Evidence for an essential role of carotenoids in the assembly of an active photosystem II

Dark-grown cells of mutant C-6D of the green alga Scenedesmus obliquus exhibit a high activity of photosystem I (PSI) but lack activity of photosystem II (PSII). These cells contain only the pigment-protein complex CPI, representing the reaction-center of PSI. Only chlorophyll a and precursors of carotenoids (lycopene, neurosporene, -carotene, -zeacarotene) could be detected in dark-grown cells by analysis using high-performance liquid chromatography.Activity of PSII and the corresponding pigment-protein complex, CPa, develop immediately upon transfer to light. Light-harvesting complexes and higher molecular forms of PSI are synthesized only in the later stages of light-induced chloroplast differentiation. During illumination the amounts of carotenoid precursors decrease and carotenes, xanthophylls and chlorophylls a and b are formed. -Carotene and lutein are synthesized without a lag-phase. Their kinetics are similar to those of CPa formation and development of PSII activity. In contrast, all other xanthophylls are synthesized only after a lag-phase of about 30 min.Inhibition of the transformation of precursors into carotenoids by nicotine prevents the light-inducible development of PSII activity and CPa formation. During illumination under anaerobic conditions no xanthophylls are synthesized but high amounts of - and -carotene accumulate. Such cells exhibit no PSII activity and show only traces of CPa. After subsequent transfer to aerobic conditions the xanthophylls are synthesized and simultaneously active PSII units are formed.The results prove that carotenoids are essential components for the assembly of active PSII units. Strong evidence is given that lutein is the absolute necessary prerequisite for this process. Whether -carotene is also an absolute necessary prerequisite for a functioning PSII unit cannot be deduced from our experiments.Abbreviations CP pigment-protein complex - HPLC high-performance liquid chromatography - LHCP light-harvesting chlorophyll-protein complex - PAGE polyacrylamide gel electrophoresis - PCV packed cell volume - PS photosystem     

The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is photosystem I,not photosystem II

Maximum quantum yields (QY) of photosynthetic electron flows through PSI and PSII were separately assessed in thylakoid membranes isolated from leaves of Cucumis sativus L. (cucumber) that had been chilled in various ways. The QY(PSI) in the thylakoids prepared from the leaves treated at 4° C in moderate light at 220 mol quanta·m^–2·s^–1 (400–700 nm) for 5 h, was about 20–30% of that in the thylakoids prepared from untreated leaves, while QY(PSII) decreased, at most, by 20% in response to the same treatment. The decrease in QY(PSI) was observed only when the leaves were chilled at temperatures below 10° C, while such a marked temperature dependency was not observed for the decrease in QY(PSII). In the chilling treatment at 4° C for 5 h, the quantum flux density that was required to induce 50% loss of QY (PSI) was ca. 50 umol quanta·m^–2·s^–1. When the chilling treatment at 4° C in the light was conducted in an atmosphere of N₂, photoinhibition of PSI was largely suppressed, while the damage to PSII was somewhat enhanced. The ferricyanide-oxidised minus ascorbate-reduced difference spectra and the light-induced absorbance changes at 700 nm obtained with the thylakoid suspension, indicated the loss of P700 to extents that corresponded to the decreases in QY(PSI). Accordingly, the decreases in QY(PSI) can largely be attributed to destruction of the PSI reaction centre itself. These results clearly show that, at least in cucumber, a typical chillingsensitive plant, PSI is much more susceptible to aerobic photoinhibition than PSII.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - P700 primary electron donor of PSI - PPFD photosynthetically active photon flux density - QY quantum yield We are grateful to invaluable comments by Prof. S. Katoh, K. Hikosaka and the members of our laboratory. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science, and Culture, Japan, to I. Terashima (#03740342 and #04640621).     

Changes in the photosynthetic characteristics and photosystem stoichiometries in wild-type and Chl <Emphasis Type="Italic">b</Emphasis>-deficient mutant rice seedlings under various irradiances

By using a wild-type rice (Oryza sativa L. cv. Norin No. 8) and the chlorophyll (Chl) b-deficient mutant derived from Norin No. 8 (chlorina 11), the present study monitored the oxygen evolution, contents of Chl a and b, β-carotene, and lutein in leaf and the contents of cytochrome f, and the reaction centres of photosystem I (PSI) and photosystem II (PSII) in thylakoids. The oxygen evolution, maximal quantum yield of PSII (F_v/F_m) and Chl concentration remained constant in both Norin No. 8 and chlorina 11 under 5 and 2% of full sunlight for six days. On the other hand, on the thylakoid level, the PSII reaction centre of chlorina 11 was more stable even under high irradiance, while approximately 40% decrease in levels of the PSII reaction centre occurred under 2% of full sunlight for six days. However, under such conditions, by regulating the stoichiometry of active PSII and PSI centres, the light absorption balance in both rice types was adjusted between the two photosystems. The present study attempted to examine whether the light absorption balance between PSII and PSI is altered to effectively conduct photosynthesis in the wild-type and Chl b-deficient mutant rice seedlings.     

Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes

Exposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide ¹⁴C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSI. Immunoblotting analysis of several PSII polypeptides such as D1/D2 reaction dimer and Cyt b₅₅₉ showed no major changes due to mild heat exposure except for the PSII core antenna polypeptide (CP43), which could reflect the reduction in PSII activity observed in light saturation studies. Similarly, haeme staining did not indicate any change in other cytochrome related polypeptides. Our results therefore clearly suggest thatin vivo exposure of rice seedlings to elevated (40 °C) temperature caused thylakoid structural disorganization, and this disorganization of some of the thylakoid complexes resulted in a loss in thylakoid photochemical function.     

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris

Conifers of the boreal zone encounter considerable combined stress of low temperature and high light during winter, when photosynthetic consumption of excitation energy is blocked. In the evergreen Pinus sylvestris L. these stresses coincided with major seasonal changes in photosystem II (PSII) organisation and pigment composition. The earliest changes occurred in September, before any freezing stress, with initial losses of chlorophyll, the D1-protein of the PSII reaction centre and of PSII light-harvesting-complex (LHC II) proteins. In October there was a transient increase in F₀, resulting from detachment of the light-harvesting antennae as reaction centres lost D1. The D1-protein content eventually decreased to 90%, reaching a minimum by December, but PSII photochemical efficiency [variable fluorescence (F_v)/maximum fluorescence (F_m)] did not reach the winter minimum until mid-February. The carotenoid composition varied seasonally with a twofold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreased from 0.9 to 0.1 from October to January. The loss of chlorophyll was complete by October and during winter much of the remaining chlorophyll was reorganised in aggregates of specific polypeptide composition, which apparently efficiently quench excitation energy through non-radiative dissipation. The timing of the autumn and winter changes indicated that xanthophyll de-epoxidation correlates with winter quenching of chlorophyll fluorescence while the drop in photochemical efficiency relates more to loss of D1-protein. In April and May recovery of the photochemistry of PSII, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occurred concomitantly. Indoor recovery of photosynthesis in winter-stressed branches under favourable conditions was completed within 3 d, with rapid increases in F₀, the epoxidation state of the xanthophylls and in light-harvesting polypeptides, followed by recovery of D1-protein content and F_v/F_m, all without net increase in chlorophyll. The fall and winter reorganisation allow Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid recovery of photosynthesis in spring.Abbreviations Elips early light-induced proteins - EPS epoxidation state - F₀ instantaneous fluorescence - F_m maximum fluorescence - F_v variable fluorescence - LHC II light-harvesting complex of PSII - LiDS lithium dodecyl sulfate This research was supported by the Swedish Natural Science Research Council. We wish to thank Dr. Adrian Clarke¹ (Department of Plant Physiology, University of Umeå, Sweden) for advice on electrophoresis, valuable discussion and providing antibodies. Dr. Stefan Jansson¹ and Dr. Torill Hundal (Department for Biochemistry, University of Stockholm, Sweden) provided antibodies. Jan Karlsson¹ helped with the HPLC, Dr. Marianna Krol gave advice on green gels and Dr. Vaughan Hurry (Cooperative Research Centre for Plant Sciences, Australian National University, Canberra, Australia) provided valuable discussion.     

Absence of lutein, violaxanthin and neoxanthin affects the functional chlorophyll antenna size of photosystem-II but not that of photosystem-I in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii double mutant npq2 lor1 lacks the beta, epsilon-carotenoids lutein and loroxanthin as well as all beta,beta-epoxycarotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2 lor1 cells are beta-carotene and zeaxanthin. The effect of these mutations on the photochemical apparatus assembly and function was investigated. In cells of the mutant strain, the content of photosystem-II (PSII) and photosystem-I (PSI) was similar to that of the wild type, but npq2 lor1 had a significantly smaller PSII light-harvesting Chl antenna size. In contrast, the Chl antenna size of PSI was not truncated in the mutant. SDS-PAGE and Western blot analysis qualitatively revealed the presence of all LHCII and LHCI apoproteins in the thylakoid membrane of the mutant. The results showed that some of the LHCII and most of the LHCI were assembled and functionally connected with PSII and PSI, respectively. Photon conversion efficiency measurements, based on the initial slope of the light-saturation curve of photosynthesis and on the yield of Chl a fluorescence in vivo, showed similar efficiencies. However, a significantly greater light intensity was required for the saturation of photosynthesis in the mutant than in the wild type. It is concluded that zeaxanthin can successfully replace lutein and violaxanthin in most of the functional light-harvesting antenna of the npq2 lor1 mutant.     

Characterization of photosystem I in rice (Oryza sativa L.) seedlings upon exposure to random positioning machine

To gain a better understanding of how photosynthesis is adapted under altered gravity forces, photosynthetic apparatus and its functioning were investigated in rice (Oryza sativa L.) seedlings grown in a random positioning machine (RPM). A decrease in fresh weight and dry weight was observed in rice seedlings grown under RPM condition. No significant changes were found in the chloroplast ultrastructure and total chlorophyll content between the RPM and control samples. Analyses of chlorophyll fluorescence and thermoluminescence demonstrate that PSII activity was unchanged under RPM condition. However, PSI activity decreased significantly under RPM condition. 77 K fluorescence emission spectra show a blue-shift and reduction of PSI fluorescence emission peak in the RPM seedlings. In addition, RPM caused a significant decrease in the amplitude of absorbance changes of P700 at 820 nm (A ₈₂₀) induced by saturated far-red light. Moreover, the PSI efficiency (Φ _I) decreased significantly under RPM condition. Immunoblot and blue native gel analyses further illustrate that accumulation of PSI proteins was greatly decreased in the RPM seedlings. Our results suggest that PSI, but not PSII, is down-regulated under RPM condition.     

Freezing of isolated thylakoid membranes in complex media. V. Inactivation and protection of electron transport reactions

When chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were frozen in media containing the predominant inorganic electrolytes of the chloroplast stroma, linear photosynthetic electron transport became progressively inhibited. After onset of freezing, both PSII- and PSI-mediated electron flow were inactivated almost to the same extent. Prolonged storage of the membranes in the frozen state increased damage to PSII relative to PSI activity. Under these conditions, a preferential injury of the water oxidation system was not observed. In thylakoids stored at 0 °C, PSI activity remained fairly unimpaired but inactivation of PSII occurred with strongest inhibition at the oxidizing side.The addition of low-molecular-weight cryoprotectants such as glycerol, sugars, certain amino acids and carbonic acids to thylakoid suspensions prior to freezing provided almost complete preservation of PSI activity and considerable but incomplete stabilization of PSII.Abbreviations BQ 1,4-benzoquinone - Chl chlorophyll - DAD 1,4-diamino-2,3,5,6-tetramethylbenzene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DMBQ 2,5-dimethyl-p-benzoquinone - DPC 1,5-diphenylcarbazide - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - MV methylviologen - PD 1,4-diaminobenzene - SOD superoxide dismutase (EC 1.15.1.1) - TMHQ tetramethyl-p-hydroquinone - TMPD N,N,N,N-tetramethyl-1,4-diaminobenzene - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol Dedicated to Professor Dr. Wilhelm Simonis, Würzburg, on the occasion of his 80th birthday     

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.     

Long-term drought stress induces structural and functional reorganization of photosystem II

Long-term drought stress on photosystem II (PSII) was studied in pea (Pisum sativum L.) seedlings. Drought stress (reduction of water content by 35–80%) led to a considerable depletion of the PSII core, and the remaining PSII complex appeared to be functional and reorganized, with a unit size (LHCP/PSII core) twofold greater than that of well-irrigated plants. By immunoblotting analysis of the PSII proteins from grana and stroma lamellae, the enhanced degradation of CP43 and D1 proteins was observed in water-stressed plants. Also, water stress caused increased phosphorylation of the PSII core and increased D1 protein synthesis. Water-stress-mediated increase in D1 synthesis did not occur when plants were exposed to photoinhibitory light. The depletion of the PSII core was essentially reversed when water-stressed plants grown at low visible irradiance were watered. We suggest that the syndrome caused by the effect of long-term water stress on photosynthesis is a combination of at least two events: a reduction in the number of active PSII centres caused by a physical destabilization of the PSII core and a PSII reorganization with enhanced D1 turnover to counteract the core depletion.Abbreviations Chl chlorophyll - CP43 and CP47 -carotene-Chla-proteins of PSII core - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - Fv/Fm the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centres are closed - LHC(P) light-harvesting complex (proteins) - Wc water content This work was supported by the Italian National Council of Research special grant RAISA, subproject 2 (paper No. 2179) on water stress B. Geiken was supported by the European program Human Capital and Mobility. We thank Dr. Roberto Barbato (Department of Biology, University of Padua, Italy) for generous gifts of various PSII antibodies.     

Chloroplast biogenesis at cold-hardening temperatures. Development of photosystem I and photosystem II activities in relation to pigment accumulation

Chloroplast biogenesis during continuous illumination at either low, cold-hardening temperatures (5°C) or non-hardening temperatures (20°C) was examined by monitoring the etioplast-chloroplast transformation with respect to pigment accumulation and the development of PSI- and PSII-associated electron transport activities in winter rye (Secale cereale L. cv Puma). Generally, chlorophyll and carotenoid accumulation during greening at 20°C were characterized by rapid initial rates in contrast to pronounced, initial lag times during biogenesis at 5°C. Although greening temperature had no effect on the sequential appearance of PSI relative to PSII, greening temperature significantly altered the pattern of appearance of PSI relative to chlorophyll accumulation. Thylakoid biogenesis under continuous illumination at 20°C imposed a pattern whereby the development of PSI activity was antiparallel to chlorophyll accumulation. In contrast, the development of PSI activity under continuous illumination at 5°C was paralllel to chlorophylll accumulation. These developmental patterns were independent of the temperature experienced during etiolation. However, rye seedlings etiolated at 20°C and subsequently subjected to continuous illumination at 5°C exhibited a 70% reduction in the maximum PSII activity (100 mol DCPIP reduced.mg Chl^-1.h^-1) attained relative to that observed for similar etiolated seedlings greened at 20°C (300 mol DCPIP reduced.mg Chl^-1.h^-1). This low temperature-induced inhibition could be alleviated by an initial 2 h exposure to continuous light at 20°C prior to greening to 5°C. Rye seedlings etiolated at 5°C attained similar maximal PSII activities (300 mol DCPIP reduced.mg Chl^-1.h^-1) regardless of the greening temperature. We suggest that the altered kinetics for pigment accumulation, the low temperature-induced change in the pattern for the appearance of PSI activity relative to chlorophyll accumulation and the differential sensitivity of 20° and 5° etiolated seedlings to greening temperature reflect an alteration in membrane organization incurred as a consequence of thylakoid assembly at low temperature.Abbreviations RH cold-hardened rye - RNH non-hardened rye - MV methylviologen - ASC ascorbate - Chl chlorophyll - DCPIP dichlorophenol indophenol     

Phosphatidylglycerol is essential for oligomerization of photosystem I reaction center

Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center.     

Relationship between biosynthesis of carotenoids and increasing complexity of photosystem I in mutant C-6D of Scenedesmus obtiquus

Dark-grown cells of the pigment mutant C-6D of Scenedesmus obliquus, strain D₃ (Gaffron 1939), contain only chlorophyll (Chl) a and carotenoid precursors. In these cells a functioning photosystem I (PSI) of basic structure was characterised by a high PSI activity and a low Chl/P700 ratio. The reaction-center complex of PSI (CPI) was shown to exist in the dark-grown cells. These findings demonstrate that the assembly of the core complex of PSI and its function are independent of the presence of carotenoids. Upon illumination, carotenoids, Ch1 b and additional Chl a were synthesized. Newly formed -carotene was shown by pigment analysis using high-performance liquid chromatography (HPLC) to be incorporated into CPI. Parallel to this process a shift of the long-wavelength fluorescence emission of PSI from 712–714 to 718–719 nm was observed. In the later stages of chloroplast differentiation, when xanthophylls and Chl b were synthesized, a higher-molecular-weight complex of PSI (CPIa) could be isolated. Pigment analysis demonstrated that CPIa contained xanthophylls and Chl b in addition to Chl a and -carotene. This indicates the formation of a light-harvesting antenna closely associated with PSI (LHCI). The addition of an LHCI to the reaction-center complex of PSI caused an increase in the absorption cross-section of PSI as shown by action spectroscopy and in-vivo fluorescence measurements. A model demonstrating the changes in the molecular organization of PSI during light-induced carotenoid biosynthesis in mutant C-6D of Scenedesmus obliquus is presented.Abbreviations Chl chlorophyll - CP chlorophyll-protein complex - LHC light-harvesting complex - HPLC high-performance liquid chromatography - PSI, II photosystem I, II - PAGE polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft and a scholarship of the Studienstiftung des deutschen Volkes to S. Römer. We thank Ms. K. Bölte for technical assistance and Mr. H. Becker for drafting the figures.     

Changes in the Carotenoid Pattern During the Synchronous Life Cycle of Scenedesmus

The wild type (WT) of Scenedesmus obliquus and a mutant lacking chlorophyll b and the light-harvesting complexes (WT-LHC₁) were synchronized by a light-dark regime. Both cultures contained the same type of carotenoids. However, concentrations and patterns of carotenoids were different during their synchronous life cycles. The concentration of total carotenoids followed more or less that of chlorophyll. The WT contained more carotenoids per cell mass, but slightly less per chlorophyll. It is discussed that part of the carotenoids of the mutant, lacking the peripheral antenna of PSII, might be located in the chlorophyll b-less apoprotein or in an enlarged core antenna of PSII. During the life cycle of Scenedesmus the carotenes are initially synthesized and most of the α-carotene is immediately oxidized to lutein which is inserted in the antennae systems of PSII and PSI. The further oxidation of lutein to loroxanthin seems to depend on both the change from dark to light, and on stages of the life cycle itself. Although the major part of β-carotene appears to be inserted in the reaction centers, a fraction of the total pool is rapidly converted to violaxanthin, following the onset of illumination. The conversion may serve to protect against photooxidation. Further conversion of violaxanthin to neoxanthin occurs to a greater extent in the mutant, WT-LHC₁. The results demonstrate (1) the close connection between the carotenoid pattern and the modulation of the photosynthetic apparatus during the life cycle of Scenedesmus and (2) the flexibility of the organism in compensating for the absence of the light-harvesting complexes of photosystems II by adjusting the carotenoid distribution.     

Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O₂ uptake rates, P700 photooxidation and the accumulation of superoxide anions (O₂ ⁻) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O₂ ⁻ generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O₂ ⁻ generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.     

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.     

Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize

Interorganellar signaling interactions are poorly understood. The maize non-chromosomal stripe (NCS) mutants provide models to study the requirement of mitochondrial function for chloroplast biogenesis and photosynthesis. Previous work suggested that the NCS6 mitochondrial mutation, a cytochrome oxidase subunit 2 (cox2) deletion, is associated with a malfunction of Photosystem I (PSI) in defective chloroplasts of mutant leaf sectors (Gu et al., 1993). We have now quantified the reductions of photosynthetic rates and PSI activity in the NCS6 defective stripes. Major reductions of the plastid-coded PsaC and nucleus-coded PsaD and PsaE PSI subunits and of their corresponding mRNAs are seen in mutant sectors; however, although thepsaA/B mRNA is greatly reduced, levels of PsaA and PsaB (the core proteins of PSI) are only slightly decreased. Levels of the PsaL subunit and its mRNA appear to be unchanged. Tested subunits of other thylakoid membrane complexes – PSII, Cyt b₆/f, and ATP synthase, have minor (or no) reductions within mutant sectors. The results suggest that specific signaling pathways sense the dysfunction of the mitochondrial electron transport chain and respond to down-regulate particular PSI mRNAs, leading to decreased PSI accumulation in the chloroplast. The reductions of both nucleus and plastid encoded components indicate that complex interorganellar signaling pathways may be involved.