Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 E · m^–2 · s^–1) and then shifted to high light (750 E·m^–2·s^–1). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.     

Photoinhibition of photosynthesis and growth responses at different light levels in psbA gene mutants of the cyanobacterium Synechococcus

Photoinhibition of photosynthesis and growth responses at diffrent light levels (10, 120 and 250 μmol m⁻² s⁻¹) were studied in psbA gene mutants R2S2C3 ( psbAI gene present) and R2K1 ( psbAIIIpsbAIII genes present) of the cyanobacterium Synechococcus sp . PCC 7942 ( Anacystis nidulans R2). Mutant R2K1 (possessing form II of the D1 protein of photosystem II) was much more resistant to photoinhibition than the mutant R2S2C3 (possessing form I of the D1 protein). At moderate inhibitory light levels (100 to 300 μmol m⁻² s⁻¹) this was largely ascribed to an increased rsistance of the photosystem II reaction cetres possessing form II of the D1 protein. However, at higher light levels the higher resistance mutant R2K1 was assigned to a higher rate of photosystem II repair, i.e. turnover of the D1 protein. Moreover, our results support the hypothesis that photoinhibition of photosystem II and photoinhibitory induced quenching are due to separate processes. Results from growth experiments show that the R2K1 mutant has a slower growth rate than the R2S2C3 mutant but shows an increased survival under high light stress conditions. It is hypothesized that high resistance to photoinhibition, though allowing a better survival under high light, is not advantageous for optimal growth.     

The Susceptibility of Photosynthesis to Photoinhibition and the Capacity of Recovery in High and Low Light Grown Cyanobacteria, Anacystis nidulans

The susceptibility of photosynthesis to photoinhibition and the rate of its recovery were studied in the cyanobacterium Anacystis nidulans grown at a low (10 micromoles per square meter per second) and a high (120 micromoles per square meter per second) photosynthetically active radiation. The rate of light limited photosynthetic O₂ evolution was measured to determine levels of photoinhibition and rates of recovery. Studies of photoinhibition and recovery with and without the translation inhibitor streptomycin demonstrated the importance of a recovery process for the susceptibility of photosynthesis to photoinhibition. We concluded that the approximately 3 times lower susceptibility to photoinhibition of high light than of low light grown cells, significantly depended on high light grown cells having an approximately 3 times higher recovery capacity than low light grown cells. It is suggested that these differences in susceptibility to photoinhibition and recovery depends on high light grown cells having a higher turnover rate of photosystem II protein(s) that is(are) the primary site(s) of photodamage, than have low light grown cells. Furthermore, we demonstrated that photoinhibition of A. nidulans may occur under physiological light conditions without visible harm to the growth of the cell culture. The results give support for the hypotheses that the net photoinhibitory damage of photosystem II results from the balance between the photoinhibitory process and the operation of a recovery process; the capacity of the latter determining significant differences in the susceptibility of photosynthesis to photoinhibition of high and low light grown A. nidulans.     

Membrane lipid composition and restoration of photosynthesis during low temperature acclimation in Synechococcus sp. strain PCC 7942

We compared temperature acclimation of the cyanobacterium Synechococcus sp. strain PCC 7942 and two psbA inactivation mutants, R2K1 and R2S2C3, following shifts from 37 to 25°C. Wild-type cultures incubated in the dark at 25°C showed no chill-induction of lipid desaturation, probably because the lipid acclimation is dependent on photosynthesis. Incubation in the light at 25°C, however, induced considerable increases in membrane lipid desaturation, and within 24 h the monoenoic fatty acids increased from about 46 to about 57%. In parallel with this desaturation the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol (MGDG/DGDG) increased. Both of these lipid changes increase the repulsive forces of the hydrophobic chains of the membrane lipids and thereby alter the physical properties of the membrane. As expected, under irradiation this temperature shift also induced a reversible replacement of the constitutive photosystem II protein, D1:1, with an alternative stress form, D1:2. Photosynthesis decreased to 42% of the control level within the initial 2 h of cold incubation, but later recovered. The D1:2 protein accumulated to high levels between 2 and 4 h after the temperature shift, when desaturation of membrane lipids and MGDG/DGDG ratio had not yet increased significantly. Much of this accumulated D1:2 protein was in a higher molecular mass form, termed D1:2^*, which is probably an unprocessed precursor form of the protein. In contrast to the wild-type cells the psbA inactivation mutants, R2K1 and R2S2C3 did not accumulate any precursor form of D1 protein either at the optimal or low growth temperature. The R2S2C3 mutant strain expresses only the constitutive D1:1 protein and suffered severe photoinhibition following the temperature shift. Nevertheless, R2S2C3 eventually recovered some photosynthetic activity, induced lipid desaturation and slowly resumed growth at 25°C, thus demonstrating acclimation to the lower growth temperature. The R2K1 mutant synthesizes only the D1:2 stress form of D1 protein and maintained oxygen evolution at a high level (ca 70% of a control rate) after the low temperature shift. Chill-induced lipid desaturation and increase in MGDG/DGDG ratio did proceed but, for unknown reasons the strain did not resume growth at the lower temperature. The physical properties of the membrane lipids were not the limiting factor for growth resumption. Our results demonstrate that in the wild-type the chill-induced desaturation of membrane lipids follows after the exchange of the two forms of the D1 proteins, but the D1 exchange results in accumulation of unprocessed D1:2^* polypeptides until the lipid composition later acclimates. We also show that the lipid desaturation process in Synechococcus sp. strain PCC 7942 is dependent upon photosynthetic activity.     

Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803

The cyanobacteria Synechocystis 6803 and 6714 contain three genes (psbA) coding for the D₁ protein. This protein is an essential subunit of photosystem II (PSII) and is the target for herbicides. We have used herbicide-resistant mutants to study the role of the two homologous copies of the psbA genes in both strains (the third copy is not expressed). Several herbicide resistance mutations map within the psbAI gene in Synechocystis 6714 (G. Ajlani et al.), Plant Mol. Biol. 13 (1989): (469–479). We have looked for mutations in copy II. Results show that in Synechocystis 6714, only psbAI contains herbicide resistance mutations. Relative expression of psbAI and psbAII has been measured by analysing the proportions of resistant and sensitive D₁ in the thylakoid membranes of the mutants. In normal growth conditions, 95% resistant D₁ and 5% sensitive D₁ were found. In high light conditions, expression of psbAII was enhanced, producing 15% sensitive D₁. This enhancement is specifically due to high light and not to the decrease of D₁ concentration caused by photoinhibition. Copy I of Synechocystis 6714 corresponds to copy 2 of Synechocystis 6803 since it was always psbA2 which was recombined in Synechocystis 6803 transformants. PSII of the transformant strains was found to be 95% resistant to herbicides as in resistant mutants of Synechocystis 6714.     

Stepwise Photoinhibition of Photosystem II : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the Q_B-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of Q_A reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified Q_B pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.     

Knockdown of the Arabidopsis thaliana chloroplast protein disulfide isomerase 6 results in reduced levels of photoinhibition and increased D1 synthesis in high light

A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild‐type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non‐photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild‐type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light‐regulated manner.     

Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II

Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI ^Q system. Over-expression was induced with 40 g/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 mol photons m^-2 s^-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 mol photons m^-2 s^-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.     

Natural Variants of Photosystem II Subunit D1 Tune Photochemical Fitness to Solar Intensity

Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn₄CaO₅ core of the water-oxidizing complex (WOC). Most cyanobacteria have 3–5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O₂ yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O₂ than either cyanobacterial D1 isoform. D1:2-PSII makes more O₂ per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S₃ state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.     

The roles of specific xanthophylls in light utilization

To evaluate the role of specific xanthophylls in light utilization, wild-type and xanthophyll mutant plants (npq1, npq2, lut2, lut2npq1 and lut2npq2) from Arabidopsis thaliana were grown under three different light regimes: 30 (low light, LL), 150 (medium light, ML) and 450 (high light, HL) μmol photons m⁻² s⁻¹. We studied the pigment content, growth rate, xanthophyll cycle activity, chlorophyll fluorescence parameters and the response to photoinhibition. All genotypes differed strongly in the growth rates and the resistance against photoinhibition. In particular, replacement of lutein (Lut) by violaxanthin (Vx) in the lut2npq1 mutant did not affect the growth at non-saturating light intensities (LL and ML), but led to a pronounced reduction of growth under HL conditions, indicating an important photoprotective role of Lut. This was further supported by a much higher sensitivity of all Lut-deficient plants to photoinhibition in comparison with the wild type. In contrast, replacement of Lut by zeaxanthin (Zx) in lut2npq2 led to a pronounced reduction of growth under all light regimes, most likely related to the permanent non-photochemical dissipation of excitation energy by Zx at Vx-binding sites and the destabilization of antenna proteins by binding of Zx to Lut-binding sites. The high susceptibility of lut2npq2 to photoinhibition in comparison with npq2 further indicated that the photoprotective function of Zx is abolished in the absence of Lut. Thus, it can be concluded from our work that neither Vx nor Zx is able to fulfil the essential photoprotective function at Lut-binding sites under in vivo conditions.     

Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus PCC7942 cells

A reporter gene assay revealed that promoters derived from Synechococcus PCC7942 (S.7942) psbAI and Synechocystis PCC6803 (S.6803) psbAII were suitable for the expression of foreign ribulose-bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) in S.7942 cells. Transformational vectors with a promoter and a foreign RuBisCO gene, cvrbc originated from Allochromatium vinosum, were constructed on a binary vector, pUC303, and introduced to S.7942 cells. When the cvrbc was expressed with the S.7942 psbAI promoter, the total RuBisCO activity increased 2.5- to 4-fold than that of the wild type cell. The S.6803 psbAII promoter increased the activity of the transformant 1.5–2 times of that of wild type cell. There was a significant increase in the rate of photosynthesis depending on the increase of RuBisCO activity. The maximum rate of photosynthesis of the transformant cell was 1.63 times higher than that of the wild type under the illumination of 400 μmol m⁻² s⁻¹, at 20 mM bicarbonate and at 30 °C. Although the photosynthesis of the higher plant is limited by the ability of photosystems under high irradiance and the high CO₂ concentration, that of the S.7942 cell is limited by the RuBisCO activity, even at high CO₂ concentrations and under high irradiance.     

Mutation K42R in Ribosomal Protein S12 Does Not Affect Susceptibility of Mycobacterium smegmatis 16S rRNA A-Site Mutants to 2-Deoxystreptamines

Recent studies have suggested that ribosomal protein S12 modulates 16S rRNA function and susceptibility to 2-deoxystreptamine aminoglycosides. To study whether the non-restrictive K42R mutation in RpsL affects 2-deoxystreptamine susceptibility in Mycobacterium smegmatis, we studied the drug susceptibility pattern of various mutants with genetic alterations in the 16S rRNA decoding A-site in the context of wild-type and mutant protein S12. RpsL K42R substitution was found not to affect the drug resistance pattern associated with mutational alterations in 16S rRNA H44.     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

High Temperature Effects on Light Sensitivity in the Two High Mountain Plant Species Soldanella alpina (L.) and Rannunculus glacialis (L.)

Abstract: The susceptibility to high temperature‐induced photoinhibition was investigated in leaves of two high mountain plant species, S. alpina and R. glacialis. In both species, PSII was similarly photoinactivated at 38 °C in the light. However, recovery from damage was much faster in S. alpina and depended on protein synthesis. In contrast, recovery was independent from protein synthesis in R. glacialis. Heat‐induced photoinactivation in both species was accompanied by: (1) a decrease in relative photosynthetic electron transport rates, (2) an increase in non‐photochemical chlorophyll fluorescence quenching, (3) a strong accumulation of zeaxanthin, (4) a marked decrease in soluble carbon metabolites and (5) an increase in lipid metabolism products, which was more pronounced in R. glacialis than in S. alpina. These results indicate that carbon assimilation was inhibited and that membranes were affected. Lipid peroxidation and possible membrane disintegration might limit the repair of damaged PSII in R. glacialis, while S. alpina appears to be protected by carotenoids and antioxidants. A marked decrease in α‐tocopherol content and an increase in reduced ascorbate indicated lipid peroxide scavenging activity in S. alpina. When zeaxanthin synthesis was impaired by DTT, photoinhibition increased and α‐tocopherol accumulated in R. glacialis. The increased susceptibility of R. glacialis leaves to light‐induced photoinhibition after growth at moderate temperature (Streb et al., 2003a) and the inability to repair heat‐induced damage might limit the distribution of R. glacialis to lower altitudes in the Alps.     

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6–10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear. To address this and other questions, potentially destabilizing substitutions were made in several predicted helices of the fourth psbA intron, Cr.psbA4, and their effects on in vitro and in vivo splicing assessed. Two-nucleotide substitutions in P4 and P7 were necessary to substantially reduce splicing of this intron in vivo, although most mutations reduced self-splicing in vitro. The P7-4,5 mutant, whose splicing was completely blocked, showed no photoautotrophic growth and synthesis of a truncated D1 (exons 1–4) polypeptide from the unspliced mRNA. Most informative was the P4′-3,4 mutant, which exhibited a 45% reduction in spliced psbA mRNA, a 28% reduction in synthesis of full-length D1, and an 18% reduction in photoautotrophic growth. These results indicate that psbA mRNA is not in great excess, and that highly efficient splicing of psbA introns, which is afforded by light conditions, is necessary for optimal photosynthetic growth.