Quality control of Photosystem II under light stress – turnover of aggregates of the D1 protein in vivo

When photodamaged under excessive light, the D1 protein is digested and removed from Photosystem (PS) II to facilitate turnover of the protein. In vitro studies have shown that part of the photodamaged D1 protein forms aggregates with surrounding polypeptides before being digested by a protease(s) in the stroma [Yamamoto Y (2001) Plant Cell Physiol 42: 121–128]. The aim of this study was to examine whether light-induced aggregation of the D1 protein also occurs in vivo. The following results were obtained: (1) PS II activity in spinach leaves was significantly inhibited by weak illumination (light intensity, 20–100 μE m⁻² s⁻¹), as monitored by chlorophyll fluorescence Fv/Fm, when the leaves were kept at higher temperatures (35–40 °C); (2) aggregation of the D1 protein, as well as cleavage of the protein, was detected in thylakoids isolated from spinach leaves that had been subjected to heat/light stress; (3) aggregates of the D1 protein disappeared after incubation of the leaves at 25 °C in the dark or under illumination with weak light. Since it is dependent on the presence of oxygen, aggregation of the D1 protein is probably induced by reactive oxygen species produced in thylakoids upon illumination at elevated temperatures. Consistent with this notion, singlet oxygen production in thylakoid samples under illumination was shown to be stimulated significantly at higher temperatures.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.     

Evidence for protection by heat-shock proteins against photoinhibition during heat-shock

The nuclear-coded 22 kd heat-shock protein (HSP-22) which is transported into the chloroplast and localized in the thylakoids was further characterized and found to be located in the grana lamellae (stacked thylakoids) as an extrinsic protein in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthetic electron flow during heat-shock of Chlamydomonas cells was light-dependent, occurring at low-light intensities (<100 W/m²) as compared with photoinhibition at 25°C (>1000 W/m²). The site of the damage was localized at the photosystem II (PS II) reaction center. The damage was drastically increased when heat-shock treatment was carried out in the presence of the 80S ribosomal translation inhibitor, cycloheximide (CHI). Pre-incubation of Chlamydomonas cells at 42°C resulted in partial protection against photoinhibition during heat-shock, as compared with cells pre-incubated at 42°C in the presence of CHI which, therefore, did not translate the heat-shock proteins. Analysis of the thylakoid polypeptides' pattern by SDS-PAGE revealed that during heat-shock in the light, thylakoid proteins became aggregated proportionally to the light intensity. Heat-shock in the presence of CHI enhanced the aggregation process which, at low light intensities, was specific to the PS II reaction center D1-protein. The results suggest that the chloroplasts HSPs prevent damage to the PS II reaction center during heat-shock in the light.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Rising CO2 Interacts with Growth Light and Growth Rate to Alter Photosystem II Photoinactivation of the Coastal Diatom Thalassiosira pseudonana

We studied the interactive effects of pCO₂ and growth light on the coastal marine diatom Thalassiosira pseudonana CCMP 1335 growing under ambient and expected end-of-the-century pCO₂ (750 ppmv), and a range of growth light from 30 to 380 µmol photons·m⁻²·s⁻¹. Elevated pCO₂ significantly stimulated the growth of T. pseudonana under sub-saturating growth light, but not under saturating to super-saturating growth light. Under ambient pCO₂ susceptibility to photoinactivation of photosystem II (σ_i) increased with increasing growth rate, but cells growing under elevated pCO₂ showed no dependence between growth rate and σ_i, so under high growth light cells under elevated pCO₂ were less susceptible to photoinactivation of photosystem II, and thus incurred a lower running cost to maintain photosystem II function. Growth light altered the contents of RbcL (RUBISCO) and PsaC (PSI) protein subunits, and the ratios among the subunits, but there were only limited effects on these and other protein pools between cells grown under ambient and elevated pCO₂.     

Effects of Low-Temperature Acclimation and Oxygen Stress on Tocopheron Production in Euglena gracilis Z

The effects of low-temperature acclimation and oxygen stress on tocopheron production were examined in the unicellular phytoflagellate Euglena gracilis Z. Cells were cultured photoheterotrophically at 27.5 ± 1°C with 5% carbon dioxide-95% air and 740 microeinsteins m⁻² s⁻¹ (photosynthetically active radiation) and served as controls. Low-temperature acclimation (12.5 ± 1°C) and high-oxygen stress (5% carbon dioxide-95% oxygen) were individually examined in the mass culturing of the algae. Chromatographic analyses demonstrated a six-to sevenfold enhancement of α-tocopherol production in temperature-stressed cells, along with a concomitant decline in the levels of α-tocotrienol and the absence of other tocopherol homologs. Oxygen-stressed cultures demonstrated the presence of high levels of α-tocopherylquinone; α-tocopheron and its homologs and precursors were absent or declined markedly. These findings are discussed in terms of the feasibility of microbial production of natural tocopherols. In addition, these results lend themselves to speculation regarding the biological role(s) of tocopherols as antioxidants and free radical scavengers in reducing photo-induced oxidative damage or lipid peroxidation toxicities or both in photosynthetically active E. gracilis Z.     

Changes in lipid composition of Photosystem 1 particles from thylakoids phosphorylated under reductive or anaerobic conditions

Changes in lipid composition of Photosystem 1 (PS 1) particles isolated from thylakoids phosphorylated under reductive or anaerobic conditions have been studied. Under reductive conditions, there was an increase in monogalactosyldiacylglycerol containing highly saturated fatty acids and phosphatidylglycerol containing transhexadecenoic fatty acid. Under anaerobic conditions, the amount of all lipid classes was increased. As we have shown earlier (S. V. Manuilskaya, O. I. Volovik, A. I. Mikhno, A. I. Polischuk and S. M. Kochubey (1990) Photosynthetica 24: 419–423) these changes were due to a co-migration of some lipid species and light-harvesting chlorophyll a/b complex LHC II from PS 2 to PS 1. These data allow us to conclude that LHC II consists of the lipoproteins containing specific lipids. Different composition of lipids co-migrating with LHC II under various conditions of phosphorylation might be caused by the variety of LHC II subpopulations transferred under each reductive condition.Abbreviations PS 1 Photosystem 1 - PS 2 Photosystem 2 - LHC II light-harvesting chlorophyll a/b protein complex II - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol     

Chilling-Induced Lipid Degradation in Cucumber (Cucumis sativa L. cv Hybrid C) Fruit

Chilling at 4°C in the dark induced lipid degradation in cucumber (Cucumis sativa L.) fruit upon rewarming at 14°C. Rates of ethane evolution by fruits rewarmed after 3 days of chilling were up to four-fold higher than those evolved by unchilled (14°C) fruits (0.02-0.05 picomoles gram fresh weight⁻¹ hour⁻¹). This potentiation of lipid peroxidation occurred prior to irreversible injury (requiring 3 to 7 days of chilling) as indicated by increases in ethylene evolution and visual observations. Decreases in unsaturation of peel tissue glycolipids were observed in fruits rewarmed after 3 days of chilling, indicating the plastids to be the site of the early phases of chilling-induced peroxidation. Losses in unsaturation of tissue phospholipids were first observed only after chilling for 7 days. Phospholipase D activity appeared to be potentiated in fruits rewarmed after 7 days of chilling as indicated by a decrease in phosphatidylcholine (and secondarily phosphatidylethanolamine) with a corresponding increase in phosphatidic acid. These results indicate that lipid peroxidation may have a role in conferring chilling injury.     

Zeaxanthin Protects Plant Photosynthesis by Modulating Chlorophyll Triplet Yield in Specific Light-harvesting Antenna Subunits

Plants are particularly prone to photo-oxidative damage caused by excess light. Photoprotection is essential for photosynthesis to proceed in oxygenic environments either by scavenging harmful reactive intermediates or preventing their accumulation to avoid photoinhibition. Carotenoids play a key role in protecting photosynthesis from the toxic effect of over-excitation; under excess light conditions, plants accumulate a specific carotenoid, zeaxanthin, that was shown to increase photoprotection. In this work we genetically dissected different components of zeaxanthin-dependent photoprotection. By using time-resolved differential spectroscopy in vivo, we identified a zeaxanthin-dependent optical signal characterized by a red shift in the carotenoid peak of the triplet-minus-singlet spectrum of leaves and pigment-binding proteins. By fractionating thylakoids into their component pigment binding complexes, the signal was found to originate from the monomeric Lhcb4–6 antenna components of Photosystem II and the Lhca1–4 subunits of Photosystem I. By analyzing mutants based on their sensitivity to excess light, the red-shifted triplet-minus-singlet signal was tightly correlated with photoprotection in the chloroplasts, suggesting the signal implies an increased efficiency of zeaxanthin in controlling chlorophyll triplet formation. Fluorescence-detected magnetic resonance analysis showed a decrease in the amplitude of signals assigned to chlorophyll triplets belonging to the monomeric antenna complexes of Photosystem II upon zeaxanthin binding; however, the amplitude of carotenoid triplet signal does not increase correspondingly. Results show that the high light-induced binding of zeaxanthin to specific proteins plays a major role in enhancing photoprotection by modulating the yield of potentially dangerous chlorophyll-excited states in vivo and preventing the production of singlet oxygen.     

Effect of preincubation temperature on in vitro light saturated photosystem I activity in thylakoids isolated from cold hardened and nonhardened rye

Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5°C or 20°C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol → methylviologen) after dark preincubation at temperatures between 0 and 60°C. Thylakoids isolated in the presence or absence of Na⁺/Mg²⁺ from 20°C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60°C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20°C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20°C grown rye, thylakoids isolated from 5°C grown rye in the presence of Na⁺/Mg²⁺ exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60°C. This was not due to damage to PSI electron transport in thylakoids isolated from 5°C grown plants since light saturated PSI activity was 60% higher in 5°C thylakoids than 20°C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5°C thylakoids could be observed after dark preincubation only when 5°C thylakoids were initially isolated in the absence of Na⁺/Mg²⁺. We suggest that 5°C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20°C thylakoids and hence cannot be increased further by dark preincubation.     

Photoinhibition of carotenoidless reaction centers from Rhodobacter sphaeroides by visible light. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides. Illumination by 1100 μEm⁻² s⁻¹ light induced only a slight effect in wild type, carotenoid containing 2.4.1. reaction centers. In contrast, illumination of reaction centers isolated from the carotenoidless R26 strain resulted in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm arising from the P⁺Q_B ⁻ → PQ_B recombination. In addition to this effect, the L, M and H protein subunits of the R26 reaction center were damaged as shown by their loss on Coomassie stained gels, which was however not accompanied by specific degradation products. Both the loss of photochemical activity and of protein subunits were suppressed in the absence of oxygen. By applying EPR spin trapping with 2,2,6,6-tetramethylpiperidine we could detect light-induced generation of singlet oxygen in the R26, but not in the 2.4.1. reaction centers. Moreover, artificial generation of singlet oxygen, also led to the loss of the L, M and H subunits. Our results provide evidence for the common hypothesis that strong illumination by visible light damages the carotenoidless reaction center via formation of singlet oxygen. This mechanism most likely proceeds through the interaction of the triplet state of reaction center chlorophyll with the ground state triplet oxygen in a similar way as occurs in Photosystem II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Time-Course Changes of Oxidative Stress Response to High-Intensity Discontinuous Training versus Moderate-Intensity Continuous Training in Masters Runners

Beneficial systemic effects of regular physical exercise have been demonstrated to reduce risks of a number of age-related disorders. Antioxidant capacity adaptations are amongst these fundamental changes in response to exercise training. However, it has been claimed that acute physical exercise performed at high intensity (>60% of maximal oxygen uptake) may result in oxidative stress, due to reactive oxygen species being generated excessively by enhanced oxygen consumption. The aim of this study was to evaluate the effect of high-intensity discontinuous training (HIDT), characterized by repeated variations of intensity and changes of redox potential, on oxidative damage. Twenty long-distance masters runners (age 47.8±7.8 yr) on the basis of the individual values of gas exchange threshold were assigned to a different 8-weeks training program: continuous moderate-intensity training (MOD, n = 10) or HIDT (n = 10). In both groups before (PRE) and after (POST) training we examined the following oxidative damage markers: thiobarbituric acid reactive substances (TBARS) as marker of lipid peroxidation; protein carbonyls (PC) as marker of protein oxidation; 8-hydroxy-2-deoxy-guanosine (8-OH-dG) as a biomarker of DNA base modifications; and total antioxidant capacity (TAC) as indicator of the overall antioxidant system. Training induced a significant (p<0.05) decrease in resting plasma TBARS concentration in both MOD (7.53±0.30 and 6.46±0.27 µM, PRE and POST respectively) and HIDT (7.21±0.32 and 5.85±0.46 µM, PRE and POST respectively). Resting urinary 8-OH-dG levels were significantly decreased in both MOD (5.50±0.66 and 4.16±0.40 ng mg⁻¹creatinine, PRE and POST respectively) and HIDT (4.52±0.50 and 3.18±0.34 ng mg⁻¹creatinine, PRE and POST respectively). Training both in MOD and HIDT did not significantly modify plasma levels of PC. Resting plasma TAC was reduced in MOD while no significant changes were observed in HIDT. In conclusion, these results suggest that in masters runners high-intensity discontinuous does not cause higher level of exercise-induced oxidative stress than continuous moderate-intensity training, inducing similar beneficial effects on redox homeostasis.     

Damage to photosystem II by lipid peroxidation products

BackgroundPhotosystem II proteins of higher plant chloroplasts are prone to oxidative stress, and most prominently the reaction center-binding D1 protein is damaged under abiotic stress. The reactive oxygen species produced under these stress conditions have been suggested to be responsible for the protein injury.Scope of reviewRecently, it has been shown that the primary and secondary products of non-enzymatic and enzymatic lipid peroxidation have a capability to modify photosystem II proteins. Here, we give an overview showing how lipid peroxidation products formed under light stress and heat stress in the thylakoid membranes cause oxidative modification of proteins in higher plant photosystem II.Major conclusionsDamage to photosystem II proteins by lipid peroxidation products represents a new mechanism underlying photoinhibition and heat inactivation.General significanceComplete characterization of photosystem II protein damage is of crucial importance because avoidance of the damage makes plants to survive under various abiotic stresses. Further physiological significance of photosystem II protein oxidation by lipid peroxidation product should have a potential relevance to plant acclimation because the oxidized proteins might serve as signaling molecules.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Repair of UV-B induced damage of Photosystem II via de novo synthesis of the D1 and D2 reaction centre subunits in Synechocystis sp. PCC 6803

The repair of ultraviolet-B radiation induced damage to the structure and function of Photosystem II was studied in the cyanobacterium Synechocystis sp. PCC 6803. UV-B irradiation of intact Synechocystis cells results in the loss of steady-state oxygen evolution, an effect accompanied by a parallel loss of both D1 and D2 protein subunits of the Photosystem II reaction centre. Transfer of the UV-irradiated cells to normal growth conditions under visible light results in partial recovery of the inhibited oxygen evolving activity and restoration of the lost D1 and D2 proteins. The extent of recovery decreases with increasing degree of damage: after 50% inhibition, the original activity is completely restored within 2 hours. In contrast, after 90–95% inhibition less than half of the original activity is regained during a 4 hour recovery period. The translation inhibitor lincomycin completely blocks the recovery process if added after the UV-B treatment, and accelerates the kinetics of activity loss if added before the onset of UV-B irradiation. Substantial retardation of recovery and acceleration of activity loss is also observed if the very low intensity short wavelength contribution (<290 nm) is not filtered out from the UV-B light source. It is concluded that in intact cells UV-B induced damage of the Photosystem II complex can be repaired. This process is the first example of simultaneous D1 and D2 protein repair in Photosystem II, and considered to function as an important defence mechanism against detrimental UV-B effects in oxygenic photosynthetic organisms. De novo synthesis of the D1 and D2 reaction centre subunits is a key step of the repair process, which itself can also be inhibited by ultraviolet light, especially by the short wavelength UV-C components, or by high doses of UV-B.     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

Photoprotective energy quenching in the red alga Porphyridium purpureum occurs at the core antenna of the photosystem II but not at its reaction center

Photosynthetic organisms have evolved light-harvesting antennae over time. In cyanobacteria, external phycobilisomes (PBSs) are the dominant antennae, whereas in green algae and higher plants, PBSs have been replaced by proteins of the Lhc family that are integrated in the membrane. Red algae represent an evolutionary intermediate between these two systems, as they employ both PBSs and membrane LHCR proteins as light-harvesting units. Understanding how red algae cope with light is not only interesting for biotechnological applications, but is also of evolutionary interest. For example, energy-dependent quenching (qE) is an essential photoprotective mechanism widely used by species from cyanobacteria to higher plants to avoid light damage; however, the quenching mechanism in red algae remains largely unexplored. Here, we used both pulse amplitude-modulated (PAM) and time-resolved chlorophyll fluorescence to characterize qE kinetics in the red alga Porphyridium purpureum. PAM traces confirmed that qE in P. purpureum is activated by a decrease in the thylakoid lumen pH, whereas time-resolved fluorescence results further revealed the quenching site and ultrafast quenching kinetics. We found that quenching exclusively takes place in the photosystem II (PSII) complexes and preferentially occurs at PSII’s core antenna rather than at its reaction center, with an overall quenching rate of 17.6 ± 3.0 ns⁻¹. In conclusion, we propose that qE in red algae is not a reaction center type of quenching, and that there might be a membrane-bound protein that resembles PsbS of higher plants or LHCSR of green algae that senses low luminal pH and triggers qE in red algae.     

Circadian expression of the light-harvesting protein of Photosystem II in etiolated bean leaves following a single red light pulse: Coordination with the capacity of the plant to form chlorophyll and the thylakoid-bound protease

The appearance of the light harvesting II (LHC II) protein in etiolated bean leaves, as monitored by immunodetection in LDS-solubilized leaf protein extracts, is under phytochrome control. A single red light pulse induces accumulation of the protein, in leaves kept in the dark thereafter, which follows circadian oscillations similar to those earlier found for Lhcb mRNA (Tavladoraki et al. (1989) Plant Physiol 90: 665–672). These oscillations are closely followed by oscillations in the capacity of the leaf to form Chlorophyll (Chl) in the light, suggesting that the synthesis of the LHC II protein and its chromophore are in close coordination. Experiments with levulinic acid showed that PChl(ide) resynthesis does not affect the LHC II level nor its oscillations, but new Chl a synthesis affects LHC II stabilization in thylakoids, implicating a proteolytic mechanism. A proteolytic activity against exogenously added LHC II was detected in thylakoids of etiolated bean leaves, which was enhanced by the light pulse. The activity, also under phytochrome control, was found to follow circadian oscillations in verse to those in the stabilization of LHC II protein in thylakoids. Such a proteolytic mechanism therefore, may account for the circadian changes observed in LHC II protein level, being implicated in pigment-protein complex assembly/stabilization during thylakoid biogenesis.Abbreviations Chl chlorophyll - CL continuous light - D dark - FR far-red light - LA levulinic acid - LHC II light-harvesting complex serving Photosystem II - PChl(ide) protochlorophyllide - PCR protochlorophyllide oxidoreductase - R red light     

Bactericidal Activity of Photocatalytic TiO2 Reaction: toward an Understanding of Its Killing Mechanism

When titanium dioxide (TiO₂) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO₂ photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells⁻¹ after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO₂. We concluded that TiO₂ photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.