**5.** *In vitro* **digestibility of endosperm proteins**

To study *in vitro* protein digestibility, the method of whole‐grain flour pepsin treatment, widely practiced in the past few years, was used [37–42]. The flour (20 mg) of transgenic sam‐ ples (kernels of transgenic plants from T<sup>1</sup> –T3 generations) and of original non‐transgenic line Zh10 was treated with 5 ml of 0.15% pepsin solution (Sigma‐Aldrich, activity: 806 units/mg of protein) in a 0.1 M potassium phosphate buffer (pH 2.0) for 120 min at 37°C with repeated shaking. The control samples were incubated in potassium phosphate buffer without pepsin addition under the same conditions. For quantitative estimation of protein digestibility, the digested and control samples were centrifuged and the pellet was incubated with a sample buffer (0.0625 M Tris·HCl, pH 6.8) under reducing conditions (see above). The samples were subjected to SDS‐PAGE (see above). After electrophoresis, the gels were scanned. The amount of protein, expressed as volume (intensity × area) of kafirin bands or of total protein bands in the lane, was quantified with the Scangel program (Dr. A.F. Ravich, Agricultural Research Institute of the South‐East Region, Saratov, Russian Federation) [41]. The digestibility value was counted as the percent ratio of the difference between protein volume in the control sample and in digested sample to the protein volume in the control sample. All experiments were performed in two replications.

It was found that transgenic plants obtained in our experiments significantly differed in digestibility of endosperm storage proteins from the original non‐transgenic line Zh10 [28]. Comparison of electrophoretic spectra before and after pepsin digestion of proteins of T1 plant #94‐2 (almost floury endosperm; **Figure 8A**, lanes 1, 2) with Zh‐10 kernels (**Figure 8A**, lanes 5, 6) revealed that in transgenic plant the amount of undigested α‐kafirin monomers and total undigested protein was significantly fewer (in 1.7–1.9 times) than in original non‐trans‐ genic line (**Table 2**). The digestibility value reached 85.4%, whereas in original line this value was about 60%, usual index for sorghum flour (**Table 3**). Remarkably, in kernels of trans‐ genic plant #94‐3‐08 (T2 generation) with thick irregularly developed vitreous endosperm (**Figure 8A**, lanes 3, 4), the differences in kafirin digestion, in comparison with original line Zh‐10 (**Figure 8A**, lanes 5, 6), were more pronounced: the amount of undigested monomers was 17.5 times fewer, and the amount of total undigested protein was 4.7 times fewer than in original line (**Table 2**). The digestibility value reached 92% (**Table 3**).

One should note considerable differences in content of kafirin oligomers between original non‐transgenic line Zh10 and transgenic plants (**Figure 8**). Decreased content of kafirin oligo‐ mers, which apparently was caused by reduction of γ‐kafirin synthesis, might be the reason of higher protein digestibility in transgenic plants.

Another examples of significantly improved kafirin digestibility in transgenic plants obtained in our experiments are presented in **Figure 8B**, where almost complete disappearance of kafi‐ rin monomers after pepsin digestion was observed in plants from T2 generation with both floury (#94‐2‐11, lanes 5, 6) and modified endosperm (#94‐2‐04, lanes 1, 2, and #94‐2‐05, lanes 3, 4). Total protein digestibility indices in 94‐2‐05 and 94‐2‐11 plants reached 74.1% and 90.7%, respectively, that significantly differed from original non‐transgenic line (**Table 3**). Remarkably, in electrophoretic spectra of digested samples of transgenic plants, one should note the polypeptides with molecular weights approx. 40 and 42 kDa. Previously, we found that these polypeptides were more prominent in electrophoretic spectra of more digestible lines than in spectra of poorly digestible ones [41]. In this study, appearance of these poly‐ peptides in transgenic samples coincides with almost complete digestion of kafirin monomers and slightly reduces total protein digestibility values (**Table 3**).

**5.** *In vitro* **digestibility of endosperm proteins**

for silencing of the γ‐kafirin gene. (A) Kernel with floury endosperm (Т<sup>3</sup>

94‐2‐05‐2; T3

94‐2‐05, T2

ples (kernels of transgenic plants from T<sup>1</sup>

and sectors of vitreous endosperm (T2

94‐3‐08; T3

endosperm (T2

100 Plant Engineering

To study *in vitro* protein digestibility, the method of whole‐grain flour pepsin treatment, widely practiced in the past few years, was used [37–42]. The flour (20 mg) of transgenic sam‐

**Figure 7.** Cross sections of kernels with different types of endosperm of transgenic sorghum plants with genetic construct

line Zheltozernoe 10 with thick vitreous endosperm (marked by arrows); (C–E) modified endosperm type with blurs

94‐2‐04, T1

94‐2‐11‐2, respectively). Bar = 1 mm.

Zh10 was treated with 5 ml of 0.15% pepsin solution (Sigma‐Aldrich, activity: 806 units/mg of protein) in a 0.1 M potassium phosphate buffer (pH 2.0) for 120 min at 37°C with repeated shaking. The control samples were incubated in potassium phosphate buffer without pepsin addition under the same conditions. For quantitative estimation of protein digestibility, the digested and control samples were centrifuged and the pellet was incubated with a sample buffer (0.0625 M Tris·HCl, pH 6.8) under reducing conditions (see above). The samples were subjected to SDS‐PAGE (see above). After electrophoresis, the gels were scanned. The amount

generations) and of original non‐transgenic line

94‐2‐05‐1); (B) kernel of original non‐transgenic

94‐6, respectively); (F–H) irregularly developed vitreous

–T3

Plants from T3 generation inherited improved digestibility of kafirins. Comparison of elec‐ trophoretic spectra of proteins obtained from plants #94‐2‐11‐2 and # 94‐2‐11‐3 (**Figure 9A**, lanes 1–4), which were characterized by almost floury or modified endosperm, with the spec‐ trum of the original line (**Figure 9A**, lanes 5, 6) before and after pepsin digestion showed that in transgenic plants, the amount of undigested α‐kafirin monomers was significantly fewer (3.4–6.0 times, respectively) (**Table 2**). Likewise, the total sum of undigested proteins was also reduced (2.9–3.2 times). The digestibility value reached 85.5–87.8%, whereas in the original line this value was 59.3%, the usual index for sorghum flour (**Table 3**).

**Figure 8.** SDS‐PAGE of endosperm proteins of kernels developed on transgenic sorghum plants with genetic construct for silencing of the γ‐kafirin gene in reducing conditions. (A) 1, 2—#94‐2 (T<sup>1</sup> generation) with almost floury endosperm; 3, 4—#94‐3‐8 (T2 generation) with thick vitreous endosperm; 5, 6—original non‐transgenic line Zheltozernoe 10 (Zh10) with normal vitreous endosperm; M—molecular weight markers (kDa; Thermo Scientific). 1, 3, 5—before, and 2, 4, 6 after pepsin digestion. Dashed arrows indicate probable kafirin oligomers. α‐kafirin monomers are indicated by brace. (B) 1, 2—#94‐2‐04; 3, 4—#94‐2‐05, both with modified endosperm, in which vitreous layer is covered by thin floury layer (**Figure 4C**); 5, 6—#94‐2‐11 with floury endosperm; 7, 8—original non‐transgenic line Zh10. 40 and 42 kDa appeared in digested samples are marked by arrows. 1, 3, 5, 7—before and 2, 4, 6, 8—after pepsin digestion (**Figure 8A** is published with the permission of the publishing house "Nauka").

Development of Transgenic Sorghum Plants with Improved *In Vitro* Kafirin Digestibility http://dx.doi.org/10.5772/intechopen.69973 103


**Figure 8.** SDS‐PAGE of endosperm proteins of kernels developed on transgenic sorghum plants with genetic construct

with normal vitreous endosperm; M—molecular weight markers (kDa; Thermo Scientific). 1, 3, 5—before, and 2, 4, 6 after pepsin digestion. Dashed arrows indicate probable kafirin oligomers. α‐kafirin monomers are indicated by brace. (B) 1, 2—#94‐2‐04; 3, 4—#94‐2‐05, both with modified endosperm, in which vitreous layer is covered by thin floury layer (**Figure 4C**); 5, 6—#94‐2‐11 with floury endosperm; 7, 8—original non‐transgenic line Zh10. 40 and 42 kDa appeared in digested samples are marked by arrows. 1, 3, 5, 7—before and 2, 4, 6, 8—after pepsin digestion (**Figure 8A** is published

generation) with thick vitreous endosperm; 5, 6—original non‐transgenic line Zheltozernoe 10 (Zh10)

generation) with almost floury endosperm;

for silencing of the γ‐kafirin gene in reducing conditions. (A) 1, 2—#94‐2 (T<sup>1</sup>

with the permission of the publishing house "Nauka").

3, 4—#94‐3‐8 (T2

102 Plant Engineering


1 c—control sample; p—pepsin treatment.

2 Values are expressed as amount of dots (intensity × mm2 ).

3 Percentage from estimated protein quantity in undigested sample.

**Table 2.** Quantitative analysis of SDS‐PAGE of total flour proteins from kernels of transgenic sorghum plants obtained by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL.


Notes: Each value is a mean from two replications. Data followed by the same letter did not differ significantly (*P* < 0.05) from plant from the same group of families according to Duncan Multiple Range Test. Protein digestibility was calculated as percent ratio of difference between total estimated protein quantity in the control and digested sample to total estimated protein quantity in the control sample.

\*\*Significant at *P <* 0.01.

**Table 3.***In vitro* protein digestibility of sorghum flour from kernels of transgenic plants obtained by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL.

Development of Transgenic Sorghum Plants with Improved *In Vitro* Kafirin Digestibility http://dx.doi.org/10.5772/intechopen.69973 105

**Plant Endosperm type Protein digestibility (%)**

**Plant Lane1 Estimated protein quantity2 Percent of undigested** 

**α‐kafirin monomers**

Zheltozernoe 10 (original line) 7 (c) 10.090·10<sup>6</sup> 19.495·10<sup>6</sup> 56.4 38.8 8 (p) 5.692·10<sup>6</sup> 7.570·10<sup>6</sup>

).

**Table 2.** Quantitative analysis of SDS‐PAGE of total flour proteins from kernels of transgenic sorghum plants obtained

**protein3**

**monomers**

**total**

**total α‐kafirin** 

94‐2 Floury 85.4 c

94‐6 Floury 85.2 c

94‐2‐05 Modified 74.1 b

94‐2‐11 Floury 90.7 cd

 94‐3‐08 Vitreous, irregular 92.0 d Zheltozernoe 10 (original non‐transgenic line) Vitreous 60.4 a *F* 71.52\*\*

94‐2‐11‐2 Modified 87.8 b

94‐2‐11‐3 Modified 85.5 b

94‐2‐04‐2 Modified 85.2 b

94‐3‐04‐1 Floury 83.1 b

94‐3‐04‐1 Modified 90.3 c

94‐3‐08‐2 Vitreous, irregular 86.2 b

 94‐3‐08‐3 Vitreous, irregular 88.3 b Zheltozernoe 10 (original non‐transgenic line) Vitreous 59.3 a *F* 68.311\*\*

Notes: Each value is a mean from two replications. Data followed by the same letter did not differ significantly (*P* < 0.05) from plant from the same group of families according to Duncan Multiple Range Test. Protein digestibility was calculated as percent ratio of difference between total estimated protein quantity in the control and digested sample to

**Table 3.***In vitro* protein digestibility of sorghum flour from kernels of transgenic plants obtained by genetic transformation

Plants from T1

Plants from T3

Т1

1

104 Plant Engineering

2

3

Т1

T2

T2

T2

Т3

Т3

Т3

Т3

Т3

Т3

Т3

\*\*Significant at *P <* 0.01.

and T2

c—control sample; p—pepsin treatment.

Values are expressed as amount of dots (intensity × mm2

Percentage from estimated protein quantity in undigested sample.

by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL.

families

total estimated protein quantity in the control sample.

with *A. tumefaciens* GV3101/pNRKAFSIL.

families

**Figure 9.** SDS‐PAGE of endosperm proteins of kernels of transgenic sorghum plants from T<sup>3</sup> families #94‐2‐11 (with modified endosperm) and (with irregular vitreous endosperm) in reducing conditions. (A) 1, 2—#94‐2‐11‐2; 3, 4—#94‐2‐11‐3; 5, 6—original non‐transgenic line Zh10; M—molecular weight markers (kDa). Dashed arrows indicate fraction of kafirin oligomers; brace—α‐kafirin monomers. 1, 3, 5—control samples; 2, 4, 6—samples after pepsin digestion. (B) 1–6—Three individual plants from #94‐3‐08 family; 7, 8—original non‐transgenic line Zh10. 1, 3, 5, 7 before and 2, 4, 6, 8—after pepsin digestion (published with the permission of the publishing house "Nauka").

Improved *in vitro* protein digestibility was observed also in plants from other T3 families: #94‐2‐04, #94‐3‐04 and #94‐3‐08 (**Table 3**). In these plants, kernels had either floury or modified endosperm (#94‐2‐04‐2; #94‐3‐04‐1) or endosperm with irregularly developed vitreous layer (#94‐3‐08). Quantitative analysis showed that the level of digestibility of endosperm proteins in these plants was 83–90%, significantly differing from the digestibility of proteins in the original non‐transgenic line.

Thus, the comparison of electrophoretic spectra of endosperm proteins before and after pep‐ sin treatment showed a high level of kafirin digestibility in transgenic sorghum plants, har‐ boring genetic construct for silencing of the γ‐kafirin gene. Such electrophoretic spectra of digested endosperm proteins are not characteristic of ordinary sorghum cultivars obtained by classical breeding [40–42] except highly digestible sorghum mutant (*hdhl*) and its hybrids [37–39]. Apparently, a decrease in the level of γ‐kafirin increases the digestibility of α‐kafirins. This increase may be due to chemical reasons (reduction of polymerization) and/or physical reasons (change in the spatial arrangement of α‐kafirins in the protein bodies that increase their availability to pepsin digestion).

Earlier it was reported on obtaining of transgenic sorghum plants carrying genetic constructs for silencing of γ‐ and α‐kafirins, which were characterized by increased *in vitro* protein digestibility [25–27]. However, electrophoretic spectra of endosperm proteins after pepsin treatment were not shown in these studies. It should be noted also that in these studies improvement of kafirin digestibility was induced by complex genetic constructs that contained inverted repeats of sev‐ eral kafirin genes (δ2, γ1, γ2; or α1, δ2, γ1, γ2). These repeats were separated by the sequence of ADH1 intron, and the constructs were driven by the maize 19‐kDa α‐zein promoter [24–26]. In another work [27], the genetic construct included the complete sequence of the γ‐kafirin gene, which was terminated by a nucleotide sequence of the self‐cleaving ribozyme of tobacco ringspot virus that should destroy γ‐kafirin mRNA. In our study [28], the effect was achieved by using a simpler genetic construct, containing inverted repeats of a short segment of the gene γ‐kafirin (307 bp) separated by *ubi1*‐intron gene, under the control of the constitutive *35S*‐promoter, which allowed us to reach apparently rather high level of silencing of a target gene.
