Isolated Pea Chloroplasts Research Articles

Cloning, characterization and import studies on protochlorophyllide reductase from wheat (Triticum aestivum)

A full-length protochlorophyllide reductase clone (pWR5) has been isolated from a dark-grown wheat (Triticum aestivum) cDNA library generated in the phage vector lambda gt10. Comparison of the sequence of pWR5 with published sequences indicates a high degree of conservation of the structure of the mature protein amongst species but with the structure of the transit peptide less highly conserved. Within the cereals, the structure of the complete preprotein shows a remarkable degree of sequence homology (98% between barley and wheat). In vitro expression of pWR5 generates a preprotein of the expected molecular mass, approx. 41 kDa. Isolated pea chloroplasts can import, process and locate the mature reductase to the thylakoid membranes. From analysis of the CNBr-cleavage fragments of the N-[3H]phenylmaleimide-treated enzyme, the substrate-protected cysteine group in the enzyme is tentatively identified as Cys-296.

Redox titration of multiple protein phosphorylations in pea chloroplast thylakoids

Redox titrations were carried out on the protein kinase reactions of isolated pea chloroplast thylakoid membranes. Of the 13 phosphoproteins observed by autoradiography, all were found to titrate with the same midpoint potential of around +40 mV. Eleven proteins, including LHCII, D1, D2, CP43, a 9 kDa and a 55 kDa protein, were phosphorylated under reducing conditions, with a midpoint potential of E m = +38 ± 4 mV, n = 0.95 ± 0.06. Two other proteins, including one at 63 kDa, were phosphorylated only under oxidizing conditions, E m = +33 ± 11 mV, n = 0.67 ± 0.09. These midpoint potentials and n values suggest that either cytochrome b 6 or else a semiquinone associated with the cytochrome b 6 f complex may be the controlling redox sensor. The ‘reverse’ redox dependence of phosphorylation of the 63 kDa and 46 kDa bands suggests that more than one redox-controlled protein kinase (or phosphatase) functions in the thylakoid membrane. We also suggest that a protein kinase (or phosphatase) is itself regulated by phosphorylation in a redox-controlled reaction.

The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae

R-phycoerythrin is the major light-harvesting pigment protein of most red algal phycobilisomes. It is composed of three pigmented polypeptide subunits, the alpha, beta, and gamma. While alpha and beta phycoerythrin subunits are each unique in the red alga Aglaothamnion neglectum, there are two different gamma subunits with distinct molecular masses. Both gamma subunits are pigmented by virtue of covalently attached linear tetrapyrroles. The amino acid sequence of one of the gamma subunits, as deduced from the nucleotide sequence of a cDNA clone, has no significant similarity to any known sequence in the data bases. This result is surprising, since the gamma subunit of phycoerythrin is thought to have a function that is similar to cyanobacterial linker polypeptides. The A. neglectum gamma subunit is synthesized as a 36-kDa precursor protein that is processed at the amino terminus to yield a 33-kDa mature protein. The amino-terminal extension was able to direct the pea small subunit of Rubisco into isolated pea chloroplasts. This result suggests that red algae transport proteins into the plastid by a mechanism similar to that of higher plants. There are significant changes in levels of mRNA encoding the gamma 33 subunit when A. neglectum is grown under different conditions of illumination and in nitrogen-deficient medium. These changes parallel those previously observed for transcripts encoding the alpha and beta phycoerythrin subunits. Hence, there may be coordinated expression of nuclear and plastid-encoded phycoerythrin subunit genes.

ATP‐dependent protein synthesis in isolated pea chloroplasts

In the presence of externally added ATP, in the dark, isolated pea chloroplasts accumulate two proteins of molecular masses of about 22 and 24 kDa which precipitate with specific antibodies raised against the D1 protein. By chasing in the light, these proteins disappeared on the fluorogram concomitant with the appearance of the precursor- and mature-sized D1 proteins. Polysome analysis indicated that the 22-kDa component is associated with membrane-bound ribosomes and is thus ascribed to a translation intermediate of the D1 protein. On the other hand, the 24-kDa component could not be found in the polysome fraction under the experimental condition used, suggesting the possibility that this component is a degradation product of the D1 protein. The conclusion from this analysis is that the synthesis and/or stable accumulation of the D1 protein requires factor(s) caused by illumination, in addition to ATP, in isolated pea chloroplasts.

The pH Dependence of Violaxanthin Deepoxidation in Isolated Pea Chloroplasts.

The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.

Chloroplast import of the precursor of the gamma subunit of pea chloroplast ATP synthase.

A cDNA clone encoding the complete precursor of the gamma subunit of the pea chloroplast ATP synthase has been isolated from a pea leaf cDNA library in lambda gt 11 following detection with antibodies to the purified gamma subunit. The cDNA insert of 1.4 kbp is smaller than transcripts of about 1.6 kb detected by northern hybridisation of RNA from both light- and darkgrown pea leaves. The cDNA encodes a polypeptide of 376 amino acid residues, of which 52 residues constitute an N-terminal presequence and 324 residues make up the mature protein. Transcription and translation of the cDNA in vitro produced a protein of 42 kDa, which was imported by isolated pea chloroplasts and processed to the mature 36 kDa subunit.

Import of the precursor of the chloroplast Rieske iron-sulphur protein by pea chloroplasts.

cDNA clones encoding the precursor of the Rieske FeS protein of the chloroplast thylakoid membrane have been isolated from a pea leaf cDNA library in lambda gt 11, following screening with antibodies to purified pea chloroplast Rieske FeS protein. The longest cDNA insert of 880 bp encodes a polypeptide of 230 amino acid residues, of which 50 residues constitute an N-terminal cleavable presequence and 180 residues make up the mature protein. Transcription and translation of the cDNA in vitro produced a polypeptide of 26 kDa which was efficiently imported by isolated pea chloroplasts and processed to the mature 20 kDa protein. Southern hybridisation to pea genomic DNA indicated the presence of a single gene encoding the Rieske FeS protein in the haploid genome.

Biophysical characterization of a transit peptide directing chloroplast protein import

We have investigated the biophysical properties of a 35 amino acid peptide representing the entire length of a chloroplastic targeting sequence. The peptide, termed gamma-tp, corresponds in sequence to the transit peptide of the gamma subunit of the chloroplast ATP synthase from Chlamydomonas reinhardtii. We found that gamma-tp blocks the import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase into isolated pea chloroplasts (KI approximately 5 microM), suggesting that it interacts with higher plant plastids in a physiological manner. We also found the gamma-tp to have a high affinity for nonpolar environments, but not to cause a general disruption of membrane integrity. Hydrophobic moment analysis suggests that the gamma-tp can adopt an amphipathic beta structure. However, circular dichroism measurements indicate that the peptide is largely a random coil, in both the presence and absence of sodium laurylsulfate micelles. In the absence of a recognizable secondary structural targeting motif, we asked whether the presence of a transit peptide on a chloroplast protein increases the protein's overall affinity for nonpolar environments. Phase-partition experiments with Triton X-114 suggest that this is not the case. These results are discussed in relation to the mechanism of protein targeting to chloroplasts.

Ribulose-1,5-bis-Phosphate Carboxylase/Oxygenase Degradation in Isolated Pea Chloroplasts Incubated in the Light or in the Dark

Summary Intact pea ( Pisum sativum L.) chloroplasts isolated mechanically were incubated in the dark or in the light. After incubation intact chloroplasts were reisolated on Percoll steps prior to analysis. A 37 kD polypeptide derived from the large subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulated during incubation in darkness. Other degradation products (45, 42, 37, and 32 kD) were detected on immunoblots from organelles incubated in the light. The catabolism of Rubisco in the chloroplasts was affected by the composition of the incubation medium.

Targeting of ricin A chain into pea chloroplasts

Achimaeric gene was constructed encoding the pre-sequence of the 33 kDa oxygen-evolving complex protein from wheat (a thylakoid lumen protein) linked to ricin A chain. The fusion protein is efficiently imported by isolated pea chloroplasts and localised partly in the stroma, with the remainder bound to the stromal surface of the thylakoids. The imported protein is fully processed by both the stromal and thylakoidal processing peptidases, indicating that partial or complete translocation across the thylakoid membrane has taken place.

Induction of oscillations in chlorophyll fluorescence by re-illumination of intact isolated pea chloroplasts

Oscillations in chlorophyll fluorescence yield were observed upon re-illumination of intact isolated pea (Pisum sativumL.) chloroplasts that had attained their maximal rate of photosynthesis and had spent a short period in darkness. The oscillations depended on the length of the previous dark period, the length of previous illumination, and the reaction temperature. This finding confirms the presence of an "oscillatory center" in the chloroplasts temselves.

Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence

The precursor form of the major-harvesting chlorophyll a/b-binding protein (pLHCP) of chloroplast thylakoids was overproduced in E. coli cells and used to study the influence of soluble factors on post-translational protein import into isolated pea chloroplasts. pLHCP solubilised in 8 M urea was not import-competent. However, if pLHCP was dialysed in the presence of soluble proteins (leaf extract) after urea treatment, import competence was gained. Dialysis of pLHCP in the presence of leaf extract alters its protease sensitivity. Stremai proteins, ovalbumin, trypsin inhibitor or chloroplast lipids could not produce import competence of pLHCP. Two components from leaf extract seem to be necessary, one of which can be mimicked by purified hsc 70, the other one requiring ATP. We conclude that soluble proteins from outside the stromal compartment are necessary for post-translational import of proteins into chloroplasts.

The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence.

The role of transit peptides in intraorganellar targeting has been studied for a chlorophyll a/b binding (CAB) polypeptide of photosystem II (PSII) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) from Pisum sativum (pea). These studies have involved in vitro import of fusion proteins into isolated pea chloroplasts. Fusion of the CAB transit peptide to RBCS mediates import to the stroma, as evidenced by assembly of RBCS with chloroplast-synthesized large subunit (RBCL) to form holoenzyme. Similarly, fusion of the RBCS transit peptide to the mature CAB polypeptide mediates import and results in integration of the processed CAB protein into the thylakoid membrane. Correct integration was indicated by association with PSII and assembly with chlorophyll to form the light-harvesting chlorophyll a/b protein complex (LHCII). We interpret these results as evidence that the CAB transit peptide is functionally equivalent to a stromal-targeting sequence and that intraorganellar sorting of the CAB protein must be determined by sequences residing within the mature protein. Our results and those of others suggest that import and integration of CAB polypeptides into the thylakoid proceeds via the stroma.

Targeting of a foreign protein into the thylakoid lumen of pea chloroplasts

A chimaeric gene was constructed which encodes the pre-sequence of the 33 kDa protein of the oxygen-evolving complex, a thylakoid lumen protein, linked to dihydrofolate reductase, a cytoplasmic protein. The encoded fusion protein is transported into the thylakoid lumen of isolated pea chloroplasts, with concomitant removal of the pre-sequence.

Import of proteins into chloroplasts and mitochondria

Although both chloroplasts and mitochondria are capable of nucleic acid and protein synthesis, their biogenesis requires the import of a large number of nuclear-encoded proteins that are synthesized on cytoplasmic ribosomes. In the case of both organelles the transfer is post-translational and energy dependent. Most of the nuclear-encoded proteins are synthesized as precursors with an amino-terminal extension known as the signal, transit or pre-sequence. Like proteins destined for the secretory pathway, targeting of nuclear-encoded chloroplast and mitochondrial proteins to the proper cellular location is mediated by these N-terminal topogenic sequences. As is the case for protein translocation across the endoplasmic reticulum or prokaryotic plasma membrane, our data suggest dual recognition of signal sequences for protein import into mitochondria or chloroplasts; first by a soluble recognition system and then by a membrane bound one. The latter has been localised to distinct regions where the inner and outer membrane of these organelles are in close contact.The in vitro import of cytoplasmically synthesized precursors into chloroplasts has been previously described. We have transcribed mRNA from a cDNA clone coding for a cytoplasmically synthesized polypeptide of the chloroplast stroma, the small subunit of ribulose 1,5,-bisphosphate carboxylase. Translation of the specific mRNA in a wheat germ cell-free system yielded a large precursor (pS), and post-translational incubation with isolated pea chloroplasts resulted in import of pS into the chloroplast stroma accompanied by conversion of pS into the mature form (S) by means of cleavage of an NH2-terminal transit sequence. Import was an energy-dependent process and required ATP hydrolysis. Nonpenetrating ATP-consuming systems had no effect on import when chloroplasts were allowed to synthesize ATP in the stroma in the dark, suggesting that the ATP exerts its effect on import within the stroma and not in the cytosol. Studies with various ionophores demonstrated that ATP hydrolysis within the stroma was not used to generate a membrane potential across membranes.

The role of phospholipids in regulating photosynthetic electron transport activities: Treatment of chloroplasts with phospholipase A 2

The involvement of phospholipids in the regulation of photosynthetic activities was studied by incubating isolated pea chloroplasts with phospholipase A 2 to remove the β-positioned fatty acyl chains of the phospholipid molecules. The treatment was effective in eliminating 50% of phosphatidylglycerol (PG) and 90% of phosphatidylcholine (PCh) of the chloroplast thylakoids, but it resulted in only a partial decrease in photosynthetic electron transport activities. As found by fluorescence induction measurements the decrease in the electron flow activity was due to the inactivation of about 20% of photosystem II (PSII) centres. However, the fluorescence induction kinetics of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts showed that the number of PSII β-like centres was proportionally increased in the membrane by the progress of phospholipase A 2 treatment, suggesting that phospholipid molecules may play an important role in regulating the association of peripheral chlorophyll a/ b containing light-harvesting complex (LHCII-peripheral) with PSII α. The specific involvement of PG and PCh in the organization and function of PSII is discussed.

Inhibition of primary photochemistry of photosystem II by copper in isolated pea chloroplasts

The light-saturated rates of electron transport of the photoreactions, H 2 O → DCIP and H 2 O→ methyl viologen in isolated pea thylakoids are inhibited in the presence of cupric chloride with an I 50 value of 7.5 μM. This inhibition is more or less equal for both the light-limited and the light-saturated rates suggesting a decrease in the concentration of active PS II reaction centres. Chlorophyll variable fluorescence ( F v ) at room temperature in the presence and absence of DCMU is inhibited by copper; the apparent I 50 value being 25 μM and 30% of F v remains insensitive to copper at a concentration which inhibits electron transport completely. When the insensitive part of Fy is subtracted out, the I 50 value becomes 7.5 μM. The rate of electron transport in H 2 O → silicomolybdate (+ DCMU) photoreaction is inhibited by copper in a manner similar to the inhibition of F v . The inhibition of manganese → DCIP and manganese → silicomolybdate (+ DCMU) rates of electron transport in heat-treated thylakoids is similar to that of H 2 O → DCIP and H 2 O → silicomolybdate (+ DCMU), respectively. These results are interpreted as that copper inhibits the primary photochemistry of only one fraction of PS II centres (α centres/B-type centres) while the other fraction (β centres/non-B-type centres) is insensitive to copper. The strongest evidence of copper inhibition of the PS II reaction centre (RC) is that copper inhibits chlorophyll fluorescence in the isolated PS II RC which contains two polypeptides, Dl and D2, one cytochrome b -559 apoprotein, 4–5 Chi a , one P-680, two pheophytin and one β-carotene molecules but no Q A or Q B . Copper binds to the isolated RC in a stoichiometry of 0.8 copper per RC.

Inhibition of corn acetyl-CoA carboxylase by cyclohexanedione and aryloxyphenoxypropionate herbicides

Cyclohexanedione (sethoxydim, clethodim) and aryloxyphenoxypropionate (haloxyfop, diclofop, fluazifop, quizalofop) herbicides exhibit selective herbicidal activity on grasses. Dicots are tolerant to these herbicide classes. The effects of haloxyfop and sethoxydim on fatty acid biosynthesis in corn ( Zea may L.) and pea ( Pisum sativum) seedling chloroplasts were examined. The incorporation of [ 14C]acetate or [ 14C]pyruvate into fatty acids in isolated corn seedling chloroplasts was inhibited 90% or greater by 10 μ M sethoxydim or 1 μ M haloxyfop. The incorporation of [ 14C]acetate into fatty acids in isolated pea chloroplasts was not inhibited by 100 μ M sethoxydim or 10 μ M haloxyfop. The effect of these herbicides on fatty acid synthetase from disrupted corn chloroplasts was assayed using [ 14C]malonyl-CoA. Sethoxydim (10 μ M) and haloxyfop (1 μ M) stimulated incorporation of [ 14C]malonyl-CoA into fatty acids by approximately 50%. Acetyl-CoA carboxylase from disrupted corn seedling chloroplasts was inhibited by sethoxydim and haloxyfop with I 50 values of 4.7 and 0.5 μ M, respectively. Other aryloxyphenoxypropionate (diclofop, fluazifop, quizalofop) and cyclohexandione (clethodim) herbicides also inhibited this enzyme. Acetyl-CoA carboxylase activity from pea seedling chloroplasts was not inhibited by 1 m M sethoxydim or 0.1 m M haloxyfop. Thus, cyclohexanedione and aryloxyphenoxypropionate herbicides are potent inhibitors of acetyl-CoA carboxylase in corn, a susceptible species; whereas the enzyme from pea, a tolerant species, was tolerant to the herbicides.

Deletion of the Carboxyl-terminal Portion of the Transit Peptide Affects Processing but Not Import or Assembly of the Small Subunit of Ribulose-1,5-bisphosphate Carboxylase

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.

Chloroplast Protein Import

The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0 degrees C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.