Possible Involvement of a Low Redox Potential Component(s) Downstream of Photosystem I in the Translational Regulation of the D1 Subunit of the Photosystem II Reaction Center in Isolated Pea Chloroplasts

Accumulation of the precursor and the mature form of the D1protein of the photosystem II reaction center in illuminatedpea chloroplasts was prevented by the addition of the inhibitorsatrazine, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 3,5-dibromo-4-hydroxybenzonitrile.Under such conditions, the compensatory accumulation of twotranslational intermediates of the D1 protein, of 22 and 24kDa, respectively, was induced by the addition of ATP, as alsoobserved in darkness in the presence of ATP [Taniguchi et al.(1993) FEBS Lett. 317: 57], suggesting that the synthesis ofthe full-length D1 protein requires a factor that is generatedby the operation of photosynthetic electron transport. The accumulationof the full-length Dl protein was induced in the light, evenin the presence of atrazine, when both 2,6-dichlorophenolindophenoland ascorbate were also present and in darkness upon the additionof dithiothreitol. Moreover, reagents with a relatively lowredox potential, namely, duroquinone and methylviologen, preventedthe accumulation. These observations suggest that the translationof the D1 protein might be regulated at specific steps duringthe elongation of the polypeptide via a redox change in a componentaround photosystem I. Results of pre-illumination experimentsindicate that the factor needed for the accumulation of D1 proteinis relatively stable and retains its activity in darkness afterexposure to light. (Received April 18, 1996; Accepted June 6, 1996)     

Light-Dependent Uptake of Pyruvate by Mesophyll Chloroplasts of a C4 Plant, Panicum miliaceum L.

Light-dependent active uptake of pyruvate was reported in mesophyllchloroplasts of a C₄ plant, Panicum miliaceum [Ohnishi and Kanai(1987) Plant Cell Physiol. 28: 1]. The present study tried toclarify the energy source of this active uptake. Preilluminationof the mesophyll chloroplasts increased over tenfold their pyruvateuptake in the light and dark. This indicates that light itselfis not essential for the enhancement. The pyruvate uptake capacity(the initial uptake rate) of the mesophyll chloroplasts increasedon illumination and reached a steady-state level after a fewminutes; this rise was faster under higher light intensities.When the chloroplasts were returned to darkness, the uptakecapacity decayed with a half-life of about 1 min; this was independentof the light intensity of preillumination. Illumination of thechloroplasts also increased the stromal pH from about 7 to 8and the stromal ATP level from about 5 to 15–25 nmol.(mg chl)^–1. The change of the former during dark-to-lightand light-to-dark transitions occurred within 2 to 5 min, whilethe change of the latter took place much faster within 1 min.The steady-state levels of the pyruvate uptake capacity andstromal pH were saturated at a light intensity of 3 µE.m^–2.s^–1,while the ATP level increased with a further increase in thelight intensity. The former two parameters also showed similarsensitivity to the inhibition by carbonylcyanide-m-chlorophenylhydrazone,while a higher concentration of the inhibitor was needed toreduce the ATP level. Nitrite at 4 mM inhibited the light-dependentpyruvate uptake and stromal alkalization but had little effecton the stromal ATP level, while 2 mM arsenate decreased thestromal ATP without significant effects on pyruvate uptake andstromal pH. The good correlation of pyruvate uptake and stromalpH suggests that the active pyruvate uptake by the mesophyllchloroplasts is primarily driven by the pH gradient across theenvelope. (Received August 15, 1986; Accepted December 8, 1986)     

Synthetic abilities of Euglena chloroplasts in darkness

Protein synthesis, normally a light-dependent process in isolated mature chloroplasts of Euglena gracilis var. bacillaris will take place in darkness if ATP and Mg2+ (ATP/Mg) are supplied. Either 5 or 10 mM ATP plus 15 mM MgCl2 are optimal and rates equal to those in the light can be obtained. Since ATP and Mg2+ are not stoichiometrically related, and since the optimal Mg2+ concentration is similar to that which stabilizes chloroplast ribosomes in vitro, it is suggested that the chloroplast is freely permeable to Mg2+ under these conditions. Protein synthesis under these conditions is not inhibited appreciably by DCMU, FCCP, cycloheximide, or by the addition of ribonuclease, but is highly sensitive to chloramphenicol. Carbon dioxide fixation is also a light-dependent process in isolated mature chloroplasts from Euglena, but addition of ATP (5 mM) and fructose bisphosphate (5 mM) plus aldolase (1.0 unit/ml) (fructose-1,6-bisphosphate/aldolase) yields CO2 fixation rates in darkness that are 43% of those normally obtained in the light. Mg2+ higher than 1.0 mM (e.g., 16 mM) is somewhat inhibitory. Chlorophyll synthesis from 5-aminolevulinate in 36 h developing chloroplasts from Euglena is also light-dependent, but addition of ATP/Mg and fructose-1,6-bis-phosphate/aldolase in darkness brings about the accumulation of a compound having the same RF on chromatography as protochlorophyllide from Barley; a subsequent brief illumination of the chloroplasts converts this compound to a compound with the RF of chlorophyll. Thus Euglena chloroplasts supplied with appropriate additions can carry out protein synthesis, carbon dioxide fixation and most of chlorophyll synthesis in darkness. This versatility is appropriate in photosynthetic organelles isolated from photo-organotrophic cells.     

Synthesis of ribulose 1,5-bisphosphate carboxylase by isolatedSorghum mesophyll chloroplasts

Chloroplasts isolated fromSorghum vulgare are active in light-dependent, organelle protein synthesis. Intact chloroplasts can use light as an energy source; photosynthetically inactive chloroplasts require the addition of ATP for this protein synthesis. Preincubation of chloroplasts in light at 25°C for 1 h depleted the endogenous templates completely; such preincubated chloroplasts translated exogenously added heterologous templates efficiently. When total cellular RNA fromChlorella protothecoides, a C₃ plant, was used as template for translation in a cell-free light-dependent system of isolated mesophyll chloroplasts fromSorghum vulgare, a C₄ type plant, polypeptides of 55 kDa (large subunit) and 15 kDa (small subunit) were detectable in the fluorographic profile of the newly synthesized proteins; these polypeptides were absent in the products obtained with endogenous RNA. Evidence for the fidelity of the system was obtained by immunological analysis of ribulose 1, 5-bisphosphate carboxylase obtained by the translation ofChlorella cellular RNAs.     

Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [(35)S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.     

Vieillissement de l'appareil photosynth?tique I. Effet synergique de la lumi?re et du vieillissement in vitro sur les changements de volume de chloroplastes isol?s d'?pinard

In view of the difficulty of studying all of the aspects ofageing of the photosynthetic apparatus in vivo and of the actionof light upon this phenomenon, the consequences of chloroplastageing were investigated in vitro, especially on the volumeof these organelles. It was found that at 20?C in a Tris-NaCl medium, chloroplastswelling occurred slowly in the dark for the 6 hr experimentalperiod, while it was activated in the light, with an optimumafter 90 min. The most characteristic feature of these 2 curvesis that they cross each other after 5 hr, indicating that, independentof the previous treatments (dark or light), the chloroplastsin vitro reach the same volume after a few hours. Special attentionwas devoted to the pH effect. In dark and light conditions,swelling was found to be maximum at pH 7.5 to 8.5 and was independentof the buffer used. There was no major difference between swellingin Tris and Tricine. A marked depression in the chloroplastvolume was observed at pH 4.5 in the dark and at pH 5.5 in thelight. Simultaneous with the swelling phenomenon, the capacity of chloroplaststo carry out light-dependent shrinkage diminished. There wasa striking parallelism between swelling rate and loss of chloroplastshrinkage capacity. The action of light is synergetic on boththese processes in that it accelerated to the same extent boththe dark-swelling and the inhibition of light-dependent shrinkingin chloroplasts incubated in the dark. (Received May 24, 1969; )     

A point mutation in atpC1 raises the redox potential of the Arabidopsis chloroplast ATP synthase gamma-subunit regulatory disulfide above the range of thioredoxin modulation

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the gamma-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the Em,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the gamma-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.     

Light-dependent regulation of the synthesis of soluble and intracytoplasmic membrane proteins of Rhodopseudomonas sphaeroides.

Cells of Rhodopseudomonas sphaeroides grown under saturating light conditions (30 W/m2) and then shifted to low light intensity (3 W/m2) required 2.5 h to adapt to the new lower light conditions. After the shift, cell growth, whole cell protein accumulation, and bacteriochlorophyll accumulation ceased immediately. Approximately midway into the adaptation period, bacteriochlorophyll synthesis commenced at a new, higher rate, which continued through the beginning of the low-light growth period until new steady-state levels were reached. Immediately after the downshift, the rate of cellular protein synthesis declined to 22% of its preshift rate. Pulse-labeling of protein throughout the adaptation period and comparison with a steady-state prelabel culture revealed that synthesis of two of the three light-harvesting proteins, as well as two additional high-molecular-weight photosynthetic membrane proteins, was derepressed three- to fivefold compared with bulk cellular protein. Finally, the synthesis of at least three soluble proteins showed light-dependent regulation after the light downshift. These results are discussed in terms of the light-dependent regulation of synthesis of the photosynthetic membrane macromolecular components and the division of protein synthesis between the photosynthetic membranes and the soluble cell phase.     

Requirements for the light-stimulated degradation of stromal proteins in isolated pea (Pisum sativum L.) chloroplasts

Chloroplasts from 17-d-old pea leaves (Pisum sativum L.) wereisolated to elucidate the requirements for the light-induceddegradation of stromal proteins. The influence of electron transportthrough the thylakoids and the influence of ATP on protein degradationwere investigated. When chloroplasts were incubated in the light(45 µmol m^–2s^–1), glutamine synthetase, thelarge subunit of ribulose-1,5-bisphosphate carboxylase and glutamatesynthase were degraded, whereas phosphoribulokinase, ferredoxin-NADP⁺reductase and the 33 kDa protein of photosystem II remainedmore stable. Major protein degradation was not observed over240 mm in darkness. The electron transport inhibitor dichlorophenyldimethylureareduced protein degradation in the light over several hours,whereas dibromothymoquinone was less effective. Inhibiting theproduction of ATP with tentoxin or by destroying the     

Comparison of the metabolic properties of plastids isolated from developing leaves or embryos of Brassica napus L

Plastids isolated from developing leaves and embryos of oilseed rape (Brassica napus L.) were incubated with substrates in the light or the dark, with or without exogenous ATP. Incorporation of HCO^-3, and carbon from a range of substrates into fatty acids and/or starch by leaf chloroplasts was absolutely light-dependent and was unaffected by provision of ATP. Incorporation of HCO^-3 into fatty acids and/or starch by embryo plastids was also light-dependent. However, the light-dependent rates attained, when expressed on a comparable basis, were less than 32% of those from Glc6P (plus ATP), which was the most effective substrate for starch and fatty acid synthesis. In the light alone the rates of carbon incorporation from Glc6P, pyruvate and acetate into fatty acids, and from Glc6P into starch by embryo plastids were less than 27% of the respective ATP-dependent (dark) rates. Light had no effect on these ATP-dependent rates of synthesis by embryo plastids. While transporter activities for both glucose and Glc6P were present in embryo plastids, leaf chloroplasts did not have the latter activity. It is concluded that light at in vivo levels can contribute energy to carbon metabolism in embryo plastids. However, this contribution is likely to be small and these plastids are therefore largely dependent upon interaction with the cytosol for the ATP, reducing power and carbon precursors that are required for maximal rates of starch and fatty acid synthesis.     

Coregulation of electron transport and Benson-Calvin cycle activity in isolated spinach chloroplasts: studies on glycerate 3-phosphate reduction

Glycerate 3-phosphate-dependent O2 evolution was measured in intact chloroplasts in the absence of CO2. At all concentrations of added glycerate 3-phosphate oxygen evolution ceased before stoichiometric amounts of oxygen were evolved. The inhibition of glycerate 3-phosphate-dependent-O2 evolution increased with increasing concentrations of substrate added. A similar response was observed in chloroplasts treated with KCN which inhibits ribulose-1,5-bisphosphate carboxylase-oxygenase. Oxygen uptake via the oxygenase activity of this enzyme is therefore not the cause of the discrepancy in stoichiometry of oxygen release in this system. The addition of NaHCO3 to chloroplasts in which oxygen evolution was inhibited by glycerate 3-phosphate caused an immediate sustained rate of oxygen evolution in the absence of KCN but not with KCN present. Simultaneous measurements of chlorophyll a fluorescence showed that qQ remained oxidized, although net O2 evolution had ceased. As O2 evolution decreased, qE and delta pH increased. Upon the addition of the NaHCO3, QA became more oxidized while delta pH and qE were decreased, suggesting that the inhibition of electron transport at high glycerate 3-phosphate concentrations was mediated by photosynthetic control via delta pH. However, the levels of ATP, ADP, ribulose 1,5-bisphosphate, and Pi concentrations and ATP/ADP ratio. The stromal glycerate 3-phosphate content declined upon illumination until O2 evolution ceased. At this time a constant stromal glycerate 3-phosphate concentration of 8-10 mM was maintained while net import of glycerate 3-phosphate into the stroma had virtually ceased. The stromal triosephosphate content remained at a constant low level throughout but the glycerate 3-phosphate level increased slightly after addition of NaHCO3. The data provided by the measurements of thylakoid reactions and stromal metabolites suggest that photosynthetic electron transport is tightly coupled to the requirements of the stroma for ATP and NADPH. Glycerate 3-phosphate reduction requires much less ATP than the operation of the complete Benson-Calvin cycle since the stoichiometry of ATP and NADPH utilization is reduced to 1:1. We conclude that thylakoid electron flow is not sufficiently flexible to maintain NADPH and ATP production in the ratio of 1:1. This situation will favor overenergization of the thylakoid membrane, increased leakiness of protons, increased electron drainage to O2, and result in progressive inhibition of noncyclic electron flow.     

Isolation of biochemically active chloroplasts from chlamydomonas

Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits.The isolated chloroplasts are active in photosynthetic O₂-evolution and CO₂-fixation, with the highest rates obtained in the presence of ATP.The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.     

Properties of the Signal Transduction Pathway in the Blue Light Response of Stomatal Guard Cells of Vicia faba and Commelina benghalensis

Blue light-dependent proton extrusion in guard cell protoplastsfrom Vicia faba and light-dependent stomatal opening in theepidermis of Commelina benghalensis are inhibited by the calmodulin(CaM) antagonist, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfononamide(W-7) and the myosin light chain kinase (MLCK) inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine (ML-7) [Shimazaki, K., Kinoshita, T.and Nishimura, M. (1992) Plant Physiol. 99: 1416]. We now suggestthat the inhibition occurs in the blue light signaling pathwaywithout affecting the proton pump. Addition of fusicoccin (FC),an activator of H⁺-ATPase, to the protoplasts and the epidermiswhose blue light-dependent proton extrusion and light-dependentstomatal opening had been inhibited by W-7 and ML-7, inducedboth proton extrusion and stomatal opening, respectively. Bluelight-dependent proton extrusion was inhibited by K-252a, awide-range inhibitor of protein kinases, and KT5926, a selectiveinhibitor of MLCK. FC induced proton extrusion in the presenceof K-252a and KT5926. In contrast, phenylmercuric acetate (PMA),carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) inhibited both the proton extrusion and stomatal opening,but FC did not induce the responses. These results suggest thatW-7, ML-7, K-252a and KT5926 inhibit the signal transductionprocess by which the perception of blue light is transducedinto activation of the proton pump in guard cells, and thatMLCK or MLCK-like protein is involved in the blue light responseof stomata. The possibility that calcium-dependent, calmodulinindependent protein kinase [Harper, J.F. et al. (1991) Science252: 951] functions rather than MLCK in the blue light responseof stomata should be noted, however. (Received July 23, 1993; Accepted September 30, 1993)     

Energy Supply for ATP-Synthase Deficient Chloroplasts of Chlamydomonas reinhardii

The mutant F54 of the unicellular green alga Chlamydomonas reinhardiiis not able to perform photophos-phorylation. Nevertheless,it grows on acetate and the chloroplasts accomplish most oftheir energy-requiring synthetic processes. However, no light-dependentchloroplast protein synthesis could be detected in intact F54chloroplasts isolated from a cell wall-deficient double mutantF54-cw-15. Exogenous ATP was not able to induce this in organelloprotein synthesis to an appreciable degree. In contrast, thestrictly ATP-dependent protein synthesis was stimulated veryefficiently by glyceraldehyde-3-phosphate, dihydroxy-acetonephosphate and glycerol-3-phosphate, but strongly inhibited by3-phosphoglycerate. These compounds can be transported acrossthe envelope membrane by the triose phosphate translocator.Pyridoxal phosphate, a specific inhibitor of the translocator,abolished the stimulation by triose phosphates. Spermidine,which activates initiation of translation in chloroplasts, enhancedtriose phosphate-stimulated protein synthesis even further.In the dark, no stimulation was observed, indicating that alight-dependent reaction was also involved in this kind of ATPproduction in chloroplasts. The results suggest that chloroplastsdefective in photophosphorylation recruit their energy via anATP shuttle which was shown in this study to import rather thanexport ATP across the chloroplast envelope. (Received August 21, 1997; Accepted November 18, 1997)     

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D-threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.     

Regulation of proton leakage from broken chloroplasts by CF0.

Measurements of proton translocation in CF₁-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF₀ is still dependent on light intensity with a first-order rate constant equal to mR₀, where R₀ is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF₀ is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF₁.CF₀ complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.     

The mechanism of the control of carbon fixation by the pH in the chloroplast stroma

The salts of several weak acids have been used to render the envelope permeable to protons. In order to investigate the role of stromal pH changes in the light regulation of CO₂ fixation, formate, octanoate, nitrite, and glyoxylate have been tried as tools to reverse the light-dependent alkalization of the stroma. For this purpose, the decrease of the stromal pH in illuminated spinach chloroplasts, as caused by the addition of these substances or by instantaneous lowering of the pH in the medium, has been compared with the corresponding decrease of CO₂ fixation and the change of stromal metabolite levels. It appears from out data that formate and octanoate are suited best to obtain a specific inhibition of CO₂ fixation by lowering the stromal pH. The measurement of the corresponding metabolite levels indicates that this inhibition is primarily due to an inhibition of fructose- and sedoheptulose bisphosphatase. It is concluded that these two enzymes are important regulatory steps for the light control of CO₂ fixation.     

Enhancement of protein synthesis in isolated chloroplasts by irradiation of fern gametophytes with blue light

Irradiation of the gametophytes of Pteridium aquilinum with blue light led to a nearly 5-fold increase in the amino acid-incorporating activity of isolated chloroplasts. The blue light effect was not due to increased synthesis of ATP or other energy donors by the chloroplasts but was probably related to an increased production of chlorophyll and photosynthetic enzymes.