Open access peer-reviewed chapter

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

Written By

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

Submitted: 09 September 2020 Reviewed: 25 January 2021 Published: 18 February 2021

DOI: 10.5772/intechopen.96204

From the Edited Volume

Grain and Seed Proteins Functionality

Edited by Jose Carlos Jimenez-Lopez

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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.


  • 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, 2, 3, 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, 17, 18, 19]; obtaining transgenic plants with the genetic constructs that induce silencing of γ- and/or α-kafirin genes [20, 21, 22, 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 resistance, starch and oil content, and health benefits in different agriculturally important crops [25, 26, 27, 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 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).

NameStructure of genetic constructionReference
pABS032Maize 19-kDa α-zein promoter; inverted repeats of gene fragments 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, separated by the intron of the ADH1 gene[20, 34, 35]
pABS166Maize 19-kDa α-zein promoter; inverted repeats of gene fragments encoding α1 (25 kDa) and γ1 (27 kDa), separated by an intron of the ADH1 gene[20, 34, 35]
pABS149Maize 19-kDa α-zein promoter; inverted repeats of gene fragments 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]
pPTN915γ-kafirin promoter; complete sequence of the γ-kafirin-1 gene (GeneBank acc. no. X62480), the sequence of the ribozyme gene of the tobacco mosaic virus as a terminator[21]
pPTN1017α-kafirin gene promoter; inverted repeats of the α-kafirin (29 kDa) gene fragment, separated by the intron of the Arabidopsis gene encoding the spliceosome D1 protein[21]
pABS042Maize 19-kDa α-zein promoter; inverted repeats of δ-kafirin 2 (18 kDa), γ-kafirin 1 (25 kDa), γ-kafirin 2 (50 kDa), and lysine α-ketoglutarate reductase gene fragments, separated by an intron of the alcohol dehydrogenase gene (ADH1)[22]
pABS044Maize 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 alcohol dehydrogenase gene (ADH1)[22, 35]
pNRKAF35S promoter; inverted repeats of the γ-kafirin 1 gene fragment (GeneBank accession no. M73688), separated by the maize ubi1-intron[23, 35]

Table 1.

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.


2. Decreased content of kafirins

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 the γ-kafirin gene [21]. Many studies using SDS-PAGE have also clearly demonstrated a decrease in the content of monomers and polymers of kafirins [20, 22, 23, 32]. In our experiments, SDS-PAGE of proteins extracted from kernels of transgenic plants of Zh10 in non-reducing conditions (without the addition of 2-mercaptoethanol, which breaks the S-S bonds and, thereby, destroys the polymers of the kafirins), showed a decreased content of γ-kafirin monomer (28 kDa), as well as 47 and 66 kDa oligomers, which are supposed to arise as a result of γ-kafirin polymerization [33]. SDS-PAGE of kafirins extracted under reducing conditions from the kernels of transgenic plants of the Avans cultivar (T1 generation) carrying the same genetic construct revealed also a noticeable decrease in the content of γ- and α-kafirins (Figure 1).

Figure 1.

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.


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 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).

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].

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

Figure 3.

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.


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 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, 38, 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].

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).

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 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.

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.

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.

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).

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].

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 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].

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.

Figure 7.

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 nos-specific 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.

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.

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].


7. Conclusions

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

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 sulfur-containing 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 endosperm in combination with a high digestibility of kafirins.



The work was funded in part by the Russian Foundation for Basic Research, grant 19-016-00117.


  1. 1. Oria MP, Hamaker BR, Shull JM. Resistance of sorghum α-, β- and γ-kafirins to pepsin digestion. J Agric Food Chem. 1995;43:2148-2153. DOI: 10.1021/jf00056a036
  2. 2. Nunes A, Correia I, Barros A, Delgadillo I. Sequential in vitro pepsin digestion of uncooked and cooked sorghum and maize samples. J. Agric. Food Chem. 2004;52:2052-2058. DOI: 10.1021/jf0348830
  3. 3. Wong JH, Lau T, Cai N, Singh J, Pedersen JF, Vensel WH, Hurkman WJ, Wilson JD, Lemaux PG, Buchanan BB. Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm. J. Cereal Sci. 2009;49:73-82. DOI: 10.1016/j.jcs.2008.07.013
  4. 4. Elkonin LA, Italianskaya JV, Fadeeva IYu, Bychkova VV, Kozhemyakin VV.In vitro protein digestibility in grain sorghum: effect of genotype and interaction with starch digestibility. Euphytica. 2013;193:327-337. DOI: 10.1007/s10681-013-0920-4
  5. 5. Zhang G, Hamaker BR. Low a-amylase starch digestibility of cooked sorghum flours and the effect of protein. Cereal Chem 1998;75:710-713. DOI: 10.1094/CCHEM.1998.75.5.710
  6. 6. Ezeogu LI, Duodu KG, Taylor JRN. Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours. J Cereal Sci. 2005;42:33-44. DOI: 10.1016/j.jcs.2005.02.002
  7. 7. Henley EC, Taylor JRN, Obukosia SD. The importance of dietary protein in human health: combating protein deficiency in Sub-Saharan Africa through transgenic biofortified sorghum. In: Taylor SL, editor. Advances in Food and Nutrition Research, Vol. 60. Burlington: Academic Press; 2010. p. 21-52. DOI: 10.1016/S1043-4526(10)60002-2
  8. 8. Duressa D, Weerasoriya D, Bean SR, Tilley M, Tesso T. Genetic basis of protein digestibility in grain sorghum. Crop Sci. 2018;58:2183-2199. DOI: 10.2135/cropsci2018.01.0038
  9. 9. Belton PS, Delgadillo I, Halford NG, Shewry PR. Kafirin structure and functionality. J. Cereal Sci. 2006;44:272-286. DOI:10.1016/j.jcs.2006.05.004
  10. 10. De Mesa-Stonestreet NJ, Alavi S, Bean SR. Sorghum proteins: the concentration, isolation, modification, and food applications of kafirins. J. Food Sci. 2010;75: 90-104. DOI: 10.1111/j.1750-3841.2010.01623.x
  11. 11. Oria MP, Hamaker BR., Axtell JD, Huang CP. A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies. Proc. Natl. Acad. Sci. USA. 2000; 97:5065-5070. DOI: 10.1073/pnas.080076297
  12. 12. Mohan DP. Chemically induced high lysine mutants in Sorghum bicolor (L.) Moench [thesis]. West Lafayette: Purdue University; 1975
  13. 13. Weaver CA, Hamaker BR, Axtell JD. Discovery of grain sorghum germplasm with high uncooked and cooked in vitro protein digestibility. Cereal Chem. 1998;75:665-670. DOI: 10.1094/CCHEM.1998.75.5.665
  14. 14. Wu Y, Yuan L, Guo X, Holding DR, Messing J. Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum. Nat. Commun. 2013;4:2217. DOI: 10.1038/ncomms3217
  15. 15. Mehlo L, Mbambo Z, Bado S, Lin J, Moagi SM, Buthelezi S, Stoychev S, Chikwamba R. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum. Mutation Res. 2013;749: 66-72. DOI: 10.1016/j.mrfmmm.2013.05.002
  16. 16. Laidlaw H, Mace E, Williams S, Sakrewski K, Mudge AM, Prentis PJ, Jordan DR, Godwin ID. Allelic variation of the β-, γ- and δ-kafirin genes in diverse Sorghum genotypes. Theor Appl Genet. 2010;121:1227-1237. DOI: 10.1007/s00122-010-1383-9
  17. 17. Cremer JE, Bean SR, Tilley MM, Ioerger BP, Ohm JB, Kaufman RC, Wilson JD, Innes DJ, Gilding EK, Godwin ID. Grain sorghum proteomics: integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants. J. Agric. Food Chem. 2014;62:9819-9831. DOI: 10.1021/jf5022847
  18. 18. Chiquito-Almanza E, Ochoa-Zarzosa A, Lуpez-Meza JE, Pecina-Quintero V, Nuñez-Colín CA, Anaya-López JL. A new allele of γ-kafirin gene coding for a protein with high lysine content in Mexican white sorghum germplasm. J. Sci. Food Agricult. 2015. DOI: 10.1002/jsfa.7513
  19. 19. Duressa D, Bean S, Amand PS, Tesso T. Identification of variant α-kafirin alleles associated with protein digestibility in grain sorghum. Crop Science. 2020;60:2467-2478. 10.1002/csc2.20198
  20. 20. da Silva LS, Taylor J, Taylor JR. Transgenic sorghum with altered kafirin synthesis: kafirin solubility, polymerization, and protein digestion. J. Agric. Food Chem. 2011;59:9265-9270. DOI: 10.1021/jf201878p
  21. 21. Kumar T, Dweikat I, Sato S, Ge Z, Nersesian N, Elthon T, Bean S, Ioerger BP, Tiley M, Clemente T. Modulation of kernel storage proteins in grain sorghum (Sorghum bicolor (L.) Moench). Plant Biotechnol. J. 2012;10: 533-544. DOI: 10.1111/j.1467-7652.2012.00685.x
  22. 22. Grootboom AW, Mkhonza N L, Mbambo Z, O’Kennedy MM, da Silva LS, Taylor J, Taylor JRN., Chikwamba R, Mehlo L. Co-suppression of synthesis of major α-kafirin sub-class together with γ-kafirin-1 and γ-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum. Plant Cell Rep. 2014;33:521-537. DOI: 10.1007/s00299-013-1556-5
  23. 23. Elkonin LA, Italianskaya JV, Domanina IV, Selivanov NY, Rakitin AL, Ravin NV. Transgenic sorghum with improved digestibility of storage proteins obtained by Agrobacterium-mediated transformation. Russ. J. Plant Physiol. 2016;63:678-689. DOI: 10.1134/S1021443716050046
  24. 24. Li A, Jia S, Yobi A, Ge Z, Sato SJ, Zhang C, Angelovici R, Clemente TE, Holding DR. Editing of an alpha-kafirin gene family increases digestibility and protein quality in sorghum. Plant Physiol. 2018;177:1425-1438. DOI: 10.1104/pp.18.00200
  25. 25. Saurabh S, Vidyarthi AS, Prasad D. RNA interference: concept to reality in crop improvement. Planta. 2014;239:543-564. DOI 10.1007/s00425-013-2019-5
  26. 26. Younis A, Siddique MI, Kim C-K, Lim K-B. RNA Interference (RNAi) induced gene silencing: a promising approach of Hi-tech plant breeding. Int. J. Biol. Sci. 2014;10:1150-1158. DOI: 10.7150/ijbs.10452
  27. 27. Guo Q , Liu Q , Smith NA, Liang G, Wang MB. RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops. Current Genomics, 2016,17:476-489. DOI: 10.2174/138920291766616052010 3117
  28. 28. Muhammad T, Zhang F, Zhang Y, Liang Y. RNA Interference: A Natural Immune System of Plants to Counteract Biotic Stressors. Cells 2019, 8, 38. DOI:10.3390/cells8010038
  29. 29. Smith N.A., Singh S.P., Wang M-B., Stoutjesdijk P., Green A., Waterhouse P.M. Total silencing by intron-spliced hairpin RNAs. Nature, 2000, 407, 319-320
  30. 30. El’konin LA, Domanina IV, Ital’yanskaya YuV. Genetic engineering as a tool for modification of seed storage proteins and improvement of nutritional value of cereal grain. Agricultural Biology. 2016;51:17-30. DOI: 10.15389/agrobiology.2016.1.17eng
  31. 31. De Barros EG, Takasaki K, Kirleis AW, Larkins BA. Nucleotide sequence of a cDNA clone encoding γ-kafirin protein from Sorghum bicolor. Plant Physiol. 1991; 97:1606-1607 DOI:10.1104/pp.97.4.1606
  32. 32. Elkonin LA, Italyanskaya JV, Panin VM, Selivanof NY. Development of transgenic sorghum plants with improved in vitro kafirin digestibility. In: Jurić S, editor. Plant Engineering. Zagreb (Chroatia): InTech, 2017; p.91-112. DOI: 10.5772/intechopen.69973
  33. 33. Nunes A, Correia I, Barros A, Delgadillo I. Characterization of kafirin and zein oligomers by preparative dodecylsulfate-polyacrylamide gel electrophoresis. J Agric Food Chem. 2005; 53:639-643. DOI: 10.1021/jf049553+
  34. 34. Da Silva LS, Jung R, Zhao ZY, Glassman K, Grootboom AW, Mehlo L, O'Kennedy MM, Taylor J, Taylor JRN. Effect of suppressing the synthesis of different kafirin sub-classes on grain endosperm texture, protein body structure and protein nutritional quality in improved sorghum lines. J. Cereal Sci. 2011; 54:160-167. DOI: 10.1016/j.jcs.2011.04.009
  35. 35. Da Silva LS. Transgenic sorghum: Effects of altered kafirin synthesis on kafirin polymerisation, protein quality, protein body structure and endosperm texture. [thesis]. Pretoria, South Africa: Department of Food Science, Faculty of Natural and Agricultural Sciences, Pretoria, South Africa University; 2012
  36. 36. Ndimba RJ, Kruger J, Mehlo L, Barnabas A, Kossmann J, Ndimba BK. A Comparative Study of Selected Physical and Biochemical Traits of Wild-Type and Transgenic Sorghum to Reveal Differences Relevant to Grain Quality. Front. Plant Sci. 2017;8:952. DOI: 10.3389/fpls.2017.00952
  37. 37. Segal G, Song R, Messing J. A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics. 2003;165:387-397.
  38. 38. Wu Y, Holding DR, Messing J. γ-Zeins are essential for endosperm modification in quality protein maize. Proc. Natl. Acad. Sci. USA. 2010;107:12810-12815. DOI: 10.1073/pnas.1004721107
  39. 39. Guo X., Yuan L., Chen H., Sato S.J., Clemente T.E., Holding D.R. Non-redundant function of zeins and their correct stoichiometric ratio drive protein body formation in maize endosperm. Plant Physiol. 2013;162,1359-1369. DOI: 10.1104/pp.113.218941
  40. 40. Huang S, Frizzi A, Florida CA, Kruger DE, Luethy MH. High lysine and high tryptophan transgenic maize resulting from the reduction of both 19- and 22-kD α-zeins. Plant Mol. Biol. 2006;61:525-535. DOI: 10.1007/s11103-006-0027-6
  41. 41. Kawakatsu T, Hirose S, Yasuda H, Takaiwa F. Reducing rice seed storage protein accumulation leads to changes in nutrient quality and storage organelle formation. Plant Physiol. 2010;154:1842-1854. DOI: 10.1104/pp.110.164343
  42. 42. Wallace JC, Lopes MA, Paiva E, Larkins BA. New methods for extraction and quantitation of zeins reveal a high content of γ-zein in modified opaque-2 maize. Plant Physiol. 1990;92:191-196. DOI: 10.1104/pp.92.1.191
  43. 43. Wu Y, Messing J. Proteome balancing of the maize seed for higher nutritional value. Front. Plant Sci. 2014;5:240. DOI: 10.3389/fpls.2014.00240
  44. 44. Schmidt MA, Barbazuk WB, Sandford M, May G, Song Z, Zhou W, Nikolau BJ, Herman EM. Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome. Plant Physiol. 2011;156:330-345. DOI: 10.1104/pp.111.173807
  45. 45. Dalakouras A, Wassenegger M, Dadami E, Ganopoulos I, Pappas ML, Papadopoulou K. Genetically modified organism-free RNA interference: exogenous application of RNA molecules in plants. Plant Physiology, 2020;182:38-50. DOI: 10.1104/pp.19.00570
  46. 46. Tuttle JR, Idris AM, Brown JK, Haigler CH, Robertson D. Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiol. 2008;148:41-50. DOI: 10.1104/pp.108.123869
  47. 47. von Born P, Bernardo-Faura M, Rubio-Somoza I. An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana. PLoS ONE. 2018;13: e0192984. DOI: 10.1371/journal.pone.0192984
  48. 48. Elkonin LA, Italyanskaya YuV. In vitro digestibility of storage endosperm proteins of transgenic sorghum plants carrying genetic construct for silencing of the gamma-kafirin gene. Advances in Current Natural Sciences 2017;12:96-100 [In Russian]
  49. 49. Elkonin LA, Panin VM, Gerashchenkov GA, Kenzhegulov OA. Improvement of sorghum grain quality using modern genetic tools. In: Current Challenges in Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Proceedings of the 5th International Scientific Conference (PlantGen2019); 24-29 June 2019; Novosibirsk: ICG; 2019. p.129-132
  50. 50. Elkonin LA, Panin VM, Kenzhegulov OA, Gerashchenkov GA. Improvement of grain sorghum nutritive properties using modern genetic and biotechnological methods. Plant Biotechnology and Breeding. 2019;2:41-48. (In Russ.). DOI: 10.30901/2658-6266-2019-3-o6
  51. 51. Geetha KB, Lending CR, Lopes MA, Wallace JC, Larkins BA. Opaque-2 modifiers increase gamma-zein synthesis and alter its spatial-distribution in maize endosperm. Plant Cell 1991;3:1207-1219. DOI: 10.1105/tpc.3.11.1207
  52. 52. Lopes MA, Takasaki K, Bostwick DE, Helentjaris T, Larkins BA. Identification of opaque 2 modifier loci in quality protein maize. Mol. Gen. Genet. 1995;247:603-613. DOI: 10.1007/BF00290352

Written By

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

Submitted: 09 September 2020 Reviewed: 25 January 2021 Published: 18 February 2021