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.     

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.     

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.     

Personal recollections of 40 years in photosynthesis research*

The major theme of my work in photosynthesis has been electron transport in green plant thylakoids. In particular, we investigated the properties and the role of the NADP-reducing flavoprotein and its possible function in cyclic electron transport, the regulation by protons of electron transport, and the redox system of ascorbate and monodehydroascorbate (the ascorbate free radical). The function of this system in providing ATP in the stoichiometric amount needed for carbon assimilation, and the regulation of the alternative transfer of electrons to NADP and to the ascorbate free radical were among the achievements of my collaborators and myself. Specifically, the early conviction that cyclic phosphorylation was essential part in photosynthesis was shattered as far as higher plants are concerned, and replaced by a modified Mehler reaction providing additional ATP to run the Calvin cycle. The situation seems to be different in unicellular green algae, where quantitatively much l arger changes of the relative size of Photosystem (PS) I and PS II antennae during the so-called state transitions have been reported, and these seem to be associated with a high activity of cyclic electron transport in state 2. Beyond the science, the friendly interactions with so many persons around the world sharing my interest in photosynthesis and in other aspects of human life have been most rewarding.     

Inhibition of photosynthetic electron transport and formation of inactive chlorophyll in winter stressed Pinus silvestris

Kinetics of fluorescence at room temperature, electron transport and photooxidation of P700 and cytochrome f have been studied in chloroplasts isolated from active and winter stressed Pinus silvestris. The winter stress induced block in the electron transport chain between the two photosystems is close to the site of plastoquinone, since winter stress and DCMU caused the same type of inhibition of the reoxidation of the primary electron acceptor Q of photosystem II. No winter inhibition of the electron transport between cytochrome f and P700 was observed. Time course studies of P700 photooxidation in chloroplasts of active and winter stressed pine have shown that the photosynthetic unit size must be about equal in the two types of chloroplasts. An apparent increase of the photosynthetic unit size was induced by winter stress, as revealed by the high chlorophyll/P700 ratio of winter stressed pine. The phenomenon is explained by the formation of photosynthetically inactive chlorophyll. Low-temperature fluorescence emission spectra were recorded when either chlorophyll a (433 nm) or chlorophyll b (477 nm) were preferentially excited. Winter stress induced the formation of a chlorophyll a fraction emitting at 673 nm. This chlorophyll is most likely derived from the chlorophyll a antennae of the two photosystems, and it probably contributes to the photosynthetically inactive pool of chlorophyll in winter stressed pine. The light harvesting chlorophyll a/b complex is relatively resistant to winter stress.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

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。     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Photosynthetic electron transport and establishment of an associated trans-thylakoid proton electrochemical gradient during development of the wheat leaf

Abstract Photosynthetic electron transport activities and the ability to generate and maintain a trans-thylakoid proton electrochemical gradient were examined during chloroplast development in 4-day-old wheat leaves grown under a diurnal light regime. Polarographic and spectropholometric studies on leaf tissue demonstrated that poorly developed chloroplasls at the leaf base could photo-oxidize water and transfer electrons from photosystem 2 to photosystem 1. The capacity for non-cyclic whole-chain electron transport increased during chloroplast development. Thylakoids isolated from the leaf base, although capable of pumping protons into the inlrathylakoid space, could not maintain a trans-membrane proton electrochemical gradient; this ability developed at later stages of chloroplast biogenesis in the leaf. The implications of these results for the energetics of the developing leaf are discussed.     

Clotrimazole对叶绿体ATPase功能的影响及其作用部位

clotrimazole能抑制 DTT+光激活的类囊体膜上Mg~(2+)—ATPase的活力。这种抑制属于可逆非竞争性抑制。进一步的实验还表明clotrimazole可以消除 9—AA光下荧光粹灭指示的正常类囊体及DCCD重组残缺膜的跨膜质子梯度。卵磷脂可以减缓 clotrimazole对9—AA荧光粹灭的抑制作用。clotrimazole还能抑制DTT加热激活的游离CF_1 Ca~(2+)—ATPase的活力。根据以上结果我们推测 clotrimazole在类囊体上可能有两个作用部位,一个在类囊体膜脂;另一个在CF_1。     

On the nature of the photosynthetic energy storage monitored by photoacoustic spectroscopy

The photosynthetic energy storage yield of uncoupled thylakoid membranes was monitored by photoacoustic spectroscopy at various measuring beam intensities. The energy storage rate as evaluated by the half-saturation measuring beam intensity (i₅₀) was inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea, by heat inactivation or by artificial electron acceptors specific for photosystem I or photosystem II; and was activated by electron donors to photosystem I. The reactions involving both photosystems were all characterized by a similar maximal energy storage yield of 16±2 percent. The data could be interpreted if we assumed that the energy storage elicited by the photosystems at 35 Hz is detected at the level of the plastoquinone pool.Abbreviations PS photosystem - Tes N-Tris [hydroxymethl] methyl-2-aminoethanesulfonic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - FeCN potassium ferricyanide - DCBQ 2,5-dichlorobenzoquinone - TMPD N,N,N-tetramethyl-p-phenilenediamine     

铜离子在光系统Ⅱ电子传递中的作用部位和方式

铜离子对PS Ⅱ电子传递有明显的抑制作用,并且不能被加入人工电子供体DPC而恢复电子传递。铜离子表现出对胰蛋白酶消化叶绿体膜后使PS Ⅱ电子传递所受抑制有加成作用,并且铜离子又可拮抗胰蛋白酶对被DCMU阻止的PS Ⅱ电子传递的部分恢复作用。因而推测铜离子在PS Ⅱ的作用部位是在DPC供电子处至PS Ⅱ作用中心之间,其作用方式可能在于钝化了参与PS Ⅱ电子传递的膜蛋白。用SDS-PAGE对叶绿体膜蛋白的分离结果,也符合于这一假设。     

Influence of electrochemical proton gradient on electron flow in photosystem I of pea leaves

Photoinduced changes in the redox state of photosystem I (PSI) primary donor, chlorophyll P700 were studied by measuring differential absorbance changes of pea leaves at 810 nm minus 870 nm (ΔA ₈₁₀). The kinetics of ΔA ₈₁₀ induced by 5-s pulses of white light were strongly affected by preillumination. In dark-adapted leaves, the light pulse caused a transient oxidation of P700 and its subsequent reduction. An identical pulse, applied after 30-s preillumination with white light, induced sequential appearance of two peaks of P700 oxidation. These kinetic differences of ΔA ₈₁₀ reflect regulatory changes of electron flow on the donor and acceptor sides of PSI induced by illumination of leaf for 20–40 s. The amplitude of ΔA ₈₁₀ second peak depended nonmonotonically on the dark interval preceding illumination: it increased with the length of dark period in the range 3–10 s and decreased upon longer dark intervals. The second wave of ΔA ₈₁₀ disappeared after the treatment with combination of ionophores preventing ΔpH and electric potential formation at the thylakoid membrane. In leaves treated with monensin eliminating ΔpH only, the ΔA ₈₁₀ signals become incompletely reversible and were characterized by slow relaxation in darkness. The results indicate an important role of electrochemical proton gradient in generation of the second wave of light-induced P700 oxidation.     

Transmembrane ferricyanide reduction by cells of the yeastSaccharomyces cerevisiae

Both respiratory-competent and respiratory-deficient yeast cells reduce external ferricyanide. The reduction is stimulated by ethanol and inhibited by the alcohol dehydrogenase inhibitor, pyrazole. The reduction of ferricyanide is not inhibited by inhibitors of mitochondrial or microsomal ferricyanide reduction. Cells in exponential-phase growth show a much higher rate of ferricyanide reduction. The reduction of ferricyanide is accompanied by increased release of protons by the yeast cells. We propose that the ferricyanide reduction is carried out by a transmembrane NADH dehydrogenase.     

Environmental and metaboliccontrol of LHCII protein phosphorylation: revealing the mechanismsfor dual regulation of the LHCII kinase

Light‐harvesting complex II (LHCII) protein phosphorylation inplant chloroplasts is under complex regulation. Combination of the invivo monitoring of LHCII protein phosphorylation (by immunoblotting)with the in vitro[γ‐³²P]ATPphosphorylation assays revealed that the basic activation/deactivationmodel of the LHCII kinase, regulated by reversible occupation/releaseof plastoquinol at the plastoquinol oxidation (Q_o) siteof the cytochrome b₆f (cyt b₆f) complex, isconsistent with, but not sufficient to explain the data obtainedwith isolated chloroplasts, leaf discs or intact leaves. Not onlythe light conditions but also the metabolic state of the entireplant, particularly the sugar metabolism, exerted a control overLHCII protein phosphorylation. Feeding of leaves with glucose (alsowith glutathione) activated the LHCII kinase in darkness. On the otherhand, independently of the basic activation/deactivationmechanism of the kinase, a strong inhibition of LHCII protein phosphorylationoccurred in vivo at increasing irradiances and even at lowlight conditions, depending on the metabolic state of the plant.Both the experiments with intact chloroplasts and the reconstitutionexperiments with isolated thylakoids to mimic LHCII kinase inhibition,disclosed that the kinase in its activated state (plastoquinol at theQ_o site of cyt b₆f complex) is protected againstinhibition by thiol reductants. However, directly upon deactivationof the kinase (release of plastoquinol from the Q_o site) itbecomes a target for inhibition by thiol reductants. Thus the twointerdependent regulatory systems of the LHCII kinase, the constantlyoccurring activation and deactivation on the one hand and the inhibitionby thiol reductants on the other, are strongly dependent on theconcentration of reducing equivalents in the chloroplast stroma.A scheme demonstrating the interconversion of activated, deactivated andinhibited states of the LHCII kinase in the chloroplast environmentof intact leaves is presented.     

黄瓜叶片光合电子传递对水分胁迫的响应

黄瓜叶片在水分胁迫下叶片相对含水量减少,类囊体室温吸收光谱的吸收峰降低,同时其NADP光还原活性、Ca^2 -ATPase活性也相应降低,全链电子传递明显受阻。类囊体膜蛋白电泳分析结果显示：类囊体膜色素蛋白复合体含量有不同程度的降低,其中PSⅡ色素蛋白复合体含量下降较多,试验结果表明水分胁迫通过限制光能的吸收,传递双及转换效率,抑制了光合电子传递过程。     

Photosystem 2 activities of hyper-accumulator Dicranopteris dichotoma Bernh from a light rare earth elements mine

The distribution of rare earth elements (REEs) in the fern Dicranopteris dichotoma Bernh plants from a light rare earth elements mine (LRM) and a non-mining (NM) area in Longnan county of Jiangxi province, China were investigated by means of inductively coupled plasma-mass spectrometry, transmission electron microscopy, and energy-dispersive X-ray microanalysis. The photosynthetic characteristics of D. dichotoma were studied by chlorophyll (Chl) a fluorescence kinetics. Contents of REEs in the lamina and the root of D. dichotoma were higher than those in soils, and were mainly distributed in lamina. A part of them was found in the chloroplast. By comparing with D. dichotoma from NM area, the efficiency of photosystem 2 photochemistry and electron transport rate were significantly enhanced in lamina of the plant from LRM because most of REEs deposits were distributed along cell wall, in vacuole, and in chloroplast. High contents of REEs in lamina did not decrease the photosynthetic activities in LRM plants of D. dichotoma. Besides, D. dichotoma could change its β-carotene content to avoid the damaging effect of high REEs content.