Proteomic Determination of Low-Molecular-Weight Glutenin Subunit Composition in Aroona Near-Isogenic Lines and Standard Wheat Cultivars

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography–tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the ‘Aroona’ cultivar and 12 ‘Aroona’ near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for ‘Aroona’ and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in ‘Aroona’ and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.


Introduction
Gluten proteins comprise 70−80% of the total wheat flour protein; give wheat dough its unique viscoelastic properties; and make it possible to produce bread, pasta, noodles, and other products [1,2]. Gluten proteins can be separated on the basis of their solubility into monomeric gliadins that dissolve in 60-80% alcohol and polymeric glutenins that are weakly acidic and alkaline-soluble [3,4]. Glutenins consist of high-molecular-weight glutenin subunits (HMW-GSs; 70-90 kDa) and low-molecular-weight glutenin subunits (LMW-GSs; 20-45 kDa) that are polymerized by inter-molecular disulfide bonds [5]. HMW-GSs are encoded by genes at the Glu-1 loci (Glu-A1, Glu-B1, and Glu-D1) on the long arms of homoeologous group 1 chromosomes, with three to five active genes in most bread wheat cultivars. LMW-GSs are encoded by genes at the Glu-3 loci (Glu-A3, Glu-B3, and

Identification of LMW-GS Alleles Using RP-HPLC
To determine the allelic composition of low-molecular-weight glutenin subunits (LMW-GSs) in 'Aroona' and 12 'Aroona' near-isogenic lines (ARILs; Table 1), we extracted their glutenin fractions and analyzed them by RP-HPLC. The separation of LMW-GSs was optimized under the following conditions: linear gradient from 25 to 45% solvent B and 0.8 mL/min flow rate for 50 min at 65 • C. We observed five HMW-GS peaks between 5 and 20 min, and LMW-GS and gliadin peaks were detected between 20 and 45 min (Figure 1). HMW-GS alleles in 'Aroona' comprise Ax1 at the Glu-A1 locus, Bx7 and By9 at the Glu-B1 locus, and Dx2 and Dy12 at the Glu-D1 locus [32], which can be identified as peaks in RP-HPLC based on hydrophobicity [33]. We identified the peaks corresponding to each LMW-GS encoded at Glu-3 loci by comparing LC peak patterns in 'Aroona' with those in 12 ARILs: six alleles (a, b, c, d, e, and f ) at Glu-A3, six (a, b, c, d, g, and h) at Glu-B3, and three (b, c, and f ) at Glu-D3 (Figure 1). Table 1. Allelic compositions of LMW-GS at the Glu-A3, Glu-B3, and Glu-D3 loci in 'Aroona' and 'Aroona' near isogenic lines (ARILs).

Comparison of LMW-GSs among 'Aroona' and Its Near-Isogenic Lines Using 2-DGE
Spot patterns for LMW-GS alleles in 2-DGE were analyzed from 'Aroona' and its 12 ARILs. Previously, LMW-GS alleles encoded at the Glu-3 locus were successfully identified in 32 Korean wheat cultivars and 11 standard wheat cultivars [23]. These results facilitated the identification of LMW-GSs alleles in 'Aroona' and its ARILs.

Discussion
LMW-GSs are encoded by genes at Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3), with copy numbers varying from 10-40. LMW-GS alleles at the Glu-3 loci determine end-use quality. To evaluate the dough-processing qualities of wheat cultivars, LMW-GS alleles at the Glu-3 loci have been ranked for dough strength in wheat cultivars from Australia [25] and New Zealand [29] and for the 'Aroona' near-isogenic lines (ARILs) [19,31]. Since the composition of LMW-GS alleles in individual cultivars has been given priority during wheat breeding, SDS-PAGE, 2-DGE, HPLC, and LMW-GS molecular marker systems have been used to define LMW-GS alleles in different wheat cultivars. Previously, one to two peaks corresponding to LMW-GSs encoded at each Glu-3 locus were identified by the composition analysis of LMW-GS in common wheat cultivars using a RP-UHPLC method [24]. In this study, two to five LMW-GS peaks in RP-HPLC corresponding to Glu-A3, -B3, and -D3 alleles were determined through comparative analysis of 'Aroona' and the 12 ARILs. The accuracy was confirmed by comparing the resulting RP-HPLC patterns with patterns in standard wheat cultivars and suggests that our improved RP-HPLC method can be used to identify LMW-GS alleles faster, easier, and more accurately than traditional methods such as SDS-PAGE. However, three Glu-A3 alleles (Glu-A3a, -A3b, and -A3c) and two Glu-D3 alleles (Glu-D3b and -D3f ) could not be discriminated. Furthermore, by integrating our 2-DGE data on LMW-GSs in this study with the composition of LMW-GS genes previously characterized in 'Aroona' and ARILs [19,28,36], we created reference maps ( Figure 3) for protein spots corresponding to each LMW-GS allele in three standard cultivars, 'Gabo' (Glu-A3b, -B3b, and -D3b), 'Aroona' (Glu-A3c, -B3b, and -D3c), and 'Orca' (Glu-A3d, -B3d, and -D3c).

Line Alleles a Active LMW-GS Genes ( b Gene Name in Chinese Wheat Germplasm)
At Glu-3A

Plant Materials
'Aroona' and its 12 near-isogenic lines differing at the Glu-A3, Glu-B3, and Glu-D3 loci ( Table 1) were provided by Dr. Marie Appelbee and Prof. Ken Shepherd, SARDI Grain Quality Research Laboratory, South Australia. The grains of 10 standard wheat cultivars used for the identification of Glu-3 alleles (Supplementary Table S1) were kindly provided by the National Bioresource Project-Wheat, Japan (NBRP-Wheat, https://shigen.nig.ac.jp/ wheat/komugi/, 12 October 2015) and by the National Plant Germplasm System of the USDA-ARS, USA (NPGS, https://www.ars-grin.gov/npgs/, 12 October 2015). The grains of the wheat cultivars were ground with a mortar and pestle to fine powders and stored at −80 • C until glutenin extraction.

Glutenin Extraction
Glutenin was extracted as previously reported [23]. Wheat flour (300 mg) was mixed with 15 mL of 50% propanol (v/v) at 65 • C for 30 min and then centrifuged at 10,000× g for 5 min. The supernatant fraction containing gliadin was removed. This extraction was repeated three times to minimize gliadin contamination. The precipitate was suspended in 1.5 mL of 50% (v/v) propanol, 80 mM Tris-HCl (pH 8.0), and 1% (w/v) dithiothreitol (DTT) at 65 • C for 30 min. After centrifugation at 10,000× g for 5 min, 1.5 mL of 80 mM Tris-HCl (pH 8.0) with 1.4% 4-vinylpyridine (v/v) was added for alkylation and incubated at 65 • C for 15 min. After centrifugation at 10,000× g for 2 min, the supernatant was transferred to a new 1.5 mL tube and stored at −4 • C overnight. Glutenin fractions were precipitated using acetone containing 15% TCA (v/v) and stored at −20 • C until use.