Na+-driven pH regulation by Na+/H+ antiporters promotes photosynthetic efficiency in cyanobacteria.

Photosynthetic organisms have developed mechanisms to regulate light reactions in response to varying light conditions. Photosynthetic electron transport leads to the formation of a ΔpH across the thylakoid membrane, which is crucial for regulating electron transport. However, other pH modulators remain to be identified, particularly in cyanobacteria. In this study, we evaluated the potential involvement of six Na+/H+ antiporters (NhaS1-NhaS6) in control of pH in the cyanobacterium Synechocystis sp. PCC 6803. Synechocystis showed a strong requirement for Na+ at high light intensities, with ΔnhaS1 and ΔnhaS2 strains unable to grow under high light conditions. We analyzed Na+ efflux-driven H+-uptake activities of NhaS1-NhaS6 in inverted membranes of Escherichia coli. Biological fractionation and immunoelectron microscopy revealed that NhaS1 localizes to both the plasma and thylakoid membranes while NhaS2 localizes to the plasma membrane. Measurement of photosynthesis activity indicated that NhaS2 promotes ATP production and electron transport from PQ to P700. Measurements of pH outside of the cells and in the cytoplasm suggested that both NhaS1 and NhaS2 are involved in plasma membrane-mediated light-dependent H+ uptake and cytoplasmic acidification. NhaS1 and NhaS2 were also found to prevent photoinhibition under high light treatment. These results indicate that H+ transport mediated by NhaS1 and NhaS2 plays a role in regulating intracellular pH and maintaining photosynthetic electron transport.

Incorporation of photosynthetically active algal chloroplasts in cultured mammalian cells towards photosynthesis in animals.

Chloroplasts are photosynthetic organelles that evolved through the endosymbiosis between cyanobacteria-like symbionts and hosts. Many studies have attempted to isolate intact chloroplasts to analyze their morphological characteristics and photosynthetic activity. Although several studies introduced isolated chloroplasts into the cells of different species, their photosynthetic activities have not been confirmed. In this study, we isolated photosynthetically active chloroplasts from the primitive red alga Cyanidioschyzon merolae and incorporated them in cultured mammalian cells via co-cultivation. The incorporated chloroplasts retained their thylakoid structure in intracellular vesicles and were maintained in the cytoplasm, surrounded by the mitochondria near the nucleus. Moreover, the incorporated chloroplasts maintained electron transport activity of photosystem II in cultured mammalian cells for at least 2 days after the incorporation. Our top-down synthetic biology-based approach may serve as a foundation for creating artificially photosynthetic animal cells.

Phylogenetic Profiling Analysis of the Phycobilisome Revealed a Novel State-Transition Regulator Gene in Synechocystis sp. PCC 6803.

Phycobilisomes play a crucial role in the light-harvesting mechanisms of cyanobacteria, red algae and glaucophytes, but the molecular mechanism of their regulation is largely unknown. In the cyanobacterium, Synechocystis sp. PCC 6803, we identified slr0244 as a phycobilisome-related gene using phylogenetic profiling analysis, a method used to predict gene function based on comparative genomics. To investigate the physiological function of the slr0244 gene, we characterized slr0244 mutants spectroscopically. Disruption of the slr0244 gene impaired state transition, a process by which the distribution of light energy absorbed by the phycobilisomes between two photosystems is regulated in response to the changes in light conditions. The Slr0244 protein seems to act in the process of state transition, somewhere at or downstream of the sensing step of the redox state of the plastoquinone (PQ) pool. These findings, together with past reports describing the interaction of this gene product with thioredoxin and glutaredoxin, suggest that the slr0244 gene is a novel state-transition regulator that integrates the redox signal of PQ pools with that of the photosystem I-reducing side. The protein has two universal stress protein (USP) motifs in tandem. The second motif has two conserved cysteine residues found in USPs of other cyanobacteria and land plants. These redox-type USPs with conserved cysteines may function as redox regulators in various photosynthetic organisms. Our study also shows the efficacy of phylogenetic profiling analysis in predicting the function of cyanobacterial genes that have not been annotated so far.

Improved capacity for the repair of photosystem II via reinforcement of the translational and antioxidation systems in Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803, translation factor EF-Tu is inactivated by reactive oxygen species (ROS) via oxidation of Cys82 and the oxidation of EF-Tu enhances the inhibition of the repair of photosystem II (PSII) by suppressing protein synthesis. In our present study, we generated transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together. Expression of EF-Tu (C82S) alone in Synechocystis enhanced the repair of PSII under strong light, with the resultant mitigation of PSII photoinhibition, but it stimulated the production of ROS. However, overexpression of superoxide dismutase and catalase, together with the expression of EF-Tu (C82S), lowered intracellular levels of ROS and enhanced the repair of PSII more significantly under strong light, via facilitation of the synthesis de novo of the D1 protein. By contrast, the activity of photosystem I was hardly affected in wild-type cells and in all the lines of transformed cells under the same strong-light conditions. Furthermore, transformed cells that overexpressed EF-Tu (C82S), superoxide dismutase, and catalase were able to survive longer under stronger light than wild-type cells. Thus, the reinforced capacity for both protein synthesis and ROS scavenging allowed both photosynthesis and cell proliferation to tolerate strong light.

Dual Redox Regulation of the DNA-Binding Activity of the Response Regulator RpaB in the Cyanobacterium Synechocystis sp. PCC 6803.

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.

Investigation of Nostoc sp. HK-01, Cell Survival over Three Years during the Tanpopo Mission.

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

Respiration Interacts With Photosynthesis Through the Acceptor Side of Photosystem I, Reflected in the Dark-to-Light Induction Kinetics of Chlorophyll Fluorescence in the Cyanobacterium Synechocystis sp. PCC 6803.

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

Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the Cyanobacterium Synechococcus elongatus PCC 7942.

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

The circadian rhythm regulator RpaA modulates photosynthetic electron transport and alters the preferable temperature range for growth in a cyanobacterium.

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.

Screening of mutants using chlorophyll fluorescence.

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.

The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

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.

Morphological and cytological observations of corolla green spots reveal the presence of functional chloroplasts in Japanese gentian.

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.

Light dependent accumulation of ß-carotene enhances photo-acclimation of Euglena gracilis.

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 ß-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 ß-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 ß-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.

Direct injection of pigment-protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC.

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.

Guard cell photosynthesis is crucial in abscisic acid-induced stomatal closure.

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.

Crassulacean Acid Metabolism Induction in Mesembryanthemum crystallinum Can Be Estimated by Non-Photochemical Quenching upon Actinic Illumination During the Dark Period.

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.

Evaluation of the Condition of Respiration and Photosynthesis by Measuring Chlorophyll Fluorescence in Cyanobacteria.

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.

Characterization of the influence of chlororespiration on the regulation of photosynthesis in the glaucophyte Cyanophora paradoxa.

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.

Estimation of photosynthesis in cyanobacteria by pulse-amplitude modulation chlorophyll fluorescence: problems and solutions.

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.