Isolation and characterization of a minor legumin and its constituent polypeptides from Pisum sativum (pea).
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ABSTRACT:
SUBMITTER: March JF
PROVIDER: S-EPMC1148942 | biostudies-other | 1988 Mar
REPOSITORIES: biostudies-other
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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: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: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:Ferritin was isolated from the seeds of pea (Pisum sativum) and lentil (Lens esculenta). The homogeneity of the phytoferritins was established by polyacrylamide-gel electrophoresis. The subunit molecular weights were respectively 20 300 and 21 400 for hte pea and lentil proteins. A neutron low-angle scattering study established the molecular weight of the oligomer as 480 000 for pea apoferritin and 510 000 for lentil apoferritin. Although the quaternary structure of 24 polypeptide chains is preserved, the phytoferritins have a larger cavity in the interior than mammalian ferritins and can thus potentially store 1.2-1.4 times as much iron. The amino acid composition of the phytoferritins show some similarities to those of mammalian apoferritins; tryptic 'fingerprinting' reveals that there are many differences in the amino acid sequence of plant and mammalian apoferritins.
| S-EPMC1183963 | biostudies-other
Project description:Micronutrient malnutrition is a global concern that affects more than two billion people worldwide. Pea (Pisum sativum) is a nutritious pulse crop with potential to assist in tackling hidden hunger. Here we report seed ionomic data of 96 diverse pea accessions collected via inductively coupled plasma mass spectrometry (ICP-MS). We found a 100 g serving of peas provides the following average percent daily value for U.S. recommendations: 8% Ca, 39% Mg, 73% Cu, 37% Fe, 63% Mn, 45% Zn, 28% K, and 43% P. Correlations were observed between the majority of minerals tested suggesting strong interrelationships between mineral concentration levels. Hierarchical clustering identified fifteen accessions with high-ranking mineral concentrations. Thirty accessions could be compared to earlier inductively coupled optical emission spectrometry (ICP-OES) data, which revealed significant differences particularly for elements at extreme low or high levels of accumulation. These results improve our understanding of the range of variation in mineral content found in peas and provide additional mineral data resources for germplasm selection.
| S-EPMC8568279 | biostudies-literature
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:The genome sequences of several legume species are now available allowing the comparison of the nitrogen (N) transporter inventories with non-legume species. A survey of the genes encoding inorganic N transporters and the sensing and assimilatory families in pea, revealed similar numbers of genes encoding the primary N assimilatory enzymes to those in other types of plants. Interestingly, we find that pea and Medicago truncatula have fewer members of the NRT2 nitrate transporter family. We suggest that this difference may result from a decreased dependency on soil nitrate acquisition, as legumes have the capacity to derive N from a symbiotic relationship with diazotrophs. Comparison with M. truncatula, indicates that only one of three NRT2s in pea is likely to be functional, possibly indicating less N uptake before nodule formation and N-fixation starts. Pea seeds are large, containing generous amounts of N-rich storage proteins providing a reserve that helps seedling establishment and this may also explain why fewer high affinity nitrate transporters are required. The capacity for nitrate accumulation in the vacuole is another component of assimilation, as it can provide a storage reservoir that supplies the plant when soil N is depleted. Comparing published pea tissue nitrate concentrations with other plants, we find that there is less accumulation of nitrate, even in non-nodulated plants, and that suggests a lower capacity for vacuolar storage. The long-distance transported form of organic N in the phloem is known to be specialized in legumes, with increased amounts of organic N molecules transported, like ureides, allantoin, asparagine and amides in pea. We suggest that, in general, the lower tissue and phloem nitrate levels compared with non-legumes may also result in less requirement for high affinity nitrate transporters. The pattern of N transporter and assimilatory enzyme distribution in pea is discussed and compared with non-legumes with the aim of identifying future breeding targets.
| S-EPMC8774688 | biostudies-literature
Project description:As a rate-limiting enzyme for chlorophyll biosynthesis, Mg-chelatase is a promising target for improving photosynthetic efficiency. It consists of CHLH, CHLD, and CHLI subunits. In pea (Pisum sativum L.), two putative CHLI genes (PsCHLI1 and PsCHLI2) were revealed recently by the whole genome sequencing, but their molecular features are not fully characterized. In this study, PsCHLI1 and PsCHLI2 cDNAs were identified by PCR-based cloning and sequencing. Phylogenetic analysis showed that PsCHLIs were derived from an ancient duplication in legumes. Both PsCHLIs were more highly expressed in leaves than in other organs and downregulated by abscisic acid and heat treatments, while PsCHLI1 was more highly expressed than PsCHLI2. PsCHLI1 and PsCHLI2 encode 422- and 417-amino acid proteins, respectively, which shared 82% amino acid identity and were located in chloroplasts. Plants with a silenced PsCHLI1 closely resembled PsCHLI1 and PsCHLI2 double-silenced plants, as both exhibited yellow leaves with barely detectable Mg-chelatase activity and chlorophyll content. Furthermore, plants with a silenced PsCHLI2 showed no obvious phenotype. In addition, the N-terminal fragment of PsCHLI1 (PsCHLI1N, Val63-Cys191) and the middle fragment of PsCHLI1 (PsCHLI1M, Gly192-Ser336) mediated the formation of homodimers and the interaction with CHLD, respectively, while active PsCHLI1 was only achieved by combining PsCHLI1N, PsCHLI1M, and the C-terminal fragment of PsCHLI1 (Ser337-Ser422). Taken together, PsCHLI1 is the key CHLI subunit, and its peptide fragments are essential for maintaining Mg-chelatase activity, which can be used to improve photosynthetic efficiency by manipulating Mg-chelatase in pea.
| S-EPMC8821089 | biostudies-literature
Project description:An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.
| S-EPMC1132980 | biostudies-other
Project description:A lipoxygenase cDNA clone, pCD45, was identified in a Pisum sativum L. (pea) seed mRNA cDNA library by hybrid-release/translation followed by immunoprecipitation with antiserum raised against lipoxygenase from Glycine max L. (soya bean). pCD45 hybrid-selected an mRNA encoding the larger of the two polypeptides of Mr approximately 95 000 that were immunoprecipitated from cell-free translation products of pea seed poly(A)-containing RNA by the G. max anti-lipoxygenase. 'Northern'-blot analysis showed the mRNA that hybridized to pCD45 to be approximately 3000 nucleotides in length. Three to five copies of the lipoxygenase gene corresponding to pCD45 were estimated to be present per haploid Pisum genome; hybridization of the cDNA insert from pCD45 to G. max DNA was also detected.
| S-EPMC1152842 | biostudies-other