Uncoupling Of Photophosphorylation Research Articles

The utilization of photophosphorylation uncoupler to improve lipid production of Chlorella, a case study using transcriptome and functional gene expression analysis to reveal its mechanism

Uncoupling of photophosphorylation appears to have positive effect on lipid production of microalgae, however, underlying mechanisms involved in the effect are still unresolved. In this study, an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), was used for the promotion of microalgal lipid accumulation. The results indicated that addition of CCCP was able to enhance the lipid productions of all tested species of Chlorella, especially when cultivated under mixotrophic condition. Based on the transcriptome and functional gene expression analyses, the functional genes involving in photosynthetic electron transport (PET), fatty acids synthesis, tricarboxylic acid (TCA) cycle, glycolysis and pentose phosphate (PP) pathway were found to be up-regulated while the genes participating in photosystem II, ATP synthesis during photosynthesis and calvin cycle were to be down-regulated by the presence of CCCP. According to these observations, the mechanism of CCCP to the promotion of lipid synthesis probably through channeling the NAD(P)H metabolic fluxes from carbon fixation towards fatty acid biosynthesis and lipid production, which was accomplished by (1) down-regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase genes that involving in CO2 fixation via calvin cycle to reduce the NADPH consumption; (2) up-regulation of genes involved in TCA cycle and PP pathway to produce more NAD(P)H. The results of this study provide a new method to promote microalgal lipid production.

Quantification of an Adverse Outcome Pathway Network by Bayesian Regression and Bayesian Network Modeling.

ABSTRACTThe adverse outcome pathway (AOP) framework has gained international recognition as a systematic approach linking mechanistic processes to toxicity endpoints. Nevertheless, successful implementation into risk assessments is still limited by the lack of quantitative AOP models (qAOPs) and assessment of uncertainties. The few published qAOP models so far are typically based on data‐demanding systems biology models. Here, we propose a less data‐demanding approach for quantification of AOPs and AOP networks, based on regression modeling and Bayesian networks (BNs). We demonstrate this approach with the proposed AOP #245, “Uncoupling of photophosphorylation leading to reduced ATP production associated growth inhibition,” using a small experimental data set from exposure of Lemna minor to the pesticide 3,5‐dichlorophenol. The AOP‐BN reflects the network structure of AOP #245 containing 2 molecular initiating events (MIEs), 3 key events (KEs), and 1 adverse outcome (AO). First, for each dose–response and response–response (KE) relationship, we quantify the causal relationship by Bayesian regression modeling. The regression models correspond to dose–response functions commonly applied in ecotoxicology. Secondly, we apply the fitted regression models with associated uncertainty to simulate 10 000 response values along the predictor gradient. Thirdly, we use the simulated values to parameterize the conditional probability tables of the BN model. The quantified AOP‐BN model can be run in several directions: 1) prognostic inference, run forward from the stressor node to predict the AO level; 2) diagnostic inference, run backward from the AO node; and 3) omnidirectionally, run from the intermediate MIEs and/or KEs. Internal validation shows that the AOP‐BN can obtain a high accuracy rate, when run is from intermediate nodes and when a low resolution is acceptable for the AO. Although the performance of this AOP‐BN is limited by the small data set, our study demonstrates a proof‐of‐concept: the combined use of Bayesian regression modeling and Bayesian network modeling for quantifying AOPs. Integr Environ Assess Manag 2021;17:147–164. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg 2+. It is also demonstrated that the increase in F V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.

Role of Carbonyl Cyanide m-Chlorophenylhydrazone in Enhancing Photobiological Hydrogen Production by Marine Green Alga Platymonas subcordiformis

We demonstrated that a significant volume of H(2) gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H(2) gas was produced without CCCP. The role of CCCP in enhancing photobiological H(2) production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water-splitting enzyme system Y) rapidly inhibited the photosystem II (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O(2) in the light. As a result, anaerobiosis during the stage of photobiological H(2) evolution was established, preventing severe O(2) inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of DeltapH across the thylakoid membrane and thus enhances the accessibility of electron and H(+) to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light-harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H(2) evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H(2) evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells.

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.

Physiological and Biochemical Processes Related to Ammonium Toxicity in Higher Plants

AbstractNitrate and ammonium have different effects on many biochemical and physiological processes in plants, and at high concentrations this can lead to markedly different growth responses. Most plant species show reduced growth, smaller leaves and a stunted root system when exposed to high ammonium concentrations, and in severe cases this leads to chlorosis. Although well known, ammonium toxicity is poorly understood and is generally considered to be the result of one or more of the following effects; (i) ammonium‐induced mineral nutrient deficiency, arising from the impaired uptake of metal ions; (ii) secondary growth inhibition arising from the acidification of the rooting medium; (iii) alterations in intracellular pH and osmotic balance; (iv) uncoupling of photophosphorylation from electron transport, following the accumulation of ammonium in leaves; and (v) altered polyamine and phytohormone metabolism.These hypotheses are reviewed in the light of the available literature and experimental evidence from own experiments. It is concluded that no mechanism on its own provides an adequate explanation of the available data.

In situ evidence that chilling in the light does not cause uncoupling of photophosphorylation or detachment of coupling factor in chilling-sensitive plants

The potential involvement of impaired photophosphorylation in the chilling sensitivity of photosynthesis in warm climate plant species has been a topic of investigation for more than two decades. With recent advances in the analysis of photosynthetic energy transduction in intact leaves, experiments are now possible that either address or avoid important uncertainties in the significance and interpretation of earlier in vitro work. Nevertheless, different laboratories using different techniques to analyze the effects of chilling in the light on photophosphorylation in intact cucumber (Cucumis sativus) leaves have come to very different conclusions regarding the role of impaired ATP formation capacity in the inhibition of net photosynthesis. In order to evaluate these discrepancies and bring this issue to a final resolution, in this investigation, we have made a detailed analysis of the decay of the flash-induced electrochromic shift and changes in chlorophyll fluorescence yield in cucumber leaves before, during and after a 5 h light-chill at chill temperatures of between 4 and 10°C. We feel that our findings address the major discrepancies in both data and interpretation as well as provide convincing evidence that photophosphorylation is not disrupted in cucumber leaves during or after light and chilling exposure. It follows that impaired photophosphorylation is not a contributing element to the inhibition of net photosynthesis that is widely observed in warm climate plants as a result of chilling in the light.

Interaction of carbonyl cyanide m-chlorophenylhydrazone with the photosystem II acceptor side

We show that CCCP, known as an uncoupler of photophosphorylation and an ADRY agent, inhibits FeCy photoreduction and coupled O 2 evolution by isolated chloroplasts equally (1 50 ∼ 2μM), but is practically without effect on the O 2 evolution coupled with SiMo reduction within the 0.2–10μM concentration range. CCCP has no effect on the nanosecond chlorophyll fluorescence in chloroplasts incubated at low light intensity, but decreases it at high light intensity. The electron transfer from reduced TMPD or duroquinol to methylviologen is resistant to CCCP. The efficiency of the CCCP inhibitory action on the FeCy photoreduction depends on the rate of electron flow, which is controlled by the light intensity. The data obtained show that CCCP is oxidized by the photosystem II donor side and is reduced by Q p, competing for electrons with FeCy and the cytochrome blf complex.

Pattern of cotolerance in marine periphyton communities established under arsenate stress

Arsenate-tolerant marine periphyton communities established under arsenate stress in a flow-through aquarium system, were investigated for cotolerance to various inhibitors of photosynthesis. A short-term test system for inhibition of periphyton photosynthesis was used to quantify the community tolerance levels. Preexposure to arsenate at 10 μM increased arsenate tolerance > 16,000-fold compared to control communities established at background levels of arsenate. A substantial cotolerance was observed only for thiophosphate which is a phosphate analog uncoupler of photophosphorylation with a mode of action similar to that of arsenate. For the uncouplers CCCP (carbonyl cyanide m-chloro-phenylhydrazone) and Primene JMT (a primary amine with a branched C 18–C 22 aliphatic chain), the inhibitor of photosynthetic electron transport DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and arsenite, there was a general tendency for a small cotolerance, although for Primene JMT and DCMU an increased sensitivity was some-times observed. The general increase in tolerance was however quantitatively insignificant compared to the specific cotolerance observed for thiophosphate, suggesting that the mode of action and tolerance mechanisms are similar for arsenate and thiophosphate, and most likely related to their phosphate analogy. We suggest that significant cotolerance is not ubiquitous nor common in periphyton, and that the emerging cotolerance pattern may be indicative of the toxicity and tolerance mechanisms.

Photophosphorylation after Chilling in the Light

The response of in situ photophosphorylation in attached cucumber (Cucumis sativus L. cv Ashley) leaves to chilling under strong illumination was investigated. A single-beam kinetic spectrophotometer fitted with a clamp-on, whole leaf cuvette was used to measure the flash-induced electrochromic absorbance change at 518 minus 540 nanometers (DeltaA(518-540)) in attached leaves. The relaxation kinetics of the electric field-indicating DeltaA(518-540) measures the rate of depolarization of the thylakoid membrane. Since this depolarization process is normally dominated by proton efflux through the coupling factor during ATP synthesis, this technique can be used, in conjuction with careful controls, as a monitor of in situ ATP formation competence. Whole, attached leaves were chilled at 5 degrees C and 1000 microeinsteins per square meter per second for up to 6 hours then rewarmed in the dark at room temperature for 30 minutes and 100% relative humidity. Leaf water potential, chlorophyll content, and the effective optical pathlength for the absorption measurements were not affected by the treatment. Light- and CO(2)-saturated leaf disc oxygen evolution and the quantum efficiency of photosynthesis were inhibited by approximately 50% after 3 hours of light chilling and by approximately 75% after 6 hours. Despite the large inhibition to net photosynthesis, the measurements of DeltaA(518-540) relaxation kinetics showed photophosphorylation to be largely unaffected by the chilling and light exposure. The amplitude of the DeltaA(518-540) measures the degree of energization of the photosynthetic membranes and was reduced significantly by chilling in the light. The cause of the decreased energization was traced to impaired turnover of photosystem II. Our measurements showed that the chilling of whole leaves in the light caused neither an uncoupling of photophosphorylation from photosynthetic electron transport nor any irreversible inhibition of the chloroplast coupling factor in situ. The sizeable inhibition in net photosynthesis observed after chilling in the light cannot, therefore, be attributed to any direct effect on photophosphorylation competence.

Uncoupling of Photophosphorylation by Triethyl Lead (Et 3Pb +) Generated from Tetraethyl Lead (Et 4Pb) in Illuminated Chloroplasts

Summary Tetraethyl lead (Et 4 Pb), which is used as an anti-knock agent in gasoline, was transformed to the toxic triethyl lead (Et 3 Pb + ) in chloroplasts of Spinacia oleracea L. by light. This transformation depends on a reactive O 2 species formed in a Mehler-type reaction during the photosynthetic electron transport. Methyl viologen, increasing the formation of superoxide free radical ions in a chloroplast suspension upon illumination, caused an enhanced generation of Et 3 Pb + from Et 4 Pb. Ascorbate, a scavenger of such reactive O 2 species, reduced the formation of 3 Pb + drastically. 3 Pb + inhibited photophosphorylation at concentrations of ≥ 10 nM, whereas O 2 evolution was remarkably enhanced around 10–100 μM. This uncoupling effect, which is comparable to that induced by CCCP (carbonyl cyanide m-chlorophenylhydrazone), was attributed to a recently demonstrated property of 3 Pb + (Hager et al., 1987), i.e. acting as a powerfull transmembrane Cl − /OH − antiporter and thereby dissipating H + and other ion or electrical gradients across membranes at nano-molar levels.

Relationship between the octanol-water partition coefficient of tertiary amines and their effect of ?selective? uncoupling of photophosphorylation

A series of tertiary amines was investigated for effects on the transmembrane proton potential difference (Δ [Formula: see text]H), on photophosphorylation and on electron-flux control related to the intrathylakoid proton potential ([Formula: see text]HI), using isolated chloroplasts ofSpinacia oleracea L. As indicated by 9-aminoacridine fluorescence and [14C]methylamine uptake, all amines studied inhibited a build-up ofΔ [Formula: see text]H and, in parallel, ATP synthesis. Even whenΔ [Formula: see text]H was low, strong[Formula: see text]H1-dependent electron-flux control was observed under the influence of tertiary amines. The strength of flux control in the presence of lowΔ [Formula: see text]H and the effectiveness of inhibition of ATP synthesis linearly increased with the lipophilicity of the amines. The most effective of the amines tested caused 50% inhibition of ATP synthesis at a concentration of 6 μM, which is about 1000-fold lower than the concentration required for inhibition by methylamine. The data presented indicate the existence of two proton domains in the thylakoid vesicles, one of them feeding the ATP-synthase, the other the sites of pH-dependent electron-flux control. It is concluded that tertiary amines develop their action in a lipophilic domain of the thylakoid membrane, in the vicinity of the ATP-synthase complex. A mechanism for 'selective' uncoupling and for the maintenance of[Formula: see text]HI-dependent electron flux control in the presence of lowΔ [Formula: see text]H is discussed.

Binding, uptake and expression of foreign DNA by cyanobacteria and isolated etioplasts

Discoveries of the uptake and expression of various Escherichia coli plasmids by the cyanobacterium Anacystis nidulans and isolated cumber etioplasts are reviewed. In particular, the binding and uptake of nick-translated (32)P-labeled plasmids and the expression of genes in the native plasmids are considered.Permeaplasts of A. nidulans 6301 and isolated EDTA-washed cucumber etioplasts exhibit binding and uptake of DNA that is unaffected by uncouplers of photophosphorylation or by dissipators of transmembrane proton graident. ATP inhibits both binding and udptake by permeaplasts or EDTA-washed etioplasts but the analog AMP-PNP (non-hydrolzable) is noninhibitory. With permeaplasts there is no effect of 20 mM Mg(2+) (in the light) upon intake, whereas with EDTA-washed etioplasts, Mg(2+) at the same concentration inhibits uptake as does 20 mM Ca(2+).The transformation of A. nidulans 6301 to ampicillin-resistance by the plasmid pBR322 is much enhanced in permeaplasts. Indeed extracts of transformed cells catalyze the hydrolosis of the β-lactam nitrocefin. Transfromation of A. nidulans to antibiotic resistance may also be achieved with the plasmids pHUB4 and pCH1. The effect of light on transformation of A. nidulans 6301 differs with different plasmids. In pBR322 transformants the expression of ribulose bisphosphate carboxylase-oxygenase (RuBisCO) is markedly elevated. In these transformants, the foreign plasmid replicates by a pathway involving chromosomal integration and dissociation.The plasmid pCS75, a derivative of pUC9 (and therefore of pBR322) containing a Pst1 insert carrying genes for the large and small (S) subunits of RuBisCO from A. nidulans, is taken up and expressed in EDTA-washed cucumber cotyledon etioplasts. Expression is evidenced by the hydrolysis of nitrocefin and immunoprecipitation of labeled S subunits of RuBisCO (utilizing etioplasts which have been labeled with (35)S-methionine after incubation with pCS75). The plasmid pUC9-CM carrying a cat gene is also expressed in cucumber etioplasts in a manner that demonstrates dependence both on the duration of etioplast washing by EDTA and plasmid concentration. Translation (as measured by (35)S-methionine incorporation) by EDTA-washed etioplasts increases with cotyledon greening. However the enhancement of translation by prior incubation of EDTA-washed plastids with pCS75 decreases to zero during 24hr of cotyledon greening. Results suggest that the expression of foreign DNA in plastids may depend critically upon their developmental state.

Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin D

Palmitic acid and gramicidin D at low concentrations uncouple photophosphorylation in a mechanism that is inconsistent with classical uncoupling in the following properties: (1) delta pH, H+ uptake, or the transmembrane electric potential is not inhibited. (2) O2 evolution is stimulated under nonphosphorylating conditions but slightly inhibited in the presence of adenosine 5'-diphosphate + inorganic phosphate (Pi). (3) Light-triggered adenosine 5'-triphosphate (ATP)-Pi exchange is hardly affected, and ATPase activity is only slightly stimulated. (4) ATP-induced delta pH formation is selectively inhibited. This characteristic uncoupling is observed only when the native coupling sites of the electron transport system are used for energization such as for methylviologen-coupled phosphorylation. With pyocyanine, which creates an artificial coupling site, 1000-fold higher gramicidin D and higher palmitic acid concentrations are required for inhibition, and the inhibition is accompanied by a decrease in delta pH. Moreover, comparison between photosystem 1 and photosystem 2 electron transport and the effects of membrane unstacking suggest that low gramicidin D preferentially inhibits photosystem 2, while palmitic acid inhibits more effectively photosystem 1 coupling sites. The inhibitory capacity of fatty acids significantly drops when the chain length is reduced below 16 hydrocarbons or upon introduction of a single double bond in the hydrocarbon chain. It is suggested that palmitic acid and gramicidin D interfere with a direct H+ transfer between specific electron transport and the ATP synthase complexes, which provides an alternative coupling mechanism in parallel with bulk to bulk delta microH+. The sites of inhibition seem to be located in chloroplast ATP synthase, photosystem 2, and the cytochrome b6f complexes.

Excitation-energy redistribution in the cryptomonad alga Cryptomonas ovata

We have studied the redistribution of excitation energy in the cryptomonad alga Cryptomonas ovata. Low-temperature fluorescence emission spectra from cells preilluminated with light 1 and light 2 show that preferential excitation of Photosystem II (PS II) leads to decreased fluorescence emission from chlorophyll (Chl) a associated with PS II relative to the emission following the preferential excitation of Photosystem I (PS I). The fluorescence change is indicative of a light-state transition by the cells. However, comparision of measurements of the kinetics of P-700 photooxidation by cells fixed with glutaraldehyde following illumination with light 1 or light 2 shows that the relative activity of PS I is lower in cells fixed in light 2. This is in contrast to the expectation for cells in State 2. Excitation spectra for the fluorescence emission from PS II Chl a show that preferential excitation of PS II leads to a decreased probability for energy transfer from phycoerythrin and Chl c 2 to PS II when compared to cells in which PS I is preferentially excited. This result is in accordance with recent picosecond time-resolved fluorescence studies (Bruce, D., Biggins, J., Charbonneau, S. and Thewalt, M. (1987) in Progress in Photosynthesis Research (Biggins, J., ed.), Vol. II, pp. 777–780, Martinus Nijhoff, Dordrecht) and we, therefore, suggest that C. ovata does not undergo a classical light-state transition. However, preferential excitation of PS II or PS I appears to cause pigment-protein conformational changes which change the probability for energy transfer from phycoerythrin to PS II, and we suggest that this may be a mechanism for photoprotection of PS II. Studies of the kinetics of excitation-energy redistribution, and of the effects of electron-transport inhibitors and uncouplers of photophosphorylation indicate that the mechanism for excitation-energy redistribution in C. ovata and phycobilisome-containing organisms may be similar.

Freezing of Isolated Thylakoid Membranes in Complex Media. IV. Stabilization of CF 1 by ATP and Sulfate

Summary Freeze-thaw treatment of isolated spinach thylakoid membranes in buffered media containing Cl-, NO3-, K+, Na+, and Mg2+ at concentrations close to those found in the stroma of intact chloroplasts resulted in inactivation of photophosphorylation. This membrane damage was mainly due to dissociation of CF1, which caused an increase in H+ permeability of the thylakoid membrane and stimulation of linear photosynthetic electron transport; simultaneously, uncoupled electron flow decreased, reflecting a partial inhibition in the capacity of the electron transport chain. The addition of ATP or sulfate at concentrations comparable to those in the chloroplast stroma in situ provided marked protection of photophosphorylation against freeze-thaw inactivation. Under these conditions, the ability to form a transthylakoid proton gradient in the light was largely maintained and uncoupling of photophosphorylation from electron transport and dissociation of peripheral membrane proteins did not occur. Obviously, both ATP and sulfate were highly effective in stabilizing CF1 binding during freezing. The lack of complete cryoprotection of photophosphorylation of isolated thylakoids by ATP and sulfate was likely due to the inability of these solutes to prevent partial damage of the electron transport chain. As an efficient cryopreservation of photophosphorylation was also reached in the presence of other essential compounds of the chloroplast stroma (sugars, amino acids, phosphate), it is concluded that in intact leaves CF1 may be sufficiently protected in the course of freeze-thawing.

Effects of the sesquiterpene lactone tetraesters thapsigargicin and thapsigargin, from roots of Thapsia garganica L., on isolated spinach chloroplasts

K. A. Santarius, G. Falsone and H. Haddad. Effects of the sesquiterpene lactone tetraesters thapsigargicin and thapsigargin, from roots of Thapsia garganica L., on isolated spinach chloroplasts. Toxicon 25, 389 – 399, 1987.—The effect of thapsigargicin and thapsigargin, extracted from the roots of Thapsia garganica L., on isolated photosynthetic membranes (thylakoids) and intact chloroplasts from spinach leaves ( Spinacia oleracea L.) was investigated. Both sesquiterpene lactone tetraesters impair membranes and organelles in an identical, chlorophyll-dependent manner. In thylakoids these compounds primarily act as inhibitors of photophosphorylation. At lower sesquiterpene lactone tetraester/chlorophyll ratios, cyclic and non-cyclic photophosphorylation, ADP-stimulated electron transport and the photosynthetic control ratio progressively decreased with increasing concentrations of thapsigargicin and thapsigargin, whereas the state 4 electron flow, the coupling efficiency of photophosphorylation, the light-induced proton gradient, and the H + flux across the membranes remained nearly unaffected. Half-maximal inhibition of photophosphorylation was obtained with 4 – 5 × 10 −7 moles sesquiterpene lactone tetraesters per mg chlorophyll. At higher sesquiterpene lactone tetraester/chlorophyll ratios, uncoupling of photophosphorylation from electron transport occurred. This was evident from stimulation of the state 4 electron flow, decline in the ADP/2e − ratio, increase in proton permeability and decrease in ΔpH, whereas the uncoupled electron transport was only little impaired. In intact chloroplasts inhibition of HCO 3 −, 3-phosphoglycerate and oxaloacetate reduction by thapsigargicin and thapsigargin was not caused by inactivation of the photochemical reactions of the thylakoid membranes but were rather due to alterations in the permeability properties of the chloroplast envelope. This was concluded from similarities in the kinetics of these reactions. It is suggested that the highly lipid soluble sesquiterpene lactone tetraesters effectively disrupt the lipid - protein associations of biomembranes.

Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.

Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.

Role of Guard Cell Chloroplasts in Stomatal Movement

High sensitivity of guard cells to DBM1B (10 -7 M) and relatively lower response to diuron suggest that energy harnessed via cyclic electron transport may be contributing, a great deal, in meeting, the energy requirements of stomatal movement in light. The effect of ammonium chloride, an uncoupler of photophosphorylation on stomatal opening also supports this. The sensitivity of guard cells to ammonium chloride is p H dependent.