Mechanistic Insights on Salicylic Acid-Induced Enhancement of Photosystem II Function in Basil Plants under Non-Stress or Mild Drought Stress.

Photosystem II (PSII) functions were investigated in basil (Ocimum basilicum L.) plants sprayed with 1 mM salicylic acid (SA) under non-stress (NS) or mild drought-stress (MiDS) conditions. Under MiDS, SA-sprayed leaves retained significantly higher (+36%) chlorophyll content compared to NS, SA-sprayed leaves. PSII efficiency in SA-sprayed leaves under NS conditions, evaluated at both low light (LL, 200 µmol photons m-2 s-1) and high light (HL, 900 µmol photons m-2 s-1), increased significantly with a parallel significant decrease in the excitation pressure at PSII (1-qL) and the excess excitation energy (EXC). This enhancement of PSII efficiency under NS conditions was induced by the mechanism of non-photochemical quenching (NPQ) that reduced singlet oxygen (1O2) production, as indicated by the reduced quantum yield of non-regulated energy loss in PSII (ΦNO). Under MiDS, the thylakoid structure of water-sprayed leaves appeared slightly dilated, and the efficiency of PSII declined, compared to NS conditions. In contrast, the thylakoid structure of SA-sprayed leaves did not change under MiDS, while PSII functionality was retained, similar to NS plants at HL. This was due to the photoprotective heat dissipation by NPQ, which was sufficient to retain the same percentage of open PSII reaction centers (qp), as in NS conditions and HL. We suggest that the redox status of the plastoquinone pool (qp) under MiDS and HL initiated the acclimation response to MiDS in SA-sprayed leaves, which retained the same electron transport rate (ETR) with control plants. Foliar spray of SA could be considered as a method to improve PSII efficiency in basil plants under NS conditions, at both LL and HL, while under MiDS and HL conditions, basil plants could retain PSII efficiency similar to control plants.

The Arabidopsis SAFEGUARD1 suppresses singlet oxygen-induced stress responses by protecting grana margins.

Singlet oxygen (1O2), the major reactive oxygen species (ROS) produced in chloroplasts, has been demonstrated recently to be a highly versatile signal that induces various stress responses. In the fluorescent (flu) mutant, its release causes seedling lethality and inhibits mature plant growth. However, these drastic phenotypes are suppressed when EXECUTER1 (EX1) is absent in the flu ex1 double mutant. We identified SAFEGUARD1 (SAFE1) in a screen of ethyl methanesulfonate (EMS) mutagenized flu ex1 plants for suppressor mutants with a flu-like phenotype. In flu ex1 safe1, all 1O2-induced responses, including transcriptional rewiring of nuclear gene expression, return to levels, such as, or even higher than, those in flu Without SAFE1, grana margins (GMs) of chloroplast thylakoids (Thys) are specifically damaged upon 1O2 generation and associate with plastoglobules (PGs). SAFE1 is localized in the chloroplast stroma, and release of 1O2 induces SAFE1 degradation via chloroplast-originated vesicles. Our paper demonstrates that flu-produced 1O2 triggers an EX1-independent signaling pathway and proves that SAFE1 suppresses this signaling pathway by protecting GMs.

A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II.

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

Singlet oxygen damages the function of Photosystem II in isolated thylakoids and in the green alga Chlorella sorokiniana.

Singlet oxygen (1O2) is an important damaging agent, which is produced during illumination by the interaction of the triplet excited state pigment molecules with molecular oxygen. In cells of photosynthetic organisms 1O2 is formed primarily in chlorophyll containing complexes, and damages pigments, lipids, proteins and other cellular constituents in their environment. A useful approach to study the physiological role of 1O2 is the utilization of external photosensitizers. In the present study, we employed a multiwell plate-based screening method in combination with chlorophyll fluorescence imaging to characterize the effect of externally produced 1O2 on the photosynthetic activity of isolated thylakoid membranes and intact Chlorella sorokiniana cells. The results show that the external 1O2 produced by the photosensitization reactions of Rose Bengal damages Photosystem II both in isolated thylakoid membranes and in intact cells in a concentration dependent manner indicating that 1O2 plays a significant role in photodamage of Photosystem II.

At the Edges of Photosynthetic Metabolic Plasticity-On the Rapidity and Extent of Changes Accompanying Salinity Stress-Induced CAM Photosynthesis Withdrawal.

The common ice plant (Mesembryanthemum crystallinum L.) is a facultative crassulacean acid metabolism (CAM) plant, and its ability to recover from stress-induced CAM has been confirmed. We analysed the photosynthetic metabolism of this plant during the 72-h response period following salinity stress removal from three perspectives. In plants under salinity stress (CAM) we found a decline of the quantum efficiencies of PSII (Y(II)) and PSI (Y(I)) by 17% and 15%, respectively, and an increase in nonphotochemical quenching (NPQ) by almost 25% in comparison to untreated control. However, 48 h after salinity stress removal, the PSII and PSI efficiencies, specifically Y(II) and Y(I), elevated nonphotochemical quenching (NPQ) and donor side limitation of PSI (YND), were restored to the level observed in control (C3 plants). Swelling of the thylakoid membranes, as well as changes in starch grain quantity and size, have been found to be components of the salinity stress response in CAM plants. Salinity stress induced an over 3-fold increase in average starch area and over 50% decline of average seed number in comparison to untreated control. However, in plants withdrawn from salinity stress, during the first 24 h of recovery, we observed chloroplast ultrastructures closely resembling those found in intact (control) ice plants. Rapid changes in photosystem functionality and chloroplast ultrastructure were accompanied by the induction of the expression (within 24 h) of structural genes related to the PSI and PSII reaction centres, including PSAA, PSAB, PSBA (D1), PSBD (D2) and cp43. Our findings describe one of the most flexible photosynthetic metabolic pathways among facultative CAM plants and reveal the extent of the plasticity of the photosynthetic metabolism and related structures in the common ice plant.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom.

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO3-). To investigate the cellular and genetic basis of diatom NO3- assimilation, we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum In NR-KO cells, N-assimilation was abolished although NO3- transport remained intact. Unassimilated NO3- accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO3- chloride channel transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO3- Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO3-, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO3- addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO3- replete and deplete conditions.

The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw.

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Glycolate Induces Redox Tuning Of Photosystem II in Vivo: Study of a Photorespiration Mutant.

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against 1O2 in vivo under photorespiratory conditions. Here, we compared the properties of QA in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of QA/QA- In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO2 conditions.

Physiological and thylakoid ultrastructural changes in cyanobacteria in response to toxic manganese concentrations.

In this study, two cyanobacterial strains (morphologically identified as Microcystis novacekii BA005 and Nostoc paludosum BA033) were exposed to different Mn concentrations: 7.0, 10.5, 15.7, 23.6 and 35.4 mg L-1 for BA005; and 15.0, 22.5, 33.7, 50.6, and 76.0 mg L-1 for BA033. Manganese toxicity was assessed by growth rate inhibition (EC50), chlorophyll a content, quantification of Mn accumulation in biomass and monitoring morphological and ultrastructural effects. The Mn EC50 values were 16 mg L-1 for BA005 and 39 mg L-1 for BA033, respectively. Reduction of chlorophyll a contents and ultrastructural changes were observed in cells exposed to Mn concentrations greater than 23.6 and 33.7 mg L-1 for BA005 and BA033. Damage to intrathylakoid spaces, increased amounts of polyphosphate granules and an increased number of carboxysomes were observed in both strains. In the context of the potential application of these strains in bioremediation approaches, BA005 was able to remove Mn almost completely from aqueous medium after 96 h exposure to an initial concentration of 10.5 mg L-1, and BA033 was capable of removing 38% when exposed to initial Mn concentration of 22.5 mg L-1. Our data shed light on how these cyanobacterial strains respond to Mn stress, as well as supporting their utility as organisms for monitoring Mn toxicity in industrial wastes and potential bioremediation application.

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO3-/CO2 as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α­carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α­carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO3-/CO2. Addition of exogenous HCO3- or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.

Carnosic Acid and Carnosol, Two Major Antioxidants of Rosemary, Act through Different Mechanisms.

Carnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary (Rosmarinus officinalis). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis (Arabidopsis thaliana) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta.

The identification of IsiA proteins binding chlorophyll d in the cyanobacterium Acaryochloris marina.

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (-F6) isolated from iron-deficient culture contained Chl d-bound PSI-IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).

The artificial humic substance HS1500 does not inhibit photosynthesis of the green alga Desmodesmus armatus in vivo but interacts with the photosynthetic apparatus of isolated spinach thylakoids in vitro.

Humic substances (HSs) can influence the growth and composition of freshwater phytoplankton assemblage. Since HSs contain many phenolic and quinonic moieties and cause growth reductions in eco-physiological field experiments, HSs are considered photosystem II herbicides. To test this specific mode of action in vivo and in vitro, respectively, we used intact cells of the green alga Desmodesmus armatus, as well as thylakoids isolated from spinach (Spinacia oleracea) as a model system for the green algal chloroplast. Photosynthetic electron transport was measured as oxygen evolution and variable chlorophyll fluorescence. The in vivo effect of the artificial humic substance HS1500 on algae consisted of no impact on photosynthesis-irradiance curves of intact green algae compared to untreated controls. In contrast, addition of HS1500 to isolated thylakoids resulted in light-induced oxygen consumption (Mehler reaction) as an in vitro effect. Fluorescence induction kinetics of HS-treated thylakoids revealed a large static quenching effect of HS1500, but no inhibitory effect on electron transport. For the case of intact algal cells, we conclude that the highly hydrophilic and rather large molecules of HS1500 are not taken up in effective quantities and, therefore, cannot interfere with photosynthesis. The in vitro tests show that HS1500 has no inhibitory effect on photosystem II but operates as a weak, oxygen-consuming Hill acceptor at photosystem I. Hence, the results indicate that eco-physiological field experiments should focus more strongly on effects of HSs on extracellular features, such as reducing and red-shifting the underwater light field or influencing nutrient availability by cation exchange within the plankton network.

Manipulation of the Xanthophyll Cycle Increases Plant Susceptibility to Sclerotinia sclerotiorum.

The xanthophyll cycle is involved in dissipating excess light energy to protect the photosynthetic apparatus in a process commonly assessed from non-photochemical quenching (NPQ) of chlorophyll fluorescence. Here, it is shown that the xanthophyll cycle is modulated by the necrotrophic pathogen Sclerotinia sclerotiorum at the early stage of infection. Incubation of Sclerotinia led to a localized increase in NPQ even at low light intensity. Further studies showed that this abnormal change in NPQ was closely correlated with a decreased pH caused by Sclerotinia-secreted oxalate, which might decrease the ATP synthase activity and lead to a deepening of thylakoid lumen acidification under continuous illumination. Furthermore, suppression (with dithiothreitol) or a defect (in the npq1-2 mutant) of violaxanthin de-epoxidase (VDE) abolished the Sclerotinia-induced NPQ increase. HPLC analysis showed that the Sclerotinia-inoculated tissue accumulated substantial quantities of zeaxanthin at the expense of violaxanthin, with a corresponding decrease in neoxanthin content. Immunoassays revealed that the decrease in these xanthophyll precursors reduced de novo abscisic acid (ABA) biosynthesis and apparently weakened tissue defense responses, including ROS induction and callose deposition, resulting in enhanced plant susceptibility to Sclerotinia. We thus propose that Sclerotinia antagonizes ABA biosynthesis to suppress host defense by manipulating the xanthophyll cycle in early pathogenesis. These findings provide a model of how photoprotective metabolites integrate into the defense responses, and expand the current knowledge of early plant-Sclerotinia interactions at infection sites.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.

Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii.

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO2 availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO2 maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO2 reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.

High light-dependent phosphorylation of photosystem II inner antenna CP29 in monocots is STN7 independent and enhances nonphotochemical quenching.

Phosphorylation of the photosystem II antenna protein CP29 has been reported to be induced by excess light and further enhanced by low temperature, increasing resistance to these stressing factors. Moreover, high light-induced CP29 phosphorylation was specifically found in monocots, both C3 and C4, which include the large majority of food crops. Recently, knockout collections have become available in rice (Oryza sativa), a model organism for monocots. In this work, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of high light-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that kinases and phosphatases involved in CP29 phosphorylation are distinct from those reported to act in State 1-State 2 transitions. In addition, we elucidated the photoprotective role of CP29 phosphorylation in reducing singlet oxygen production and enhancing excess energy dissipation. We thus established, in monocots, a mechanistic connection between phosphorylation of CP29 and nonphotochemical quenching, two processes so far considered independent from one another.

The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex.

Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b6/f (Cyt b6/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b6/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700(+) indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b6/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b6/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins.

Growth inhibition and possible mechanism of oleamide against the toxin-producing cyanobacterium Microcystis aeruginosa NIES-843.

Oleamide, a fatty acid derivative, shows inhibitory effect against the bloom-forming cyanobacterium Microcystis aeruginosa. The EC50 of oleamide on the growth of M. aeruginosa NIES-843 was 8.60 ± 1.20 mg/L. In order to elucidate the possible mechanism of toxicity of oleamide against M. aeruginosa, chlorophyll fluorescence transient, cellular ultrastructure, fatty acids composition and the transcription of the mcyB gene involved in microcystins synthesis were studied. The results of chlorophyll fluorescence transient showed that oleamide could destruct the electron accepting side of the photosystem II of M. aeruginosa NIES-843. Cellular ultrastructure examination indicated that the destruction of fatty acid constituents, the distortion of thylakoid membrane and the loss of integrity of cell membrane were associated with oleamide treatment and concentration. The damage of cellular membrane increased the release of microcystins from intact cells into the medium. Results presented in this study provide new information on the possible mechanisms involved and potential utilization of oleamide as an algicide in cyanobacterial bloom control.

[THE EFFECT OF ACID RAIN ON ULTRASTRUCTURE AND FUNCTIONAL PARAMETERS OF PHOTOSYNTHETIC APPARATUS OF PEA LEAVES].

The effects of simulated acid rain (SAR) on the ultrastructure and functional parameters of the photosynthetic apparatus were studied using 14-day-old pea leaves as test system. Pea plants were sprayed with an aqueous solution containing NaNO3(0.2 mM) and Na2SO4(0.2 mM) (pH 5.6, a control variant), or with the same solution, which was acidified to pH 2.5 (acid variant). Functional characteristics were determined by chlorophyll fluorescence analysis. Acid rain application caused reduction in the efficiency of the photosynthetic electron transport by 25%, which was accompanied by an increase by 85% in the quantum yield of thermal dissipation of excess light quanta. Ultrastructural changes in chloroplast were registered by transmission electron microscopy (TEM) after two days of the SAR-treatment of pea leaves. In this case, the changes in the structure of grana, heterogeneity of thylakoids packaging in granum, namely, the increase of intra-thylakoid gaps and thickness of granal thylakoids compared to the control were found. The migration of protein complexes in thylakoid membranes of chloroplasts isolated from leaves treated with SAR was suppressed. It was shown also that carbonic anhydrase activity was inhibited in chloroplast preparations isolated from SAR-treated pea leaves. We proposed a hypothesis on the possible inactivation of thylakoid carbonic anhydrase under SAR and its involvement in the inhibition of photochemical activity of chloroplasts. The data obtained allows to suggest that acid rains negatively affect the photosynthetic apparatus disrupting the membrane system of chloroplast.