Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum.

Kafirins are the major storage proteins in sorghum (Sorghum bicolor) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the k1C family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the k1C genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple k1C family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all k1C genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.

The α' subunit of ß-conglycinin and various glycinin subunits of soy are not required to modulate hepatic lipid metabolism in rats.

PURPOSE: This study examined the effect of soy proteins with depletion of different subunits of the two major storage proteins, ß-conglycinin and glycinin, on hepatic lipids and proteins involved in lipid metabolism in rats, since the bioactive component of soy responsible for lipid-lowering is unclear. METHODS: Weanling Sprague Dawley rats were fed diets containing either 20% casein protein in the absence (casein) or presence (casein + ISF) of isoflavones or 20% alcohol-washed soy protein isolate (SPI) or 20% soy protein concentrates derived from a conventional (Haro) or 2 soybean lines lacking the α' subunit of ß-conglycinin and the A1-3 (1TF) or A1-5 (1a) subunits of glycinin. After 8 weeks, the rats were necropsied and liver proteins and lipids were extracted and analysed. RESULTS: The results showed that soy protein diets reduced lipid droplet accumulation and content in the liver compared to casein diets. The soy protein diets also decreased the level of hepatic mature SREBP-1 and FAS in males, with significant decreases in diets 1TF and 1a compared to the casein diets. The effect of the soy protein diets on female hepatic mature SREBP-1, FAS, and HMGCR was confounded since casein + ISF decreased these levels compared to casein alone perhaps muting the decrease by soy protein. A reduction in both phosphorylated and total STAT3 in female livers by ISF may account for the gender difference in mechanism in the regulation and protein expression of the lipid modulators. CONCLUSIONS: Overall, soy protein deficient in the α' subunit of ß-conglycinin and A1-5 subunits of glycinin maintain similar hypolipidemic function compared to the conventional soy protein. The exact bioactive component(s) warrant identification.

Integration of transcriptomic and proteomic analysis towards understanding the systems biology of root hairs.

Plants and other multicellular organisms consist of many types of specialized cells. Systems-wide exploration of large-scale information from singe cell level is essential to understand how cell works. Root hairs, tubular-shaped outgrowths from root epidermal cells, play important roles in the acquisition of nutrients and water, in the interaction with microbe, and in plant anchorage, and represent an ideal model to study the biology of a single cell type. Single cell sampling combined with omics approaches has been applied to study plant root hairs. This review emphasizes the integration of omics approaches towards understanding the systems biology of root hairs, unraveling the common and plant species-specific properties of root hairs, as well as the concordance of protein and transcript abundance. Understanding plant root hair biology by mining the integrated omics data will provide a way to know how a single cell differentiates, elongates, and functions, which might help molecularly modify crops for developing sustainable agriculture practices.

Allergenicity assessment strategy for novel food proteins and protein sources.

To solve the future food insecurity problem, alternative and sustainable protein sources (e.g. insects, rapeseed, fava bean and algae) are now being explored for the production of food and feed. To approve these novel protein sources for future food a comprehensive risk assessment is needed according to the European food legislation. Allergenicity risk assessment might pose some major difficulties, since detailed guidance on how to assess the allergenic potential of novel foods is not available. At present, the approach relies mostly on the guidance of allergenicity assessment for genetically modified (GM) plant foods. The most recent one was proposed by EFSA (2010 and 2011); "weight-of-evidence approach". However this guidance is difficult to interpret, not completely applicable or validated for novel foods and therefore needs some adjustments. In this paper we propose a conceptual strategy which is based on the "weight-of-evidence approach" for food derived from GM plants and other strategies that were previously published in the literature. This strategy will give more guidance on how to assess the allergenicity of novel food proteins and protein sources.

Influence of soil cover and N and K fertilization on the quality of biofortified QPM in the humid tropics.

BACKGROUND: In the humid tropics, unfavorable conditions present challenges to smallholder farmers attempting to meet food demands. The objective of this study was to evaluate the influence of alley cropping and addition of potassium and nitrogen on the productivity and nutritional value of quality protein maize (QPM). The experimental design consisted of randomized blocks with four replicates in a 5 × 2 factorial scheme, with five treatments, Gliricidia + Acacia (GA), Gliricidia + Clitoria (GC), Leucaena + Acacia (LA), Leucaena + Clitoria (LC) and bare soil (BS), in two cropping systems, one with addition of nitrogen and potassium (NK) and one without. RESULTS: The grain yield of LC + NK was significantly higher than that of all other treatments except GC + NK and LA + NK, and six times higher than that of BS + NK. The protein content of LC + NK was higher than that of the treatments without residue. CONCLUSION: Although the mulching of tree legumes increased the yield and quality of food for smallholder agriculture, achieving this outcome requires eliminating potentially negative interactions when combining trees and crops in addition to enhancing the availability and uptake of nutrients. © 2015 Society of Chemical Industry.

A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy.

StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

Isolation of salt stress-related genes from Aspergillus glaucus CCHA by random overexpression in Escherichia coli.

The halotolerant fungus Aspergillus glaucus CCHA was isolated from the surface of wild vegetation around a saltern with the salinity range being 0-31%. Here, a full-length cDNA library of A. glaucus under salt stress was constructed to identify genes related to salt tolerance, and one hundred clones were randomly selected for sequencing and bioinformatics analysis. Among these, 82 putative sequences were functionally annotated as being involved in signal transduction, osmolyte synthesis and transport, or regulation of transcription. Subsequently, the cDNA library was transformed into E. coli cells to screen for putative salt stress-related clones. Five putative positive clones were obtained from E. coli cells grown on LB agar containing 1 M NaCl, on which they showed rapid growth compared to the empty vector control line. Analysis of transgenic Arabidopsis thaliana lines overexpressing CCHA-2142 demonstrated that the gene conferred increased salt tolerance to plants as well by protecting the cellular membranes, suppressing the inhibition of chlorophyll biosynthesis. These results highlight the utility of this A. glaucus cDNA library as a tool for isolating and characterizing genes related to salt tolerance. Furthermore, the identified genes can be used for the study of the underlying biology of halotolerance.

Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR® real-time PCR.

BACKGROUND: Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect-resistant EE-1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties. RESULTS: End-point and real-time polymerase chain reaction (PCR) methods were used to detect EE-1 brinjal. In end-point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3' transgene integration sequence, primers specific for the event EE-1 brinjal were designed. These primers were used for end-point single, multiplex and SYBR-based real-time PCR. End-point single PCR showed that the designed primers were highly specific to event EE-1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE-1 brinjal genomic DNA. The limits of detection and quantification for SYBR-based real-time PCR assay were 10 and 100 copies respectively. CONCLUSION: The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations.

LeMAPK4 participated in cold-induced ethylene production in tomato fruit.

BACKGROUND: Mitogen-activated protein kinase (MAPK, EC 2.7.11.24) cascade from several plant species has been shown to be activated during response to abiotic stress. Ethylene plays an important role in fruit tolerance to environmental stress. However, the mechanisms by which MAPK regulates defence systems in fruit and the relationship between MAPK and ethylene remain to be determined. RESULTS: MAPK inhibitor significantly decreased the chilling tolerance of tomato (Lycopersicon esculentum cv. Lichun) fruit during cold storage. Moreover, decreases in ethylene content, LeACS2 expression and activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and ACC oxidase (ACO, EC 1.14.17.4) due to MAPK inhibitor occurred within 24 h after cold treatment. Upon treatment with cold and ethephon, the ethylene content, activities of ACS and ACO and expression of LeACS2, LeACO1 and LeMAPK4 increased. CONCLUSION: The results showed the regulation of MAPK in ethylene biosynthesis to protect tomato fruit from cold stress. In addition, the participation of LeMAPK4 in cold-induced ethylene biosynthesis in tomato fruit was indicated.

Metabolic and bioprocess engineering for production of selenized yeast with increased content of seleno-methylselenocysteine.

Specific Se-metabolites have been recognized to be the main elements responsible for beneficial effects of Se-enriched diet, and Se-methylselenocysteine (SeMCys) is thought to be among the most effective ones. Here we show that an engineered Saccharomyces cerevisiae strain, expressing a codon optimized heterologous selenocysteine methyltransferase and endowed with high intracellular levels of S-adenosyl-methionine, was able to accumulate SeMCys at levels higher than commercial selenized yeasts. A fine tuned carbon- and sulfate-limited fed-batch bioprocess was crucial to achieve good yields of biomass and SeMCys. Through the coupling of metabolic and bioprocess engineering we achieved a ∼24-fold increase in SeMCys, compared to certified reference material of selenized yeast. In addition, we investigated the interplay between sulfur and selenium metabolism and the possibility that redox imbalance occurred along with intracellular accumulation of Se. Collectively, our data show how the combination of metabolic and bioprocess engineering can be used for the production of selenized yeast enriched with beneficial Se-metabolites.

Isolation, purification, crystallization and preliminary crystallographic studies of a chitinase from Crocus vernus.

A chitinase has been isolated and purified from Crocus vernus corms. N-terminal amino-acid sequence analysis of the approximately 30 kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=172.3, b=37.1, c=126.4 Å, ß=127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1 Å.

Effects of genetically modified maize expressing Cry1Ab and EPSPS proteins on Japanese quail.

A 49-d feeding study was conducted to evaluate the effects of the genetically modified (GM) maize strain C0030.3.5 on Japanese quails (Coturnix japonica) in terms of body performance and egg quality. Furthermore, the bodily fats of transgenic proteins in the Japanese quails were investigated. The results showed that the parameters body weight, hematology, serum chemistry, relative organ weight, and histopathological appearance were normal in male and female quails that consumed GM diets, and no differences could be attributed to the varying diets in regard to the laying performances or nutrient egg compositions between the groups. Furthermore, the transgenic Cry1Ab and EPSPS proteins were undetectable by Western blot in the blood, organ, fecal, and whole egg samples of quails fed a diet containing GM maize. The results obtained after 49 d suggested that consumption of C0030.3.5 transgenic feed did not adversely affect quail health or egg quality, and there was no evidence of transgenic protein translocation to the blood, tissues, feces, and eggs. Based on the different parameters assessed, C0030.3.5 transgenic maize is a safe food source for quails that does not differ in quality from non-GM maize.

Nutritional evaluation and transcriptome analyses of short-time germinated seeds in soybean (Glycine max L. Merri.).

Germination is a common practice for nutrition improvement in many crops. In soybean, the nutrient value and genome-wide gene expression pattern of whole seeds germinated for short-time has not been fully investigated. In this study, protein content (PC), water soluble protein content (WSPC), isoflavone compositions were evaluated at 0 and 36 h after germination (HAG), respectively. The results showed that at 36HAG, PC was slightly decreased (P > 0.05) in ZD41, J58 and JHD, WSPC and free isoflavone (aglycones: daidzein, genistein, and glycitein) were significantly increased (P < 0.05), while total isoflavone content was unchanged. Transcriptomic analysis identified 5240, 6840 and 15,766 DEGs in different time point comparisons, respectively. GO and KEGG analysis showed that photosynthesis process was significantly activated from 18HAG, and alternative splicing might play an important role during germination in a complex manner. Response to hydrogen peroxide (H2O2) was found to be down regulated significantly from 18 to 36HAG, suggesting that H2O2 might play an important role in germination. Expression pattern analysis showed the synthesis of storage proteins was slowing down, while the genes coding for protein degradation (peptidase and protease) were up regulated as time went by during germination. For genes involved in isoflavone metabolism pathway, UGT (7-O-glucosyltransferase) coding genes were significantly up regulated (40 up-DEGs vs 27 down-DEGs), while MAT (7-O-glucoside-6''-O-malonyltransferase) coding genes were down regulated, which might explain the increase of aglycones after germination. This study provided a universal transcriptomic atlas for whole soybean seeds germination in terms of nutrition and gene regulation mechanism.

Cloning, expression, characterization, and computational approach for cross-reactivity prediction of manganese superoxide dismutase allergen from pistachio nut.

BACKGROUND: Tree nut allergy is one of the common potentially life-threatening food allergies in children and adults. Recombinant food allergens offer new perspectives to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of food allergies. So far, superoxide dismutase (s) has been identified as a panallergen and studied in different allergenic sources. Manganese Superoxide Dismutase (MnSOD) has also been reported in pistachio that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression, and purification of MnSOD from pistachio nut. METHODS: The pistachio MnSOD was cloned and expressed in E. coli BL21 (DE3) using a vector pET-32b (+). A recombinant protein was purified by metal precipitation. The protein immunoreactivity was evaluated using patients' IgE binding by means of ELISA and immunoblotting assays. RESULTS: The MnSOD gene from pistachio was successfully cloned and expressed in E. coli. The purified pistachio MnSOD was recognized by IgE in 10 (40%) out of the 25 sera tested. Our results also showed that this protein might trigger some cross-reactions toward IgE antibodies and thus could be considered as a panallergen. CONCLUSIONS: For the first time recombinant manganese superoxide dismutase from nut source was expressed as a possible allergen. This pistachio allergen could be a possible basis for cross-reactivity with MnSOD from other sources.

Drought stress affects the protein and dietary fiber content of wholemeal wheat flour in wheat/Aegilops addition lines.

Wild relatives of wheat, such as Aegilops spp. are potential sources of genes conferring tolerance to drought stress. As drought stress affects seed composition, the main goal of the present study was to determine the effects of drought stress on the content and composition of the grain storage protein (gliadin (Gli), glutenin (Glu), unextractable polymeric proteins (UPP%) and dietary fiber (arabinoxylan, ß-glucan) components of hexaploid bread wheat (T. aestivum) lines containing added chromosomes from Ae. biuncialis or Ae. geniculata. Both Aegilops parents have higher contents of protein and ß-glucan and higher proportions of water-soluble arabinoxylans (determined as pentosans) than wheat when grown under both well-watered and drought stress conditions. In general, drought stress resulted in increased contents of protein and total pentosans in the addition lines, while the ß-glucan content decreased in many of the addition lines. The differences found between the wheat/Aegilops addition lines and wheat parents under well-watered conditions were also manifested under drought stress conditions: Namely, elevated ß-glucan content was found in addition lines containing chromosomes 5Ug, 7Ug and 7Mb, while chromosomes 1Ub and 1Mg affected the proportion of polymeric proteins (determined as Glu/Gli and UPP%, respectively) under both well-watered and drought stress conditions. Furthermore, the addition of chromosome 6Mg decreased the WE-pentosan content under both conditions. The grain composition of the Aegilops accessions was more stable under drought stress than that of wheat, and wheat lines with the added Aegilops chromosomes 2Mg and 5Mg also had more stable grain protein and pentosan contents. The negative effects of drought stress on both the physical and compositional properties of wheat were also reduced by the addition of these. These results suggest that the stability of the grain composition could be improved under drought stress conditions by the intraspecific hybridization of wheat with its wild relatives.

miRNAs play important roles in aroma weakening during the shelf life of 'Nanguo' pear after cold storage.

Cold storage is commonly employed to delay senescence in 'Nanguo' pear after harvest. However, this technique also causes fruit aroma weakening. MicroRNAs are regulators of gene expression at the post-transcriptional level that play important roles in plant development and in eliciting responses to abiotic environmental stressors. In this study, the miRNA transcript profiles of the fruit on the first day (C0, LT0) move in and out of cold storage and the optimum tasting period (COTP, LTOTP) during shelf life at room temperature and after cold storage were analyzed, respectively. 314 known miRNAs were identified in 'Nanguo' pear; 176 and 135 miRNAs were significantly differentially expressed on the C0 vs. LT0 and on the COTP vs. LTOTP, respectively. After prediction the target genes of these differentially expressed miRNAs, 9 s-lipoxygenase (LOX2S), 13 s-lipoxygenase (LOX1_5), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH1) were found differentially expressed, which were the key genes during aroma formation. The expression pattern of these target genes and the related miRNAs were identified by RT-PCR. mdm-miR172a-h, mdm-miR159a/b, mdm-miR160a-e, mdm-miR395a-i, mdm-miR399a/b/c, mdm/ppe-miR535a/b, and mdm-miR7120a/b may negatively regulate the target genes expression. These results indicate that miRNAs may play key roles in aroma weakening in cold storage 'Nanguo' pear and provide valuable information for studying the molecular mechanisms of miRNAs in the aroma weakening of fruit due to low temperature.

Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare).

Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6kDa-peptide and 88kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88kDa-peptide of ONG2-I. The peptides between 6kDa and 88kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice.

Sequence organization of a pea chloroplast DNA gene coding for a 34,500-dalton protein.

A gene (PGII), which codes for a 34.5-kilodalton protein, has been isolated and cloned from pea chloroplast DNA. The production of its 1.2-kilobase mRNA is photodependent. The direction of transcription has been determined, the site of initiation of transcription has been found, and an in vitro protein product has been produced. The gene, including the 5' and 3'-flanking regions, has been sequenced. It shows ca. 95% homology to the photosystem II thylakoid membrane protein, photogene 32, from spinach and tobacco. There are no intervening sequences. The 5'-flanking region suggests similarities with Escherichia coli promoters. The 5'-flanking region is remarkably conserved among pea, spinach, and tobacco DNA.

[Detection of transgenic proteins in maize flour marketed in Bogotá, Colombia]. / Detección de proteínas transgénicas en harinas de maíz comercializadas en Bogotá, Colombia.

Objective To detect the presence or absence of transgenic proteins derived from GM crops in maize flour marketed in Bogota D.C., Colombia. Methods 11 extraction protocols for total protein were evaluated in 17 precooked flour, two uncooked and three positive controls. Subsequently, the presence of 7 transgenic proteins (CP4-EPSPS, Cry1Ab, Cry1Ac, Cry1F, Cry2A, Cry34Ab1 and Cry3Bb1) using commercial ELISA kits was determined. Results It was determined that the best protocol for total protein extraction was buffer with Triton X-100, which allowed obtaining protein concentrations greater than 0.5 mg per gram of flour and does not generate interference with the ELISA technique. Four transgenic proteins were detected: CP4EPSPS, Cry1F, Cry1Ab and Cry34Ab1 in precooked and uncooked flour with percentages varying between 20 and 100 %. Conclusion Seven of the 19 maize flours contain traces of transgenic protein (B2,B8,A3,O3,O1,C1 and C2) that provide resistance to lepidopterans and coleopterans, and tolerance to glyphosate herbicide, (CP4EPSPS- Cry1Ab, Cry1F, Cry34Ab1 and Cry3Bb1). All detected events are approved for human consumption in Colombia, according to the Ministry of Health and Social Protection.

Tailoring Grain Storage Reserves for a Healthier Rice Diet and its Comparative Status with Other Cereals.

A global rise of diet-related noncommunicable diseases calls for a focus on diet-based nutritional intervention across the entire socioeconomic consumer spectrum. We review recent reports in the area of healthier rice aimed at developing rice grains with improved dietary fiber compositions (increased amounts of nonstarch polysaccharides and resistant starch), and less digestible starch (higher amylose and phospholipid complex in the endosperm) resulting in reduced glycemic impact upon grain consumption. We furthermore elaborate on the interconnections of elevated amounts of protein and a balanced composition of essential amino acids. The importance of a nutritious aleurone layer and its role in lipid storage and micronutrient composition is discussed briefly in the context of brown rice benefits. We identify gene targets for precision breeding that will facilitate the production of rice grains and rice-based products to mitigate the impact of nutrition-related preventable diseases.