph, except for a different extension time that was 1 min and 30 seconds. Positive plants were multiplied up to T4 generation, in order to obtain as many homozygous plants as possible to submit to proteomic analyses. Negative plants, corresponding to the null segregant plants, namely those transgenic plants that have lost the transgene by segregation, were also selected, multiplied up to the T4 generation, and used as control.
Plants, either wild type and transgenic lines, included the null segregant genotypes, were grown together in a growth chamber. T4 generation plants, previously screened by PCR, were used for proteome analyses, by pooling half seeds of PCR positive plants. Since we were interested only in the presence/absence of the transgenic proteins, we did not use formal replicates, but extracted proteins from each of at least four different positive lines obtained from transformation with each of the three transgenes, and compared to as many biological replicates of the null segregant.
Extraction of glutenin subunits
Seeds from the durum wheat cultivar Svevo as well from its transgenic lines (included null segregants) were crushed and 50 mg of flour were washed three times with 1 mL of 50% (v/v) propanol in order to remove the soluble protein fraction . In case of extraction of total glutenin subunits, the pellet was eventually extracted with a solution (1 mg: 10 μl) of 50% propanol containing 50 mM Tris-HCl pH 8.8, 1 mM EDTA, 10 mM iodoacetamide or 1.4% of 4-vynilpyridine, 1% (w/v) DTT for 1 h at 70°C. After centrifugation at 13,000 rpm for 15 min, four volumes of cold acetone were added to the recovered supernatant and kept overnight at -20°C to precipitate glutenin subunits. After centrifugation at 13,000 rpm for 15 min, the precipitated proteins were collected and dried in a Savant centrifuge.
In the case of the 42K-N23T protein, since tags were not added, in order to facilitate the identification of differences between null segregant and transformed genotypes, we used as a control a protein fraction enriched in B-type low-molecular weight glutenin subunits (similar in structure to the 42K-N23T protein) that was obtained according to .
2D electrophoretic analysis (IEF vs SDS-PAGE) of LMW-GS
Quantification of proteins prior to isoelectric focusing (IEF) was performed with the 2-D quant Kit Assay (GE Healthcare).
Total glutenin subunits or B subunits of LMW-GS were suspended in 250 μl of a solution composed of 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1.2% (v/v) Destreak Reagent, 0.5% (v/v) IPG buffer pH 3-10 and 6–11 for at least 2 hours. IEF was performed with the IPGphor™ Isoelectric Focusing System (Amersham Pharmacia Biotech) and was carried out on immobilized pH gradient (IPG) strips (18 cm, 1 mm) pH 3-10 (for plants transformed with B1133-WT and B1133-T23N genes) and pH 6-11 (for plants transformed with the 42 K-N23T gene). The strips were hydrated with samples overnight (12.30 h) at room temperature. IEF was performed at 90,000 volt-hours at 20°C. After focusing, the strips were equilibrated for 30 min at room temperature in a solution of 6 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, 50 mM Tris-HCl, pH 6.8, and 1% (w/v) DTT. Strips were then loaded on the top of a 1 mm thick by 18 cm SDS polyacrylamide gel, T 11%, C 1.28%, by using the Protean Plus Dodeca cell (Bio-Rad). Electrophoretic separation was carried out at 40 mA/gel, with cooling at 10°C. Gels were stained with Coomassie Blue G250  and destained in water before image acquisition.
The gel analyses were performed using the software SameSpots Progenesis (vers. 4.5.4293.47197, Nonlinear Dynamics, UK). This software includes statistical analyses such as ANOVA (p ≤ 0.05), and determination of False Discovery Rate (FDR, q ≤ 0.05).
Western blotting for amino acid sequencing
A 9 cm × 7 cm gel piece, corresponding to the region of interest, namely that including proteins in the molecular weight and pI ranges corresponding to the transgenic proteins, was cut out of the unstained 2D gel and electroblotted on Sequi-blot PVDF (polyvinylidene difluoride) membranes (Bio-Rad, Hercules, CA), previously wetted in methanol and rinsed with deionized water for 5 minutes before soaking in electroblot buffer (10 mM CAPS [3-cyclohexylamino-1-propanesulfonic acid], pH 11). Filter paper (Whatman 3 MM) was also soaked in electroblot buffer before use. Gel pieces were soaked in electroblot buffer for 5-10 minutes. Western blottings were performed using a Mini Trans Blot Cell module (Bio-Rad). Transfer was performed for 1 hour at 4°C, at a constant voltage of 100 V. The transfer stack was then dismantled and the membrane was rinsed with distilled water for 5 minutes before staining with Coomassie blue (0,025% (w/v) Coomassie R-250, 40% (v/v) methanol) for 5 minutes. The membranes were then de-stained for 5 minutes in 50% (v/v) methanol, briefly rinsed in distilled water and allowed to air dry at room temperature. Spots of interest were excised using a clean razor blade, and amino acid sequencing was performed essentially according to the procedure reported by Tao and Kasarda , but using an Applied Biosystems Procise 492 sequencer.
For immunoblotting experiments, the gel pieces containing the region of interest were incubated in transfer buffer (25 mM Tris- HCl, pH 8,0, 192 mM glycine and 0,04% SDS) for 15 minutes. The blotting was performed in the Mini Trans Blot Cell apparatus (Bio-Rad) using a PVDF membrane (Bio-Rad) according to the manufacturer’s protocol. After transferring, the membrane was saturated in 100 ml of Blocking solution (10 mM Tris-HCl pH 8, 150 mM NaCl, 01% Tween20 and 5% non-fat dry milk) at room temperature, on an orbital shaker, for 2 h. The membrane was then washed twice in washing buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl and 0.2% Tween20) and incubated overnight with an anti-Flag-tag polyclonal antibody (Sigma-Aldrich). After removal from the incubation buffer, the membrane was washed extensively and incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Merck Millipore) at room temperature, for 1 hour. The antigen-antibody complex was detected using the “Western blotting Luminol reagent” kit (Santa Cruz Biotechnology, Inc.) following the manufacturer’s procedure.
Mass spectrometry analysis
Coomassie Brilliant Blue stained bands were cut from the polyacrylamide gels and stored in 1.5 mL Eppendorf tubes. Immediately prior to enzymatic digestion, the gel pieces (1-3) were placed into the wells of a 96-well reaction plate that was positioned in an automated xyz robot (DigestPro, Intavis, Langenfeld, Germany) that automatically destained, reduced, alkylated and digested the proteins in the gel plugs with either chymotrypsin, thermolysin or trypsin. Twenty μg of the selected enzyme was used for each 96-well sample plate and digestion was performed at 35°C. At the end of the digestion period, the instrument eluted the samples of enzymatically cleaved peptides into a 96-well receiving plate that was then inserted into the autosampler interfaced with the QSTAR pulsar i hybrid quadrupole-TOF mass spectrometer (Applied Biosystems/MDX Sciex, Toronto, Canada) configured with an electrospray ionization (ESI) source (Protana, Odessa Denmark). Mass spectrometric analysis was performed as previously described . When sufficient material was available the samples were reanalyzed using the data obtained from the first mass spec analysis to form an exclusion list to allow previously unidentified spectra to be analyzed.
The resulting data were searched, using Mascot (http://www.matrixscience.com/) and X!Tandem (http://www.thegpm.org/) against a database containing 11,589 wheat protein sequences from NCBI T. aestivum plus a list of common laboratory contaminants (http://www.thegpm.org/) as well as expected sequences from the mutant and wild type expressed proteins. The results of the searches were combined, analyzed and visualized using Scaffold version 4.075 (http://www.proteomesoftware.com).