Regeneration of photosystem II and phycobilin pigments in photoorganotrophically grown Anabaena variabilis

Regeneration of photosynthetic activity and phycobilin pigmentswas studied with cells of Anabaena variabilis lacking photosystemII activity and phycobilin pigments. Regeneration was achievedonly when the cells were incubated in the presence of nitrateor nitrite. The addition of ammonium salts or urea was far lesseffective. Nitrate-directed regeneration was independent oflight and inhibited by chlorate. Dark-regenerated cells, however,differed from light-regenerated ones in that the former wereincapable of excitation transfer from phycocyanin to pigmentsystemII chlorophyll a, although they emitted fluorescence of pigmentsystem II chlorophyll a origin, if illuminated by the lightabsorbed by chlorophyll. The regeneration process inAnabaenacells is assumed to consist of two steps: [1] light-independent,nitratesupported synthesis of phycobilin pigments and photosystemII integrity, followed by [2] light-directed formation of excitationtransfer from phycocyanin to pigment system II chlorophyll a.An antibiotic study revealed that the former is associated withprotein synthesis, while the latter isnot. ¹ Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164, Japan. (Received November 19, 1975; )     

Photodynamic effects of hypericin on photosynthetic electron transport and fluorescence of Anacystis nidulans (Synechococcus 6301)

We investigated the photodynamic action of hypericin, a natural naphthodianthrone, on photosynthetic electron transport and fluorescence of the cyanobacterium Anacystis nidulans (Synechococcus 6301). The most drastic effect was the inactivation of photosynthetic oxygen evolution in the presence of the electron acceptor phenyl-p-benzoquinone in aerobic cells which required 1 hypericin/5 chlorophyll a for half-maximal effect. Anaerobic A. nidulans was only partially inactivated and variable chlorophyll a fluorescence remained unperturbed suggesting that photoreaction center II was not a target. Further, hypericin, stimulated photoinduced oxygen uptake in the presence of methylviologen in aerobic cells. This action was less specific than the inactivation of oxygen evolution (1 hypericin/0.5–0.7 chlorophyll a for half-maximal effect). Results point to the involvement of molecular oxygen in two ways. Type I mechanism (Henderson BW and Dougherty TJ (1992) Photochem Photobiol 55: 145–157) in which ground state oxygen reacts with excited substrate triplets appears probable for the inactivation of oxygen evolution. On the other hand, Type II mechanism in which excited oxygen singlets react with ground state substrate molecules appears probable in the stimulation of methylviologen mediated oxygen uptake.Abbreviations Chl chlorophyll - DAD diaminodurene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Hepes N-[2-hydroxyethyl]-N-[ethanesulfonic acid] - MV methyl viologen - PBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PS I, PS II Photosystems I and II - RC I, RC II reaction centers of PS I and PS II     

Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina

Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96±0.04 Chl a, 53.21±1.00 Chl d, and 5.48±0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01±0.06 Chl a and 62.96±2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

温室栽培对软枣猕猴桃叶片形态及叶绿素荧光特性的影响

【目的】深入研究温室栽培条件下软枣猕猴桃叶片的形态与叶绿素荧光特性,探明温室与露地栽培环境下叶片光形态建成的差异。【方法】以软枣猕猴桃品种‘佳绿’和‘魁绿’的5年生植株为试材,测定温室及露地栽培条件下不同叶龄叶片的叶绿素相对含量及叶绿素荧光参数,分析这些参数在温室与露地栽培条件下的差异。【结果】温室与露地栽培软枣猕猴桃的光形态建成均处于1~40 d叶龄间,不同栽培条件下的同一品种荧光特性趋于一致;花期后,不同栽培条件下的同一品种荧光特性差异较大。温室栽培的软枣猕猴桃叶片叶面积较大,叶绿素b含量较高,趋于阴生叶特性,以吸收光能为基础的性能指数等显著低于露地栽培,对光能的吸收捕获能力较强,但热耗散较高,用于电子传递的能量低于露地栽培,叶绿素荧光参数表现出对环境的适应性变化。【结论】软枣猕猴桃叶片的光形态建成时间为展叶后1~40 d,即在花期之前完成,不同栽培环境无明显差异;温室栽培软枣猕猴桃叶片的光形态建成与露地栽培具显著差异,叶面积明显增大;温室栽培在一定程度上改变了叶片的叶绿素荧光特性,降低了光合性能。     

Fate of the porphyrin cofactors during the light-dependent turnover of catalase and of the photosystem II reaction-center protein D1 in mature rye leaves

The apoprotein of the enzyme catalase (EC 1.11.1.6) was shown to exhibit a light-dependent turnover in leaves. Present results indicate that photoinactivation of the enzyme was not accompanied by a synchronous destruction and new synthesis of its heme moiety. In rye (Secale cereale L.) leaves the catalase content was not depleted in light when porphyrin synthesis was inhibited by gabaculine. Photoinactivation of purified bovine liver or rye leaf catalase in vitro was not accompanied by concomitant damage to the heme groups. Both the incorporation of -[³H]aminolevulinic acid ([³H]ALA) into catalase-heme and its apparent turnover increased with irradiance. However, the apparent half-life of the catalase-heme was much longer than that of its apoprotein. It is probable that not only degradation but also an exchange with the free heme pool contributed to the apparent turnover of radioactivity of the catalase-heme. Part of the chlorophyll (Chl) associated with photosystem II (PS II) had a preferential light-induced turnover, and repair of PS II appeared to require new Chl synthesis also in mature green rye leaves. The activity of PS II, indicated by the ratio of variable to maximal fluorescence (F_v/F_m), rapidly declined in the presence of gabaculine in light and the reaction-center proteins D1 and D2 were depleted. When segments of mature green rye leaves were labeled with [³H]ALA and incorporation into Chl-protein complexes analysed after electrophoretic separation in the presence of Deriphat, the highest radioactivity was observed in the core complex of PS II, while PS I and the light-harvesting complex of PS II (LHC II) were unlabeled. In greening etiolated leaves highest incorporation was observed in LHC II. Both the incorporation of [³H]ALA into the PS II core complex of green rye leaves and its turnover increased with irradiance. However, the apparent half-life of the PS II-bound labeled porphyrin compounds (mainly Chl) was considerably longer than that of the reaction-center protein D1 under identical conditions.Abbreviations ALA -aminolevulinic acid - CII Core complex of PS II - Chl chlorophyll - DMSO dimethyl sulfoxide - F_v/F_m ratio of variable to maximal chlorophyll fluorescence - LHC light-harvesting complex - PAR photosynthetically active radiation We thank the Deutsche Forschungsgemeinschaft for financial support. Technical assistence by B. Kramer and Ch. van Oijen is greatly appreciated. We are grateful to Dr. Johanningmeier and Dr. Godde (Lehrstuhl für Biochemie der Pflanzen, Universität Bochum, Germany) for providing antisera against the D1 and D2 proteins and Dr. M. Schmidt (Botanisches Institut, Universität Frankfurt am Main, Germany) for valuable advice. Deriphat 160 was kindly supplied by Henkel Corp., Hoboken, N.J., USA.     

Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging

Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [³²P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.     

Light- and temperature-induced changes in the distribution of excitation energy between Photosystem I and Photosystem II in spinach leaves

The influence of light quality and temperature on the distribution of the absorbed quanta between Photosystem I (PS I) and Photosystem II (PS II) in spinach leaves has been studied from the characteristics of chlorophyll fluorescence at 77 K. Leaves were preilluminated at different temperatures with either PS I light (to establish State 1) or with PS II light (to establish State 2), then cooled to 77 K and measured for fluorescence. In State 1, energy distribution appeared to be unaffected by temperature. A transition to State 2 resulted in an increase in PS I fluorescence and a decrease in the PS II fluorescence, indicating that a larger fraction of energy becomes redistributed to PS I. However, the extent of this redistribution varied: it was only small at 5°C to 20°C, but it largely increased at temperatures exceeding 20°C. This variation in the extent was related to a change in the mechanism of the state transition: at 15°C only the ‘initial’ distribution of energy was affected, while at 35°C an additional increase in the spill-over constant, k_{T (II → I)}, was included. It is assumed that under physiological conditions k_{T (II → I)} is under the control of temperature rather than of light quality, whereby in leaves adapted to high physiological temperatures, the probability of energy spill-over from closed PS II centres to PS I is enhanced. In darkened leaves, the spill-over constant has been manipulated by preincubation at different temperatures. Then, the light-induced ‘energization’ of thylakoid membranes has been tested by measuring the light-induced electrochromic absorbance change at 515 nm (and light-induced light-scattering changes) in these leaves. The flash-induced 515 nm signal as well as the initial peak during a 1 s illumination were not affected by energy distribution. However, the amplitude of the pseudo-steady-state signal (as established during 1 s illumination) was considerably enhanced in leaves in which a larger fraction of the absorbed energy is distributed to PS I at the expense of PS II excitation. The results have been interpreted in such a way that an increase in energy spill-over from PS II to PS I favours a cyclic electron transport around PS I. It is discussed that changes in energy distribution (via spill-over) may serve to maintain a suitable balance between non-cyclic and cyclic electron transport in vivo.     

Loss of photosystem II induced by a nitrate deficiency in photoorganotrophically grown Anabaena variabilis

When photoorganotrophically trained cells of Anabaena variabiliswere grown in nitrate-free medium, they lost the activity ofphotosynthetic oxygen evolution and became devoid of phycobilinpigments. These cells (H cells) lacked the fluorescence emissioncharacteristic of photosystem II chlorophyll, and their lamellarfragments failed to photoreduce DPIP even in the presence ofdiphenylcarbazide as the electron donor, suggesting that theloss of photosynthetic oxygen evolution in H cells is primarilydue to degeneration of an integral part of photosystem II. These characteristics of H cells closely resembled those ofheterocysts differentiated from normal, vegetative cells in[i] the deficiency of phycobilin pigments, [ii] the loss ofphotosystem II activity, [iii] the photoorganotrophic mode ofcell growth, depending upon the organic substances furnishedexternally or provided from neighboring vegetative cells, and[iv] the manner of transformation from normal cells, both typesof cells being induced in the absence of nitrate. In spite ofsuch similarity, light and electron microscopic observationsrevealed that H cells differed significantly from heterocysts.Furthermore, the readiness with which H cells resumed photosystemII activity and the independence of this resumption from cellgrowth exclude the possibility that the nutritional enrichmentof heterocysts was responsible for the loss of photosyntheticactivity. ¹Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164. (Received May 14, 1975; )     

State 1-state 2 transition in leaves and its association with ATP-induced chlorophyll fluorescence quenching

By using chlorophyll fluorescence, a study has been made of changes in spillover of excitation energy from Photosystem (PS) II to PS I associated with the State 1–State 2 transition in intact pea and barley leaves and in isolated envelope-free chloroplasts treated with ATP. (1) In pea leaves, illumination with light preferentially absorbed by PS II (Light 2) led to a condition of maximum spillover (state 2) while light preferentially absorbed by PS I induced minimum spillover condition (State 1) as judged from the redox state of Q and low-temperature emission spectra. The State 1–State 2 transitions took several minutes to occur, with the time increasing when the temperature was lowered from 19 to 6°C. (2) In contrast to the wild type, leaves of a chlorophyll b-less mutant barley did not exhibit a State 1–State 2 transition, suggesting the involvement of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex in spillover changes in higher plants. (3) Spillover in isolated pea chloroplasts was increased by treatment with ATP either (a) in Light 2 in the absence of an electron acceptor or (b) in the dark in the presence of NADPH and ferredoxin. These observations can be interpreted in terms of the model that a more reduced state of plastoquinone activates the protein kinase which catalyzes phosphorylation of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981). Nature 291, 25–29). This process was found to be very temperature sensitive. (4) Pea chloroplasts illuminated in the presence of ATP seemed to exhibit a slight decrease in the degree of thylakoid stacking, and an increased intermixing of the two photosystems. (5) The possible mechanism by which protein phosphorylation regulates the State 1–State 2 changes in intact leaves is presented in terms of changes in the spatial relationship of two photosystems resulting from alteration in membrane organization.     

Light-induced increase in initial chlorophyll fluorescence Fo level and the reversible inactivation of PS II reaction centers in soybean leaves

With a portable PAM-2000 fluorometer it was observed that responses of initial chlorophyll fluorescence F_o level to strong light were different in various plant species examined. When the photochemical efficiency of Photosystem II, F_v/F_m, declined, F_o increased significantly in leaves of some plants such as soybean and cotton, while F_o decreased remarkably in other plants such as wheat and barley. In order to explore the mechanism of the increase in F_o in soybean leaves, the change in D1 protein amount and effects of lincomycin and far-red light on these fluorescence parameters were observed by SDS–PAGE combined with gel scanning and chlorophyll fluorescence analysis. The following results were obtained. (1) The amount of inactive PS II reaction centers increased under strong light and decreased during subsequent dark recovery [Hong and Xu (1997) Chinese Sci Bull 42(8): 684–689]. (2) No net loss of D1 protein occurred after strong light treatment. (3) Lincomycin taken up through petioles following strong light treatment had no significant effect on D1 protein level and the decay of F_o in the dark. (4) Far-red light applied after strong light treatment could largely attenuate the increase in F_o and accelerate F_o decay in the dark. Based on these results, it is deduced that the increase in F_o under strong light is mainly due to reversible inactivation of part of PS II reaction centers, rather than the net loss of D1 protein and that reversible inactivation of PS II is prevalent in some plants.     

Involvement of the light-harvesting complex in cation regulation of excitation energy distribution in chloroplasts

A highly purified light-harvesting pigment-protein complex (LHC) was obtained by fractionation of cation-depleted chloroplast membranes using the nonionic detergent, Triton X-100. The isolated LHC had a chlorophyll $\text{a}\text{b}$ ratio of 1.2 and exhibited no photochemical activity. SDS-polyacrylamide gel electrophoresis of the LHC revealed three polypeptides in the molecular weight classes of 23, 25, and 30 × 10³. Antibodies were prepared against the LHC and their specificity was established. The effect of the α-LHC (antibodies to LHC) on salt-mediated changes in PS I and PS II photochemistry, Chl α fluorescence inductions, and 77 °K fluorescence emission spectra was investigated. The results show that: (i) The Mg²⁺-induced 20% decrease in photosystem I (PS I) quantum yield observed in control chloroplasts was blocked by the presence of the α-LHC antibody, (ii) The Mg²⁺-induced 70% increase in photosystem II (PS II) quantum yield of control chloroplasts was reduced 35% for plastids in the presence of α-LHC antibody, (iii) The Mg²⁺-induced increase in room-temperature variable fluorescence was reduced 60% by α-LHC antibody, (iv) The Mg²⁺-induced increase in the $\text{F685}\text{F730}$ emission peak ratio at 77 °K was inhibited 50% in the presence of α-LHC antibody. These results provide direct evidence for the involvement of the light-harvesting complex in cation regulation of energy redistribution between the photosystems. The fact that the α-LHC antibody does not fully block Mg²⁺-induced PS II increases or chlorophyll fluorescence increases supports the concept that Mg²⁺ has two mechanisms of action: one effect on energy distribution and a second direct effect on photosystem II centers.     

Low-Intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts

The fluorescence decays of barley chloroplasts have been measured by single-photon counting with tunable picosecond dye laser excitation. The fluorescence decays of dark-adapted chloroplasts are best fitted to a sum of three exponential lifetime components with lifetimes of 112, 380 and 2214 ps. The relative magnitude of each component is shown to be dependent on the excitation wavelength and collected emission wavelength. The excitation wavelength dependence is correlated with the Photosystem (PS) I and PS II action study of Ried [36] and with the measured pigment distributions in the photosynthetic unit [37,41]. Experiments varying the single excitation pulse intensity from 10⁸ to 10¹² photons/cm² pulse show that our results are not distorted by singlet-singlet annihilation. Unflowed samples where the cloroplasts are under constant illumination show 2-fold increases in quantum yield of fluorescence primarily in the two longer lifetime components. Theoretical calculations of Shipman [31] on an isolated reaction center with a homogeneous antenna are discussed and the principles extended to discussion of the measured barley chloroplast fluorescence decay components in terms of photosynthetic unit light-harvesting array models and earlier experimental work. Our data support a photosynthetic unit model in which 70–90% of the photons absorbed are quenched by either PS I or efficiently quenching PS II in a process where the fluorescence lifetime is 100 ps. The origin of the intermediate 380 ps. component is probably due to excitation transfer to a PS II reaction center in a redox state which quenches less efficiently.     

Functional characterization of the PS II–LHC II supercomplex isolated by a direct method from spinach thylakoid membranes

Recently, a novel procedure to isolate a highly pure and active Photosystem II preparation directly from thylakoid membranes, referred to as PS II–LHC II supercomplex, was reported [Eshaghi et al. (1999) FEBS Lett 446: 23–26]. In addition to the reaction center core proteins, the supercomplex contains all the extrinsic proteins of the oxygen evolving complex and a set of chlorophyll a/b binding proteins. In this paper, the functional properties of this isolated supercomplex are further characterized by using EPR spectroscopy, thermoluminescence, fluorescence relaxation kinetics and flash induced oxygen yield measurements. The PS II–LHC II supercomplex contains, in addition to Q_A and Q_B, a small pool of plastoquinone (PQ). Although the isolated complex is no longer membrane bound, it has preserved functional characteristics of a well defined PS II preparation with the exception of some modification of Q_B sites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex

A Synechococcus sp. strain PCC 7002 psaAB::cat mutant has been constructed by deletional interposon mutagenesis of the psaA and psaB genes through selection and segregation under low-light conditions. This strain can grow photoheterotrophically with glycerol as carbon source with a doubling time of 25 h at low light intensity (10 E m^–2 s^–1). No Photosystem I (PS I)-associated chlorophyll fluorescence emission peak was detected in the psaAB::cat mutant. The chlorophyll content of the psaAB::cat mutant was approximately 20% that of the wild-type strain on a per cell basis. In the absence of the PsaA and PsaB proteins, several other PS I proteins do not accumulate to normal levels. Assembly of the peripheral PS I proteins PsaC,PsaD, PsaE, and PsaL is dependent on the presence of the PsaA and PsaB heterodimer core. The precursor form of PsaF may be inserted into the thylakoid membrane but is not processed to its mature form in the absence of PsaA and PsaB. The absence of PS I reaction centers has no apparent effect on Photosystem II (PS II) assembly and activity. Although the mutant exhibited somewhat greater fluorescence emission from phycocyanin, most of the light energy absorbed by phycobilisomes was efficiently transferred to the PS II reaction centers in the absence of the PS I. No light state transition could be detected in the psaAB::cat strain; in the absence of PS I, cells remain in state 1. Development of this relatively light-tolerant strain lacking PS I provides an important new tool for the genetic manipulation of PS I and further demonstrates the utility of Synechococcus sp. PCC 7002 for structural and functional analyses of the PS I reaction center.Abbreviations ATCC American type culture collection - Chl chlorophyll - DCMU 3-(3,4-dichlorophyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - PCC Pasteur culture collection - PS I Photosystem I - PS II Photosystem II - SDS sodium dodecyl sulfate     

Uncoupling of detectable O2 evolution from the apparent S-state transitions in Photosystem II by lauroylcholine chloride: Possible implications in the photosynthetic water-splitting mechanism

Recently we have introduced the use of choline / fatty acid derived compounds, in particular lauroylcholine chloride (LCC), to probe selectively Photosystem II (PS II) structure and function (Wydrzynski, T. and Huggins, B.J. (1983) in The Oxygen-Evolving System of Photosynthesis (Inoue, Y., Crofts, A.R., Govindjee, Murata, N., Renger, G. and Satoh, K., eds.), pp. 265–272, Academic Press Tokyo, Japan). In this paper we report an unusual condition in thylakoid membrane samples at relatively low amounts of LCC in which detectable O₂ evolution cannot be measured, yet electron flow through PS II is near normal without added electron donors. LCC does not appear to interfere with the O₂ yield measurements directly nor act as an electron donor itself after the Tris block. Under this condition, steady state and flash O₂ yield measurements show no O₂ release or uptake, while steady-state ferricyanide photoreduction and the variable component of the chlorophyll a fluorescence transient remains at more than 50% of the control. The photoreduction of the primary quinone acceptor, Q_A, measured by microsecond range chlorophyll a fluorescence continues for a minimum of 200 single turnover excitation light flashes. Most importantly, the yield of the 35 |gms component of the chlorophyll a delayed fluorescence remains at approx. 65% of the control and oscillates with a normal period four over two cycles, indicating the normal cycling of the S-state transitions in PS II. Thus, it appears that PS II can operate normally without detectable O₂ evolution. The question remains as to whether water is still being photooxidized under this condition without the release of the dioxygen product, or whether there is another source of electrons. The results are interpreted in terms of the possible existence of an additional water binding component (termed ‘H’) in PS II and a concerted oxidation reaction mechanism for photosynthetic water splitting.     

Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants

Chloroplast thylakoid protein phosphorylation produces changes in light-harvesting properties and in membrane structure as revealed by freeze-fracture electron microscopy. Protein phosphorylation resulted in an increase in the 77 °K fluorescence signal at 735 nm relative to that at 685 nm. In addition, a decrease in connectivity between Photosystem II centers (PS II) and a dynamic quenching of the room temperature variable fluorescence was observed upon phosphorylation. Accompanying these fluorescence changes was a 23% decrease in the amount of stacked membranes. Microscopic analyses indicated that 8.0-nm particles fracturing on the P-face moved from the stacked into the unstacked regions upon phosphorylation. The movement of the 8.0-nm particles was accompanied by the appearance of chlorophyll b and 25 to 29 kD polypeptides in isolated stroma lamellae fractions. We conclude that phosphorylation of a population of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complexes (LHC) in grana partitions causes the migration of these pigment proteins from the PS II-rich appressed membranes into the Photosystem I (PS I) enriched unstacked regions. This increases the absorptive cross section of PS I. In addition, we suggest that the mobile population of LHC functions to interconnect PS II centers in grana partitions; removal of this population of LHC upon phosphorylation limits PS II → PS II energy transfer and thereby favors spillover of energy from PS II to PS I.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis