The Rubisco subunit binding protein

Chloroplasts contain an abundant soluble protein that binds non-covalently newly synthesized large and small subunits of the enzyme ribulose bisphosphate carboxylase-oxygenase. This binding protein has been purified from Pisum sativum and Hordeum vulgare in the form of a dodecamer consisting of equal amounts of two types of subunit. These subunits are synthesized as higher molecular mass precursors by cytoplasmic ribosomes before import into the chloroplast. Antibodies raised against the purified binding protein from Pisum sativum detect polypeptides not only in extracts of plastids from several plant species but also in cell extracts of several bacterial species. The oligomeric binding protein dissociates reversibly into monomeric subunits in the presence of 1–5 mmol/liter MgATP. For one type of subunit the cDNA sequence has been isolated and determined and reveals homology with certain bacterial proteins.These observations are discussed in relation to the idea that the binding protein is an example of a general class of proteins termed "molecular chaperones" which are required for the correct assembly of certain oligomeric proteins such as the carboxylase from their subunits.Abbreviations BP Binding protein - Rubisco Ribulose bisphosphate carboxylase-oxygenase     

A storage role for albumins in pea cotyledons

Abstract It is widely held that the albumins of pea seeds are mainly enzyme proteins. In the present study of Pisum sativum cv. Greenfeast, certain major polypeptides confined to the albumin fraction from the cotyledons were shown to be degraded in vivo following germination, thus functioning as a reserve of amino nitrogen and carbon skeletons. A general definition of seed storage proteins is proposed, to encompass any protein present in major quantity in the seed which is degraded following imbibition, and for which no other function may be demonstrated.     

The use of a multiple antigen peptide to detect a minichromosome maintenance protein in pea (Pisum sativum)

A 13-residue oligopeptide corresponding to a conserved region of the MCM family of proteins was synthesised as a multiple antigen peptide in which eight copies of the peptide were conjugated to an oligo-lysine core. The multiple antigen peptide was used for raising antibodies. Western blots of the polypeptides present in the DNA polymerase–primase complex from pea (Pisum sativum L.) were challenged with the antibodies which, even at a dilution of 1:5000, clearly revealed a polypeptide of approximately 62 kDa.     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Iron Deficiency Induced Changes on Electron Transport Rate in Pisum Sativum Chloroplasts

Iron deficiency induced decrease in the rate of whole electron transport chain in chloroplasts of pea (Pisum sativum L.). Such reduction was mainly due to the loss of photosystem (PS) 2 activity. The same result was obtained when the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) was evaluated. The loss in PS 2 activity was primarily due to a loss of 33, 23 and 17 kDa polypeptides. In contrast, iron deficiency induced the synthesis of 28 and 29 kDa polypeptides.     

Genome size in wild Pisum species

Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fulvum and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum Kleine Rheinländerin). Total variation of EB-DNA between samples covered 97.7% to 114.9% of the P. sativum value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P. fulvum was homogeneous in genome size (108.9% of P. sativum). Wide variation was observed between samples in P. abyssinicum (100.9–109.7%), P. elatius (97.7–114.9%) and P. humile (98.3–111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately.     

Excessive iron accumulation in the pea mutants dgl and brz: subcellular localization of iron and ferritin

The pea (Pisum sativum L.) mutants dgl and brz are defective in the regulation of iron uptake. Enhanced proton extrusion and constitutively high Fe(III) reductase activities in the roots lead to an accumulation of iron and other divalent cations in different organs of the mutants. Ultrastructural investigations of the basal leaflets of the mutants revealed in the cytoplasm, in mitochondria and especially in, or close to the endoplasmic reticulum numerous electron-dense particles which were absent in the corresponding wild type plants DGV and Sparkle. By means of electron-spectroscopic imaging and electron-energy-loss spectroscopy it could be shown that these electron-dense particles consist mainly of iron. Some of the iron deposits were immunocytochemically identified as the iron-storage protein ferritin. It is suggested that the precipitation of excessive iron in the dgl and brz mutant leaves in the form of electron-dense iron particles combined with the accumulation of ferritin is part of the plant defense mechanism against Fe-mediated oxidative stress. Received: 17 February 1998 / Accepted: 4 July 1998     

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta ³⁵S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.     

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

Nearest neighbour relationships within the LHC2-PS2 complex were investigated by using the reversible crosslinking agent dithiobis(succinimidyl propionate) (DSP). This was accomplished by treating PS2-enriched membranes, prepared from chloroplasts of Pisum sativum, with the crosslinker followed by diagonal electrophoresis of the solubilised polypeptides.Analysis of the off-diagonal spot patterns produced by crosslinker cleavage and second dimension electrophoresis was made on the basis of: staining with Coomassie blue or silver, labelling with [³⁵S]-methionine, and sensitivity to 1 M NaCl washing. It was concluded that LHC2 polypeptides crosslinked with several components of the PS2 complex and that the extrinsic polypeptides associated with water oxidation, having approximate molecular weights of 16 and 23 kDa, crosslink to form homodimers. The latter finding suggests that there may be more than one copy of each of these polypeptides per PS2 complex.Abbreviations DMSO dimethylsulphoxide - DSP dithiobis(succinimidylpropionate) - DCPIP dichlorophenolindophenol - SDS sodium dodecylsulphate - PS2 photosystem 2 - LHC2 light harvesting chlorophyll a/b complex associated with photosystem 2 - MES 2[N-morpholino] ethanesulphonic acid     

Immunolocalization of lipoxygenase in pea (Pisum sativum L.) carpels

Summary Polyclonal antibodies against a part of pea (Pisum sativum L.) LOXG protein have been raised to study the pattern of distribution of related lipoxygenases in pea carpels. The antiserum recognized three lipoxygenase polypeptides in carpels. One of them became undetectable 24 hours after fruit development induction, suggesting that it may correspond to the protein derived from loxg cDNA. Immunolocalization experiments showed that lipoxygenase protein was present only in pod tissues: it was abundant in the mesocarp and, from the day of anthesis, in the endocarp layers. Lipoxygenase distribution is regulated throughout development. The association of lipoxygenase with cells in which processes of expansion and growth will potentially take place support a role in pod growth and development.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IgG immunoglobulin G - GA₃ gibberellic acid - LOX lipoxygenase - PAGE polyacrylamide gel - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulfate - Tris 2-amino-2-hydroxymethyl-1,3-propanediol     

Cyclic AMP-dependent phosphorylation of pea proteins induced by forskolin

An increase in the level of phosphorylation of low-molecular-weight polypeptides in pea (Pisum sativum L.) leaves and changes in the content of some polypeptides after 10-min forskolin action in situ were demonstrated. A total level of protein phosphorylation in the homogenate of forskolin-treated leaves diminished after 25 min of its incubation in vitro. Using a highly specific inhibitor of cAMP-dependent protein kinases and cAMP treatments, we established that forskolin-induced change in the phosphorylation level of some polypeptides was cAMP-dependent.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 27–35.Original Russian Text Copyright © 2005 by Karimova, Tyrykina, Zakharova.     

Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Variation in Oidium neolycopersici development on host and non-host plant species and their tissue defence responses

Detailed studies of the infection processes of Oidium neolycopersici (tomato powdery mildew) and plant tissue responses were carried out on 10 Lycopersicon spp. accessions and one of each species of Cucumis sativus, Datura stramonium, Lactuca sativa, Petunia hybrida and Pisum sativum with different levels of resistance. Germination of O. neolycopersici conidia was not inhibited by the host plant. However, in the early stages of O. neolycopersici infection significant differences in conidial germ tube development on resistant and susceptible plant lines were observed. The first substantial differences appeared after 24 h post inoculation (hpi). Mycelia and conidiophores developed on susceptible lines; however, there was no mycelial development on resistant plant lines. The most frequent resistant response in Lycopersicon species was the necrotic (hypersensitive) reaction (HR), occasionally followed by pathogen development. The completely resistant accession L. hirsutum (LA 1347) showed only a limited number of necrotic host cells per infection site (2%). In Oidium resistant tomato lines OR 4061 and OR 960008 the existence of adult resistance was detected. This phenomenon occurred mainly in accession OR 4061. Rapid development and profuse sporulation of O. neolycopersici was observed on juvenile plants (6–8 wk old), however this was in contrast to the slow development and sporadic sporulation observed on 4 month old plants. Evidence of posthaustorial resistance was observed in the interaction of O. neolycopersici with non-host species (Lactuca sativa and Pisum sativum.) This was in contrast to Datura stramonium and Petunia hybrida, where development of powdery mildew was delayed at a later stage in the infection cycle. With the exception of Pisum sativum, the necrotic (hypersensitive) response was observed often.     

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.)

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.     

Residual effect of germination temperature on the growth of peas

Summary In Pisum sativum there is a marked difference in the final height of the plants depending on the temperature at which the seed is germinated. Significant differences were also found in such parameters as growth rate, node of first flower, seeds per plant, pods per plant as well as flowers per plant.     

Identification of DNA amplification fingerprinting (DAF) markers close to the symbiosis-ineffective sym31 mutation of pea (Pisum sativum L.)

 We demonstrate efficient genome mapping through a combination of bulked segregant analysis (BSA) with DNA amplification fingerprinting (DAF). Two sets of 64 octamer DAF primers, along with two PCR programs of low- and high-annealing temperatures (30°C and 55°C, respectively), appeared to be enough to locate molecular markers within 2–5 cM of a gene of interest. This approach allowed the rapid identification of four BSA markers linked to the pea (Pisum sativum L.) Sym31 gene, which is responsible for bacteroid and symbiosome differentiation. Three of these markers are shown to be tightly linked to the sym31 mutation. Two markers flanking the Sym31 gene, A21-310 and B1-277, cover a 4–5 cM interval of pea linkage group 3. Both markers were converted to sequence-characterized amplified regions (SCARs). The flanking markers may be potential tools for marker-assisted selection or for positional cloning of the Sym31 gene. Received: 2 July 1998 / Accepted: 8 October 1998     

Reduced rhizosphere colonization ability ofAgrobacterium tumefaciens chromosomal virulence (chv) mutants

Mutations in the chromosomal virulence (chv) region ofA. tumefaciens strain A723 reduce virulence, motility, and ability of the bacteria to bind to plant cells. We conducted experiments to assess the ability ofchv mutants to colonize the rhizosphere ofPisum sativum. The mutation had no effect on ability of bacteria to grow with a defined number of root cap cells as the sole carbon and nitrogen source. Ten days after inoculation, there were up to 103-fold more wild type thanchv mutant bacteria present in the rhizosphere of inoculated plants.     

On heliotropism in tendrils of Pisum sativum: A response to infrared irradiation

Summary In 1876, Darwin observed that tendrils of Pisum sativum oriented the long axis of their circumnutational sweep to keep it approximately normal to the direction of the sun's rays. Using various types of irradiation sources, the present study confirms Darwin's findings. Although blue light slightly increases the rate of circumnutation, only infrared irradiation (i.e. heat), effects the reorientation. The response can therefore be termed Diathermotropic. Diathermotropism in circumnutating tendrils is probably a device to bend the leaves of the elongating weak-stemmed plant away from the shade and toward the sun.     

Resistance of pea roots to endomycorrhizal fungus or Rhizobium correlates with enhanced levels of endogenous salicylic acid

The analysis of SA accumulation in roots of plant symbiotic mutants revealed two independent phenomena associated with the inability of either the plant or the microsymbiont to form a compatible symbiosis. SA accumulation in roots of the wild type and symbiosis-resistant P2 (Nod^-, MYC^-) Pisum sativum genotypes was induced upon interaction with Glomus mosseae. The amplitude of this accumulation was higher in P2 plants and increased with time, an effect that was not observed in roots of the wild-type, an effect that was not observed in roots of the wild-type P. sativum genotype. Likewise, Rhizobium leguminosarum wild type or a mutant blocked in Nod factor biosynthesis induced SA accumulation in P2, whereas SA accumulation in roots of the wild-type plant was dependent on the inability of the bacterium to produce Nod factors. These results suggest that the sym30 gene, which is mutated in P2 plants, could be implicated in a common pathway that leads to the suppression of an SA-dependent defence mechanism in legume plants against Rhizobium and endomycorrhizal fungi, thus allowing establishment of symbiosis.