Identification and characterization of high‐molecular‐weight secalins from triticale seeds by capillary zone electrophoresis

Abstract

A rapid and reliable method for separation and characterization of the variability of high-molecular-weight secalin subunits (HMW-SS) in hexaploid triticale (x Triticosecale Wittmack) by CZE has been developed. In this method, a mixture of two poly(ethylene oxide) polymers differing in molecular weight and a high concentration of ACN in isoelectric buffer was applied as the running electrolyte. For dynamic coating of the capillary inner wall, a low-concentration mixture of poly(vinylpyrrolidone) and hydroxypropylmethylcellulose was employed. Wide allelic variations in rye HMW-SS composition, including some novel x- and y-type HMW-SS, were detected by CZE. The CZE electropherograms of HMW-SS showed two groups of peaks in accordance with y- and x-type subunits, with migration times of 8.0-8.8 and 11.0-13.3 min, respectively. HMW-SS differed in migration times from the simultaneously resolved HMW glutenin subunits, but frequently had very similar electrophoretic mobilities during separation by SDS-PAGE. Each of the two rye subunits 2r and 6.5r detected by SDS-PAGE represents in fact two subunits (5.1r or 5.2r, and 6.4r or 6.5r, respectively). After analyzing 106 European triticale cultivars, 12 HMW-SS were identified (six x-type and six y-type). They form six allelic variants of these subunits. The simultaneous separation and identification of triticale HMW glutenin and secalin subunits by CZE is an efficient alternative to SDS-PAGE and should facilitate breeding of valuable cultivars.

Rheological characterisation of gluten from triticale (x Triticosecale Wittmack)

“…There are some essential differences between triticale and common wheat in the number of fractions and molecular masses of α ‐, γ ‐, and ω ‐gliadins as well as HMW‐GS, and LMW‐GS . Allelic variations of HMW‐GS of wheat result in large differences in bread‐making properties.…”

Molecular, Physicochemical and Rheological Characteristics of Introgressive Triticale/Triticum monococcum ssp. monococcum Lines with Wheat 1D/1A Chromosome Substitution

“…The presence of both wheat and rye HMW proteins interferes with the identification of individual HMW subunits in triticale by the SDS-PAGE method (wheat and rye HMW proteins have similar molecular weights and electrophoretic mobilities, but appear at varying levels). As revealed in earlier investigations by Salmanowicz [32,34], triticale HMW-GS and -SS can be easily discriminated by CZE. As shown in Figure 4, from four to eight major protein peaks with migration times in the range of 7.8 to 13.2 min are observed in the CZE electrophoretic profiles of the four different triticale groups.…”

“…Extraction of HMW-SS and HMW-GS was performed based on the method of Salmanowicz [34]. In total, 150 mg of flour was used for sequential extraction of albumins and globulins (two 5-min extractions with 0.4 M NaCl + 0.067 M HKNaPO 4 (pH 7.6) at RT), gliadins (two 10-min extractions using 70% ( v / v ) ethanol), monomeric prolamins (two 15-min extractions using 50% isopropanol), and glutenins (a single extraction for 30 min with 0.05 M Tris-HCl buffer (pH 7.5) containing 50% v / v 1-propanol, 2 M urea and 1% w / v DTT at 60 °C under nitrogen).…”

“…The HMW protein separations were carried out according to Salmanowicz [34], with some modifications. A solution containing 75 mM IDA, 0.15% PEO with a molecular weight of 8,000,000, 26 mM SB3-12, and 20% ( v / v ) acetonitrile (AcN) was used as the separation buffer.…”

Variation of High-Molecular-Weight Secalin Subunit Composition in Rye (Secale cereale L.) Inbred Lines

“…In the analyzed lines, 9 tentative HMW-SS compositions were detected. Six rye HMW-SS 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.…”