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Cyanobacterial strains can grow within a specific temperature range that approximately corresponds to their natural habitat. However, how the preferable temperature range for growth (PTRG) is determined at the molecular level remains unclear. In this study, we detected a PTRG upshift in a mutant strain of Synechococcus elongatus PCC 7942 lacking the circadian rhythm regulator RpaA. Subsequent analyses revealed that RpaA decreases the electron transport from photosystem I to NADPH. The change in electron transport likely inhibits H2 O2 generation under high-temperature conditions and contributes to the observed PTRG upshift in rpaA-deficient cells. The importance of the effects of the circadian rhythm regulator on the PTRG is discussed.

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator 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 PIIper 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 the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the 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 was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

The response regulator RpaB plays a central role in transcriptional regulation of photosynthesis-related genes in cyanobacteria. RpaB is phosphorylated by its cognate histidine kinase Hik33 and functions as both an activator and a repressor under low-light conditions, whereas its phosphorylation level and DNA-binding activity promptly decrease upon the upshift of photon flux density, causing changes in the gene expression profile. In this study, we assessed the possibility of redox regulation of the DNA-binding activity of RpaB in Synechocystis sp. PCC 6803 by the addition of inhibitors of photosynthetic electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, or the reducing agent dithiothreitol under different photon flux densities. Analysis of the phosphorylation level of RpaB revealed that reduction of QA and increase in the availability of reducing equivalents at the acceptor side of photosystem I (PSI) can independently trigger dephosphorylation. The redox-state-dependent regulation by an unidentified thiol other than Cys59 of RpaB is prerequisite for the phosphorylation-dependent regulation of the DNA-binding activity. Environmental signals, recognized by Hik33, and metabolic signals recognized as the availability of reducing equivalents, must be integrated at the master regulator RpaB, in order to attain the flexible regulation of acclimatory responses.

We discovered that pigments including carotenoids and (bacterio)chlorophylls in pigment-protein complexes, membrane fragments, and chlorosomes suspended in water could be injected directly into C18 HPLC and analyzed without any other treatments. We applied this method to LH1-RC and chromatophores of purple bacteria, chlorosomes of green sulfur bacteria, thylakoid membranes of cyanobacteria, and PSII and thylakoid membranes of spinach. HPLC elution profiles and pigment composition were the same as those of the conventional extraction method. The principle of this method might be that samples are first trapped on top of column, followed by the immediate extraction of the pigments with the HPLC eluent and their separation using the C18 column, as usual. In the conventional extraction method, pigments are first extracted with organic solvents, followed by evaporation of the solvents. The dried pigments are then dissolved in organic solvents and injected into C18 HPLC after filtration. The advantages of this method include the preventions of pigment isomerization and oxidation and the possibility of injecting all samples. Its drawbacks include the accumulation of denatured proteins at the top of column, causing increased HPLC pressure. The use of a guard column might solve this problem. Many factors, such as samples, column, and HPLC systems, may affect this method. Nevertheless, we think that some samples can be analyzed using this method.

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.

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.

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.

Gentian is an important ornamental flower in Japan. The corolla of the majority of cultivated Japanese gentians have green spots, which are rarely encountered in flowers of other angiosperms. Little information is available on the functional traits of the green spots. In this study, we characterized the green spots in the Japanese gentian corolla using a number of microscopic techniques. Opto-digital microscopy revealed that a single visible green spot is composed of approximately 100 epidermal cells. The epidermal cells of a green spot formed a dome-like structure and the cell lumen contained many green structures that were granular and approximately 5 μm in diameter. The green structures emitted red autofluorescence when irradiated with 488 nm excitation light. Transmission electron microscopy revealed that the green structures contained typical thylakoids and grana, thus indicating they are chloroplasts. No grana were observed and the thylakoids had collapsed in the plastids of epidermal cells surrounding green spots. To estimate the rate of photosynthetic electron transfer of the green spots, we measured chlorophyll fluorescence using the MICROSCOPY version of an Imaging-PAM (pulse-amplitude-modulated) fluorometer. Under actinic light of 449 μmol m-2 s-1, substantial electron flow through photosystem II was observed. Observation of green spot formation during corolla development revealed that immature green spots formed at an early bud stage and developed to maturity associated with chloroplast degradation in the surrounding epidermal cells. These results confirmed that the Japanese gentian corolla contains functional chloroplasts in restricted areas of epidermal cells and indicated that a sophisticated program for differential regulation of chloroplast formation and degradation is operative in the epidermis.

The pmgA-disrupted (ΔpmgA) 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 ΔpmgA mutant derived from a single colony. When the ΔpmgA 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 ΔpmgA 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.

Reactive oxygen species (ROS) are ubiquitous signaling molecules involved in diverse physiological processes, including stomatal closure. Photosynthetic electron transport (PET) is the main source of ROS generation in plants, but whether it functions in guard cell signaling remains unclear. Here, we assessed whether PET functions in abscisic acid (ABA) signaling in guard cells. ABA‐elicited ROS were localized to guard cell chloroplasts in Arabidopsis thaliana, Commelina benghalensis, and Vicia faba in the light and abolished by the PET inhibitors 3‐(3, 4‐dichlorophenyl)‐1, 1‐dimethylurea and 2, 5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone. These inhibitors reduced ABA‐induced stomatal closure in all three species, as well as in the NADPH oxidase‐lacking mutant atrboh D/F. However, an NADPH oxidase inhibitor did not fully eliminate ABA‐induced ROS in the chloroplasts, and ABA‐induced ROS were still observed in the guard cell chloroplasts of atrboh D/F. This study demonstrates that ROS generated through PET act as signaling molecules in ABA‐induced stomatal closure and that this occurs in concert with ROS derived through NADPH oxidase.

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.

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.

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.

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 nblA, 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.

SummarySigma factors encoded by the nucleus of plants confer promoter specificity on the bacterial-type RNApolymerase in chloroplasts. We previously showed that transcripts of OsSIG1, which encodes one such sigmafactor in rice, accumulaterelatively late during leaf development. We havenow isolated and characterizedtwoallelic mutants of OsSIG1, in which OsSIG1 is disrupted by insertion of the retrotransposon Tos17, in order tocharacterize the functions of OsSIG1. The OsSIG1 )/ plants were found to be fertile but they manifested anapproximately one-third reduction in the chlorophyll content of mature leaves. Quantitative RT-PCR andnorthern blot analyses of chloroplast gene expression revealed that the abundance of transcripts derived fromthe 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) ofphotosystemI.Analysisofchlorophyllfluorescencealsorevealedasubstantialreductionintherateofelectrontransfer from photosystem II to photosystem I in the OsSIG1 mutants. Our results thus indicate that OsSIG1plays an important role in the maintenance of photosynthetic activity in mature chloroplasts of rice byregulating expression of chloroplast genes for components of photosystem I.Keywords: sigma factor, photosystem I, chloroplast, RNA polymerase, transcription, rice.IntroductionGene expression in plastids is mediated by at least two dif-ferent transcriptional systems based on a plastid-encodedRNA polymerase (PEP) and a nucleus-encoded RNApolymerase (NEP; Shiina et al., 2005; Hajdukiewicz et al.,1997; Hedtke et al., 1997). Plastid-encoded RNA polymeraseis a multisubunit eubacterial-type RNA polymerase, with thecore subunits being encoded by the plastid genes rpoA,rpoB, rpoC1 and rpoC2. Nucleus-encoded RNA polymeraseis a single-subunit bacteriophage-type enzyme that isencoded by a nuclear gene and is similar to a mitochondrialRNA polymerase. Analysis of PEP-deficient plants hassuggested that PEP functions predominantly in the expres-sion of photosynthetic genes and that NEP transcribesnon-photosynthetic genes in plastids (Liere and Maliga,1999). Plastid-encoded RNA polymerase requires sigmafactors, which are encoded by the nuclear genome, forpromoter recognition and initiation of transcription atspecific genes. To date, six sigma factors (SIG1, SIG2,SIG3, SIG4, SIG5 and SIG6) have been identified andcharacterized in the model dicotyledonous plant Arabid-opsis thaliana (Fujiwara et al., 2000; Isono et al., 1997;Tanaka et al., 1997). For the model monocotyledonousplant rice (Oryza sativa), six sigma factor genes, OsSIG1(Os-SigA), OsSIG2A, OsSIG2B, OsSIG3, OsSIG5 and Os-SIG6, have also been isolated (Kasai et al., 2004; Kubotaet al., 2007; Tozawa et al., 1998) or predicted from the

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.

In acclimation to changing light environments, photosynthetic organisms modulate the ratio of two photosynthetic reaction centers (photosystem I and photosystem II). Two mutants, which could not modulate photosystem stoichiometry upon the shift to high light, were isolated from mutants created by random transposon mutagenesis. Measurements of chlorophyll fluorescence and analysis of the reaction center subunits of photosystem I through Western blotting in these mutants revealed that the content of photosystem I could not be suppressed under high-light condition. In these mutants, transposon was inserted to sll1961, which is homologous to the gene of gluconate operon transcriptional repressor (GntR) in Bacillus anthracis A2012. 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 novel transcriptional regulator, Sll1961, and its possible target protein, Sll1773, may be responsible for the regulation of photosystem stoichiometry in response to high light.

In order to avoid the photodamage, cyanobacteria regulate the distribution of light energy absorbed by phycobilisome antenna either to photosystem II (PSII) 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. 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 (PAM) fluorometer. Non-photochemical quenching (qN), 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 qN 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., Copyright notice. c2002 Journal of Biological Chemistry. All rights reserved. Source published by American Physical Society.

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (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 QA to QB. 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.

Oxygen evolution is inhibited when leaves of chilling-sensitive plants like cucumber are treated at 0 degrees 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 S(2)Q(B)(-) 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 degrees 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.

Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-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 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) 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 Ca(2+). 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 CaCl(2). 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 CaCl(2)-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.

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.

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 micromol x m(-2) x 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.

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 μE m−2 s−1) cells were transferred to high light (HL [200μE m−2s−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 ofpmgA 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 ofpmgA 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.

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. HvLhcb1 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 HvLhcb1 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.

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 Ala500 and Val501 on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.

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 Chi 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.

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, Chl content was used as an index of disease severity. As leaf Chl content was lowered, leaf absorptance, maximal quantum yield of photosynthesis on an absorbed quantum basis (φo2,max) and light-saturated rate of photosynthesis (Pmax) 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 φo2,maxin 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 Pmaxhas been never reported for b-less mutants. Possible mechanisms causing these changes in photosynthetic properties to the infected leaves are discussed.

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.

The photosynthetic apparatus of a mutant of Chlamydomonas reinhardtii , hf -9, impaired in fatty acid desaturation at the ω6 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:l(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.

Photoinhibition was denned originally as the decrease in photosynthetic activity that occurs upon excess illumination. The site of photoinhibiti on has generally been considered to be located in PSII. However, a novel type of photoinhibition has recently been characterized in chillingsensitive plants. This photoinhibition occurs under relatively weak illumination at chilling temperatures and the main site of damage is in PSI. The photoinhibition of PSI 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.

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.

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, FX, FA and FB,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 FA/FB. This indicates the increase in the charge recombination between P-700+ and A0−. It is concluded that weak-light treatment of cucumber leaves at chilling temperature destroys FX, FA and FB and possibly A1. This gives the molecular basis for the mechanism of selective PSI photodamage that was recently reported [Sonoike and Terashima (1994) Planta 194, 287–293].

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.

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.

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 α , Z β and Z γ 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.

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.

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.

Chloroplasts are descendents of a cyanobacterial endosymbiont, but many chloroplast protein genes of endosymbiont origin are encoded by the nucleus. The chloroplast-cyanobacteria 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.

(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.

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.

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 c6, 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 c6, 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 c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.

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.

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.

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 (PhiII) and the quantum yield of open reaction centers of photosystem II (Fv'/Fm') were decreased. Furthermore, an increase in non-photochemical 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.

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 μM 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 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 (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.

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. The isolated Photosystem I reaction center complexes of S. elongatus are still highly resistant to heat

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.