Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds.
Ontology highlight
ABSTRACT:
SUBMITTER: Casey R
PROVIDER: S-EPMC1186401 | biostudies-other | 1979 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundProanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied.ResultsPAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. 'Courier' and 'Solido' PAs were primarily prodelphinidin-types, whereas the PAs from 'LAN3017' were mainly the procyanidin-type. The mean degree of polymerization of 'LAN3017' PAs was also higher than those from 'Courier' and 'Solido'. Next-generation sequencing of 'Courier' seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis.ConclusionSignificant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR.
| S-EPMC4175280 | biostudies-literature
Project description:Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an ;acidic' subunit (mol.wt. about 40000) and a ;basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the ;basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0-5.3 for the ;acidic' subunits, and 8.3-8.7 for the ;basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The ;basic' and ;acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.
| S-EPMC1161378 | biostudies-other
Project description:The purification and characterization of a minor legumin species from Pisum sativum is described. Electrophoretic data indicate that it corresponds to a legumin subunit pair previously designated L1. The beta-polypeptides of the minor legumin have a phenylalanine N-terminus. This is the first time that an amino acid other than glycine has been reported as the N-terminus of the basic polypeptides from legumin-like proteins from any plant species. Sequence analyses of the isolated alpha- and beta-polypeptides of the minor legumin show that it does not correspond to any of the three legumin gene families that have previously been defined on the basis of DNA hybridizations and genetic analyses.
| S-EPMC1148942 | biostudies-other
Project description:Low phytic acid (lpa) crops are low in phytic acid and high in inorganic phosphorus (Pi). In this study, two lpa pea genotypes, 1-150-81, 1-2347-144, and their progenitor CDC Bronco were grown in field trials for two years. The lpa genotypes were lower in IP? and higher in Pi when compared to CDC Bronco. The total P concentration was similar in lpa genotypes and CDC Bronco throughout the seed development. The action of myo-inositol phosphate synthase (MIPS) (EC 5.5.1.4) is the first and rate-limiting step in the phytic acid biosynthesis pathway. Aiming at understanding the genetic basis of the lpa mutation in the pea, a 1530 bp open reading frame of MIPS was amplified from CDC Bronco and the lpa genotypes. Sequencing results showed no difference in coding sequence in MIPS between CDC Bronco and lpa genotypes. Transcription levels of MIPS were relatively lower at 49 days after flowering (DAF) than at 14 DAF for CDC Bronco and lpa lines. This study elucidated the rate and accumulation of phosphorus compounds in lpa genotypes. The data also demonstrated that mutation in MIPS was not responsible for the lpa trait in these pea lines.
| S-EPMC4844337 | biostudies-literature
Project description:Crop plants can become contaminated with human pathogenic bacteria in agro-production systems. Some of the transmission routes of human pathogens to growing plants are well explored such as water, manure and soil, whereas others are less explored such as seeds. Fenugreek seeds contaminated with the entero-hemorrhagic Escherichia coli O104:H4 were suspected to be the principle vectors for transmission of the pathogen to sprouts at the food-borne disease outbreak in Hamburg and surrounding area in 2011. In this study we raised the questions of whether cells of the entero-aggregative E. coli O104:H4 strain 55989 is capable of colonizing developing plants from seeds and if it would be possible that, via plant internalization, these cells can reach the developing embryonic tissue of the next generation of seeds. To address these questions, we followed the fate of strain 55989 and of two other E. coli strains from artificially contaminated seeds to growing plants, and from developing flower tissue to mature seeds upon proximate introductions to the plant reproductive organs. Escherichia coli strains differing in origin, adherence properties to epithelial cells, and virulence profile were used in our experimentation to relate eventual differences in seed and plant colonization to typical E. coli properties. Experiments were conducted under realistic growth circumstances in greenhouse and open field settings. Entero-aggregative E. coli strain 55989 and the two other E. coli strains were able to colonize the root compartment of pea plants from inoculated seeds. In roots and rhizosphere soil, the strains could persist until the senescent stage of plant growth, when seeds had ripened. Colonization of the above-soil parts was only temporary at the start of plant growth for all three E. coli strains and, therefore, the conclusion was drawn that translocation of E. coli cells via the vascular tissue of the stems to developing pea seeds seems unlikely under circumstances realistic for agricultural practices. Proximate introductions of cells of E. coli strains to developing flowers also did not result in internal seed contamination, indicating that internal seed contamination with E. coli is an unlikely event. The fact that all three E. coli strains showed stronger preference for the root-soil zones of growing pea plants than for the above soil plant compartments, in spite of their differences in clinical behaviour and origin, indicate that E. coli in general will colonize root compartments of crop plants in production systems.
| S-EPMC7565074 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2010-03-01 | GSE20110 | GEO
Project description:A genomic clone from pea (Pisum sativum L.) contains all of one gene encoding a 'minor' (B-type) legumin polypeptide, and most of a second very similar gene. The two genes, designated LegJ and LegK, are arranged in tandem, separated by approx. 6 kb. A complete sequence of gene LegJ and its flanking sequences is given, with as much of the sequence of gene LegK as is present on the genomic clone. Hybridization of 3' flanking sequence probes to seed mRNA, and sequence comparisons with cDNA species, suggested that gene LegJ, and probably gene LegK, was expressed. The partial amino acid sequences of 'minor' legumin alpha- and beta-polypeptides were used to confirm the identity of these genes. The transciption start in gene LegJ was mapped. The 5' flanking sequence of gene LegJ contains a sequence conserved in legumin genes from pea and other species, which is likely to have functional significance in control of gene expression. Sequence comparisons with legumin genes and cDNA species from Vicia faba and soya bean show that separation of legumin genes into A- and B-type subfamilies occurred before separation of the Viciae and Glycinae tribes.
| S-EPMC1148808 | biostudies-other
Project description:Phylogenetic relationships of the Abyssinian pea (Pisum sativum ssp. abyssinicum) to other subspecies and species in the genus were investigated to test between different hypotheses regarding its origin and domestication. An extensive sample of the Pisum sativum ssp. sativum germplasm was investigated, including groups a-1, a-2, b, c, and d as identified by Kwon et al. (2012). A broad sample of P. fulvum but relatively few P. s. ssp. elatius accessions were analyzed. Partial sequences of 18 genes were compared and these results combined with comparisons of additional genes done by others and available in the literature. In total, 54 genes or gene fragment sequences were involved in the study. The observed affinities between alleles in P. ssp. sativum, P. s. ssp. abyssinicum, P. s. ssp. elatius, and P. fulvum clearly demonstrated a close relationship among the three P. sativum subspecies and rejected the hypothesis that the Abyssinian pea was formed by hybridization between one of the P. sativum subspecies and P. fulvum. If hybridization were involved in the generation of the Abyssinian pea, it must have been between P. s. ssp. sativum and P. s. ssp. elatius, although the Abyssinian pea possesses a considerable number of highly unique alleles, implying that the actual P. s. ssp. elatius germplasm involved in such a hybridization has yet to be tested or that the hybridization occurred much longer ago than the postulated 4000 years bp. Analysis of the P. s. ssp. abyssinicum alleles in genomic regions thought to contain genes critical for domestication indicated that the indehiscent pod trait was independently developed in the Abyssinian pea, whereas the loss of seed dormancy was either derived from P. s. ssp. sativum or at least partially developed before the P. s. ssp. abyssinicum lineage diverged from that leading to P. s. ssp. sativum.
| S-EPMC5915832 | biostudies-literature
Project description:Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.
| S-EPMC7013545 | biostudies-literature
Project description:Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.
| S-EPMC2826372 | biostudies-literature