Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation

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Quantifying and monitoring functional Photosystem II and the stoichiometry of the two photosystems in leaf segments: Approaches and approximations

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O2 yield per single-turnover flash in CO2-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O2 molecule after four flashes. However, the gross O2 yield per single-turnover flash (multiplied by four) could overestimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700? (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field

Modelling the effect of vertical mixing on bottle incubations for determining in situ phytoplankton dynamics. I. Growth rates

Reliable estimates of in situ phytoplankton growth rates are central to understanding the dynamics of aquatic ecosystems. A common approach for estimating in situ growth rates is to incubate natural phytoplankton assemblages in clear bottles at fixed depths or irradiance levels and measure the change in chlorophyll a (Chl) over the incubation period (typically 24 h). Using a modelling approach, we investigate the accuracy of these Chl-based methods focussing on 2 aspects: (1) in a freely mixing surface layer, the cells are typically not in balanced growth, and with photoacclimation, changes in Chl may yield different growth rates than changes in carbon; and (2) the in vitro methods neglect any vertical movement due to turbulence and its effect on the cells' light history. The growth rates thus strongly depend on the incubation depth and are not necessarily representative of the depth-integrated in situ growth rate in the freely mixing surface layer. We employ an individual based turbulence and photosynthesis model, which also accounts for photoacclimation and photo - inhibition, to show that the in vitro Chl-based growth rate can differ both from its carbon-based in vitro equivalent and from the in situ value by up to 100%, depending on turbulence intensity, optical depth of the mixing layer, and incubation depth within the layer. We make recommendations for choosing the best depth for single-depth incubations. Furthermore we demonstrate that, if incubation bottles are being oscillated up and down through the water column, these systematic errors can be significantly reduced. In the present study, we focus on Chl-based methods only, while productivity measurements using carbon-based techniques (e.g. 14C) are discussed in Ross et al. (2011; Mar Ecol Prog Ser 435:33-45). © Inter-Research 2011

Resveratrol Prevents High Fluence Red Light-Emitting Diode Reactive Oxygen Species-Mediated Photoinhibition of Human Skin Fibroblast Migration.

BackgroundSkin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL) is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease.ObjectiveThe goal of our study was to investigate the how reactive oxygen species (ROS) free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed.MethodsHigh fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR). For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2) on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2.ResultsHigh fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158.4% relative to matched controls, respectively. These LED-RL associated increases in ROS were prevented by pretreating cells with 0.0001% or 0.001% resveratrol. Next, we quantified the effect of hydrogen peroxide (H2O2)-associated ROS on fibroblast migration speed, and found that while H2O2-associated ROS significantly decreased relative fibroblast migration speed, pretreatment with 0.0001% or 0.001% resveratrol significantly prevented the decreases in migration speed. Furthermore, we found that LED-RL at 480, 640 and 800 J/cm2 decreased fibroblast migration speed to 83.0%, 74.4%, and 68.6% relative to matched controls, respectively. We hypothesized that these decreases in fibroblast migration speed were due to associated increases in ROS generation. Pretreatment with 0.0001% and 0.001% resveratrol prevented the LED-RL associated decreases in migration speed.ConclusionHigh fluence LED-RL increases ROS and is associated with decreased fibroblast migration speed. We provide mechanistic support that the decreased migration speed associated with high fluence LED-RL is mediated by ROS, by demonstrating that resveratrol prevents high fluence LED-RL associated migration speed change. These data lend support to an increasing scientific body of evidence that high fluence LED-RL has anti-fibrotic properties. We hypothesize that our findings may result in a greater understanding of the fundamental mechanisms underlying visible light interaction with skin and we anticipate clinicians and other researchers may utilize these pathways for patient benefit

Modelling the effect of vertical mixing on bottle incubations for determining in situ phytoplankton dynamics. II. Primary production

The estimation of in situ phytoplankton primary production is pivotal to many questions in biological oceanography and marine ecology both in a local and global context. Applications range from earth system modelling, the characterisation of aquatic ecosystem dynamics, or the local management of water quality. A common approach for estimating in situ primary production is to incubate natural phytoplankton assemblages in clear bottles at a range of fixed depths and to measure the uptake of carbon (14C) during the incubation period (typically 24 h). One of the main concerns with using fixed-depth bottle incubations is whether stranding samples at fixed depths biases the measured CO2 fixation relative to the 'true' in situ mixed conditions. Here we employ an individual based turbulence and photosynthesis model, which also accounts for photoacclimation and -inhibition, to examine whether the in vitro productivity estimates obtained from fixed-depth incubations are representative of the in situ productivity in a freely mixing water column. While previous work suggested that in vitro estimates could either over- or underestimate the in situ productivity, we show that the errors due to arresting the incubation bottles at fixed depths are indeed minimal. We present possible explanations for how previous authors could have arrived at contradictory results and discuss whether they might be artefacts related to the particular sampling protocol used. We discuss the errors associated with chlorophyll-based incubation methods for determining in situ phytoplankton growth rates in Ross et al. (2011; Mar Ecol Prog Ser 435:13-31). © Inter-Research 2011

Photoinhibition Of Hardened And Non-hardened Rye (secale Cereale L Cv Musketeer) Studied With Isolated Thylakoids, Isolated Mesophyll Cells, And Intact Leaves

Photoinhibition of photosynthesis has been shown to be more severe when combined with other stresses. This suggests a possible correlation between the capacity to grow at low temperature and an increased resistance to photoinhibition at low temperature. Previous work has shown with chlorophyll a fluorescence that rye plants (Secale cereale L. cv Musketeer) acclimated to low temperature were more resistant to photoinhibition than non-hardened rye plants. The goal of the present study was to assess whether the morphological changes observed at the leaf level and the structural changes observed at the thylakoid level during the cold hardening process were responsible for the increased resistance of cold hardened rye to photoinhibition.;Photoinhibition was monitored in hardened and non-hardened winter rye at the leaf level, with isolated mesophyll cells, and with isolated thylakoids. They were exposed to high photon fluxes at 20 and at 5{dollar}\sp\circ{dollar}C and recovery from photoinhibition was assessed upon return to lower photon fluxes. Room temperature chlorophyll a fluorescence, light limited CO{dollar}\sb2{dollar} fixation rates, electron transport rates, photoacoustic spectroscopy and atrazine binding were measured during and following photoinhibition.;Cold-hardened isolated rye cells were more resistant to photoinhibition when monitored with chlorophyll a fluorescence. However, CO{dollar}\sb2{dollar} fixation rates showed similar extent of photoinhibition in cold hardened and non-hardened isolated rye cells. Photoinhibition was similar at 5 and at 20{dollar}\sp\circ{dollar}C but the recovery from photoinhibition was slower when occurring at 5{dollar}\sp\circ{dollar}C. Both groups of cells showed similar rates of recovery. In contrast, cold hardened isolated rye thylakoids were more susceptible to photoinhibition which mainly affected Photosystem II activity.;Leaf morphological differences and structural changes occurring at the thylakoid level during cold hardening of winter rye are not responsible for its increased resistance to photoinhibition. Photoinhibition as monitored with chlorophyll a fluorescence showed different trends and different kinetics compared to CO{dollar}\sb2{dollar} fixation. CO{dollar}\sb2{dollar} fixation data showed that regulatory mechanisms also play a role during photoinhibition and subsequent recovery. Therefore, CO{dollar}\sb2{dollar} fixation should be monitored in intact leaves before the increased resistance of cold hardened winter rye is conclusively proven

Photorespiration: metabolic pathways and their role in stress protection

Photorespiration results from the oxygenase reaction catalysed by ribulose-1,5-bisphosphate carboxylase/ oxygenase. In this reaction glycollate-2-phosphate is produced and subsequently metabolized in the photorespiratory pathway to form the Calvin cycle intermediate glycerate-3-phosphate. During this metabolic process, CO2 and NH3 are produced and ATP and reducing equivalents are consumed, thus making photorespiration a wasteful process. However, precisely because of this ine¤ciency, photorespiration could serve as an energy sink preventing the overreduction of the photosynthetic electron transport chain and photoinhibition, especially under stress conditions that lead to reduced rates of photosynthetic CO2 assimilation. Furthermore, photorespiration provides metabolites for other metabolic processes, e.g. glycine for the synthesis of glutathione, which is also involved in stress protection. In this review, we describe the use of photorespiratory mutants to study the control and regulation of photorespiratory pathways. In addition, we discuss the possible role of photorespiration under stress conditions, such as drought, high salt concentrations and high light intensities encountered by alpine plants

Photodamage to Oxygen Evolving Complex - An Initial Event in Photoinhibition of Photosystem II.

Photosystem II (PSII) of oxygenic photosynthesis is susceptible to photoinhibition. Photoinhibition is defined as light induced damage resulting in turnover of the D1 protein subunit of the reaction center of PSII. Both visible and ultraviolet (UV) light cause photoinhibition. Photoinhibition induced by UV light damages the oxygen evolving complex (OEC) via absorption of UV photons by the Mn ion(s) of OEC. Under visible light, most of the earlier hypotheses assume that photoinhibition occurs when the rate of photon absorption by PSII antenna exceeds the use of the absorbed energy in photosynthesis. However, photoinhibition occurs at all light intensities with the same efficiency per photon. The aim of my thesis work was to build a model of photoinhibition that fits the experimental features of photoinhibition. I studied the role of electron transfer reactions of PSII in photoinhibition and found that changing the electron transfer rate had only minor influence on photoinhibition if light intensity was kept constant. Furthermore, quenching of antenna excitations protected less efficiently than it would protect if antenna chlorophylls were the only photoreceptors of photoinhibition. To identify photoreceptors of photoinhibition, I measured the action spectrum of photoinhibition. The action spectrum showed resemblance to the absorption spectra of Mn model compounds suggesting that the Mn cluster of OEC acts as a photoreceptor of photoinhibition under visible light, too. The role of Mn in photoinhibition was further supported by experiments showing that during photoinhibition OEC is damaged before electron transfer activity at the acceptor side of PSII is lost. Mn enzymes were found to be photosensitive under visible and UV light indicating that Mn-containing compounds, including OEC, are capable of functioning as photosensitizers both in visible and UV light. The experimental results above led to the Mn hypothesis of the mechanism of continuous-light-induced photoinhibition. According to the Mn hypothesis, excitation of Mn of OEC results in inhibition of electron donation from OEC to the oxidized primary donor P680+ both under UV and visible light. P680 is oxidized by photons absorbed by chlorophyll, and if not reduced by OEC, P680+ may cause harmful oxidation of other PSII components. Photoinhibition was also induced with intense laser pulses and it was found that the photoinhibitory efficiency increased in proportion to the square of pulse intensity suggesting that laser-pulse-induced photoinhibition is a two-photon reaction. I further developed the Mn hypothesis suggesting that the initial event in photoinhibition under both continuous and pulsed light is the same: Mn excitation that leads to the inhibition of electron donation from OEC to P680+. Under laser-pulse-illumination, another Mn-mediated inhibitory photoreaction occurs within the duration of the same pulse, whereas under continuous light, secondary damage is chlorophyll mediated. A mathematical model based on the Mn hypothesis was found to explain photoinhibition under continuous light, under flash illumination and under the combination of these two.Siirretty Doriast

Photoinhibition of Photosystem II. Kinetics, Photoprotection and Mechanism

Photosystem II (PSII) is susceptible to light-induced damage defined as photoinhibition. In natural conditions, plants are capable of repairing the photoinhibited PSII by on-going degradation and re-synthesis of the D1 reaction centre protein of PSII. Photoinhibition is induced by both visible and ultraviolet light and photoinhibition occurs under all light intensities with the same efficiency per photon. In my thesis work, I studied the reaction kinetics and mechanism of photoinhibition of PSII, as well as photoprotection in leaves of higher plants. Action spectroscopy was used to identify photoreceptors of photoinhibition. I found that the action spectrum of photoinhibition in vivo shows resemblance to the absorption spectra of manganese model compounds of the oxygen evolving complex (OEC) suggesting a role for manganese as a photoreceptor of photoinhibition under UV and visible light. In order to study the protective effect of non-photochemical quenching, the action spectrum was measured from leaves of wild type Arabidopsis thaliana and two mutants impaired in nonphotochemical quenching of chlorophyll a excitations. The findings of action spectroscopy and simulations of chlorophyll-based photoinhibition mechanisms suggested that quenching of antenna excitations protects less efficiently than would be expected if antenna chlorophylls were the only photoreceptors of photoinhibition. The reaction kinetics of prolonged photoinhibition was studied in leaves of Cucurbita maxima and Capsicum annuum. The results indicated that photoinhibitory decrease in both the oxygen evolution activity and ratio of variable to maximum fluorescence follows firstorder kinetics in vivo. The persistence of first-order kinetics suggests that already photoinhibited reaction centres do not protect against photoinhibition and that the mechanism of photoinhibition does not have a reversible intermediate. When Cucurbita maxima leaves were photoinhibited with saturating single-turnover flashes and continuous light, the light response curve of photoinhibition was found to be essentially a straight line with both types of illumination, suggesting that similar photoinhibition mechanisms might function during illumination with continuous light and during illumination with short flashes.Siirretty Doriast