CDNA cloning, primary structure and gene expression for H-protein, a component of the glycine-cleavage system (glycine decarboxylase) of pea (Pisum sativum) leaf mitochondria.
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ABSTRACT:
SUBMITTER: Macherel D
PROVIDER: S-EPMC1131509 | biostudies-other | 1990 Jun
REPOSITORIES: biostudies-other
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Project description:H-protein is the lipoyl-protein component of the glycine decarboxylase complex, which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration in plants. We have isolated and characterized the gene (gdcH) encoding the H-protein in pea (Pisum sativum L.). The H-protein gene is distributed in a stretch of about 1.55 kbp and contains three introns (75, 64 and 185 bp) located in the coding region. No intervening sequences were detected in the 5' and 3' non-coding regions. This intron-exon structure contrasts with the preliminary H-protein gene structures reported for human and chicken, where these genes (dispersed on 13 and 8 kbp genomic fragments respectively) are composed of five highly conserved exons and are interrupted by four long introns. Two main transcription sites were detected by primer extension of RNA. The first transcriptional initiation site was assigned the +1 position and correlated with a putative TATA box located at position -26. The second transcriptional start site was not correlated with a putative TATA box, but may be regulated by an 'initiator' element described by Smale & Baltimore [(1989) Cell (Cambridge, Mass.) 57, 103-113] which contains, within itself, the transcription start site. The presence of two potential promoters may be related to the specialized overexpression pattern of H-protein in leaves, in order to support photorespiration.
| S-EPMC1132945 | biostudies-other
Project description:The aim of the present work was to further determine how the T-protein of the glycine-cleavage system and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the glycine-cleavage system was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.
| S-EPMC1137213 | biostudies-other
Project description:Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, ?-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.
| S-EPMC3295397 | biostudies-literature
Project description:Aimed at gene-based markers design, we generated and analyzed transcriptome sequencing datasets for six pea (Pisum sativum L.) genetic lines that have not previously been massively genotyped. Five cDNA libraries obtained from nodules or nodulated roots of genetic lines Finale, Frisson, Sparkle, Sprint-2 and NGB1238 were sequenced using a versatile 3'-RNA-seq protocol called MACE (Massive Analysis of cDNA Ends). MACE delivers a single next-generation sequence from the 3'-end of each individual cDNA molecule that precisely quantifies the respective transcripts. Since the contig generated from the 3'-end of the cDNA by assembling all sequences encompasses the highly polymorphic 3'-untranslated region (3'-UTR), MACE efficiently detects single nucleotide variants (SNVs). Mapping MACE reads to the reference nodule transcriptome assembly of the pea line SGE (Transcriptome Shotgun Assembly GDTM00000000.1) resulted in characterization of over 34,000 polymorphic sites in more than 9700 contigs. Several of these SNVs were located within recognition sequences of restriction endonucleases which allowed the design of co-dominant CAPS markers for the particular transcript. Cleaned reads of sequenced libraries are available from European Nucleotide Archive (http://www.ebi.ac.uk/) under accessions PRJEB18101, PRJEB18102, PRJEB18103, PRJEB18104, PRJEB17691.
| S-EPMC5192247 | biostudies-literature
Project description:High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.
| S-EPMC1135205 | biostudies-other
Project description:Soybean Dwarf Virus (SbDV) is an important plant pathogen, causing economic losses in soybean. In North America, indigenous strains of SbDV mainly infect clover, with occasional outbreaks in soybean. To evaluate the risk of a US clover strain of SbDV adapting to other plant hosts, the clover isolate SbDV-MD6 was serially transmitted to pea and soybean by aphid vectors. Sequence analysis of SbDV-MD6 from pea and soybean passages identified 11 non-synonymous mutations in soybean, and six mutations in pea. Increasing virus titers with each sequential transmission indicated that SbDV-MD6 was able to adapt to the plant host. However, aphid transmission efficiency on soybean decreased until the virus was no longer transmissible. Our results clearly demonstrated that the clover strain of SbDV-MD6 is able to adapt to soybean crops. However, mutations that improve replication and/or movement may have trade-off effects resulting in decreased vector transmission.
| S-EPMC5490830 | biostudies-literature
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
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: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: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