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

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

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

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

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

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

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

Photosystem I (PSI), one of the two photosystems that drive electron transfer in oxygenic photosynthesis, is a very large pigment-protein complex with more than 100 cofactors. Crystal structure at 3.3 A in higher plants, and that at 2.5 A in cyanobacteria, gives us a detailed image of proteins and binding cofactors. However, most of the accumulated information is regarded as a snapshot of PSI. In spite of its general static image, components of PSI must be newly synthesized and assembled during initial synthesis, acclimation processes, and recovery from photoinhibition. Some factors required for such processes are common between higher plants, algae and cyanobacteria, but others are not. For the true comprehension of PSI, it is important to understand the dynamic nature of PSI including assembly or degradation. In this chapter, “four Ws and one H” of the PSI assembly are summarized.

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

The photosynthetic apparatus of a mutant of Chlamydomonas reinhardtii , hf -9, impaired in fatty acid desaturation at the ω6 position of fatty acids of chloroplasts was investigated. Measurement of photosynthetic activities revealed that both PS I and PS II activities were reduced in hf -9. However, little alteration occurred in the contents and subunit assemblies of the PS I complex, PS II core complex and light-harvesting complex of PS II. Lipids bound to these chlorophyll-protein (CP) complexes in hf -9 were shown to contain decreased levels of 16:4(4,7,10,13) and 18:3(9,12,15), with accumulation of 16:l(7) and 18:1(9), as compared with in the parent. Highly unsaturated fatty acids of chloroplast lipids may be required for the normal functions of PS I and PS II, by associating with these complexes.

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

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

The 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.

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

Chlorophyll fluorescence induction kinetics of cyanobacteria is known to reflect various cellular metabolisms. A simple mathematical method was developed to quantify the kinetic similarity of the chlorophyll fluorescence, and applied to cyanobacterial mutants involved in the regulation of photosystem stoichiometry. We showed that simple sum of the squared deviation between two fluorescence kinetics could be used as a measure of similarity. Appropriate weighting function enabled us to characterize the kinetics further. When the mutant having similar fluorescence kinetics to that of a certain photosystem stoichiometry mutant was listed according to the similarity distance, other photosystem stoichiometry mutants ranked high in the list. Apparently, the mutants with similar fluorescence induction kinetics share the common phenotype among them, thus opening the way to reveal the function of genes by chlorophyll fluorescence induction kinetics.

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.

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

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.

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

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

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.

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

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.

Cette etude a pour but d'analyser les variations d'absorption du P-700 induites par le traitement de particules de photosysteme 1 de Spinacia oleracea par des detergents. La relation entre les coefficients d'absorption apparents et l'importance relative des photoblanchiments a 680 et 700 nm est etudiee

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.