Isolated Spinach Chloroplasts Research Articles

Native uridine 5 ′-diphosphate–sulfoquinovose synthase, SQD1, from spinach purifies as a 250-kDa complex

Sulfoquinovosyldiacylglycerol is a polar lipid present in photosynthetic membranes. It contributes to the negative surface charge of the membrane and plays a pivotal role under phosphate stress. The SQD1 protein is the key enzyme involved in the formation of the sulfolipid head group precursor, uridine 5 ′-diphosphate (UDP)–sulfoquinovose, from UDP–glucose and sulfite. A cDNA encoding the spinach SQD1 protein was isolated and functionally expressed in Escherichia coli. The recombinant enzyme was compared to the native enzyme purified from isolated spinach chloroplasts. While the K m for UDP–glucose was indistinguishable for the two forms, the K m for sulfite was more than fourfold lower (<5 μM) for the native enzyme. Sizing by gel filtration indicated that the native form purified as a large complex of approximately 250 kDa, which is more than twice as large as the calculated size for the homodimer. It is proposed that in vivo SQD1 forms a complex with accessory proteins.

Superoxide as an Obligatory, Catalytic Intermediate in Photosynthetic Reduction of Oxygen by Adrenaline and Dopamine

The superoxide anion radical is known to be the first product of photosynthetic reduction of oxygen mediated by a variety of electron carriers. The effectiveness of many of these electron carriers as herbicides, and the toxicity of the superoxide they produce, have been suggested to rule out oxygen reduction as a physiological component of normal photosynthesis. Here results with isolated spinach chloroplasts are presented that demonstrate that the related catecholamines adrenaline and dopamine mediate photosynthetic reduction of oxygen. Complete inhibition by added superoxide dismutase of light-dependent oxygen uptake by isolated chloroplasts and of the electron transport it supports indicates that superoxide is an obligatory catalytic intermediate, not a product, in adrenaline- and dopamine-mediated oxygen reduction. These compounds might function as chemical analogues of a proposed natural mediator, or oxygen-reducing factor, that allows oxygen reduction to participate in energy transduction in photosynthesis. The identity of the putative natural mediator and the role of oxygen reduction in photosynthesis are discussed. The fully oxidized form of adrenaline, adrenochrome, also acts as a mediator of photosynthetic oxygen uptake, but only by reducing oxygen to superoxide.

Effect of lichen metabolites on thylakoid electron transport and photophosphorylation in isolated spinach chloroplasts.

Investigation of the lichen Parmotrema tinctorum led to the isolation of several known compounds. Among them, lecanorin (1), methyl-beta-orcinol carboxylate, methyl orsellinate, orcinol, and methyl haematommate (3) caused significant inhibition of the radicle growth and germination of seedlings of Amaranthus hypochondriacus and Echinochloa crusgalli. In addition, lecanorin (1) and gyrophoric acid (2) significantly inhibited the light-dependent synthesis of ATP and uncoupled electron transfer on the reducing side of photosystem II in freshly lysed, illuminated spinach chloroplasts. The targets of 1 and 2 were located at the water-splitting enzyme level and in one of the redox enzymes in the range of electron transport from P(680) to Q(A), respectively.

Photosynlhetic Inhibitory Activity of Dihydro-β-agarofurans Sesquiterpenes from Maytenus dislicha and Maytenus boaria (Celastraceae)

Abstract The effects of 9β-benzoyloxy-la, 2α, 6β, 8α, 15-pentaacetoxy-dihydro-β-agarofuran and 9β furoyloxy-1α, 6β, 8α-triacetoxy-dihydro-β-agarofuran, major phytogrowth inhibitors isolated from the aerial parts of Maytenus disticha (Celastraceae) and seeds of Maytenus boaria (Celastaraceae), respectively, on different photosynthetic activities of isolated spinach chloroplasts have been investigated. Photophosphorylation and electron transport (basal, phosphorylating and uncoupled) were inhibited in a concentration dependent manner by both compounds, therefore acting as Hill reaction inhibitors. The site of action of these natural compounds was located in the span from P680 to QA. 9β-benzoyloxy-1,2,6,8,15-pentaacetoxydihydro-β-agarofuran was one order of magnitude more potent (I50 = 2.6 μм) than 9β-furoyloxy-1,6,8,-triacetoxydihydro-β-agarofuran, suggesting that the substitution at C-9 and the acetoxy groups at carbons 2 and 15 are important structural requirements for the displayed inhibitory activity.

Phytotoxic and photosynthetic activities of maduramicin and maduramicin methyl ester.

The polyether antibiotic maduramicin and its methyl ester derivative inhibited photophosphorylation and proton uptake in isolated spinach chloroplasts. Both compounds also enhanced basal and phosphorylating electron transport and stimulated Mg(2+)-dependent ATPase activity, therefore, they behave as uncouplers of photophosphorylation being the methyl ester derivative more potent than the parent compound. On the other hand, maduramicin inhibited germination and radicle elongation of several crop and weed species. In addition, the antibiotic caused phytotoxic injury and fresh weight reduction to 4-to-6 week old seedlings of two weed and two crop species when applied at 10(-4) M by foliar application in the greenhouse.

Impairment of Photosystem II Donor Side by the Natural Product Odoratol

The effect of odoratol, a natural protolimonoid, isolated from Cedrela odorata (Meliaceae) and two of its derivatives on different photosynthetic reactions of isolated spinach chloroplasts was investigated. This natural product is an inhibitor (150 μM) of oxygen evolution. Polarographic analyses of the photosynthetic partial redox reactions indicate that (a) photosystem I (PSI) activity is unaffected by odoratol, (b) there is a substantial inhibition of the electron flow of uncoupled photosystem II (PSII) as measured from water to silicomolybdate or diaminodurene, and (c) the electron flow from diphenyl carbazide to dichlorophenolindophenol of Tris-washed chloroplasts was insensitive. Collectively, these data suggest that the site of action of odoratol is located at the donor side of PSII. Comparison of chlorophyll a fluorescence induction curves of chloroplasts with authenticated donor side damage and those obtained from odoratol-treated samples further supports this interpretation. Comparative analyses ...

Effect of encecalin, euparin and demethylencecalin on thylakoid electron transport and photophosphorylation in isolated spinach chloroplasts

The major phytotoxic compounds (encecalin, euparin and demethylencecalin) isolated from Helianthella quinquenervis (Hook) A Gray (Asteraceae) were evaluated on different photosynthetic activities in chloroplasts isolated from spinach leaves. ATP synthesis, proton uptake and electron flow (basal, phosphorylating and uncoupled) were inhibited by encecalin and demethylencecalin in a concentration dependent manner, therefore acting as Hill reaction inhibitors. Encecalin and demethylencecalin did not affect photosystem I (electron transport from diaminodurene to methylviologen), but they inhibited photosystem II (from water to 2,5-dibromo-3-methyl-6-isopropyl-1,4-p-benzoquinone). Since these compounds inhibited electron flow in the photosystem II partial reactions from water to silicomolybdate and from diphenylcarbazide to dichlorophenol-indophenol, the site of inhibition was located in the span from P680 to QA of the electron transport chain. Euparin, inhibited ATP synthesis, proton uptake and basal and phosphorylating electron transports, but it has not effect on uncoupled electron flow from water to methylviologen. Mg2+-ATPase activity from bound membrane thylakoid chloroplasts was also inhibited by this compound. These results suggested that euparin inhibited phosphorylation in chloroplasts, acting as an energy-transfer inhibitor. © 1998 Society of Chemical Industry.

Transport of CtpA protein from the cyanobacterium Synechocystis 6803 across the thylakoid membrane in chloroplasts.

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C-terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast-targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the SecA protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec-dependent thylakoid transport route by high concentrations of the precursor of the 33-kDa subunit of the oxygen-evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23-kDa subunit of the oxygen-evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the deltapH-dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway.

Photoinhibition of Cyclic Photophosphorylation and Light-Induced pH Changes in Isolated Spinach Chloroplasts

As a result of comparative investigations of the photoinhibition dynamics of cyclic photophosphorilation CPP) and light-induced pH changes of isolated spinach chloroplasts at room- and low temperature (+1¼C) it was established that the extent and rate of these photoinhibition processes are dependent on temperature. The light-induced pH-change characteristics are inhibited completely during illumination with strong light (100000 1x) at low temperature and room temperature for 30 min. CPP is more sensitive to strong light effects, complete inhibition is obtained in 20 min at low temperature and in 10 min at room temperature. The light-induced pH changes are only inhibited by 50%. The inhibition kinetics of cyclic photophosphorylation in anaerobic and aerobic conditions point out that the acceptor side of Photosystem I is probably involved in photodamage. The results suggest that betaine stabilizes the cyclic photophosphorylation with spinach chloroplasts under the light treatment.

Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro, is targeted to envelope membranes of isolated spinach chloroplasts. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations. The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli. In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E. coli. To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter-exchange mode of transport. Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator. An apparent KM for ATP of 28 microM was determined which is similar to values reported for isolated plastids. The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.

The protein kinase, protein phosphatase and inhibitor protein of nitrate reductase are ubiquitous in higher plants and independent of nitrate reductase expression and turnover

Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (<3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5′-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca2+/Mg2+-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with γ-[32P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.

Sorting of Nuclear-encoded Chloroplast Membrane Proteins to the Envelope and the Thylakoid Membrane

The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CF0II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF0II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.

Identification and localization of the first glutaredoxin in leaves of a higher plant

Glutaredoxin(thioltransferase) has been identified and purified to homogeneity from spinach leaves. Its cytosolic localization was demonstrated by chromatographic and immunological analysis of extracts from isolated spinach chloroplasts and mitochondria, respectively. Spinach glutaredoxin shows a significant crossreactivity with antibodies raised against E. coli glutaredoxin and possesses a specific thioltransferase activity comparable to that of the E. coli protein. Minor thioltransferase activities (less than 10% of total leaf activity) have been observed in spinach chloroplasts which are probably due to the presence of trypsin inhibitor and thioredoxins (TRf and TRm).

Immunological Characterization and Chloroplast Localization of the Tryptophan Biosynthetic Enzymes of the Flowering Plant Arabidopsis thaliana

In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.

Photosystem II Inhibition bys-Triazines Having Hydrophilic Amino Groups

Photosystem II inhibition by s-triazines having hydrophilic amino groups was examined in isolated spinach chloroplasts. Among them, the hydroxyalkyl- and alkoxyalkylamino derivatives were potent inhibitors. The hydroxyalkylamino-s-triazines seemed to interact with the binding site in a manner different from that of other triazines, since they needed a time to build up to constant inhibition and showed a different thermoluminescence glow peak.

Regulation of NADP‐Dependent Glyceraldehyde 3‐Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

AbstractActivation of NAD(P)‐glyceraldehyde 3‐phosphate dehydrogenase (NADP‐GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3‐phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3‐phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time‐course of activation appears to depend upon the actual concentration of 1,3‐bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The Ka values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the Ka value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP‐GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered Kavalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP‐GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.

Photobiotechnology: Application of photosynthesis to the production of renewable fuels and chemicals

Sustained hydrogen photoevolution fromChlamydomonas reinhardtii and C.moewusii was measured under an anoxic, CO2-containing atmosphere. It has been discovered that light intensity-and temperature influence the partitioning of reductant between the hydrogen photoevolution pathway and the Calvin cycle. Under low incident light intensity (1-3 Wm-2) or low temperature (≈ 0‡C), the flow of photosynthetic reductant to the Calvin cycle was reduced, and reductant was partitioned to the hydrogen pathway as evidenced by sustained H2 photoevolution. Under saturating light (25 W m-2) and moderate temperature (20 ± 5‡C), the Calvin cycle became the absolute sink for reductant with the exception of a burst of H2 occurring at light on. A novel photobiophysical phenomenon was observed in isolated spinach chloroplasts that were metalized by precipitating colloidal platinum onto the surface of the thylakoid membranes. A two-point irradiation and detection system was constructed in which a continuous beam helium-neon laser (λ = 632.8nm) was used to irradiate the platinized chloroplasts at varying perpendicular distances from a single linear platinum electrode in pressure contact with the platinized chloroplasts. No external voltage bias was applied to the system. The key objective of the experiments reported in this report was to measure the relative photoconductivity of the chloroplast-metal composite matrix.

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level Fj are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.

Electron transfer between Photosystem II and the cytochrome b/F complex: mechanistic and structural implications

Electron transfer between Photosystem II (PSII) centers, cytochrome b/f complexes and Photosystem I (PSI) centers have been studied in isolated spinach chloroplasts in the presence of ferricyanide. The analysis of the reduction of cytochrome b h under saturating illumination shows that about 2/3 of cytochrome b/f complexes are rapidly accessible to PSII-formed plastoquinol (< 10ms), while for the remaining fraction, cytochrome b h is reduced in the 100 ms range. These results are interpreted according to a model proposed by Joliot et al. (Joliot, P., Lavergne, J. and Béal, D. (1992) Biochim. Biophys. Acta 1101, 1–12), in which the diffusion of plastoquinone is restricted to domains, including an average of 4 PSII centers. Cytochrome b/f complexes included in the grana regions are involved in the linear electron flow from PSII to PSI; long-range electron transfer reactions are exclusively mediated by plastocyanin. Under flash excitation, the rate of electron transfer from PSII to PSI is limited neither by the diffusion nor by the binding of plastoquinol to cytochrome b/f complexes, but by electron transfer processes occurring within the cytochrome b/f complexes. The rate of these limiting processes is very likely controlled by the redox state of the high potential chain (cytochrome F and Rieske protein). Electron transfer through the cytochrome b/f complexes is discussed in terms of a Q-cycle or a semiquinone cycle mechanism.