Ascorbate biosynthesis and function in photoprotection

Ascorbate (vitamin C) can reach very high concentrations in chloroplasts (20-300 mM). The pool size in leaves and chloroplasts increases during acclimation to high light intensity and the highest concentrations recorded are in high alpine plants. Multiple functions for ascorbate in photosynthesis have been proposed, including scavenging of active oxygen species generated by oxygen photoreduction and photorespiration, regeneration of alpha-tocopherol from alpha-tocopheryl radicals, cofactor for violaxanthin de-epoxidase and donation of electrons to photosystem II. Hydrogen peroxide scavenging is catalysed by ascorbate peroxidase (Mehler peroxidase reaction) and the subsequent regeneration of ascorbate by reductant derived from photosystem I allows electron flow in addition to that used for CO2 assimilation. Ascorbate is synthesized from guanosine diphosphate-mannose via L-galactose and L-galactono-1,4-lactone. The last step, catalysed by L-galactono-1,4-lactone dehydrogenase, is located on the inner mitochondrial membrane and uses cytochrome c as electron acceptor. L-galactono-1,4-lactone oxidation to ascorbate by intact leaves is faster in high-light acclimated leaves and is also enhanced by high light, suggesting that this step contributes to the control of pool size by light. Ascorbate-deficient Arabidopsis thaliana vtc mutants are hypersensitive to a number of oxidative stresses including ozone and ultraviolet B radiation. Further investigation of these mutants shows that they have reduced zeaxanthin-dependent non-photochemical quenching, confirming that ascorbate is the cofactor for violaxanthin de-epoxidase and that availability of thylakoid lumen ascorbate could limit this reaction. The vtc mutants are also more sensitive to photo-oxidation imposed by combined high light and salt treatments.     

N,N-diethylhydroxylamine: a new electron donor to photosystem II

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. Diethylhydroxylamine supports dichlorophenolindophenol photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II before the herbicide binding site. The stimulation of the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed that the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamine. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations with diethylhydroxylamine either in light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that diethylhydroxylamine is a useful electron donor to the photosystem II.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of UV-B radiation on photosynthetic primary reactions in spinach chloroplasts

Spinach ( Spinacia oleracea L. cv. Matador) chloroplasts were irradiated with several levels of UV-B radiation. Measurements which reflect characteristic steps of photosynthetic electron transport were made to localize the site of impairment of photosynthesis by UV-B radiation.
Variable fluorescence, the μs-kinetics of the 320 nm absorption changes and also oxygen evolution were substantially reduced in chloroplasts irradiated with UV-B. It was not possible to restore the amplitude of the 320 nm absorption changes nor the signal of the transmembrane electric field measured at 520 nm by adding the photo-system II donor couple hydroquinone/ascorbate to UV-B treated chloroplast samples. This indicates that impairment of photosystem II activity is not caused by selective inhibition of the water-splitting enzyme system Y, but rather is due to blockage of photosystem II reaction centers. Photosystem 1 is inferred to be highly resistant to UV-B radiation.
These results suggest that the reaction centers of photosystem II are transformed into dissipative sinks for excitation energy by action of UV-B radiation.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

Production of reactive oxygen species by photosystem II

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment-protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.     

Effects of Some Chemicals on the Oxygen Evolution and Oxygen Uptake in Isolated Wheat Chloroplasts Irradiated without the Addition of an Oxidant

The oxygen exchange, obtained when isolated chloroplasts of Triticum aestivum, wheat, are irradiated without the addition of a Hill oxidant has been investigated using an oxygen electrode. Ascorbate, catalase, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone(DBMIB), diethyldithio-carbamate (DEDT), dichlorophenylmethylurea (DCMU), and potassium cyanide were added to the Chloroplasts in order to investigate the oxygen exchange. At least two oxygen uptake reactions, one sensitive to catalase and one catalase-insensitive, appeared upon irradiation. Hydrogen peroxide was the product of the oxygen uptake in the former process, and water was the reductant. The formation of hydrogen peroxide was probably associated with photosystem I. The other oxygen consuming reaction was found to be insensitive to both catalase and potassium cyanide. After the chloroplasts had been treated with DCMU, it was possible to show that the catalase-insensitive oxygen uptake was localized in photosystem I, and that a cyclic electron transport system or some endogenous reductant (-s) acted in the oxygen uptake. Addition of ascorbate or DEDT to the chloroplasts led to an enhanced oxygen uptake in 710 nm light. This was probably due to the effect of these compounds on the superoxide radical ion formed in photosystem I. The stimulated oxygen uptake was only weakly affected by catalase, indicating that hydrogen peroxide was not a product of this oxygen uptake. Addition of DEDT and potassium cyanide inhibited (strongly respectively weakly) the oxygen uptake when photosystem II was functioning. The effect of these compounds was probably due to an inhibition of the electron transport at the plastocyanin. DBMIB inhibited the oxygen uptake reactions and the cooperation between the two photosystems. The cooperation between the photosystems was also studied in DCMU-treated chloroplasts. The reactions in photosystem II, measured as oxygen evolution, were more inhibited than the coupling between the photosystems. The oxygen “gush” appearing upon irradiation in light of 650 nm was not affected by a DBMIB-treatment, showing that the oxygen evolution was due to the reduction of plastoquinone. The reoxidation in the dark of the plastoquinone pool was stimulated by DBMIB and potassium cyanide indicating that an oxygen uptake could be associated with plastoquinone. The sites of interaction of oxygen with the electron transport pathways in chloroplasts, and the different reductants for the oxygen consuming reactions are discussed.     

D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

A pronounced light-induced zeaxanthin formation accompanied by an unusually slight increase in non-photochemical quenching: a study with barley leaves treated with methyl viologen at moderate light

Light-induced deepoxidation of violaxanthin to antheraxanthin and zeaxanthin in plants is associated with the induction of pronounced xanthophyll-dependent non-photochemical quenching (NPQ). To date, a misbalance between a high amount of zeaxanthin in thylakoid membranes and low NPQ has been explained by an absence of lumen acidification (e.g. when NPQ is measured in the dark after high light stress). In this study, we report that this misbalance can also be observed under moderate light. We found this result (deepoxidation state, DEPS, above 55% and NPQ0.9) in barley leaves treated with 10 μM methyl viologen (MV) under white light (100 μmol photons m⁻² s⁻¹, photosynthetically active radiation (PAR), growth irradiance). The addition of MV at this moderate light did not accelerate electron transport in thylakoid membranes, and induced only slight oxidative stress (no lipid peroxidation, almost unchanged maximum yield of photosystem II photochemistry, a decrease in activity of ascorbate peroxidase, and an increase in that of glutathion reductase). We suggest that, in leaves treated under the conditions used here, the lumen acidification induced by light-limited electron transport in thylakoid membranes was high enough to activate violaxanthin deepoxidase, but not sufficiently high to form the expected number of zeaxanthin-dependent quenching centers in photosystem II antennae.     

The inhibition of photosynthetic electron transport in spinach chloroplasts by low osmolarity.

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.     

Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled "Regulation of Electron Transport in Chloroplasts".     

PHOTOSYNTHETIC ELECTRON TRANSPORT IN CELL-FREE EXTRACTS OF DIVERSE PHYTOPLANKTON1,2

A simple and rapid procedure for preparing thylakoid membranes that are active in photosynthetic electron transport from diverse phytoplankton species is described. The method requires disruption of algal cells with glass beads, exposure to mild hypotonic stress, and subsequent enrichment of the thylakoid membranes by differential centrifugation. Isolated thylakoid membranes were assayed for photosynthetic electron transport activity by measuring rates of oxygen consumption and oxygen production, using a variety of electron donors and acceptors. In the dinoflagellate Gonyaulax polyedra Stein, a relatively broad pH optimum between 7.0 and 8.0 was determined for the whole chain electron transport from water to methyl viologen. The preparation maintained maximum activity for 45 min following the preparation. The assay for photosystem I activity in G. polyedra, determined as electron flow from ascorbate/2,6-dichlorophenolindophenol to methyl viologen, had a somewhat narrower pH optimum around 8.0. Rates of whole chain photosynthetic electron transport on a per cell and on a per chlorophyll a basis were shown to decrease dramatically with cell age in batch cultures of G. polyedra. Using the procedures optimized for G. polyedra, reproducible rates of electron transport on a per cell chlorophyll a basis were also measured in cultures of the dinoflagellate Glenodinium sp., the diatom Nitzschia closterium (Ehrenberg 1839) Wm. Smith 1853 and the chrysophyte Monochrysis lutheri Droop {= Pavlova lutheri (Droop) Green}. Other electron transport assays applied to G. polyedra, and that resulted in comparable rates to those found in other algal groups, include the photosystem II assay from water to diaminodurene/ferricyanide and the photosystem I assay from durohydroquinone to methyl viologen.     

Reprint of: Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products

Illumination of a suspension of thylakoids with light at high intensity causes inhibition of the photosystem II electron transport activity and loss from the membrane of the D1 protein of the photosystem II reaction center. Impairment of the electron transport activity and depletion of D1 protein from the thylakoid membrane of pea were investigated with reference to the presence or absence of oxygen in the suspension. The breakdown products of the D1 protein were identified by immunoblotting with anti-D1 polyclonal antibodies which were proven to recognize mainly the C-terminal region of the protein. The results obtained show that (i) the light-induced inactivation of the photosystem II electron transport activity under anaerobic conditions is faster than in the presence of oxygen; (ii) depletion of D1 protein is observed on a longer time scale with respect to loss of electron transport activity and is faster when photoinhibition is performed in the presence of oxygen; (iii) C-terminal fragments of D1 are only observed when photoinhibition is carried out anaerobically and are mainly localized in the stroma-exposed regions; and (iv) the fragments observed after anaerobic photoinhibition are quickly degraded on further illumination of the thylakoid suspension in the presence of oxygen.     

Ascorbate in thylakoid lumen as an endogenous electron donor to Photosystem II: Protection of thylakoids from photoinhibition and regeneration of ascorbate in stroma by dehydroascorbate reductase

Photoinhibition of the electron transport activity from tyrosine Z (Y_Z) in PS II to NADP⁺in Tris-treated thylakoids was suppressed by electron donation with either diphenylcarbazide or ascorbate (AsA) during the photoinhibition treatment. This suggests that AsA prevents donor side-induced photoinhibition in vivo as an endogenous donor. AsA in the lumen is photooxidized to monodehydroascorbate (MDA) in Tris-treated thylakoids, as detected by electron spin resonance spectrometry, but not in oxygenic thylakoids. Redox analysis of pyridine nucleotide in the presence of either MDA reductase or dehydroascorbate (DHA) reductase showed that the MDA photoproduced in the lumen is disproportionated to AsA and DHA, and the DHA leaking into the stroma is reduced to AsA by DHA reductase. No leakage of MDA through the thylakoid membrane was observed. Thus, the DHA-reducing enzyme system is indispensable in maintaining AsA concentrations in chloroplasts.     

Photoinhibition of Chloroplast Reactions. II. Multiple Effects

Ultraviolet light inhibits the photoreduction of 2,6-dichlorophenolindo-phenol or nicotinamide adenine dinucleotide phosphate with water as the electron donor (evolution of oxygen) but not the photoreduction of nicotinamide adenine dinucleotide phosphate with ascorbate as the electron donor. It inhibits photophosphorylation associated with either system. Experiments undertaken to test whether plastoquinone is the site of UV inhibition yielded inconclusive results.

Visible light (> 420 mμ) causes the loss of all chloroplast activities, photosystem I being more sensitive than system II. The data suggests 2 modes of action for visible light. The one sensitized by system II results in damage resembling that of UV light. The other, sensitized by system I, results in the destruction of the reaction center of this system.

     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.     

Effect of cadmium on photosystem II activity in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: alteration of O–J–I–P fluorescence transients indicating the change of apparent activation energies within photosystem II

In this study, we evaluated how cadmium inhibitory effect on photosystem II and I electron transport may affect light energy conversion into electron transport by photosystem II. To induce cadmium effect on the photosynthetic apparatus, we exposed Chlamydomonas reinhardtii 24 h to 0–4.62 μM Cd²⁺. By evaluating the half time of fluorescence transients O–J–I–P at different temperatures (20–30°C), we were able to determine the photosystem II apparent activation energies for different reduction steps of photosystem II, indicated by the O–J–I–P fluorescence transients. The decrease of the apparent activation energies for PSII electron transport was found to be strongly related to the cadmium-induced inhibition of photosynthetic electron transport. We found a strong correlation between the photosystem II apparent activation energies and photosystem II oxygen evolution rate and photosystem I activity. Different levels of cadmium inhibition at photosystem II water-splitting system and photosystem I activity showed that photosystem II apparent activation energies are strongly dependent to photosystem II donor and acceptor sides. Therefore, the oxido-reduction state of whole photosystem II and I electron transport chain affects the conversion of light energy from antenna complex to photosystem II electron transport.