Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten‐free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α‐, γ‐, ω‐gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP‐HPLC and mass spectrometry (LC‐MS/MS), whereas gluten immunogenicity was assayed by an anti‐gliadin 33‐mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ‐ and α‐gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω‐gliadins and γ‐gliadins, and three of these also silenced α‐gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC‐MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α‐ and ω‐gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.     

Conductive Li3.08Cr0.02Si0.09V0.9O4 Anode Material: Novel “Zero‐Strain” Characteristic and Superior Electrochemical Li+ Storage

“Zero‐strain” compounds are ideal energy‐storage materials for long‐term cycling because they present negligible volume change and significantly reduce the mechanically induced deterioration during charging–discharging. However, the explored “zero‐strain” compounds are very limited, and their energy densities are low. Here, γ phase Li_3.08Cr_0.02Si_0.09V_0.9O₄ (γ‐LCSVO) is explored as an anode compound for lithium‐ion batteries, and surprisingly its “zero‐strain” Li⁺ storage during Li⁺ insertion–extraction is found through using various state‐of‐the‐art characterization techniques. Li⁺ sequentially inserts into the 4c(1) and 8d sites of γ‐LCSVO, but its maximum unit‐cell volume variation is only ≈0.18%, the smallest among the explored “zero‐strain” compounds. Its mean strain originating from Li⁺ insertion is only 0.07%. Consequently, both γ‐LCSVO nanowires (γ‐LCSVO‐NW) and micrometer‐sized particles (γ‐LCSVO‐MP) exhibit excellent cycling stability with 90.1% and 95.5% capacity retention after as long as 2000 cycles at 10C, respectively. Moreover, γ‐LCSVO‐NW and γ‐LCSVO‐MP respectively deliver large reversible capacities of 445.7 and 305.8 mAh g^?1 at 0.1C, and retain 251.2 and 78.4 mAh g^?1 at 10C. Additionally, γ‐LCSVO shows a suitably safe operating potential of ≈1.0 V, significantly lower than that of the famous “zero‐strain” Li₄Ti₅O₁₂ (≈1.6 V). These merits demonstrate that γ‐LCSVO can be a practical anode compound for stable, high‐energy, fast‐charging, and safe Li⁺ storage.     

Gluten quality of bread wheat is associated with activity of RabD GTPases

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER‐to‐Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down‐regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT‐PCR to be down‐regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE‐HPLC found that a smaller proportion of HMW‐GS and large proportion of LMW‐GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS‐PAGE was seen in the transgenic grain.     

Diversifying crops for food and nutrition security – a case of teff

There are more than 50000 known edible plants in the world, yet two‐thirds of global plant‐derived food is provided by only three major cereals – maize (Zea mays), wheat (Triticum aestivum) and rice (Oryza sativa). The dominance of this triad, now considered truly global food commodities, has led to a decline in the number of crop species contributing to global food supplies. Our dependence on only a few crop species limits our capability to deal with challenges posed by the adverse effects of climate change and the consequences of dietary imbalance. Emerging evidence suggests that climate change will cause shifts in crop production and yield loss due to more unpredictable and hostile weather patterns. One solution to this problem is through the wider use of underutilised (also called orphan or minor) crops to diversify agricultural systems and food sources. In addition to being highly nutritious, underutilised crops are resilient in natural and agricultural conditions, making them a suitable surrogate to the major crops. One such crop is teff [Eragrostis tef (Zucc.) Trotter], a warm‐season annual cereal with the tiniest grain in the world. Native to Ethiopia and often the sustenance for local small farmers, teff thrives in both moisture‐stressed and waterlogged soil conditions, making it a dependable staple within and beyond its current centre of origin. Today, teff is deemed a healthy wheat alternative in the West and is sought‐after by health aficionados and those with coeliac disease or gluten sensitivity. The blooming market for healthy food is breathing new life into this underutilised crop, which has received relatively limited attention from mainstream research perhaps due to its ‘orphan crop’ status. This review presents the past, present and future of an ancient grain with a potential beyond its size.     

γ‐Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ‐Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ‐Tubulin Ring Complexes (γ‐TuRCs). While the subunits that constitute γ‐Tubulin Small Complexes (γ‐TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ‐TuRC‐specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ‐TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ‐TuRCs on astral MTs. γ‐TuRCs locate along the length of astral MTs, and depletion of γ‐TuRC‐specific proteins increases MT dynamics and causes the plus‐end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down‐regulation rescues spindle orientation defects induced by γ‐TuRC depletion. Therefore, we propose a role for γ‐TuRCs in regulating spindle positioning by controlling the stability of astral MTs.     

Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species

The γ‐tocopherol methyltransferase (γ‐TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ‐TMT in regulating abiotic stress within chloroplasts. The At γ‐tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high‐level expression of γ‐TMT converted most of γ‐tocopherol to α‐tocopherol in transplastomic seeds (~10‐fold higher) in the absence of abiotic stress. When grown in 400 mm NaCl, α‐tocopherol content in transplastomic TMT leaves increased up to 8.2‐fold and 2.4‐fold higher than wild‐type leaves. Likewise, under heavy metal stress, α‐tocopherol content in the TMT leaves increased up to 7.5‐fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3‐fold within 48 h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild‐type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α‐tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.     

Overexpression of serine acetyltransferase in maize leaves increases seed‐specific methionine‐rich zeins

Maize kernels do not contain enough of the essential sulphur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40‐fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high‐Met zeins during kernel development.     

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.     

An engineered C‐terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N‐terminal half of the Zea mays prolamin γ‐zein forms interchain disulfide bonds that facilitate the formation of ER‐located protein bodies. Wild‐type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C‐terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Δ418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild‐type protein. Moreover, Δ418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.     

Cereal asparagine synthetase genes

Asparagine synthetase catalyses the transfer of an amino group from glutamine to aspartate to form glutamate and asparagine. The accumulation of free (nonprotein) asparagine in crops has implications for food safety because free asparagine is the precursor for acrylamide, a carcinogenic contaminant that forms during high‐temperature cooking and processing. Here we review publicly available genome data for asparagine synthetase genes from species of the Pooideae subfamily, including bread wheat and related wheat species (Triticum and Aegilops spp.), barley (Hordeum vulgare) and rye (Secale cereale) of the Triticeae tribe. Also from the Pooideae subfamily: brachypodium (Brachypodium dIstachyon) of the Brachypodiae tribe. More diverse species are also included, comprising sorghum (Sorghum bicolor) and maize (Zea mays) of the Panicoideae subfamily and rice (Oryza sativa) of the Ehrhartoideae subfamily. The asparagine synthetase gene families of the Triticeae species each comprise five genes per genome, with the genes assigned to four groups: 1, 2, 3 (subdivided into 3.1 and 3.2) and 4. Each species has a single gene per genome in each group, except that some bread wheat varieties (genomes AABBDD) and emmer wheat (Triticum dicoccoides; genomes AABB) lack a group 2 gene in the B genome. This raises questions about the ancestry of cultivated pasta wheat and the B genome donor of bread wheat, suggesting that the hybridisation event that gave rise to hexaploid bread wheat occurred more than once. In phylogenetic analyses, genes from the other species cluster with the Triticeae genes, but brachypodium, sorghum and maize lack a group 2 gene, while rice has only two genes, one group 3 and one group 4. This means that TaASN2, the most highly expressed asparagine synthetase gene in wheat grain, has no equivalent in maize, rice, sorghum or brachypodium. An evolutionary pathway is proposed in which a series of gene duplications gave rise to the five genes found in modern Triticeae species.     

Suppression of α‐amylase genes improves quality of rice grain ripened under high temperature

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism‐related genes. Here we report the involvement of a starch‐hydrolyzing enzyme, α‐amylase, in high temperature‐triggered grain chalkiness. In developing seeds, high temperature induced the expression of α‐amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α‐amylase activity, while it decreased an α‐amylase‐repressing plant hormone, ABA, suggesting starch to be degraded by α‐amylase in developing grains under elevated temperature. Furthermore, RNAi‐mediated suppression of α‐amylase genes in ripening seeds resulted in fewer chalky grains under high‐temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α‐amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.     

Viscoelastic properties of wheat gluten in a molecular dynamics study

Wheat (Triticum spp.) gluten consists mainly of intrinsincally disordered storage proteins (glutenins and gliadins) that can form megadalton-sized networks. These networks are responsible for the unique viscoelastic properties of wheat dough and affect the quality of bread. These properties have not yet been studied by molecular level simulations. Here, we use a newly developed α-C-based coarse-grained model to study ∼ 4000-residue systems. The corresponding time-dependent properties are studied through shear and axial deformations. We measure the response force to the deformation, the number of entanglements and cavities, the mobility of residues, the number of the inter-chain bonds, etc. Glutenins are shown to influence the mechanics of gluten much more than gliadins. Our simulations are consistent with the existing ideas about gluten elasticity and emphasize the role of entanglements and hydrogen bonding. We also demonstrate that the storage proteins in maize and rice lead to weaker elasticity which points to the unique properties of wheat gluten.     

γ‐Tubulin complex in Trypanosoma brucei: molecular composition,subunit interdependence and requirement for axonemal central pair protein assembly

γ‐Tubulin complex constitutes a key component of the microtubule‐organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ‐tubulin small complex (γTuSC) composed of γ‐tubulin, gamma‐tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ‐tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ‐tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ‐tubulin complex in T. brucei is composed of γ‐tubulin and three GCP proteins, GCP2‐GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ‐tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.     

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Poly‐γ‐glutamate (γ‐PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the γ‐PGA synthesis ywsC‐ywtAB genes in its chromosome, cannot produce γ‐PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in γ‐PGA‐producing B. subtilis mutant strains. Mutant B. subtilis PGA6‐2 stably produces high levels of γ‐PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6‐2 is not only a γ‐PGA producer, but it is also a candidate for the genetic and metabolic engineering of γ‐PGA production. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Suppression,disaggregation, and modulation of γ‐Synuclein fibrillation pathway by green tea polyphenol EGCG

Oligomerization of γ‐Synuclein is known to have implications for both neurodegeneration and cancer. Although it is known to co‐exist with the fibrillar deposits of α‐Synuclein (Lewy bodies), a hallmark in Parkinson's disease (PD), the effect of potential therapeutic modulators on the fibrillation pathway of γ‐Syn remains unexplored. By a combined use of various biophysical tools and cytotoxicity assays we demonstrate that the flavonoid epigallocatechin‐3‐gallate (EGCG) significantly suppresses γ‐Syn fibrillation by affecting its nucleation and binds with the unstructured, nucleus forming oligomers of γ‐Syn to modulate the pathway to form α‐helical containing higher‐order oligomers (~158 kDa and ~ 670 kDa) that are SDS‐resistant and conformationally restrained in nature. Seeding studies reveal that these oligomers although “on‐pathway” in nature, are kinetically retarded and rate‐limiting species that slows down fibril elongation. We observe that EGCG also disaggregates the protofibrils and mature γ‐Syn fibrils into similar SDS‐resistant oligomers. Steady‐state and time‐resolved fluorescence spectroscopy and isothermal titration calorimetry (ITC) reveal a weak non‐covalent interaction between EGCG and γ‐Syn with the dissociation constant in the mM range (K_d ~ 2–10 mM). Interestingly, while EGCG‐generated oligomers completely rescue the breast cancer (MCF‐7) cells from γ‐Syn toxicity, it reduces the viability of neuroblastoma (SH‐SY5Y) cells. However, the disaggregated oligomers of γ‐Syn are more toxic than the disaggregated fibrils for MCF‐7cells. These findings throw light on EGCG‐mediated modulation of γ‐Syn fibrillation and suggest that investigation on the effects of such modulators on γ‐Syn fibrillation is critical in identifying effective therapeutic strategies using small molecule modulators of synucleopathies.     

The involvement of wheat U‐box E3 ubiquitin ligase TaPUB1 in salt stress tolerance

U‐box E3 ubiquitin ligases play important roles in the ubiquitin/26S proteasome machinery and in abiotic stress responses. TaPUB1‐overexpressing wheat (Triticum aestivum L.) were generated to evaluate its function in salt tolerance. These plants had more salt stress tolerance during seedling and flowering stages, whereas the TaPUB1‐RNA interference (RNAi)‐mediated knock‐down transgenic wheat showed more salt stress sensitivity than the wild type (WT). TaPUB1 overexpression upregulated the expression of genes related to ion channels and increased the net root Na⁺ efflux, but decreased the net K⁺ efflux and H⁺ influx, thereby maintaining a low cytosolic Na⁺/K⁺ ratio, compared with the WT. However, RNAi‐mediated knock‐down plants showed the opposite response to salt stress. TaPUB1 could induce the expression of some genes that improved the antioxidant capacity of plants under salt stress. TaPUB1 also interacted with TaMP (Triticum aestivum α‐mannosidase protein), a regulator playing an important role in salt response in yeast and in plants. Thus, low cytosolic Na⁺/K⁺ ratios and better antioxidant enzyme activities could be maintained in wheat with overexpression of TaPUB1 under salt stress. Therefore, we conclude that the U‐box E3 ubiquitin ligase TaPUB1 positively regulates salt stress tolerance in wheat.     

Eukaryotic protein production in designed storage organelles

Background  

Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.