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Expression of the pea S-adenosylmethionine decarboxylase gene is involved in developmental and environmental responses.

A cDNA, able to complement the S-adenosyl-L-methionine decarboxylase (SAMdC; EC 4.1.1.50)-defective yeast strain Y342, has been isolated from pea (Pisum sativum L.). Expression of the SAMdC gene was characterised during pea development. Northern analysis showed a differential expression of the pea SAMdC gene in vegetative and reproductive tissues. The highest SAMdC mRNA levels were found in undifferentiated callus and tissues with high rates of cell division, and at the onset of fruit development. SAMdC expression was also induced in senescing ovaries, probably in relation to an accumulation of spermine during ovary senescence. Finally, the levels of SAMdC transcripts in leaves and shoots were differentially affected by ozone treatment.

Adenosylmethionine Decarboxylase↗

Downstream elements from the pea albumin 1 gene confer sulfur responsiveness on a reporter gene.

The levels of mRNAs for some of the sulfur-rich proteins in seeds are regulated by the level of sulfur supplied to the plants. In peas, there is a mechanism that lowers the level of mRNA for legumin and pea albumin 1 (PA1) when plants are grown under sulfur-deficient conditions. This mechanism acts after transcription initiation. In this study, a gene encoding PA1 was expressed in leaves of transgenic tobacco. Expression of the gene was controlled by the level of sulfur supplied to the plants, mimicking the behaviour of the intact gene in peas. A gene encoding a different high-sulfur protein, ovalbumin, was unresponsive to sulfur status and was used as a reporter gene to test defined regions of the PA1 gene for sulfur responsiveness. These constructs, together with a set of PA1 gene deletions, were tested in transgenic tobacco and yielded the following observations: the PA1 gene was sensitive to sulfur status in the leaf as well as the seed; intron processing of the PA1 transcript was not required for sensitivity to sulfur stress; both the coding region and the 3' flanking regions of the PA1 gene contained sequences which conferred sensitivity to sulfur stress; the sulfur-responsive sequence in the 3' region was contained within a 134-nucleotide segment downstream of the end of the coding sequence. We conclude that there are at least two downstream elements which confer sensitivity to sulfur supply.

Albumins↗

Cadmium effects on populations of root nuclei in two pea genotypes inoculated or not with the arbuscular mycorrhizal fungus Glomus mosseae.

Plants possess a broad range of strategies to cope with cadmium (Cd) stress, including the arbuscular mycorrhizal (AM) symbiosis. In cell responses towards Cd, the contribution of changes in ploidy levels is still unclear. We used flow cytometry to investigate if nuclear ploidy changes are involved in response mechanisms toward Cd and to analyze the effect of the symbiotic status on populations of nuclei. The impact of Cd was investigated in roots of two pea (Pisum sativum L.) genotypes differing in their Cd-sensitivity (Cd-sensitive VIR4788 and Cd-tolerant VIR7128). In pea seedlings grown under hydropony, 25 and 250 microM Cd concentrations lead to an increase in 4 C together with a decrease in 2 C nuclei. The same genotypes, grown in soil/sand substrate, were inoculated or not with the AM fungus Glomus mosseae BEG12 and treated or not with Cd at transplanting (Cd1) or 2 weeks after (Cd2). The Cd2 increased the proportion of 6 and 8 C nuclei in the mycorrhizal VIR4788 and in the non-mycorrhizal VIR7128 genotypes. Thus, changes in ploidy levels reflect pea responses towards Cd, which are modulated by the symbiotic interaction. The Cd-induced increase in ploidy may account for changes in DNA transcription and/or translation.

Cadmium↗

Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

Base Sequence↗

Constitutive expression of pea Lhcb 1-2 in tobacco affects plant development, morphology and photosynthetic capacity.

Lhcb1-2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1-2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 degrees C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 degrees C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1-2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

Amino Acid Sequence↗

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum).

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

Actins↗

Solubilization and properties of GDP-fucose: xyloglucan 1,2-alpha-L-fucosyltransferase from pea epicotyl membranes.

GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.

Chromatography, Gel↗

Chloroplast and cytoplasmic enzymes: isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases.

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.

Amino Acid Sequence↗

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.

Anions↗

Glucokinase of pea seeds.

1. Glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2) was extracted from pea seeds and purified by fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and Sephadex. 2. The relative rates of phosphorylation of glucose, mannose and fructose (final concentration 5 mM) were 100, 64 and 11. 3. The Km for glucose of pea-seed glucokinase was 70 muM and the Km for mannose was 0.5 mM. The Km for fructose was much higher (30 mM). 4. Mg2+ ions were essential for activity. Mn2+ could partially replace Mg2+. 5. Enzyme activity was not inhibited by glucose 6-phosphate. A number of other metabolites had no effect on glucokinase activity. 6. Pea-seed glucokinase was inhibited by relatively low concentrations of ADP.

Adenosine Diphosphate↗

Studies on phytohemagglutinins. XXVII. A study of the pea lectin binding site.

Under defined mild conditions the reaction of the pea lectin with 2-nitrophenylsulfenyl chloride results in sulfenylation of only 2 of the 10 tryptophan residues of the lectin molecule with simultaneous loss of biological activity. Both sulfenylated tryptophan residues belong to the two heavy subunits of the lectin. Enzymic hydrolysis and separation of the tryptic peptides yields only one homogeneous yellow peptide containing the modified tryptophan residue. The isolated peptide has the following sequence (NPS, nitrophenylsulfenyl): HAsp-Val-Val-Pro-Glu-(2-NPS-Trp)-Val-ArgOH. The octapeptide is either directly a part of the pea lectin binding site or it plays an important role in maintaining the tertiary structure of the binding site. According to the amino acid composition and amino acid sequence, the octapeptide isolated from the pea lectin is almost identical with that part of the peptide chain of concanavalin A near to which the location of the sugar binding site is supposed to be.

Amino Acid Sequence↗

Beta-phenylethylamine (PEA): an endogenous anxiogen? Three series of experimental data.

Like the anxiogenic drugs caffeine, pentylenetetrazole, and yohimbine, the endogenous neuroactive monoamine beta-phenylethylamine (PEA) is effective in three tests for anxiogens in mice. In a social interaction test it reduced both the number and duration of contacts. In a conflict situation test (a dark-light chamber) it reduced the number of transitions between dark and light compartments. Diazepam, a standard anxiolytic, prevented both effects of PEA. Intracerebroventricular administration of PEA induced generalized clonic seizures which were antagonized by various anxiolytics but not by the tested doses of butyrophenone neuroleptics and standard anticonvulsants effective in other tests for convulsants.

Animals↗

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins.

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2-acetamido-2-deoxy-beta-D-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-beta-D-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal alpha-D-mannopyranosyl groups.

Glycopeptides↗

Cleavage of the precursor of pea chloroplast cytochrome f by leader peptidase from Escherichia coli.

Leader peptidase from Escherichia coli was able to process the precursor of pea cytochrome f synthesised in vitro. N-Terminal sequencing established that cleavage by leader peptidase generated the same mature sequence as in pea chloroplasts. Processing by leader peptidase was much more efficient co-translationally rather than post-translationally, and the extent of post-translational processing declined with time suggesting that the cytochrome f precursor folded to an uncleavable conformation. Detergent extracts of pea thylakoid membranes were unable to process the cytochrome f precursor co- or post-translationally.

Amino Acid Sequence↗

Improved method for the analysis of alpha-galactosides in pea seeds by capillary zone electrophoresis. Comparison with high-performance liquid chromatography-triple-pulsed amperometric detection.

A capillary zone electrophoretic (CZE) method is described for the analysis of the raffinose family of oligosaccharides (RFO) in pea seeds. Extraction of RFO was carried out in 80% ethanol and the extract was passed through a Sep-Pak C18 cartridge. This proved to be an improvement on the currently accepted purification technique. High-quality electrophoregrams were obtained which allowed the separation and quantitation of sucrose, raffinose, stachyose and verbascose The CZE results were compared with those obtained by anion-exchange high-performance liquid chromatography coupled to a triple-pulsed amperometric detection (HPAC-PAD). The samples were obtained from four pea strains which were near-isogenic except for genes at the r and rb loci. A high degree of precision and reproducibility was obtained for the RFO compositions of all the pea strains. No statistically significant differences (p < or equal to 0.05) were found between the two analytical techniques using paired Student-t tests.

Chromatography, High Pressure Liquid↗

Xyloglucan oligosaccharide alpha-L-fucosidase activity from growing pea stems and germinating nasturtium seeds.

[14C]Fucose-labelled xyloglucan (XG) was synthesized from tamarind seed XG by incubating it with GDP-[14C]fucose plus solubilized pea fucosyltransferase, and [14C]fucose-labelled XG nonasaccharide was prepared from the parent hemicellulose by partial hydrolysis with fungal cellulase. alpha-L-Fucosidase activity was readily detected in crude enzyme extracts of growing regions of etiolated pea stems (Pisum sativum) and in cotyledons of germinating nasturtium seedlings (Tropaeolum majus) using the fucosylated XG-nonasaccharide as substrate. Both enzymes showed little activity against intact fucosylated XG and they were totally inactive against p-nitrophenyl-alpha-L-fucoside. Auxin treatment of pea stems, which greatly increased the activity of endo-1,4-beta-glucanases that hydrolyse XG in apical growing regions, failed to result in a similar increase in XG-nonasaccharide alpha-fucosidase activity. However, germination of nasturtium seed, which resulted in a large increase in endo-1,4-beta-glucanase (XG-ase) activity in the cotyledons, was accompanied by comparable increases in XG-alpha-fucosidase activity.

Carbohydrate Sequence↗

Transfer of PCDD/PCDF from contaminated soils into carrots, lettuce and peas.

In a field experiment, the PCDD/PCDF transfer pathways from soil into carrots, lettuce and peas has been investigated. PCDD/PCDF contamination levels in soil varied between 5 ng I-TEq/kg on the control plot and 56 ng I-TEq/kg on the contaminated plot. PCDD/PCDF levels in carrots were threefold higher in the contaminated plot than in the control plot, which was a result of a tenfold increase in the PCDD/PCDF levels of the peel. PCDD/PCDF levels in lettuce and peas were not higher when grown on the contaminated plot and were much lower than in carrots, which indicates that the PCDD/PCDF in lettuce and peas from both plots are of atmospheric origin.

Air Pollutants↗

Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase.

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP+ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653-658) the total sequence is not known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713-722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme is characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.

Amino Acid Sequence↗