Variation of High-Molecular-Weight Secalin Subunit Composition in

Oct 13, 2014 - Extractability and chromatographic separation of rye ( Secale cereale L.) flour proteins. Lore Redant , Joke Buggenhout , Kristof Brijs...
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Variation of High-Molecular-Weight Secalin Subunit Composition in Rye (Secale cereale L.) Inbred Lines Bolesław P. Salmanowicz,*,†,§ Monika Langner,†,§ and Helena Kubicka-Matusiewicz‡ †

Institute of Plant Genetics, Polish Academy of Sciences, PL 60-479 Poznań, Poland Center for Biological Diversity Conservation in Powsin, Polish Academy of Sciences Botanical Garden, PL 02-973 Warsaw, Poland

‡

ABSTRACT: In this study, identification and characterization of the rye HMW secalin subunit (HMW-SS) composition in 68 inbred rye (Secale cereale L.) lines was performed by capillary zone electrophoresis (CZE). The HMW-SS were separated in an uncoated fused-silica capillary using an isoelectric iminodiacetic buffer in combination with poly(ethylene oxide), lauryl sulfobetaine, and acetonitrile as the separation buffer. The separations of the nonalkylated HMW-SS provided very good resolution and high reproducibility. Generally, the x-type rye HMW-SS were more abundant and have longer migration times than the y-type subunits. Both types of rye HMW-SS were separated into the major protein peak and one or two minor peaks. In total, seven x-type HMW-SS, five of which were newly identified subunits, and six y-type subunits, four of which were new, were distinguished on the basis of their CZE migration times. The migration order of the rye HMW-SS using CZE differed considerably from the relative electrophoretic mobilities in the SDS-PAGE gels. KEYWORDS: allelic variation, Glu-R1 locus, capillary zone electrophoresis, HMW secalins, rye

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which is located on the long arms of chromosome 1R.15,16 This locus consists of two paralogous alleles of duplication origin that encode the x- and the y-types of the subunits. Consequently, a Glu-R1x and Glu-R1y designation for these genes has been proposed.17 Despite partial homology, the rye HMW-SS differ significantly from wheat HMW glutenin subunits (HMW-GS) with respect to their structural and quantitative parameters, which are especially important for the formation and properties of wheat gluten.17,18 The incorporation of the locus Glu-R1 into wheat has negatively impacted the wheat’s processing quality.19,20 Individual rye cultivars exhibit significant differences in bread-making quality, which is determined by the composition, quantity, and structure of the storage proteins, starches, and pentosans, in addition to genotypic-environmental interactions.21,22 Most rye cultivars are mixtures of genotypes, as rye is an open-pollinating species, and its varieties typically consist of outcrosses. An earlier study of HMW secalins in different rye cultivars and populations using SDS-PAGE analysis revealed high genetic variation and considerable diversity between the subunit band patterns of the different cultivars because of the intravarietal heterogeneity.10,11,23,24 In contrast with other cereals,24−26 this genetic diversity among rye cultivars presents problems with regard to the correct characterization of HMW subunits in the various cultivars and populations. The present work describes detection of allelic variation of the rye HMW secalin subunits in inbred lines by capillary zone electrophoresis (CZE) analysis. The HMW-SS isolated from rye inbread lines had not been previously separated and characterized by CZE method. A better knowledge of the rye

INTRODUCTION Rye (Secale cereale (S. cereale) L.) is one of the most important crops in the world due to its nutrient value, as it is well-adapted to climatically less favorable conditions and acidic soils of low productivity.1 Rye is predominantly cultivated in Europe, especially in the eastern, central, and northern regions of the continent. Rye grain is mainly used for animal feed, ethanol, and, to a lesser extent, bread and pasta.1,2 The global production of this cereal totaled approximately 16.5 million tons in 2013, and nearly 90% of its production was in Europe.3 Although the consumption of rye breads has declined over the past several decades, the consumption of rye-based products currently exhibits an increasing trend due to the inherently high nutritional quality attributed to the amino acid composition and dietary fiber found in rye.4−6 In addition, rye is a valuable genetic resource for wheat and triticale breeding as a source of favorable agronomic traits such as disease resistance and high yielding capacity. On the other hand, an introgression of some rye genes encoding HMW secalins into wheat has a negative influence on bread-making quality.7,8 The analysis of storage proteins is well-known to be a powerful tool for the identification and differentiation of cereal genotypes.9,10 The storage proteins of rye, usually termed secalins, are a complex and highly polymorphic mixture of polypeptides. Rye secalins are classified into four major types of proteins based on their electrophoresis mobilities in SDSPAGE gels. Three of them, the high-molecular-weight secalin subunits (HMW-SS), the 40,000 Da (40 kDa) γ-secalins, and the ω-secalins with molecular weights ranging from 45 to 50 kDa, are partially homologous with their corresponding protein types in wheat.11−13 The fourth type, the 75 kDa γ-secalins, are the most abundant group of secalins, accounting for greater than 50% of the total proteins in rye, but they do not have analogues in other cereals.11,14 The HMW-SS are found in an aggregated state and are encoded at the locus Glu-R1 (or Sec-3), © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10535

April 17, 2014 September 22, 2014 October 12, 2014 October 13, 2014 dx.doi.org/10.1021/jf502926q | J. Agric. Food Chem. 2014, 62, 10535−10541

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storage protein diversity and of the relationships between the considerable numbers of inbred lines should aid in the development of breeding programs that efficiently utilize the available rye germplasm. The high resolution and separation efficiency of the presented CZE method facilitated the rapid identification of the rye HMW-SS compositions in the lines. The qualitative compositions of the rye HMW-SS in the inbred lines determined by CZE in this study were then compared with SDS-PAGE data.

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Table 1. Sixty-eight Rye Inbred Lines Used in This Study with Determined HMW-SS Compositions cultivar/variety Pancerne

MATERIALS AND METHODS

Dankowskie Selekcyjne

Plant Materials. The experiment was performed on 68 inbred lines of winter rye (S. cereale L.) from generation S20, which were obtained by self-pollination and from the selection of 14 rye cultivars and two inbred forms (Table 1). Simultaneously grains from six rye cultivars (cv., accession number, origin country (Everest, 30206, FRA; Buriatskaya, 30308, RUS; Lekkeroggen, 30660, GER; Ponsi, 31004, SWE; Radosinska Record, 30143, SVK; Stel, 31032, SWE)) obtained from the National Centre for Plant Genetic Resources, Radzików, Poland and three standard triticale cultivars Binova (2*/13 + 16/5.1r + 6.4r), Eldorado (1/7 + 8/2r + 5.3r), and Titan (2*/7 + 18/1.2r + 6.5r) were analyzed. The inbred rye lines were obtained by self-pollination of the individual plants and of the sisters bred within the cultivars and the bred forms for 11 years. For this purpose, two ears from each plant and one ear from each of the two plants of a given variety or form were placed under a tomophane isolator. In the wax maturity phase, the isolators containing ears were cut off and the grains from the ears were manually collected. In the subsequent year, the seeds from the isolated plants from a given variety and a bred form were sown separately. This cycle was repeated for 5 years to obtain phenotypically equalized inbred lines of winter rye, which were subjected to self-pollination and selection for the subsequent 5 years. Using this approach, we obtained individual inbred lines of winter rye of genetically defined morphological features. Then, sets of 100 kernels from each of the selected inbred lines were sown every second year in plots of 1 m2. Prior to flowering, the plants were protected with canvas sheets that were spread on metal frames, which were then removed from plants representing a given line after flowering. The ears were manually cut at full maturity and were threshed in a laboratory threshing machine. Then, the seed germination energies and abilities were evaluated on the basis of observations of 100 kernels from each line sown in three batches. If the ability of the kernels to germinate was greater than 80% for a given inbred line, the seeds were dried to a moisture content of 7−8% in a chamber containing air with approximately 20% relative humidity and at 20 °C. Finally, the seeds from a given line were cleaned and sealed under a vacuum in laminated foil sheets. The samples of seeds representing the studied lines were stored in this form at −25 °C. Reagents and Chemical Materials. Iminodiacetic acid (IDA), (hydroxylpropyl)methylcellulose (HPMC), acetonitrile (AcN), poly(vinylpyrrolidone) (PVP; Mr ∼ 360,000), dithiothreitol (DTT), urea, and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (lauryl sulphobetaine, SB3-12) were purchased from Sigma (St. Louis, MO, USA). Poly(ethylene oxide) (PEO) with a molecular weight of 8,000,000 Da and acrylamide were acquired from Aldrich (Milwaukee, WI, USA). Sodium phosphate monobasic and dibasic, potassium phosphate monobasic, and sodium hydroxide were the products of J. T. Baker (Phillipsburg, NJ, USA). All chemicals were of electrophoresis or analytical grade. All solutions were prepared in deionized 18 MΩ·m water using a Milli-Q system (Millipore, Bedford, MA, USA). The solutions were filtered through a 0.5 μm Millipore membrane filter before being injected into the capillary. Apparatus. The CZE experiments were performed on a Beckman Coulter P/ACE system MDQ capillary electrophoresis instrument. The system was equipped with a high-voltage power supply, diode array detector, and 32 Karate ver. 8.0 software (Beckman Coulter) for

Forms short-stem from Jeleniec

Smolickie

Dankowskie Zlote

Chrobre

Wojcieszyckie

Omka Zelandzkie Uniwersalne Garczynskie Karlik Mikulickie Kamalinskaja4 Vjatka

no. of inbred lines

HMW-SS compositions

description of inbred lines

6

5.1r + 6.4r

L4, L420/97, D855/1, Mbj/4, H9/3, L96/4, L428/97w, L428/97n L228, L318/2 L427 L520, L140/3/7 L305, L230/2/9, L103, L18/1, L410/04, L147/1, L305/2, Ll1/94 L298/97, L169/1/10 BcL148bp/10, L9/99/8 L299, L310, L522, Mns/1, L518, L517,

2 2 1 2 8

5.2*r + 6.5r 5.1r 1.9r + 5.4r heterozygosity 5.1r + 6.4r

1 1 2 6

5.2*r + 6.5r 2r + 7r heterozygosity 5.1r + 6.4r

3 1 1 5

2r + 6.6r 5.3*r +7r heterozygosity 5.1r + 6.4r

2 1 1

2r + 7r 2.1r + 5.4r 5.3*r + 7r

L299/L1, J74/3, L237/94/1 H32 kz/2 Bj1, L186/2/2, bj/98, chlp/n, L185/2/1 L145, Nsegr/1/11/12 L68/7/8 L59

1 1 1 2 2 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1

5.2*r + 6.5r 6.1r + 7.2r heterozygosity 5.1r + 6.4r 5.2*r + 6.5r 5.3*r + 7 r heterozygosity 5.1r + 6.4r 2r + 7r heterozygosity 2r + 7r heterozygosity 5.1r + 6.4r 2r + 7r 2r + 6.6r 2r + 7r 5.1r + 6.4r 5.2*r + 6.5r 2.1r + 5.4r 2r + 6.6r

L176/1 lo segr L29/3/2 L208, kpl/2/18 L338, L155 M15/3 L154/6 L267/2/25 CH7/99 S67p/94/4 127 sl/2/1 Ch5/99 Ch2/99 L233, L260/94 8/segr/5 L128 L245 L127bk/97 pc-wch/95

system control and data handling. The separations were performed using uncoated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with internal diameter of 50 μm and 31.2 cm in total length (detection at 10.2 cm from the capillary outlet). The temperature was maintained at 40 °C during all runs. Extraction of HMW Secalin Subunits from Rye Flour. HMW secalin subunits were extracted from 50 mg of rye flour. First, twice preextraction was performed with 0.5 mL of 0.4 M NaCl and 0.067 M HKNaPO4 (pH 7.6) at room temperature to remove the albumins and globulins (mixing each time on a vortex mixer for 10 min and centrifuging for 5 min at 12500g). The 40 kDa γ- and ω-secalins were removed by two 10 min extractions using 70% (v/v) ethanol, followed by centrifugation at 15000g for 10 min. Then, the HMW-SS and 75 kDa γ-secalins were isolated from the sediment by an extraction for 30 min with 0.05 M Tris buffer (titrated by HCl to pH 7.5) containing 50% (v/v) 1-propanol, 2 M urea, and 1% (w/v) DTT at 60 °C under 10536

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Figure 1. SDS-PAGE patterns of rye high-molecular-weight secalin subunits (HMW-SS) from analyzed inbred lines (A) and cultivars (B). Panel A: lane 1, standard triticale cv. Vision (2*/6.8 + 20*/5.2r + 9.5r); lane 2, standard triticale cv. Eldorado (1/7 + 8/2r + 5.3r); lane 3, L428/97n (5.2*r + 6.5r); lane 4, L427 (1.9r + 5.4r); lane 5, L127bk/97 (2.1r + 5.4r); lane 6, L228 (5.1r); lane 7, L298/97 (5.2*r + 6.5r); lane 8, L260/94 (2r + 6.6r); lane 9, Ch2/99 (2r + 7r); lane 10, L59 (5.3*r + 7r); lane 11, lo segr (6.1r + 7.2r); lane 12, H9/3 (5.1r + 6.4r); lane 13, standard triticale cv. Binova (2*/13 + 16/5.1r + 6.4r); lane 14, standard triticale cv. Titan (2*/7 + 18/1.2r + 6.5r). Panel B: lanes 1−4, rye cv. Ponsi; lanes 5−8, rye cv. Stel; lanes 9−12, rye cv. Everest. separations were performed with a constant voltage of 10 kV at 40 °C. The samples were injected hydrodynamically under low pressure (3.45 Pa) for 10 s into the anodic end. The separation buffer and dynamic coating of the capillary wall solution were stored in a refrigerator at 4 °C after being degassed with a vacuum system in an ultrasonic tank. Proteins were detected by UV absorbance measurements at 200 nm. The HMW-SS extracts were filtered through a 0.20 μm PVDF syringe filter prior to CZE analysis. The particular HMW-SS were identified through comparisons of single and mixed samples and the results obtained from the SDS-PAGE separations. Three protein separations were performed by CZE for each analytical assay. Statistical Analysis. The resolution was calculated using the halfwidth method. The mean differences were compared using an unpaired Student’s t test. All statistical analyses were performed using Statistica software (version 10.0 PL, StatSoft Polska).

nitrogen. This mixture was then centrifuged at 13000g for 15 min. The obtained supernatant was aliquoted between two tubes, and half of the volume of the reduced proteins was alkylated with 4-vinylpyridine at 60 °C for 30 min. Then, the alkylated and nonalkylated HMW proteins were precipitated by the addition of 1-propanol to a final concentration of 60% (v/v), and the samples were stored at 4 °C overnight. The precipitated HMW-SS were collected after centrifugation for 8 min at 15000g. Next, the HMW-SS precipitates were redissolved in 75 μL of a solution containing 40% (v/v) AcN + 0.1% (v/ v) trifluoroacetic acid (TFA) and 2 M urea, vortexed for 30 min at 60 °C, and centrifuged again for 10 min at 15000g. All samples were used for SDS-PAGE and CZE analyses within 24 h of extraction. Separation of Rye HMW-SS by SDS-PAGE. The alkylated HMW secalin subunits that were obtained from the rye samples and the triticale cultivars (as protein standards) were separated by electrophoresis on a vertical SDS-PAGE gel using a Protean II xi cell unit (Bio-Rad, Hercules, CA, USA) and the discontinuous TrisHCl−glycine buffer system of Laemmli.27 A 10 μL aliquot of a protein sample was loaded onto the upper 4.5% gel, and separation was performed on 12.0% (m/v; 1.35% C) polyacrylamide in resolving solution at 240 V for 45 min after the tracking dye migrated off the gel. The gels were stained overnight with Coomassie Brilliant Blue G-250. The designation of the rye HMW-GS in the inbred lines was performed according to McIntosh et al.28 Separation of Rye HMW-SS by CZE. The HMW protein separations were conducted according to Salmanowicz29 with some modifications. A solution containing 75 mM IDA (pH 2.7), 0.15% PEO with a molecular weight of 8,000,000 Da, 26 mM SB3-12, and 15% (v/v) AcN was used as the separation buffer. A buffer containing 0.1 M IDA, 0.2% (m/v) PVP with a Mw of 0.36 × 106, 0.05% (m/v) HPMC, and 20% (v/v) ACN was used to dynamically coat the capillary wall. Conditioning comprised a rinsing sequence of 1.0 M NaOH, ultrapure water, 0.1 M HCl, ultrapure water, and buffer (all at 275.7 kPa and for 2 min). For the first use on a day, the capillary was additionally flushed with 1 M HCl for 20 min, followed by a 5 min rinse with Milli-Q water and 15 min with separation buffer. The

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RESULTS AND DISCUSSION SDS-PAGE Analysis of Rye HMW-SS. Alkylated rye HMW protein extracts from 68 inbred lines were used for the identification of allelic variation of HMW-SS compositions by one-dimensional SDS-PAGE. Figure 1A shows the electrophoretic patterns of the rye HMW secalin subunits detected in the selected inbred lines and two standard triticale cultivars. The presented patterns revealed wide variability in the qualitative composition of these subunits. Also earlier conducted analysis of storage proteins in 11 inbred lines showed a difference in the number of bands and their molecular masses.30 The SDS-PAGE gel patterns for the particular inbred lines showed two major protein bands for the rye HMW-SS: a more slowly moving x-subunit and a more quickly moving y-subunit. A total of 59 analyzed lines appeared to show genetic homogeneity, while the remaining 9 lines were found to be heterogeneous. It was difficult to make exact identifications of 10537

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the subunits because of the very similar electrophoretic mobilities of the rye HMW-SS in the SDS-PAGE gel. The nomenclature commonly used for wheat was used to mark the protein bands in the separating gels: numeration of the bands in the order of their migration in the gel in reference to the bands of the standard wheat HMW glutenin subunits using Arabic numerals with the letter “r” at the end. In the analyzed lines, 9 tentative HMW-SS compositions were detected. Six rye HMWSS isoforms have similar electrophoretic mobilities, as previously described isoforms from triticale cultivars,29 and four of them were marked as x-type (2r, 5.1r, 5.2r, and 5.3r), and two as y-type (6.4r and 6.5r). Moreover, three new x-type (1.9r, 2.1r and 6.1r) rye HMW-SS isoforms and four new ytype (5.4r, 6.6r, 7r, and 7.2r) subunits were detected in the gels. In 30 analyzed rye inbred lines, the triticale HMW-SS composition 5.1r + 6.4r was identified (Table 1). A majority of these lines (25 lines) had been selected from the 4 initial rye cultivars (cvs Pancerne, Dańkowskie Selekcyjne, Jeleniec, and Smolickie). Optimization of CZE Separation Conditions. The HMW fraction from the inbred line D855/1 containing the composition 5.1r + 6.4r was used as a model protein mixture for the development of the CZE separation method. Until now, an isoelectric buffer system composed of IDA in conjunction with 0.3 M PEO, 26 mM SB3-12, and 30% AcN has been applied as the running buffer for the separation of the triticale HMW-SS together with wheat HMW glutenin subunits in uncoated fused-silica capillaries.30 Good solubility of rye HMW-SS in low-concentration 15% (v/v) AcN in conjunction with 2 M urea in comparison with 40% AcN used earlier for redissolution of the mixture of the wheat and rye HMW proteins in triticale cultivars29 enabled the development of a more efficient method of separation of these classes of proteins. The resolution was determined to be optimum at a concentration of 80 mM IDA in the running buffer containing 0.15% PEO, 26 mM SB3-12, and 15% AcN. This modified procedure maintained satisfactory peak shapes and separation efficiencies for the major peaks of wheat HMW-GS at (4.6−6.8) × 105 plates/m. The alkylated rye HMW-SS isoforms migrated in the capillary faster than the nonalkylated isoforms (tm of major xtype peaks, 10.8 and 12.5 min, respectively), but the reproducibilities and separation efficiencies of the individual protein peaks were less satisfactory (not presented). The relative standard deviation (RSD) values for the migration times (tm) of peaks representing the major x-type and y-type peaks of the alkylated subunits ranged from 1.04% to 1.66%, whereas those of the nonalkylated subunits ranged from 0.49% to 0.84%. Detection of Rye HMW Secalin Subunits in Inbred Lines by CZE. In contrast to the challenging rye HMW-SS identifications on the SDS-PAGE gels due to the very similar electrophoretic mobilities and molecular masses of the particular subunits, the CZE separation of these subunits enabled the correct identification of the specific subunits on the basis of their peak migration times within 16 min. The CZE profiles of nine selected inbred rye lines with detected various HMW-SS compositions are presented in Figure 2A−I. The presented electropherograms of the nonalkylated proteins indicated satisfactory separation of all rye HMW-SS isoforms. The HMW-SS that were isolated from the homogeneity inbred lines were separated into two individual peaks representing xand y-type subunits, with the exception of two lines that contained only an x-type subunit (Figure 2E). In most cases, a

Figure 2. CZE electropherograms of nonalkylated rye HMW-SS from selected inbread lines with different subunits compositions: (A) L427 (1.9r + 5.4r), (B) L260/94 (2r + 6.6r), (C) Ch2/99 (2r + 7r), (D) L127bk/97 (2.1r + 5.4r), (E) L228 (5.1r), (F) H9/3 (5.1r + 6.4r), (G) L298/97 (5.2*r + 6.5r), (H) L59 (5.3*r + 7r), and (I) lo segr (6.1r + 7.2r). Proteins were separated at 10.0 kV and 40 °C with a 75 mM isoelectric IDA buffer, containing of 0.15 M PEO, 26 mM SB3-12, and 15% AcN. Prior to the separation, the capillary was rinsed with 0.1 M IDA solution containing 0.2% PVP, 0.05% HPMC, and 20% AcN.

major peak and one or two minor peaks for each HMW-SS were observed. The multiple detected CZE peaks for individual HMW-SS indicated various posttranslational modifications of these subunits. Additionally, the multiple peaks for wheat HMW-GS and triticale HMW-SS have previously been detected via CZE25,26,29,31 and capillary isoelectric focusing.32 The migration order of the rye HMW-SS in the capillaries differed from the relative electrophoretic mobilities in the SDSPAGE gels. The differences in the identifications of the HMWSS compositions determined by SDS-PAGE and CZE methods could be caused by the various intensities following the staining of the individual HMW secalin bands in SDS-PAGE gels, owing to considerable quantitative differences in these subunits revealed by CZE analysis. Moreover, rye HMW-SS possess similar molecular masses, which makes the identification of particular subunits on the basis of SDS-PAGE patterns in comparison with CZE profiles very difficult. In total, seven xtype and six y-type HMW-SS were designated via the CZE method (Table 1). The rye HMW-SS were separated in the capillary within two time ranges, namely, the y-type subunits with shorter migration times (8.21−9.18 min) and the x-type subunits with longer migration times (11.68−14.06 min) 10538

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Table 2. Means of Migration Times (tm) of Individual x- and y-Type HMW Secalin Subunits in Rye Inbred Lines Determined by CZE Method x-type

1.9r

2r

2.1r

n tm (min) y-type

1 13.53 ± 0.15 5.4r

13 11.64 ± 0.12 6.4r

2 12.01 ± 0.11 6.5r

n tm (min)

3 8.74 ± 0.10

30 8.21 ± 0.08

5.1r 32 12.33 ± 0.13 6.6r

7 8.84 ± 0.09

6 8.46 ± 0.06

5.2*r

5.3*r

6.1r

7 12.63 ± 0.10

3 13.74 ± 0.16

1 14.06 ± 0.18

7r

7.2r

10 8.98 ± 0.06

1 9.18 ± 0.09

compositions 1.9r + 5.4r and 6.1r + 7.2r occurred very rarely (at 1.5%). Detection of HMW Secalin Subunits in Rye Cultivars. The separation of rye HMW secalin subunits from six cultivars was performed using SDS-PAGE and CZE methods. The SDSPAGE patterns of the rye HMW-SS obtained from four selected single grains descending from three cultivars are shown in Figure 1B. The obtained SDS-PAGE electropherograms revealed considerable genetic variations in the rye HMW-SS and resulted in highly diverse subunit band patterns. The majority of the lanes in the SDS gel showed four protein bands (two x-type and two y-type subunits) with different protein staining intensities. The correct identification of particular x + y HMW-SS subunit pairs was therefore not possible. The presence of two x-type and two y-type HMW-SS on SDSPAGE gels for the individual rye cultivars has also been presented before in other works.10,17,25,34 The occurrence of two to four inbred lines that were selected earlier from individual initial rye cultivars differing in their HMW-SS compositions confirmed the high level of rye polymorphism (Table 1). The CZE analysis of the rye HMW secalin fraction from single grains of six cultivars fully confirmed the works of other authors11,23,24 who identified the multilines (mixtures of genotypes) of most rye cultivars. Generally, the HMW-SS fractions from the analyzed rye cultivars were separated into four major peaks representing two x- and two y-type subunits (Figure 3A,B). Moreover, there often occurred considerable quantitative differences in the peak areas for the particular x + y subunit pairs. Although certain single seeds of the rye cultivars presented very characteristic CZE profiles of the HMW-SS, generally the high levels of heterogeneity and heterozygosity made cultivar identification very difficult, which is in contrast to

(Table 2). Both the detected rye x-type and y-type HMW-SS migrated in the capillary with time ranges similar to those of the triticale HMW glutenin subunits (HMW-GS), whereas the typical rye HMW-SS exhibited a wider range of migration times than the triticale HMW-SS.26,29 The lack of considerable distinction between the rye HMW-SS migration times from the European triticale cultivars,33 from which they earlier were obtained, can only be demonstrated based on the limited utilization of rye germplasm for the creation of this synthetic species. Generally, the x-type rye HMW-SS in the analyzed lines were more abundant than the y-type subunits. Seven x-type HMWSS were distinguished on the basis of their CZE migration times (Table 1). Using the SDS-PAGE electrophoretic mobilities of wheat HMW-GS bands as points of reference,26 the following names were assigned to the rye HMW-SS: 1.9r, 2r, 2.1r, 5.1r, 5.2*r, 5.3*r, and 6.1r. Only two x-type subunits earlier identified in the triticale rye cultivars, 2r and 5.1r, were also detected in the presented CZE study. However, the remaining five rye subunits were new and had not been described before. New subunits 5.2*r and 5.3*r possessed SDSPAGE electrophoretic mobilities that were similar to those of the earlier detected triticale subunits 5.2r and 5.3r,29 respectively, but they had considerably different migration times using CZE analysis. The relative migration order of the xtype rye HMW-SS encoded by genes on the Glu-R1 locus was 6.1r > 5.3*r > 1.9r > 5.2*r > 5.1r > 2.1r > 2r (from the slowest to the fastest). In summary, the presented CZE data did not fully confirm the classification of the specific encoded HMW-SS alleles based on the SDS-PAGE analysis. Six rye y-type HMW-SS were distinguished in the analyzed material based on their migration times via the CZE method, of which four were new and had not been previously described (Table 1). According to their relative electrophoretic mobilities in the SDS-PAGE gels, the following names were assigned to these subunits: 5.4r, 6.4r, 6.5r, 6.6r, 7r, and 7.2r. The y-type subunits 7r and 7.2r, the smallest of the subunits as determined by SDS-PAGE, exhibited the longest migration times, 8.98 and 9.18 min, respectively (Table 2). Additionally, most analyzed rye samples contained y-type subunit 6.4r, which possessed the shortest tm of 8.21 min. The relative migration order of the ytype rye HMW-SS encoded by genes on the Glu-R1 locus was 7.2r > 7r > 6.5r > 5.4r > 6.6r > 6.4r (from the slowest to the fastest). The following eight new HMW secalin subunit pairs 1.9r + 5.4r (Glu-R1h), 2r + 6.6r (Glu-R1i), 2r + 7r (Glu-R1j), 2.1r + 5.4r (Glu-R1k), 5.1r + Null (Glu-R1l), 5.2*r + 6.5r (Glu-R1m), 5.3*r + 7r (Glu-R1n), and 6.1r + 7.2r (Glu-R1p) were identified on the basis of the CZE analysis performed in this study and after regarding the nomenclature for rye allelic variants (pairs of x + y). The rye HMW-SS allelic compositions 5.1r + 6.4r and 2r + 7r were the most frequently identified (41.1% and 10.3%, respectively) in the analyzed inbred lines, whereas the

Figure 3. CZE electropherograms of rye HMW-SS from two selected cultivars: (A) cv. Lekkeroggen (5.1r + 6.4r and 2r + 7r) and (B) cv. Buriatskaya (1.9r + 5.4r and 6.1r + 7.2r). Separation conditions were as described in Figure 2. 10539

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rye/wheat blends and feeding value for chicks of whole meals and breads. J. Agric. Food Chem. 2001, 49, 2446−2453. (9) Branlard, G.; Dardevet, M.; Amiour, N.; Igrejas, G. Allelic diversity of HMW and LMW glutenin subunits and omega-gliadins in French bread wheat (Triticum aestivum L.). Genet. Resour. Crop Evol. 2003, 50, 669−679. (10) Ribeiro, M.; Seabra, L.; Ramos, A.; Santos, S.; Pinto-Carnide, O.; Carvalho, C.; Igrejas, G. Polymorphism of the storage proteins in Portuguese rye (Secale cereale L.) populations. Hereditas 2012, 149, 72−74. (11) Shewry, P. R.; Parmar, S.; Miflin, B. J. Extraction, separation and polymorphism of the prolamin storage proteins (secalins) of rye. Cereal Chem. 1983, 60, 1−6. (12) Shewry, P. R.; Kreis, M.; Burgess, S. R.; Parmar, S.; Miflin, B. J. The synthesis and deposition of the prolamin storage proteins (secalins) of rye. Planta 1983, 159, 439−445. (13) Gellrich, C.; Schieberle, P.; Wieser, H. Biochemical characterization and quantification of the storage protein (secalin) types in rye flour. Cereal Chem. 2003, 80, 102−109. (14) Shewry, P. R.; Field, J. M.; Lew, E. J.-L.; Kasadra, D. D. The purification and characterization of two groups of storage proteins (secalins) from rye (Secale cereale L.). J. Exp. Bot. 1982, 33, 261−268. (15) Lawrence, G. J.; Shepherd, R. W. Inheritance of glutenin protein subunits of wheat. Mapping the nucleolus organiser region, seed protein loci and isozyme loci on chromosome 1R in rye. Theor. Appl. Genet. 1981, 60, 333−337. (16) Singh, N. K.; Shepherd, K. W. Mapping of the genes controlling high-molecular-weight glutenin subunits of rye on the long arm of chromosome 1R. Genet. Res. 1984, 44, 117−123. (17) De Bustos, A.; Jouve, N. Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale. Theor. Appl. Genet. 2003, 107, 74−83. (18) Koehler, P.; Wieser, H. Comparative studies of high Mr subunits of rye and wheat. III. Localisation of cysteine residues. J. Cereal Sci. 2000, 32, 189−197. (19) Shewry, P. R.; Bechtel, D. B. Morphology and chemistry of the rye grain. In Rye: Production, chemistry and technology, 2nd ed.; Bushuk, W., Ed.; AACC: St. Paul, MN, USA, 2001; pp 69−121. (20) Lukaszewski, A. Cytogenetically engineered rye chromosomes 1R to improve bread-making quality of hexaploid triticale. Crop Sci. 2006, 46, 2183−2194. (21) Hansen, H. B.; Møller, B.; Andersen, S. B.; Jørgensen, J. R.; Hansen, Å. Grain characteristics, chemical composition, and functional properties, of rye (Secale cereale L.) as influenced by genotype and harvest year. J. Agric. Food Chem. 2004, 52, 2282−2291. (22) Hansen, H. B.; Rasmussen, C. V.; Knudsen, K. E. B.; Hansen, Å. Effects of genotype and harvest year on content and composition of dietary fibre in rye (Secale cereale L.) grain. J. Sci. Food Agric. 2003, 83, 76−85. (23) Kubiczek, R. P.; Huebner, F. R.; Bietz, J. A. Reversed-phase high performance liquid chromatography of secalins: Application to rye cultivar identification. J. Cereal Sci. 1993, 17, 191−201. (24) Petrovičová, L.; Gálová, Z.; Chňapek, M.; Kečkešová, M. Protein polymorphism in genetic resources of rye using SDS-PAGE. J. Microbiol. Biotechnol. Food Sci. 2013, 2, 1531−1539. (25) Yan, Y.; Yu, J.; Jiang, Y.; Hu, Y.; Cai, M.; Hsam, S. L. K.; Zeller, F. J. Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers. Electrophoresis 2003, 24, 1429− 1436. (26) Salmanowicz, B. P. Detection of high molecular weight glutenin subunits in triticale (× Triticosecale Wittm.) cultivars by capillary zone electrophoresis. J. Agric. Food Chem. 2008, 56, 9355−9361. (27) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680− 685. (28) McIntosh, R. A.; Yamazaki, Y.; Devos, K. M.; Dubcovsky, J.; Rogers, W. J.; Appels, R. Catalogue of gene symbols for wheat. Annu. Wheat Newsl. 2003, 49, 246−282.

the case of identifying cultivars of wheat, triticale, and barley.35,36 In conclusion, it has been proven that HMW secalin subunits from rye inbred lines can be rapidly (below 15 min) separated via CZE method with both high resolution and good reproducibility. Using selective preparation of HMW proteins precipitated by addition of 1-propanol to a final concentration of 60% (v/v), 75 kDa γ-secalins from HMW protein extract were eliminated, which have migration times similar to those of HMW-SS during the CZE separation. The HMW-SS were separated into two major peaks (x-type and y-type subunits), with the exception of two lines that contained only the x-type subunit. The CZE analysis of rye HMW secalin fraction fully confirmed the occurrence of the mixture of genotypes in single grains of rye cultivars. A knowledge of the HMW secalin subunit composition of the particular rye samples enables breeders the utilization of rye germplasm in a wider range for the creation of new genotypes of triticale and wheat species with desirable properties. Fast and more accurate characterization of rye HMW-SS by CZE is an efficient alternative to identification of these proteins by the standard SDS-PAGE analysis.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

B.P.S. and M.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ABBEVIATIONS USED HMW-SS, high molecular weight secalin subunit; CZE, capillary zone electrophoresis; HPMC, hydroxypropylmethylcellulose; IDA, iminodiacetic acid; PEO, poly(ethylene oxide); PVP, poly(vinylpyrrolidone); AcN, acetonitrile; SB3-12, Ndodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate

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REFERENCES

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