Measurement of the inorganic pyrophosphate in tissues of Pisum sativum L.

Purified pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) was used to measure the inorganic pyrophosphate in unfractionated extracts of tissues of Pisum sativum L. The fructose 1,6-bisphosphate produced by the above enzyme was measured by coupling to NADH oxidation via aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). Amounts of pyrophosphate as low as 1 nmol could be measured. The contents of pyrophosphate in the developing embryo of pea, and in the apical 2 cm of the roots, were appreciable; 9.4 and 8.9 nmol g^-1 fresh weight, respectively. The possibility that pyrophosphate acts in vivo as an energy source for pyrophosphate: fructose 6-phosphate 1-phosphotransferase and for UDPglucose pyrophosphorylase (EC 2.7.7.9) is considered.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Summary Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.In honour of Prof. P. van Duijn     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.     

Soluble starch synthases and starch branching enzymes from cotyledons of smooth- and wrinkled-seeded lines of Pisum sativum L.

Soluble starch synthase and branching enzyme were purified from 18-day-old cotyledons of the smooth-seeded pea cultivar Alaska (RR) and wrinkled-seeded pea cultivar Progress #9 (rr) by DEAE-cellulose chromatography. Two coeluting peaks of primed and citrate-stimulated starch synthase activity and a major and minor peak of branching enzyme activity were observed in Alaska. However, in Progress #9, only one peak of synthase activity was found. When crude extracts of Progress #9 were centrifuged, over 70% of the starch synthase activity was recovered in the pelleted fraction, and additional washings of the pellet released no further activity. The addition of purified starch granules to Alaska crude extracts also resulted in the recovery of a greater proportion of synthase activity in pelleted fractions. The two peaks of branching enzyme activity in Alaska differed in their stimulation of phosphorylase, amylose branching activity, and activity in various buffers. The DEAE-cellulose profile of Progress #9 showed no distinct peak of branching enzyme and less than 10% of the total activity found in Alaska. The association of one form of soluble starch synthase with the pelleted fraction and the greatly reduced levels of branching enzyme provide a partial explanation for the appearance of high-amylose starch in Progress #9 cotyledons.     

Ethylene metabolism in Pisum sativum L.

The possible role of C₂H₄ metabolism in mediating the responses of plants to C₂H₄ is re-examined. It is demonstrated that (i) the effects of inhibitors upon C₂H₄ action do not correspond with their effects on metabolism, (ii) elicitors of C₂H₄ effects do not have appropriate effects on C₂H₄ metabolism, (iii) inhibitors of C₂H₄ metabolism do not affect the response of plants to C₂H₄. It is concluded that metabolism of C₂H₄ is not linked to the mode of action of the growth regulator.Abbreviations DTC sodium diethyldithiocarbamate - FW fresh weight     

Genome size variation in Pisum sativum.

Pisum sativum L. is one of the plant species where infraspecific genome size variation, up to 1.29-fold between cultivars, has been reported. The present investigation deals with a Feulgen cytophotometric analysis of this phenomenon in 25 wild accessions, landraces, and cultivars of widely different geographic origin. Differences between accessions were maximally 1.054-fold in single experiments but proved to be nonreproducible upon repeated measurements. Seedlings of the same accession often differed significantly, up to 1.056-fold, but values from root and shoot tips in one individual were not significantly correlated, indicating the absence of true genome size variation between plants. Upon calibration against Allium cepa a 1C value of 4.42 pg is estimated for Pisum sativum. Altogether the data suggest that, contrary to the divergence in the literature data and recent reports on DNA content variation, the pea has a stable genome size.     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.     

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30–40% of the measured rate of CO₂ fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2–5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.Abbreviation GOGAT l-Glutamine:2-oxoglutarate aminotransferase     

A gamma-pyronyl-triterpenoid saponin from Pisum sativum.

A new triterpenoid saponin was isolated from Pisum sativum and characterized as 3-O-[alpha-L-rhamnopyranosyl-(1----2)-beta-D-galactopyranosyl(1----2)-be ta- D-glucuronopyranosyl(1----)]-22-O-[3'-hydroxy-2'-methyl-5',6'-dihy dro-4'- pyrone(6'----)]-3 beta, 22 beta, 24-trihydroxyolean-12-ene. The name chromosaponin I is proposed. Chromosaponin I yielded soyasaponin I, known as phytochrome inhibitor, during extraction, but the latter was not found in the free form in this plant.     

Lipoxygenase heterogeneity in Pisum sativum

Antibodies raised against two pea (Pisum sativum L. cv. Birte) seed lipoxygenases have been used to analyze lipoxygenase heterogeneity in seeds and in other organs. At least seven different polypeptides were identified in vivo; five of these were identified as precursors synthesized in vitro. The developmental appearance of the seed polypeptides has been analyzed and early and late forms were identified. Limited N-terminal sequence data indicated further heterogeneity when compared with sequences predicted from cDNAs.Abbreviations cDNA complementary DNA - DAF days after flowering - HPLC high-performance liquid chromatography - Ig immunoglobulin - kb kilobase - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SSC 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0 This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute. We acknowledge financial support from the Commission of the European Communities Biotechnology Action Programme; grant No. 0063-UK.     

Carnitine acyltransferases in chloroplasts of Pisum sativum L.

Carnitine-acetyltransferase (EC 2.3.1.7) and carnitine-palmitoyltransferase (EC 2.3.1.21) activities were shown to be present in chloroplasts of green pea leaves and possibly to occur in leaf mitochondrial and peroxisomal fractions. A role for the enzymes in the transfer of acyl groups across membranes is suggested.     

Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

Involvement of mammalian bilitranslocase-like protein(s) in chlorophyll catabolism of Pisum sativum L. tissues

Putative pea bilin and cyclic tetrapyrrole transporter proteins were identified by means of an antibody raised against a bilirubin-interacting aminoacidic sequence of mammalian bilitranslocase (TC No. 2.A.65.1.1). The immunochemical approach showed the presence of several proteins mostly in leaf microsomal, chloroplast and tonoplast vesicles. In these membrane fractions, electrogenic bromosulfalein transport activity was also monitored, being specifically inhibited by anti-bilitranslocase sequence antibody. Moreover, the inhibition of transport activity in pea leaf chloroplast vesicles, by both the synthetic cyclic tetrapyrrole chlorophyllin and the heme catabolite biliverdin, supports the involvement of some of these proteins in the transport of linear/cyclic tetrapyrroles during chlorophyll metabolism. Immunochemical localization in chloroplast sub-compartments revealed that these putative bilitranslocase-like transporters are restricted to the thylakoids only, suggesting their preferential implication in the uptake of cyclic tetrapyrrolic intermediates from the stroma during chlorophyll biosynthesis. Finally, the presence of a conserved bilin-binding sequence in different proteins (enzymes and transporters) from divergent species is discussed in an evolutionary context.     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.