Isolated Pea Thylakoids Research Articles

Picosecond time-resolved study on the nature of high-energy-state quenching in isolated pea thylakoids different localization of zeaxanthin dependent and independent quenching mechanisms

The influence of the transthylakoid proton gradient on the kinetics of picosecond fluorescence decay was examined using isolated pea thylakoids having high or low zeaxanthin contents. Fluorescence lifetime measurements were performed with open (Fo) and closed (Fm) PS II reaction centers. Zeaxanthin formation in membrane energized isolated thylakoids led to a marked decrease of the average fluorescence lifetime at both Fm and Fo. In contrast, when zeaxanthin synthesis was blocked by the inhibitor DTT, the fluorescence lifetime decrease was less pronounced in the Fm state and totally missing in the Fo state. Samples containing the uncoupler ammonium chloride did not exhinit any zeaxanthin influence on the fluorescence decay kinetics. By detailed kinetic analysis of the fluorescence data based on the exciton/radical pair equilibrium model it was possible to separately locate and quantify the effects of zeaxanthin, on the one hand, and the proton gradient, on the other hand, in terms of rate constants of individual primary processes within PS II. It is shown that the enhanced non-photochemical fluorescence quenching (NPQ) in the presence of zeaxanthin mainly originates in the antenna, while without zeaxanthin smaller changes in the Fm state are owing to changes in processes located at the reaction center. Possible mechanisms of zeaxanthin dependent and independent nonphotochemical fluorescence quenching in open and closed PS II are discussed.

Effects of Potassium-(picrate)-(18-crown-6) on the Photosynthetic Electron Transport

Abstract The effects of potassium-(picrate)-(18-crown-6) on the electron transport of photosystem II was investigated in isolated pea thylakoids. Low concentrations of the compound inhibited the fast decay of fluorescence yield associated with electron transfer between the primary (QA) and secondary (QB) quinone electron acceptor and increased the intermediary level of fluorescence to the Fmax level. The decay half-time of fluorescence yield measured in the presence of DCMU(S2QA - charge recombination) decreased from about 1.8 s to about 0.3 s in thylakoids treated with potassium-(picrate)-(18-crown-6). While the inhibition of electron transport by DCMU gave rise to the appearance of a thermoluminescence band at about + 10°C (S2QA - charge recombination) addition of potassium-(picrate)-(18-crown-6) resulted in a thermoluminescence band at about -10°C. Increasing concentrations of potassium-(picrate)-(18-crown-6) diminished the fluorescence yield and the -10°C TL band and abolished the Signal IIS and Signal IIf EPR signals of the tyrosine-D and tyrosine-Z electron donors, respectively. The phenolic-type inhibitor, potassium picrate had the same effect on thermoluminescence and on the tyrosine EPR signals. It is concluded that potassium-(picrate)-(18-crown-6) is a phenolic type inhibitor owing to its picrate constituent. At low concentrations picrate and potassium-(picrate)-18-crown) not only block the electron transport between QA and QB but they probably decrease the midpoint redox potential of QA, as well. At high concentrations they also inhibit the light-induced oxidation of the tyrosine-D and tyrosine-Z donors.

CytochromefEncoded by the Chloroplast Genome Is Imported into Thylakoids via the SecA-Dependent Pathway

In vitroimport of the precursor of tobacco cytochromef,which is is encoded by the chloroplast genome, into isolated pea thylakoids was analyzed. Upon incubation with thylakoids and a stromal fraction, the precursor of cytochromefwas efficiently imported into thylakoids. The imported cytochromefwas tightly integrated into the thylakoid membrane. Insertion of cytochromefinto the thylakoid membrane was blocked by nigericin, sodium azide, and antibodies against pea chloroplast SecA. These results suggest that cytochromefutilizes the bacterial-type SecA-dependent pathway to be imported into thylakoids.

The role of phospholipids in regulating photosynthetic electron transport activities: Treatment of thylakoids with phospholipase C

The involvement of phospholipids in the regulation of photosynthetic electron transport activities was studied by incubating isolated pea thylakoids with phospholipase C to remove the head-group of phospholipid molecules. The treatment was effective in eliminating 40-50% of chloroplast phospholipids and resulted in a drastic decrease of photosynthetic electron transport. Measurements of whole electron transport (H2O→methylviologen) and Photosystem II activity (H2O→p-benzoquinone) demonstrated that the decrease of electron flow was due to the inactivation of Photosystem II centers. The variable part of fluorescence induction measured in the absence of electron acceptor was decreased by the progress of phospholipase C hydrolysis and part of the signal could be restored on addition of 3-(3',4'-dicholorophenyl)-1,1-dimethylurea. The B and Q bands of thermoluminescence corresponding to S2S3QB (-) and S2S3QA (-) charge recombination, respectively, was also decreased with a concomitant increase of the C band, which originated from the tyrosine D(+)QA (-) charge recombination. These results suggest that phospholipid molecules play an important role in maintaining the membrane organization and thus maintaining the electron transport activity of Photosystem II complexes.

The △pH-driven, ATP-independent Protein Translocation Mechanism in the Chloroplast Thylakoid Membrane: KINETICS AND ENERGETICS

Previous studies have shown that proteins are transported across the chloroplast thylakoid membrane by two very different mechanisms, one of which requires stromal factors and ATP, whereas the other mechanism is ATP independent but completely reliant on the thylakoidal delta pH. We have examined the role of the delta pH in the latter mechanism by simultaneously monitoring the magnitude of delta pH (by 9-aminoacridine fluorescence quenching) and the rate of import of the 23-kDa photosystem II protein into isolated pea thylakoids. We show that protein import can take place, at low but significant rates, at very low values of delta pH (in the region of 1.2-1.4), and that plots of the rate of protein import against proton concentration gradient are probably hyperbolic in nature. There is no evidence for a threshold level of delta pH which is required to drive translocation of the 23-kDa protein. Addition of uncouplers midway during import incubations results in a rapid and complete inhibition of translocation, showing that the continuous presence of the delta pH is required for translocation to take place. During import into intact chloroplasts, the intermediate-size 23-kDa protein substrate for the thylakoidal protein transport machinery is found only in the stromal fraction at all values of delta pH, suggesting that the initial interaction with the machinery is relatively weak, reversible and delta pH-independent. We therefore propose that the delta pH is required for both the initiation and completion of translocation; these roles are in marked contrast to the roles of protonmotive force in mitochondrial and sec-dependent bacterial protein transport.

Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimisation of in vitro assays.

In vitro assays for the import of proteins by isolated pea thylakoids have been refined and optimised with respect to (a) the method of thylakoid preparation, (b) the concentration of thylakoids in the import assay, and (c) the pH and temperature of the import assay. As a result, the 23 kDa and 16 kDa proteins of the photosynthetic oxygen-evolving complex are imported with efficiencies approaching 100%; import of the third oxygen-evolving complex protein is also observed, albeit with lower efficiencies. We have also demonstrated import of three further thylakoid proteins: plastocyanin, the CFoII subunit of the ATP synthase, and the photosystem I subunit, PSI-N, using this import assay. Import of plastocyanin, PSI-N and the 33 kDa oxygen-evolving complex protein subunit requires the presence of stromal extract whereas the other three proteins are efficiently imported in the absence of added soluble proteins. Import into isolated barley thylakoids was achieved under identical assay conditions, although with somewhat lower efficiency than into pea thylakoids.

Phosphopeptides as Substrates for Thylakoid Protein Phosphatase Activity

Lack of a suitable substrate has been a major obstacle in studying the chloroplastic thylakoid membrane protein phosphatase activity. In this study, the suitability of synthetic phosphopeptides for this purpose was investigated. Phosphothreonine-containing phosphopeptides mimicking the N-terminal phosphorylation site of the major thylakoid phosphoprotein, the light-harvesting chlorophyll a/ b-binding protein (LHCP-II), were dephosphorylated by isolated pea thylakoid membranes. Phosphopeptides representing unrelated sequences or in which the target phosphothreonine had been changed to a phosphoserine were not dephosphorylated. The dephosphorylation of phosphopeptides by thylakoid membranes was similar to the dephosphorylation of endogenous LHCP-II in its pH-dependence profile, sensitivity to inhibitors, and bivalent cation requirement. The same phosphopeptide analogs of the LHCP-II phosphorylation site inhibited endogenous LHCP-II dephosphorylation in isolated thylakoids, whereas the dephospho-analogs and nonsubstrate phosphopeptides had no effect. Collectively, these results suggest that phosphopeptides mimicking a thylakoid phosphoprotein dephosphorylation site can be exploited for further study of the thylakoid protein phosphatase activity.

Integration of a cyanobacterial protein involved in nitrate reduction (narB) into isolated Synechococcus but not into pea thylakoid membranes.

Chimeric genes comprised of Rubisco small subunit transit peptide fused in frame with full-length and truncated sequences of a nitrate reductase (narB) structural gene of Synechococcus were constructed. Fusion proteins were synthesized in a rabbit reticulocyte system. In thylakoido integration of synthetic proteins resulted in the association of the full-length narB-coded protein to the Synechococcus photosynthetic membranes. The membrane-associated protein was sensitive to trypsin treatment but could not be removed by washing in the presence of NaBr. Trypsin pretreatment of thylakoids abolished the capability for association. The association of the narB-coded protein with thylakoids might require another membrane protein whose identity is not known. It is proposed that the Synechococcus narB polypeptide is a peripheral, membrane bound protein anchored to the thylakoids via a short hydrophobic domain while the major part of the protein resides on the outer side of the thylakoid membranes. The chimeric narB proteins were processed and imported by intact pea chloroplasts in vitro; however, the mature proteins were found localized in the stroma and not in the thylakoid membrane fraction. Similarly, the attempt to integrate the protein in vitro into isolated pea thylakoid membranes failed although these membranes incorporate early light-inducible proteins.

Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.

Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol and targeted into the thylakoid lumen by bipartite presequences. In this report, we describe conditions for the efficient import of each of these proteins by isolated pea thylakoids. Import of the 33-kDa protein requires both light and stromal extract. The probable function of the stromal extract is to provide stromal processing peptidase to remove the first "envelope transit" signal of the presequence. Import of the 23-kDa protein is also driven by light, but stromal extract is not required for import; furthermore, efficient import is still observed if the precursor is modified to completely block cleavage by residual stromal processing peptidase activity. The intermediate form of the 23-kDa protein, which is generated by incubation of the precursor protein with stromal processing peptidase, is also efficiently imported. The results indicate that the thylakoidal protein transport system can import both the precursor and intermediate forms of the 23-kDa protein, but probably only the intermediate form of the 33-kDa protein.

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.

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.

The kinetics of adenine nucleotide binding to chloroplast ATPase, CF 0-CF 1, during the illumination and post illumination periodi in isolated pea thylakoids

The steady-state binding of [2- 3H]ADP to thylakoid membrane protonmotive ATPase (CF 0-CF 1) was studied using a quench technique similar to that developed by Strotmann, Bickel-Sandkotter and Shoshan (FEBS Lett. 101 (1979) 316–320). The amount of adenine nucleotide bound in the light and post-illumination dark period is shown to depend on the efficaency of uncoupling when the reaction is quenched. Previous observations that post-illumination binding of [2- 3H]ADP displays a fast and a slow phase of binding were confirmed only when uncoupling efficiency was relatively low during quenching. When uncoupling efficiency is increased, the fast phase of dark [2- 3H]ADP binding is abolished, and correspondingly more nucleotide appears to be bound upon quenching in the light. The previous assignment of the observed kinetic phases to different species of enzyme-nucleotide complex has been reassessed in the light of this new data.

The effect of lipid saturation on membrane structure and function in isolated pea thylakoids

Traditionally, investigations into the role of different lipid classes in supporting the function of membrane proteins in chloroplasts have adopted two approaches, namely reconstituted protein-lipid systems (e.g. Van Walraven el ul., 1984) and selective removal of lipids from the membrane by exogenous lipases (e.g. Rawyler & Siegenthaler, 1981; Jordan et al., 1983). However, both of these approaches have serious limitations (Thomas et al., 1985). This study presents an alternative method which selectively modifies the degree of saturation of membrane lipids in isolated pea thylakoids using a water-soluble palladium( I I ) sulphonated alizarine catalyst (Vigh et ul., 1985). The effect of lipid saturation on membrane organization and stability to hightemperature stress was subsequently investigated. Gas-liquid chromatography analysis of the fatty acid composition of pea thylakoids showed a marked reduction of the C-18 double bond index from 5.2 to 1 .1 by a 30 min catalytic hydrogenation. Freeze-fracture electron micrographs of control and hydrogenated thylakoids showed that a decrease in the degree of saturation of membrane lipids resulted in the appearance of some gel-phase-separated lipid. Differential scanning calorimetry of thylakoids monitors membrane endothermic transitions that have been proposed to correspond to the breakdown or dissociation of protein complexes (Cramer et al., 1981). Hydrogenation of thylakoid lipids resulted in an increased stability of membrane protein complexes to heat stress, displacing the threshold of the breakdown/ dissociation temperature from 30 to 40 C. The effect of hydrogenation upon the stability of membrane protein complexes to heat stress was investigated by monitoring Photosystem I 1 (PS 11) activity with chlorophyll fluorescence kinetics. Fig. 1 presents the effect of 5 min thermal incubation on the PS 11 activity of control and hydrogenated thylakoids. Clearly hydrogenation results in an increased stability to heat stress, confirming the calorimetry results presented above. Catalytic hydrogenation of thylakoid membranes produces a modification of lipid saturation in vivo and provides a useful tool by which lipid-protein interactions may be studied. In this investigation reducing the C-I8 double bond index from 5.2 to 1 . 1 resulted in gel-phase separation of some lipids. This was accompanied by an increased stability of the protein complexes, and in par-

Light-dependent degradation of the QB -protein in isolated pea thylakoids

The 32 000-dalton Q(B)-protein of photosystem II (PS II) is rapidly damaged and removed from isolated pea thylakoids during incubation in the light resulting in a loss of photosynthetic electron flow through PS II. This in vitro photoinhibition is similar to that previously reported with intact Chlamydomonas cells. The damage occurs at a faster rate in vitro, however, due to the inability of isolated thylakoids to synthesize replacement Q(B)-protein. The removal of the damaged Q(B)-protein does not require any soluble components of the chloroplast stroma and is unaffected by the protease inhibitors phenyl-methylsulfonylfluoride or antipain. Unlike the effect of trypsin, no low mol. wt. membrane-bound or soluble fragments of the labelled Q(B)-protein could be identified either by autoradiography or immunologically using polyclonal antibodies specific for the Q(B)-protein. The lightinduced damage to the Q(B)-protein (indicated by a loss of Q(B) functional activity), preceded the removal of the protein from the membrane. We conclude that photodamage of the Q(B)-protein generates a conformational change which renders the protein susceptible to attack by a highly efficient, intrinsic membrane protease.

Influence of structural and physical properties of the thylakoid membrane on QA - oxidation

Using isolated pea thylakoids, the relative rate of QA (-) oxidation has been estimated under various conditions, from the restoration of the induction curves following a dark period and from light 1-induced changes in modulated chlorophyll fluorescence excited by light 2.Alterations of QinfA (sup-) oxidation rates were observed under conditions which affected the degree of thylakoid stacking, the lipid fluidity and the integrity of the membranes. The results are discussed in terms of the interactions between QA (-) and the plastoquinone pool with particular emphasis on lateral diffusion.

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Analysis of chlorophyll fluorescence quenching by DBMIB as a means of investigating the consequences of thylakoid membrane phosphorylation

The effect of Mg 2+ concentration and phosphorylation of the light harvesting chlorophyll a b protein on the ability of DBMIB to quench chlorophyll fluorescence of isolated pea thylakoids has been studied. Over a wide range of Mg 2+ concentrations (5−0.33 mM), the observed changes in fluorescence yield are mirrored by similar changes in the quenching ability of DBMIB, indicating that the cation-induced phenomenon involves alterations in radiative lifetimes. In contrast, phosphorylation at 10 mM Mg 2+ brings about a lowering of the chlorophyll fluorescence yield, while having no effect on the quenching capacity of DBMIB. This result can be interpreted as a phosphorylation-induced decrease in PS II absorption cross-section. At Mg 2+ levels between 5 and 1 mM, phosphorylation leads to a change in the quenching of fluorescence by DBMIB, when compared with non-phosphorylated thylakoids. At these cation levels, the degree of DBMIB-induced quenching cannot wholly account for the observed changes in chlorophyll fluorescence due to phosphorylation. It is concluded that the phosphorylation- and Mg 2+-induced changes in fluorescence yield are independent but inter-related processes which involve surface charge screening as emphasised by the change in cation sensitivity of the DBMIB quenching before and after phosphorylation.

Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll [formula omitted] phosphorylation

The effect of Mg 2+ concentration and phosphorylation of light-harvesting chlorophyll a b- protein on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg 2+ concentration from 3 to 0.4 mM decreases only the variable fluorescence ( F v) and the area above the induction curve while at the same time increasing the slow exponential component of the rise ( β max). (2) A further decrease in Mg 2+ concentration from 0.4 to 0 mM decreases the initial ( F 0) fluorescence level such that the ratio F v F m increases slightly as does the area above the induction curve and β max. (3) Thylakoid membranes, phosphorylated at 5 mM Mg 2+, show an equal decrease in F v and F 0, no change in the area above the induction curve and an increase in β max. At 2 mM Mg 2+, however, phosphorylation induced a more extensive quenching of F v so that the F v F m ratio was lowered and the area above the induction curve decreased while β max increased. (4) When phosphorylated membranes were subsequently suspended in an Mg 2+-free medium the effect on F 0 due to phosphorylation was found to be additive to that due to the absence of Mg 2+. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg 2+ levels. At high Mg 2+ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg 2+ levels there is an additional effect of increased spillover from Photosystem II to I.

Oligomycin Effects on ATPase and Photophosphorylation of Pea Chloroplast Thylakoid Membranes

Oligomycin inhibited the membrane-bound, Ca(2+)-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg(2+)-dependent (light- and dithiothreitol (DTT)-activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition.Sensitivity of isolated pea thylakoid Mg(2+)-dependent ATPase to oligomycin was found during summer months but not during the winter. This was traced to a seasonal variation in relative humidity with values of 20% or less during the winter, leading to lack of inhibition in the isolated chloroplasts. The effect of low relative humidity during growth could be reversed by placing plants for 17 to 20 hours in a chamber at 50% relative humidity. The optimum humidity range for sensitivity to oligomycin was between 40 and 60%.With chloroplasts potentially sensitive to oligomycin due to "permissive" growth conditions, the inhibition was modulated by a number of parameters during chloroplast activation or ATPase assay. Preincubation in high concentrations of KCl diminished oligomycin sensitivity, and this effect was reversed by valinomycin. The greatest sensitivity, as well as the highest ATPase rates, occurred only if thylakoids were activated by light and DTT at a relatively high temperature (35 to 40 degrees C). Controlling the levels of uncouplers and running the reaction in darkness rather than in the light, both of which diminished the degree of membrane energization, increased the sensitivity to oligomycin. It is possible that accessibility of the oligomycin binding site is diminished by membrane energization. These results are contrasted with those from studies of variability in sensitivity of yeast mitochondria to oligomycin.