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transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together.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.

Chloroplast translation factor EF-Tu of Arabidopsis thaliana can be inactivated via oxidation of a specific cysteine residue.

Translational elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803. However, the sensitivity to ROS of chloroplast-localized EF-Tu (cpEF-Tu) of plants remains to be elucidated. In the present study, we generated a recombinant cpEF-Tu protein of Arabidopsis thaliana and examined its sensitivity to ROS in vitro. In cpEF-Tu that lacked a bound nucleotide, one of the two cysteine residues, Cys149 and Cys451, in the mature protein was sensitive to oxidation by H2O2, with the resultant formation of sulfenic acid. The translational activity of cpEF-Tu, as determined with an in vitro translation system, derived from Escherichia coli, that had been reconstituted without EF-Tu, decreased with the oxidation of a cysteine residue. Replacement of Cys149 with an alanine residue rendered cpEF-Tu insensitive to inactivation by H2O2, indicating that Cys149 might be the target of oxidation. In contrast, cpEF-Tu that had bound either GDP or GTP was less sensitive to oxidation by H2O2 than nucleotide-free cpEF-Tu. The addition of thioredoxin f1, a major thioredoxin in the Arabidopsis chloroplast, to oxidized cpEF-Tu allowed the reduction of Cys149 and the reactivation of cpEF-Tu, suggesting that the oxidation of cpEF-Tu might be a reversible regulatory mechanism that suppresses the chloroplast translation system in a redox-dependent manner.

Light-inducible expression of translation factor EF-Tu during acclimation to strong light enhances the repair of photosystem II.

In photosynthetic organisms, the repair of photosystem II (PSII) is enhanced after acclimation to strong light, with the resultant mitigation of photoinhibition of PSII. We previously reported that oxidation of translation elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, depresses the repair of PSII in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the role of EF-Tu in the repair of PSII after acclimation of Synechocystis to strong light. In cells that had been grown under strong light, both the repair of PSII and the synthesis of proteins de novo were enhanced under strong light, with the resultant mitigation of photoinhibition of PSII. Moreover, levels of EF-Tu were elevated, whereas levels of other components of the translation machinery, such as translation factor EF-G and ribosomal proteins L2 and S12, did not change significantly. The expression of the gene for EF-Tu was induced by light, as monitored at the transcriptional level. Elevation of the level of EF-Tu was strongly correlated with the subsequent enhancement of PSII repair in cells that had been grown under light at various intensities. Furthermore, overexpression of EF-Tu in Synechocystis enhanced protein synthesis and PSII repair under strong light, even after cell culture under nonacclimating conditions. These observations suggest that elevation of the level of EF-Tu might be a critical factor in enhancing the capacity for repair of PSII that develops during acclimation to strong light.

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 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.

Optimizing the timing of 3.6 mg Pegfilgrastim Administration for Dose-Dense Chemotherapy in Japanese Patients with Breast Cancer.

Perioperative dose-dense chemotherapy (DDCT) with pegfilgrastim (Peg) prophylaxis is a standard treatment for high-risk breast cancer. We explored the optimal timing of administration of 3.6 mg Peg, the dose approved in Japan. In the phase II feasibility study of DDCT (adriamycin+cyclophosphamide or epirubicin+cyclophosphamide followed by paclitaxel) for breast cancer, we investigated the feasibility, safety, neutrophil transition, and optimal timing of Peg treatment by administering Peg at days 2, 3, and 4 post-chemotherapy (P2, P3, and P4 groups, respectively). Among the 52 women enrolled, 13 were aged > 60 years. The anthracycline sequence was administered to P2 (n=33), P3 (n=5), and P4 (n=14) patients, and the taxane sequence to P2 (n=38) and P3 (n=6) patients. Both sequences showed no interaction between Peg administration timing and treatment discontinuation, treatment delay, or dose reduction. However, the relative dose intensity (RDI) was significantly different among the groups. The neutrophil count transition differed significantly among the groups receiving the anthracycline sequence. However, the neutrophil count remained in the appropriate range for both sequences in the P2 group. The timing of Peg administration did not substantially affect the feasibility or safety of DDCT. Postoperative day 2 might be the optimal timing for DDCT.

Long-Chain Saturated Fatty Acids, Palmitic and Stearic Acids, Enhance the Repair of Photosystem II.

Free fatty acids (FFA) generated in cyanobacterial cells can be utilized for the biodiesel that is required for our sustainable future. The combination of FFA and strong light induces severe photoinhibition of photosystem II (PSII), which suppresses the production of FFA in cyanobacterial cells. In the present study, we examined the effects of exogenously added FFA on the photoinhibition of PSII in Synechocystis sp. PCC 6803. The addition of lauric acid (12:0) to cells accelerated the photoinhibition of PSII by inhibiting the repair of PSII and the de novo synthesis of D1. α-Linolenic acid (18:3) affected both the repair of and photodamage to PSII. Surprisingly, palmitic (16:0) and stearic acids (18:0) enhanced the repair of PSII by accelerating the de novo synthesis of D1 with the mitigation of the photoinhibition of PSII. Our results show chemical potential of FFA in the regulation of PSII without genetic manipulation.

Overexpression of Orange Carotenoid Protein Protects the Repair of PSII under Strong Light in Synechocystis sp. PCC 6803.

Orange carotenoid protein (OCP) plays a vital role in the thermal dissipation of excitation energy in the photosynthetic machinery of the cyanobacterium Synechocystis sp. PCC 6803. To clarify the role of OCP in the protection of PSII from strong light, we generated an OCP-overexpressing strain of Synechocystis and examined the effects of overexpression on the photoinhibition of PSII. In OCP-overexpressing cells, thermal dissipation of energy was enhanced and the extent of photoinhibition of PSII was reduced. However, photodamage to PSII, as monitored in the presence of lincomycin, was unaffected, suggesting that overexpressed OCP protects the repair of PSII. Furthermore, the synthesis de novo of proteins in thylakoid membranes, such as the D1 protein which is required for the repair of PSII, was enhanced in OCP-overexpressing cells under strong light, while the production of singlet oxygen was suppressed. Thus, the enhanced thermal dissipation of energy via overexpressed OCP might support the repair of PSII by protecting protein synthesis from oxidative damage by singlet oxygen under strong light, with the resultant mitigation of photoinhibition of PSII.

Oxidation of Translation Factor EF-Tu Inhibits the Repair of Photosystem II.

The repair of photosystem II (PSII) is particularly sensitive to oxidative stress and the inhibition of repair is associated with oxidative damage to the translational elongation system in the cyanobacterium Synechocystis sp. PCC 6803. However, the molecular mechanisms underlying this inhibition are unknown. We previously demonstrated in vitro that EF-Tu, a translation factor that delivers aminoacyl-tRNA to the ribosome, is inactivated by reactive oxygen species via oxidation of the Cys residue Cys-82. In this study, we examined the physiological role of the oxidation of EF-Tu in Synechocystis Under strong light, EF-Tu was rapidly oxidized to yield oxidized monomers in vivo. We generated a Synechocystis transformant that expressed mutated EF-Tu in which Cys-82 had been replaced with a Ser residue. Under strong light, the de novo synthesis of proteins that are required for PSII repair, such as D1, was enhanced in the transformant and photoinhibition of PSII was alleviated. However, photodamage to PSII, measured in the presence of lincomycin, was similar between the transformant and wild-type cells, suggesting that expression of mutated EF-Tu might enhance the repair of PSII. Alleviating photoinhibition through mutation of EF-Tu did not alter cell growth under strong light, perhaps due to the enhanced production of reactive oxygen species. These observations suggest that the oxidation of EF-Tu under strong light inhibits PSII repair, resulting in the stimulation of photoinhibition.

The LexA transcription factor regulates fatty acid biosynthetic genes in the cyanobacterium Synechocystis sp. PCC 6803.

Specific transcription factors have been identified in various heterotrophic bacterial species that regulate the sets of genes required for fatty acid metabolism. Here, we report that expression of the fab genes, encoding fatty acid biosynthetic enzymes, is regulated by the global regulator LexA in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803. Sll1626, an ortholog of the well-known LexA repressor involved in the SOS response in heterotrophic bacteria, was isolated from crude extracts of Synechocystis by DNA affinity chromatography, reflecting its binding to the upstream region of the acpP-fabF and fabI genes. An electrophoresis mobility shift assay revealed that the recombinant LexA protein can bind to the upstream region of each fab gene tested (fabD, fabH, fabF, fabG, fabZ and fabI). Quantitative RT-PCR analysis of the wild type and a lexA-disrupted mutant strain suggested that LexA acts as a repressor of the fab genes involved in initiation of fatty acid biosynthesis (fabD, fabH and fabF) and the first reductive step in the subsequent elongation cycle (fabG) under normal growth conditions. Under nitrogen-depleted conditions, downregulation of fab gene expression is partly achieved through an increase in LexA-repressing activity. In contrast, under phosphate-depleted conditions, fab gene expression is upregulated, probably due to the loss of repression by LexA. We further demonstrate that elimination of LexA largely increases the production of fatty acids in strains modified to secrete free fatty acids.

ATP is a driving force in the repair of photosystem II during photoinhibition.

Repair of photosystem II (PSII) during photoinhibition involves replacement of photodamaged D1 protein by newly synthesized D1 protein. In this review, we summarize evidence for the indispensability of ATP in the degradation and synthesis of D1 during the repair of PSII. Synthesis of one molecule of the D1 protein consumes more than 1,300 molecules of ATP equivalents. The degradation of photodamaged D1 by FtsH protease also consumes approximately 240 molecules of ATP. In addition, ATP is required for several other aspects of the repair of PSII, such as transcription of psbA genes. These requirements for ATP during the repair of PSII have been demonstrated by experiments showing that the synthesis of D1 and the repair of PSII are interrupted by inhibitors of ATP synthase and uncouplers of ATP synthesis, as well as by mutation of components of ATP synthase. We discuss the contribution of cyclic electron transport around photosystem I to the repair of PSII. Furthermore, we introduce new terms relevant to the regulation of the PSII repair, namely, "ATP-dependent regulation" and "redox-dependent regulation," and we discuss the possible contribution of the ATP-dependent regulation of PSII repair under environmental stress.

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

Disturbance of cell-size determination by forced overproduction of sulfoquinovosyl diacylglycerol in the cyanobacterium Synechococcus elongatus PCC 7942.

Sulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria or their descendants, plastids at species-dependent levels. We investigated the physiological significance of the intrinsic SQDG content in the cyanobacterium Synechococcus elongatus PCC 7942, with the use of its mutant, in which the genes for SQDG synthesis, sqdB and sqdX, were overexpressed. The mutant showed a 1.3-fold higher content of SQDG (23.6 mol% relative to total cellular lipids, cf., 17.1 mol% in the control strain) with much less remarkable effects on the other lipid classes. Simultaneously observed were 1.6- to 1.9-fold enhanced mRNA levels for the genes responsible for the synthesis of the lipids other than SQDG, as if to compensate for the SQDG overproduction. Meanwhile, the mutant showed no injury to cell growth, however, cell length was increased (6.1 ± 2.3, cf., 3.8 ± 0.8 µm in the control strain). Accordingly with this, a wide range of genes responsible for cell division were 1.6-2.4-fold more highly expressed in the mutant. These results suggested that a regulatory mechanism for lipid homeostasis functions in the mutant, and that SQDG has to be kept from surpassing the intrinsic content in S. elongatus for repression of the abnormal expression of cell division-related genes and, inevitably, for normal cell division.

Overexpressed Superoxide Dismutase and Catalase Act Synergistically to Protect the Repair of PSII during Photoinhibition in Synechococcus elongatus PCC 7942.

The repair of PSII under strong light is particularly sensitive to reactive oxygen species (ROS), such as the superoxide radical and hydrogen peroxide, and these ROS are efficiently scavenged by superoxide dismutase (SOD) and catalase. In the present study, we generated transformants of the cyanobacterium Synechococcus elongatus PCC 7942 that overexpressed an iron superoxide dismutase (Fe-SOD) from Synechocystis sp. PCC 6803; a highly active catalase (VktA) from Vibrio rumoiensis; and both enzymes together. Then we examined the sensitivity of PSII to photoinhibition in the three strains. In cells that overexpressed either Fe-SOD or VktA, PSII was more tolerant to strong light than it was in wild-type cells. Moreover, in cells that overexpressed both Fe-SOD and VktA, PSII was even more tolerant to strong light. However, the rate of photodamage to PSII, as monitored in the presence of chloramphenicol, was similar in all three transformant strains and in wild-type cells, suggesting that the overexpression of these ROS-scavenging enzymes might not protect PSII from photodamage but might protect the repair of PSII. Under strong light, intracellular levels of ROS fell significantly, and the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, was enhanced. Our observations suggest that overexpressed Fe-SOD and VktA might act synergistically to alleviate the photoinhibition of PSII by reducing intracellular levels of ROS, with resultant protection of the repair of PSII from oxidative inhibition.

A Feed-Forward Loop Consisting of the Response Regulator RpaB and the Small RNA PsrR1 Controls Light Acclimation of Photosystem I Gene Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) have been suggested to be the two most critical factors for this response in Synechocystis sp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of the psrR1 gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence of psrR1 in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and the psrR1 gene is repressed by the binding of RpaB under low-light (LL) conditions. A decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression of psrR1 gene expression. Accumulating PsrR1 molecules then prevent translation from pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions.

Moderate Heat Stress Stimulates Repair of Photosystem II During Photoinhibition in Synechocystis sp. PCC 6803.

Examination of the effects of high temperature on the photoinhibition of photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 revealed that the extent of photoinhibition of PSII was lower at moderately high temperatures (35-42 °C) than at 30 °C. Photodamage to PSII, as determined in the presence of chloramphenicol, which blocks the repair of PSII, was accelerated at the moderately high temperatures but the effects of repair were greater than those of photodamage. The synthesis de novo of the D1 protein, which is essential for the repair of PSII, was enhanced at 38 °C. Electron transport and the synthesis of ATP were also enhanced at 38 °C, while levels of reactive oxygen species fell. Inhibition of the Calvin-Benson cycle with glycolaldehyde abolished the enhancement of repair of PSII at 38 °C, suggesting that an increase in the activity of the Calvin-Benson cycle might be required for the enhancement of repair at moderately high temperatures. The synthesis de novo of metabolic intermediates of the Calvin-Benson cycle, such as 3-phosphoglycerate, was also enhanced at 38 °C. We propose that moderate heat stress might enhance the repair of PSII by stimulating the synthesis of ATP and depressing the production of reactive oxygen species, via the stimulation of electron transport and suppression of the accumulation of excess electrons on the acceptor side of photosystem I, which might be driven by an increase in the activity of the Calvin-Benson cycle.

Zeaxanthin and Echinenone Protect the Repair of Photosystem II from Inhibition by Singlet Oxygen in Synechocystis sp. PCC 6803.

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.

Analysis of spontaneous suppressor mutants from the photomixotrophically grown pmgA-disrupted mutant in the cyanobacterium Synechocystis sp. PCC 6803.

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.

Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery.

When photosynthetic organisms are exposed to abiotic stress, their photosynthetic activity is significantly depressed. In particular, photosystem II (PSII) in the photosynthetic machinery is readily inactivated under strong light and this phenomenon is referred to as photoinhibition of PSII. Other types of abiotic stress act synergistically with light stress to accelerate photoinhibition. Recent studies of photoinhibition have revealed that light stress damages PSII directly, whereas other abiotic stresses act exclusively to inhibit the repair of PSII after light-induced damage (photodamage). Such inhibition of repair is associated with suppression, by reactive oxygen species (ROS), of the synthesis of proteins de novo and, in particular, of the D1 protein, and also with the reduced efficiency of repair under stress conditions. Gene-technological improvements in the tolerance of photosynthetic organisms to various abiotic stresses have been achieved via protection of the repair system from ROS and, also, by enhancing the efficiency of repair via facilitation of the turnover of the D1 protein in PSII. In this review, we summarize the current status of research on photoinhibition as it relates to the effects of abiotic stress and we discuss successful strategies that enhance the activity of the repair machinery. In addition, we propose several potential methods for activating the repair system by gene-technological methods.

Light Influences the Growth, Pigment Synthesis, Photosynthesis Capacity, and Antioxidant Activities in Scenedesmus falcatus.

Light plays a significant role in microalgae cultivation, significantly influencing critical parameters, including biomass production, pigment content, and the accumulation of metabolic compounds. This study was intricately designed to optimize light intensities, explicitly targeting enhancing growth, pigmentation, and antioxidative properties in the green microalga, Scenedesmus falcatus (KU.B1). Additionally, the study delved into the photosynthetic efficiency in light responses of S. falcatus. The cultivation of S. falcatus was conducted in TRIS-acetate-phosphate medium (TAP medium) under different light intensities of 100, 500, and 1000 µmol photons m-2·s-1 within a photoperiodic cycle of 12 h of light and 12 h of dark. Results indicated a gradual increase in the growth of S. falcatus under high light conditions at 1000 µmol photons m-2·s-1, reaching a maximum optical density of 1.33 ± 0.03 and a total chlorophyll content of 22.67 ± 0.2 µg/ml at 120 h. Conversely, a slower growth rate was observed under low light at 100 µmol photons m-2·s-1. However, noteworthy reductions in the maximum quantum yield (Fv/Fm) and actual quantum yield (Y(II)) were observed under 1000 µmol photons m-2·s-1, reflecting a decline in algal photosynthetic efficiency. Interestingly, these changes under 1000 µmol photons m-2·s-1 were concurrent with a significant accumulation of a high amount of beta-carotene (919.83 ± 26.33 mg/g sample), lutein (34.56 ± 0.19 mg/g sample), and canthaxanthin (24.00 ± 0.38 mg/g sample) within algal cells. Nevertheless, it was noted that antioxidant activities and levels of total phenolic compounds (TPCs) decreased under high light at 1000 µmol photons m-2·s-1, with IC50 of DPPH assay recorded at 218.00 ± 4.24 and TPC at 230.83 ± 86.75 mg of GAE/g. The findings suggested that the elevated light intensity at 1000 µmol photons m-2·s-1 enhanced the growth and facilitated the accumulation of valuable carotenoid pigment in S. falcatus, presenting potential applications in the functional food and carotenoid industry.