Inhibition and uncoupling of photosynthetic electron transport by diterpene lactone amide derivatives

Nine diterpene lactone amide derivatives 1-9 were synthesized from 6-oxovouacapan-7beta,17beta-lactone, which was obtained from 6alpha,7beta-dihydroxyvouacapan-17beta-oic acid isolated from Pterodon polygalaeflorus Benth., and tested for their activity on photosynthetic electron transport. Amide derivatives 3-5 behaved as electron transport chain inhibitors; they inhibited the photophosphorylation and uncoupled non-cyclic electron transport from water to methylviologen (MV). Furthermore, 4 and 5 enhanced the basal electron rate acting as uncouplers. Compound 6 behaved as an uncoupler; it enhanced the light-activated Mg2+-ATPase and basal electron flow, without affecting the uncoupled non-cyclic electron transport. Compounds 1-2 and 7-9 were less active or inactive. Compounds 3-5 did not affect photosystem I (PSI); they inhibited photosystem II (PSII) from water to 2,6-dichlorophenol indophenol (DCPIP). Compound 4 inhibited PSII from water to silicomolybdate (SiMo), but it had no effect on the reaction from diphenylcarbazide (DPC) to DCPIP indicating that its inhibition site was at the water splitting enzyme complex (OEC). Compounds 3 and 5 inhibited PSII from water to DCPIP without any effect from water to SiMo, therefore they inhibited the acceptor site of PSII. Chlorophyll a fluorescence kinetics confirmed the behaviour of 3-5.     

Three cytotoxic Annonaceous acetogenins from the seeds of Annona squamosa

Three new Annonaceous acetogenins, squamocin-IV (1), squamocin-V (2) and squamoxinone-E (3), together with seven other known Annonaceous acetogenins (4–10) were isolated from the seeds of Annona squamosa. The structures of all of the isolates were established and characterized through spectral and chemical methods. The new Annonaceous acetogenins 1–3 were evaluated for their cytotoxicity against the Hep-G2, SMMC-7721, BEL-7402, BGC-803 and H460 human cancer cell lines. Compound 3 exhibited the best cytotoxic activity, with IC₅₀ values of 0.103, 0.687, 4.19, 0.43 and 6.56 μg/mL, respectively for the cell lines above; meanwhile, Compound 1 showed selective cytotoxic activity against H460, with an IC₅₀ value of 0.049 μg/mL. The first mono-ACG (2) composed of 38 carbons was discovered.     

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme.     

Novel cytotoxic monotetrahydrofuranic Annonaceous acetogenins from Annona montana

Four new monotetrahydrofuranic Annonaceous acetogenins, montalicins G (1) and H (2) and monlicins A (3) and B (4), and two new linear acetogenins, (+)-monhexocin (5) and (-)-monhexocin (6), as well as three known compounds, murisolin (7), 4-deoxyannomontacin (8), and muricatacin (9), were isolated from the seeds of Annona montana by high performance liquid chromatographic (HPLC) method. The absolute stereochemical structures of new isolates were elucidated and characterized by spectral and chemical methods. Interestingly, these compounds show special cytotoxicity against human hepatoma cells, Hep G2.     

Analysis of donors of electrons to photosystem I and cyclic electron flow by redox kinetics of P700 in chloroplasts of isolated bundle sheath strands of maize

Bundle sheath chloroplasts of NADP-malic enzyme (NADP-ME) type C₄ species have a high demand for ATP, while being deficient in linear electron flow and oxidation of water by photosystem II (PSII). To evaluate electron donors to photosystem I (PSI) and possible pathways of cyclic electron flow (CEF1) in isolated bundle sheath strands of maize (Zea mays L.), an NADP-ME species, light-induced redox kinetics of the reaction center chlorophyll of PSI (P700) were followed under aerobic conditions. Donors of electrons to CEF1 are needed to compensate for electrons lost from the cycle. When stromal electron donors to CEF1 are generated during pre-illumination with actinic light (AL), they retard the subsequent rate of oxidation of P700 by far-red light. Ascorbate was more effective than malate in generating stromal electron donors by AL. The generation of stromal donors by ascorbate was inhibited by DCMU, showing ascorbate donates electrons to the oxidizing side of PSII. The inhibitors of NADPH dehydrogenase (NDH), amytal and rotenone, accelerated the oxidation rate of P700 by far-red light after AL, indicating donation of electrons to the intersystem from stromal donors via NDH. These inhibitors, however, did not affect the steady-state level of P700⁺ under AL, which represents a balance of input and output of electrons in P700. In contrast, antimycin A, the inhibitor of the ferredoxin-plastoquinone reductase-dependent CEF1, substantially lowered the level of P700⁺ under AL. Thus, the primary pathway of ATP generation by CEF1 may be through ferredoxin-plastoquinone, while function of CEF1 via NDH may be restricted by low levels of ferredoxin-NADP reductase. NDH may contribute to redox poising of CEF1, or function to generate ATP in linear electron flow to O₂ via PSI, utilizing NADPH generated from malate by chloroplastic NADP-ME.     

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.     

Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803

We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 micromol x m(-2) x s(-1)), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light. (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII. These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.     

Effects of water stress on major photosystem II gene expression and protein metabolism in barley leaves

Although it has long been recognized that water deficit in plants reduces photosystem (PS) II mRNAs and proteins, the detailed mechanisms behind this have not been thoroughly elucidated. In the present study, effects of water stress in barley leaves on degradation of major PSII mRNA and dissociation and migration of PSII proteins were investigated. The results indicated that (1) the steady-state levels of major PSII mRNAs and proteins declined with increasing water stress, as a consequence of increased degradation; under severe water stress, the half-lives of D1 and D2 proteins decreased from 12–14 h to 7–8 h and the half-lives of psbA and psbD mRNA decreased from above 16 to 6–10 h; (2) monomerization of PSII were increased during water stress. Severe water stress accelerated turnover of PSII and inhibited PSII activities.     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.     

Tricolorin A, a potent natural uncoupler and inhibitor of photosystem II acceptor side of spinach chloroplasts

Tricolorin A, (11 S )-11-hydroxyhexadecanoic acid 11- O - α - l - rhamnopyranosyl-(1↠3)- O - α - l -{2- O -(2 S -methylbutanoyl)-4- O -(2 S -methylbutanoyl)}-rhamnopyranosil-(1↠2)- O - β - d -glucopyranosil-(1↠2)- β -fucopyranoside-(1,3'-lactone), the major phytogrowth inhibitor isolated from Ipomoea tricolor Cav. (Convolvulaceae) was found to be a potent uncoupler (U₅₀=0.33 μ M ) of photophosphorylation in spinach chloroplasts. Tricolorin A inhibited H⁺-uptake and adenosine 5'-triphosphate (ATP) synthesis, and stimulated basal and phosphorylating electron flows. Using a combination of two well-known fluorescent ΔpH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine, the uncoupling behavior of tricolorin A was also demonstrated for submitochondrial particles. Polarographic data showed that high concentrations (20 μ M ) of tricolorin A inhibited photosystem II (PSII) electron flow at the level of plastoquinone B (Q_B). Chlorophyll (Chl) a fluorescence analysis showed that tricolorin A induced accumulation of Q_A⁻ and strongly decreased the electron transport capacity, suggesting that the target of this molecule was located at the Q_B level. The macrocyclic lactone-type structure of this allelopathic agent proved to be an important structural requirement for uncoupling activity since its hydrolysis caused loss of the inhibitory potential.     

Photoinactivation of ascorbate peroxidase in isolated tobacco chloroplasts: Galdieria partita APX maintains the electron flux through the water-water cycle in transplastomic tobacco plants

We evaluated the H2O2-scavenging activity of the water-water cycle (WWC) in illuminated intact chloroplasts isolated from tobacco leaves. Illumination under conditions that limited photosynthesis [red light (>640 nm), 250 micromol photons m(-2) s(-1) in the absence of HCO3-] caused chloroplasts to take up O2 and accumulate H2O2. Concomitant with the O2 uptake, both ascorbate peroxidase (APX) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) lost their activities. However, superoxide dismutase (SOD), monodehydroascorbate radical reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities remained unaffected. The extent to which the photosynthetic linear electron flow decreased was small compared with the decline in APX activity. Therefore, the loss of APX activity lowered the electron flux through the WWC, as evidenced by a decrease in relative electron flux through PSII [Phi(PSII)xPFD]. To verify these interpretations, we created a transplastomic tobacco line in which an H2O2-insensitive APX from the red alga, Galdieria partita, was overproduced in the chloroplasts. In intact transplastomic chloroplasts which were illuminated under conditions that limited photosynthesis, neither O2 uptake nor H2O2 accumulation occurred. Furthermore, the electron flux through the WWC and the activity of GAPDH were maintained. The present work is the first report of APX inactivation by endogenous H2O2 in intact chloroplasts.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

Changes in cyclic and respiratory electron transport by the movement of phycobilisomes in the cyanobacterium Synechocystis sp. strain PCC 6803

Phycobilisomes (PBS) are the major accessory light-harvesting complexes in cyanobacteria and their mobility affects the light energy distribution between the two photosystems. We investigated the effect of PBS mobility on state transitions, photosynthetic and respiratory electron transport, and various fluorescence parameters in Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBS to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBS at PSII inhibited the increase in cyclic electron flow, photochemical and non-photochemical quenching, and decrease in respiration that occurred during the movement of PBS from PSII to PSI. In contrast, the immobilization of PBS at PSI inhibited the increase in respiration and photochemical quenching and decrease in cyclic electron flow and non-photochemical quenching that occurred when PBS moved from PSI to PSII. Linear electron transport did not change during PBS movement but increased or decreased significantly during longer illumination with far-red or green light, respectively. This implies that PBS movement is completed in a short time but it takes longer for the overall photosynthetic reactions to be tuned to a new state.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Ameliorative role of nitric oxide on H2O2 toxicity to a chlorophycean alga Scenedesmus obliquus

A concentration-dependent toxicity of hydrogen peroxide (H(2)O(2)) was observed on growth yield, chlorophyll a content and chlorophyll fluorescence characteristics of the green microalga Scenedesmus obliquus under laboratory batch culture conditions. The addition of sodium nitroprusside, a nitric oxide (NO) donor, in combination with H(2)O(2) prevented chlorophyll losses, and the inhibition level of growth yield, maximum quantum yield of photosystem II (PSII) and the light-adapted quantum yield of PSII were significantly reduced. The antioxidant compounds, penicillamine and thiourea also reduced the damage caused by H(2)O(2) exposure. The protective actions of sodium nitroprusside were, however, arrested in cultures where sodium nitroprusside was supplemented in combination with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a specific scavenger of NO. The NO(3)(-)-grown Scenedesmus depicted less sensitivity to H(2)O(2) toxicity with respect to the quantum yields of PSII as compared to its NH(4)(1)-grown counterpart. The role of NO in providing protection against H(2)O(2) toxicity to the processes under study was discussed.     

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.     

The involvement of photosystem II-generated H2O2 in photoinhibition.

The involvement of H2O2 generated by photosystem II (PSII) in the process of photoinhibition of thylakoids with a functional oxygen-evolving complex (OEC) was investigated. The rate of photoinhibition was decreased to the rate of loss of activity in the dark when bovine Fe-catalase was present during the photoinhibitory illumination. Photoinhibition was accelerated for both Cl(-)-depleted and Cl(-)-sufficient thylakoids when KCN was present to inhibit the thylakoid-bound Fe-catalase. We propose that these preparations become photoinhibited by reactions with H2O2 produced via oxidation of water by the Cl(-)-depleted OEC and by reduction of O2 at the QB site when PSII is illuminated without an electron acceptor.