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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.

In cyanobacteria, the photosynthetic prokaryotes, direct interaction between photosynthesis and respiration exists at plastoquinone (PQ) pool, which is shared by the two electron transport chains. Another possible point of intersection of the two electron transport chains is NADPH, which is the major electron donor to the respiratory chain as well as the final product of the photosynthetic chain. Here, we showed that the redox state of NADPH in the dark affected chlorophyll fluorescence induction in the cyanobacterium Synechocystis sp. PCC 6803 in a quantitative manner. Accumulation of the reduced NADPH in the dark due to the defect in type 1 NAD(P)H dehydrogenase complex in the respiratory chain resulted in the faster rise to the peak in the dark-to-light induction of chlorophyll fluorescence, while depletion of NADPH due to the defect in pentose phosphate pathway resulted in the delayed appearance of the initial peak in the induction kinetics. There was a strong correlation between the dark level of NADPH determined by its fluorescence and the peak position of the induction kinetics of chlorophyll fluorescence. These results indicate that photosynthesis interacts with respiration through NADPH, which enable us to monitor the redox condition of the acceptor side of photosystem I by simple measurements of chlorophyll fluorescence induction in cyanobacteria.

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.

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.

Carotenoids are essential components of photosynthetic organisms including land plants, algae, cyanobacteria, and photosynthetic bacteria. Although the light-mediated regulation of carotenoid biosynthesis, including the light/dark cycle as well as the dependence of carotenoid biosynthesis–related gene translation on light wavelength, has been investigated in land plants, these aspects have not been studied in microalgae. Here, we investigated carotenoid biosynthesis in Euglena gracilis and found that zeaxanthin accumulates in the dark. The major carotenoid species in E. gracilis, namely β-carotene, neoxanthin, diadinoxanthin and diatoxanthin, accumulated corresponding to the duration of light irradiation under the light/dark cycle, although the translation of carotenoid biosynthesis genes hardly changed. Irradiation with either blue or red light (3 μmol photons m−2 s−1) caused a 1.3-fold increase in β-carotene content compared with the dark control. Blue-light irradiation (300 μmol photons m−2 s−1) caused an increase in the cellular content of both zeaxanthin and all trans-diatoxanthin, and this increase was proportional to blue-light intensity. In addition, pre-irradiation with blue light of 3 or 30 μmol photons m−2 s−1 enhanced the photosynthetic activity and tolerance to high-light stress. These findings suggest that the accumulation of β-carotene is regulated by the intensity of light, which may contribute to the acclimation of E. gracilis to the light environment in day night conditions.

Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis. Furthermore, chlorophyll fluorescence from cyanobacteria reflects the condition of respiration, since cyanobacterial photosynthesis shares several components of electron transport chain with respiration. This protocol presents the method to monitor the condition of both photosynthesis and respiration in cyanobacteria simply by measuring chlorophyll fluorescence in the dark and in the light with pulse amplitude modulation (PAM) chlorophyll fluorometer.

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.

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., This is a pre-copy-editing, author-produced PDF of an article accepted for publication in MICROBIOLOGY-SGM following peer review. The definitive publisher-authenticated version American Society for Microbiology, MICROBIOLOGY-SGM 155 (2009), 989-996; DOI 10.1099/mic.0.024018-0 online at: http://mic.sgmjournals.org/cgi/content/abstract/155/3/989

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.

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.

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-maltoside, 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 degrees C 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 degrees C was reactivated by following illumination even at 41 degrees C 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.

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.

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.

The recovery process after chilling-induced photoinhibition of photosystem I (PSI) was studied in leaves of a chilling-sensitive plant, cucumber (Cucumis sativus 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.

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.

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 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].

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.

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.

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°C showed an emission spectrum having a single peak at 690 nm, whereas the Z-band emitting at -160°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.

(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.

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.

In chilling sensitive plants such as cucumber, tomato and common bean, the site of chilling damage under moderate light is Photosystem I (PSI) [1,2]. Upon the treatment of cucumber leaves at 4°C for 5 h under the photon flux density at 100–200 µmol/m2s, the PSI activity determined in vitro as electron transfer from an artificial electron donor, DAD, to NADP+ decreased to about 20% while more than 80% of the PSII activity remained [3]. The damage, which was induced by the reactive oxygen species produced at the acceptor side of PSI [4,5], involved the destruction of iron-sulfur centers [6] and the degradation of a PSI reaction center subunit, PsaB protein [4,5,7]. Similar selective photoinhibition of PSI was observed also in another chilling sensitive plant, common bean [8]. Although PSI in chilling tolerant plant can he also photoinhibited after the isolation of thylakoid membranes [9], extent of PSI photoinhibition in vivo was relatively small in chilling tolerant plants unless the leaves were poisoned with KCN [10]. One exception was potato, in which absorption change due to P-700, the reaction center chlorophyll of PSI, determined in vivo was reported to decrease with only a small change in PSII [11]. To understand the relation between chilling sensitivity of plants and photoinhibition of PSI, we compared the absorption change of P-700 determined in vivo and in vitro after the chilling treatment of cucumber leaves. It was concluded that apparent decrease of P-700 determined in vivo, possibly duc to the enhanced cyclic electron flow around PSI, can be induced upon chilling treatment with little change in the extent of P-700 determined in vitro.

It was recently shown that the site of photoinhibition in leaves ofCucumis sativus L. at low temperatures is Photosystem I (PSI), not PSII (I. Terashima et al. 1994, Planta193, 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°C for 5 h to less than 100 μmol·m−2s−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, A1 or Fx, is the first site of inactivation. When the photon flux density during the treatment was increased to 220 μmol·m−2s−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.

Photosystem I (PS I) reaction center complex consists of two large subunits which carry antenna chlorophyll a, P-700 and early electron acceptors and several small subunits, one of which contains the iron-sulfur centers, FA and FB. Because the PS I complexes are usually isolated with detergents, careful evaluation of detergent-effects, such as solubilization of chlorophyll a, or inactivation of P-700, is essential for determination of stoichiometry of subunit polypeptides and functional constituents in the complex. Recently, we have shown that the differential extinction coefficient of P-700 is markedly affected by sodium dodecyl sulfate (SDS) which induces a band-shift of chlorophyll a molecules at 690 nm [1]. Herein, we report effects of other detergents on the extinction coefficient of P-700 and provide a simple method to estimate the extinction coefficient in PS I preparations treated with various detergents. Using the extinction values thus determined, stoichiometry of chlorophyll a and the two large subunits in PS I reaction center complexes were estimated.

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.

A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CP1-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS. In contrast to CP1-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60-kDa polypeptide and hence is called CP60. The yield of CP60 was maximal with 1-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents. The absorption spectrum of CP60, which retained more than half of the chlorophyll alpha molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature. CP60 emitted a fluorescence band at 717 to 725 nm at 77 degrees K. The temperature dependence of the far red band of CP60 was essentially the same as that of CP1-e between 77 and 273 degrees K. No photoresponse of P700 was detected in CP60. The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CP1-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60-kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex. It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry.

Stoichiometries of photosystem I (PSI) and photosystem II (PSII) reaction centers in a cultivar of rice, Norin No. 8, and three chlorophyll ^-deficient mutants derived from the cultivar were investigated. Quantitation of PSI by photooxidation of P-700 and chromatographic assay of vitamin Kt 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 photoreductio n of QA, 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 alb 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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0144-2) contains supplementary material, which is available to authorized users.