Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

Involvement of a rice mutation in storage protein biogenesis in endosperm and its genomic location.

MAIN CONCLUSION: A mutation was first found to cause the great generation of glutelin precursors (proglutelins) in rice (Oryza sativa L.) endosperm, and thus referred to as GPGG1. The GPGG1 was involved in synthesis and compartmentation of storage proteins. The PPR-like gene in GPGG1-mapped region was determined as its candidate gene. In the wild type rice, glutelins and prolamins are synthesized on respective subdomains of rough endoplasmic reticulum (ER) and intracellularly compartmentalized into different storage protein bodies. In this study, a storage protein mutant was obtained and characterized by the great generation of proglutelins combining with the lacking of 13 kD prolamins. A dominant genic-mutation, referred to as GPGG1, was clarified to result in the proteinous alteration. Novel saccular composite-ER was shown to act in the synthesis of proglutelins and 14 kD prolamins in the mutant. Additionally, a series of organelles including newly occurring several compartments were shown to function in the transfer, trans-plasmalemmal transport, delivery, deposition and degradation of storage proteins in the mutant. The GPGG1 gene was mapped to a 67.256 kb region of chromosome 12, the pentatricopeptide repeat (PPR)-like gene in this region was detected to contain mutational sites.

Influence on Accumulation Levels and Subcellular Localization of Prolamins by Fusion with the Functional Peptide in Transgenic Rice Seeds.

To exploit the rice seed-based oral vaccine against Sjögren's syndrome, altered peptide ligand of N-terminal 1 (N1-APL7) from its M3 muscarinic acetylcholine receptor (M3R) autoantigen was expressed as fusion protein with the representative four types of rice prolamins (16 kDa, 14 kDa, 13 kDa, and 10 kDa prolamins) under the control of the individual native prolamin promoter. The 10kD:N1-APL7 and 14kD:N1-APL7 accumulated at high levels (287 and 58 µg/grain), respectively, whereas production levels of the remaining ones were remarkably low. Co-expression of these fusion proteins did not enhance the accumulation level of N1-APL7 in an additive manner. Downregulation of endogenous seed storage proteins by RNAi-mediated suppression also did not lead to substantial elevation of the co-expressed prolamin:N1-APL7 products. When transgenic rice seeds were subjected to in vitro proteolysis with pepsin, the 10kD:N1-APL7 was digested more quickly than the endogenous 10 kDa prolamin and the 14kD:N1-APL7 deposited in PB-Is. This difference could be explained by the finding that the 10kD:N1-APL7 was unexpectedly localized in the PB-IIs containing glutelins. These results indicated that not only accumulation level but also subcellular localization of inherent prolamins were highly influenced by the liked N1-APL7 peptide.

Nitrogen-dependent binding of the transcription factor PBF1 contributes to the balance of protein and carbohydrate storage in maize endosperm.

Fluctuations in nitrogen (N) availability influence protein and starch levels in maize (Zea mays) seeds, yet the underlying mechanism is not well understood. Here, we report that N limitation impacted the expression of many key genes in N and carbon (C) metabolism in the developing endosperm of maize. Notably, the promoter regions of those genes were enriched for P-box sequences, the binding motif of the transcription factor prolamin-box binding factor 1 (PBF1). Loss of PBF1 altered accumulation of starch and proteins in endosperm. Under different N conditions, PBF1 protein levels remained stable but PBF1 bound different sets of target genes, especially genes related to the biosynthesis and accumulation of N and C storage products. Upon N-starvation, the absence of PBF1 from the promoters of some zein genes coincided with their reduced expression, suggesting that PBF1 promotes zein accumulation in the endosperm. In addition, PBF1 repressed the expression of sugary1 (Su1) and starch branching enzyme 2b (Sbe2b) under normal N supply, suggesting that, under N-deficiency, PBF1 redirects the flow of C skeletons for zein toward the formation of C compounds. Overall, our study demonstrates that PBF1 modulates C and N metabolism during endosperm development in an N-dependent manner.

Analysis of oat seed transcriptome with regards to proteins involved in celiac disease.

Oat (Avena sativa L.) is considered to be a healthy food. In contrast to other grain crops, oat is high in protein, lipids, dietary fiber, antioxidants, and uniquely in avenanthramides. The question of whether it can also be consumed by people suffering from celiac disease is still unresolved. The main aim of this study was to extract and sequence genes for potentially harmful avenins, globulins, and α-amylase/trypsin inhibitors in six oat varieties and to establish their variability using PacBio sequencing technology of enriched libraries. The results were compared with sequences of the genes already present in databases. In total, 21 avenin, 75 globulin, and 25 α-amylase/trypsin inhibitor genes were identified and mapped in the hexaploid oat chromosomes. In all of the three gene families, only marginal sequence differences were found between the oat varieties within the individual genes. Avenin epitopes were found in all four types of avenin genes occurring in all oat varieties tested within this study. However, the number of avenin genes was nearly four times lower than of globulin genes and, on the protein level, formed only 10% of storage proteins. Therefore, the question of whether oat is safe to celiac disease people is a question of boundary values.

Wheat α-gliadin and high-molecular-weight glutenin subunit accumulate in different storage compartments of transgenic soybean seed.

Wheat seed storage proteins (prolamins) are important for the grain quality because they provide a characteristic texture to wheat flour products. In wheat endosperm cells, prolamins are transported from the Endoplasmic reticulum to Protein storage vacuoles through two distinct pathways-a conventional pathway passing through the Golgi apparatus and an unconventional Golgi-bypassing pathway during which prolamins accumulate in the ER lumen, forming Protein bodies. Unfortunately, transport studies conducted previously achieved limited success because of the seed-specificity of the latter pathway and the multigene architecture of prolamins. To overcome this difficulty, we expressed either of the two families of wheat prolamins, namely α-gliadin or High-molecular-weight subunit of glutenin, in soybean seed, which naturally lacks prolamin-like proteins. SDS-PAGE analysis indicated the successful expression of recombinant wheat prolamins in transgenic soybean seeds. Their accumulation states were quite different-α-gliadin accumulated with partial fragmentation whereas the HMW-glutenin subunit formed disulfide-crosslinked polymers without fragmentation. Immunoelectron microscopy of seed sections revealed that α-gliadin was transported to PSVs whereas HMW-glutenin was deposited in novel ER-derived compartments distinct from PSVs. Observation of a developmental stage of seed cells showed the involvement of post-Golgi Prevacuolar compartments in the transport of α-gliadin. In a similar stage of cells, deposits of HMW-glutenin surrounded by membranes studded with ribosomes were observed confirming the accumulation of this prolamin as ER-derived PBs. Subcellular fractionation analysis supported the electron microscopy observations. Our results should help in better understanding of molecular events during the transport of prolamins in wheat.

Genome-wide identification, characteristics and expression of the prolamin genes in Thinopyrum elongatum.

BACKGROUND: Prolamins, unique to Gramineae (grasses), play a key role in the human diet. Thinopyrum elongatum (syn. Agropyron elongatum or Lophopyrum elongatum), a grass of the Triticeae family with a diploid E genome (2n = 2x = 14), is genetically well-characterized, but little is known about its prolamin genes and the relationships with homologous loci in the Triticeae species. RESULTS: In this study, a total of 19 α-gliadin, 9 γ-gliadin, 19 ω-gliadin, 2 high-molecular-weight glutenin subunit (HMW-GS), and 5 low-molecular-weight glutenin subunit (LMW-GS) genes were identified in the Th. elongatum genome. Micro-synteny and phylogenetic analysis revealed dynamic changes of prolamin gene regions and genetic affinities among Th. elongatum, Triticum aestivum, T. urartu and Aegilops tauschii. The Th. elongatum genome, like the B subgenome of T. aestivum, only contained celiac disease epitope DQ8-glia-α1/DQ8.5-glia-α1, which provided a theoretical basis for the low gluten toxicity wheat breeding. The transcriptome data of Th. elongatum exhibited differential expression in quantity and pattern in the same subfamily or different subfamilies. Dough rheological properties of T. aestivum-Th. elongatum disomic substitution (DS) line 1E(1D) showed higher peak height values than that of their parents, and DS6E(6D) exhibited fewer α-gliadins, which indicates the potential usage for wheat quality breeding. CONCLUSIONS: Overall, this study provided a comprehensive overview of the prolamin gene family in Th. elongatum, and suggested a promising use of this species in the generation of improved wheat breeds intended for the human diet.

Effects of an Additional Cysteine Residue of Avenin-like b Protein by Site-Directed Mutagenesis on Dough Properties in Wheat (Triticum aestivum L.).

Avenin-like b protein is rich in cysteine residues, providing the possibility to form intermolecular disulfide bonds and then participate in glutenin polymerization. Site-directed mutagenesis was adopted to produce mutant avenin-like b gene encoding mutant avenin-like b protein, in which one tyrosine codon at the C-terminal is substituted by a cysteine codon. Compared with the control lines, both transgenic lines with wild-type and mutant avenin-like b genes demonstrated superior dough properties. While compared within the transgenic lines, the mutant lines showed relative weaker dough strength and decreased sodium-dodecyl-sulfate sedimentation volumes (from 69.7 mL in line WT alb-1 to 41.0 mL in line Mut alb-4). These inferior dough properties were accompanied by the lower contents of large-sized glutenin polymers, the decreased particle diameters of glutenin macropolymer (GMP), due to the lower content of intermolecular ß-sheets (from 39.48% for line WT alb-2 to 30.21% for line Mut alb-3) and the varied contents of disulfide bonds (from 137.37 µmol/g for line WT alb-1 to 105.49 µmol/g for line Mut alb-4) in wheat dough. The extra cysteine might alter the original disulfide bond structure, allowing cysteine residue usually involved in an intermolecular disulfide bond to become available for an intrachain disulfide bond. Avenin-like b proteins were detected in glutenin macropolymers, providing further evidence for this protein to participate in the polymerization of glutenin. This is the first time to investigate the effect of a specific cysteine residue in the avenin-like b protein on flour quality.

Development of Decreased-Gluten Wheat Enabled by Determination of the Genetic Basis of lys3a Barley.

Celiac disease is the most common food-induced enteropathy in humans, with a prevalence of approximately 1% worldwide. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as gluten, found in wheat (Triticum aestivum). Related prolamins are present in barley (Hordeum vulgare) and rye (Secale cereale). The incidence of both celiac disease and a related condition called nonceliac gluten sensitivity is increasing. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here, we used bulked segregant RNA sequencing and map-based cloning to identify the genetic lesion underlying a recessive, low-prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild-type barley gene and then used targeting-induced local lesions in genomes to identify induced single-nucleotide polymorphisms in the three homeologs of the corresponding wheat gene. Combining inactivating mutations in the three subgenomes of hexaploid bread wheat in a single wheat line lowered gliadin and low-molecular-weight glutenin accumulation by 50% to 60% and increased free and protein-bound lysine by 33%.

New insights into the evolution of wheat avenin-like proteins in wild emmer wheat (Triticum dicoccoides).

Fifteen full-length wheat grain avenin-like protein coding genes (TaALP) were identified on chromosome arms 7AS, 4AL, and 7DS of bread wheat with each containing five genes. Besides the a- and b-type ALPs, a c type was identified in the current paper. Both a and b types have two subunits, named x and y types. The five genes on each of the three chromosome arms consisted of two x-type genes, two y-type genes, and one c-type gene. The a-type genes were typically of 520 bp in length, whereas the b types were of 850 bp in length, and the c type was of 470 bp in length. The ALP gene transcript levels were significantly up-regulated in Blumeria graminis f. sp. tritici (Bgt)-infected wheat grain caryopsis at early grain filling. Wild emmer wheat [(WEW), Triticum dicoccoides] populations were focused on in our paper to identify allelic variations of ALP genes and to study the influence of natural selection on certain alleles. Consequently, 25 alleles were identified for TdALP-bx-7AS, 13 alleles were identified for TdALP-ax-7AS, 7 alleles were identified for TdALP-ay-7AS, and 4 alleles were identified for TdALP-ax-4AL Correlation studies on TdALP gene diversity and ecological stresses suggested that environmental factors contribute to the ALP polymorphism formation in WEW. Many allelic variants of ALPs in the endosperm of WEW are not present in bread wheat and therefore could be utilized in breeding bread wheat varieties for better quality and elite plant defense characteristics.

RNA-Binding Protein RBP-P Is Required for Glutelin and Prolamine mRNA Localization in Rice Endosperm Cells.

In developing rice (Oryza sativa) endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins.

Analysis of novel high-molecular-weight prolamins from Leymus multicaulis (Kar. et Kir.) Tzvelev and L. chinensis (Trin. ex Bunge) Tzvelev.

Nine novel high-molecular-weight prolamins (HMW-prolamins) were isolated from Leymus multicaulis and L. chinensis. Based on the structure of the repetitive domains, all nine genes were classified as D-hordeins but not high-molecular-weight glutenin subunits (HMW-GSs) that have been previously isolated in Leymus spp. Four genes, Lmul 1.2, 2.4, 2.7, and Lchi 2.5 were verified by bacterial expression, whereas the other five sequences (1.3 types) were classified as pseudogenes. The four Leymus D-hordein proteins had longer N-termini than those of Hordeum spp. [116/118 vs. 110 amino acid (AA) residues], whereas three (Lmul 1.2, 2.4, and 2.7) contained shorter N-termini than those of the Ps. juncea (116 vs. 118 AA residues). Furthermore, Lmul 1.2 was identified as the smallest D-hordein, and Lmul 1.2 and 2.7 had an additional cysteines. Phylogenetic analysis supported that the nine D-hordeins of Leymus formed two independent clades, with all the 1.3 types clustered with Ps. juncea Ns 1.3, whereas the others were clustered together with the D-hordeins from Hordeum and Ps. juncea and the HMW-GSs from Leymus. Within the clade of four D-hordein genes and HMW-GSs, the HMW-GSs of Leymus formed a separated branch that served as an intermediate between the D-hordeins of Ps. juncea and Leymus. These novel D-hordeins may be potentially utilized in the improvement of food processing properties particularly those relating to extra cysteine residues. The findings of the present study also provide basic information for understanding the HMW-prolamins among Triticeae species, as well as expand the sources of D-hordeins from Hordeum to Leymus.

Dynamic Evolution of α-Gliadin Prolamin Gene Family in Homeologous Genomes of Hexaploid Wheat.

Wheat Gli-2 loci encode complex groups of α-gliadin prolamins that are important for breadmaking, but also major triggers of celiac disease (CD). Elucidation of α-gliadin evolution provides knowledge to produce wheat with better end-use properties and reduced immunogenic potential. The Gli-2 loci contain a large number of tandemly duplicated genes and highly repetitive DNA, making sequence assembly of their genomic regions challenging. Here, we constructed high-quality sequences spanning the three wheat homeologous α-gliadin loci by aligning PacBio-based sequence contigs with BioNano genome maps. A total of 47 α-gliadin genes were identified with only 26 encoding intact full-length protein products. Analyses of α-gliadin loci and phylogenetic tree reconstruction indicate significant duplications of α-gliadin genes in the last ~2.5 million years after the divergence of the A, B and D genomes, supporting its rapid lineage-independent expansion in different Triticeae genomes. We showed that dramatic divergence in expression of α-gliadin genes could not be attributed to sequence variations in the promoter regions. The study also provided insights into the evolution of CD epitopes and identified a single indel event in the hexaploid wheat D genome that likely resulted in the generation of the highly toxic 33-mer CD epitope.

Genetic diversity of avenin-like b genes in Aegilops tauschii Coss.

Avenin-like storage proteins influence the rheological properties and processing quality in common wheat, and the discovery of new alleles will benefit wheat quality improvement. In this study, 13 avenin-like b alleles (TaALPb7D-A-M) were discovered in 108 Aegilops tauschii Coss. accessions. Ten alleles were reported for the first time, while the remaining three alleles were the same as alleles in other species. A total of 15 nucleotide changes were detected in the 13 alleles, resulting in only 11 amino acid changes because of synonymous mutations. Alleles TaALPb7D-E, TaALPb7D-G, and TaALPb7D-J encoded the same protein. These polymorphic sites existed in the N-terminus, Repetitive region (Left), Repetitive region (Right) and C-terminus domains, with no polymorphisms in the signal peptide sequence nor in those encoding the 18 conserved cysteine residues. Phylogenetic analysis divided the TaALPb7Ds into four clades. The Ae. tauschii alleles were distributed in all four clades, while the alleles derived from common wheat, TaALPb7D-G and TaALPb7D-C, belonged to clade III and IV, respectively. Alleles TaALPb7D-G and TaALPb7D-C were the most widely distributed, being present in nine and six countries, respectively. Iran and Turkey exhibited the highest genetic diversity with respect to TaALPb7D alleles, accessions from these countries carrying seven and six alleles, respectively, which implied that these countries were the centers of origin of the avenin-like b gene. The new alleles discovered and the phylogenetic analysis of avenin-like b genes will provide breeding materials and a theoretical basis for wheat quality improvement.

Compensatory rebalancing of rice prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies.

KEY MESSAGE: Bioactive peptide was produced by fusion to rice prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between prolamins within PB-Is. Peptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. Use of seed storage protein as a fusion carrier is reasonable strategy for production of such small size bioactive peptides. In this study, to examine the efficacy of various rice prolamins deposited in ER-derived protein bodies (PB-Is), the APL12 from the Glucose-6-phosphate isomerase (GPI325-339) was expressed by fusion to four types of representative prolamins under the control of the individual native promoters. When the 14 and 16 kDa Cys-rich prolamins, which were localized in middle layer of PB-Is, were used for production of the APL12, they highly accumulated in transgenic rice seeds (~ 200 µg/grain). By contrast, fusion to the 10 and 13 kDa prolamins, which were localized in the core and outermost layer of PB-Is, resulted in lower levels of accumulation (~ 40 µg/grain). These results suggest that accumulation levels were highly affected by their deposition sites. Next, when different prolamin/APL12 fusion proteins were co-expressed to increase accumulation levels, they could not be increased so much as their expected additive levels. High accumulation of one type prolamin/APL12 led to reduction of other type(s) prolamin/APL12 to maintain the limited amounts of prolamins that can be deposited in PB-Is. Moreover, suppression of endogenous seed proteins by RNA interference also did not significantly enhance the accumulation levels of prolamin/APL12. These findings suggest that there may be compensatory rebalancing mechanism that controls the accumulation levels of prolamins deposited within PB-Is.

New insights into structural organization and gene duplication in a 1.75-Mb genomic region harboring the α-gliadin gene family in Aegilops tauschii, the source of wheat D genome.

Among the wheat prolamins important for its end-use traits, α-gliadins are the most abundant, and are also a major cause of food-related allergies and intolerances. Previous studies of various wheat species estimated that between 25 and 150 α-gliadin genes reside in the Gli-2 locus regions. To better understand the evolution of this complex gene family, the DNA sequence of a 1.75-Mb genomic region spanning the Gli-2 locus was analyzed in the diploid grass, Aegilops tauschii, the ancestral source of D genome in hexaploid bread wheat. Comparison with orthologous regions from rice, sorghum, and Brachypodium revealed rapid and dynamic changes only occurring to the Ae. tauschii Gli-2 region, including insertions of high numbers of non-syntenic genes and a high rate of tandem gene duplications, the latter of which have given rise to 12 copies of α-gliadin genes clustered within a 550-kb region. Among them, five copies have undergone pseudogenization by various mutation events. Insights into the evolutionary relationship of the duplicated α-gliadin genes were obtained from their genomic organization, transcription patterns, transposable element insertions and phylogenetic analyses. An ancestral glutamate-like receptor (GLR) gene encoding putative amino acid sensor in all four grass species has duplicated only in Ae. tauschii and generated three more copies that are interspersed with the α-gliadin genes. Phylogenetic inference and different gene expression patterns support functional divergence of the Ae. tauschii GLR copies after duplication. Our results suggest that the duplicates of α-gliadin and GLR genes have likely taken different evolutionary paths; conservation for the former and neofunctionalization for the latter.

Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat.

The wheat avenin-like proteins (ALP) are considered atypical gluten constituents and have shown positive effects on dough properties revealed using a transgenic approach. However, to date the genetic architecture of ALP genes is unclear, making it impossible to be utilized in wheat breeding. In the current study, three genes of type-b ALPs were identified and mapped to chromosomes 7AS, 4AL and 7DS. The coding gene sequence of both TaALP-7A and TaALP-7D was 855 bp long, encoding two identical homologous 284 amino acid long proteins. TaALP-4A was 858 bp long, encoding a 285 amino acid protein variant. Three alleles were identified for TaALP-7A and four for TaALP-4A. TaALP-7A alleles were of two types: type-1, which includes TaALP-7A1 andTaALP-7A2, encodes mature proteins, while type-2, represented byTaALP-7A3, contains a stop codon in the coding region and thus does not encode a mature protein. Dough quality testing of 102 wheat cultivars established a highly significant association of the type-1 TaALP-7A allele with better wheat processing quality. This allelic effects were confirmed among a range of commercial wheat cultivars. Our research makes the ALP be the first of such genetic variation source that can be readily utilized in wheat breeding.

Teff, an Orphan Cereal in the Chloridoideae, Provides Insights into the Evolution of Storage Proteins in Grasses.

Seed storage proteins (SSP) in cereals provide essential nutrition for humans and animals. Genes encoding these proteins have undergone rapid evolution in different grass species. To better understand the degree of divergence, we analyzed this gene family in the subfamily Chloridoideae, where the genome of teff (Eragrostis tef) has been sequenced. We find gene duplications, deletions, and rapid mutations in protein-coding sequences. The main SSPs in teff, like other grasses, are prolamins, here called eragrostins. Teff has γ- and δ-prolamins, but has no ß-prolamins. One δ-type prolamin (δ1) in teff has higher methionine (33%) levels than in maize (23-25%). The other δ-type prolamin (δ2) has reduced methionine residues (<10%) and is phylogenetically closer to α prolamins. Prolamin δ2 in teff represents an intermediate between δ and α types that appears to have been lost in maize and other Panicoideae, and was replaced by the expansion of α-prolamins. Teff also has considerably larger numbers of α-prolamin genes, which we further divide into five sub-groups, where α2 and α5 represent the most abundant α-prolamins both in number and in expression. In addition, indolines that determine kernel softness are present in teff and the panicoid cereal called foxtail millet (Setaria italica) but not in sorghum or maize, indicating that these genes were only recently lost in some members of the Panicoideae Moreover, this study provides not only information on the evolution of SSPs in the grass family but also the importance of α-globulins in protein aggregation and germplasm divergence.

Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice protoplasts.

In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants.

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain.

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7% reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.