Thf1 interacts with PS I and stabilizes the PS I complex in Synechococcus sp. PCC7942

Thylakoid formation1 protein (Thf1) is a multifunctional protein that is conserved in all photosynthetic organisms. In this study, we used the model cyanobacterium Synechococcus sp. PCC7942 (hereafter Synechococcus) to show that the level of Thf1 is altered in response to various stress conditions. Although this protein has been reported to be involved in thylakoid formation, the thylakoid membrane in the thf1 deletion strain (ΔThf1) was not affected. Compared with the WT, ΔThf1 showed reduced PS II activity, with increased levels of D1 under high light (HL) conditions, which was resulted from blocked D1 degradation by the FtsH protease and thus inhibits PS II repair. PS I was found to be more seriously affected than PS II in ΔThf1, even under low light conditions, suggesting that PS I damage could be the primary effect of thf1 deletion in Synechococcus. Further analysis revealed that the ΔThf1 mutant had a lower PS I subunit content and lower PS I stability under HL conditions. Further sucrose gradient fractionation of the membrane protein complexes and crosslinking and immunoblot analysis indicated that Thf1 interacts with PS I. Together, our results reveal that Thf1 interacts with PS I and thereby stabilizes PS I in Synechococcus.     

Two forms of the Photosystem II D1 protein alter energy dissipation and state transitions in the cyanobacterium Synechococcus sp. PCC 7942

Synechococcus sp. PCC 7942 (Anacystis nidulans R2) contains two forms of the Photosystem II reaction centre protein D1, which differ in 25 of 360 amino acids. D1: 1 predominates under low light but is transiently replaced by D1:2 upon shifts to higher light. Mutant cells containing only D1:1 have lower photochemical energy capture efficiency and decreased resistance to photoinhibition, compared to cells containing D1:2. We show that when dark-adapted or under low to moderate light, cells with D1:1 have higher non-photochemical quenching of PS II fluorescence (higher q_N) than do cells with D1:2. This is reflected in the 77 K chlorophyll emission spectra, with lower Photosystem II fluorescence at 697–698 nm in cells containing D1:1 than in cells with D1:2. This difference in quenching of Photosystem II fluorescence occurs upon excitation of both chlorophyll at 435 nm and phycobilisomes at 570 nm. Measurement of time-resolved room temperature fluorescence shows that Photosystem II fluorescence related to charge stabilization is quenched more rapidly in cells containing D1:1 than in those with D1:2. Cells containing D1:1 appear generally shifted towards State II, with PS II down-regulated, while cells with D1:2 tend towards State I. In these cyanobacteria electron transport away from PS II remains non-saturated even under photoinhibitory levels of light. Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical dissipation of excess energy into the electron transport chain. D1:1 confers capacity for extreme State II which may be of benefit under low and variable light.Abbreviations D1 the atrazine-binding 32 kDa protein of the PS II reaction centre core - D1:1 the D1 protein constitutively expressed during acclimated growth in Synechococcus sp. PCC 7942 - D1:2 an alternate form of the D1 protein induced under excess excitation in Synechococcus sp. PCC 7942 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Fo minimal fluorescence in the dark-adapted state - Fo minimal fluorescence in a light-adapted state - F_M maximum fluorescence with all quenching mechanisms at a minimum, measured in presence of DCMU - F_M maximal fluorescence in a light-adapted state, measured with a saturating flash - F_Mdark maximal fluorescence in the dark-adapted state - F_V variable fluorescence in a light-adapted state (F_M-Fo) - PAM pulse amplitude modulated fluorometer - q_N non-photochemical quenching of PS II fluorescence - q_N (dark) q_N in the dark adapted state - q_P photochemical quenching of fluorescence     

Hydroxy‐plastochromanol and plastoquinone‐C as singlet oxygen products during photo‐oxidative stress in Arabidopsis

In the present study, we have shown that hydroxy‐plastochromanol and plastoquinone‐C, the hydroxy derivatives of plastochromanol and plastoquinone‐9, respectively, are specifically formed from the parent compounds upon action of singlet oxygen and can be regarded as stable, specific, natural products of singlet oxygen action during photo‐oxidative stress in vivo. The presented data indicate that plastoquinone‐C formation dominates mainly during relatively short periods of high light stress where efficient production of singlet oxygen takes place, whereas hydroxy‐plastochromanol is rather formed under conditions of long‐term, less pronounced generation of singlet oxygen. An interesting observation was that hydroxy‐plastochromanol is formed even at very low light conditions (5–10 μmol photons m^?2 s^?1), indicating that singlet oxygen is generated not only during high light stress but also its formation by photosystem II is inseparably connected with the functioning of this photosystem even at the lowest light intensities.     

Expression of the CMV‐CP Gene in Synechocystis 6803 Affects Cyanobacterial Photosynthesis

Previous work has shown that the presence of excess coat protein (CP) of cucumber mosaic virus (CMV) in the chloroplasts was related with mosaic symptoms. However, whether these mosaic symptoms are directly induced by the interaction between CP and chloroplasts is unknown. To directly demonstrate the interaction between CP and the chloroplast, Synechocystis sp. PCC 6803 was used as the chloroplast model. The cDNA encoding the CMV‐CP was cloned in a cyanobacterial shuttle vector (pKT‐CP) and transferred to Synechocystis sp. PCC 6803. The CP was expressed in the cyanobacterium with the psbA promoter. The expression of CMV‐CP hindered the growth of transgenic cyanobacterium cells and decreased its photosynthetic rate and the PS II activity. The transgenic cells showed increased fluorescence (F) from the phycobilisome terminal emitters and increased fluorescence (F) from PS II. The absorption spectra at room temperature showed the Chl and the phycocyanin absorption peak of the mutant strain significantly decreased. These results showed that CP may directly affect the cyanobacterium cells and decreased its photosynthesis, especially the PS II activity. These data might provide new evidence for mosaic symptoms being directly induced by the interaction between CP and chloroplasts.     

The role of chloroplast-encoded protein biosynthesis on the rate of D1 protein degradation in Dunaliella salina

Mechanistic aspects of the Photosystem II (PS II) damage and repair cycle in Dunaliella salina were investigated. The work addressed the role of chloroplast-encoded protein biosynthesis on the rate of the D1 protein (chloroplast psbA gene product) degradation, following photoinhibition of PS II under in vivo conditions. Cells were grown under different light-intensities and the rate of D1 photodamage and degradation was measured via pulse-chase measurements with (³⁵S)sulfate. It is shown that no detectable difference exists in the rate of D1 degradation in D. salina, measured in the presence or absence of lincomycin, a chloroplast protein biosynthesis inhibitor. The results suggest that de novo D1 biosynthesis does not play a role in the regulation of D1 degradation. In low-light (100 mol photons m^–2 s^–1) grown cells, the rate of photodamage to D1 did not exceed the rate of its degradation and replacement. In high-light (2200 mol photons m^–1 s^–1) grown cells, the rate of D1 photodamage was faster than the rate of its degradation, resulting in a significant accumulation of photoinactivated PS II centers in the chloroplast thylakoids (chronic photoinhibition). The latter was coincident with the appearance of a 160 kD complex that contained photodamaged D1. Electron micrographs of D. salina thylakoids revealed extensive grana stacks in the thylakoid membrane of low-light grown cells. Only rudimentary appressions consisting of simple membrane pairings were found in the high-light grown cells. The results are discussed in terms of the regulation of D1 degradation in chloroplasts under in vivo conditions.Abbreviations Chl chlorophyll - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - HL high light - LL low light - Linc lincomycin     

Phosphorylation of PS II polypeptides inhibits D1 protein-degradation and increases PS II stability

To study the significance of Photosystem (PS) II phosphorylation for the turnover of the D1 protein, phosphorylation was compared with the synthesis and content of the D1 protein in intact chloroplasts. As shown by radioactive labelling with [³²P_i] phosphorylation of PS II polypeptides was saturated at light intensities of 125 mol m^-2 s^-1. Under steady state conditions, in intact chloroplasts D1 protein, once it was phosphorylated, was neither dephosphorylated nor degraded in the light. D1 protein-synthesis was measured as incorporation of [¹⁴C] leucine. As shown by non-denaturing gel-electrophoresis followed by SDS-PAGE newly synthesised D1 protein was assembled to intact PS II-centres and no free D1 protein could be detected. D1 protein-synthesis was saturated at light intensities of 500 mol m^-2 s^-1. The content of D1 protein stayed stable even after illumination with 5000 mol m^-2 s^-1 showing that D1 protein-degradation was saturated at the same light intensities. The difference in the light saturation points of phosphorylation and of D1 protein-turnover indicates a complex regulation of D1 protein-turnover by phosphorylation. Separation of the phosphorylated and dephosphorylated D1 protein by LiDS-gelelectrophoresis combined with radioactive pulse-labelling with [¹⁴C] leucine and [³²P_i] revealed that D1 protein, synthesised under steady state conditions in the light, did not become phosphorylated but instead was rapidly degraded whereas the phosphorylated form of the D1 protein was not a good substrate for degradation. According to these observations phosphorylation of the D1 protein creates a pool of PS II centres which is not involved in D1 to these observations phosphorylation of the D1 protein creates a pool of PS II centres which is not involved in D1 protein-turnover. Fractionation of thylakoid membranes confirms that the phosphorylated, non-turning over pool of PS II-centres was located in the central regions of the grana, whereas PS II-centres involved in D1 protein-turnover were found exclusively in the stroma-lamellae and in the grana-margins.Abbreviations chl chlorophyll - F_v yield of variable fluorescence, difference between F_m, the maximal fluorescence yield at saturating light, when all reaction-centres are closed, and F_o, the fluorescence yield in the dark, when all reaction-centres are open - LHC light harvesting complex - PFD photon flux density - PS photosystem     

Ammonium tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 and the role of the psbA multigene family

Ammonium is one of the major nutrients for plants, and a ubiquitous intermediate in plant metabolism, but it is also known to be toxic to many organisms, in particular to plants and oxygenic photosynthetic microorganisms. Although previous studies revealed a link between ammonium toxicity and photodamage in cyanobacteria under in vivo conditions, ammonium‐induced photodamage of photosystem II (PSII) has not yet been investigated with isolated thylakoid membranes. We show here that ammonium directly accelerated photodamage of PSII in Synechocystis sp. strain PCC6803, rather than affecting the repair of photodamaged PSII. Using isolated thylakoid membranes, it could be demonstrated that ammonium‐induced photodamage of PSII primarily occurred at the oxygen evolution complex, which has a known binding site for ammonium. Wild‐type Synechocystis PCC6803 cells can tolerate relatively high concentrations of ammonium because of efficient PSII repair. Ammonium tolerance requires all three psbA genes since mutants of any of the three single psbA genes are more sensitive to ammonium than wild‐type cells. Even the poorly expressed psbA1 gene, whose expression was studied in some detail, plays a detectable role in ammonium tolerance.     

Microaerophilic degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains

Aim: The goal of this study was to compare the degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0·04 mg l^?1 dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l^?1) conditions. Methods and Results: Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60‐fold slower than under fully aerobic conditions. Only the breakdown product, 4‐nitro‐2,4‐diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l^?1) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. Conclusions: The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. Impact and Significance of the Study: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.     

Synthesis of rigid tryptophan mimetics by the diastereoselective Pictet–Spengler reaction of β3‐homo‐tryptophan derivatives with chiral α‐amino aldehydes

The Pictet–Spengler (PS) cyclizations of β³‐hTrp derivatives as arylethylamine substrates were performed with L‐α‐amino and D‐α‐amino aldehydes as carbonyl components. During the PS reaction, a new stereogenic center was created, and the mixture of cis/trans 1,3‐disubstituted 1,2,3,4‐tetrahydro‐β‐carbolines was obtained. The ratio of cis/trans diastereomers depends on the stereogenic centre of used amino aldehyde and the size of substituents. It was confirmed by 1H and 2D NMR (ROESY) spectra. The conformations of cyclic products were studied by 2D NMR ROESY spectra. Products of the PS condensation after removal of protecting group(s) can be incorporated into a peptide chain as tryptophan mimetics with the possibility of the β‐turn induction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors

Irradiation of Spinach oleracea intact leaf tissue and of mesophyll protoplasts of Valerianella locusta at 20° C with strong light resulted in severe (40–80%) inhibition of photosynthesis, measured as photosystem II electron transport activity in isolated thylakoids or as fluorescence parameter F_V/F_M on intact leaf disks. No net degradation of the D1 protein of photosystem II was seen under these conditions. However, in the presence of streptomycin, an inhibitor of chloroplast protein synthesis, net D1 degradation (up to about 80%) did occur with a half-time of 4–6h, and photoinhibition was enhanced. Thylakoid ultrastructure remained stable during photoinhibition, even when substantial degradation of D1 took place in the presence of streptomycin. When leaf disks were irradiated at 2°C, streptomycin did not influence the degree of photoinhibition, and net Dl degradation did not occur. These results suggest that in excess (photoinhibitory) light at 20°C, turnover (coordinated degradation and synthesis) of D1 diminished the degree of photoinhibition. The observed photoinhibition is thought to be due to the accumulation of inactive photosystem II reaction centres still containing D1. In the presence of streptomycin, the Dl protein was degraded (probably in the previously inactivated centres), but restoration of active centres via D1 synthesis was blocked, leading to more severe photoinhibition. Low temperature (2°C), by restricting both degradation and resynthesis of D1, favoured the accumulation of inactive centres. Streptomycin and chloramphenicol (another inhibitor of chloroplast protein synthesis) were tested for side-effects on photosynthesis. Strong inhibitory effects of chloramphenicol, but much less severe effects of streptomycin were observed.     

Photosystem I shows a higher tolerance to sorbitol‐induced osmotic stress than photosystem II in the intertidal macro‐algae Ulva prolifera (Chlorophyta)

The photosynthetic performance of the desiccation‐tolerant, intertidal macro‐algae Ulva prolifera was significantly affected by sorbitol‐induced osmotic stress. Our results showed that photosynthetic activity decreased significantly with increases in sorbitol concentration. Although the partial activity of both photosystem I (PS I) and photosystem II (PS II) was able to recover after 30 min of rehydration, the activity of PS II decreased more rapidly than PS I. At 4 M sorbitol concentration, the activity of PS II was almost 0 while that of PS I was still at about one third of normal levels. Following prolonged treatment with 1 and 2 M sorbitol, the activity of PS I and PS II decreased slowly, suggesting that the effects of moderate concentrations of sorbitol on PS I and PS II were gradual. Interestingly, an increase in non‐photochemical quenching occurred under these conditions in response to moderate osmotic stress, whereas it declined significantly under severe osmotic stress. These results suggest that photoprotection in U. prolifera could also be induced by moderate osmotic stress. In addition, the oxidation of PS I was significantly affected by osmotic stress. P700⁺ in the thalli treated with high concentrations of sorbitol could still be reduced, as PS II was inhibited by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), but it could not be fully oxidized. This observation may be caused by the higher quantum yield of non‐photochemical energy dissipation in PS I due to acceptor‐side limitation (Y(NA)) during rehydration in seawater containing DCMU.     

High diversity of ammonia‐oxidizing archaea in permanent and seasonal oxygen‐deficient waters of the eastern South Pacific

The community structure of putative aerobic ammonia‐oxidizing archaea (AOA) was explored in two oxygen‐deficient ecosystems of the eastern South Pacific: the oxygen minimum zone off Peru and northern Chile (11°S–20°S), where permanent suboxic and low‐ammonium conditions are found at intermediate depths, and the continental shelf off central Chile (36°S), where seasonal oxygen‐deficient and relatively high‐ammonium conditions develop in the water column, particularly during the upwelling season. The AOA community composition based on the ammonia monooxygenase subunit A (amoA) genes changed according to the oxygen concentration in the water column and the ecosystem studied, showing a higher diversity in the seasonal low‐oxygen waters. The majority of the archaeal amoA genotypes was affiliated to the uncultured clusters A (64%) and B (35%), with Cluster A AOA being mainly associated with higher oxygen and ammonium concentrations and Cluster B AOA with permanent oxygen‐ and ammonium‐poor waters. Q‐PCR assays revealed that AOA are an abundant community (up to 10⁵amoA copies ml^?1), while bacterial amoA genes from β proteobacteria were undetected. Our results thus suggest that a diverse uncultured AOA community, for which, therefore, we do not have any physiological information, to date, is an important component of the nitrifying community in oxygen‐deficient marine ecosystems, and particularly in rich coastal upwelling ones.     

Structure‐based design of novel Chlamydomonas reinhardtii D1‐D2 photosynthetic proteins for herbicide monitoring

The D1‐D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, Q_A and Q_B, the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of Q_B binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1‐D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three‐dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1‐D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.     

Light acclimation involves dynamic re‐organization of the pigment–protein megacomplexes in non‐appressed thylakoid domains

Thylakoid energy metabolism is crucial for plant growth, development and acclimation. Non‐appressed thylakoids harbor several high molecular mass pigment–protein megacomplexes that have flexible compositions depending upon the environmental cues. This composition is important for dynamic energy balancing in photosystems (PS) I and II. We analysed the megacomplexes of Arabidopsis wild type (WT) plants and of several thylakoid regulatory mutants. The stn7 mutant, which is defective in phosphorylation of the light‐harvesting complex (LHC) II, possessed a megacomplex composition that was strikingly different from that of the WT. Of the nine megacomplexes in total for the non‐appressed thylakoids, the largest megacomplex in particular was less abundant in the stn7 mutant under standard growth conditions. This megacomplex contains both PSI and PSII and was recently shown to allow energy spillover between PSII and PSI (Nat. Commun., 6, 2015, 6675). The dynamics of the megacomplex composition was addressed by exposing plants to different light conditions prior to thylakoid isolation. The megacomplex pattern in the WT was highly dynamic. Under darkness or far red light it showed low levels of LHCII phosphorylation and resembled the stn7 pattern; under low light, which triggers LHCII phosphorylation, it resembled that of the tap38/pph1 phosphatase mutant. In contrast, solubilization of the entire thylakoid network with dodecyl maltoside, which efficiently solubilizes pigment–protein complexes from all thylakoid compartments, revealed that the pigment–protein composition remained stable despite the changing light conditions or mutations that affected LHCII (de)phosphorylation. We conclude that the composition of pigment–protein megacomplexes specifically in non‐appressed thylakoids undergoes redox‐dependent changes, thus facilitating maintenance of the excitation balance between the two photosystems upon changes in light conditions.     

New prenyllipid metabolites identified in Arabidopsis during photo‐oxidative stress

In the present study, we have identified new prenyllipid metabolites formed during high light stress in Arabidopsis thaliana, whose origin and function remained unknown so far. It was found that plastoquinone‐C accumulates mainly in the reduced form under high light conditions, as well as during short‐term excess light illumination both in the wild‐type and tocopherol biosynthetic vte1 mutant, suggesting that plastoquinone‐C, a singlet oxygen‐derived prenyllipid, is reduced in chloroplasts by photosystem II or enzymatically, outside thylakoids. Plastoquinone‐B, a fatty acid ester of plastoquinone‐C, was identified for the first time in Arabidopsis in high light grown wild‐type plants and during short‐time, excess light illumination of the wild‐type plants and the vte1 mutant. The gene expression analysis showed that vte2 gene is most pronouncedly up‐regulated among the prenyllipid biosynthetic genes under high light and induction of its expression is mainly caused by an increased level of singlet oxygen, as was demonstrated in experiments with D₂O‐treated plants under excess light conditions.     

Inhibition of CO2-fixation and its effect on the activity of Photosystem II,on D1-protein synthesis and phosphorylation

To study the effects of limitations in the Calvin-cycle on Photosystem (PS) II function and on its repair by D1-protein turnover, glycerinaldehyde (DLGA) was applied to 1 h dark-adapted pea leaves via the petiole. The application resulted in a 90% inhibition of photosynthetic oxygen evolution after 90 min illumination at either 120 or 500 µmol m^–2 s^–1. In the control leaves an increase of light-dependent oxygen production to 147 and 171% was observed after 90 min illumination. According to chlorophyll fluorescence quenching analysis the inhibition of photosynthetic electron transport by DLGA led to a substantial increase in the reduction state of the primary quinone acceptor of PS II, QA, and to a rise in membrane energetisation. However, PS II functionality was hardly affected by DLGA at the low light intensity as indicated by the constant high yield of variable fluorescence, Fv/Fm. Only at 500 µmol m^–2 s^–1 a 15% loss of Fv/Fm was observed in the presence of DLGA indicating that inactivated PS II centres had accumulated. The control leaves also showed a slight loss of Fv/Fm which did not affect photosynthetic electron transport due to a faster reoxidation of QA. The relative stability of PS II function in the presence of DLGA could not be ascribed to an increased repair by the rapid turnover of the D1-protein. Radioactive pulse-labelling studies with [14C] leucine in combination with immunological determination of the protein content revealed that both synthesis and degradation of the protein were inhibited in DLGA-treated leaves whereas in the control leaves a stimulation of D1-protein turnover was observed. The changes of D1-protein turnover could be explained by differences in the occupancy state of the QB-binding niche. A relation between the phosphorylation status of the PS II polypeptides and the turnover of the D1-protein could not be established. As shown by radioactive labelling with [32P]i, addition of DLGA led to an increase in the phosphorylation level of the PS II polypeptides D1 and D2 at the low light intensity when compared to the non-treated control. At the higher light intensity the phosphorylation level of the PS II polypeptides in control and DLGA-treated leaves were identical in spite of the substantial differences in D1-protein turnover.