**1. Introduction**

16 Will-be-set-by-IN-TECH

462 Gel Electrophoresis – Advanced Techniques

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Seed storage proteins of cereals constitute the basis of mankind nutrition. However, climate changes, especially, increased droughts that are distinctly observed in many regions all over the globe, hamper sustainable production of traditional cereals, such as wheat, maize and barley, and dictates necessity to cultivate drought resistant and heat tolerant crops. Among these crops, the grain sorghum, owing to its ability for sustainable grain production in conditions of minimal level of precipitation, takes one of the leading places. However, application of sorghum grain for food and feed purposes is limited by its relatively low nutritive value in comparison with other cereals.

One of the reasons of poor nutritive value of sorghum grain is the resistance of its seed storage proteins (kafirins) to protease digestion. Kafirins are alcohol-soluble prolamin proteins making up to 80% of endosperm sorghum proteins (Hamaker et al., 1995). As well as other prolamins, sorghum kafirins contain high levels of proline and glutamine and are deposited in protein bodies of endosperm cells during kernel development. According to differences in solubility in aqueous *tert*-butanol solutions, molecular weight, structure and immunochemical similarity to zeins (maize prolamins) the kafirins were classified into α-, βand γ-kafirins (Shull et al., 1991; for review, see: Belton et al., 2006). The α-kafirins are highly hydrophobic prolamin proteins (soluble in 40-90% aqueous *tert*-butanol solutions), they comprise 66-84% of total kafirins, depending on the endosperm type (vitreous or opaque). By SDS-PAGE the α-kafirins usually are resolved into two proteins, 25 kDa and 23 kDa. The γ-kafirin accounts for 9-21% of total kafirins depending on the endosperm type (Waterson et al., 1993). According to immunochemical data, the γ-kafirin is a protein with molecular mass of 28 kDa (Shull et al., 1991) although the sequence of the γ-kafirin gene corresponds to the protein with molecular mass of about 20 kDa (De Barros et al., 1991). The β-kafirin, in different endosperm types, accounts for about 7-13% of the total kafirins, and is resolved by the SDS-PAGE into three bands of 20, 18 and 16 kDa (Shull et al., 1991; 1992) or produced one band of 20kDa (El Nour et al., 1998); such variability, perhaps, is due to genotype differences.

One of the main characteristic features of kafirin proteins is their ability to form olygo- or polymers of high molecular weight. These oligomers comprise α- and γ-kafirins linked

Gel Electrophoresis as a Tool to Study Polymorphism and

sterility-inducing cytoplasm.

Nutritive Value of the Seed Storage Proteins in the Grain Sorghum 465

Line, F1 hybrid1 Grain color Endosperm type VIR-120 white floury Pishchevoe-614 (P-614) light-brown semi-vitreous Volzhskoe-4 light-brown floury Volzhskoe-4 waxy (V-4w) pink semi-vitreous Karlikovoe beloe (KB) white semi-vitreous Milo-10 yellow floury KVV-45 white semi-vitreous KVV-97 white vitreous KVV-3 white semi-vitreous KP-70 creamy semi-vitreous Тopaz creamy semi-vitreous О-1237 white semi-vitreous Sudzern svetlyi (Sud) creamy semi-vitreous F5 [M35-1A] Pishchevoe-614/KVV-45 white semi-vitreous А2 Karlikovoe beloe/Pishchevoe-614 (A2 KB/P-614) light-brown semi-vitreous A2 Karlikovoe beloe/KP-70 (A2 KB/P-614) white-yellowish semi-vitreous М35-1A Karlikovoe beloe /KVV-45 (M35-1A KB/KVV-45) white semi-vitreous А2 KVV-97/Pishchevoe-614 light-brown semi-vitreous А2 Sudzern svetlyi/Topaz (A2 Sud/Topaz) creamy semi-vitreous А2 О-1237/ Pishchevoe-614 (А2 О-1237/P-614) light-brown semi-vitreous

In parenthesis: brief designation used in the paper. F1 hybrids were obtained using male-sterile counterparts of fertile lines; they are designated as A2 or М35-1А depending on the type of male

Department, N.I. Vavilov Institute of Plant Production, St. Petersburg, Russia).

For quantitative estimation of kafirin digestibility the SDS-PAGE banding patterns were scanned by laser densitometer ULTROSCAN XL (LKB-Pharmacia) with wavelength 633 nm. The protein quantity in each fraction was expressed as the area (mm2) of the appropriate peak on densitogram, which was calculated by Software LKB 2222 (Version 3.00). In some experiments, the SDS-PAGE banding patterns were analyzed by Scangel program (developed by Dr. A.F. Ravich). The protein quantity in each fraction and in each lane of electrophoregramm was expressed as the amount of dots in the appropriate protein band. Experiments were performed in two replications. The data on digestibility of kafirins (the ratio of protein peak area before and after pepsin digestion) were subjected to variance analysis using the program Agros (Version 2.09; Dr. S. Martynov, Wheat Genetic Resources

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

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)

above, and, after that, the protein spectrum of the sample was studied by SDS-PAGE.

Table 1. The grain sorghum entries used in this investigation

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 (γ- and βkafirins) 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 γ-kafirin plays an important role in resistance of sorghum seed storage proteins to protease digestion, namely, γ-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 α-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 F1 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.
