Phosphoproteins and protein-kinase activity in isolated envelopes of pea (Pisum sativum L.) chloroplasts

A protein kinase was found in envelope membranes of purified pea (Pisum sativum L.) chloroplasts. Separation of the two envelope membranes showed that most of the enzyme activity was localized in the outer envelope. The kinase was activated by Mg²⁺ and inhibited by ADP and pyrophosphate. It showed no response to changes in pH in the physiological range (pH 7-8) or conventional protein substrates. Up to ten phosphorylated proteins could be detected in the envelope-membrane fraction. The molecular weights of these proteins, as determined by polyacrylamide-gel electrophoresis were: two proteins higher than 145 kDa, 97, 86, 62, 55, 46, 34 and 14 kDa. The 86-kDa band being the most pronounced. Experiments with separated inner and outer envelopes showed that most labeled proteins are also localized in the outer-envelope fraction. The results indicate a major function of the outer envelope in the communication between the chloroplast and the parent cell.     

Import and localisation of nucleoside diphosphate kinase 2 in chloroplasts

Nucleoside diphosphate kinases (NDPKs) are key enzymes that are involved in the homeostasis of nucleoside triphosphates (NTPs). Different isoforms exist, which are found in diverse cell compartments, for example the cytosol, mitochondria, and plant chloroplasts. NDPK2 of Pisum sativum has been shown to be localised in chloroplasts. Two forms of different size have been reported in plastids and it has been speculated that they function in distinct suborganellar compartments. We investigated the import behaviour and localisation of these two isoforms. Our results indicate that they do not differ in their route of entry into the organelle and both forms end up in the chloroplast stroma.     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.     

Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts

Isolated outer envelope membrane from pea (Pisum sativum L.) chloroplasts can be used in vitro to study binding and partial translocation of precursor proteins destined for the inside of the organelle. Efficient binding to a receptor protein on the outside of the membrane vesicle and generation of a translocation intermediate depends strictly on the presence of ATP. Protease treatment of the translocation intermediate demonstrates its insertion into the membrane. The membrane-inserted precursor protein cannot be extracted by 1 M NaCl and is also NaOH resistant to a large extent. Mild solubilization of outer envelope membranes by detergent resulted in the isolation of a complex which still contained the precursor protein. We have identified a constitutively expressed homologue hsc 70 as part of this membrane complex. Antibodies against hsp 70 (inducible heat shock protein 70) were able to immuno-precipitate the complex bound precursor protein. A second protein of 86 kDa molecular weight (OEP 86) from the outer envelope membrane was also identified as a major component of this complex.     

Molecular cloning,sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.     

Molecular cloning and analysis of a cDNA coding for nucleoside diphosphate kinase from ryegrass

A full-length cDNA, LpNDPK, encoding ryegrass nucleoside diphosphate kinase (EC 2.7.4.6) has been cloned and sequenced. The nucleotide sequence of the clone contains an open reading frame of 450 nucleotides encoding a protein of 150 amino acid residues with a calculated molecular mass of 16.5 kDa and a P_i of 6.62. The LpNDPK encoded protein possesses substantial homology with nucleoside diphosphate kinases (NDPKs) isolated and cloned form other sources; the highest identity (86 percnt;) was observed with NDPK from sugarcane (Saccharum officinarum). Amino acid comparisons with other NDPKs show that the presented ryegrass NDPK sequence also contains several motifs and specific residues crucial for catalytic activity which are highly conserved among other NDPKs. RT-PCR expression analysis using primers covering the coding region of LpNDPK revealed that the ryegrass NDPK gene is equally expressed in stem, leaf, and flower tissue.     

Nucleoside Diphosphate Kinase Is a Possible Component of the Ethylene Signal Transduction Pathway

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.     

Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum.

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [gamma-32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino-acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali-stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.     

Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic -helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning -helices. Some of these -helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.Abbreviations bp base pairs - kDa kilodaltons - M_r relative moleculas mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We wish to thank Dr D. Pappin and R. Jakes (AFRC Sequencing Laboratory, Department of Biochemistry, University of Leeds, UK) for performing the N-terminal sequence determinations and are greatful to Dr J. S. Gantt (Botany Department, University of Georgia, Athens, USA) for a pea leaf cDNA library and to Professor J. C. Gray (University of Cambridge, Department of Botany, Cambridge, UK) for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Science and Engineering Research Council and the Royal Society. D.L.W. was the recipient of the Royal Society Rosenheim research fellowship and K.F. was supported by a fellowship from the Studienstiftung des deutschen Volkes.     

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.     

Pea dehydrins: identification,characterisation and expression

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species.The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum.A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins.B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water.During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.     

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

Proteome-level changes in the roots of Pisum sativum in response to salinity

We initiated a proteomics-based approach to identify root proteins affected by salinity in pea (Pisum sativum cv. Cutlass). Salinity stress was imposed either on 2-wk old pea plants by watering with salt water over 6 wk or by germinating and growing pea seeds for 7 days in Petri dishes. Concentrations of NaCl above 75 mM had significant negative effects on growth and development of peas in both systems. Salinity-induced root proteome-level changes in pea were investigated by 2-D electrophoresis of proteins from control, 75 and 150 mM NaCl-treated plants and seedlings. The majority of the protein spots visualised showed reproducible abundance in root protein extracts from whole plants and seedlings. Of these proteins, 35 spots that exhibited significant changes in abundance due to NaCl treatment were selected for identification using ESI-Q-TOF MS/MS. The identities of these proteins, which include pathogenesis-related (PR) 10 proteins, antioxidant enzymes such as superoxide dismutase (SOD) as well as nucleoside diphosphate kinase (NDPK) are presented, and the roles of some of them in mediating responses of pea to salinity are discussed. This is the first report of salinity-induced changes in the root proteome of pea that suggests a potential role for PR10 proteins in salinity stress responses. Our findings also suggest the possible existence of a novel signal transduction pathway involving SOD, H₂O₂, NDPK and PR10 proteins with a potentially crucial role in abiotic stress responses.