Genetic Variation of Storage Proteins in Sorghum Grain: Studies by Isoelectric Focusing and High-Performance Liquid Chromatography

Cereal Chem. 63(5):420-427 The genetic variability of the storage proteins in grain of several inbreds silver stain. RP-HPLC was performed on a large pore column by gradient and hybrids of sorghum (Sorghum bicolor (L.) Moench) and varieties elution with increasing concentration of acetonitrile in water. Both representing different races was investigated by separating extracted separations indicated that kafirins and ASGs from the same genotype proteins by isoelectric focusing (IEF) and reversed-phase highcontain mostly the same proteins but with quantitive differences in performance liquid chromatography (RP-HPLC). The kafirins were amounts. Grains of different inbreds usually contained some different extracted from the ground grain with 60% tert-butanol and the reduced kafirin and ASG proteins; the hybrids contained proteins inherited from alcohol-soluble glutelins (ASGs) were extracted with 60% tert-butanol both parents, with those from the female predominating. Representatives containing 5% 2-mercaptoethanol. IEF was conducted with reducedof the different sorghum races varied in their component alcohol-soluble alkylated proteins in thin polyacrylamide gels containing 2% pH 6-8 proteins, with those most widely removed from the geographical center of ampholines and 8M urea, and the resolved proteins were visualized with origin showing the greatest differences. Sorghum (Sorghum bicolor (L.) Moench) is a major food grain hybrid lines of sorghum were similar. However, Paulis and Wall in many arid parts of the world and is the second largest feed grain (1979) found that electrophoretic patterns of kafirins from widely crop in the United States (Wall and Ross 1970). Although sorghum different genetic sources differed markedly. Taylor and Schussler is grown mainly for the energy stored in its starch, the grain is also (1984) demonstrated that the patterns of kafirins separated by an important source of protein where it is a major diet or animal polyacrylamide gel electrophroresis in an acidic buffer varied with feed component. Varietal differences in sorghum's protein and the sorghum cultivar, but the resolution of component proteins amino acid composition have been examined (Virupaksha and was not maximized in their system. Paulis and Wall (1979) Sastry 1968). The protein is generally deficient in lysine, but observed that the electrophoretic patterns of the alcohol-soluble mutant lines having increased lysine content were discovered by fraction of reduced glutelins were similar to those of the kafirin Axtell et al (1974). However, very little information is available patterns derived from the same cultivar. They postulated that the concerning the genetic regulation of variability of sorghum polypeptide components of the kafirins and alcohol-soluble proteins. The poor solubility and hydrophobic nature of these glutelins were mostly identical. proteins cause problems in isolation and separation of the major The present study found that both IEF and RP-HPLC were storage proteins, contributing to this difficulty. Two relatively new effective methods of demonstrating genetic variations of kafirins approaches to resolving sorghum proteins, isoelectric focusing and alcohol-soluble glutelins extracted from selected inbreds used (IEF) and reversed-phase high-performance liquid chromatography for development of hybrids in the United States. The use of RP(RP-HPLC), were used to successfully resolve alcohol-soluble HPLC for separation of cereal proteins was pioneered by Bietz sorghum storage proteins in this study. (1983). The relationship of kafirins extracted from hybrids to those The proteins of sorghum endosperm are heterogeneous and vary of the parental sorghum inbreds was also determined in the present in properties and amino acid composition (Virupaksha and Sastry study. Because the U.S. sorghums are derived from several races 1968). The lysine-rich albumins and globulins, which are soluble in grown in various parts of the world (De Wet and Harlan 1971), the saline solutions, are minor components. The alcohol-soluble diversity of sorghum proteins from selected varieties representing lysine-deficient kafirins are the major protein components; these different races was also examined. The classification of races in this are most effectively extracted from meal with 60% tertiary butanol study is that of Harlan and De Wet (1972). (Jones and Beckwith 1970) and can be resolved into several components by gel electrophoresis (Sastry and Virupaksha 1967, MATERIALS AND METHODS Jones and Beckwith 1970). Most of the remaining protein, designated glutelin, is soluble in dilute alkali and was shown by Sorghum Grain Extraction Beckwith (1972) to consist mainly of polypeptide chains linked by Inbred TX 414, inbred CK 60 A-line, the hybrid RS 626, derived disulfide bonds into larger molecules. Reduction of disulfides from the cross of these lines and inbred TX 415, inbred Redlan releases the polypeptide chains which, when dissolved in A-line, and hybrid RS 671, derived from the latter two inbreds, surfactants or urea, were shown to be heterogeneous by were used to investigate protein variation in inbred and hybrid polyacrylamide gel electrophoresis (Beckwith 1972). Some lines. A-line refers to male-sterile plants used as the female in reduced glutelin polypeptides are soluble in 60% tert-butanol and crosses to form hybrids. Selected varieties of different races have amino acid compositions and electrophoretic mobilities like included Standard Yellow Milo (race Caffra-Caudatum) from the kafirins (Paulis and Wall 1979). South Africa; Pink Kafir (race Caffra), Spur Feterita (race Jones and Beckwith (1970) concluded that the electrophoretic Caudatum), and Hegari (race Caudatum) from East Africa; Shallu patterns of kafirins extracted from grains of three different U.S. (race Guinea) from West Africa; White Durra (race Durra) from India; and Manchu Brown Kaoliang (race Nervosa) from China. Presentaddress: Nuclear Research Laboratory, Indian Institute of Agriculture, New The grain was ground in a Udy cyclone sample mill to pass Delhi, 11012 India. through a 40-mesh screen. The meal was defatted upon extraction Northern Regional Research Center, Agricultural Research Service, U.S. with 10 volumes/ weight petroleum ether by stirring at 4' C for 1 hr. Department of Agriculture, Peoria, IL 61604. The mention of firm names or trade products does not imply that they are endorsed After filtering on a Buchner funnel, the meal was air-dried. To or recommended by the U.S. Department of Agriculture over other firms or similar obtain kafirins, 3.5 g of defatted meal was extracted with 35 ml of products not mentioned. 60% tert-butanol in water (Jones and Beckwith 1970) at room This article is in the public domain and not copyrightable. It may be freely temperature for 2 hr on a reciprocating shaker. After reprinted with customary crediting of the source. American Association of centrifugation in an International model MC centrifuge at 2,600 Cereal Chemists, Inc., 1986. rpm (1,500 X g) for 20 min and removal of the supernatant, the 420 CEREAL CHEMISTRY residue was resuspended in 35 ml 60% tert-butanol and extracted RP-HPLC separation of proteins was achieved on a further for 1 hr. After centrifugation the resulting supernatant was SynChropak RP-P (C18) 250 X 4.1 mm 300 A reversed-phase combined with the previous one to yield the kafirin extract. column (SynChropak, Inc., Linden, IN) by a modification of the The alcohol-soluble glutelin (ASG) was next extracted from the method of Bietz (1983). Gradient elution was attained by a Spectra residual meal with 35 ml of 60% tertiary butanol containing 5% Physics model 8700 pump and gradient system, a Spectra Physics 2-mercaptoethanol (2-ME) by shaking for 2 hr at room model 8500 Dynamic mixer and a WISP 710B auto sample temperature. After centrifugation and removal of the supernatant, injector. Reduced proteins in 50 41 60% tert-butanol 5% 2-ME the residue was shaken with 35 ml of the same solvent for 1 hr. The extracts were automatically injected onto the column. Proteins supernatants were combined to yield the ASG extract. Extractions were eluted at a flow rate of 1 ml/ min with a continuous linear were performed in duplicate. gradient formed by mixing solvent A and solvent B, in which Nitrogen contents of the extracts were determined by a solvent B increased from 45 to 60% of the mixture over 60 min at semimicro-Kjeldahl procedure (AACC 1983). Protein was 70' C; solvent A was water plus 0.1% trifluoroacetic acid, and calculated by multiplication of the nitrogen content by 6.25. solvent B was acetonitrile and 0.1% trifluoroacetic acid. Protein peaks in the eluent were measured at 210 nm with a Beckman 165 Reduction and Alkylation of Proteins variable dual wavelength detector. Chromatograms were recorded Ten milliliters of the kafirin and ASG extracts was removed on a Houston Omniscribe recorder, and data were transferred to, from each of the separate extracts for HPLC. To the 10 ml of stored in, and integrated by means of a Modcomp MAX 4 kafirin extract 0.53 ml of 2-ME was added so that the solution was computer system. Elution of proteins was also followed by 5% 2-ME, and the solution was allowed to stand overnight at room monitoring at 280 nm, but these data were not recorded on the temperature in order to reduce protein disulfide bonds to permit computer. comparison with the reduced ASG. To remove any particulates, Auxillary computer programs were used to analyze the data and the extracts were centrifuged at 20,000 rpm with an L8-M plot composite elution patterns. Especially valuable was the Beckman ultracentrifuge for 20 min and the supernatants carefully computer program CHROCP (Butterfield 1984) for addition or decanted and used for injection on the columns, subtraction of chromatographic data. In the manual mode, the The remaining protein extracts were placed in separate Viscose operator enters any value for K in the formula A KB = C, where casing bags, dialyzed against 20 L of distilled water with four A is data set 1, B is data set 2, and C is the resultant difference changes over 48 hr at 40 C, and lyophilized. To reduce disulfide curve. When the entered value of K is negative and adjusted to bonds, 2 mg of lyophilized protein extract was added in 1.5-ml relative total areas of A and B, curve C may represent the addition polypropylene centrifuge tubes to 100 Al of solution containing 8M of A and B in any ratio, such as the 2:1 female-to-male ratio of urea and 1% 2-ME at pH 8.0. Reduction proceeded overnight at genes contributed to cereal endosperm proteins. room temperature. Liberated sulfhydryls were alkylated by adding 20 y I of an aqueous solution containing 7.1% acrylonitrile in 8M RESULTS urea. After I hr, alkylation was terminated by acidification by adding 10 b1 of pH 3.1 aluminum lactate buffer in 8M urea (Paulis Protein Composition and Wall 1977). After each treatment, the solutions were agitated The protein contents of grains of inbred TX 414, inbred CK 60 with a vortex mixer. After acidification the tubes were centrifuged A-line, and their hybrid RS 626, and those of inbred TX 415, with an Eppendorf centrifuge for 20 min. IEF was performed with inbred Redlan A-line, and their hybrid RS 671 are compared in the supernatant protein solutions. Table I. The hybrid seeds were lower in protein content than either parental inbred. Hybrids generally exhibit lower protein contents Protein Separations than inbreds or open-pollinated varieties do (Miller et al 1964) IEF was conducted on an LKB 2117 Multiphor unit with because of their higher starch content. Protein contents of grains saturated aspartic acid and 0.1 M lysine for the anodic and cathodic representing seven different races of sorghums varied considerably solutions, respectively. The IEF polyacrylamide gel slab (2 X 125 X but were generally quite high (Table II). 260 mm) was composed of 5% acrylamide and 0.183% N,N'Analyses of duplicate kafirin or ASG extracts for percent of methylene bisacrylamide with 2% pH 6-8 LKB ampholines in 8M total proteins (Tables I and II) varied slightly but the sum of the urea, which was polymerized by 0.02% ammonium persulfate. two extractions (K + ASG) was fairly consistent, suggesting that Paper wicks were immersed in the reduced-alkylated protein some kafirin forms disulfide cross-linked ASG. No consistent solution, excess solution was removed, and the wicks were applied relationship was established in these studies between the percent of to the gel surface. Focusing was carried out at 13 W constant power protein constituted by kafirins or ASGs to the total amount of for 4 hr at 40 C with an EC model 500 power supply. Gels were protein in seeds of inbreds or hybrids (Table I) or in grains of the fixed in a solution of 120 ml of methanol, 40 g of trichloroacetic different races (Table II). It has been observed that some highacid, and 240 ml of water overnight and stained with silver using a protein lines of grain had protein more deficient in lysine, Bio-Rad (Richmond, CA) reagent kit by following the indicating higher levels of kafirins and ASGs (Deyoe and manufacturer's directions for 2-mm gels. Densitometric analyses Shellenberger 1965). Our results may reflect the difficulty of of the gels were conducted on a Zeineh recording scanning quantitatively extracting kafirin and ASG in the presence of densitometer using a soft red laser light source (633 nm). tannins (Jambunathan and Mertz 1973). Taylor et al (1984) TABLE I Distribution of Protein Fractions in Grains of Hybrids and Their Parents Protein Content (%, N X 6.25) Kafirin ASGe Kafirin + ASG Genotype (as is basis)a (% of protein)b (% of protein)b (% of protein)b Hybrid RS 626 10.4 ± 0.3 19.4 ± 0.5 26.1 ± 4.0 45.5 Inbred CK 60-A-line (male-sterile) 14.6 ± 0.1 28.8 ± 8.4 25.3 ± 4.4 54.1 Inbred TX 414 13.6 ± 0.4 16.8 ± 8.4 25.3 ± 5.9 42.1 Hybrid RS 671 10.2 ± 0.2 30.2 ± 6.8 23.4 ± 2.3 53.6 Inbred Redlan A-line (male-sterile) 15.4 ± 0.2 25.0 ± 0.5 22.1 ± 4.8 47.2 Inbred TX 415 15.7 ± 0.3 19.6 ± 0.1 22.9 ± 5.0 42.5 'Averages of duplicate analyses. 'Averages of duplicate extractions. Each extract analyzed twice. CASG = alcohol-soluble glutelin. Vol. 63, No. 5,1986 421 analyzed 41 sorghum cultivars grown under different conditions corn (Nucca et al 1978) and are consistent with the conclusions of and found little correlation between the total kafirin plus ASG and Taylor and Schussler (1984) that sorghum prolamin compositions protein content, even though they added polyvinylpyrrolidone to generally vary with genotype. The IEF pattern of the kafirin from their extraction solutions to minimize the effect of tannins on hybrid RS 626 (Fig. 1, 1 K) contains bands present in and unique to protein solubility. either of the parent strains CK 60 A-line or TX 414. For example, kafirin bands from CK 60-A-line (Fig. 1, 2K) at 4.7 and 6.2 cm are IEF Patterns and RP-HPLC Separations absent in TX 414 extracts (Fig. 1, 3K). Kafirins at 5.6 and 6.7 cm of Inbred and Hybrid Sorghum Kafirins and ASGs from TX 414 are absent in the CK 60-A-line kafirin IEF Figure 1 illustrates the IEF patterns of alcohol-soluble proteins separation. However, consistent with the fact that the female strain from typical inbred and hybrid seed extracted from lines used to CK 60 A-line contributes two sets of chromosomes to the produce commercial grain. The kafirins of each of the inbreds CK endosperm, whereas the male contributes only one set, the 60 A-line, TX 414, and Redlan A-line (2K, 3K, and 5K, intensities of bands resembling those in CK 60 A-line are greater respectively, in Fig. 1) showed significantly different patterns than those from TX 414. A similar relationship was observed for which would assist identification of these inbreds. In contrast, the the IEF patterns of the kafirins extracted from Redlan A-line and pattern of TX 414 kafirin is similar to kafirin from TX 415 (Fig. 1, TX 415 and the derived hybrid from their cross, RS 671 (5K, 6K, 6K). This similarity might be anticipated, as both TX 414 and and 4K, Fig. 1). TX 415 were selected from a cross of SA 7078 and TX 09. These In an earlier study, Paulis and Wall (1979) demonstrated that observations are similar to those for IEF patterns of prolamins of electrophoretic patterns of sorghum alcohol-soluble glutelins and TABLE II Distribution of Protein Fractions in Grains of Genotypes from Different Sorghum Races Protein Content Kafirin ASGc Kafirin + ASG Genotype (%)a (% of protein)b (% of protein)' (% of protein)b Standard Yellow Milo 16.8 ± 0.3 20.8 ± 1.4 23.1 ± 5.6 43.9 Pink Kafir 10.1 ± 0.2 18.9 ± 0.6 23.6 ± 3.1 42.5 Spur Feterita 15.6 ± 0.4 28.8 ± 0.7 26.0 ± 2.4 54.8 Hegari 11.1 ± 0.3 24.9 ± 2.6 25.9 ± 6.1 50.8 Shallu 14.7 ± 0.1 21.4± 3.6 19.8 ± 1.8 41.2 White Durra 13.7 ± 0.4 22.1 ± 0.2 22.4 ± 7.1 44.5 Manchu Brown Kaoliang 16.3 ± 0.3 17.6 ± 4.1 20.3 ± 4.9 37.9 Av 14.0 22.1 23.0 45.1 a Average of duplicate analyses. hAverages of duplicate extractions. Each extract analyzed twice. c ASG = alcohol-soluble glutelin.