Presence Of Lincomycin Research Articles

Photoinhibition in benthic diatom assemblages under light stress

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 70:87-92 (2013) - DOI: https://doi.org/10.3354/ame01648 NOTE Photoinhibition in benthic diatom assemblages under light stress P. Cartaxana1,*, N. Domingues1,2, S. Cruz3, B. Jesus2,4, M. Laviale3, J. Serôdio3, J. Marques da Silva2 1Centro de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal 2Departamento de Biologia Vegetal and Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG), Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal 3Departamento de Biologia and CESAM – Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal 4LUNAM université, Université de Nantes, Mer Molécules Santé EA 2160, Faculté des Sciences et des Techniques, BP 92208, 44322 Nantes cedex 3, France *Email: pcartaxana@fc.ul.pt ABSTRACT: Microphytobenthos are frequently subjected to light intensities higher than those required to saturate photosynthesis, which consequently can cause photoinhibition. Photosystem II (PSII) protein D1 (the main target of photoinhibition) and xanthophyll cycle pigments were quantified in epipelic benthic diatom assemblages under high irradiance, in the presence of inhibitors and promoters of photoprotection mechanisms. Levels of D1 protein were significantly lower under high irradiance (1 h, 1500 µmol photons m–2 s–1) than under low light (80 µmol photons m–2 s–1), corresponding to a photoinhibition of 22 to 29%. Photoinhibition increased to 44 and 80% in the presence of lincomycin (inhibitor of chloroplast-protein synthesis) and dithiothreitol (inhibitor of the xanthophyll cycle), respectively. High light treatment had no significant effect on D1 protein concentrations in the presence of added glutathione and ascorbate, scavengers of reactive oxygen species (ROS). In contrast, the ROS promoter methylviologen increased photoinhibition to 63%. Under light stress, the functional stability of PSII reaction centres of the studied epipelic benthic diatoms was more dependent on xanthophyll cycle activation than on D1 protein recycling mechanisms, and our results substantiate the role of antioxidants in photoprotection via ROS scavenging. KEY WORDS: Microphytobenthos · Xanthophyll cycle · D1 protein · Reactive oxygen species · Photoprotection · Antioxidants Full text in pdf format PreviousCite this article as: Cartaxana P, Domingues N, Cruz S, Jesus B, Laviale M, Serôdio J, Marques da Silva J (2013) Photoinhibition in benthic diatom assemblages under light stress. Aquat Microb Ecol 70:87-92. https://doi.org/10.3354/ame01648 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 70, No. 1. Online publication date: August 06, 2013 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2013 Inter-Research.

Large‐scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

Magnetic field protects plants against high light by slowing down production of singlet oxygen

Recombination of the primary radical pair of photosystem II (PSII) of photosynthesis may produce the triplet state of the primary donor of PSII. Triplet formation is potentially harmful because chlorophyll triplets can react with molecular oxygen to produce the reactive singlet oxygen (¹O₂). The yield of ¹O₂ is expected to be directly proportional to the triplet yield and the triplet yield of charge recombination can be lowered with a magnetic field of 100-300 mT. In this study, we illuminated intact pumpkin leaves with strong light in the presence and absence of a magnetic field and found that the magnetic field protects against photoinhibition of PSII. The result suggests that radical pair recombination is responsible for significant part of ¹O₂ production in the chloroplast. The magnetic field effect vanished if leaves were illuminated in the presence of lincomycin, an inhibitor of chloroplast protein synthesis, or if isolated thylakoid membranes were exposed to light. These data, in turn, indicate that ¹O₂ produced by the recombination of the primary charge pair is not directly involved in photoinactivation of PSII but instead damages PSII by inhibiting the repair of photoinhibited PSII. We also found that an Arabidopsis thaliana mutant lacking α-tocopherol, a scavenger of ¹O₂, is more sensitive to photoinhibition than the wild-type in the absence but not in the presence of lincomycin, confirming that the target of ¹O₂ is the repair mechanism.

D1 protein turnover is involved in protection of Photosystem II against UV-B induced damage in the cyanobacterium Arthrospira ( Spirulina) platensis

By using two strains of Arthrospira (Spirulina) platensis, an economically important filamentous cyanobacterium, we compared the impairment of PSII activity and loss of D1 protein content under UV-B radiation. Our study showed that UV-B radiation induced a gradual loss of the oxygen-evolving activity to about 56% after 180 min UV-B irradiation both in strains 439 and D-0083, which have been kept under indoor and an outdoor culturing conditions, respectively for a prolonged period of time. The loss of oxygen evolution was accelerated in both strains in the presence of lincomycin, an inhibitor of protein synthesis, and the amount of D1 protein showed a decrease comparable to that of oxygen evolution during the UV-B exposure. However, the UV-B induced loss of oxygen-evolving activity and D1 protein amount was largely prevented when A. platensis cells were exposed to UV-B irradiance supplemented with visible light. Comparison of the two strains also showed a smaller extent of D1 protein synthesis dependent PSII repair in the indoor strain. Our results show that turnover of the D1 protein is an important defense mechanism to counteract the UV-B induced damage of PSII in A. platensis, and also that visible light plays an important role in maintaining the function of PSII under simultaneous exposure to UV-B and visible light.

The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development

Developing seedlings of the Arabidopsis gun1 (genomes uncoupled1) mutant, which is defective in retrograde plastid-to-nucleus signalling, show several previously unrecognized mutant phenotypes. gun1 seedlings accumulated less anthocyanin than wild-type seedlings when grown in the presence of 2% (w/v) sucrose, due to lower amounts of transcripts of early anthocyanin biosynthesis genes in gun1. Norflurazon and lincomycin, which induce retrograde signalling, further decreased the anthocyanin content of sucrose-treated seedlings, and altered the temporal pattern of anthocyanin accumulation. Lincomycin treatment altered the spatial pattern of sucrose-induced anthocyanin accumulation, suggesting that plastids provide information for the regulation of anthocyanin biosynthesis in Arabidopsis seedlings. The temporal pattern of accumulation of LHCB1 transcripts differed between wild-type and gun1 seedlings, and gun1 seedlings were more sensitive to sucrose suppression of LHCB1 transcript accumulation than wild-type seedlings. Growth and development of gun1 seedlings was more sensitive to exogenous 2% sucrose than wild-type seedlings and, in the presence of lincomycin, cotyledon expansion was enhanced in gun1 seedlings compared to the wild type. gun1 seedlings were more sensitive than wild-type seedlings to the inhibition of seedling growth and development by abscisic acid. These observations clearly implicate GUN1 and plastid signalling in the regulation of seedling development and anthocyanin biosynthesis, and indicate a complex interplay between sucrose and plastid signalling pathways.

De Novo Biosynthesis of Fatty Acids Plays Critical Roles in the Response of the Photosynthetic Machinery to Low Temperature in Arabidopsis

The Arabidopsis thaliana kas3 mutant was isolated based on the hypersensitivity of PSII to low temperature using a Chl fluorescence imaging technique. Chl content was lower in kas3 seedlings cultured at 23 degrees C than in the wild type, but PSII activity was only mildly affected. However, after the chilling treatment at 4 degrees C for 7 d, PSII activity was severely impaired in kas3. PSII was more sensitive to light at 4 degrees C in the presence of lincomycin, suggesting that the kas3 mutation accelerates at least the PSII photodamage. The kas3 mutation causes an amino acid alteration in 3-ketoacyl-ACP synthase III (KasIII), leading to the partial loss of the de novo synthesis pathway for fatty acids in plastids. Consequently, the total fatty acid level was reduced to 75% of the wild-type level in kas3 at 23 degrees C and was further reduced to 60% at 4 degrees C. The composition of fatty acids was also slightly affected in kas3 at both 4 and 23 degrees C. Consistent with the results of the electron transport analysis, the chilling treatment also destabilized PsaA and cytochrome (Cyt) f and D1 in kas3. An analysis of double mutants with pgr1 conditionally defective in Cyt b(6)f activity and with var2 defective in FtsH protease suggested that the kas3 mutation has pleiotropic effects on chloroplast function, probably impacting both the Cyt b(6)f activity and translation in chloroplasts at 23 degrees C. The full activity of KasIII is required for the biogenesis of the intact electron transport machinery in thylakoid membranes and is especially important for the process of responding to low temperature.

Function of ROC4 in the Efficient Repair of Photodamaged Photosystem II in Arabidopsis†

ROC4 is the only cyclophilin in the chloroplast stroma. Here, we used the T-DNA knockout mutant of roc4 to study the physiological role of ROC4 in vivo in Arabidopsis thaliana. Our results showed that ROC4 is not required for the biogenesis and functional operation of photosystem II (PSII). However, growth in greenhouse and PSII activity, as detected by photoinhibition measurements showed increased sensitivity to high light irradiance in the mutant. In the presence of chloroplast protein synthesis inhibitor lincomycin, which blocks de novo protein synthesis and thus the repair of PSII, wild-type and mutant plants showed a similar extent of inactivation of PSII activity. The recovery of PSII activity in roc4 leaves from photoinhibition is also impaired compared with that of wild-type plants. Immunoblot analysis showed that the degradation of PSII reaction center proteins occurred at a similar rate in the presence of lincomycin in wild-type and mutant plants. Thus, these results suggest that ROC4 functions in the repair of photodamaged PSII.

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

Differential regulation of psbA and psbD gene expression, and the role of the different D1 protein copies in the cyanobacterium Thermosynechococcus elongatus BP-1

In Thermosynechococcus elongatus BP-1 , which is the preferred organism in recent structural studies of PSII, three psbA and two psbD genes code for three D1 and one D2 protein isoforms, respectively. The regulation and function of these genes and protein products is largely unknown. Therefore, we used quantitative RT-PCR to follow changes in the mRNA level of the respective genes, in combination with biophysical measurements to detect changes in the electron transport activity of Photosystem II under exposure to different visible and UV light, and temperature conditions. In cells which are acclimated to 40 μmol m − 2 s − 1 growth light conditions at 40 °C the main populations of the psbA and psbD transcripts arise from the psbA1 and psbD1 genes, respectively. When the temperature is raised to 60 °C psbA1 becomes the single dominating psbA mRNA species. Upon exposure of the cells to 500 μmol m − 2 s − 1 intensity visible light psbA3 replaces psbA1 as the dominating psbA mRNA species, and psbD2 increases at the expense of psbD1. UV-B radiation also increases the abundance of psbA3, and psbD2 at the expense of psbA1 and psbD1, respectively. From the different extent of total D1 protein loss in the absence and presence of lincomycin it was estimated that the PsbA3 protein isoform replaces PsbA1 in about 65% of PSII centers after 2 h of high light acclimation. Under the conditions of different psbA transcript distributions chlorophyll fluorescence and thermoluminescence measurements were applied to monitor charge recombination characteristics of the S 2Q A − and S 2Q B − states. We obtained faster decay of flash-induced chlorophyll fluorescence in the presence of DCMU, as well as lower peak temperature of the Q and B thermoluminescence bands when PsbA3 replaced PsbA1 as the main D1 protein isoform. The relevance of dynamic changes in the abundance of psbA and psbD transcript levels, as well as D1 protein isoforms in the acclimation of T. elongatus to changing environmental conditions is discussed.

A Bacterial Transgene for Catalase Protects Translation of D1 Protein during Exposure of Salt-Stressed Tobacco Leaves to Strong Light

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum 'Xanthi') and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [(35)S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplast's translational machinery to salt stress by scavenging hydrogen peroxide.

광 스트레스에 대한 고추 잎의 광합성 반응과 광 보호 메커니즘

Photoinhibition and photoprotection of PSII in the leaves of hot pepper (Capsicum annuum L.) grown in Hoagland solution and Tap water were compared. Though changes in the rates of <TEX>$O_2$</TEX> evolution as a function of photon fluence rate (PFR) were comparable, the rates of respiration in the dark was 3 times higher in the Hoagland solution grown leaves than in the Tap-water grown ones. Compared to Hoagland solution grown plane, PSIIs of Tap water grown pepper leaves were more susceptible to photoinhibitory light treatment. In order to inactivate functional PSII to the same extents, Hoagland solution grown plants required almost 2-fold high light <TEX>$(1600{\mu}molm^{-2}s^-)$</TEX> treatment than those of Tap water <TEX>$(900{\mu}molm^{-2}s^-)$</TEX>. Interestingly, the remaining fraction of PSII in Hoagland grown pepper was able to survive under prolonged illumination in the presence of lincomycin, which probably means that the growth condition of plant seemed to have an effect on the recovery of PSII from light stress. When PSII was severly photoinactivated at a chilling temperature, recovery was observed only if the residual functional PSII were not inhibited with DCMU, Nigericin and MV during recovery. In conclusion, PSIIs grown in the Hoagland solution was more resistant to excess light than in the Tap water grown one and the recovery of PSII from photodamage was more efficient in Hoagland grown pepper leaves than Tap water grown one, which means that the increased dark respiration may play a important role in the protection of PSII from photoinhibition by helping repair photosynthetic proteins (in particular, the D1 protein of PSII) degraded by photoinhibition.

Quantitative analysis of lincomycin in animal tissues and bovine milk by liquid chromatography electrospray ionization tandem mass spectrometry

A sensitive method for determining lincomycin in bovine milk, animal muscles and organs using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) is presented. Milk and homogenized animal tissues were extracted with acetonitrile twice after addition of an appropriate amount of clindamycin, a lincosamide analogue as the internal standard. The combined extracts were finally made up to 10 ml with distilled water and partitioned with hexane to remove the animal fats prior to analysis. Analytes in the extracts were separated on a reversed phase C18 column ( 250 mm×2.1 mm, 5 μm) using a mobile phase of a 3:7 (v/v) mixture of 0.1% formic acid in acetonitrile and an ammonium formate buffer (ammonium formate:formic acid:acetonitrile:water, 1:5:50:950, v/v/v/v) running at a flow rate of 0.2 ml min −1. Presence of lincomycin was confirmed by the presence of two characteristic product ions at m/ z 126.1 and 359.2 within a defined retention time window from the precursor ion at m/ z 407.2, whilst quantification was based on the relative ratio of the sum of the peak areas at m/ z 126.1 and 359.2 for lincomycin to that of the internal standard (peaks at m/ z 126.1 and 377.2) with reference to the respective ratios of the calibration standards. The validated method that was found to have linear responses in the calibration range from 25 to 3000 μg kg −1 and satisfactory intra-day and inter-day accuracy (94.4–107.8%) and precision (1.3–7.8%) at concentrations ranging from 100 to 1500 μg kg −1 has been applied to real samples and matrix spiked samples. It is considered robust and suitable for analysis of lincomycin in milk and animal tissues.

Contribution of photosynthetic electron transport, heat dissipation, and recovery of photoinactivated photosystem II to photoprotection at different temperatures in Chenopodium album leaves.

Temperature dependence of photoinhibition and photoprotective mechanisms (10-35 degrees C) was investigated for Chenopodium album leaves grown at 25 degrees C under 500 micro mol quanta m(-2) s(-1). The fraction of active photosystem II (PSII) was determined after photoinhibitory treatment at different temperatures in the presence and absence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis. In the absence of lincomycin, leaves were more tolerant to photoinhibition at high (25-35 degrees C) than at low (11-15 degrees C) temperatures. In the presence of lincomycin, the variation in the tolerance to photoinactivation became relatively small. The rate constant of photoinactivation (k(pi)) was stable at 25-35 degrees C and increased by 50% with temperature decrease from 25 to 11 degrees C. The rate constant of recovery of inactivated PSII (k(rec)) was more sensitive to temperature; it was very low at 11 degrees C and increased by an order of magnitude at 35 degrees C. We conclude that the recovery of photoinactivated PSII plays an essential role in photoprotection at 11-35 degrees C. Partitioning of light energy to various photoprotective mechanisms was further analyzed to reveal the factor responsible for k(pi). The fraction of energy utilized in photochemistry was lower at lower temperatures. Although the fraction of heat dissipation increased with decreasing temperatures, the excess energy that is neither utilized by photochemistry nor dissipated by heat dissipation was found to be greater at lower temperatures. The k(pi) value was strongly correlated with the excess energy, suggesting that the excess energy determines the rate of photoinactivation.

Isolation and partial characterization of a Synechococcus sp. PCC 7942 mutant resistant to a natural herbicide

Mutants of Synechococcus sp. PCC 7942 resistant to a natural photosystem II-inhibitory herbicide, oscillatorin, were isolated from a random mutagenesis with N-methyl- N′-nitro- N-nitrosoguanidine. These mutants had elevated growth rates and photosynthetic activity relative to the wild type, and exhibited a high degree of resistance to phenolic-type but not to the atrazine-class of herbicides. Examination of dichlorophenol indophenol photo-reductions in the wild type with water and diphenyl carbazide electron donors revealed that oscillatorin preferentially affects the water oxidizing complex. A comparative analysis of the content of photosynthesis associated proteins revealed that in one such mutant designated Osc r1, PsbO, the manganese stabilizing protein, was highly expressed even under manganese and calcium deficient growth conditions. The amount of PsbA was also higher in the mutant. Solubilizing Mn from the water oxidizing complex was less inhibitory to photosystem II activity in mutant Osc r1. While wild type showed significant photoinhibition in the presence of lincomycin, mutant Osc r1 was much more resistant. Our results suggest that over-expression of PsbO in the mutant Osc r1 and related stability and integrity of water oxidizing complex confers resistance to the oxidative stress caused by oscillatorin.

A Synechococcus sp. PCC 7942 mutant with a higher tolerance towards bentazone.

In this article we describe the partial characterization of a Synechococcus sp. PCC 7942 mutant Mu1 with an enhanced resistance towards the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). The mutant was derived from a random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NSG) and exhibited superior growth rates, pigment content and overall photosynthetic activities under regular growth conditions compared to wild type. Whereas Synechococcus PCC 7942 wild type showed significant photoinhibition, especially in the presence of lincomycin, Mu1 was much more robust. A comparative analysis of the content of several photosynthesis-associated proteins revealed that Mu1 had an increased expression of PsbO on mRNA and protein level and that PsbO is tightly bound to Photosystem II, relative to wild type. This result was substantiated by mass spectrometer measurements of photosynthetic water oxidation revealing a higher stability and integrity of the water oxidizing complex in Mu1 cells grown under regular or calcium deficient conditions. Therefore, our results give rise to the possibility that the overexpression of PsbO in mutant Mu1 confers resistance to reactive oxygen species (ROS) formed as a consequence of bentazone binding to the acceptor side of PS II. In addition, we observed a significantly higher tolerance towards bentazone in iron depleted wild type cells, conditions under which the IdiA protein becomes expressed in highly elevated amounts. As we have previously shown, IdiA preferentially protects the acceptor site of PS II against oxidative stress, especially under iron limitation. Thus, it is likely that IdiA due to its topology interferes with bentazone binding or protects PS II against ROS generated in the presence of bentazone.

A Critical Role for the Var2 FtsH Homologue of Arabidopsis thaliana in the Photosystem II Repair Cycle in Vivo

Using a var2-2 mutant of Arabidopsis thaliana, which lacks a homologue of the zinc-metalloprotease, FtsH, we demonstrate that this protease is required for the efficient turnover of the D1 polypeptide of photosystem II and protection against photoinhibition in vivo. We show that var2-2 leaves are much more susceptible to light-induced photosystem II photoinhibition than wild-type leaves. Furthermore, the rate of photosystem II photoinhibition in untreated var2-2 leaves is equivalent to that of var2-2 and wild-type leaves, which have been treated with lincomycin, an inhibitor of the photosystem II repair cycle at the level of D1 synthesis. This is in contrast to untreated wild-type leaves, which show a much slower rate of photosystem II photoinhibition due to an efficient photosystem II repair cycle. The recovery of var2-2 leaves from photosystem II photoinhibition is also impaired relative to wild-type. Using Western blot analysis in the presence of lincomycin we show that the D1 polypeptide remains stable in leaves of the var2-2 mutant under photoinhibitory conditions that lead to D1 degradation in wild-type leaves and that the abundance of DegP2 is not affected by the var2-2 mutation. We conclude, therefore, that the Var2 FtsH homologue is required for the cleavage of the D1 polypeptide in vivo. In addition, we identify a conserved lumenal domain in Var2 that is unique to FtsH homologues from oxygenic phototrophs.

Photoinactivation of photosystem II complexes and photoprotection by non-functional neighbours in Capsicum annuum L. leaves.

Leaf segments from Capsicum annuum plants grown at 100 micromol photons m(-2) s(-1) (low light) or 500 micromol photons m(-2) s(-1) (high light) were illuminated at three irradiances and three temperatures for several hours. At various times, the remaining fraction (f) of functional photosystem II (PS II) complexes was measured by a chlorophyll fluorescence parameter (1/Fo -1/Fm, where Fo and Fm are the fluorescence yields corresponding to open and closed PS II traps, respectively), which was in turn calibrated by the oxygen yield per saturating single-turnover flash. During illumination of leaf segments in the presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis, the decline of f from 1.0 to about 0.3 was mono-exponential. Thereafter, f declined much more slowly, the remaining fraction (approximately equals 0.2) being able to survive prolonged illumination. The results can be interpreted as being in support of the hypothesis that photoinactivated PS II complexes photoprotect functional neighbours (G. Oquist et al. 1992, Planta 186: 450-460), provided it is assumed that a photoinactivated PS II is initially only a weak quencher of excitation energy, but becomes a much stronger quencher during prolonged illumination when a substantial fraction of PS II complexes has also been photoinactivated. In the absence of lincomycin, photoinactivation and repair of PS II occur in parallel, allowing f to reach a steady-state value that is determined by the treatment irradiance, temperature and growth irradiance. The results obtained in the presence and absence of lincomycin are analysed according to a simple kinetic model which formally incorporates a conversion from weak to strong quenchers, yielding the rate coefficients of photoinactivation and of repair for various conditions, as well as gaining an insight into the influence off on the rate coefficient of photoinactivation. They demonstrate that the method is a convenient alternative to the use of radiolabelled amino acids for quantifying photoinactivation and repair of PS II in leaves.

A last-ditch defence against high-light stress: photoinactivated photosystem II complexes protect functional neighbours in Capsicum annuum L. leaves

Leaf segments from Capsicum annuum plants grown in 100 (low light) or 500 (high light) µmol m?2 s?1 were illuminated at three irradiances and three temperatures for several hours. At various times, the remaining fraction (f ) of functional photosystem II (PS II) complexes was measured by a chlorophyll fluorescence parameter (1/Fo ? 1/Fm, where Fo and Fm are the fluorescence yields corresponding to open and closed PS II traps, respectively), which was in turn calibrated by the oxygen yield per saturating single-turnover flash. During illumination of leaf segments in the presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis, the decline of f from 1.0 to about 0.3 was mono-exponential. Thereafter, f declined much more slowly, the remaining fraction (~0.2) being able to survive prolonged illumination. The results can be interpreted as being in support of the hypothesis that photoinactivated PS II complexes photoprotect functional neighbours [Oquist et al. (1992) Planta 186: 450-460]. In the absence of lincomycin, photoinactivation and repair of PS II occur in parallel, allowing f to reach a steady-state value. The results obtained in the presence and absence of lincomycin are analysed according to a simple kinetic model which formally incorporates a conversion from weak to strong quenchers, yielding the (intrinsic) rate coefficient of photoinactivation and the rate coefficient of repair, as well as gaining an insight into the influence of f on the operational rate coefficient of photoinactivation.

The repair of photosystem II is the site of regulation by environmental stresses

The photosynthetic machinery, in particular the photosystem II complex (PSII), is rapidly damaged by light, but it is also rapidly repaired by light in vivo. Therefore, the extent of photodamage in living organisms is a result of balance between the photo-induced damage to PSII and the repair of the photodamaged PSII. We developed a method to separately measure the damage and repair processes in the cyanobacterium Synechocystis sp. PCC 6803. The initial rate of strong light-induced inactivation of PSII was measured in the presence of lincomycin which blocks the repair process by inhibiting the protein synthesis, whereas the initial rates of weak light-induced recovery of PSII activity was measured after switching the light intensity from strong to weak. We observed that the initial rate of photodamage was proportional to light intensity and this proportionality was not affected by the presence of DCMU or reactive oxygen species (ROS) such as superoxide radicals and H2O2 and by stress conditions due to high salt and cold. In contrast, the initial rate of repair was fast at low intensities of light and was saturated at 300 µE m-2 s-1, and this rate of repair at the saturation level was depressed by the environmental factors. Based on these observations we conclude that the photodamage to PSII depends solely on light intensity without regulation and that the repair of the PSII is the site of regulation by environments. The results of Northern blotting and Western blotting analyses suggest that the major site of inhibitory action by environmental factors is the translation of psbA genes.

A very early event of photoinactivation of photsystem II at low temperature

When the photosynthetic machinery is irridiated by strong light the photosystem II (PSII) complex is photodamaged although PSII is repaired by a process which requires protein synthesis. We investigated the photoinactivation and recovery of PSII at low temperatures in Synechocystis sp. PCC 6803 cells and a mutant defective in unsaturation of membrane lipids. When the cells were exposed to light of 2000 µE m-2 s-2 at 0°C and then were incubated at 34°C in darkness, approximately 50% of the PSII activity recovered after 60 min. This recovery occurred in the presence of lincomycin which inhibits translation of D1 protein suggesting that the recovery in darkness did not involve synthesis de novo of proteins. This observation together with other results lead us to hypothesize of the involvement of an intermediately photoinactivated PSII prior to the stable photodamaged PSII, which is currently well discussed.