The photosynthetic apparatus of C3 and CAM-induced Mesembryanthemum crystallinum L.

Accompanying the CAM induction of Mesembryanthemum crystallinum L. grown in high salinity there are changes in the enzymes of carbon metabolism. However, there are no changes in the electron transport activities, Chla/b ratios or in the distribution of chlorophyll amongst the various pigment-protein complexes of isolated thylakoids. Hence with CAM induction there are no changes in the photochemical apparatus of M. crystallinum thylakoids. Despite comparable amounts of chlorophylla/b-proteins of photosystem II to those found in typical C₃ sun plants, both the C₃ and CAM M. crystallinum chloroplasts have relatively more photosystem II, and, concommitantly, less photosystem I complex. This is consistent with greater fluorescence emission at 685 and 695 nm, and lower emission at 735 nm (measured at 77 K) than typically found for C₃ plants, whether sun or shade species. Photoinhibition of isolated C₃ and CAM thylakoids by white light led to comparable decreases in electron transport capacities and fluorescence emission at 77 K with photosystem II being more affected than PSI. We suggest however, that the presence of more core PSII complexes relative to PSI complexes in this CAM-inducible plant, may provide an additional strategy to mitigate photoinhibition in the short-term.     

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the expolasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 Å. The corresponding histogram for heterocyst thylakoids lacks the 100 Å size class, but has a very large peak at about 55 Å with a shoulder at 75 Å. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 Å for vegetative cells and 64 Å for heterocysts. The thylakoids of both cell types have about 5600 particles/μm² on the P-face. On the E-face, the density drops from 939 particles/μm² on vegetative cell thylakoids to 715 particles/μm² on heterocyst thylakoids. The data suggest that the 100 Å E-face particle of vegetative cell thylakoids is a PSII complex. The 55 Å EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 Å EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.     

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica.

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the exoplasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 A. The corresponding histogram for heterocyst thylakoids lacks the 100 A size class, but has a very large peak at about 55 A with a shoulder at 75 A. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 A for vegetative cells and 64 A for heterocysts. The thylakoids of both cell types have about 5600 particles/micrometers2 on the P-face. On the E-face, the density drops from 939 particles/micrometers2 on vegetative cell thylakoids to 715 particles/micrometers2 on heterocyst thylakoids. The data suggest that the 100 A E-face particle of vegetative cell thylakoids is a PSII complex. The 55 A EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 A EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.     

The State of Photosystem I Complexes in the Central Regions of Pea Granal Thylakoids

Grana-core and grana-margin fragments were obtained from pea (Pisum sativum L.) thylakoids, and both fractions contained photosystem I (PSI) complexes. The yield of these fractions exhibited variations for the plants grown during various periods of the summer season. Low-temperature fluorescence spectra, excitation spectra of long-wave fluorescence, and P700 kinetic characteristics were recorded for these fractions. PSI complexes in central granal regions were associated with PSII and the light-harvesting complexes of PSII, which followed from the excitation spectra of long-wave fluorescence and the kinetic characteristics of P700 light oxidation and dark reduction. The characteristics of the margin regions were changed depending on the fraction yield. If the yield was low, marginal fragments contained mainly PSI complexes. When the yield increased, PSI associates with PSII appeared. A spatial distribution and state of PSI complexes in granal thylakoids are discussed as related to the size and composition of the light-harvesting antenna.     

Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cells known as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocysts contain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cell into a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapse fluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSII degraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The other phycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified by selective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation, and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in the heterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased our understanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments and provided details of the energetic requirements for diazotrophic growth.The most essential pigment-protein complexes for oxygenic photosynthesis are PSI and PSII, which are embedded in the thylakoid membranes of chloroplasts and cyanobacteria. Cooperation between PSI and PSII achieves light-driven noncyclic electron transport from the oxidative splitting of water to the reduction of ferredoxin and is accompanied by the generation of a proton gradient for ATP synthesis. Phycobilisomes (PBS), another pigment-protein complex, are attached to the stromal side of the thylakoid membrane in cyanobacteria and red algae; they work as light-harvesting antennae to transfer electronic excitation energy mainly to PSII and, in some cases, to PSI (Gantt 1994). The integration of these pigment-protein complexes changes in response to light conditions, nutrient status, and developmental stage (Fujita et al., 1994; Grossman et al., 1994; Wolk et al., 1994).Some cyanobacteria, including Anabaena variabilis, are able to grow diazotrophically using the nitrogen-fixing enzyme nitrogenase. Because nitrogenase is sensitive to oxygen, oxygenic photosynthesis is not readily compatible with diazotrophic growth. When this filamentous cyanobacterium is grown under fixed nitrogen-deficient conditions, approximately 1 in 10 to 20 vegetative cells differentiates into a heterocyst, in which oxygenic photosynthesis is suppressed and nitrogenase becomes operative (Haselkorn, 1978; Wolk et al., 1994). The other vegetative cells continue oxygenic photosynthesis. The differentiation of heterocysts from chains of vegetative cells has been studied extensively (Golden and Yoon, 2003; Toyoshima et al., 2010). The abundances of PSII and PBS decrease during the transition. PSI appears to persist in the heterocyst to produce ATP by cyclic electron transport, because nitrogen fixation demands a large amount of ATP (Wolk et al., 1994). However, the mechanisms by which PBS and PSII are degraded during heterocyst differentiation remain unclear, and whether the amount of PSI per cell changes is unknown.The PBS of A. variabilis contain three types of phycobiliproteins, pigment-protein complexes with distinct absorption and fluorescence spectra. The core PBS contains allophycocyanin (APC), which absorbs around 654 nm (Ying and Xie, 1998); the core is most closely connected to PSII. More peripherally in the PBS, the so-called rod contains phycoerythrocyanin (PEC) and phycocyanin (PC), which absorb maximally around 575 and 604 to 620 nm, respectively (Switalski and Sauer, 1984; Zhang et al., 1998). Photon energy is absorbed by PEC, then transferred downhill through PC and APC and finally to PSII. The structure of PBS is probably optimized not only for efficient energy transfer to PSII and/or PSI but also for transformation and/or degradation under various nutrient conditions. However, the order in which these subunits degrade during heterocyst differentiation remains unknown. One strategy to address this question is to isolate heterocysts at several stages during differentiation and quantify their proteomes via mass spectrometry. However, such isolation procedures work well only when there is a good understanding of the properties of cells at different stages. Ideally, noninvasive methods should be used to understand changes in the integrity of PSII and PBS in intact cells in filaments.In principle, time-lapse microscopic observations can clarify the process of differentiation from a vegetative cell into a mature heterocyst. Spectral microscopy is an ideal tool to analyze physiological state and/or amounts of pigment-protein complexes under various conditions. Acquiring microscopic fluorescence spectra of individual cells is a natural extension of laser scanning confocal fluorescence microscopy, which has been applied to several types of cyanobacterial cells, including heterocysts (Peterson et al., 1981; Ying et al., 2002; Wolf and Schüssler, 2005; Kumazaki et al., 2007; Vermaas et al., 2008; Sukenik et al., 2009; Bordowitz and Montgomery, 2010; Collins et al., 2012, Sugiura and Itoh, 2012). Microscopic fluorescence spectra reflect the concentration of pigment-protein complexes and the energy transfer dynamics between photosynthetic pigments. However, to date, there have been no thorough time-lapse investigations of the fluorescence spectra of heterocysts and vegetative cells during the differentiation process.In this study, we investigated the dynamic changes in thylakoid membranes of A. variabilis during heterocyst differentiation. Our unique microscopic system can acquire fluorescence spectra from an entire linearly illuminated region with about 2-nm wavelength resolution in a single exposure (Kumazaki et al., 2007). Heterocyst formation was induced by transferring vegetative cell filaments from fixed-nitrogen-sufficient incubation medium to nitrogen-deprived medium. We conducted long-term observations (60–96 h) on identical filaments. Another unique feature of our setup is that it uses a near-infrared (NIR) excitation laser source. Our previous microspectroscopic study of chloroplasts of a higher plant, maize (Zea mays), and a green alga (Parachlorella kessleri) showed that continuous wave (CW) laser light emitting at 785 to 820 nm excited PSI with high selectivity under the one-photon excitation (OPE) mode. This enabled us to observe highly PSI-rich fluorescence spectra and images with signals around 710 to 740 nm, even at approximately 295 K (Hasegawa et al., 2010, 2011). We used this technique to quantify PSI in individual heterocysts compared with its parental and contiguous vegetative cells. Pigment fluorescence under OPE qualitatively differed from that under two-photon excitation (TPE) using a pulsed NIR laser (typically achieved with picosecond or femtosecond pulses), because TPE using 800 to 830 nm resulted in spectra with contributions from PBS, PSII, and PSI, as typically observed by visible light excitation (Kumazaki et al., 2007; Hasegawa et al., 2010, 2011). The advantages of our microscopic system are the high wavelength resolution and coverage of the entire fluorescence spectrum, the availability of fluorescence spectra at several differentiation stages, and the multiple excitation modes with different selectivities for pigment-protein complexes. Together, these analyses allowed us to characterize spectral decomposition and to understand the time dependence of different pigment-protein complexes, even at a physiological temperature. Microscopic absorption spectra were also obtained from single cells. These data were tentatively used to estimate the absolute concentrations of PSI and PSII in heterocysts and vegetative cells.     

Interaction of phycobilisomes with photosystem II dimers and photosystem I monomers and trimers in the cyanobacterium Spirulina platensis.

Distribution of phycobilisomes between photosystem I (PSI) and photosystem II (PSII) complexes in the cyanobacterium Spirulina platensis has been studied by analysis of the action spectra of H2 and O2 photoevolution and by analysis of the 77 K fluorescence excitation and emission spectra of the photosystems. PSI monomers and trimers were spectrally discriminated in the cell by the unique 760 nm low-temperature fluorescence, emitted by the trimers under reductive conditions. The phycobilisome-specific 625 nm peak was observed in the action spectra of both PSI and PSII, as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 695 nm (PSII), 730 nm (PSI monomers), and 760 nm (PSI trimers). The contributions of phycobilisomes to the absorption, action, and excitation spectra were derived from the in vivo absorption coefficients of phycobiliproteins and of chlorophyll. Analyzing the sum of PSI and PSII action spectra against the absorption spectrum and estimating the P700:P680 reaction center ratio of 5.7 in Spirulina, we calculated that PSII contained only 5% of the total chlorophyll, while PSI carried the greatest part, about 95%. Quantitative analysis of the obtained data showed that about 20% of phycobilisomes in Spirulina cells are bound to PSII, while 60% of phycobilisomes transfer the energy to PSI trimers, and the remaining 20% are associated with PSI monomers. A relevant model of organization of phycobilisomes and chlorophyll pigment-protein complexes in Spirulina is proposed. It is suggested that phycobilisomes are connected with PSII dimers, PSI trimers, and coupled PSI monomers.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

Far-red light-regulated efficient energy transfer from phycobilisomes to photosystem I in the red microalga Galdieria sulphuraria and photosystems-related heterogeneity of phycobilisome population

Phycobilisomes (PBS) are the major photosynthetic antenna complexes in cyanobacteria and red algae. In the red microalga Galdieria sulphuraria, action spectra measured separately for photosynthetic activities of photosystem I (PSI) and photosystem II (PSII) demonstrate that PBS fraction attributed to PSI is more sensitive to stress conditions and upon nitrogen starvation disappears from the cell earlier than the fraction of PBS coupled to PSII. Preillumination of the cells by actinic far-red light primarily absorbed by PSI caused an increase in the amplitude of the PBS low-temperature fluorescence emission that was accompanied by the decrease in PBS region of the PSI 77 K fluorescence excitation spectrum. Under the same conditions, fluorescence excitation spectrum of PSII remained unchanged. The amplitude of P700 photooxidation in PBS-absorbed light at physiological temperature was found to match the fluorescence changes observed at 77 K. The far-red light adaptations were reversible within 2-5min. It is suggested that the short-term fluorescence alterations observed in far-red light are triggered by the redox state of P700 and correspond to the temporal detachment of the PBS antenna from the core complexes of PSI. Furthermore, the absence of any change in the 77 K fluorescence excitation cross-section of PSII suggests that light energy transfer from PBS to PSI in G. sulphuraria is direct and does not occur through PSII. Finally, a novel photoprotective role of PBS in red algae is discussed.     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue–green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.     

Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice.

The stability of chlorophyll-protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO(2) assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll-protein complexes of PSI was greater than in chlorophyll-protein complexes of PSII. The fluorescence yields for all chlorophyll-protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll-protein complex was different and the order for the stability of chlorophyll-protein complexes during leaf senescence was: LHCII>CP43>CP47>LHCI>PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll-protein complexes.     

Correlation of photosystem-II complexes with exoplasmatic freeze-fracture particles of thylakoids of the cyanobacterium Synechococcus sp.

The supramolecular structure of the exoplasmic freeze-fracture particles of thylakoids of the thermophilic cyanobacterium Synechococcus sp. is compared with that of isolated photosystem-II complexes. The in-situ EF particles are scattered on the thylakoids or organized in rows of variable length; the latter aligned particles measure 10 nmx20 nm and are separated perpendicular to their long axis into two parts. We propose that they represent dimers composed of two monomeric 10-nm EF particles side by side. Isolated photosystem (PS)II particles correspond in size to the monomeric 10-nm EF particles as analysed by negative contrast and freeze-fracture electron microscopy. Dimeric PSII particles, very similar to the in-situ 10 nmx20 nm EF particles, are obtained after incorporation of purified PSII complexes into liposomes made from phospholipid and cholesterol. Each monomeric complex consists of the reaction center, the water-splitting system, the chlorophyll antennae and phycobilisome-binding polypeptides. We propose that the dimeric complexes bind one hemidiscoidal phycobilisome at their domains exposed to the external side of the thylakoids. The implications of this arrangement of the PSII-phycobilisome complexes within the thylakoids upon excitation-energy distribution are discussed.Abbreviations EF exoplasmic fracture face - LDS lithium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PS photosystem - SDS sodium dodecyl sulfate - SPC-buffer 0.5 M sucrose, 0.5 M K₂HPO₄/KH₂PO₄, 0.3 M Nacitrate, pH 7.0 This study is dedicated to Professor W. Nultsch on the occasion of his 60th birthday.     

Influence of thylakoid membrane lipids on the structure and function of the plant photosystem II core complex

Main conclusion

MGDG leads to a dimerization of isolated, monomeric PSII core complexes. SQDG and PG induce a detachment of CP43 from the PSII core, thereby disturbing the intrinsic PSII electron transport. The influence of the four thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure and function of isolated monomeric photosystem (PS) II core complexes was investigated. Incubation with the negatively charged lipids SQDG and PG led to a loss of the long-wavelength 77 K fluorescence emission at 693 nm that is associated with the inner antenna proteins. The neutral galactolipids DGDG and MGDG had no or only minor effects on the fluorescence emission spectra of the PSII core complexes, respectively. Pigment analysis, absorption and 77 K fluorescence excitation spectroscopy showed that incubation with SQDG and PG led to an exposure of chlorophyll molecules to the surrounding medium followed by conversion to pheophytin under acidic conditions. Size-exclusion chromatography and polypeptide analysis corroborated the findings of the spectroscopic measurements and pigment analysis. They showed that the negatively charged lipid SQDG led to a dissociation of the inner antenna protein CP43 and the 27- and 25-kDa apoproteins of the light-harvesting complex II, that were also associated with a part of the PSII core complexes used in the present study. Incubation of PSII core complexes with MGDG, on the other hand, induced an almost complete dimerization of the monomeric PSII. Measurements of the fast PSII fluorescence induction demonstrated that MGDG and DGDG only had a minor influence on the reduction kinetics of plastoquinone Q_A and the artificial PSII electron acceptor 2,5-dimethyl-p-benzoquinone (DMBQ). SQDG and, to a lesser extent, PG perturbed the intrinsic PSII electron transport significantly.     

Organization of the Marginal Regions of Pea Granal Thylakoids

Thylakoids of pea chloroplasts isolated from plants grown during various time intervals from June to August were subjected to fragmentation. Using a modified procedure, a fraction of larger particles was separated from those previously considered as fragments of intergranal thylakoids. The particles of the fraction isolated were identified as fragments of marginal regions of granal thylakoids (margins). The relative yield of these fragments depended on the time interval of plant growth. Two types of low-temperature fluorescence spectra corresponding to a high and low yield of the fraction were detected. The characteristics of the first one were a high fluorescence intensity in the short-wave region and the presence of bands with maxima at 687 and 696 nm emitted by photosystem II (PSII). The ratio of PSII to PSI complexes (PSII/PSI) in the fractions characterized by a low and high yield varied from 1 to 5. The analysis of excitation spectra of long-wave fluorescence of PSI showed that PSI complexes in the margin fragments obtained at a low fraction yield were depleted in chlorophyll forms with a 682-nm absorption maximum and enriched in those with a 668-nm maximum. Since an increase in the yield of the margin-fragment fraction is due to an increased unstacking of granal thylakoids, the differences in the characteristics of fragments obtained with a low and a high yield reflect the changes in the composition of granal thylakoids in the direction from the margin to the centrum, that is, a decrease in the relative content of PSI complexes and alterations in the composition and size of its light-harvesting antenna. The consistency between the data obtained and the present view concerning the different functions of PSI located in different thylakoid regions is discussed.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Heat stress induces an inhibition of excitation energy transfer from phycobilisomes to photosystem II but not to photosystem I in a cyanobacterium Spirulina platensis.

The effects of high temperature (30-52.5 degrees C) on excitation energy transfer from phycobilisomes (PBS) to photosystem I (PSI) and photosystem II (PSII) in a cyanobacterium Spirulina platensis grown at 30 degrees C were studied by measuring 77 K chlorophyll (Chl) fluorescence emission spectra. Heat stress had a significant effect on 77 K Chl fluorescence emission spectra excited either at 436 or 580 nm. In order to reveal what parts of the photosynthetic apparatus were responsible for the changes in the related Chl fluorescence emission peaks, we fitted the emission spectra by Gaussian components according to the assignments of emission bands to different components of the photosynthetic apparatus. The 643 and 664 nm emissions originate from C-phycocyanin (CPC) and allophycocyanin (APC), respectively. The 685 and 695 nm emissions originate mainly from the core antenna complexes of PSII, CP43 and CP47, respectively. The 725 and 751 nm band is most effectively produced by PSI. There was no significant change in F725 and F751 during heat stress, suggesting that heat stress had no effects on excitation energy transfer from PBS to PSI. On the other hand, heat stress induced an increase in the ratio of Chl fluorescence yield of PBS to PSII, indicating that heat stress inhibits excitation energy transfer from PBS to PSII. However, this inhibition was not associated with an inhibition of excitation energy transfer from CPC to APC since no significant changes in F643 occurred at high temperatures. A dramatic enhancement of F664 occurring at 52.5 degrees C indicates that excitation energy transfer from APC to the PSII core complexes is suppressed at this temperature, possibly due to the structural changes within the PBS core but not to a detachment of PBS from PSII, resulting in an inhibition of excitation energy transfer from APC to PSII core complexes (CP47 + CP43). A decrease in F685 and F695 in heat-stressed cells with excitation at 436 nm seems to suggest that heat stress did not inhibit excitation energy transfer from the Chl a binding proteins CP47 and CP43 to the PSII reaction center and the decreased Chl fluorescence yields from CP43 and CP47 could be explained by the inhibition of the energy transfer from APC to PSII core complexes (CP47 + CP43).     

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: Characterization of the rbcL deletion mutant of tobacco

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q_A and Q_B, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F₀ ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O₂ in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.     

不同叶龄黄瓜叶片叶绿素蛋白质复合物组分的比较研究

用SDS — 聚丙烯酰胺凝胶电泳的方法,对比分析了老、嫩黄瓜叶片叶绿素蛋白质复合物之间的差异,发现嫩黄瓜叶片中缺少1条属光系统I的CPIb带。从低温荧光发射光谱观察到,嫩黄瓜的光系统I相对高于光系统Ⅱ,而老黄瓜则相反。指出在叶绿体发育过程中首先形成光系统I,以后是光系统Ⅱ。我们还注意到,叶片中的F685/F735比值与叶绿素蛋白质复合物中的单体／寡聚体比值之间呈正相关关系。     

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150–160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.