A lipoxygenase is the main lipid body protein in cucumber and soybean cotyledons during the stage of triglyceride mobilization.

The 90-kDa lipid body protein characterized earlier by its high expression during the stage of fat degradation was identified as a form of lipoxygenase. This organelle form was compared with lipoxygenase species purified from the cytosol. It is further shown that the antibodies raised against the lipid body membrane lipoxygenase from cucumber cotyledons cross-react with both cytosolic and lipid body lipoxygenase from soybean.     

Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs

We have characterized two cDNA clones for 24 kDa soybean oleosin, the seed oil body membrane protein. Differences in the predicted amino acid sequences of the two clones and the presence of a doublet on immunoblots indicate that 24 kDa oleosin exists in at least two isoforms in soybean. The predicted amino acid sequence also contains a unique carboxy terminal region that is dominated by a series of different tandem amino acid repeats.     

RFLP analysis of soybean seed protein and oil content

Summary The objectives of this study were to present an expanded soybean RFLP map and to identify quantitative trait loci (QTL) in soybean [Glycine max (L.) Merr.] for seed protein and oil content. The study population was formed from a cross between a G. max experimental line (A81-356022) and a G. soja Sieb. and Zucc. plant introduction (PI 468916). A total of 252 markers was mapped in the population, forming 31 linkage groups. Protein and oil content were measured on seed harvested from a replicated trial of 60 F₂-derived lines in the F₃ generation (F₂₃ lines). Each F₂₃ line was genotyped with 243 RFLP, five isozyme, one storage protein, and three morphological markers. Significant (P<0.01) associations were found between the segregation of markers and seed protein and oil content. Segregation of individual markers explained up to 43% of the total variation for specific traits. All G. max alleles at significant loci for oil content were associated with greater oil content than G. soja alleles. All G. soja alleles at significant loci for protein content were associated with greater protein content than G. max alleles.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Biotransformation of soybean oil to a self-healing biopolymer

Soybean oil in the presence of mineral salts, peptone, carbohydrates, lipase, and lipopeptide transformed to a soft tissue-like material after 10-day incubation at 37°C with shaking at 200 rpm. When damaged, the resultant soft tissue self-healed in an aqueous medium. Analysis revealed that during the self-assembly process, components present in the aqueous medium were entangled in the soft tissue; upon damage, release of these components accelerated healing. Superficial damage resulted in smooth healing, whereas deep damage gave uneven healing similar to deep skin wounds.     

A genome-wide association study of seed protein and oil content in soybean

Background

Association analysis is an alternative to conventional family-based methods to detect the location of gene(s) or quantitative trait loci (QTL) and provides relatively high resolution in terms of defining the genome position of a gene or QTL. Seed protein and oil concentration are quantitative traits which are determined by the interaction among many genes with small to moderate genetic effects and their interaction with the environment. In this study, a genome-wide association study (GWAS) was performed to identify quantitative trait loci (QTL) controlling seed protein and oil concentration in 298 soybean germplasm accessions exhibiting a wide range of seed protein and oil content.

Results

A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped using various methods including Illumina Infinium and GoldenGate assays and 31,954 markers with minor allele frequency >0.10 were used to estimate linkage disequilibrium (LD) in heterochromatic and euchromatic regions. In euchromatic regions, the mean LD (r ² ) rapidly declined to 0.2 within 360 Kbp, whereas the mean LD declined to 0.2 at 9,600 Kbp in heterochromatic regions. The GWAS results identified 40 SNPs in 17 different genomic regions significantly associated with seed protein. Of these, the five SNPs with the highest associations and seven adjacent SNPs were located in the 27.6-30.0 Mbp region of Gm20. A major seed protein QTL has been previously mapped to the same location and potential candidate genes have recently been identified in this region. The GWAS results also detected 25 SNPs in 13 different genomic regions associated with seed oil. Of these markers, seven SNPs had a significant association with both protein and oil.

Conclusions

This research indicated that GWAS not only identified most of the previously reported QTL controlling seed protein and oil, but also resulted in narrower genomic regions than the regions reported as containing these QTL. The narrower GWAS-defined genome regions will allow more precise marker-assisted allele selection and will expedite positional cloning of the causal gene(s).     

Proteomic analysis of oil body membrane proteins accompanying the onset of desiccation phase during sunflower seed development

A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20–30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20–30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.     

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds.     

Isolation of soybean protein P34 from oil bodies using hydrophobic interaction chromatography

Background  

Soybeans play a prominent role in allergologic research due to the high incidence of allergic reactions. For detailed studies on specific proteins it is necessary to have access to a large amount of pure substance.     

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5′ cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species.     

The onset and duration of mobilization affect the regeneration in the rat muscle

The effects of different mobilization protocols for muscle regeneration after myotoxin injury was compared in the rat tibialis anterior (TA) muscle. Adult Wistar rats were divided into control (C); mobilized (M); injury (I); injury + late mobilization (LM) and injury + early mobilization (EM) groups. Muscle injury was induced by intramuscular lidocaine injection. The exercised animals were mobilized for 5 and 8 days during 15 and 45 minutes/session. The swimming started 1 hour or 3 days after injury. All animals were killed 8 days after the injury, together with the control group, when the TA muscles were weighted and excised. Cross sections were obtained by cryostat and submitted to Toluidine Blue stain. Qualitative morphological characterization of muscle regeneration and quantitative analysis of muscle fiber and non-muscle fiber area density were performed. The I and late mobilization groups showed decreased muscle mass when compared to all other groups. All injured animals showed signs of muscle fiber damage, although signs of early regenerated muscle fibers were more evident in injury + mobilization groups. Only the EM groups submitted to 45 minutes of exercise had increased muscle fiber and decreased non-muscle fiber area density values when compared to I group (p<0.05). Conclusion: the regeneration process is related to the onset of exercise, since animals submitted to early mobilization showed improved regeneration when comparted to LM groups. Besides, the length of session is also important for accelerating the regeneration process, as it was observed that 45 minutes was better than 15 minutes duration.     

Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping

Decapping is a key step in mRNA turnover. However, the composition and regulation of the human decapping complex is poorly understood. Here, we identify three proteins that exist in complex with the decapping enzyme subunits hDcp2 and hDcp1: hEdc3, Rck/p54, and a protein in decapping we name Hedls. Hedls is important in decapping because it enhances the activity of the catalytic hDcp2 subunit and promotes complex formation between hDcp2 and hDcp1. Specific decapping factors interact with the mRNA decay activators hUpf1 and TTP, and TTP enhances decapping of a target AU-rich element (ARE) RNA in vitro. Each decapping protein localizes in cytoplasmic processing bodies (PBs), and overexpression of Hedls produces aberrant PBs and concomitant accumulation of a deadenylated ARE-mediated mRNA decay intermediate. These observations suggest that multiple proteins involved in human decapping are important subunits of PBs and are activated on ARE-mRNAs by the protein TTP.     

Heat processing of soybean kernel and its effect on lysine availability and protein solubility

Soybean kernels of cultivars Bosa and ZPS 015 were used in the experiment. The contents of available lysine as well as water and salt soluble proteins, were analysed in fresh soybean kernels, soybean products made after the processes of dry extrusion, micronisation, microwave toasting and autoclaving. Utilizing a technological procedure of processing, kernels were exposed to temperatures from 57 to 150°C. The duration of exposure of the soybean kernels to the increased temperatures, ranged from 25-30 seconds in dry extrusion to 30 minutes in autoclaving. All treatments were subjected to different sources of heat, causing different thermodynamic processes to take place in kernels and change their chemical composition; i.e. nutritive quality. The content of water and salt soluble proteins decreased under the influence of higher temperatures in the course of all treatments of processing. The drop of solubility already was drastically effected by temperatures of 100°C in dry extrusion, while there was a gradual decrease in other treatments. The content of available lysine was determined by the modified Carpenter methods with DNFB. The processes of micronisation and microwave toasting showed the greatest effect on the reduction of lysine availability. Dry extrusion and autoclaving, performed within closed systems — in which the increased moisture content has a special effect — resulted in significantly smaller changes of the available lysine content.     

Spatial and temporal changes in lipase activity sites during oil body mobilization in protoplasts from sunflower seedling cotyledons

Hydrolysis of triacylglycerols (TAGs) catalyzed by lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) action, is the principal biochemical event during oil body mobilization in germinating oilseeds. Employing a fluorescence microscopic technique developed in the author’s laboratory, a shift in the intracellular lipase activity has been demonstrated in the protoplasts of sunflower seedling cotyledons during seed germination. Lipase activity is primarily confined to protein storage vacuoles (PSVs) in 1 d old seedling cotyledons. At 2 d old stage, a relocalization of lipase activity begins and activity can be observed both on PSVs and oil bodies. At later stages of development (3–6 d), smaller PSVs coalesce into a large vegetative vacuole devoid of lipase activity. During this phase, lipase activity is confined to oil bodies only and maximum activity is detected in 4 d old seedlings, coinciding with maximum rate of lipolysis. Thus, present investigations on protoplasts from seedling cotyledons provide evidence for intracellular shift in lipase activity to sites of TAG hydrolysis (oil bodies) and also show a structural and functional reorganization of PSVs.     

Selectively hydrogenated soybean oil with conjugated linoleic acid modifies body composition and plasma lipids in rats

The present study examined effects of a selectively hydrogenated soybean oil (SHSO) containing about 21% CLA on body composition, adipose depots and organ weights, and plasma lipid profiles in rats. Male Sprague Dawley rats were fed for 6 weeks a purified diet containing 0%, 1%, 3%, and 5% of SHSO. Different levels of SHSO supplementation did not significantly affect growth performance, although there was a trend toward decreased body weight gain with increasing dietary SHSO levels. The weights of inguinal, epididymal, and retroperitoneal adipose depot, but not mesenteric, were significantly influenced by dietary SHSO supplementation (P < 0.05, P < 0.01 and P < 0.001, respectively). Although the absolute weight of body protein in the control rats was higher in SHSO-fed rats, the effect on absolute weight of body protein is diluted and eliminated when the data are adjusted for eviscerated carcass weight as a percentage base. Therefore, as dietary SHSO level increased, body protein as a percentage of carcass weight increased (P < 0.05), although as dietary SHSO level increased, body fat proportion in carcass decreased (P < 0.01). Plasma triglycerides (TG) and total cholesterol (TC) concentrations were beneficially decreased, and HDL-cholesterol (HDL-C) to TC ratio was also beneficially increased by SHSO supplementation (P < 0.05, P < 0.001, and P < 0.01, respectively). However, plasma HDL-C concentration undesirably decreased with dietary SHSO supplementation (P < 0.05). The present study observed that body composition and plasma lipids were beneficially modulated by SHSO supplementation at least 3% levels (0.6% of CLA), and suggested that SHSO is a useful fat source because of the high level of CLA.     

Continuous processing of fusion protein expressed as an Escherichia coli inclusion body

In this study we develop the components of an integrated process for the continuous extraction and purification of a histidine-tagged fusion protein expressed as an inclusion body in Escherichia coli. Lac21 was selected as a model peptide and was expressed as a fusion to ketosteroid isomerase. A purification strategy was developed on a 1-ml batch column before successful scale-up and transfer to a continuous purification system, having a bed volume of 240 ml. Preliminary experiments proved cleavage of the fusion protein. The use of chemical extraction and continuous chromatography gives a flowsheet far superior to the traditional methods for inclusion body processing.