Etiolated Pea Shoots Research Articles

Mitochondrial сomplexome of etiolated pea shoots

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

Identification of a novel triterpenoid saponin from Pisum sativum as a specific inhibitor of the diguanylate cyclase of Acetobacter xylinum.

A specific and highly potent inhibitor of diguanylate cyclase, the key regulatory enzyme of the cellulose synthesizing apparatus in the bacterium Acetobacter xylinum, was isolated from extracts of etiolated pea shoots (Pisum sativum). The inhibitor has been purified by a multistep procedure, and sufficient amounts of highly purified compound (3-8 mg) for spectral analysis were obtained. The structure of this compound was established as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1--> 2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside. The structure was elucidated on the basis of susceptibility to various enzymes, chemical and spectral methods, such as GC-MS, FAB-MS, and the following types of 2D-NMR: COSY, ROESY, TOCSEY, HMQC, HMBC analyses. An identical or a very similar compound with identical biological activity was also isolated from A. xylinum, strongly suggesting that at least certain aspects of cellulose synthesis in the bacteria and in higher plants may be regulated in a similar manner. The content of this saponin in etiolated plants was about 0.04 mumol (g fresh tissue)-1.

Oxaloacetate and Malate Transport by Plant Mitochondria

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10(-3). From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.

Effect of Red Light on the Synthesis of Wound Ethylene in Etiolated Pea Shoots

Apical segments from 7-day-old etiolated pea seedlings (Pisum sativum L. cv. Vorbote) produced 3 to 4 nl C(2)H(4) h(-1)g(-1) f.w. during the first 5 hours after excision. This formation of wound ethylene was due to a sharp increase of ACC concentration in the segments after exision. A 15 min red light pulse (approx. 0.22 W m(-2)) applied 3 hours before excision prevented the increase of ACC and inhibited wound ethylene formation. The lower ACC level was not caused by a higher rate of metabolism of ACC to MACC and also the capacity for the conversion of ACC to ethylene was not affected by the red light pulse. We therefore relate the lower ethylene formation after red light to an inhibition of wound ACC formation. The red light effect on wound ethylene production was reversible by far red light, so it can be explained as a phytochrome effect. Exogenous ethylene had a similar effect on the synthesis of wound ethylene as red light. Possible relations between these two effects are discussed.

Effect of Light on Ethylene Production: Red Light Enhancement of 1-Aminocyclopropane-1-carboxylic Acid Concentration in Etiolated Pea Shoots

Summary Seven day old etiolated pea seedlings (Pisum sativum L. cv Vorbote) were illuminated with red light (3.3 Wm-2) for 15 min. After different time intervals ethylene production in intact seedlings, and ACC content and ACC-mediated ethylene production («ACC-oxidase» activity) in crude extracts were measured. The red light pulse resulted in an increased ACC content (maximum after 3 h) followed by a rise in ACC oxidase activity and of ethylene production in intact seedlings. The red light effects were reversed by 15 min far red (0.3 Wm-2) illumination suggesting that synthesis of ACC is, in this case, regulated via phytochrome.

Effect of Neutral Salts on Spectral Characteristics of Undegraded Phytochrome Partially Purified from Etiolated Pea Shoots

Effect of Neutral Salts on Spectral Characteristics of Undegraded Phytochrome Partially Purified from Etiolated Pea Shoots

The Role of Peroxidase in Ethylene Formation from 1-Aminocyclopropane-1-carboxylic Acid

Summary Activity of ethylene formation from 1-aminocyclopropane-1-carboxylic acid (ACC) was compared to peroxidase (POD) activity in crude extracts of etiolated pea shoots ( Pisum sativum L.) as well as in Sephadex and Concanavalin A-Sepharose (ConA) fractions of these extracts. In crude extracts the stability of POD activity was higher than that of ethylene formation. Mercaptoethanol and sodiumazide inhibited both activities, but other inhibitors of ethylene formation (hydroxylammoniumchloride, catalase, EDTA, KCN, CuSO 4 , CoCl 2 ) had a weak or no effect at all on POD activity. High-molecular weight fractions (FI) from Sephadex of pea crude extracts formed ethylene from ACC and exhibited POD activity. By mixing high molecular weight fractions with low molecular weight fractions from the Sephadex column ethylene formation was stimulated. A similar effect could be reached by substituting FI fractions with horse radish peroxidase (HRP), but ethylene production was very low. After ConA fractionation the highest ethylene formation was found to be in the POD containing eluate, but in addition we found ethylene formation in effluent fractions which showed no POD activity. Mn 2+ otherwise has a stimulating effect on ethylene production from Sephadex fractions as well as from HRP but not on the crude extract. It appears that peroxidase itself does not play a major role in decarboxylation or ring cleavage of ACC, but that it might be involved in a more complex way by providing radicals, H 2 O 2 or otherwise unknown factors.

Enzymatic ethylene formation from 1-aminocyclopropane-1-carboxylic acid by manganese, a protein fraction and a cofactor of etiolated pea shoots

The cofactor of enzymatic, 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation was concentrated on cation exchange columns. When chelators of cations were added to the homogenates, cofactor activity was lost. Cofactor fractions were partly resistant to oxidation at 600° C. Mn(2+) substituted for the cofactor in ethylene formation from 1-aminocyclopropane-1-carboxylic acid by a protein fraction isolated from etiolated pea shoots. In addition, Mn(2+) enhanced the stimulatory effect of the concentrated cofactor. The elution volume for the cofactor on a Sephadex G-25 column was lower than that of MnCl2. In paper electrophoresis the cofactor migrated to the cathode at pH 10.8 and 2.2. The RF of cofactor on cellulose plates developed in butanol: acetic acid: H2O was 0.4. After cellulose chromatography, cofactor activity had to be reconstituted by the addition of MnCl2. Chelators, anti-oxidants, and catalase were inhibitors of Mn(2+)-cofactor-dependent ethylene formation. The protein necessary for 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation in vitro was seperated from 95-98% of the total protein in homogenates by DE-52 cellulose chromatography and (NH4)2SO4-fractionation.

Isolation and characterization of NADH-glutamate synthase from pea (Pisum sativum L.).

Both ferredoxin-glutamate synthase (EC 1.4.7.1) and NADH-glutamate synthase (EC 1.4.1.14) were isolated separately on DEAE-cellulose chromatography from etiolated pea shoots. The latter enzyme was purified 1,400-fold by ammonium sulfate fractionation and column chromatographies of DEAE-cellulose, Sephadex G-200 and blue-Sepharose. The enzyme had a molecular weight of 220,000 and an isoelectric point of 4.3. The optimum pH was 7.6. Apparent Km values for l-glutamine, 2-oxoglutarate and NADH were 400, 37 and 4 µm, respectively. The enzyme had its absorption maxima at 275, 375 and 440 nm, suggesting that pea NADH-glutamate synthase is a flavoprotein. It showed NADH-diaphorase activity toward ferricyanide and 2,6-dichlorophenol indophenol as the electron acceptor. Sulfhydryl reagents, metal-chelating reagents, phthalein acids and azaserine were strong inhibitors. Ammonium and phosphate ions enhanced the enzyme activity.

Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots

A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0° resulted in very rapid binding of both Pr and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The amount of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of Pr and Pfr in the bound form was the same as that in 12,000 x g supernatant. After a brief exposure of the segments to red light at 0° and a subsequent dark incubation at 30° in Tris-HCL buffer containing dithiothreitol or EDTA, which bot inhibit Pfr decay, the contents of phytochrome in the mitochondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed by far-red light. The increase of the phytochrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of Δ (ΔA) per mg protein.

Protein Kinase Activity Associated with Isolated Ribosomes from Peas and Lemna

Protein kinase (ATP:protein phosphokinase) has been found associated with ribosomes prepared from 5-day-old etiolated pea shoots and sterile cultures of Lemna minor. The enzyme catalyzes the phosphorylation of ribosomal proteins in vitro and may be involved in the formation of phosphoproteins in the ribosomes in vivo. Protein kinase sediments with ribosomes during sucrose density gradient centrifugation through a buffer containing 0.02 m KCl. Seventy per cent of the enzymic activity dissociates from the ribosomes in 0.3 m KCl, but the remaining protein kinase remains firmly bound in 0.7 m KCl. Cyclic AMP does not modify the activity of these protein kinases in vitro. Benzyladenine inhibits the protein kinase, but only at high concentrations of this cytokinin.

Metabolism of Tritiated Gibberellin A9 by Shoots of Dark-grown Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A(9) ((3)H-GA(9)) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA(20), GA(10), 2,3-dihydro-GA(31), and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GA(30) and 2,3-dihydro-GA(31) were produced within 5 hours following (3)H-GA(9) application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after (3)H-GA(9) application. Levels of GA(10) increased with time, and since no GA(10) was produced during the purification procedures, GA(10) was, in all probability, produced from (3)H-GA(9) within the plant tissue. The radioactive interconversion products produced by pea from (3)H-GA(9) have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of (3)H-GA(9) with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu).

Cyclic nucleotide-independent protein kinase from pea shoots

Protein kinase has been isolated from 6-day old etiolated pea shoots. Crude homogenates contain endogenous protein substrates for the kinase. Casein or phosvitin, but not histone, can serve as substrates for assay. DEAE-cellulose columns distinguish several forms of protein kinase activity. Cyclic nucleotides do not modify the activity of these protein kinases in vitro .

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.

Indole-3-methanol—a metabolite of indole-3-acetic acid in pea seedlings

Degradation of indole-3-acetic acid was investigated in etiolated pea shoots; the study was limited to indolic metabolites. The products formed were fractionated by column chromatography and identified by thin-layer chromatography and chemical methods. The pathway of indole-3-acetic acid degradation involving indole-3-aldehyde was found to be more significant than stated in literature, and indole-3-methanol was established as the major indolic metabolite.

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.

Untersuchungen über Auxinoxydasen

In an analysis of the β-indolyl-acetic acid (IAA) oxidase system of etiolated pea shoots, three different peroxidases were separated and purified several hundredfold by column chromatography. Each of these “multiple peroxidases” requires only the presence of a phenolic cofactor for IAA oxidation. Although the addition of manganous ion alone is without effect, in the presence of the phenol it increases the rate of IAA oxidation by two of the peroxidases while inhibiting the third. These results are contradictory to theories which postulate that manganese ions or enzymic activities additional to that of peroxidase are essential for the function of an IAA oxidase.

THE EFFECTS OF ETHIONINE ON SOME PLANT GROWTH SYSTEMS

Ethionine (CH3∙CH2∙S∙CH2∙CH2∙CH∙NH2COOH) was tested on six plant growth systems: (a) growth of lateral buds on decapitated dwarf beans, (b) rooting of excised, etiolated bean hypocotyls, (c) linear growth and final cell size of primary roots of wheat, (d) growth of hypocotyl and roots of flax seedlings, (e) growth of sections from etiolated pea shoots, and (f) expansion of leaf disks from etiolated beans. Ethionine stimulated growth in each of these systems with the possible exception of (a) and (d). The work on the rooting of etiolated bean hypocotyls includes the description of a new rooting test.Pretreatment of pea sections with manganese lowered or prevented the stimulatory effect of ethionine.Expansion of etiolated bean leaf disks was not increased by ethylenediaminetetraacetic acid, penicillin G, and the ‘antiauxins’ α (2,4-dichlorophenoxy)- isobutyric acid, α(p-chlorophenoxy)isobutyric acid, and 2,3,5-tri-iodobenzoic acid whereas maleic hydrazide, o-chlorophenoxyisobutyric acid, m-methyl phenoxyacetic acid, and 2,4,6-trichlorophenoxyacetic acid (TCPAA) stimulated expansion. TCPAA, at 10 mg/liter, completely replaced the stimulatory effect of cobalt on light-treated disks. The peculiar dose–response curve and some morphogenetic effects with TCPAA are described.Ethionine, cobalt, and TCPAA all inhibit chlorophyll synthesis in leaf disks exposed to light. TCPAA also causes a loss of yellow pigment in disks kept in the dark.Depending upon the test system, ethionine simulates the effects of auxin or ‘antiauxins’. Metabolic studies with ethionine are briefly reviewed. Some connections between the known effects of ethionine and theories of auxin action, and the possible significance of ethionine as an inhibitor of adaptive enzyme synthesis, are pointed out.