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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.

Chlorophyll fluorescence has been widely used for the estimation of photosynthesis or its regulatory mechanisms. Chlorophyll fluorescence measurements are the methods with non-destructive nature and do not require contact between plant materials and fluorometers. Furthermore, the measuring process is very rapid. These characteristics of chlorophyll fluorescence measurements make them a suitable tool to screen mutants of photosynthesis-related genes. Furthermore, it has been shown that genes with a wide range of functions can be also analyzed by chlorophyll fluorescence through metabolic interactions. In this short review, we would like to first introduce the basic principle of the chlorophyll fluorescence measurements, and then explore the advantages and limitation of various screening methods. The emphasis is on the possibility of chlorophyll fluorescence measurements to screen mutants defective in metabolisms other than photosynthesis.

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.

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.

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.

The survival of the terrestrial cyanobacterium

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.

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 C

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.

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.

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

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

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

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.

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.

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

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.

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.

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.

The morphology of the pyrenoid and the physiology of the CO2-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd. bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of Cr. insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO2 (low values of K0.5(CO2)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cd. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2–3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204: 269–276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups.

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

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.

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.