**3.** *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.

Fig. 1. Electrophoretic patterns of sorghum seed storage proteins before (1, 3, 5) and after (2, 4, 6) pepsin digestion*.* Lanes 1, 2 – Volzhskoe-4; 3,4 – Pishchevoe-614; 5,6 – F5 [M35-1A] Pishchevoe-614/KVV-45; M – molecular weight markers (kDa). α, β, γ – individual kafirin fractions. Gels were stained with Coomassie Brilliant Blue R-250.

In electrophoretic spectra of sorghum lines subjected to pepsin digestion, one could clearly distinguish the γ- (28 kDa), α1 (25 kDa) and α2 (23 kDa) kafirins and one or several bands of β-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 α1 and γ-kafirins were the most abundant in all lines and hybrids tested: 24-37% and 10-13% of all endosperm proteins, respectively; β-kafirins represent relatively small fractions

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)

SDS-PAGE spectra of the seed storage proteins of a number of lines used in our

Fig. 1. Electrophoretic patterns of sorghum seed storage proteins before (1, 3, 5) and after (2, 4, 6) pepsin digestion*.* Lanes 1, 2 – Volzhskoe-4; 3,4 – Pishchevoe-614; 5,6 – F5 [M35-1A]

α, β, γ – individual kafirin fractions. Gels were stained with Coomassie Brilliant Blue R-250.

In electrophoretic spectra of sorghum lines subjected to pepsin digestion, one could clearly distinguish the γ- (28 kDa), α1 (25 kDa) and α2 (23 kDa) kafirins and one or several bands of β-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 α1 and γ-kafirins were the most abundant in all lines and hybrids tested: 24-37% and 10-13% of all endosperm proteins, respectively; β-kafirins represent relatively small fractions

Pishchevoe-614/KVV-45; M – molecular weight markers (kDa).

investigations, before and after pepsin digestion, are shown on Figures 1 and 2.

for about 15 hr. Gels were stained with AgNO3 solution.

**3.** *In vitro* **kafirin digestibility** 


(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.

Table 2. Relative content of different kafirin fractions in some sorghum lines and F1 hybrids (Italianskaya et al., 2009)

After pepsin digestion the amount of protein in kafirin fractions substantially reduced (Figs. 1; 2). Different sorghum lines and cultivars differed significantly by this trait. For example, among the entries presented in 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 γ- and α-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 α1 and γ-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, and other 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 β-kafirin fractions in majority of lines. This fact contradicts to hypothesis that explains poor kafirin digestibility by formation of S-S bonds because β-kafirins as well as γ-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

Gel Electrophoresis as a Tool to Study Polymorphism and

Nutritive Value of the Seed Storage Proteins in the Grain Sorghum 469

Fig. 3. Densitograms of electrophoretic spectra of endosperm proteins of sorghum line KVV-45 (a, b) and cultivar Volzhskoe-4 (c, d) before (a, c) and after (b, d) pepsin digestion. α1, α2,

In order to explore the genetic basis of kafirin digestibility, we studied the expression of this trait in the F1 hybrids between parental lines differing by resistance to pepsin digestion. Comparison of kafirin digestibility in the F1 hybrids and their parental lines showed that different hybrid combinations had different mode of inheritance of resistance to pepsin

β and γ-kafirin fractions are indicated.

affect (Table 4).

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; M – molecular 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. The SDS-PAGE banding patterns were scanned and analyzed by Scangel program (developed by Dr. A.F. Ravich)

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

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; M – molecular weight markers (kDa). di- and trimers of kafirins are indicated by arrows, and , respectively. Gels were stained with Coomassie Brilliant Blue R-250.

digestion 1,2 VIR-120 9769124 897710 9.2 90.8 3,4 Volzhskoe-4w 7285338 2692241 37.0 63.0 5,6 KVV-45 16465667 3554046 21.6 78.4 7,8 KVV-97 26995517 9483915 35.1 64.9 9,10 KVV-3 12242662 5571704 45.5 54.5

13,14 KP-70 14462063 3651537 25.2 74.8 Table 3. Densitometry of electrophoretic patterns of seed storage proteins shown in Figure 2.

The SDS-PAGE banding patterns were scanned and analyzed by Scangel program

beloe <sup>13897393</sup> <sup>4335642</sup>31.2 68.8

Amount of undigested protein, %

Digestibility, %

Line Total amount of dots in

the lanes

control after pepsin

Lane number

11,12 Karlikovoe

(developed by Dr. A.F. Ravich)

kDa) was observed after the cooking process (Duodu et al., 2003; Nunes et al., 2004).

Fig. 3. Densitograms of electrophoretic spectra of endosperm proteins of sorghum line KVV-45 (a, b) and cultivar Volzhskoe-4 (c, d) before (a, c) and after (b, d) pepsin digestion. α1, α2, β and γ-kafirin fractions are indicated.

In order to explore the genetic basis of kafirin digestibility, we studied the expression of this trait in the F1 hybrids between parental lines differing by resistance to pepsin digestion. Comparison of kafirin digestibility in the F1 hybrids and their parental lines showed that different hybrid combinations had different mode of inheritance of resistance to pepsin affect (Table 4).

Gel Electrophoresis as a Tool to Study Polymorphism and

Nutritive Value of the Seed Storage Proteins in the Grain Sorghum 471

Fig. 4. Densitograms of endosperm proteins electrophoretic spectra of F1 hybrids and their parental lines after pepsin digestion: a – A2 KVV-114, b – F1 A2 KVV-114/V-4w, c – V-4w, d – A2 KB, e – F1 A2 KB/KP-70, f – KP-70. Fractions of di- and trimers of kafirin proteins

(45kDa and 66 kDa) are shown by arrows, and , respectively.


1 Mean from two replications. Data followed by the same letter did not differ significantly (*p*<0.05) according to Duncan Multiple Range Test.

\* Significant at *p<*0.05.

Table 4. *In vitro* protein digestibility of endosperm proteins in F1 sorghum hybrids and their parental lines

The F1 hybrids A2 KB/P-614, A2 KB/KP-70 and A2 KVV-97/P-614 had significantly lower kafirin digestibility than parental lines, which were characterized by its relatively high level. The reasons of such negative heterosis are unclear. Perhaps, genetic factors conditioning relatively high kafirin digestibility of KP-70, KB and P-614 are recessive and locate in different loci. At the same time, the F1 hybrid M35-1A KB/KVV-45 did not differ from parental lines and retained high level of kafirin digestibility of the line KVV-45. Perhaps, high digestibility of KVV-45 contrary to other lines may be controlled by any dominant gene(s). 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 F1 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 F1 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 F1 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.

KVV-45 24.4 24.6 32.2 24.5 a M35-1A Karlikovoe beloe /KVV-45 36.2 33.9 34.2 26.8 ab Karlikovoe beloe 21.3 37.2 26.0 32.1 bcd А2 Karlikovoe beloe /KP-70 39.5 51.5 42.3 41.6 g KP-70 22.4 29.5 22.3 26.1 a А2 Karlikovoe beloe/Pishchevoe-614 41.1 51.3 42.3 40.4 efg Pishchevoe-614 53.4 64.5 34.7 33.7 cd А2 KVV-97/Pishchevoe-614 48.9 55.7 44.3 40.5 fg KVV-97 40.4 30.3 20.4 34.2 d

*F* 0.05 14.76\* LSD 0.05 5.4 1 Mean from two replications. Data followed by the same letter did not differ significantly (*p*<0.05)

Table 4. *In vitro* protein digestibility of endosperm proteins in F1 sorghum hybrids and their

The F1 hybrids A2 KB/P-614, A2 KB/KP-70 and A2 KVV-97/P-614 had significantly lower kafirin digestibility than parental lines, which were characterized by its relatively high level. The reasons of such negative heterosis are unclear. Perhaps, genetic factors conditioning relatively high kafirin digestibility of KP-70, KB and P-614 are recessive and locate in different loci. At the same time, the F1 hybrid M35-1A KB/KVV-45 did not differ from parental lines and retained high level of kafirin digestibility of the line KVV-45. Perhaps, high digestibility of KVV-45 contrary to other lines may be controlled by any dominant gene(s). 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

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 F1 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 F1 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 F1 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.

Amount of undigested protein, percent from untreated sample1 <sup>γ</sup> <sup>α</sup>1 <sup>β</sup> Total

proteins

Line, F1 hybrid1

according to Duncan Multiple Range Test.

\* Significant at *p<*0.05.

parental lines

breeding.

Fig. 4. Densitograms of endosperm proteins electrophoretic spectra of F1 hybrids and their parental lines after pepsin digestion: a – A2 KVV-114, b – F1 A2 KVV-114/V-4w, c – V-4w, d – A2 KB, e – F1 A2 KB/KP-70, f – KP-70. Fractions of di- and trimers of kafirin proteins (45kDa and 66 kDa) are shown by arrows, and , respectively.

Gel Electrophoresis as a Tool to Study Polymorphism and

endosperm and their digestibility.

Blue G-250.

Nutritive Value of the Seed Storage Proteins in the Grain Sorghum 473

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 F1 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

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, 10 – without this procedure*.* Lanes 1-4 – Sudzern svetlyi; 5-8 – F1 A2 Sudzern svetlyi/Topaz; 9-12 – Topaz; M – molecular weight markers (kDa). Gels were stained with Coomassie Brilliant

In addition to variation of a number of β-kafirin fractions in different sorghum entries described above, we have revealed polymorphism of the α-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 α1 fraction was composed from three polypeptides: α1-1, α1-2, and α1-3; α2 fraction was composed from two polypeptides: α2-1 and α2-2 (Fig. 7, lanes 1-3). We hypothesized that this polymorphism could be used in studies of genetic structure of

**5. Kafirins as the markers of endosperm genetic structure** 

endosperm in apomixis research in sorghum.

Fig. 5. Densitograms of endosperm proteins electrophoretic spectra of the line P-614 (a) and F1 hybrid A2 KVV-97/P-614 (b) after pepsin digestion.

#### **4. 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 F1 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.

Fig. 5. Densitograms of endosperm proteins electrophoretic spectra of the line P-614 (a) and

In order to found out dependence of sorghum protein digestibility on starch digestibility the flour of several lines and F1 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

F1 hybrid A2 KVV-97/P-614 (b) after pepsin digestion.

**4. Interaction of starch and protein digestibility** 

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 F1 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, 10 – without this procedure*.* Lanes 1-4 – Sudzern svetlyi; 5-8 – F1 A2 Sudzern svetlyi/Topaz; 9-12 – Topaz; M – molecular weight markers (kDa). Gels were stained with Coomassie Brilliant Blue G-250.
