Activation of a pea membrane protein kinase by calcium ions

Membranes from the buds of Pisum sativum L. contain a protein kinase which is activated 5- to 15-fold by micromolar levels of calcium. Best calcium activations were found with light-membrane fractions, and on density gradients these band at a similar position to the plasma membrane. Other heavier membranes, however, also contain a calcium-dependent protein kinase. The activity of the calcium-dependent protein kinase is inhibited by added phospholipids and phospholipase, in contrast to protein-kinase C. Calcium-dependent protein-kinase activity can be inhibited by 40% by low concentrations of the calmodulin inhibitor, trifluoperazine, but inhibitions are detected only after prior incubation of the membranes for some hours in ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid. Substantial calcium-dependent protein-kinase activity remains uninhibited by trifluoperazine indicating that there may be calmodulin-dependent and calmodulin-independent, but calcium-activated, protein kinases in pea membranes. The calcium-activated protein kinase seems to be intrinsically bound to membranes and only slight or partial solubilisation is obtained by the detergents nonidet P-40, (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate or octyl glucose. Better solubilisation is obtained by acetone treatment. There is some retention of calcium activation after partial solubilisation. A calcium-independent protein kinase has also been detected in membrane preparations; it has a substrate specificity different from that the calcium-dependent enzyme. Our results indicate, therefore, that there may be at least three protein kinases attached to pea shoot membranes.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - TFP trifluoperazine     

Distribution of calmodulin in pea seedlings: Immunocytochemical localization in plumules and root apices

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in young etiolated pea (Pisum sativum L.) seedlings. A fairly uniform staining was seen in the nucleoplasm and background cytoplasm of most cell types. Cell walls and nucleoli were not stained. In addition, patterned staining reactions were seen in many cells. In cells of the plumule, punctate staining of the cytoplasm was common, and in part this stain appeared to be associated with the plastids. A very distinctive staining of amyloplasts was seen in the columella of the root cap. Staining associated with cytoskeletal elements could be shown in division stages. By metaphase, staining of the spindle region was quite evident. In epidermal cells of the stem and along the underside of the leaf there was an intense staining of the vacuolar contents. Guard cells lacked this vacuolar stain. Vacuolar staining was sometimes seen in cells of the stele, but the most distinctive pattern in the stele was associated with young conducting cells of the xylem. These staining patterns are consistent with the idea that the interactions of plastids and the cytoskeletal system may be one of the Ca²⁺-mediated steps in the response of plants to environmental stimuli. Nuclear functions may also be controlled, at least in part, by Ca²⁺.     

Distribution of glucose/mannose-specific isolectins in pea (Pisum sativum L.) seedlings

We report on the distribution and initial characterization of glucose/mannose-specific isolectins of 4- and 7-d-old pea (Pisum sativum L.) seedlings grown with or without nitrate supply. Particular attention was payed to root lectin, which probably functions as a determinant of host-plant specificity during the infection of pea roots by Rhizobium leguminosarum bv. viciae. A pair of seedling cotyledons yielded 545±49 g of affinity-purified lectin, approx. 25% more lectin than did dry seeds. Shoots and roots of 4-d-old seedlings contained 100-fold less lectin than cotyledons, whereas only traces of lectin could be found in shoots and roots from 7-d-old seedlings. Polypeptides with a subunit structure similar to the precursor of the pea seed lectin could be demonstrated in cotyledons, shoots and roots. Chromatofocusing and isoelectric focusing showed that seed and non-seed isolectin differ in composition. An isolectin with an isoelectric point at pH 7.2 appeared to be a typical pea seed isolectin, whereas an isolectin focusing at pH 6.1 was the major non-seed lectin. The latter isolectin was also found in root cell-wall extracts, detached root hairs and root-surface washings. All non-seed isolectins were cross-reactive with rabbit antiserum raised against the seed isolectin with an isolectric point at pH 6.1. A protein similar to this acidic glucose/mannose-specific seed isolectin possibly represents the major lectin to be encountered by Rhizobium leguminosarum bv. viciae in the pea rhizosphere and at the root surface. Growth of pea seedlings in a nitrate-rich medium neither affected the distribution of isolectins nor their hemagglutination activity; however, the yield of affinity-purified root lectin was significantly reduced whereas shoot lectin yield slightly increased. Agglutination-inhibition tests demonstrated an overall similar sugar-binding specificity for pea seed and non-seed lectin. However root lectin from seedlings grown with or without nitrate supplement, and shoot lectin from nitrate-supplied seedlings showed a slightly different spectrum of sugar binding. The absorption spectra obtained by circular dichroism of seed and root lectin in the presence of a hapten also differed. These data indicate that nutritional conditions may affect the sugar-binding activity of non-seed isolectin, and that despite their similarities, seed and non-seed isolectins have different properties that may reflect tissue-specialization.Abbreviations IEF isoelectric focusing - MW molecular weight - pI isoelectric point - Psl1, Psl2 and Psl3 pea isolectins - SDSPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis The authors wish to thank Professors L. Kanarek and M. van Poucke for helpful discussions.     

Isolation of plasma-membrane-bound calcium/calmodulin-regulated protein kinase from pea using Western blotting

Membranes isolated from pea buds contain protein-kinase activity which is greatly activated by low concentrations of calcium ions. This paper describes a simple purification of this enzyme with a novel means of detecting enzyme activity by Western blotting. The purified enzyme appears to autophosphorylate primarily on serine residues, is activated by bovine calmodulin and additional evidence from phase partitioning indicate most of this enzyme to be located in the plasma membrane.Abbreviations PAGE polyacrylamide-gel electrophoresis - SDS sodium dodecyl sulphate     

Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings

The diversity of abundant mRNA sequences in various parts of 4-d etiolated pea seedlings (Pisum sativum L. var. Rondo CB) was compared by a cell-free translation of the mRNAs in the presence of [³⁵S]methionine and by an analysis of the products by two-dimensional electrofocussing/ electrophoresis (2D separation). The various parts of the seedlings were also examined for the pattern of protein synthesis in vivo. Proteins were labeled by injection of [³⁵S]methionine into the cotyledons, followed by 2D separation of the products. Over 95% of the abundant mRNA sequences and newly synthesized abundant polypeptides were shared by all parts of etiolated seedlings, including the cotyledons. However, a few distinct differences were observed when comparing mRNAs of roots and shoots; the most prominent among these were a group of six abundant mRNA sequences found exclusively in shoots. Only about 30% of the polypeptides synthesized on isolated RNA could be traced in equivalent positions on the gels as the polypeptides synthesized in vivo. Analysis of total RNA from light-grown pea seedlings showed the appearance of some twenty-five translation products not found with total RNA from etiolated seedlings, while about nine other translation products disappeared. At least ten of the light-induced RNA sequences were also present after growth in low-intensity red light (>600 nm) and are therefore thought to be controlled by the phytochrome system. Comparison of 11-d light-grown pea plants with 4-d light-grown seedlings did not reveal additional translatable RNA sequences, indicating that the major morphogenetic changes that occur after 4 d are not accompanied by significant changes in the pattern of abundant RNA sequences.     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

Light-enhanced perception of gravity in stems of intact pea seedlings

Dark-grown, 6-d-old pea seedlings (Pisum sativum L. cv. Alaska) do not respond gravitropically to brief (approx. 3 min) horizontal presentations, but seedlings given a pulse of red light (R) 16–24 h earlier respond to such stimuli by vigorous curvature of the epicotyl. With continuous horizontal stimulation (approx. 100 min), the kinetics and extent of the gravitropic response are almost identical in irradiated and dark-control plants. Prior R thus increases graviperception without altering the rate-limiting steps underlying the generation of curvature. This effect of R on graviperception develops slowly; seedlings studied only a few hours after R show differences in the kinetics of the gravitropic response, but not in presentation time. Neither the kinetics nor the extent of gravitropic curvature should be used as criteria for establishing changes in primary processes in gravitropism.     

Changes in respiration of mitochondria isolated from cotyledons of ethylene-treated pea seedlings

Treatment of intact, germinating pea (Pisum sativum L. cv. Homesteader) seedlings with ethylene enhanced the cyanide-resistant respiration of mitochondria isolated from the cotyledons. The level of enhancement depended on the concentration of ethylene. Thus, exposure to 0.9 l·l^-1 of ethylene in air for days 4–6 of germination had little effect on cyanide-resistant respiration, while exposure to 130 l·l^-1 increased it from 10 to 50 nmol O₂·min^-1·(mg protein)^-1. The length of exposure to ethylene also affected the degree of enhancement. According to some literature data, lipoxygenase (EC 1.13.11.12) activity can be mistaken for cyanide-resistant respiration, but in our preparations of purified pea mitochondria ethylene had no effect on lipoxygenase activity, nor did the gas disrupt the outer mitochondrial membrane. Bahr and Bonner plots of respiration in the presence of salicylhydroxamic acid (SHAM) indicated that ethylene did not affect respiration proceeding via the cytochrome pathway. Thus, increases in total respiration in mitochondria from cotyledons of ethylene-treated pea seedlings reflect increases in cyanide-resistant respiration.Abbreviations Cyt c cytochrome c - SHAM salicylhydroxamic acid     

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

Effect of gibberellic acid on ion uptake selectivity in pea seedlings

Gibberellic acid reduced the uptake of nitrogen and phosphorus relative to the cations, a common reponse in the three pea cultivars studied. In addition, in the cv. Progress, it increased the uptake of calcium relative to magnesium and potassium. No effect in the proportion in which cations are absorbed was noticeable in the other two varieties. Ion uptake is modified by gibberellic acid through its influence on the sink strength of the shoot, the size and geometry of the root system, and the selectivity in uptake. The overall effect may result in a stimulation or an inhibition, depending on the ion considered and the pea cultivar.Abbreviation GA₃ gibberellic acid     

Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1^-1 [-³²P]ATP whereas incubation with 50 mol·1^-1 [-³²P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1^-1 ATP, and around 40 mol·1^-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-³²P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1^-1). The ADP-dependent appearance of ³²P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of ³²P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1^-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.Abbreviations LHCP ligh-harvesting chlorophyll-a/b-binding protein - S_0.5 concentration giving half-maximal phosphorylation - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.     

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE diethylaminoethyl - EPR electron paramagnetic resonance - NIR nitrite reductase - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Immunolocalization of myosin in intact and wounded cells of the green algaErnodesmis verticillata (Kützing) Børgesen

Using in-vivo assays at least two classes of ethylene-binding site are shown to exist in pea epicotyls. These classes appear to have identical affinities for ethylene with a K_D of 6 · 10^–11-8 · 10^–11 M; the fast associating class has high and appropriate affinities for physiologically active analogues of ethylene, and CO₂ is without effect upon bfinding. Experiments involving suppression of endogenous ethylene production demonstrate that previous work may have underestimated both the amount of binding sites present and the affinity of such sites for ethylene. The apparent inhibition of binding by silver appears to be the consequence of a stimulation of endogenous ethylene production. The possible role of the sites as ethylene receptors is discussed.Abbreviations AOA amino oxyacetic acid - K_D dissociation constant We are grateful to the Biotechnology Action Programme of the European Economic Communities for support for this work.     

Soluble and microsomal glutatione S-transferase activities in pea seedlings (Pisum sativum L.)

Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olefinic double bond of cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean.The pea transferases had pH-optima between pH 7.4 and 7.8 K_m-values were 0.1–0.4 mM and 1–4 mM for cinnamic acid and glutathione, respectively. V-values were between 2–15 nmol mg^-1 protein x min.Chromatography on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000.The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[]pyrene with glutathione.Abbreviations GSH glutathione - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDMU l-Chloro-2,2-bis-(4-chlorophenyl)-ethylene     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated