Gel Electrophoresis as a Tool to Study Polymorphism and Nutritive Value of the Seed Storage Proteins in the Grain Sorghum

value of sorghum grain. These lines may be used in breeding programs for developing new CMS-lines and F 1 hybrids. In addition, these lines (for example, KVV-45) may be used in future investigations on molecular organization of genes encoding structure and/or deposition of kafirins, their cloning and transfer into other sorghum lines by methods of classical genetics or genetic engineering. Gel electrophoresis of the flour subjected to amylolitic enzyme action has demonstrated that starch digestion decreases content of kafirin polymers and reduces subsequent kafirin digestion by pepsin. This finding may explain the reduced nutrient value of sorghum grain, in comparison with other cereals. These data point on the complex mode of interactions of storage proteins and starch in sorghum endosperm.

together by disulphide (S-S) bonds, which are formed by sulphur-containing amino acids (Nunes et al., 2005). In the native state, both mono-and oligomers are present, while in 'reduced' extracts (i.e. with addition of 5% 2-mercaptoethanol that destroys S-S bonds) only monomers were detected (El Nour et al., 1998).
The causes of the poor kafirin digestibility appear to be multi-factorial (Duodu et al., 2003). Among these factors are chemical structure of kafirin molecules, some of which ( -andkafirins) are abundant with sulfur-containing amino acids that are capable to form S-S bonds, resistant to protease digestion; interactions of kafirins with non-protein components such as polyphenols and polysaccharides; and spatial organization of different kafirins in the protein bodies of endosperm cells.
Among the methods that were developed for investigation of sorghum protein digestibility (Pedersen & Eggum, 1983;Mertz et al., 1984;Aboubacar et al., 2003), pepsin digestion of the flour proteins with subsequent gel electrophoresis is the most informative. This method, originally applied by B. Hamaker and co-workers (Weaver et al., 1998;Aboubacar et al., 2001) has been used in a number of studies (Nunes et al., 2004;Wong et al., 2010). Application of this method allowed to isolate sorghum lines with high protein digestibility (Weaver et al., 1998) and to find out that -kafirin plays an important role in resistance of sorghum seed storage proteins to protease digestion, namely, -kafirin forms a disulfidebound enzyme-resistant layer at the periphery of protein bodies that restricts access of proteases to the inferior-located and more easily digested -kafirins (Oria et al., 2000).
In our investigations (Italianskaya et al., 2009), we studied the protein digestibility in different sorghum lines and hybrids using this method and revealed significant polymorphism for in vitro kafirin digestibility as well as the strong genetic bases of this trait. In this paper, we summarize the results of these studies, which allowed isolating sorghum lines and F 1 hybrids with increased nutritive value. In addition, we demonstrate that kafirin polymorphism may be used in genetic experiments, namely, in determination of genetic structure of endosperm in sorghum.

Material and methods
In vitro protein digestibility was studied in 10 lines and seven F 1 hybrids of the grain sorghum (Sorghum bicolor (L.) Moench) (Table 1).
To study in vitro protein digestibility the modified method of whole-grain flour pepsin treatment was used (Oria et al. 1995). For each variety 25 mg of flour was treated with 5 ml of 0.15% pepsin solution (P7000 Sigma-Aldrich) in the 0.1 M potassium-phosphate buffer (pH 2.0) for 120 min at 37 0 С with repeated shaking. Analysis of seed storage protein (kafirin) spectra was performed before and after pepsin treatment by SDS-PAG electrophoresis (SDS-PAGE) in reducing conditions. SDS-PAGE was carried out in the 12.5% (w/v) acrylamide separating gel (0.375 M TRIS·HCl, pH 8.8) and 4% stacking gel (0.125 M TRIS, pH 6.8) according to modified Laemmli method (Laemmli, 1970). SDS-reducing buffer: 62.5 mM TRIS·HCl, pH 6.8, 20% glycerol, 2% SDS, 5% -mercaptoethanol; running buffer: 25.0 mM TRIS·HCl, 192 mM glycine, 0.1% SDS, pH 8.3; spacer thikness 1.00 mm. Gels were electrophoresed at 20-23 ma for about 5 hr. Gels were stained with Coomassie Brilliant Blue G-250 or R-250 (Diezel et al, 1972 In some lines and hybrids, the dependence of in vitro protein digestibility from in vitro starch digestibility was studied. In this experiment, the flour, firstly, was subjected to amylolitic enzyme treatment according to the method of B.V. McCleary (McCleary et al., 2002) using Megazyme Resistant Starch Kit (Megazyme Co, Ireland). The pellet remained after removal of solubilised starch was used for pepsin treatment according to the method described above, and, after that, the protein spectrum of the sample was studied by SDS-PAGE.
In order to use kafirins as markers of genetic structure of endosperm the modified technique of SDS-PAGE was applied. In these experiments, AS-1a line of the grain sorghum, which is characterized by a low frequency of parthenogenic embryo formation (Elkonin et al., 2012) was used. Emasculated panicles of this line were pollinated with the pollen of the line Volzhskoe-4w homozygous for dominant gene Rs, conditioning purple color of coleoptiles, seedling leaves and stem. To study the origin of the kernels (apomictic or sexual) with the aid of the kafirin polymorphism, the kernels were split into two parts. The part with an embryo was put in a tray on a moisture filter paper to study the phenotypic traits of a seedling (expression of the Rs gene). Another part was used in SDS-PAGE to study its kafirin spectrum. In these experiments, gels were electrophoresed at constant voltage (70 V) for about 15 hr. Gels were stained with AgNO 3 solution.

In vitro kafirin digestibility
SDS-PAGE spectra of the seed storage proteins of a number of lines used in our investigations, before and after pepsin digestion, are shown on Figures 1 and 2. In electrophoretic spectra of sorghum lines subjected to pepsin digestion, one could clearly distinguish the -(28 kDa), 1 (25 kDa) and 2 (23 kDa) kafirins and one or several bands of -kafirin fractions (Fig. 1). These electrophoretic patterns correspond to kafirin spectra previously described in the literature (Shull et al., 1991;El Nour et al., 1998;Nunes et al., 2004). In our previous investigations (Table 2) we determined the relative content of different kafirin fractions and observed significant variation among different cultivars. The 1 and -kafirins were the most abundant in all lines and hybrids tested: 24-37% and 10-13% of all endosperm proteins, respectively; -kafirins represent relatively small fractions www.intechopen.com (4-10%) that is in concordance with the literature data (Shull et al., 1991;Waterson et al., 1993). 1 Relative content of each fraction is expressed as percentage of its peak area from the total endosperm proteins peak area sum. Mean data of two replications.  Figure 2 the highest digestibility level had VIR-120 -90.8% (lanes 1 and 2), while the kafirins of line KVV-3 (lanes 9 and 10) were the most resistant to pepsin digestion (54.5% digestibility level) (Table 3).
In our previous study (Italianskaya et al., 2009), we observed significantly higher variation among the lines. For example, in the cultivar Volzhskoe-4 (V-4, registered standard), the amount of undigested -and -kafirins after pepsin digestion was 80% and 73% from their initial contents, respectively. The total amount of undigested kafirins in cv. V-4 was 70% (digestibility level was 30%). At the same time, in the line KVV-45, the total amount of undigested proteins was 37% (digestibility level was 63%). Percentage of undigested 1 and -kafirins in the line KVV-45 was only 25% and 30%, respectively. The differences in kafirin spectra between this line and cv. V-4 before and after pepsin treatment are clearly seen in the Figure 3. Further investigation confirmed a high level of protein digestibility in this line (78.4%) (Table 3). Perhaps, the line KVV-45 contains mutation(s) in the genes encoding structure or deposition of kafirin molecules and, therefore, is of a great interest for future experiments.
Remarkably, in subsequent investigation it was found that in the line Topaz the digestibility level was even higher than in the KVV-45 and reached 89% (see chapter 4). This value is sufficiently high; it corresponds to digestibility level of whole grain flour protein of the best condenced-tannin-free sorghum entries (Axtell et al., 1981, andother reports, as cited in Duodu et al., 2003). One should expect that this line would have high nutritive value.
One should note high digestibility of the -kafirin fractions in majority of lines. This fact contradicts to hypothesis that explains poor kafirin digestibility by formation of S-S bonds because -kafirins as well as -kafirins contain a high amount of cystein, a sulfur-containing amino acid (Belton et al, 2006). In addition, in all lines, the polypeptides with molecular weight approx. 42 and 46 kDa were prominent in electrophoretic spectra after pepsin digestion. These polypeptides, perhaps, represent kafirin dimers, which were formed as a result of association of kafirin monomers. Earlier, the formation of similar polypeptides (45 kDa) was observed after the cooking process (Duodu et al., 2003;Nunes et al., 2004). Fig. 2. Electrophoretic patterns of sorghum seed storage proteins before (1,3,5,7,9,11,13) and after (2,4,6,8,10,12,14) pepsin digestion. Lanes 1, 2 -VIR-120; 3, 4 -Volzhskoe-4w; 5, 6 -KVV-45; 7, 8 -KVV-97; 9, 10 -KVV-3; 11, 12 -Karlikovoe beloe; 13, 14 -KP-70; Mmolecular weight markers (kDa). di-and trimers of kafirins are indicated by arrows, and , respectively. Gels were stained with Coomassie Brilliant Blue R-250.  Table 3. Densitometry of electrophoretic patterns of seed storage proteins shown in Figure 2. In order to explore the genetic basis of kafirin digestibility, we studied the expression of this trait in the F 1 hybrids between parental lines differing by resistance to pepsin digestion. Comparison of kafirin digestibility in the F 1 hybrids and their parental lines showed that different hybrid combinations had different mode of inheritance of resistance to pepsin affect (Table 4).  . This hybrid as well as the line KVV-45, is of great importance for fundamental investigation of factors influencing seed storage protein digestibility in sorghum (kafirin gene structure, structural organization of protein bodies and others) and for practical breeding.
Strong effect of genotype was also found on spectrum of high-molecular weight kafirins that were observed after pepsin digestion (Fig. 4). In some lines and F 1 hybrids two peaks diand trimers) were found (Fig. 4, A-C), while in others only one peak (trimers) was seen (Fig.  4, D-F). Remarkably, densitograms of the F 1 hybrids in the peak area clearly resembled parental ones. One should note that while the peaks corresponding to trimers were observed in electrophoretic spectra already before pepsin treatment and their amount usually reduced after that, the dimers (45 kDa) were observed only after pepsin action. In some entries kafirin polymers were highly resistant to pepsin digestion, as in the KVV-45, while in others, as in the line P-614 and F 1 hybrid A2 KVV-97/P-614 (Fig. 5, A,B), these peaks were faint or almost absent. These data point on the genetic bases of formation of these molecules, which affect nutritive value of sorghum grain.

Interaction of starch and protein digestibility
In order to found out dependence of sorghum protein digestibility on starch digestibility the flour of several lines and F 1 hybrids was subjected to pepsin action after removal of digestible starch by the amylolytic enzymes treatment, and then was studied by SDSelectrophoresis for the presence of undigested proteins. It was found that after action of amylolitic enzymes the amount of protein in the kafirin fractions significantly increases ( Fig.  6): in the lanes 3, 7 and 11 (samples after amylolitic enzyme action) almost all the protein is concentrated in the kafirin fractions, in comparison with the lanes 1, 5 and 9 (samples without amylolitic enzyme action). However, contrary to expectation that removal of starch will favor to kafirin digestion, the pepsin treatment of the samples treated before it with amylolitic enzymes (lanes 4, 8 and 12) were digested significantly fewer than samples digested by pepsin only (lanes 2, 6 and 10). Gel densitometry confirmed this visual conclusion (Table 5). Such phenomenon was observed in all F1 hybrids studied (A2 Sud/Topaz, A2 O-1237/P-614, M35-1A KB/KVV-45) and their parental lines. Perhaps, partially digested starch molecules may interact with kafirin molecules by any physical or, probably, chemical way and prevent their protease digestion. One should not exclude that similar process might take place in in vivo conditions and thus decrease sorghum protein digestibility and reduce its nutritive value.
In addition, it was found that after amylolitic enzyme treatment the amount of di-and trimer fractions significantly reduced in comparison with the non-fermented control samples. In the F 1 hybrid A2 Sud/Topaz their amount was significantly fewer even in comparison with pepsin treatment only. Such a reduction of kafirin oligomers may be also responsible in increase of the level of kafirin monomers. These data testify that starch molecules might participate in formation of kafirin oligomer molecules. They are important for understanding the factors influencing kafirin and starch interactions in sorghum endosperm and their digestibility. Fig. 6. Electrophoretic patterns of sorghum seed storage proteins from the flour before (1,3,5,7,9,11) and after (2, 4, 6, 8, 10, 12) pepsin digestion; lanes 3, 4, 7, 8, 11, 12 -after removal of soluble starch by amylolitic enzymes before pepsin digestion; lanes 1, 2, 5, 6, 9, 10without this procedure. Lanes 1-4 -Sudzern svetlyi; 5-8 -F 1 A2 Sudzern svetlyi/Topaz; 9-12 -Topaz; M -molecular weight markers (kDa). Gels were stained with Coomassie Brilliant Blue G-250.

Kafirins as the markers of endosperm genetic structure
In addition to variation of a number of -kafirin fractions in different sorghum entries described above, we have revealed polymorphism of the -kafirins. The line Volzhskoe-4w (V-4w) that is used as a tester line to distinguish the hybrid seedlings from the maternal ones, possessed specific kafirin spectrum, which was rarely observed in other sorghum lines and cultivars. The 1 fraction was composed from three polypeptides: 1-1, 1-2, and 1-3; 2 fraction was composed from two polypeptides: 2-1 and 2-2 (Fig. 7, lanes 1-3). We hypothesized that this polymorphism could be used in studies of genetic structure of endosperm in apomixis research in sorghum.
To test this possibility we used the AS-1a line, which is characterized by ability for development of aposporous embryo sacs and parthenogenetic embryos (Elkonin et al., 2012). Gel electrophoresis showed that kafirin spectrum of this line differs from V-4w (Fig.  7). Two polypeptides were observed in the 1 fraction ( 1 and 1-2), the 1-2 was in trace amount, and 1-3 was absent; the 2 fraction did not subdivide into two polypeptides (Fig.  7, lanes 4-6).

Genotype
Experimental treatment Mean data of two replications; data followed by the same letter did not differ significantly (p<0.05) according to Duncan Multiple Range Test; *, and *** significant at p<0.05, and p<0.001, respectively. Table 5. Densitometry of seed storage proteins electrophoretic patterns of F 1 A2 Sudzern/Topaz and its parents after treatment with pepsin and/or -amylase and amyloglucosidase In order to use this polymorphism for identification of seeds formed via apomixis, the kernels obtained by pollination of emasculated panicles of AS-1a with the pollen of V-4w were split into two parts. The part with an embryo was used to study the phenotypic traits of a seedling. Another part was used in SDS-PAGE to study its kafirin spectrum. In the case of autonomous endosperm development, no V-4w proteins should be found in the kafirin spectra of the kernels yielded maternal seedlings, while in the case of pseudogamous endosperm development, in the electrophretic spectra of these kernels, the SDS-PAGE must reveal V-4w proteins. It was found that kafirin spectra of kernels, which yielded maternal seedlings (Fig. 7, lanes 11,12) did not differ from the spectrum of AS-1a line (Fig. 7, lanes  4-6), while in the spectra of the kernels, which yielded hybrid seedlings the 1-3 protein was clearly distinguished (Fig. 7, lanes 7-10). These data support the results of our cyto-embryological observations of autonomous endosperm development in the AS-1a line (Elkonin et al., 2012) and are in accordance with the literature data on other sorghum lines with apomictic potentials (Rao et al., 1978;Wu et al., 1994;Ping et al., 2004).  -3), AS-1а (4-6) and of the kernels, which were set on emasculated panicles of AS-1a pollinated with the Volzhskoe-4w pollen and yielded the F 1 hybrid seedlings (7-10) and maternal plants (11-12); М -molecular weight marker (kDa). Gels were stained with AgNO 3 .

Conclusion
Summarizing, the results of our investigation demonstrate that gel electrophoresis of the seed storage proteins is a powerful instrument in researches on sorghum genetics and breeding that have both fundamental and applied orientation. It allowed to isolate of sorghum lines with individual kafirin fractions more sensitive to protease action, and, therefore, with increased protein digestibility -one of the main trait characterizing the nutritive value of sorghum grain. These lines may be used in breeding programs for developing new CMS-lines and F 1 hybrids. In addition, these lines (for example, KVV-45) may be used in future investigations on molecular organization of genes encoding structure and/or deposition of kafirins, their cloning and transfer into other sorghum lines by methods of classical genetics or genetic engineering.
Gel electrophoresis of the flour subjected to amylolitic enzyme action has demonstrated that starch digestion decreases content of kafirin polymers and reduces subsequent kafirin digestion by pepsin. This finding may explain the reduced nutrient value of sorghum grain, in comparison with other cereals. These data point on the complex mode of interactions of storage proteins and starch in sorghum endosperm.
Gel electrophoresis of the seed storage proteins allowed to determine genetic structure of endosperm in sorghum kernels with parthenogenic embryos developing in the line AS-1a with apomictic potentials and may be used in development of sorghum lines with high frequency and stable expression of this trait.

Acknowledgement
Authors are grateful to Dr. Alexander Ravich for the Software Scangel. This work was funded partly by the Russian Foundation for Basic Researches, grant 10-04-00475. As a basic concept, gel electrophoresis is a biotechnology technique in which macromolecules such as DNA, RNA or protein are fractionated according to their physical properties such as molecular weight or charge. These molecules are forced through a porous gel matrix under electric field enabling uncounted applications and uses. Delivered between your hands, a second book of this Gel electrophoresis series (Gel Electrophoresis-Advanced Techniques) covers a part, but not all, applications of this versatile technique in both medical and life science fields. We try to keep the contents of the book crisp and comprehensive, and hope that it will receive overwhelming interest and deliver benefits and valuable information to the readers.