**3. Improvement of** *in vitro* **protein digestibility**

The main goal of experiments on silencing of kafirin genes is to improve seed storage protein digestibility. Herewith, depending on the structure of the genetic construct, suppression of certain subclasses of kafirins, and the cultivars used in experiments, the level of digestibility varied significantly.

For example, transgenic plants of cv. Tx430 carrying the ABS166 genetic construct containing inverted repeats of several kafirin genes (α, γ, δ) separated by the intron sequence of the alcohol dehydrogenase gene (*ADH1*) and controlled by the 19-kDa α-zein promoter from maize were characterized by improved *in vitro* protein digestibility. Pepsin treatment of the raw flour and flour that underwent the cooking procedure resulted in 78% and 61% digestibility, respectively, while in the non-transgenic control these indicators varied within 40–50% and 34–40%, respectively [34, 35]. The genetic construct for the silencing of δ- and γ-kafirins (ABS149) also improved the digestibility of raw flour, but did not affect the digestibility of the cooked flour.

Subsequently, new transgenic plants were obtained in the sorghum public line P898012 using other genetic constructs ABS042 and ABS044, created during the ABS (Africa Biofortified Sorghum) project [22]. In these plants, an improvement in the digestibility of flour subjected to the cooking procedure was recorded: from 28% in the control to 39% (for the ABS042 construct for silencing γ- and δ-kafirins), and up to 59% (for the ABS044 construct for silencing α-, γ- and δ-kafirins).

Analysis of ultrastructure of protein bodied showed that in transgenic lines with α-kafirin silencing protein bodies were irregular in shape and had invaginations similar to P721Q mutant [34, 35]. In transgenic lines with γ-kafirin silencing, a diameter of protein bodies was reduced in comparison with original non-transgenic line [36]. In addition, in one of the studied lines, 42–1, protein bodies were highly irregular in shape, with deep invaginations present at the periphery, while in the line 42–2, the protein bodies had small peripheral indentations that gave the boundary region a cracked appearance.

In the experiments of T. Kumar et al. [20] the genetic constructs pPTN915 and pPTN1017 designed for the induction of silencing γ- or α-kafirin, respectively, were also introduced into the genome of the Tx430 line through agrobacterial transformation. *In vitro* digestibility of proteins extracted from the flour of transgenic kernels with silencing of γ-kafirin, subjected to cooking procedure, did not differ from the non-transgenic control, while the silencing of α-kafirin by pPTN1017 improved the *in vitro* protein digestibility of flour subjected to cooking.

Transgenic plants of cv. Zh10 obtained in our experiments carrying the genetic construct pNRKAF for silencing *γ-KAFIRIN-1* gene, also had a significantly

*Grain and Seed Proteins Functionality*

**2. Decreased content of kafirins**

introducing a genetic construct capable to induce *γ-KAFIRIN-1* silencing. For silencing the *γ-KAFIRIN-1* gene we used the construct pNRKAF [23] that consisted of segment of its nucleotide sequence ([31], GeneBank accession no. M73688) in forward and inverted orientation, which was separated by the sequence of the maize *ubi1*-intron. This construct was driven by the *35S* promoter. Such a construct should suppress the expression of the *γ-KAFIRIN-1* gene using RNA interference. A decrease in the level of γ-kafirin should have "stripped" the protein bodies in

In this chapter, we describe phenotypic effects of RNAi-silencing of kafirin genes in two sorghum cultivars – Zheltozernoe-10 (Zh10) and Avans, which contain pNRKAF genetic construct introduced by agrobacterial transformation, as well as characteristic features of other sorghum lines carrying similar genetic constructs for

The primary effect of the functioning of genetic constructs for RNA silencing of kafirin genes is a decreased level of transcripts of these genes. Such an effect was shown for the pPTN915 genetic construct, designed to suppress the expression of

*SDS-PAGE of kafirins from kernels of transgenic plants from the T1 generation of the RNAi mutant, cv. Avans, isolated under reducing conditions (with the addition of 2-mercaptoethanol). 1 – Original non-transgenic cv. Avans; 2–7 – Individual plants from the T1 family: 2–6 – Plants with a floury endosperm, containing ubi1-intron; 7 – Plant with a vitreous endosperm, not containing ubi1-intron; M – Molecular mass markers. Kafirins were extracted according to [20]. The arrow marks* α*-kafirin; the dotted arrow marks* γ*-kafirin.*

transgenic plants and facilitated the digestion of α-kafirins.

silencing kafirin genes created by other research groups (**Table 1**).

**146**

**Figure 1.**

improved *in vitro* digestibility of flour proteins [22]. Comparison of electrophoretic spectra before and after pepsin digestion showed that in the transgenic plants the amount of undigested monomers of α-kafirin and total undigested protein was significantly less (1.7–1.9 times) than in the original non-transgenic line. The digestibility level reached 85.4%, while in the original line this value was about 60%. It is noteworthy that in the kernels of transgenic plant No. 94–3-08 (T2) with a thick vitreous endosperm, the differences in the digestion of kafirins were more pronounced: the amount of undigested monomers was 17.5 times less, and the amount of total undigested protein was 4.7 times less than in the original line, while the level of digestibility reached 92%.

Plants from the T3 generation inherited the improved digestibility of kafirins. In these plants, kernels had either a modified type of endosperm with reduced vitreous endosperm, or an endosperm with a well-defined vitreous layer. The level of digestibility of endosperm proteins in these plants was 83–90%, significantly higher than that of the original non-transgenic line (**Figure 2**). Apparently, a decrease in the level of γ-kafirin increases the digestibility of α-kafirins. This increase may be due to chemical reasons (decrease in the amount of polymers) and/or physical reasons (changes in the spatial arrangement of α-kafirins in protein bodies, which increase their availability for cleavage by pepsin). The effect of increased digestibility of kafirins was also observed in plants from the T4 generation; however, in some cases it disappeared, possibly due to the instability of the introduced genetic construct, or due to its silencing (see below).

After experiments with the model cv. Zh10, we set the task of obtaining RNAi mutants with improved digestibility of kafirins in the new commercial cultivar Avans, which is characterized by a number of agronomically valuable traits. The analysis of the *in vitro* digestibility of proteins from kernels that set on one of the transgenic plants (#1–1) obtained by *Agrobacterium*-mediated genetic transformation with the strain carrying pNRKAF genetic construct showed a significantly higher level of digestibility compared to the original non-transgenic cultivar (**Figure 3**) (93% vs. 57%, according

#### **Figure 2.**

*Electrophoretic spectra of proteins from the flour of transgenic plants from T3 family #94–3-08 with normal vitreous endosperm. 1–6 – Individual plants from T3 generation; 7, 8 – Original non-transgenic line Zh10. 1, 3, 5, 7 – Before, 2, 4, 6, 8 – After pepsin digestion. M - molecular mass markers (kDa). [23].*

**149**

**Figure 4.**

*silencing, and original cv. Avans (B).*

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

to the densitometry of SDS-PAGE patterns). A high level of kafirin digestibility was

*Electrophoretic spectra of proteins from the flour of sorghum cv. Avans (1–3), transgenic plant #1–1 (4–6) and non-transgenic plants #5–1 (7–9) and #6–4 (10–12). M - molecular mass markers (kDa). 3, 6, 9, 12 – Before,* 

An important consequence of the functioning of genetic constructs for silencing kafirin genes is a change in the texture of endosperm: in transgenic plants, in most cases, there is complete or partial loss of the vitreous layer, as a result of which the kernels contain only floury endosperm [21, 22, 34, 35]. In our experiments, the

*Cross sections of the kernels of the RNAi mutant #1-1 (A) carrying genetic construct pNRKAF for RNAi* 

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

observed also in the next generation, T1.

*1, 2, 4, 5, 7, 8, 10, 11 – After pepsin digestion.*

**Figure 3.**

**4. Modification of endosperm texture**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

#### **Figure 3.**

*Grain and Seed Proteins Functionality*

the level of digestibility reached 92%.

construct, or due to its silencing (see below).

improved *in vitro* digestibility of flour proteins [22]. Comparison of electrophoretic spectra before and after pepsin digestion showed that in the transgenic plants the amount of undigested monomers of α-kafirin and total undigested protein was significantly less (1.7–1.9 times) than in the original non-transgenic line. The digestibility level reached 85.4%, while in the original line this value was about 60%. It is noteworthy that in the kernels of transgenic plant No. 94–3-08 (T2) with a thick vitreous endosperm, the differences in the digestion of kafirins were more pronounced: the amount of undigested monomers was 17.5 times less, and the amount of total undigested protein was 4.7 times less than in the original line, while

Plants from the T3 generation inherited the improved digestibility of kafirins. In these plants, kernels had either a modified type of endosperm with reduced vitreous endosperm, or an endosperm with a well-defined vitreous layer. The level of digestibility of endosperm proteins in these plants was 83–90%, significantly higher than that of the original non-transgenic line (**Figure 2**). Apparently, a decrease in the level of γ-kafirin increases the digestibility of α-kafirins. This increase may be due to chemical reasons (decrease in the amount of polymers) and/or physical reasons (changes in the spatial arrangement of α-kafirins in protein bodies, which increase their availability for cleavage by pepsin). The effect of increased digestibility of kafirins was also observed in plants from the T4 generation; however, in some cases it disappeared, possibly due to the instability of the introduced genetic

After experiments with the model cv. Zh10, we set the task of obtaining RNAi mutants with improved digestibility of kafirins in the new commercial cultivar Avans, which is characterized by a number of agronomically valuable traits. The analysis of the *in vitro* digestibility of proteins from kernels that set on one of the transgenic plants (#1–1) obtained by *Agrobacterium*-mediated genetic transformation with the strain carrying pNRKAF genetic construct showed a significantly higher level of digestibility compared to the original non-transgenic cultivar (**Figure 3**) (93% vs. 57%, according

*Electrophoretic spectra of proteins from the flour of transgenic plants from T3 family #94–3-08 with normal vitreous endosperm. 1–6 – Individual plants from T3 generation; 7, 8 – Original non-transgenic line Zh10. 1, 3,* 

*5, 7 – Before, 2, 4, 6, 8 – After pepsin digestion. M - molecular mass markers (kDa). [23].*

**148**

**Figure 2.**

*Electrophoretic spectra of proteins from the flour of sorghum cv. Avans (1–3), transgenic plant #1–1 (4–6) and non-transgenic plants #5–1 (7–9) and #6–4 (10–12). M - molecular mass markers (kDa). 3, 6, 9, 12 – Before, 1, 2, 4, 5, 7, 8, 10, 11 – After pepsin digestion.*

to the densitometry of SDS-PAGE patterns). A high level of kafirin digestibility was observed also in the next generation, T1.

#### **4. Modification of endosperm texture**

An important consequence of the functioning of genetic constructs for silencing kafirin genes is a change in the texture of endosperm: in transgenic plants, in most cases, there is complete or partial loss of the vitreous layer, as a result of which the kernels contain only floury endosperm [21, 22, 34, 35]. In our experiments, the

#### **Figure 4.**

*Cross sections of the kernels of the RNAi mutant #1-1 (A) carrying genetic construct pNRKAF for RNAi silencing, and original cv. Avans (B).*

RNAi mutant #1–1 of cv. Avans, had also a floury type of endosperm (**Figure 4**). It should be noted that, in similar experiments in maize, silencing of different zein genes also resulted in reduction of the vitreous endosperm and formation of kernels with floury endosperm [37–39]. It was shown that γ-zein gene plays an important role in the formation of the floury endosperm, and silencing of this gene modified the structure of protein bodies and their connection with starch grains that result in formation of floury endosperm [38].

Unfortunately, the presence of floury endosperm is a significant disadvantage of the obtained lines, since the absence of a vitreous layer increases the fragility of the kernels and reduces its resistance to fungal diseases. It should be noted that the floury (opaque) type of endosperm is characteristic of the P721Q mutant and many

#### **Figure 5.**

*Longitudinal sections of kernels of the original non-transgenic line Zheltozernoe-10 (A) and transgenic plants carrying pNRKAF genetic construct for RNA silencing of the γ-KAFIRIN-1 gene (B-F), differing in the degree of development of the vitreous endosperm. Vitreous endosperm is marked with white arrows.*

**151**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

breeding lines with improved digestibility of kafirins derived from it. Overcoming

In this regard, the transgenic plants of cv. Zh10 with the genetic construction pNRKAF for *γ-KAFIRIN-1* gene silencing are of special interest, since in most cases they had sectors or of the vitreous endosperm in the kernels, or the vitreous endosperm formed a continuous thin layer along the periphery of the kernels (**Figure 5**). It is noteworthy that the formation of the vitreous endosperm in such kernels did not reduce the digestibility of kafirins. Moreover, a plant (94–3-08) was found in T2, in whose progeny (T3, kernels is shown in **Figure 5F**) a high level of kafirin digestibility (88–90%) was combined with normal vitreous endosperm [23]. The fact of obtaining such plants shows that an increase in the digestibility of sorghum kafirins may not be associated with the reduction of the vitreous layer and formation of floury endosperm. Further investigation of these plants is needed to understand the role of the γ-kafirin in development of hard endosperm

Previously, transgenic plants with inclusions of vitreous endosperm surrounded by a floury endosperm were also observed in the transgenic plants of cv. Tx430, which contains a genetic construct for silencing α- and γ-kafirins [35]. At the same time, co-suppression of the δ-kafirin and γ-kafirin subclasses did not change the endosperm type in this cultivar. Apparently, the formation of different types of endosperm is due to the peculiarities of the expression of genetic constructs in the

In this regard, it should be noted that the nucleotide sequence that we used in the genetic construct pNRKAF was homologous not only to the γ-*KAFIRIN-1* gene located in the chromosome 2 of the sorghum genome but also to the locus of the chromosome 9 encoding bi-functional protease inhibitor protein (Pfam: PF00234) belonging to the LTP-family (lipid transfer proteins) [23]. It is possible that a higher kafirin digestibility in plant 94–3-08 and its progeny could be due mainly to the suppression of the synthesis of the protease inhibitor, which did not entail a change

These data indicate a possible effect of protease inhibitors on the digestibility of proteins in sorghum flour, which remains poorly understood. Purposeful designing of genetic constructs for RNA-silencing of protease inhibitors and their introduction into sorghum genome can help to obtain lines with improved digestibility of

An important consequence of silencing of the prolamine genes in cereals is an increase in the synthesis of other proteins, including those with a higher content of essential amino acids. For example, in transgenic maize plants with α-zein silencing, a double content of tryptophan and lysine was observed [40]. In rice, it was shown that silencing of 13 kDa prolamine increases the total lysine content up to 56% as a result of a compensatory increase in the synthesis of lysine-rich glutelin, globulins, and chaperones [41]. A significant increase in the lysine content (up to 3.3 g / 100 g of protein, compared to 2.1 g/100 g of protein in the non-transgenic control) was found in transgenic sorghum plants carrying complex genetic constructs for RNA silencing of kafirins (ABS032, ABS149) [35]. However, these genetic constructs carried, along with the fragments of the kafirin genes, the fragments of the lysine ketoglutarate reductase gene, which controls the catabolism of free lysine. This fact does not allow drawing a conclusion on the effect of kafirin silencing on the

kafirins, in which the endosperm could be of the usual vitreous type.

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

in sorghum.

genome of the recipient line.

in the texture of the endosperm.

**5. Increased synthesis of other proteins**

increase in the lysine content in sorghum.

this correlation is an extremely difficult and urgent task [8].

#### *RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

breeding lines with improved digestibility of kafirins derived from it. Overcoming this correlation is an extremely difficult and urgent task [8].

In this regard, the transgenic plants of cv. Zh10 with the genetic construction pNRKAF for *γ-KAFIRIN-1* gene silencing are of special interest, since in most cases they had sectors or of the vitreous endosperm in the kernels, or the vitreous endosperm formed a continuous thin layer along the periphery of the kernels (**Figure 5**). It is noteworthy that the formation of the vitreous endosperm in such kernels did not reduce the digestibility of kafirins. Moreover, a plant (94–3-08) was found in T2, in whose progeny (T3, kernels is shown in **Figure 5F**) a high level of kafirin digestibility (88–90%) was combined with normal vitreous endosperm [23]. The fact of obtaining such plants shows that an increase in the digestibility of sorghum kafirins may not be associated with the reduction of the vitreous layer and formation of floury endosperm. Further investigation of these plants is needed to understand the role of the γ-kafirin in development of hard endosperm in sorghum.

Previously, transgenic plants with inclusions of vitreous endosperm surrounded by a floury endosperm were also observed in the transgenic plants of cv. Tx430, which contains a genetic construct for silencing α- and γ-kafirins [35]. At the same time, co-suppression of the δ-kafirin and γ-kafirin subclasses did not change the endosperm type in this cultivar. Apparently, the formation of different types of endosperm is due to the peculiarities of the expression of genetic constructs in the genome of the recipient line.

In this regard, it should be noted that the nucleotide sequence that we used in the genetic construct pNRKAF was homologous not only to the γ-*KAFIRIN-1* gene located in the chromosome 2 of the sorghum genome but also to the locus of the chromosome 9 encoding bi-functional protease inhibitor protein (Pfam: PF00234) belonging to the LTP-family (lipid transfer proteins) [23]. It is possible that a higher kafirin digestibility in plant 94–3-08 and its progeny could be due mainly to the suppression of the synthesis of the protease inhibitor, which did not entail a change in the texture of the endosperm.

These data indicate a possible effect of protease inhibitors on the digestibility of proteins in sorghum flour, which remains poorly understood. Purposeful designing of genetic constructs for RNA-silencing of protease inhibitors and their introduction into sorghum genome can help to obtain lines with improved digestibility of kafirins, in which the endosperm could be of the usual vitreous type.

## **5. Increased synthesis of other proteins**

An important consequence of silencing of the prolamine genes in cereals is an increase in the synthesis of other proteins, including those with a higher content of essential amino acids. For example, in transgenic maize plants with α-zein silencing, a double content of tryptophan and lysine was observed [40]. In rice, it was shown that silencing of 13 kDa prolamine increases the total lysine content up to 56% as a result of a compensatory increase in the synthesis of lysine-rich glutelin, globulins, and chaperones [41]. A significant increase in the lysine content (up to 3.3 g / 100 g of protein, compared to 2.1 g/100 g of protein in the non-transgenic control) was found in transgenic sorghum plants carrying complex genetic constructs for RNA silencing of kafirins (ABS032, ABS149) [35]. However, these genetic constructs carried, along with the fragments of the kafirin genes, the fragments of the lysine ketoglutarate reductase gene, which controls the catabolism of free lysine. This fact does not allow drawing a conclusion on the effect of kafirin silencing on the increase in the lysine content in sorghum.

*Grain and Seed Proteins Functionality*

formation of floury endosperm [38].

RNAi mutant #1–1 of cv. Avans, had also a floury type of endosperm (**Figure 4**). It should be noted that, in similar experiments in maize, silencing of different zein genes also resulted in reduction of the vitreous endosperm and formation of kernels with floury endosperm [37–39]. It was shown that γ-zein gene plays an important role in the formation of the floury endosperm, and silencing of this gene modified the structure of protein bodies and their connection with starch grains that result in

Unfortunately, the presence of floury endosperm is a significant disadvantage of the obtained lines, since the absence of a vitreous layer increases the fragility of the kernels and reduces its resistance to fungal diseases. It should be noted that the floury (opaque) type of endosperm is characteristic of the P721Q mutant and many

*Longitudinal sections of kernels of the original non-transgenic line Zheltozernoe-10 (A) and transgenic plants carrying pNRKAF genetic construct for RNA silencing of the γ-KAFIRIN-1 gene (B-F), differing in the degree* 

*of development of the vitreous endosperm. Vitreous endosperm is marked with white arrows.*

**150**

**Figure 5.**

In the transgenic plants obtained in our experiments with a high *in vitro* kafirin digestibility, the total amino acid content in the kernels of plants of the T2 generation decreased by 22.8–40.2% as compared with the original, non-transgenic line [23]. At the same time, the relative content of the two main essential amino acids, lysine and threonine, has increased significantly. The proportion of lysine increased 1.6–1.7 times: from 1.54% of the total amino acid content in the flour of the original non-transgenic line to 2.41–2.63% in transgenic plants. This increase, combined with a significant decrease in the total level of amino acids, was apparently caused by a decrease in the content of α-kafirins, which are poor in lysine and threonine, while the synthesis of other proteins was not impaired. Accordingly, the relative proportions of lysine and threonine increased. It is possible that suppression of the synthesis of γ-kafirin prevents the accumulation of α-kafirins but does not affect the synthesis of other proteins richer in lysine and threonine. The appearance of new proteins in transgenic sorghum plants carrying a genetic construct for silencing α-kafirin gene was described by T. Kumar et al. [21].

It is noteworthy that in the transgenic plants of the cv. Avans with a construct for silencing γ-*KAFIRIN-1* (RNAi mutant #1–1), along with a decrease in the content of γ- and α-kafirins (**Figure 1**), an increase in the content of a number of globulins occurs, possibly resulting from the re-balancing of the proteome of the kernels (**Figure 6**).

Protein rebalancing in the endosperm is a frequent phenomenon in transgenic plants with genetic constructs for RNA silencing of seed storage proteins. In maize, it was suggested that a compensatory mechanism, which is sensitive to the protein

#### **Figure 6.**

*Electrophoretic spectra of globulins from the kernels of transgenic plants from T1 generation of RNAi mutant #1–1. 1 – Original non-transgenic cv. Avans; 2–7 individual T1 plants; M – Molecular mass markers (kDa). Globulins were extracted according to [42].*

**153**

**Figure 7.**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

content exists in the kernels; and a violation of zein synthesis in developing kernels enhances the translation of other mRNAs [43]. It is noteworthy that in transgenic soybean plants with suppressed synthesis of the main storage proteins, the seeds retained an almost identical level of total protein characteristic of untransformed soybean varieties [44]. These data suggest that restoration of proteome balance may be quite common phenomenon, providing a constant supply of nitrogen during

In our experiments, we found that the offspring of transgenic plants with a high *in vitro* digestibility of endosperm proteins sometimes lose this trait. Even different panicles of the same plant had different digestibility values. Such instability is an interesting phenomenon, which may be caused by silencing of introduced genetic construct possibly by RNA-dependent DNA methylation that is characteristic to hairpin genetic constructs [45], or by environmental factors, such as temperature, soil moisture, air humidity, etc. It has been reported that temperature causes a significant impact on RNAi-silencing [46]. It was also shown that mRNA degradation induced by microRNA and translation inhibition, depends on the temperature of plant growth [47]. Consequently, the efficiency of inhibition of kafirin synthesis by RNAi-silencing may be sensitive to plant growing conditions, and this was really

*PCR analysis of plants from the offspring of the RNAi mutant (#1–1, cv. Avans) carrying the genetic construct for silencing γ-KAFIRIN-1, with primers to the nos-promoter (A) and ubi1-intron (B). 1 – Original non-transgenic cv. Avans; 2-4 (A) and 2-5 (B) – individual T1 plants (A: #2, #3, #4; B: #1, #2, #3, #4, respectively); 5–14(A) and 6–15 (B) – Plants from another experiment; 15 (A), 16 (B) – A. tumefaciens GV3101/pNRKAF; 16 (A), 17 (B) – DNA markers; 17 (A), 18 (B) – Negative control (no DNA). The nosspecific primers amplified the 202 bp fragment (A). The ubi1-intron specific primers amplified the 588 bp* 

*fragment (B). The arrows mark the products of DNA amplification in plant #3.*

**6. Instability of the genetic construct for RNA silencing**

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

seed maturation.

shown in our experiments [48].

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

content exists in the kernels; and a violation of zein synthesis in developing kernels enhances the translation of other mRNAs [43]. It is noteworthy that in transgenic soybean plants with suppressed synthesis of the main storage proteins, the seeds retained an almost identical level of total protein characteristic of untransformed soybean varieties [44]. These data suggest that restoration of proteome balance may be quite common phenomenon, providing a constant supply of nitrogen during seed maturation.

### **6. Instability of the genetic construct for RNA silencing**

In our experiments, we found that the offspring of transgenic plants with a high *in vitro* digestibility of endosperm proteins sometimes lose this trait. Even different panicles of the same plant had different digestibility values. Such instability is an interesting phenomenon, which may be caused by silencing of introduced genetic construct possibly by RNA-dependent DNA methylation that is characteristic to hairpin genetic constructs [45], or by environmental factors, such as temperature, soil moisture, air humidity, etc. It has been reported that temperature causes a significant impact on RNAi-silencing [46]. It was also shown that mRNA degradation induced by microRNA and translation inhibition, depends on the temperature of plant growth [47]. Consequently, the efficiency of inhibition of kafirin synthesis by RNAi-silencing may be sensitive to plant growing conditions, and this was really shown in our experiments [48].

#### **Figure 7.**

*Grain and Seed Proteins Functionality*

(**Figure 6**).

α-kafirin gene was described by T. Kumar et al. [21].

In the transgenic plants obtained in our experiments with a high *in vitro* kafirin digestibility, the total amino acid content in the kernels of plants of the T2 generation decreased by 22.8–40.2% as compared with the original, non-transgenic line [23]. At the same time, the relative content of the two main essential amino acids, lysine and threonine, has increased significantly. The proportion of lysine increased 1.6–1.7 times: from 1.54% of the total amino acid content in the flour of the original non-transgenic line to 2.41–2.63% in transgenic plants. This increase, combined with a significant decrease in the total level of amino acids, was apparently caused by a decrease in the content of α-kafirins, which are poor in lysine and threonine, while the synthesis of other proteins was not impaired. Accordingly, the relative proportions of lysine and threonine increased. It is possible that suppression of the synthesis of γ-kafirin prevents the accumulation of α-kafirins but does not affect the synthesis of other proteins richer in lysine and threonine. The appearance of new proteins in transgenic sorghum plants carrying a genetic construct for silencing

It is noteworthy that in the transgenic plants of the cv. Avans with a construct for silencing γ-*KAFIRIN-1* (RNAi mutant #1–1), along with a decrease in the content of γ- and α-kafirins (**Figure 1**), an increase in the content of a number of globulins occurs, possibly resulting from the re-balancing of the proteome of the kernels

Protein rebalancing in the endosperm is a frequent phenomenon in transgenic plants with genetic constructs for RNA silencing of seed storage proteins. In maize, it was suggested that a compensatory mechanism, which is sensitive to the protein

*Electrophoretic spectra of globulins from the kernels of transgenic plants from T1 generation of RNAi mutant #1–1. 1 – Original non-transgenic cv. Avans; 2–7 individual T1 plants; M – Molecular mass markers (kDa).* 

**152**

**Figure 6.**

*Globulins were extracted according to [42].*

*PCR analysis of plants from the offspring of the RNAi mutant (#1–1, cv. Avans) carrying the genetic construct for silencing γ-KAFIRIN-1, with primers to the nos-promoter (A) and ubi1-intron (B). 1 – Original non-transgenic cv. Avans; 2-4 (A) and 2-5 (B) – individual T1 plants (A: #2, #3, #4; B: #1, #2, #3, #4, respectively); 5–14(A) and 6–15 (B) – Plants from another experiment; 15 (A), 16 (B) – A. tumefaciens GV3101/pNRKAF; 16 (A), 17 (B) – DNA markers; 17 (A), 18 (B) – Negative control (no DNA). The nosspecific primers amplified the 202 bp fragment (A). The ubi1-intron specific primers amplified the 588 bp fragment (B). The arrows mark the products of DNA amplification in plant #3.*

#### **Figure 8.**

*PCR analysis of transgenic sorghum plants (T4 generation) carrying a genetic construct pNRKAF [23] with primers to the ubi1-intron (A) and nos-promoter (B). 1 (A, B) – Original non-transgenic line Zh10; 2–-14 (A), 2-12 (B) – DNA of individual transgenic plants from the T4 families; 15 (A), 14 (B) – A. tumefaciens GV3101/pNRKAF (positive control); 16 (A), 14 (B) – DNA markers; 15 (A) – Negative control (no DNA). The ubi1-intron specific primers amplified the 267 bp fragment (A). The nos-specific primers amplified the 202 bp fragment (B). Amplified gene-specific fragments are marked with arrows [50].*

In addition to instability at the epigenetic level, we have found the genetic instability of introduced construct for RNAi-silencing. In this regard, analysis of the progeny of the RNAi mutant #1–1 (cv. Avans), carrying a construct for silencing γ-*KAFIRIN-1*, is indicative. Of the 4 studied T1 plants grown in the experimental field plot, all plants were transgenic, because carried the *nos*-promoter driving the expression of the marker gene *bar*, located in T-DNA of pNRKAF, along with a genetic construct for the γ-*KAFIRIN-1* gene silencing (**Figure 7A**). At the same time, one of these plants (#3) lacked the *ubi1*-intron, which is a part of the genetic construct for silencing (**Figure 7B**). All kernels developed in the panicle of plant #3 had the vitreous type of endosperm, characteristic to the original cultivar (**Figure 4A**), while in the panicles of other plants, in which the *ubi1-*intron was present, the kernels had a floury type of endosperm (**Figure 4B**), characteristic for transgenic plants with γ-kafirin silencing.

In addition, in Zh10 transgenic plants from the T4 families with high digestibility of kafirins, probable elimination of the *nos*-promoter, which controls the expression of the marker gene *bar* in the pNRKAF genetic construct [23] was found [49]. **Figure 8** clearly shows that in the plants from the T4 families, amplification of the *ubi1-*intron fragment was observed, while amplification of the *nos-*promoter located in the construct in front of the marker gene *bar* was absent. Thus, these plants probably turned out to be functionally marker-free transgenic plants. This fact is of significant interest, since the presence of marker genes in the genetic constructs hinders the practical use of transgenic lines in practical plant breeding.

**155**

**Author details**

**Acknowledgements**

grant 19-016-00117.

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

The research findings presented in this chapter provide strong evidence that RNA interference can be used for the improvement of the nutritional value of grain sorghum. RNAi mutants are characterized by significantly improved digestibility of kafirins and higher content of essential amino acids, in particular lysine. In some cases, these mutants retain vitreous endosperm that is highly important for grain

Nevertheless, in most cases the kernels with suppressed synthesis of γ- or α-kafirins have floury endosperm that strongly reduces their use in sorghum breeding. Such a correlation between the traits of high digestibility of kafirins and the floury type of endosperm, which was originally observed in the P721Q mutant and lines created on its basis is a serious problem (see review [8]). In maize, the correlation between the floury endosperm and the increased lysine content was disrupted using modifier genes that enhanced the accumulation of γ-zein [42, 51, 52]. However, in sorghum, an increase in the synthesis of γ-kafirin may decrease the level of kafirin digestibility due to a high content of sulfurcontaining amino acids, which contribute to the polymerization of kafirins. Possibly, one of the ways to solve this problem may be down-regulation of genes that encode protease inhibitors, which can also affect the level of digestion of kafirins by exogenous proteases. In this case, the resulting lines would have a hard

hardiness and in ensuring the resistance of kernels to fungal diseases.

endosperm in combination with a high digestibility of kafirins.

Lev A. Elkonin\*, Valery M. Panin, Odissey A. Kenzhegulov and Saule Kh. Sarsenova

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The work was funded in part by the Russian Foundation for Basic Research,

Federal Agricultural Research Centre of South-East, Saratov, Russia

\*Address all correspondence to: lelkonin@gmail.com

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

**7. Conclusions**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*
