The survival of the terrestrial cyanobacterium Nostoc sp. HK-01 was tested as part of the Tanpopo mission experiment, which was conducted both outside and inside the International Space Station (ISS). The selection of Nostoc sp. HK-01 was based on the results of on-ground experiments that demonstrated that the cyanobacterium can survive simulated space environments. This study verified cell survival after exposure to the outside environment in low Earth orbit (LEO). We examined the cellular tolerance of Nostoc sp. HK-01 simultaneously outside and inside of the ISS over a 3-year period. After the experiments were conducted, we confirmed cell viability by fluorescein diacetate (FDA). Cell growth abilities for 3 years without sunlight in space-vacuum-exposed cells were not significantly different from those of cells kept in the dark of control cells in the ISS and on the ground. Though a few light-exposed cells in space vacuum survived outside the ISS after 3 years as judged by FDA staining assay, the survival could not be verified by testing the growth ability due to an insufficient number of cells. To the best of our knowledge, this is the first pure strain of Nostoc sp. HK-01 that survived in a space environment on the inside and outside of the ISS with and without sunlight for more than 3 years (1126 days).

<title>Abstract</title>
NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the co-activator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PII  per se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which phenotype was not observed in the mutant defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the PSII photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain photosynthetic capacity.

Mesembryanthemum crystallinum, which switches the mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) upon high salt stress, was shown here to exhibit diurnal changes in not only the CO2 fixation pathway but also Chl fluorescence parameters under CAM-induced conditions. We conducted comprehensive time course measurements of M. crystallinum leaf Chl fluorescence using the same leaf throughout the CAM induction period. By doing so, we were able to distinguish the effect of CAM induction from that of photoinhibition and avoid the possible effects of differences in foliar age. We found that the diurnal change in the status of electron transfer could be ascribed to the formation of a proton gradient across thylakoid membranes presumably resulting from diurnal changes in the ATP/ADP ratio reported earlier. The electron transport by actinic illumination thus became limited at the step of plastoquinol oxidation by the Cyt b6/f complex in the 'night' period upon CAM induction, resulting in high levels of non-photochemical quenching. The actinically induced non-photochemical quenching in the 'night' period correlated well with the degree of CAM induction. Chl fluorescence parameters, such as NPQ or qN, could be used as a simple indexing system for the CAM induction.

Cyanobacteria are photosynthetic prokaryotes and widely used for photosynthetic research as model organisms. Partly due to their prokaryotic nature, however, estimation of photosynthesis by chlorophyll fluorescence measurements is sometimes problematic in cyanobacteria. For example, plastoquinone pool is reduced in the dark-acclimated samples in many cyanobacterial species so that conventional protocol developed for land plants cannot be directly applied for cyanobacteria. Even for the estimation of the simplest chlorophyll fluorescence parameter, F (v)/F (m), some additional protocol such as addition of DCMU or illumination of weak blue light is necessary. In this review, those problems in the measurements of chlorophyll fluorescence in cyanobacteria are introduced, and solutions to those problems are given.

Structural variation in the stroma-grana (SG) arrangement of the thylakoid membranes, such as changes in the thickness of the grana stacks and in the ratio between grana and inter-grana thylakoid, is often observed. Broadly, such alterations are considered acclimation to changes in growth and the environment. However, the relation of thylakoid morphology to plant growth and photosynthesis remains obscure. Here, we report changes in the thylakoid during leaf development under a fixed light condition. Histological studies on the chloroplasts of fresh green Arabidopsis leaves have shown that characteristically shaped thylakoid membranes lacking the inter-grana region, referred to hereafter as isolated-grana (IG), occurred adjacent to highly ordered, large grana layers. This morphology was restored to conventional SG thylakoid membranes with the removal of bolting stems from reproductive plants. Statistical analysis showed a negative correlation between the incidences of IG-type chloroplasts in mesophyll cells and the rates of leaf growth. Fluorescence parameters calculated from pulse-amplitude modulated fluorometry measurements and CO2 assimilation data showed that the IG thylakoids had a photosynthetic ability that was equivalent to that of the SG thylakoids under moderate light. However, clear differences were observed in the chlorophyll a/b ratio. The IG thylakoids were apparently an acclimated phenotype to the internal condition of source leaves. The idea is supported by the fact that the life span of the IG thylakoids increased significantly in the later developing leaves. In conclusion, the heterogeneous state of thylakoid membranes is likely important in maintaining photosynthesis during the reproductive phase of growth.

Glaucophytes are primary symbiotic algae with unique plastids called cyanelles, whose structure is most similar to ancestral cyanobacteria among plastids in photosynthetic organisms. Here we compare the regulation of photosynthesis in glaucophyte with that in cyanobacteria in the aim of elucidating the changes caused by the symbiosis in the interaction between photosynthetic electron transfer and other metabolic pathways. Chlorophyll fluorescence measurements of the glaucophyte Cyanophora paradoxa NIES-547 indicated that plastoquinone (PQ) pool in photosynthetic electron transfer was reduced in the dark by chlororespiration. The levels of nonphotochemical quenching of chlorophyll fluorescence was high in the dark but decreased under low light, and increased again under high light. This type of concave light dependence was quite similar to that observed in cyanobacteria. Moreover, the addition of ionophore hardly affected nonphotochemical quenching, suggesting state transition as a main component of the regulatory system in C. paradoxa. These results suggest that cyanelles of C. paradoxa retain many of the characteristics observed in their ancestral cyanobacteria. From the viewpoint of metabolic interactions, C. paradoxa is the primary symbiotic algae most similar to cyanobacteria than other lineages of photosynthetic organisms.

Although the photosynthetic reaction center is well conserved among different cyanobacterial species, the modes of metabolism, e.g. respiratory, nitrogen and carbon metabolism and their mutual interaction, are quite diverse. To explore such uniformity and diversity among cyanobacteria, here we compare the influence of the light environment on the condition of photosynthetic electron transport through Chl fluorescence measurement of six cyanobacterial species grown under the same photon flux densities and at the same temperature. In the dark or under weak light, up to growth light, a large difference in the plastoquinone (PQ) redox condition was observed among different cyanobacterial species. The observed difference indicates that the degree of interaction between respiratory electron transfer and photosynthetic electron transfer differs among different cyanobacterial species. The variation could not be ascribed to the phylogenetic differences but possibly to the light environment of the original habitat. On the other hand, changes in the redox condition of PQ were essentially identical among different species at photon flux densities higher than the growth light. We further analyzed the response to high light by using a typical energy allocation model and found that 'non-regulated' thermal dissipation was increased under high-light conditions in all cyanobacterial species tested. We assume that such 'non-regulated' thermal dissipation may be an important 'regulatory' mechanism in the acclimation of cyanobacterial cells to high-light conditions.

Estimation of photosynthesis by Chl fluorescence measurement of cyanobacteria is always problematic due to the interference from respiratory electron transfer and from phycocyanin fluorescence. The interference from respiratory electron transfer could be avoided by the use of DCMU or background illumination by blue light, which oxidizes the plastoquinone pool that tends to be reduced by respiration. On the other hand, the precise estimation of photosynthesis in cells with a different phycobilisome content by Chl fluorescence measurement is difficult. By subtracting the basal fluorescence due to the phycobilisome and PSI, it becomes possible to estimate the precise maximum quantum yield of PSII in cyanobacteria. Estimated basal fluorescence accounted for 60% of the minimum fluorescence, resulting in a large difference between the 'apparent' yield and 'true' yield under high phycocyanin conditions. The calculated value of the 'true' maximum quantum yield of PSII was around 0.8, which was similar to the value observed in land plants. The results suggest that the cause of the apparent low yield reported in cyanobacteria is mainly ascribed to the interference from phycocyanin fluorescence. We also found that the 'true' maximum quantum yield of PSII decreased under nitrogen-deficient conditions, suggesting the impairment of the PSII reaction center, while the 'apparent' maximum quantum yield showed a marginal change under the same conditions. Due to the high contribution of phycocyanin fluorescence in cyanobacteria, it is essential to eliminate the influence of the change in phycocyanin content on Chl fluorescence measurement and to evaluate the 'true' photosynthetic condition.

The pmgA-disrupted (Delta pmgA) mutant in the cyanobacterium Synechocystis sp. PCC 6803 suffers severe growth inhibition under photomixotrophic conditions. In order to elucidate the key factors enabling the cells to grow under photomixotrophic conditions, we isolated spontaneous suppressor mutants from the Delta pmgA mutant derived from a single colony. When the Delta pmgA mutant was spread on a BG11 agar plate supplemented with glucose, colonies of suppressor mutants appeared after the bleaching of the background cells. We identified the mutation site of these suppressor mutants and found that 11 mutants out of 13 had a mutation in genes related to the type 1 NAD(P)H dehydrogenase (NDH-1) complex. Among them, eight mutants had mutations within the ndhF3 (sll1732) gene: R32stop, W62stop, V147I, G266V, G354W, G586C, and deletion of 7 bp within the coding region. One mutant had one base insertion in the putative -10 box of the ndhC (slr1279) gene, leading to the decrease in the transcripts of the ndhCKJ operon. Two mutants had one base insertion and deletion in the coding region of cupA (sll1734), which is co-transcribed with ndhF3 and ndhD3 and comprises together a form of NDH-1 complex (NDH-1MS complex) involved in inducible high-affinity CO2 uptake. The results indicate that the loss of the activity of this complex effectively rescues the Delta pmgA mutant under photomixotrophic condition with 1 % CO2. However, little difference among WT and mutants was observed in the activities ascribed to the NDH-1MS complex, i.e., CO2 uptake and cyclic electron transport. This may suggest that the NDH-1MS complex has the third, currently unknown function under photomixotrophic conditions.

Background: Most organisms, especially photoautotrophs, alter their behaviours in response to day-night alternations adaptively because of their great reliance on light. Upon light-to-dark transition, dramatic and universal decreases in transcription level of the majority of the genes in the genome of the unicellular cyanobacterium, Synechococcus elongatus PCC 7942 are observed. Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.
Results: However, against this general assumption, we obtained evidence that the rapid and dynamic dark repression is an active process. Although the addition of photosynthesis inhibitors to cells exposed to light mimicked transcription profiles in the dark, it did not significantly affect the cellular level of ATP. By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation. This observation indicates that Synechococcus actively downregulates genome-wide transcription in the dark. Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.
Conclusions: Dark repression appears to enable cells to enter into nocturnal dormancy as a feed-forward process, which would be advantageous for their survival under periodic nocturnal conditions.

Carotenoids are important components of antioxidative systems in photosynthetic organisms. We investigated the roles of zeaxanthin and echinenone in the protection of PSII from photoinhibition in Synechocystis sp. PCC 6803, using mutants of the cyanobacterium that lack these carotenoids. The activity of PSII in mutant cells deficient in either zeaxanthin or echinenone was more sensitive to strong light than the activity in wild-type cells, and the activity in mutant cells deficient in both carotenoids was hypersensitive to strong light, indicating that the absence of these carotenoids increased the extent of photoinhibition. Nonetheless, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, was unaffected by the absence of either carotenoid, suggesting that these carotenoids might act by protecting the repair of PSII. Knockout of the gene for the so-called orange carotenoid protein (OCP), in which the 3'-hydroxyechinenone cofactor, a derivative of echinenone, is responsible for the thermal dissipation of excitation energy, increased the extent of photoinhibition but did not affect photodamage, suggesting that thermal dissipation also protects the repair of PSII. In mutant cells lacking OCP, as well as those lacking zeaxanthin and echinenone, the production of singlet oxygen was stimulated and the synthesis de novo of various proteins, including the D1 protein, was markedly suppressed under strong light. These observations suggest that the carotenoids and thermal dissipation might protect the repair of photodamaged PSII by depressing the levels of singlet oxygen that inhibits protein synthesis.

In cyanobacteria, photosynthesis and respiration share some components of electron transport chain. To explore the interaction between photosynthesis and respiration, we monitored the change in the yield of chlorophyll fluorescence due to state transition in ndh genes disruptants, deficient in NAD(P)H dehydrogenase (NDH-1) complexes serving for respiration or for carbon concentrating mechanism (CCM). The disruption of ndh genes essential for respiration resulted in low levels of chlorophyll fluorescence quenching in the dark (NPQ(Dark)) as well as in the low light (NPQ(LL)). The lowered NPQ(Dark) and NPQ(LL) in these ndh genes disruptants could be ascribed to the oxidation of the PQ pool due to the poor electron supply from NDH-1 complexes in respiratory electron transport. On the other hand, only NPQ(LL) decreased upon disruption of the ndh genes essential for CCM. We propose that, in the disruptants of these ndh genes, the PQ pool is oxidized in the light through the increased photosystem I content, resulting in the lowered NPQ(LL). Apparently, the two different subsets of ndh genes affect photosynthetic electron transport although in totally different manners. It is also suggested that monitoring state transition is a simple method to evaluate the condition of photosynthesis, respiration and CCM. (C) 2015 Elsevier B.V. All rights reserved.

HvLhcb1 a major light-harvesting chlorophyll a/b-binding protein in barley, is a critical player in sustainable growth under Fe deficiency. Here, we demonstrate that Fe deficiency induces phosphorylation of HvLhcb1 proteins leading to their migration from grana stacks to stroma thylakoid membranes. HvLhcbl remained phosphorylated even in the dark and apparently independently of state transition, which represents a mechanism for short-term acclimation. Our data suggest that the constitutive phosphorylation-triggered translocation of HvLhcbl under Fe deficiency contributes to optimization of the excitation balance between photosystem II and photosystem I, the latter of which is a main target of Fe deficiency. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

In Synechocystis sp. PCC 6803, the disruption of the ndhF1 gene (slr0844), which encodes a subunit of one of the NDH-1 complexes (NDH-1L complex) serving for respiratory electron transfer, causes the largest change in Chl fluorescence induction kinetics among the kinetics of 750 disruptants searched in the Fluorome, the cyanobacterial Chl fluorescence database. The cause of the explicit phenotype of the ndhF1 disruptant was examined by measurements of the photosynthetic rate, Chl fluorescence and state transition. The results demonstrate that the defects in respiratory electron transfer obviously have great impact on Chl fluorescence in cyanobacteria. The inactivation of NDH-1L complexes involving electron transfer from NDH-1 to plastoquinone (PQ) would result in the oxidation of the PQ pool, leading to the transition to State 1, where the yield of Chl fluorescence is high. Apparently, respiration, although its rate is far lower than that of photosynthesis, could affect Chl fluorescence through the state transition as leverage. The disruption of the ndhF1 gene caused lower oxygen-evolving activity but the estimated electron transport rate from Chl fluorescence measurements was faster in the mutant than in the wild-type cells. The discrepancy could be ascribed to the decreased level of non-photochemical quenching due to state transition. One must be cautious when using the Chl fluorescence parameter to estimate photosynthesis in mutants defective in state transition. © 2013 The Author.

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1-2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient-sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi-starved mgd1-2 leaves, biogenesis of thylakoid-like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress-induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light-harvesting/ photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1-2 mutant. Moreover, the reduced expression of nuclear-and plastid-encoded photosynthetic genes observed in the mgd1-2 mutant under Pi-sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid- and nuclear-encoded photosynthetic genes, independently of photosynthesis.

Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1-and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation.

The photoinhibition of Photosystem I (PSI) drew less attention compared with that of Photosystem II (PSII). This could be ascribed to several reasons, e.g. limited combinations of plant species and environmental conditions that cause PSI photoinhibition, the non-regulatory aspect of PSI photoinhibition, and methodological difficulty to determine the accurate activity of PSI under stress conditions. However, the photoinhibition of PSI could be more dangerous than that of PSII because of the very slow recovery rate of PSI. This article is intended to introduce such characteristics of PSI photoinhibition with special emphasis on the relationship between two photosystems as well as the protective mechanism of PSI in vivo. Although the photoinhibition of PSI could be induced only in specific conditions and specific plant species in intact leaves, PSI itself is quite susceptible to photoinhibition in isolated thylakoid membranes. PSI seems to be well protected from photoinhibition in vivo in many plant species and many environmental conditions. This is quite understandable because photoinhibition of PSI is not only irreversible but also the potential cause of many secondary damages. This point would be different from the case of PSII photoinhibition, which could be regarded as one of the regulatory mechanisms under stressed as well as non-stressed conditions.

Because of the high demand for iron of the photosynthetic apparatus in thylakoid membranes, iron deficiency primarily affects the electron transfer between the two photosystems (PSI and PSII), resulting in photooxidative damage in plants. However, in barley, PSII is protected against photoinhibition, and the plant survives even with a low iron content in its chlorotic leaves. In this study, we report an adaptation mechanism of the photosynthetic apparatus in barley to iron deficiency, which is concomitant with the remodeling of a PSII antenna system. Transcriptome analysis revealed that long-term iron deficiency induced the expression of two genes of the major light-harvesting Chl a/b-binding protein of PSII (LHCII), namely HvLhcb1.11 and HvLhcb1.12. Chl fluorescence analysis of the wild type and Lhcb1-less chlorina mutants clearly showed that non-photochemical quenching (NPQ) of the wild type was increased by approximately 200% by iron deficiency, whereas NPQ of chlorina mutants did not change significantly under iron deficiency. The mutant showed severe photodamage in young leaves under prolonged iron deficiency, suggesting that the HvLhcb1 protein is essential for both thermal dissipation and photoprotection in iron-deficient barley. Analysis of thylakoid protein complexes revealed that the proportion of the monomeric form of Lhcb1 significantly increased in barley grown under iron-deficient conditions. We hypothesize that alteration of the HvLhcb1 subpopulations modifies the organization of LHCII in the thylakoid membranes, which is a key step for thermal dissipation to compensate for excess excitation energy and thereby protect the photosystems from serious damage in iron-deficient barley leaves.

We developed here the quantitative and objective method to analyze chlorophyll fluorescence from the cyanobacterium Synechocystis sp. PCC 6803 in the aim of systematic examination of gene function. The overall similarity of the chlorophyll fluorescence induction kinetics was evaluated for 499 mutants. Mutants of 333 genes showed the difference in the fluorescence kinetics from that of wild type, indicating the wide interaction of photosynthesis with other metabolisms. Hierarchical clustering of the similarity of the mutants enables us to group together the mutants having defect in the regulation of photosystem stoichiometry as well as those having defects in respiration or other functions. Furthermore, wild-type cells treated with inhibitors of respiration and mutants of genes involved in respiration shared similar induction kinetics. Apparently, quantitative comparison of the induction kinetics could be useful to analyze the function of genes as well as to predict the target sites of various chemicals.

Chloroplasts are descendents of a cyanobacterial endosymbiont, but many chloroplast protein genes of endosymbiont origin are encoded by the nucleus. The chloroplastcyanobacteria relationship is a typical target of orthogenomics, an analytical method that focuses on the relationship of orthologous genes. Here, we present results of a pilot study of functional orthogenomics, combining bioinformatic and experimental analyses, to identify nuclear-encoded chloroplast proteins of endosymbiont origin (CPRENDOs). Phylogenetic profiling based on complete clustering of all proteins in 17 organisms, including eight cyanobacteria and two photosynthetic eukaryotes, was used to deduce 65 protein groups that are conserved in all oxygenic autotrophs analyzed but not in non-oxygenic organisms. With the exception of 28 well-characterized protein groups, 56 Arabidopsis proteins and 43 Synechocystis proteins in the 37 conserved homolog groups were analyzed. Green fluorescent protein (GFP) targeting experiments indicated that 54 Arabidopsis proteins were targeted to plastids. Expression of 39 Arabidopsis genes was promoted by light. Among the 40 disruptants of Synechocystis, 22 showed phenotypes related to photosynthesis. Arabidopsis mutants in 21 groups, including those reported previously, showed phenotypes. Characteristics of pulse amplitude modulation fluorescence were markedly different in corresponding mutants of Arabidopsis and Synechocystis in most cases. We conclude that phylogenetic profiling is useful in finding CPRENDOs, but the physiological functions of orthologous genes may be different in chloroplasts and cyanobacteria.

The water-water cycle is the electron flow through scavenging enzymes for the reactive species of oxygen in chloroplasts, and is proposed to play a role in alternative electron sink in photosynthesis. Here we showed that the water-water cycle is impaired in the T-DNA insertion mutant of AtHMA1 gene encoding a Cu transporting ATPase in chloroplasts. Chlorophyll fluorescence under steady state was not affected in hma1, indicating that photosynthetic electron transport under normal condition was not impaired. Under electron acceptor limited conditions, however, hma1 showed distinguished phenotype in chlorophyll fluorescence characteristics. The most severe phenotype of hma1 could be observed in high (0.1%) CO(2) concentrations, indicating that hma1 has the defect other than photorespiration. The transient increase of chlorophyll fluorescence upon the cessation of the actinic light as well as the NPQ induction of chlorophyll fluorescence revealed that the two pathways of cyclic electron flow around PSI, NDH-pathway and FQR-pathway, are both intact in hma1. Based on the NPQ induction under 0% oxygen condition, we conclude that the water-water cycle is impaired in hma1, presumably due to the decreased level of Cu/Zn SOD in the mutant. Under high CO(2) condition, hma1 exhibited slightly higher NPQ induction than wild type plants, while this increase of NPQ in hma1 was suppressed when hma1 was crossed with crr2 having a defect in NDH-mediated PSI cyclic electron flow. We propose that the water-water cycle and NDH-mediated pathways might be regulated compensationally with each other especially when photorespiration is suppressed. (C) 2008 Elsevier B.V. All rights reserved.

Downregulation of photosystem I (PSI) content is an essential process for cyanobacteria to grow under high-light (HL) conditions. In a pmgA (sll 968) mutant of Synechocystis sp. PCC 6803, the levels of PSI content, chlorophyll and transcripts of the psaAB genes encoding reaction-centre subunits of PSI could not be maintained low during HL incubation, although the causal relationship among these phenotypes remains unknown. In this study, we modulated the activity of psaAB transcription or that of chlorophyll synthesis to estimate their contribution to the regulation of PSI content under HL conditions. Analysis of the psaAB-OX strain, in which the psaAB genes were overexpressed under HL conditions, revealed that the amount of psaAB transcript could not affect PSI content by itself. Suppression of chlorophyll synthesis by an inhibitor, laevulinic acid, in the pmgA mutant revealed that chlorophyll availability could be a determinant of PSI content under HL. It was also suggested that chlorophyll content under HL conditions is mainly regulated at the level of 5-aminolaevulinic acid synthesis. We conclude that, upon the shift to HL conditions, activities of psaAB transcription and of 5-aminolaevulinic acid synthesis are strictly downregulated by regulatory mechanism(s) independent of PmgA during the first 6 h, and then a PmgA-mediated regulatory mechanism becomes active after 6 h onward of HL incubation to maintain these activities at a low level.

The sll1961 gene was reported to encode a regulatory factor of photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803. We here show that the sll1961 gene is also essential for the phycobilisome degradation during nitrogen starvation. The defect in phycobilisome degradation was observed in the sll1961 mutant despite the increased expression of nhlA, a gene involved in phycobilisome degradation during nitrogen starvation. Photosystem stoichiometry is not affected by nitrogen starvation in the sll1961 mutant nor in the wild-type. The results indicate the presence of a novel pathway for phycobilisome degradation control independent of nblA expression. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sigma factors encoded by the nucleus of plants confer promoter specificity on the bacterial-type RNA polymerase in chloroplasts. We previously showed that transcripts of OsSIG1, which encodes one such sigma factor in rice, accumulate relatively late during leaf development. We have now isolated and characterized two allelic mutants of OsSIG1, in which OsSIG1 is disrupted by insertion of the retrotransposon Tos17, in order to characterize the functions of OsSIG1. The OsSIG1(-/-) plants were found to be fertile but they manifested an approximately one-third reduction in the chlorophyll content of mature leaves. Quantitative RT-PCR and northern blot analyses of chloroplast gene expression revealed that the abundance of transcripts derived from the psaA operon was markedly reduced in OsSIG1(-/-) plants compared with that in wild-type homozygotes. This effect was accompanied by a reduction in the abundance of the core protein complex (PsaA-PsaB) of photosystem I. Analysis of chlorophyll fluorescence also revealed a substantial reduction in the rate of electron transfer from photosystem II to photosystem I in the OsSIG1 mutants. Our results thus indicate that OsSIG1 plays an important role in the maintenance of photosynthetic activity in mature chloroplasts of rice by regulating expression of chloroplast genes for components of photosystem I.

Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin - Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c(6), which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3- fold those in the wild- type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2- fold those in the wild- type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3- fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c(6), the 1 - qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c(6). Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c(6) gene.

Two ecotypes of Japanese beech (Fagus crenata Blume), the Pacific Ocean type (PAO) and the Japan Sea type (JAS), show different responses to high solar irradiance. When PAO and JAS saplings were grown in continuous high-light (H), leaves of JAS became pale green. To elucidate this phenomenon, we investigated in vivo photochemistry based on pigment concentrations of Photosystem (PS) I and PS II and Western blot analysis. In JAS-H leaves, the amount of D1-protein decreased, resulting in decreases in the maximal quantum yield of PS II (F-v/F-m) and electron transport rate, whereas PAO-H leaves maintained high activities. The PS I photochemistry determined by measurement of P-700 photo-oxidation showed that the intersystem electron pool size was 1.4 times greater in JAS-H leaves than in PAO-H leaves. Furthermore, the re-reduction kinetics of P-700(+) showed that cyclic electron transport around PS I was 1.2 times faster in PAO-H leaves than in JAS-H leaves. Analysis of the area over the fluorescence induction kinetics indicated that the relative abundance of the PS II alpha center increased in PAO-H leaves, whereas JAS leaves were observed to have low acclimation capacity to high light. These results demonstrate that PAO leaves possess acclimation mechanisms to continuous high light, whereas JAS leaves are more vulnerable to continuous high light, resulting in reduced leaf longevity owing to photoinhibition caused by increases in the intersystem electron pool size and suppression of photochemistry at the level of PS I and PS II.

Since chlorophyll fluorescence reflects the redox state of photosynthetic electron transport chain, monitoring of chlorophyll fluorescence has been successfully applied for the screening of photosynthesis-related genes. Here we report that the mutants having a defect in the regulation of photosystem stoichiometry could be identified through the simple comparison of the induction kinetics of chlorophyll fluorescence. We made a library containing 500 mutants in the cyanobacterium Synechocystis sp. PCC 6803 with transposon-mediated gene disruption, and the mutants were used for the measurement of chlorophyll fluorescence kinetics for 45 s. We picked up two genes, pmgA and sll1961, which are involved in the modulation of photosystem stoichiometry. The disruptants of the two genes share common characteristics in their fluorescence kinetics, and we searched for mutants that showed such characteristics. Out of six mutants identified so far, five showed a different photosystem stoichiometry under high-light conditions. Thus, categorization based on the similarity of fluorescence kinetics is an excellent way to identify the function of genes.

Photosynthetic parameters of the nadk2 mutant of Arabidopsis thaliana, which is defective in chloroplast NAD kinase, were investigated. In this plant, the effective efficiency of photosynthetic electron transport (Phi II) and the quantum yield of open reaction centers of photosystem II (Fv'/Fm') were decreased. Furthermore, an increase in nonphotochemical quenching attributed to energy dissipation from the xanthophyll cycle was observed. The mutant showed an aberrant de-epoxidation state of xanthophyll cycle carotenoids and had a high level of zeaxanthin even under low light conditions. These results indicate that chloroplast NAD kinase, catalyzing phosphorylation of NAD, is essential for the proper photosynthetic machinery of PSII and the xanthophyll cycle.

One of the most powerful techniques for attributing functions to genes in uni- and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits. Deletion of nearly half of the yeast genes not essential for growth affects these morphological traits. Similar morphological phenotypes are caused by deletions of functionally related genes, enabling a functional assignment of a locus to a specific cellular pathway. The high-dimensional phenotypic analysis of defined yeast mutant strains provides another step toward attributing gene function to all of the genes in the yeast genome.

In acclimation to changing light environments, photosynthetic organisms modulate the ratio of two photosynthetic reaction centers ( photosystem IIPSI] and photosystem II). One mutant, which could not modulate photosystem stoichiometry upon the shift to high light, was isolated from mutants created by random transposon mutagenesis. Measurements of chlorophyll fluorescence and analysis of the reaction center subunits of PSI through western blotting in this mutant revealed that the content of PSI could not be suppressed under high-light condition. In the mutant, transposon was inserted to the sll1961 gene encoding a putative transcriptional regulator. DNA microarray analysis revealed that the expression of sll1773 was drastically induced in the sll1961 mutant upon exposure to high light for 3 h. Our results demonstrate that a transcriptional regulator, Sll1961, and its possible target proteins, including Sll1773, may be responsible for the regulation of photosystem stoichiometry in response to high light.

To avoid the photodamage, cyanobacteria regulate the distribution of light energy absorbed by phycobilisome antenna either to photosystem II or to photosystem I ( PSI) upon high light acclimation by the process so-called state transition. We found that an alternative PSI subunit, PsaK2 (sll0629 gene product), is involved in this process in the cyanobacterium Synechocystis sp. PCC 6803. An examination of the subunit composition of the purified PSI reaction center complexes revealed that PsaK2 subunit was absent in the PSI complexes under low light condition, but was incorporated into the complexes during acclimation to high light. The growth of the psaK2 mutant on solid medium was inhibited under high light condition. We determined the photosynthetic characteristics of the wild type strain and the two mutants, the psaK1 ( ssr0390) mutant and the psaK2 mutant, using pulse amplitude modulation fluorometer. Non-photochemical quenching, which reflects the energy transfer from phycobilisome to PSI in cyanobacteria, was higher in high light grown cells than in low light grown cells, both in the wild type and the psaK1 mutant. However, this change of non-photochemical quenching during acclimation to high light was not observed in the psaK2 mutant. Thus, PsaK2 subunit is involved in the energy transfer from phycobilisome to PSI under high light condition. The role of PsaK2 in state transition under high light condition was also confirmed by chlorophyll fluorescence emission spectra determined at 77 K. The results suggest that PsaK2-dependent state transition is essential for the growth of this cyanobacterium under high light condition.

Pirin is a recently identified protein in eukaryotes as a transcription cofactor or as an apoptosis-related protein. Although Pirin is highly conserved from bacteria to human, there have been no reports on prokaryotic Pirin orthologs. We show here that pirA (sll1773) encoding an ortholog of Pirin together with an adjacent gene, pirB (ssl3389), was upregulated under high salinity and some other stress conditions in a cyanobacterium Synechocystis sp. PCC 6803. Induction of the pirAB genes was not related to cell death and disruption of pirA did not affect the gene expression profile. Expression of the pirAB genes was negatively regulated by a LysR family transcriptional regulator encoded by pirR (slr1871) located immediately upstream of pirAB in the divergent direction. DNA microarray analysis indicated that PirR repressed expression of closely located ORFs, slr1870 and mutS (sll1772), in addition to pirAB and pirR itself. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Photosystem (PS) II activity of a sulfoquinovosyl diacylgiycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Oxygen evolution is inhibited when leaves of chilling-sensitive plants like cucumber are treated at 0°C in the dark. The activity is restored by moderate illumination at room temperature. We examined the changes in the redox state of the Mn-cluster in cucumber leaves in the processes of dark-chilling inhibition and subsequent light-induced reactivation by means of thermoluminescence (TL). A TL B-band arising from S2QB - charge recombination in PSII was observed upon single-flash illumination of untreated leaves, whereas four flashes were required to yield the B-band after dark-chilling treatment for 24 h. This three-step delay indicates that over-reduced states of the Mn-cluster such as the S-2 state were formed during the treatment. Fitting analysis of the flash-number dependence of the TL intensities showed that the Mn-cluster was more reduced with a longer period of the treatment and that S-3 was the lowest S-state detectable in the dark-chilled leaves. Measurements of the Mn content by atomic absorption spectroscopy showed that Mn atoms were gradually released from PSII during the dark-chilling treatment but re-bound to PSII by illumination at 30°C. Thus, dark-chilling inhibition of oxygen evolution can be ascribed to the disintegration of the Mn-cluster due to its over-reduction. The observation of the S-3 state in the present in vivo system strongly suggests that S-3, which has been observed only by addition of exogenous reductants into in vitro preparations, is indeed a redox intermediate of the Mn-cluster in the processes of its disintegration and photoactivation. © 2003 Elsevier Science B.V. All rights reserved.

To examine the role of sulfoquinovosyl diacylglycerol (SQDG) in thylakoid membranes, we compared the structural and functional properties of photosystem II (PSII) between a mutant of Chlamydomonas reinhardtii defective in SQDG (hf-2) and the wild type. The PSII core complex of hf-2, as compared with that of the wild type, showed structural fragility when solubilized with a detergent, dodecyl beta-D-altoside, suggesting that the physical properties of the PSII complex were altered by the loss of SQDG. On the other hand, exposure of the cells to 41 degreesC for 120 min in the dark decreased the PSII activity to 70% and 50% of the initial levels in the wild type and hf-2, respectively, which implies that the PSII activity, in the absence of SQDG, becomes less stable under heat-stress conditions. PSII inactivated to 60% of the initial level by dark incubation at 41 degreesC was reactivated by following illumination even at 41 degreesC to more than 90% in the wild type, but only to 70% in hf-2. These results suggest that PSII inactivated by heat recovers through some mechanism dependent on light, and that SQDG participates in functioning of the mechanism. The conformational disorder of PSII caused by the defect in SQDG might be correlated with the increased susceptibility of its activity to heat-stress.

Whole-genome DNA microarrays were used to evaluate the effect of the redox state of the photosynthetic electron transport chain on gene expression in Synechocystis sp. strain PCC 6803. Two specific inhibitors of electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), were added to the cultures, and changes in accumulation of transcripts were examined. About 140 genes were highlighted as reproducibly affected by the change in the redox state of the photosynthetic electron transport chain. It was shown that some stress-responsive genes but not photosynthetic genes were under the control of the redox state of the plastoquinone pool in Synechocystis sp. strain PCC 6803.

Oxygen-evolving photosystem 11 (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni2(+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 mumol O-2 (mg Chl)(-1) h(-1) in the presence of Ca2+ using ferricyanide as the electron acceptor, while its activity was 680-720 mumol O-2 (mg Chl)(-1) h(-1) in the absence of Ca2+ and Cl- ions. The activity was 710-820 mumol O-2 (mg Chl)(-1) h(-1) independent of the presence or absence of Ca2+ and Cl when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B), was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca2+. These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl2- The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl2-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.

The recovery process after chilling-induced photoinhibition of photosystem I (PSI) was studied in leaves of a chilling-sensitive plant, cucumber (Cucumis sativits L. cv. Nanshin). Determination of chlorophyll content, photosystem (PS) activities in vivo and in vitro, and the amount OF reaction-center subunits of PSI revealed that: (i) The content of chlorophyll decreased to 70% of the original level gradually from 1 to 3 days after exposure to a low temperature. (ii) The amount of functional PSI per unit leaf area was reduced to 30% of the initial level by the chilling treatment. The amount of functional PSI gradually increased during the next 6 days but only to 50% of the original level. (iii) When expressed on a chlorophyll basis, however, the amount of functional PSI recovered to 90% of the original level 6 days after the treatment. (iv) The residual amount of chlorophyll on the third day after the treatment closely correlated with the amount of functional PSI at that point. These results indicate that the decrease in chlorophyll content at a normal growth temperature after chilling treatment is a consequence of the degradation of irreversibly damaged PSI complexes. Immunoblot analysis confirmed that PsaAB protein, the reaction-center subunits of PSI, was degraded during the 3 days after chilling treatment. Some characteristics of the chilling injury frequently reported, i.e. irreversibility of the injury and development of visible symptoms at room temperature, can be explained as a consequence of the chilling-induced photoinhibition of PSI.

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf -2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by approximate to 40% and also a lower growth rate than those of the wild-type. Results of genetical analysis of hf -2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf -2 cells restored the lowered PSII activity to the same level as that of wild-type cells, and also enabled the mutant to grow even in the presence of 135 nm 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf -2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.

We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 mu mol m(-2) s(-1)), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light, (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII, These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.

The morphology of the pyrenoid and the physiology of the CO2-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd, bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of CI, insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO2 (low values of K-0.5(CO2)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cn. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2-3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204. 269-276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups.

The role of chilling temperatures on photoinhibition of photosystems I and II (PSI and PSII) under weak light has been examined in cucumber, a chilling-sensitive plant. The extent of PSII photoinhibition, determined by pulse-modulated fluorescence in vivo, is closely related to the redox state of the PSII electron acceptor Q(A), measured as a fluorescence parameter, 1-q(P). On the other hand, the extent of PSI photoinhibition, which is only observed in chilling-sensitive plants at chilling temperatures, cannot be related to the redox state of Q(A), suggesting that the underlying mechanism is different from that of PSII photoinhibition. Chilling treatment at low photon flux densities is found to enhance cyclic electron flow around PSI. Both PSI photoinhibition and enhanced cyclic electron flow show similar temperature dependence, with the threshold temperature at 10 degrees C. (C) 1999 Elsevier Science S.A. All rights reserved.

Previously, we identified a novel gene, pmgA, as an essential factor to support photomixotrophic growth of Synechocystis species PCC 6803 and reported that a strain in which pmgA was deleted grew better than the wild type under photoautotrophic conditions. To gain insight into the role of pmgA, we investigated the mutant phenotype of pmgA in detail. When low-light-grown (20 mu E m(-2) s(-1)) cells were transferred to high light (HL [200 mu E m(-2) s(-1)]), pmgA mutants failed to respond in the manner typically associated with Synechocystis. Specifically, mutants lost their ability to suppress accumulation of chlorophyll and photosystem I and, consequently, could not modulate photosystem stoichiometry. These phenotypes seem to result in enhanced rates of photosynthesis and growth during short-term exposure to HL. Moreover, mixed-culture experiments clearly demonstrated that loss of pmgA function was selected against during longer-term exposure to HL, suggesting that pmgA is involved in acquisition of resistance to HL stress. Finally, early induction of pmgA expression detected by reverse transcriptase-PCR upon the shift to HL led us to conclude that pmgA is the first gene identified, to our knowledge, as a specific regulatory factor for HL acclimation.

Physiological and morphological characteristics related to the CO2-concentrating mechanism (CCM) were examined in several species of the free-living, unicellular volvocalean genus Chloromonas (Chlorophyta), which differs morphologically from the genus Chlamydomonas only by lacking pyrenoids. The absence of pyrenoids in the chloroplasts of Chloromonas (Cr.) rosae UTEX 1337, Cr. serbinowii UTEX 492, Cr. clatharata UTEX 1970, Cr. rosae SAG 26.90, and Cr. palmelloides SAG 32.86 was confirmed by light and electron microscopy. In addition, immunogold electron microscopy demonstrated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were distributed almost evenly throughout the chloroplasts in all five Chloromonas strains. However, Chloromonas exhibited two types of physiological characteristics related to the CCM depending on the species or strains examined. Chloromonas rosae UTEX 1337 and Cr. serbinowii had high photosynthetic affinities for CO2 in cells grown in culture medium bubbled with air (low-CO2 cells), compared with those grown in medium bubbled with 5% CO2 (high-CO2 cells), indicating the presence of the low-CO2-inducible CCM. In addition, these two Chloromonas strains exhibited low-CO2-inducible carbonic anhydrase (CA; EC 4.2.1.1) activity and seemed to have small intracellular inorganic carbon pools. Therefore, it appears that Cr. rosae UTEX 1337 and Cr. serbinowii possess the CCM as in pyrenoid-containing microalgae such as Chlamydomonas reinhardtii. By contrast, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides showed low photosynthetic affinities for CO2 when grown under both CO2 conditions. Moreover, these three strains exhibited an apparent absence of intracellular inorganic carbon pools and lacked low-CO2-inducible CA activity. Thus, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides, like other pyrenoid-less algae (lichen photobionts) reported previously, seem to lack the CCM. The present study is the first demonstration of the CCM in pyrenoid-less algae, indicating that pyrenoids or accumulation of Rubisco in the chloroplasts are not always essential for the CCM in algae. Focusing on this type of CCM in pyrenoid-less algae, the physiological and evolutionary significance of pyrenoid absence is discussed.

At chilling temperatures, plants suffer damage to photosynthesis. The sites and the mechanisms involved in this damage differ under different chilling conditions. The current status of our understanding of this damage is reviewed, and how chilling temperatures affect photosynthesis is discussed with emphasis on the role of light and the phase separation of membrane lipids.

We examined photosynthetic properties of Eupatorium makinoi leaves infected by a geminivirus. Since a major symptom of the geminivirus infection is variegation or yellowing of leaves, Chi content was used as an index of disease severity. As leaf Chi content was lowered, leaf absorptance, maximal quantum yield of photosynthesis on an absorbed quantum basis (phi O-2,(max)) and light-saturated rate of photosynthesis (P-max) decreased. The share of energy allocated to PS II, which can be estimated from fluorescence parameters and oxygen evolution rate, was about 30% lower in the infected yellow leaves than in uninfected leaves. Analyses of the composition of thylakoid polypeptides by gel electrophoresis showed preferential loss of LHC II. The lower phi O-2,(max) in the infected leaves was, thus, attributed to the decreased energy allocation to PS II. These features were largely consistent with those of b-less mutants, but lowered P-max has been never reported for b-less mutants. Possible mechanisms causing these changes in photosynthetic properties to the infected leaves are discussed.

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala(500) and Val(501) on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.

Mature first leaves of Phaseolus vulgaris L. were exposed to low partial pressures of CO2 (7, 6 and 0 Pa CO2) for 24 h. After exposure of leaves to 6 Pa CO2 for 24 h, there was a reduction in the carbon exchange rate (CER) at all partial pressures of CO2 at which measurements were made, After exposure to 7 Pa CO2, the CER decreased only at high partial pressures of CO2. The rates of electron transport from water to methyl viologen, through the whole chain, decreased in parallel with the decrease in CER measured at 90 Pa CO2. One site of inhibition in leaves exposed for 24 h to 6 Pa CO2 appeared to be the intersystem electron-transport chain since there were no significant changes in the activities of PSI and PSII, as determined from the level of P-700 and measurement of fluorescence, respectively, Another inhibitory phenomenon appeared to be a negative change in the activation state of Rubisco, while the level of Rubisco was unaffected by the exposure to 6 Pa CO2. These decreases in photosynthetic activity caused by depletion of CO2 explains at least in part, the inhibition of photosynthesis that is caused by rain treatment [Ishibashi and Terashima (1995) Plant Cell Environ. 18: 431].

Continuous wetness of leaves in the light causes a reduction in the carbon exchange rate (CER) in Phaseolus vulgaris L. [Ishibashi and Terashima (1995) Plant Cell Environ. 18: 431]. In this study, we investigated the initial cause of photoinhibition upon application of water, designated rain treatment, and we found a large decrease in the rate of electron transport through the whole chain from water to methyl viologen via PSII and PSI. In spite of the decrease in the rate of electron transport, there was no decrease in the activity of either PSI or PSII when these activities were measured separately. The intactness of PSI was also confirmed by the absence of any change in the photooxidizable amount of P-700, the reaction centre of PSI, and the intactness of PSII was confirmed by measurements of Chl fluorescence. The results suggest that the inhibition by the rain treatment, which occurs at the site between PSI and PSII, might be a novel type of photoinhibition, unlike the conventional types of photoinhibition that involve PSI and PSII.

A mutant of Chlamydomonas reinhardtii designated as hf-9 is impaired in fatty acid desaturation of chloroplasts, and showed lowered unsaturation levels of chloroplast lipids, as compared with the parent (Sate et al., Eur. J. Biochem., 230 (1995) 987-993). The effects of temperature on photosynthesis were compared between hf-9 and the parent for investigation whether or not unsaturation levels of chloroplast lipids are correlated with the thermal properties of photosynthesis. Growth rates determined by turbidity were higher in the parent than in hf-9 at both 10 and 24 degrees C, while similar for the parent and hf-9 at 39 degrees C. The cells grown at 24 degrees C revealed that both activities of CO2-dependent oxygen evolution and photosystem II were higher in the parent than in hf-9 in the range between 7 and 40 degrees C. In contrast, hf-9 surpassed the parent in both activities at 45 degrees C. Optimal temperatures for both activities were at around 35 degrees C and 40 degrees C in the parent and hf-9, respectively. Incubation of the cells at 41 and 45 degrees C demonstrated that the activity of photosystem II in hf-9 was more tolerant to the high temperatures than that in the parent. These results suggest that lowered unsaturation levels of chloroplast lipids contributed to high temperature tolerance of photosystem II, and eventually to that of photosynthesis.

The photosynthetic apparatus of a mutant of Chlamydomonas reinhardtii, hf-9, impaired in fatty acid desaturation at the omega 6 position of fatty acids of chloroplasts was investigated. Measurement of photosynthetic activities revealed that both PS I and PS II activities were reduced in hf-9. However, little alteration occurred in the contents and subunit assemblies of the PS I complex, PS II core complex and light-harvesting complex of PS II. Lipids bound to these chlorophyll-protein (CP) complexes in hf-9 were shown to contain decreased levels of 16:4(4,7,10,13) and 18:3(9,12,15), with accumulation of 16:1(7) and 18:1(9), as compared with in the parent. Highly unsaturated fatty acids of chloroplast lipids may be required for the normal functions of PS I and PS II, by associating with these complexes.

To clarify the way in which the light available for growth affects respiration in leaves of sun and shade plants, we examined the respiratory properties of mature leaves of Spinacia oleracea L., a sun species, and of Alocasia macrorrhiza (L.) G. Don., a shade species, that had been grown at various irradiances. In leaves of S. oleracea, the respiratory rates, on a dry mass basis, decreased with time during the night, and the higher was the growth irradiance during the day, the higher was the respiratory rate. The marked decreases in the respiratory rate during the night were accompanied by decreases in the concentration of carbohydrates in the leaves, By contrast, the respiratory rates of leaves of A. macrorrhiza were virtually constant throughout the night and the absolute rates were lower than those of S. oleracea even though the absolute value of the concentration of carbohydrates and its decrease at night resembled to those in S. oleracea, The maximum activities of respiratory enzymes were also similar to those in S. oleracea. However, the leaves of A. macrorrhiza contained less soluble protein than those of S. oleracea, These results suggest that, in S. oleracea, the concentration of carbohydrates might determine the respiratory rate while such is not the case in A. macrorrhiza. The lower respiratory rates in A. macrorrhiza might be due to a lower demand for ATP.

Photoinhibition was defined originally as the decrease in photosynthetic activity that occurs upon excess illumination. The site of photoinhibition has generally been considered to be located in PSII. However, a novel type of photoinhibition has recently been characterized in chilling-sensitive plants. This photoinhibition occurs under relatively weak illumination at chilling temperatures and the main site of damage is in PSI. The photoinhibition of PST is initiated by the inactivation of the acceptor side, with the subsequent destruction of the reaction center and the degradation of the product of the psaB gene, which is one of the two major subunit polypeptides of the PSI reaction center complex. Chilling and oxidative stress (the presence of reactive species of oxygen) are characteristic requirements for the photoinhibition of PSI in vivo.

Stoichiometries of photosystem I (PSI) and photosystem II (PSII) reaction centers in a cultivar of rice, Norin No, 8, and three chlorophyll b-deficient mutants derived from the cultivar were investigated. Quantitation of PSI by photooxidation of P-700 and chromatographic assay of vitamin K-1 showed that, on the basis of chlorophyll, the mutants have higher concentrations of PSI than the wildtype rice, Greater increases were observed in the PSII contents measured by photoreduction of Q(A), binding of a radioactive herbicide and atomic absorption spectroscopy of Mn. Consequently, the PSII to PSI ratio increased from 1.1-1.3 in the wild-type rice to 1.8 in chlorina 2, which contains no Chi b, and to 2.0-3.3 in chlorina 11 and chlorina 14, which have chlorophyll a/b ratios of 9 and 13, respectively. Measurement of oxygen evolution with saturating single-turnover flashes revealed that, whereas at most 20% of PSII centers are inactive in oxygen evolution in the wildtype rice, the non-functional PSII centers amount to about 50% in the three mutant strains, The fluorescence induction kinetics was also analyzed to estimate proportions of the inactive PSII in the mutants. The data obtained suggest that plants have an ability to adjust the stoichiometry of the two photosystems and the functional organization of PSII in response to the genetically induced deficiency of chlorophyll b.

Specific degradation of psaB gene product, one of the two large subunits of photosystem I (PS I) reaction-center, was observed during photoinhibition of PS I. In the in vitro photoinhibition using spinach thylakoid membranes, the degradation of psaB gene product gave rise to fragments of 51 kDa and 45 kDa. n-Propyl gallate, a scavenger of active oxygen species, suppressed the generation of these fragments. Addition of methyl viologen, which protects PS I from photoinhibition but increases the production of superoxide, suppressed the generation of 51 kDa fragment but increased the generation of 45 kDa fragment. It was concluded that interaction of active oxygen species with reduced electron acceptor in PS I results in inactivation of PS I and in generation of 51 kDa fragment. On the other hand, 45 kDa fragment was generated solely by the presence of active oxygen species independent of the inhibition of PS I activity.

The photosynthetic apparatus was characterized in a mutant of Chlamydomonas reinhardtii, hf-2, defective in the synthesis of a chloroplast-specific lipid, sulfoquinovosyl diacylglycerol (SQui-acyl(2)Gro). hf-2 showed reduced photosystem II (PSII) activity with little effect on photosystem I (PSI) activity, as compared with the parent. PAGE in the presence of dodecyl beta-D-maltoside (DodGlc(2)) of C. reinhardtii thylakoid membranes was used to isolate chlorophyll-protein complexes without chlorophyll (Chl) release in order to examine lipid species bound to these complexes. The four complexes obtained were shown to be the PSI complex, the PSII core complex and the two groups of the light-harvesting complex of PSII by analyses of 77-K emission spectra of Chl fluorescence and of subunit compositions. Lipid analysis of Chl-protein complexes in the parent revealed the localization of SQui-acyl(2)Gro in the PSII core complex and the two groups of the light-harvesting complex of PSII, but not in the PSI complex. These results suggest that SQui-acyl(2)Gro is responsible for PSII activity by associating with the core and light-harvesting complexes of PSII.

The site of photoinhibition at low temperatures in leaves of a chilling-sensitive plant, cucumber, is photosystem I [Terashima et al. (1994) Planta 193: 300]. As described herein, selective photoinhibition of PSI can also be induced in isolated thylakoid membranes in vitro. Inhibition was observed both at chilling temperatures and at 25 degrees C, and not only in the thylakoid membranes isolated from cucumber, but also in those isolated from a chilling-tolerant plant, spinach. Comparison of these observations in vitro to the earlier results in vivo indicates that (1) photoinhibition of PSI is a universal phenomenon; (2) a mechanism exists to protect PSI in vivo; and (3) the protective mechanism is chilling-sensitive in cucumber. The chilling-sensitive component seems to be lost during the isolation of thylakoid membranes. Very weak light (10-20 mu umol m(-2) s(-1)) was sufficient to cause the inhibition of PSI. About 80% of the oxygen-evolving activity by PSII was maintained even after the activity of PSI had decreased by more than 70%. This is the first report of the selective photoinhibition of PSI in vitro.

The activity of photosystem (PS) I in cucumber leaves was selectively inhibited by weak illumination at chilling temperatures with almost no loss of P-700 content and PSII activity, The sites of inactivation in the reducing side of PSI were determined by EPR and flash photolysis, Measurement by EPR showed the destruction of iron-sulfur centers, F-X, F-A and F-B, in parallel with the loss of quantum yield of electron transfer from diaminodurene to NADP(+). Flash photolysis showed the increases in the triplet states of P-700 and antenna pigments, along with the decrease in the electron transfer from P-700 to F-A/F-B. This indicates the increase in the charge recombination between P-700(+) and A(0)(-). It is concluded that weak-light treatment of cucumber leaves at chilling temperature destroys F-X, F-A and F-B and possibly A(1). This gives the molecular basis for the mechanism of selective PSI photodamage that was recently reported [Sonoike and Terashima (1994) Planta 194, 287-293].

The PsaC protein binds two 4Fe-4S centers, F-A and F-B, in the photosystem I (PSI) protein complex. In the T398 strain of Anabaena variabilis ATCC 29413, the psaC gene encoding this protein has been insertionally inactivated by the introduction of a neomycin resistance gene cartridge in the coding region. Photosystem I complex was purified through native gel electrophoresis of beta-dodecyl maltoside solubilized thylakoid membranes from wild-type and T398 strains of Anabaena 29413. The PSI complex from T398 strain retained functionally active P700, the reaction center chlorophylls. Interestingly, purified PSI complex from T398 cells lacked the PsaD, PsaE, and PsaL polypeptides. Western analysis with polyclonal antibodies raised against these proteins indicated that the two stromal exposed polypeptides, PsaD and PsaE, are absent in isolated thylakoid membranes from T398 cells. The PsaL polypeptide could be detected at a level comparable to that in wild-type thylakoid membranes, although it is absent in the PSI preparation from the mutant. These observations suggest that the PsaC protein is essential for the stable association of PsaD and PsaE, two hydrophilic, extrinsic polypeptides. Moreover, PsaL, a hydrophobic protein is loosely associated with PSI and is lost during the isolation of the PSI complex. (C) 1994 Academic Press, Inc.

It was recently shown that the site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is Photosystem I (PSI), not PSII (I. Terashima et al. 1994, Planta 193, 300-306). In the present study, the mechanisms of this PSI photoinhibition in vivo were examined. By lowering the photon flux density during the photoinhibitory treatment of leaves at 4 degrees C for 5 h to less than 100 mu mol.m(-2).S-1, We were able to separate the steps of the destruction of the electron-transfer components. Although P-700, the reaction-center chlorophyll, was almost intact in this low-light treatment, the quantum yield of the electron transfer through PSI and photochemically induced absorption change at 701 nm were markedly inhibited. This, along with the results from the measurements of the light-induced absorption changes in the presence of various concentrations of methyl viologen, an artificial electron acceptor, indicates that the component on the acceptor side of the PSI, A(1) or F-x, is the first site of inactivation. When the photon flux density during the treatment was increased to 220 mu mol.m(-2)s(-1), the destruction of P-700 itself was also observed. Furthermore, the partial degradation of the chlorophyll-binding large subunits was observed in photoinhibited leaves. This degradation of the subunits was not detected when the treatment was carried out under nitrogen atmosphere, the condition in which the electron transfer is not inhibited. Thus, the photoinhibitory processes in the reaction center of PSI go through three steps, the inactivation of the acceptor side, the destruction of the reaction-center chlorophyll and the degradation of the reaction center subunit(s). The similarities and the differences between the mechanisms of PSI photoinhibition and those of PSII photoinhibition are discussed.

We have determined the nucleotide sequence of the psaAB gene cluster from the filamentous cyanobacterium Anabaena variabilis ATCC 29413. These genes encode the Photosystem I reaction center proteins, which are highly homologous to similar proteins in other cyanobacteria and higher plants.

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-degrees-C in moderate light at 220 mumol 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-degrees-C, while such a marked temperature dependency was not observed for the decrease in QY(PSII). In the chilling treatment at 4-degrees-C for 5 h, the quantum flux density that was required to induce 50% loss of QY(PSI) was ca. 50 mumol quanta m-2.s-1. When the chilling treatment at 4-degrees-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 chilling-sensitive plant, PSI is much more susceptible to aerobic photoinhibition than PSII.

The PsaC protein binds two 4Fe-4S centers, FA and FB, in the photosystem I (PSI) protein complex. In the T398 strain of Anabaena variabilis ATCC 29413, the psaC gene encoding this protein has been insertionally inactivated by the introduction of a neomycin resistance gene cartridge in the coding region. Photosystem I complex was purified through native gel electrophoresis of β-dodecyl maltoside solubilized thylakoid membranes from wild-type and T398 strains of Anabaena 29413. The PSI complex from T398 strain retained functionally active P700, the reaction center chlorophylls. Interestingly, purified PSI complex from T398 cells lacked the PsaD, PsaE, and PsaL polypeptides. Western analysis with polyclonal antibodies raised against these proteins indicated that the two stromal exposed polypeptides, PsaD and PsaE, are absent in isolated thylakoid membranes from T398 cells. The PsaL polypeptide could be detected at a level comparable to that in wild-type thylakoid membranes, although it is absent in the PSI preparation from the mutant. These observations suggest that the PsaC protein is essential for the stable association of PsaD and PsaE, two hydrophilic, extrinsic polypeptides. Moreover, PsaL, a hydrophobic protein is loosely associated with PSI and is lost during the isolation of the PSI complex. © 1994 Academic Press.

Treatment of whole and fractionated plant tissues with hydrophilic and hydrophobic solvent mixtures of varied volume ratio liberates two chlorophyll (Chl) a' molecules from the photosystem (PS)I core when the solvent hydrophilicity exceeds a critical level, whereas only one molecule is extracted in hydrophobic media. The PSI core proteins, PSI-A and PSI-B, which form a heterodimer, appear to bind one Chl a' molecule each, in local environments significantly different regarding their hydrophilicity or hydrophobicity.

Photosystem I (PS I) reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus with nonionic detergents, digitonin or sucrose monolaurate, contained eight small subunit polypeptides. Two of the small polypeptides were identified by analysis of their N-terminal amino-acid sequences as the psaF and psaE gene products. Treatment with a cationic detergent, cetyltrimethylammonium bromide, resulted in depletion of five small subunits including the psaF gene product. Five PS I complexes isolated with an anionic detergent, sodium dodecylsulfate, contained zero to four small subunits but were all depleted of the psaF polypeptide. The function of the psaF gene product was examined by measuring reduction kinetics of flash-oxidized P-700 in the presence of different concentrations of cytochrome c-553. Oxidized P-700 was rapidly reduced by the reduced cytochrome in all the PS I complexes that contained, at least, the psaC and psaD polypeptides and the second-order rate constants of electron transfer from cytochrome c-553 to P-700 were essentially the same between PS I complexes that contained the psaF polypeptide and those that lost this polypeptide. Thus, the psaF polypeptide is not required for the bimolecular reaction between P-700 and cytochrome c-553. Mg2+ had a moderate stimulating effect on the rate of P-700 reduction whether PS I complexes were associated with the psaF gene product or not. The function of this subunit polypeptide is discussed. © 1993.

Function of a subunit polypeptide (the psaE gene product) of Photosystem I (PS I) reaction center complexes was investigated by comparing the reactivity of the reduced iron-sulfur centers (FA/FB)- with ferredoxin among Synechococcus PS I complexes which had been variously depleted of this polypeptide. Ferredoxin at or below 1 μM can accept electrons from (FA/FB)- effectively competing with the back reaction between P-700+ and (FA/FB)- in the thylakoid membranes and PS I complexes that contained all the eight small subunits. The high reactivity of (FA/FB)- with low concentrations of ferredoxin was observed in PS I complexes which contain only the products of psaC, psaD and psaE genes but not in complexes which carry the psaC, psaD, psaL and psaK gene products but no psaE gene product. Varied amounts of the psaE gene product were extracted by treatment with different concentrations of a cationic detergent, dodecyltrimethylammonium bromide, and 2.5 M NaCl. The solubilized polypeptide was then reconstituted to the depleted complexes. The magnitudes of the back reaction that could be suppressed by addition of ferredoxin at or below 1 μM were well correlated to the amounts of the psaE polypeptide remained bound or rebound to the complexes. It is concluded that the product of the psaE gene has a role to promote the interaction between the terminal bound electron acceptor and ferredoxin. A high autooxidizability of (FA/FB)- and contrasting effects of lipophilic cations and anions on the rate of the back reaction from (FA/FB)- to P-700+ were also reported. © 1993.

Thermoluminescence (TL) emission below 77 K from photosynthetic pigment protein complexes and purified pigments was examined using liquid He. At least four TL components emitting at around 20, 50, 70 and 90 K were resolved on the glow curve from thylakoids. The 20, 50 and 70 K bands are newly observed TL components and designated as Zα, Zβ and Zγ bands, respectively. The 90 K band was found to be a different expression of the well-know Z band which was reported as the 110 K band in literatures. These TL bands were evidenced not to be related with charge separation and subsequent electron transfer in reaction centers but originate from light-harvesting chlorophyll (Chl) a and b by the following observations: (1) red light, which causes charge separation in reaction centers, was ineffective in inducing these TL components
(2) isolated LHCI and LHCII showed higher TL intensities than isolated PS I core and PS II core complexes
and (3) purified Chl a and Chl b in solid state exhibited essentially the same TL bands. Chl-Chl and Chl-ligand interactions in proteins have been discussed as possible chemical identities of the energy trap responsible for the TL bands. © 1993.

Function of a subunit polypeptide (the psaE gene product) of Photosystem I (PS I) reaction center complexes was investigated by comparing the reactivity of the reduced iron-sulfur centers (F(A)/F(B))- with ferredoxin among Synechococcus PS I complexes which had been variously depleted of this polypeptide. Ferredoxin at or below 1 muM can accept electrons from (F(A)/F(B))- effectively competing with the back reaction between P-700+ and (F(A)/F(B))- in the thylakoid membranes and PS I complexes that contained all the eight small subunits. The high reactivity of (F(A)/F(B))- with low concentrations of ferredoxin was observed in PS I complexes which contain only the products of psaC, psaD and psaE genes but not in complexes which carry the psaC, psaD, psaL and psaK gene products but no psaE gene product. Varied amounts of the psaE gene product were extracted by treatment with different concentrations of a cationic detergent, dodecyltrimethylammonium bromide, and 2.5 M NaCl. The solubilized polypeptide was then reconstituted to the depleted complexes. The magnitudes of the back reaction that could be suppressed by addition of ferredoxin at or below 1 muM were well correlated to the amounts of the psaE polypeptide remained bound or rebound to the complexes. It is concluded that the product of the psaE gene has a role to promote the interaction between the terminal bound electron acceptor and ferredoxin. A high autooxidizability of (F(A)/F(B))- and contrasting effects of lipophilic cations and anions on the rate of the back reaction from (F(A)/F(B))- to P-700+ were also reported.

Photosystem I (PS I) reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus with nonionic detergents, digitonin or sucrose monolaurate, contained eight small subunit polypeptides. Two of the small polypeptides were identified by analysis of their N-terminal amino-acid sequences as the psaF and psaE gene products. Treatment with a cationic detergent, cetyltrimethylammonium bromide, resulted in depletion of five small subunits including the psaF gene product. Five PS I complexes isolated with an anionic detergent, sodium dodecylsulfate, contained zero to four small subunits but were all depleted of the psaF polypeptide. The function of the psaF gene product was examined by measuring reduction kinetics of flash-oxidized P-700 in the presence of different concentrations of cytochrome c-553. Oxidized P-700 was rapidly reduced by the reduced cytochrome in all the PS I complexes that contained, at least, the psaC and psaD polypeptides and the second-order rate constants of electron transfer from cytochrome c-553 to P-700 were essentially the same between PS I complexes that contained the psaF polypeptide and those that lost this polypeptide. Thus, the psaF polypeptide is not required for the bimolecular reaction between P-700 and cytochrome c-553. Mg2+ had a moderate stimulating effect on the rate of P-700 reduction whether PS I complexes were associated with the psaF gene product or not. The function of this subunit polypeptide is discussed.

Thermoluminescence (TL) emission below 77 K from photosynthetic pigment protein complexes and purified pigments was examined using liquid He. At least four TL components emitting at around 20, 50, 70 and 90 K were resolved on the glow curve from thylakoids. The 20, 50 and 70 K bands are newly observed TL components and designated as Z(alpha), Z(beta) and Z(gamma) bands, respectively. The 90 K band was found to be a different expression of the well-known Z band which was reported as the 110 K band in literatures. These TL bands were evidenced not to be related with charge separation and subsequent electron transfer in reaction centers but originate from light-harvesting chlorophyll (Chl) a and b by the following observations: (1) red light, which causes charge separation in reaction centers, was ineffective in inducing these TL components; (2) isolated LHCI and LHCII showed higher TL intensities than isolated PS I core and PS II core complexes; and (3) purified Chl a and Chl b in solid state exhibited essentially the same TL bands. Chl-Chl and Chl-ligand interactions in proteins have been discussed as possible chemical identities of the energy trap responsible for the TL bands.

A novel protein component of 3.5 kDa was detected in photosystem I complexes prepared from several cyanobacteria, viz. Synechococcus vulcanus, Synechococcus elongatus BP-1, Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. The complete amino acid sequence of this component was determined by direct protein sequencing. The sequences of the 3.5 kDa proteins from these four organisms were highly homologous to each other, featuring a hydrophobic domain in the middle. The cyanobacterial consensus sequence exhibits significant homology to the presumed product of ORF32 in the chloroplast DNA of liverwort (Marchantia polymorpha), but no homologous ORF is present in the chloroplast DNA of tobacco or rice. Since this protein appears to interact strongly with the PS I reaction center complex, it may play some role in the function and maintenance of the structure of PS I.

The psaI gene encoding the 5.2 kDa protein component (PsaI) of the photosystem I complex was cloned from the cyanobacterium Anabaena 29413. The gene is present in single copy in this cyanobacterial genome. The nucleotide sequence of a 500 bp region of the cloned DNA revealed the presence of an open reading frame encoding a 46 amino acid long polypeptide. The N-terminal 11 residues are absent in the mature polypeptide and thus represents the first identified cleavable presequence on the PsaI protein. We suggest that this presequence directs the N-terminus of the protein to the thylakoid lumen.

Emission spectra of two components of thermoluminescence from photosynthetic systems were measured using a two-dimensional photoncounter. Application of this detector system enormously improved the signal to noise (S/N) ratio and enabled the determination of an emission spectrum of a specific thermoluminescence component. The B-band emitting at 30-degrees-C showed an emission spectrum having a single peak at 690 nm, whereas the Z-band emitting at -160-degrees-C showed a spectrum having two peaks, the major band at 740 nm and a satellite band at 690 nm. It was revealed that the longer wavelength component arises from photosystem I and the shorter wavelength component from photosystem II. Light harvesting chlorophyll a/b protein complexes (LHCI and LHCII) were also found to emit the Z-band at 740 and 690 nm, respectively. The advantage of a two-dimensional photoncounter for the determination of emission spectra of weak luminescence from large-surface biological samples is discussed.

To examine the effects of chilling of leaves of cucumber (Cucumis sativus L.) in moderate light on the coupling state of thylakoids in situ, changes in fluorescence, changes in light scattering and flash-induced changes in absorbance at 518 nm were examined in intact leaves. After chilling of leaves at 5-degrees-C in the light for 5 h, the non-photochemical quenching of fluorescence, a measure of energisation of thylakoids, was largely suppressed. The treatment also caused a suppression of light-induced changes in the light scattering by leaves, which depends on the formation of a pH gradient across thylakoid membranes. When thylakoids were prepared by very gentle methods from the leaves chilled in the light, through a step of preparation of intact chloroplasts, the transport of electrons from H2O to ferricyanide was uncoupled, being insensitive to an uncoupler, methylamine.
These data provide consistent evidence that the thylakoids are uncoupled in situ by the chilling of leaves in the light and, as a consequence of the uncoupling, the energisation of the membranes is suppressed. However, the decay of the flash-induced change in absorbance at 518 nm in leaves was not markedly accelerated by the treatment. The thylakoids isolated from leaves chilled in the light, which were in the uncoupled state, also did not show a rapid decay, unless an efficient uncoupler such as gramicidin was added. These results suggest that even a considerable uncoupling of thylakoids, brought about by chilling of leaves in the light, is not sufficient to cause a marked acceleration of the decay of the flash-induced change in absorbance at 518 nm. Therefore, analysis at 518 nm is not always a sensitive method for assessing the coupling state of thylakoids.

(1) Treatment of oxygen-evolving Photosystem II membrane fragments (PS II membranes) with a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) led to immobilization of all the extrinsic 33 kDa protein molecules without any significant effects on the oxygen-evolving activity and oscillation patterns of flash-induced oxygen evolution and thermoluminescence B band. (2) With increasing concentration of EDC, the chlorophyll-binding 47 kDa protein decreased in parallel with the 33 kDa protein, yielding a crosslinked product consisting of one each of the two proteins. The results, which indicate that the two proteins are present in equimolar amounts in PS II membranes, are consistent with the stoichiometry of one copy of the 33 kDa protein per PS II unit. (3) The total immobilization of the 33 kDa protein stabilized 40 to 60% of the oxygen-evolving activity against urea/NaCl-, CaCl2- and MgCl2-wash, which otherwise solubilize the three extrinsic proteins and strongly inactivate oxygen evolution. The result implies that extraction of the extrinsic proteins may not be the sole cause of the inactivation of oxygen evolution by these treatments. (4) The crosslinking of the 33 kDa protein with EDC had no protecting effect against Tris-, NH2OH- and pH 9.0-treatments. However, the stability of oxygen evolution at alkaline pH levels was slightly but significantly increased by treatment of PS II membranes with dithiobis(succinimidylpropionate), which specifically modifies amino groups.

Emission spectra of two thermoluminescence (TL) components emitted from the photosynthetic apparatus were determined by the use of an imaging photon detector system. The following results were obtained: (i) The TL B-band emitted from whole thylakoids and PS II core complex showed the same emission spectrum peaking at about 690 nm (uncorrected for wavelength dependence of the photocathode) with a broad tailing in far-red region. This spectrum agrees with the reported emission spectrum of delayed luminescence, consistent with the view that the TL B-band arises from thermally stimulated recombination of charge pairs in PS II. (ii) The emission spectrum of TL Z-band was variant, depending on the PS I/PS II ratio in the sample. This was because the Z-band consists of two spectral components emitting at about 740 nm and 690 nm (also uncorrected for the wavelength dependence of the photocathode)
the former originates exclusively from PS I and the latter from PS II. (iii) Light-harvesting chlorophyll protein complexes (LHCI and LHCII) emit the respective spectral components of Z-band more strongly than do their respective core complexes, indicating that reaction center photochemistry is not involved in energy storage for the Z-band. (iv) A methanol extract of thylakoids emitted only a weak Z-band at 690 nm, but the formation of a chlorophyll aggregate markedly enhanced the Z-band intensity, concomitant with a shift in emission maximum to 740 nm. The mechanism of energy storage for Z-band is discussed in relation to the local chlorophyll concentration. © 1991 Elsevier Science Publishers B.V. All rights reserved.

Emission spectra of two thermoluminescence (TL) components emitted from the photosynthetic apparatus were determined by the use of an imaging photon detector system. The following results were obtained: (i) The TL B-band emitted from whole thylakoids and PS 11 core complex showed the same emission spectrum peaking at about 690 nm (uncorrected for wavelength dependence of the photocathode) with a broad tailing in far-red region. This spectrum agrees with the reported emission spectrum of delayed luminescence, consistent with the view that the TL B-band arises from thermally stimulated recombination of charge pairs in PS II. (ii) The emission spectrum of TL Z-band was variant, depending on the PS I/PS II ratio in the sample. This was because the Z-band consists of two spectral components emitting at about 740 nm and 690 nm (also uncorrected for the wavelength dependence of the photocathode); the former originates exclusively from PS I and the latter from PS II. (iii) Light-harvesting chlorophyll protein complexes (LHCI and LHCII) emit the respective spectral components of Z-band more strongly than do their respective core complexes, indicating that reaction center photochemistry is not involved in energy storage for the Z-band. (iv) A methanol extract of thylakoids emitted only a weak Z-band at 690 nm, but the formation of a chlorophyll aggregate markedly enhanced the Z-band intensity, concomitant with a shift in emission maximum to 740 nm. The mechanism of energy storage for Z-band is discussed in relation to the local chlorophyll concentration.

(1) Treatment of oxygen-evolving Photosystem II membrane fragments (PS II membranes) with a zero-length crosslinker, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) led to immobilization of all the extrinsic 33 kDa protein molecules without any significant effects on the oxygen-evolving activity and oscillation patterns of flash-induced oxygen evolution and thermoluminescence B band. (2) With increasing concentration of EDC, the chlorophyll-binding 47 kDa protein decreased in parallel with the 33 kDa protein, yielding a crosslinked product consisting of one each of the two proteins. The results, which indicate that the two proteins are present in equimolar amounts in PS II membranes, are consistent with the stoichiometry of one copy of the 33 kDa protein per PS II unit. (3) The total immobilization of the 33 kDa protein stabilized 40 to 60% of the oxygen-evolving activity against urea/NaCl−, CaCl2− and MgCl2-wash, which otherwise solubilize the three extrinsic proteins and strongly inactivate oxygen evolution. The result implies that extraction of the extrinsic proteins may not be the sole cause of the inactivation of oxygen evolution by these treatments. (4) The crosslinking of the 33 kDa protein with EDC had no protecting effect against Tris-, NH2OH- and pH 9.0-treatments. However, the stability of oxygen evolution at alkaline pH levels was slightly but significantly increased by treatment of PS II membranes with dithiobis(suc-cinimidylpropionate), which specifically modifies amino groups. © 1991, Elsevier Science Publishers B.V. All rights reserved. All rights reserved.

Stabilities of iron-sulfur centers and reaction center chlorophyll P-700 in Photosystem I reaction center complex (CP1-a), isolated by sodium dodecyl sulfate treatment from the thermophilic cyanobacterium Synechococcus elongatus, were studied by EPR and optical spectroscopy. P-700 was destroyed by treatment at temperatures above 80℃ for 5 minutes with a half inactivation temperature of 93℃. The three iron-sulfur centers F_A, F_B and F_X showed similar thermal stabilities and were half inactivated at about 70℃. Thus, the isolated Photosystem I reaction center complexes of S.elongatus are still highly resistant to heat.

Treatment of spinach Photosystem I particles with detergents induced an apparent blue shift of chlorophyll a at 690 nm in the difference spectrum of P-700. As a consequence of the band shift, the differential absorption coefficient of P-700 was increased from 63 to 87 mM^<-1>・cm^<-1>. A curve was presented, with which changes in the apparent differential absorption coefficient of P-700 can be estimated by measuring magnitudes of light-induced absorption decreases at 680 nm and 700 nm. The curve was compared with that for cyanobacterial preparations and the mechanism of the detergent-induced band shift was discussed.

The magnitude of flash-induced bleaching at 700 nm in the thylakoid membranes isolated from the thermophilic cyanobacterium Synechococcus sp. was affected neither by addition of 4 mM methyl viologen, nor by treatment of the membranes with 0.1% sodium dodecyl sulfate (SDS) for 1 h, but appreciably increased on addition of methyl viologen to the SDS-treated membranes. Detailed studies on the light-minus-dark difference spectrum of P-700 revealed that methyl viologen induces a shift-type change consisting of a bleaching at 695 nm and a positive band at 685 nm, and consequently increases the magnitude of the 700 nm bleaching without affecting P-700 photooxidation in the SDS-treated membranes. Other bipyridinium dyes, 1,1′-trimethylene-2,2′-bipyridinium dibromide and 1,1′-trimethylene-5,5′-dimethyl-2,2′-bipyridinium dibromide were equally effective as methyl viologen, and a prolonged treatment of the membranes with SDS caused a similar band shift in the difference spectrum of P-700. The band shift is closely associated with P-700 oxidation because, on redox titration, the magnitude of the band shift varied in parallel to the amount of P-700 oxidized by light. Methyl viologen also induced the band shift in the chemically oxidized-minus-reduced difference spectrum of P-700 in the SDS-treated membranes. Thus, the band shift is not related to reduction of a bound electron acceptor. As a consequence of the band shift, the oxidized-minus-reduced differential extinction coefficient of P-700 in the 700 nm region was increased by 40%. The extinction coefficient was 64 mM-1 · cm-1 at 701 nm in the thylakoid membranes, whereas the 1-h treated membranes with added methyl viologen, or a Photosystem I reaction center complex prepared by SDS-gel electrophoresis, showed the extinction value of 84-86 mM-1 · cm-1 at 698 nm. Two different models for the band shift are discussed. © 1988.