**Abstract**

Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of *γ-KAFIRIN-1* gene in sorghum cv. Avans. To obtain RNAi mutants with *γ-KAFIRIN-1* gene silencing we used *Agrobacterium*-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved *in vitro* protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of *ubi1*-intron that is a constituent part of the genetic construct for RNAi silencing, or *nos*-promotor governing expression of the marker gene (*bar*) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.

**Keywords:** kafirins, *in vitro* protein digestibility, RNAi-mutants, endosperm, *Sorghum bicolor* (L.) Moench

### **1. Introduction**

Grain sorghum is one of the most promising and relatively poorly studied agricultural crops. With its high drought tolerance, sorghum is capable of producing high grain yields in conditions of minimal moisture supply. This crop is of special

importance in the regions regularly exposed to drought, where the stable production of traditional cereals – wheat, maize, barley – is challenging. Moreover, due to the global warming of climate the importance of this crop will steadily increase. Sorghum is already one of the five most important cereal crops cultivated on the Earth. In addition, sorghum grain is gluten-free and can serve as a source of protein for people with celiac disease who are forced to follow a gluten-free diet.

At the same time, compared with other cereals, sorghum grain has a number of significant disadvantages: its storage proteins (kafirins), the content of which reaches 14–16% in some lines and varieties, are poorly digestible by proteases (pepsin, trypsin) [1–4]. The resistance of kafirins to proteolytic digestion reduces the digestibility of starch, which accumulates in significant amounts in sorghum grain (up to 70–75%) since undigested proteins reduce the availability of amylolytic enzymes to starch grains [3, 5, 6]. In addition, the kafirins have low content of indispensible amino acids – lysine, threonine, and tryptophan – and therefore are characterized by low nutritional value [7, 8]. In this regard, increasing the functionality of proteins in sorghum grain, improving their nutritional value is a very urgent problem that has both applied and fundamental importance.

The resistance of kafirins to proteolytic digestion is caused by several factors [9, 10]. Among them are the chemical composition of kafirins, some of which (γ- and β-kafirins) are abundant with sulfur-containing amino acids capable of forming intra- and intermolecular disulfide bonds, hardening protein molecules, and promoting the formation of oligo- and polymers resistant to protease digestion; interaction of kafirins with non-kafirin proteins and non-protein components, in particular, with tannins, which reduce the proteases activity, and with polysaccharides of starch grains; spatial organization of different kafirins in protein bodies of endosperm cells. It was hypothesized that γ-kafirin, which occupies the outer layer of protein bodies and which is the most resistant to proteolytic digestion, prevents the digestion of the α-kafirins – main storage proteins, located inside the protein bodies [11].

An important argument in favor of this hypothesis was the data obtained in the study of the P721Q mutant, induced by chemical mutagenesis and characterized by increased digestibility of kafirins, and the lines derived from this mutant [12, 13]. In this mutant, the protein bodies of endosperm cells have an irregular shape with invaginations. Moreover, γ-kafirin was located only at the bottom of such invaginations, without forming a continuous layer that impedes the access of proteases to α-kafirins [11, 13]. This mutation leads to the formation of kernels with a floury type of endosperm and an increased lysine content, and therefore was denoted with the symbol *hdhl* (*high digestibility high lysine*). Subsequent studies, however, revealed that the P721Q mutant has a point mutation in the signal sequence of one of the 10 copies of the gene encoding the 22 kDa α-kafirin [14]. This sequence is responsible for the packaging of α-kafirin inside the protein body. It was hypothesized that this mutation decreases the accumulation of α-kafirin in protein bodies that leads to a change in their ultrastructure and increases their sensitivity to the action of proteases [14].

To solve the problem of poor digestibility of kafirins various genetic and biotechnological approaches may be used: experimental induction of mutants with impaired synthesis or altered amino acid composition of kafirins [15]; identification of naturally occurring allelic variants of kafirins [16–19]; obtaining transgenic plants with the genetic constructs that induce silencing of γ- and/or α-kafirin genes [20–23]; editing the nucleotide sequences of kafirin genes in order to obtain lines with complete or partial knockout of these genes [24].

RNA interference (RNAi) technology is an effective genetic tool for gene silencing that was used to obtain metabolically engineered plants with improved virus

**145**

**Table 1.**

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

resistance, starch and oil content, and health benefits in different agriculturally important crops [25–28]. The proposed RNA silencing mechanism starts with the production of 20 to 25 bp small interfering RNAs (siRNAs), which are produced from genetic constructs encoding hairpin RNAs (hpRNA). A typical hpRNA construct is comprised of a sense and an antisense sequence of a portion of target gene mRNA as inverted repeats, and these inverted repeats are separated by a non-complementary spacer region. In most genetic constructs, a spliceable intron is used as spacer because it significantly improves RNA silencing efficiency in plants [29]. The sense and antisense sequences in the transcribed RNA are complementary to each other and form a hpRNA, which is processed by Dicer-like proteins (DCL). The DCL proteins generate siRNAs from a hpRNA precursor. One strand of the siRNA duplex is incorporated into an Argonaute (AGO) protein forming an RNA-induced silencing complex (RISC). The siRNA molecule guides the RISC to the complementary region

of single-stranded RNA, and the AGO protein then cleaves the target mRNA.

of seed storage proteins in different crops including wheat, rice and maize (for review see: [30]). These experiments contributed to obtaining new information on the mechanisms of protein body formation, as well as the role of various classes of prolamins and glutenins in the development of endosperm and the technological

The purpose of our investigations was to obtain the grain sorghum lines with improved digestibility of kafirins using RNA interference technology by

pABS032 Maize 19-kDa α-zein promoter; inverted repeats of gene fragments

pABS166 Maize 19-kDa α-zein promoter; inverted repeats of gene fragments

pABS149 Maize 19-kDa α-zein promoter; inverted repeats of gene fragments

pPTN915 γ-kafirin promoter; complete sequence of the γ-kafirin-1 gene (GeneBank

pPTN1017 α-kafirin gene promoter; inverted repeats of the α-kafirin (29 kDa) gene

pABS042 Maize 19-kDa α-zein promoter; inverted repeats of δ-kafirin 2 (18 kDa),

pABS044 Maize 19-kDa α-zein promoter; inverted repeats of δ-kafirin 2 (18 kDa), γ-kafirin 1 (25 kDa), γ-kafirin 2 (50 kDa), α-kafirin-A1, and lysine α-ketoglutarate reductase gene fragments, separated by an intron of the

pNRKAF *35S* promoter; inverted repeats of the γ-kafirin 1 gene fragment (GeneBank accession no. M73688), separated by the maize *ubi1-*intron

*Genetic constructs specially designed to induce RNA silencing of kafirin genes. The molecular masses of kafirins* 

separated by the intron of the *ADH1* gene

**Name Structure of genetic construction Reference**

encoding α1 (25 kDa) and γ1 (27 kDa), separated by an intron of the *ADH1*

acc. no. X62480), the sequence of the ribozyme gene of the tobacco mosaic

fragment, separated by the intron of the Arabidopsis gene encoding the

γ-kafirin 1 (25 kDa), γ-kafirin 2 (50 kDa), and lysine α-ketoglutarate reductase gene fragments, separated by an intron of the alcohol

encoding γ1 (27 kDa), γ2 (50 kDa), and δ2 (15 kDa) kafirins, lysine α-ketoglutarate reductase, separated by an intron of the *ADH1* gene

[20, 34, 35]

[20, 34, 35]

[20, 34, 35]

[21]

[21]

[22]

[22, 35]

[23, 35]

encoding α-A1 (25kDa), α-B1 (19kDa), α-B2 (22kDa), γ1 (27 kDa), γ2 (50 kDa) and δ2 (15 kDa) kafirins, and lysine α-ketoglutarate reductase,

RNA interference technology has been intensively used to suppress the synthesis

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

properties of flour and dough.

gene

virus as a terminator

spliceosome D1 protein

dehydrogenase gene (*ADH1*)

*are given in accordance with the author's description.*

alcohol dehydrogenase gene (*ADH1*)

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

resistance, starch and oil content, and health benefits in different agriculturally important crops [25–28]. The proposed RNA silencing mechanism starts with the production of 20 to 25 bp small interfering RNAs (siRNAs), which are produced from genetic constructs encoding hairpin RNAs (hpRNA). A typical hpRNA construct is comprised of a sense and an antisense sequence of a portion of target gene mRNA as inverted repeats, and these inverted repeats are separated by a non-complementary spacer region. In most genetic constructs, a spliceable intron is used as spacer because it significantly improves RNA silencing efficiency in plants [29]. The sense and antisense sequences in the transcribed RNA are complementary to each other and form a hpRNA, which is processed by Dicer-like proteins (DCL). The DCL proteins generate siRNAs from a hpRNA precursor. One strand of the siRNA duplex is incorporated into an Argonaute (AGO) protein forming an RNA-induced silencing complex (RISC). The siRNA molecule guides the RISC to the complementary region of single-stranded RNA, and the AGO protein then cleaves the target mRNA.

RNA interference technology has been intensively used to suppress the synthesis of seed storage proteins in different crops including wheat, rice and maize (for review see: [30]). These experiments contributed to obtaining new information on the mechanisms of protein body formation, as well as the role of various classes of prolamins and glutenins in the development of endosperm and the technological properties of flour and dough.


The purpose of our investigations was to obtain the grain sorghum lines with improved digestibility of kafirins using RNA interference technology by

#### **Table 1.**

*Grain and Seed Proteins Functionality*

importance in the regions regularly exposed to drought, where the stable production of traditional cereals – wheat, maize, barley – is challenging. Moreover, due to the global warming of climate the importance of this crop will steadily increase. Sorghum is already one of the five most important cereal crops cultivated on the Earth. In addition, sorghum grain is gluten-free and can serve as a source of protein

At the same time, compared with other cereals, sorghum grain has a number of significant disadvantages: its storage proteins (kafirins), the content of which reaches 14–16% in some lines and varieties, are poorly digestible by proteases (pepsin, trypsin) [1–4]. The resistance of kafirins to proteolytic digestion reduces the digestibility of starch, which accumulates in significant amounts in sorghum grain (up to 70–75%) since undigested proteins reduce the availability of amylolytic enzymes to starch grains [3, 5, 6]. In addition, the kafirins have low content of indispensible amino acids – lysine, threonine, and tryptophan – and therefore are characterized by low nutritional value [7, 8]. In this regard, increasing the functionality of proteins in sorghum grain, improving their nutritional value is a very urgent

The resistance of kafirins to proteolytic digestion is caused by several factors [9, 10]. Among them are the chemical composition of kafirins, some of which (γ- and β-kafirins) are abundant with sulfur-containing amino acids capable of forming intra- and intermolecular disulfide bonds, hardening protein molecules, and promoting the formation of oligo- and polymers resistant to protease digestion; interaction of kafirins with non-kafirin proteins and non-protein components, in particular, with tannins, which reduce the proteases activity, and with polysaccharides of starch grains; spatial organization of different kafirins in protein bodies of endosperm cells. It was hypothesized that γ-kafirin, which occupies the outer layer of protein bodies and which is the most resistant to proteolytic digestion, prevents the digestion of the α-kafirins – main storage proteins, located inside the protein

An important argument in favor of this hypothesis was the data obtained in the study of the P721Q mutant, induced by chemical mutagenesis and characterized by increased digestibility of kafirins, and the lines derived from this mutant [12, 13]. In this mutant, the protein bodies of endosperm cells have an irregular shape with invaginations. Moreover, γ-kafirin was located only at the bottom of such invaginations, without forming a continuous layer that impedes the access of proteases to α-kafirins [11, 13]. This mutation leads to the formation of kernels with a floury type of endosperm and an increased lysine content, and therefore was denoted with the symbol *hdhl* (*high digestibility high lysine*). Subsequent studies, however, revealed that the P721Q mutant has a point mutation in the signal sequence of one of the 10 copies of the gene encoding the 22 kDa α-kafirin [14]. This sequence is responsible for the packaging of α-kafirin inside the protein body. It was hypothesized that this mutation decreases the accumulation of α-kafirin in protein bodies that leads to a change in their ultrastructure and increases their sensitivity to the

To solve the problem of poor digestibility of kafirins various genetic and biotechnological approaches may be used: experimental induction of mutants with impaired synthesis or altered amino acid composition of kafirins [15]; identification of naturally occurring allelic variants of kafirins [16–19]; obtaining transgenic plants with the genetic constructs that induce silencing of γ- and/or α-kafirin genes [20–23]; editing the nucleotide sequences of kafirin genes in order to obtain lines

RNA interference (RNAi) technology is an effective genetic tool for gene silencing that was used to obtain metabolically engineered plants with improved virus

with complete or partial knockout of these genes [24].

for people with celiac disease who are forced to follow a gluten-free diet.

problem that has both applied and fundamental importance.

**144**

bodies [11].

action of proteases [14].

*Genetic constructs specially designed to induce RNA silencing of kafirin genes. The molecular masses of kafirins are given in accordance with the author's description.*

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 transgenic plants and facilitated the digestion of α-kafirins.

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