Isolated Pea Chloroplasts Research Articles

Isolation of nuclear encoded plastid ribosomal protein cDNAs.

A pea leaf cDNA library was constructed in the expression vector lambda gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500-5,500 daltons smaller than their precursors.

In vitro activity of sorghum-selective fluorophenyl urea herbicides

The use of N-cyclopropyl- N′-(2-fluorophenyl) urea as a selective herbicide in grain sorghum has recently been disclosed (U.S.P. 4,344,916). Evaluation of analogs of this compound has included two assays on isolated pea chloroplasts—photosynthetic electron transport and competition for atrazine binding sites. Of all the analogs studied in at least one of these assays, the most active in vitro were the N-cyclopropyl-, N-n-butyl-, and N-n-pentyl-derivatives of 2,5-difluorophenyl urea. The two in vitro assays correlated well with each other, and binding activity demonstrated a strong correlation with whole-plant phytotoxicity following postemergence application. Several postulated sorghum metabolites of N-cyclopropyl- N′-(2-fluorophenyl) urea showed weak or no activity in vitro, as would be expected from the compoud's selectivity properties.

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities.

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H+/e- ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W X m-2) ("strong" light) the H+/e- ratio was 3. At low intensity (0.9 W X m-2) ("weak" light) the H+/e- ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H+/e- ratio due to underestimation of the initial rate of H+ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45 degrees C induces the expression of 10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36 degrees C to 42 degrees C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)RNAs endure in the cells at 42 degrees C up to 5 h but are no longer translated in vivo, whereas some poly(A)RNAs persist and are translated. As in pea, a poly(A)RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)RNA coding for the 22-kd HSP appears after 1 h at 36 degrees C. Its synthesis increases with the temperature of incubation up to 42 degrees C, although it decreases after 2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26 degrees C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.

Changing Kinetic Properties of Fructose-1,6-bisphosphatase from Pea Chloroplasts during Photosynthetic Induction

After dark-light transitions, there is a delay in photosynthetic CO(2) fixation by isolated pea chloroplasts in the range of some minutes. In order to assess the physiological significance of light modulation of enzyme activity in the control of induction, we made estimates of the kinetic parameters of fructose-1,6-bisphosphatase immediately upon release from pea chloroplasts in the dark and after illumination for various time periods. The Michaelis constant for fructose-1,6-bisphosphate decreased and maximal velocities increased during induction. It seems likely that light activation of this enzyme is one of the factors contributing to the overcoming of the lag period in photosynthetic CO(2) fixation.

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl − in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P +-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl −-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl −-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II f and II vf. The rate of reduction of P +-680 in the Cl −-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z + in the Cl −-depleted system. Under steady-state conditions, the reduction half-time of Z + in uninhibited systems was about 450 μs, while in the Cl −-depleted chloroplasts, the reduction of Z + was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P +-680 observed following the third flash on excitation of Cl −-depleted chloroplasts was delayed by two flashed when low concentrations of NH 2OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH 2OH or NH 2NH 2 are uninhibited by Cl − depletion. Addition of NH 2OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH 2OH oxidation present in Cl −-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl − depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z + is reduced directly by Mn of the complex. In terms of the S-state model, Cl − depletion appears to inhibit the advancement of the mechanism beyond S 2, but not to inhibit the transitions from S 0 to S 1, or from S 1 to S 2.

Factors Permitting Prolonged Translation by Isolated Pea Chloroplasts

The following parameters were found to prolong the time-course of translation by isolated pea (Pisum sativum, cv Progress No. 9) chloroplasts: addition of other amino acids (an effect synergistic with sufficient free Mg(2+)), use of lower light intensities, and additions of inorganic phosphate and ATP. In a chloroplast system which includes these parameters, active translation usually extends to almost an hour. The total amount of leucine incorporated is routinely 60 to 100 nanomoles/milligram chlorophyll and often 200 nanomoles/milligram chlorophyll. Accurate estimation of the amount of amino acid incorporated depends on supplying the labeled amino acid at a concentration sufficient to overcome isotope dilution effects from endogenous pools. Approximately 39 thylakoid and 60 stroma polypeptides were visible on autoradiographs after labeling with [(35)S]methionine. Label in a few of the polypeptide bands was increased or decreased by specific changes in the reaction conditions. Due to the long period of activity and the large number of labeled products, this chloroplast system should be useful for future studies of chloroplast translation.

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts (Pisum sativum L. cv. Progress #9). A 1600‐fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)−1 min−1. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The Km for oxidized glutathione was 11 μM and the Km for NADPH was 1.7 μM. Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 mM. The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H2O2 by reducing dehydroascorbate for ascorbate‐linked reduction of H2O2. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.

Cyanoacrylate Inhibitors of the Hill Reaction III. Stereochemical and Electronic Aspects of Inhibitor Binding

Ethoxyethyl 3-octylamino-2-cyanoacrylate and related compounds in which the amino group was replaced by N-CH3, S, O and CH2 were synthesized and their activity as inhibitors of the Hill reaction in isolated pea chloroplasts determined. All compounds showed moderate to high activity but there was no obvious correlation between activity and the electronic character of the ester carbonyl group. The stereochemistry of the various inhibitor molecules was deduced from the PMR spectra and the possible influence of stereochemistry on Hill inhibitory activity discussed. Replacement of the olefinic proton in the 2-cyanoacrylates with a β-alkyl substituent was examined and a specific relationship between the length of the alkyl chain and activity was observed.

Cyanoacrylate Inhibitors of Photosynthetic Electron Transport. Nature of the Interaction with the Receptor Site

The inhibitory activities (pI50 values) of a series of 3-alkylamino-2-cyanoacrylic acid ester derivatives in relation to photosypthetic electron transport in isolated pea chloroplast suspensions are reported. Assuming such pI50 values reflect the relative binding affinities for the inhibitor receptor site, the results suggest that several groups in these molecules interact specifically with the binding domain so that a multi-point attachment is formed. Stereochemical factors appear to play a significant role in the interaction.

Photodynamic Damage to Isolated Chloroplasts: A Possible Model for in vivo Effects of Photosynthetic Inhibitor Herbicides

The breakdown of chlorophylls, carotenoids, and linolenic acid together with the formation of malondialdehyde and ethane was followed in isolated pea chloroplast membranes. Breakdown was enhanced by light, oxygen, D2O and rose bengal, but retarded by crocetin. The results are discussed in relationship to the role of singlet oxygen in promoting damage in vivo.

Synthesis of components of the cytochrome b‐f complex by isolated pea chloroplasts

Three components of the cytochrome b-f complex, cytochrome f, cytochrome b-563 and a 15.2-kDa polypeptide, were labelled with radioactive amino acids in isolated pea chloroplasts incubated in the light with added Mg-ATP. The assembly of cytochrome b-563 (19.5 kDa) into the cytochrome complex required the presence of added Mg-ATP whereas cytochrome f (37.3 kDa) and the 15.2-kDa polypeptide were assembled in its absence. Incorporation of [35S]methionine into the polypeptide chain of cytochrome b-563 and the 15.2-kDa component was confirmed by peptide mapping of the products of partial digestion with papain.

Newly synthesized proteins are degraded by an ATP-stimulated proteolytic process in isolated pea chloroplasts

Up to 22% of the [ 3H]leucine-labeled proteins synthesized chloroplasts in the light was degraded during subsequent incubation for 20–40 min. The degradation of these radioactive proteins was more rapid in the light than in the dark and was at least 2-fold greater in the presence of 5 mM ATP in light or darkness. Exogenous amino acids did not influence degradation rates, although they promoted protein synthesis. Overall, proteins from thylakoid and stromal fractions were degraded at comparable rates. Analysis by electrophoresis in denaturing polyacrylamide gels revealed that many proteins decreased in both fractions. Certain low molecular mass stromal proteins were lost almost completely during a 90 min incubation in the presence of ATP, while others were unaffected or decreased only slightly. Thus Chloroplasts, like eukaryotic and prokaryotic cells and mithochondria, contain an ATP-stimulated proteolytic system.

Cyanoacrylate inhibitors of the Hill reaction. I. Nature of the inhibitor/receptor site interaction.

A series of alkyl 3-alkylamino-2-cyanoacrylates was synthesized and assayed as inhibitors of the Hill reaction in isolated pea chloroplasts. The results suggest that there is a single centre in these molecules, possibly the carbonyl group, which specifically interacts with the inhibitor receptor site and that maximum activity is associated with an asymmetric distribution about this centre of lipophilic alkyl groups which are involved in non-specific binding to discrete hydrophobic regions.

Light-driven DNA biosynthesis in isolated pea chloroplasts

Isolated chloroplasts from Pisum sativum leaves catalyze [ 3H]thymidine incorporation into acid-insoluble material using light as the sole energy source. Neither ribonucleotides, deoxyribonucleotides nor deoxyribonucleosides (other than [ 3H]thymidine) are required. However, it is necessary that the plastids be intact and photosynthetically competent, as little synthesis occurs in lysed preparations or in the presence of photophosphorylation inhibitors. Thymidine incorporation is markedly reduced by the DNA synthesis inhibitors rifampicin, nalidixic acid, ethidium bromide and N-ethylmaleimide. Plant age is an important factor, since rates of synthesis are 3–10-times higher in plastids isolated from young plants (6–8 days old) than in chloroplasts from older ones (9–14 days old). The maximum rates using plastids from young leaves of 60–70 pmd/mg chlorophyll/h are 30–60-times greater than those previously reported [1].

A Mg 2+ requirement for rapid ATP-driven protein synthesis by intact pea chloroplasts

Protein synthesis in the dark by chloroplasts isolated from young pea plants, driven by ATP, was slow unless Mg 2+ was added. Maximal stimulation occured when the added Mg 2+ was equimolar with the added ATP. The amount of incorporation of [ 3H] leucine into protein in the dark exceeded that found using light as the source of energy. When present in excess over the Mg 2+ level, ATP inhibited protein synthesis, largely by shortening the time course of the reaction. A low level of free Mg 2+ in the medium may have an important role in maintaining active protein synthesis by isolated pea chloroplasts. No indication was found of a need for light-activation of the enzymes of protein synthesis.

Characterization of Dicarboxylate Stimulation of Ammonia, Glutamine, and 2-Oxoglutarate-Dependent O2 Evolution in Isolated Pea Chloroplasts

Intact isolated chloroplasts from pea (Pisum sativum) leaves carried out light-dependent (NH(3), 2-oxoglutarate) and (glutamine, 2-oxoglutarate)-dependent O(2) evolution at rates of 3.3 +/- 0.7 (n = 7) and 6.0 +/- 0.4 (n = 5) micromoles per milligram chlorophyll per hour, respectively. Malate stimulated the rate of (NH(3), 2-oxoglutarate)-dependent O(2) evolution 2.1 +/- 0.5 (n = 7)-fold in the absence of glutamine, and 3.3 +/- 0.4 (n = 11)-fold in the presence of glutamine. Malate also stimulated (glutamine, 2-oxoglutarate)-dependent O(2) evolution in the presence of high concentrations of glutamine. The affinity (K(1/2)) of (NH(3), glutamine, 2-oxoglutarate)-dependent O(2) evolution for 2-oxoglutarate was estimated at 200 to 250 micromolar in the absence of malate and 50 to 80 micromolar when malate (0.5 millimolar) was present. In contrast to malate and various other dicarboxylates, aspartate, glutarate, and glutamate did not stimulate (NH(3), glutamine, 2-oxoglutarate)-dependent O(2) evolution in isolated pea chloroplasts. Using both in vitro assays and reconstituted chloroplast systems, malate was shown to have no effect on the activities of either glutamine synthetase or glutamate synthase.The concentration of malate required for maximal stimulation of O(2) evolution was dependent on the concentration of 2-oxoglutarate present. However, the small extent of the competition between malate and 2-oxoglutarate for uptake was not consistent with that predicted by the current ;single carrier' model proposed for the uptake of dicarboxylates into chloroplasts.

Crystalline arrays of ribosomes in isolated pea chloroplasts

Crystalline arrays of ribosomes in isolated pea chloroplasts

Crystalline arrays of ribosomes in isolated pea chloroplasts

Abstract Transmission electron microscopy of chloroplasts isolated by osmotic lysis of pea leaf protoplasts has revealed crystalline arrays of ribosomal particles associated with the thylakoid membranes. Optical diffraction techniques have established the crystallinity of the arrays and an image‐enhancement technique has given an indication of ribosomal macrostructure. A model of crystal‐packing is presented. This apparently artefactual induction of ribosome crystals should provide a valuable approach towards the elucidation of the details of the structure of chloroplast ribosomes.

Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts.

Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.