Cooperation of photosystem I with the plastoquinone pool in oxygen reduction in higher plant chloroplasts

The possible functions of a light-induced electron transfer to oxygen in the photosynthetic electron transport chain of higher plant chloroplasts are considered. The thermodynamic preconditions, as well as the experimental data about the participations of ferredoxin, the components of photosystems I and II, and plastoquinone in oxygen reduction are examined. It is concluded that, even in the presence of ferredoxin and ferredoxin + NADP⁺, oxygen reduction is carried out mainly by the membrane-bound carriers of the photosynthetic electron transport chain. The hypothesis is put forward that most superoxides, which are produced by reduction of O₂ molecules by the intramembrane components of the acceptor side of photosystem I, are reduced within the membrane by the plastohydroquinone molecules to the hydrogen peroxide. It is assumed that the H₂O₂ molecules that originate as the result of this process serve for signaling about the redox state of the plastoquinone pool. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 1, pp. 137–144.     

Oxygen reduction in a plastoquinone pool of isolated pea thylakoids

Oxygen uptake in isolated pea thylakoids in the presence of an inhibitor of plastoquinol oxidation by b ₆/f-complex dinitrophenylether of 2-iodo-4-nitrothymol (DNP-INT) was studied. The rate of oxygen uptake in the absence of DNP-INT had a distinct maximum at pH 5.0 followed by a decline to pH 6.5 and posterior slow rise, while in the presence of an inhibitor it increased at an increasing pH from 4.5 to 6.5 and then kept close to the rate in its absence up to pH 8.5. Gramicidin D substantially affected the oxygen uptake rate in the absence of DNP-INT, and only slightly in its presence. Such differences pointed to the presence of special oxygen reduction site(s) in photosynthetic electron transport chain `before' cytochrome complex. Oxygen uptake in membrane fragments of Photosystem II (BBY-particles) was low and did not depend on pH. This did not support the participation of Q_B in oxygen reduction in DNP-INT-treated thylakoids. Oxygen uptake in thylakoids in the presence of DNP-INT was inhibited by DCMU as well as by catalase in whole pH range. The catalase effect indicated that oxygen uptake was the result of dioxygen reduction by electrons derived from water, and that H₂O₂ was a final product of this reduction. Photoreduction of Cyt c in the presence of DNP-INT was partly inhibited by superoxide dismutase (SOD), and this pointed to superoxide formation. The latter was confirmed by a rise of the oxygen uptake rate in the presence of ascorbate and by suppression of this rise by SOD. Both tests showed that the detectable superoxide radicals averaged 20–25% of potentially formed superoxide radicals the quantity of which was calculated from the oxygen uptake rate. The obtained data implies that the oxygen reduction takes place in a plastoquinone pool and occurs mainly inside the membrane, where superoxide can be consumed in concomitant reactions. A scheme for oxygen reduction in a plastoquinone pool in thylakoid membranes is proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m²·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

The role of ascorbate in the protection of thylakoids against photoinactivation

The inactivation of electron transport upon preillumination of isolated, stroma free thylakoids has been studied. Inactivation is defined here as the loss of activity which is not reversed upon relaxation of qE. It was found that both PS 2 and PS 1 dependent electron transport were inactivated, whilst the coupling of ATP synthesis to electron transport was not affected. The inactivation concerned both the transfer of excitation energy to the reaction centres, and the reaction centres themselves. Ascorbate protected against photoinactivation of the electron transport from H₂O to NADP or to methylviologen, much less the electron transport depending only on PS 1. The protection by ascorbate required its well known action as a cofactor of de-epoxidation of violaxanthin and the consequent formation of qE: under conditions where de-epoxidation was inhibited (presence of DTT or uncouplers) qE was also suppressed and ascorbate protection was abolished. Ascorbate did not p rotect the thylakoids against inactivation caused by H₂O₂in the dark.The latter was shown to concern mostly PS 2 electron transport.     

Interaction of electron acceptors with thylakoids from halophytic and non-halophytic species

Thylakoid membranes isolated from halophytic species showed differences in their interactions with ionic and lipophilic electron acceptors when compared to thylakoids from non-halophytes. FeCN was considerably less efficient as electron acceptor with halophyte thylakoids, supporting much lower rates of O₂ evolution and having a lower affinity. FeCN accepted electrons at a different, DMMIB insensitive, site with these thylakoids. 1,4-Benzo-quinones with less positive midpoint potentials were less effective in accepting electrons from halophyte thylakoids compared to nonhalophyte thylakoids, also reflected in lower rates of O₂ evolution and lower affinity. Considering the lipolphilic nature and the fact that there was no apparent change in the site donating electrons to the quinones, an alteration in the midpoint potential of this site by about +100mV is postulated for the halophyte thylakoids.Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - Cyt b₆/f cytochrome b₆/f complex - DBMIB 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone - DCBQ 2,6-dichloro-1,4-benzoquinone - DCIP 2,6-dichlorophenol-indolphenol - DMBQ 2,5-dimethyl-1,4-benzoquinone - E_m7 midpoint redox potential at pH 7.0, FeCN-K₃Fe(CN)₆ - HNQ 5-hydroxy-1,4-naphthoquinone - MV methylviologen - NQ 1,4-naphthoquinone - PBQ phenyl-1,4-benzoquinone - PC plastocyanin - PQ plastoquinone     

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dark reoxidation of the plastoquinone-pool is mediated by the low-potential form of cytochrome b-559 in spinach thylakoids

We have found that in isolated spinach thylakoids, plastoquinone-pool (PQ-pool), after its photoreduction, undergoes dark-reoxidation with the half-time of _1/2 = 43 ± 3 s. To explain the observed rates of PQ-pool reoxidation, a nonenzymatic plastoquinol (PQH₂) autoxidation under molecular oxygen and an enzymatic oxidation by the low-potential form of cytochrome b-559 (cyt. b-559LP), as the postulated PQ-oxidase in chlororespiration, were investigated. It was found that the autoxidation rate of PQH₂ in organic solvents and liposomes was too low to account for the observed oxidation rate of PQH₂ in thylakoids. The rate of cyt. b-559LP autoxidation in isolated Photosystem II was found to be similar (_1/2 = 26 ± 5 s) to that of the PQ-pool. This suggests that the LP form of cyt. b-559 is probably responsible for the PQ-oxidase activity observed during chlororespiration.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25+/-3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 micromol/m(2).s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

Cytochrome c Oxidase (Heme aa 3) from Paracoccus denitrificans: Analysis of Mutations in Putative Proton Channels of Subunit I

One of the challenging features of energy-transducing terminal oxidases, like the aa ₃ cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen. As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis. The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.     

Spectrophotometric measurement of plastoquinone photoreduction in the blue-green alga,Phormidium uncinatum

The redox state of plastoquinone was measured in vivo in the blue-green alga, Phormidium uncinatum by means of a double beam UV-spectrophotometer. The difference in absorbance of the oxidized and the reduced forms of plastoquinone was amplified, and stored and averaged in a computer. The redox state was changed by two alternating actinic light beams. When one actinic wavelength was kept constant at 700 nm (PSI) variation of the other yielded an action spectrum representing photosystem II. The inhibitors of the photosynthetic electron transport chain, DCMU and DBMIB, reduced the difference in absorbance between the oxidized and reduced forms of plastoquinone.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea     

Thiol redox state and related enzymes in sclerotium-forming filamentous phytopathogenic fungi

Thiol redox state (TRS) reduced and oxidized components form profiles characteristic of each of the four main types of differentiation in the sclerotiogenic phytopathogenic fungi: loose, terminal, lateral-chained, and lateral-simple, represented by Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor, respectively. A common feature of these fungi is that as their undifferentiated mycelium enters the differentiated state, it is accompanied by a decrease in the low oxidative stress-associated total reduced thiols and/or by an increase of the high oxidative stress-associated total oxidized thiols either in the sclerotial mycelial substrate or in its corresponding sclerotium, indicating a relationship between TRS-related oxidative stress and sclerotial differentiation. Moreover, the four studied sclerotium types exhibit high activities of TRS-related antioxidant enzymes, indicating the existence of antioxidant protection of the hyphae of the sclerotium medulla until conditions become appropriate for sclerotium germination.     

Function of plastoquinones B and C as electron acceptors in Photosystem II and fatty acid analysis of plastoquinone B

We have found that in petroleum-ether extracted tobacco thylakoids, plastoquinone A (PQ-A) and plastoquinone C (PQ-C) had similar efficiency in restoration of oxygen-evolving activity, while plastoquinone B (PQ-B), which is a fatty acid ester of PQ-C, was about 50% less effective. This indicates that apart from PQ-A, PQ-C and to a smaller extent PQ-B may function as electron acceptors of Photosystem II (PS II). The DCMU inhibition curves for PQ-C and PQ-B were biphasic and an initial slow decline was followed by a sharp decrease in oxygen evolution yield with a 50% inhibition (I50) at 0.25 M DCMU. In the case of PQ-A (I50 = 0.20 M DCMU), the activity decreased gradually without the sharp transition. The corresponding inhibition curve for unextracted thylakoids, where all the native prenylquinones are present, shows an intermediate shape between PQ-A and PQ-C but with a higher I50, equal to 0.32 M, suggesting that the contribution of PQ-C as an electron acceptor of Photosystem II might be significant in thylakoid membranes with natural prenyllipid composition. -Tocopherol quinone showed no activity in the restoration of oxygen evolution in extracted thylakoids, indicating that it cannot accept electrons from PS II. The fatty acid composition of PQ-B isolated from maple leaves showed a high degree of saturated fatty acids like myristic and palmitic acid, and its unique composition indicates that it is a natural component of the thylakoid membrane.     

Multiparametric protocol for the determination of thiol redox state in living matter

Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. However, these TRS parameters can estimate the GSSG/GSH potential, which might be useful for indicating abnormalities in redox metabolism. Nonetheless, evaluation of the multiparameric nature of TRS is required for a more accurate assessment of its physiological role. The present protocol extends the partial assessment of TRS by current methodologies. It measures 15 key parameters of TRS by two modular subprotocols: one for the glutathione (GSH)- and cysteine (CSH)-based nonprotein (NP) thiols/mixed disulfides (i.e., GSH, GSSG, GSSNP, CSH, CSSNP, NPSH, NPSSNP, NP_xSH_NPSSNP, NP_xSH_NPSH), and the other for their protein (P) thiols/mixed disulfides (i.e., PSH, PSSG, PSSC, PSSNP, PSSP, NP_xSH_PSSNP). The protocol eliminates autoxidation of GSH and CSH (and thus overestimation of GSSG and CSSNP). Its modularity allows the determination GSH and GSSG also by other published specific assays. The protocol uses three assays; two are based on the photometric reagents 4,4′-dithiopyridine (DTP) and ninhydrin (NHD), and the third on the fluorometric reagent o-phthaldialdehyde (OPT). The initial assays employing these reagents have been extensively modified and redesigned for increased specificity, sensitivity, and simplicity. TRS parameter values and their standard errors are estimated automatically by sets of Excel-adapted algebraic equations. Protocol sensitivity for NPSH, PSH, NPSSNP, PSSP, PSSNP, CSH, CSSNP, PSSC, NP_xSH_NPSSNP, and NP_xSH_NPSH is 1 nmol –SH/CSH, for GSSNP 0.2 nmol, for GSH and GSSG 0.4 nmol, and for PSSG 0.6 nmol. The protocol was applied on human plasma, a sample of high clinical value, and can be also applied in any organism.     

花生下胚轴组织H~ 分泌与质膜氧化还原系统的某些特性

花生幼苗下胚轴细胞表面存在氧化NADH与还原Fe(CN)_6~(3-)的氧化还原系统(redox system),它的活性随反应介质pH的上升而增加,氧是该系统的天然电子受体,受DCCD抑制。在NADH与Fe(CN)_6~(3-)被氧化与还原的同时,组织的H~ 分泌明显受到促进,并随反应介质pH上升而增加。质膜H~ -ATPase专一抑制剂Na_3VO_4(0.1 mmol/L)对组织H~ 分泌抑制绝对量基本上不受介质pH的影响,表明质膜H~ -ATPase与redox system共同参与该组织的H~ 分泌,部分redox system支配的H~ 分泌是依赖氧的。     

Regulation of the Ca-independent phospholipase A2 in liver mitochondria by changes in the energetic state

The effect of electron transport chain redox status on activity of the mitochondrial Ca²⁺-independent phospholipase A₂ (iPLA₂) has been examined. When oxidizing NAD-linked substrates, the enzyme is not active unless deenergization occurs. Uncoupler, rotenone, antimycin A, and cyanide are equally effective at upregulating the enzyme, while oligomycin is ineffective. Thenoyltrifluoroacetone causes deenergization and activates the enzyme, but only if succinate is the respiratory substrate. These findings show that the mitochondrial iPLA₂ responds to the energetic state overall, rather than to the redox status of individual electron transport chain complexes. With NAD-linked substrates, and using rotenone to deenergize, iPLA₂ activation can be reversed by adding succinate to reestablish a membrane potential. For this purpose, ascorbate plus N,N,N′N′-tetramethyl-phenylenediamine can be used instead of succinate and is equally effective. With succinate as substrate, the membrane potential can be reduced in a graded and stable fashion by adding increasing concentrations of malonate, which is a competitive inhibitor of succinate utilization. A partial and stable activation of the iPLA₂ accompanies partial deenergization. These findings suggest that in addition to the several functions that have been proposed, the mitochondrial iPLA₂ may help to coordinate local capillary blood flow with changing energy demands.     

Effects of Prolonged UV-B Enhanced Fluorescent Radiation on Some Marine Microalgae

The eukaryotic unicellular microalgae Chlorella salina, Dicrateria inornata, and Isochrysis galbana were grown under control (fluorescent 20 W m^–2) and UV-B enhanced (UV-B^E, 0.5 W m^–2) fluorescent radiation. The growth rate showed marginal increase under UV-B^E. Decrease in protein content was observed in Dicrateria cells but in Chlorella an initial increase up to 4 d and in Isochrysis an increase at days 4 and 5 was noted. The chlorophyll a content showed marked increase in Chlorella and Isochrysis but in Dicrateria a decline was found. UV-B^E reduced the photosynthetic activity in all three species, but the reduction was larger in Chlorella and Dicrateria. Fluorescence excitation spectra for F₆₈₂ in Chlorella cells grown for 5 d under UV-B^E showed reduction in all peaks. In contrast to this, in Dicrateria and Isochrysis cells, the 530 and 590 nm excitation peaks increased with an appreciable decrease in the 466 nm peak. SDS-PAGE analysis revealed significant decrease in the contents of 47, 33, and 23 kDa polypeptides in Chlorella cells. In Dicrateria cells, significant loss in the content of 55, 38, and 18 kDa polypeptides was observed. The content of low molecular mass polypeptides (15 kDa) remained unaffected. Isochrysis cells were more stable in preserving the content of thylakoid polypeptides.