Karenia brevis Extract Induces Cellular Entry through Distinct Mechanisms in Phagocytic RAW 264.7 Macrophages versus Non-Phagocytic Vero Cells.

Marine algae extracts are an important area of potential drug discovery; however, nearly all studies to date have used non-fluorescent-based methods to determine changes in target cell activity. Many of the most robust immunological and cellular analyses rely on fluorescent probes and readouts, which can be problematic when the algae extract is fluorescent itself. In this study, we identified the fluorescent spectrum of an isolated extract from the marine dinoflagellate Karenia brevis, which included two fluorescing components: chlorophyll α and pheophytin α. When excited at 405 nm and 664 nm, the extract emitted fluorescence at 676 nm and 696 nm, respectively. The extract and its fluorescing components, chlorophyll α and pheophytin α, entered phagocytic RAW 264.7 macrophages and non-phagocytic Vero kidney cells through distinct mechanisms. When incubated with the extract and its main components, both the RAW 264.7 macrophages and the Vero cells accumulated fluorescence as early as 30 min and continued through 48 h. Vero kidney cells accumulated the K. brevis fluorescent extract through a dynamin-independent and acidified endosomal-dependent mechanism. RAW 264.7 macrophages accumulated fluorescent extract through a dynamin-independent, acidified endosomal-independent mechanism, which supports accumulation through phagocytosis. Furthermore, RAW 264.7 macrophages downregulated cell-surface expression of CD206 in response to extract stimulation indicating activation of phagocytic responses and potential immunosuppression of these immune cells. This study represents the first characterization of the cellular update of K. brevis extracts in phagocytic versus non-phagocytic cells. The data suggest the importance of understanding cellular uptake of fluorescing algae extracts and their mechanism of action for future drug discovery efforts.

Phytoplankton bloom stages estimated from chlorophyll pigment proportions suggest delayed summer production in low sea ice years in the northern Bering Sea.

Decreased sea ice cover in the northern Bering Sea has altered annual phytoplankton phenology owing to an expansion of open water duration and its impact on ocean stratification. Limitations of satellite remote sensing such as the inability to detect bloom activity throughout the water column, under ice, and in cloudy conditions dictate the need for shipboard based measurements to provide more information on bloom dynamics. In this study, we adapted remote sensing land cover classification techniques to provide a new means to determine bloom stage from shipboard samples. Specifically, we used multiyear satellite time series of chlorophyll a to determine whether in-situ blooms were actively growing or mature (i.e., past-peak) at the time of field sampling. Field observations of chlorophyll a and pheophytin (degraded and oxidized chlorophyll products) were used to calculate pheophytin proportions, i.e., (Pheophytin/(Chlorophyll a + Pheophytin)) and empirically determine whether the bloom was growing or mature based on remotely sensed bloom stages. Data collected at 13 north Bering Sea stations each July from 2013-2019 supported a pheophytin proportion of 28% as the best empirical threshold to distinguish a growing vs. mature bloom stage. One outcome was that low vs. high sea ice years resulted in significantly different pheophytin proportions in July; in years with low winter-to-spring ice, more blooms with growing status were observed, compared to later stage, more mature blooms following springs with abundant seasonal sea ice. The detection of growing blooms in July following low ice years suggests that changes in the timing of the spring bloom triggers cascading effects on mid-summer production.

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins.

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Chlorophyll and Pheophytin Dephytylating Enzymes Required for Efficient Repair of PSII in Synechococcus elongatus PCC 7942.

The Chlorophyll Dephytylase1 (CLD1) and pheophytinase (PPH) proteins of Arabidopsis thaliana are homologous proteins characterized respectively as a dephytylase for chlorophylls (Chls) and pheophytin a (Phein a) and a Phein a-specific dephytylase. Three genes encoding CLD1/PPH homologs (dphA1, dphA2 and dphA3) were found in the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and shown to be conserved in most cyanobacteria. His6-tagged DphA1, DphA2 and DphA3 proteins were expressed in Escherichia coli, purified to near homogeneity, and shown to exhibit significant levels of dephytylase activity for Chl a and Phein a. Each DphA protein showed similar dephytylase activities for Chl a and Phein a, but the three proteins were distinct in their kinetic properties, with DphA3 showing the highest and lowest Vmax and Km values, respectively, among the three. Transcription of dphA1 and dphA3 was enhanced under high-light conditions, whereas that of dphA2 was not affected by the light conditions. None of the dphA single mutants of S. elongatus showed profound growth defects under low (50 µmol photons m-2 s-1) or high (400 µmol photons m-2 s-1) light conditions. The triple dphA mutant did not show obvious growth defects under these conditions, either, but under illumination of 1,000 µmol photons m-2 s-1, the mutant showed more profound growth retardation compared with wild type (WT). The repair of photodamaged photosystem II (PSII) was much slower in the triple mutant than in WT. These results revealed that dephytylation of Chl a or Phein a or of both is required for efficient repair of photodamaged PSII.

Pigment-modified reaction centers of Chloroflexus aurantiacus: chemical exchange of bacteriopheophytins with plant-type pheophytins.

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina.

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c6 ) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A0 , A1 , and three Fe4 S4 clusters, FX , FA , and FB . Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A0 is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

How Can We Predict Accurate Electrochromic Shifts for Biochromophores? A Case Study on the Photosynthetic Reaction Center.

Protein-embedded chromophores are responsible for light harvesting, excitation energy transfer, and charge separation in photosynthesis. A critical part of the photosynthetic apparatus are reaction centers (RCs), which comprise groups of (bacterio)chlorophyll and (bacterio)pheophytin molecules that transform the excitation energy derived from light absorption into charge separation. The lowest excitation energies of individual pigments (site energies) are key for understanding photosynthetic systems, and form a prime target for quantum chemistry. A major theoretical challenge is to accurately describe the electrochromic (Stark) shifts in site energies produced by the inhomogeneous electric field of the protein matrix. Here, we present large-scale quantum mechanics/molecular mechanics calculations of electrochromic shifts for the RC chromophores of photosystem II (PSII) using various quantum chemical methods evaluated against the domain-based local pair natural orbital (DLPNO) implementation of the similarity-transformed equation of motion coupled cluster theory with single and double excitations (STEOM-CCSD). We show that certain range-separated density functionals (ωΒ97, ωΒ97X-V, ωΒ2PLYP, and LC-BLYP) correctly reproduce RC site energy shifts with time-dependent density functional theory (TD-DFT). The popular CAM-B3LYP functional underestimates the shifts and is not recommended. Global hybrid functionals are too insensitive to the environment and should be avoided, while nonhybrid functionals are strictly nonapplicable. Among the applicable approximate coupled cluster methods, the canonical versions of CC2 and ADC(2) were found to deviate significantly from the reference results both for the description of the lowest excited state and for the electrochromic shifts. By contrast, their spin-component-scaled (SCS) and particularly the scale-opposite-spin (SOS) variants compare well with the reference DLPNO-STEOM-CCSD and the best range-separated DFT methods. The emergence of RC excitation asymmetry is discussed in terms of intrinsic and protein electrostatic potentials. In addition, we evaluate a minimal structural scaffold of PSII, the D1-D2-CytB559 RC complex often employed in experimental studies, and show that it would have the same site energy distribution of RC chromophores as the full PSII supercomplex, but only under the unlikely conditions that the core protein organization and cofactor arrangement remain identical to those of the intact enzyme.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Modulation of drug resistance in Staphylococcus aureus by 132-hydroxy-(132-R/S)-pheophytin isolated from Sargassum polyceratium.

Efflux pumps are integral parts of the bacterial plasma membrane that are responsible for many cases of antibiotic resistance. Modulators of drug resistance are regarded as the most suitable new antibacterial therapies. We evaluated the extracts of Sargassum polyceratium and the isolated compound pheophytin (Sp-1) for antibiotic modifying activity in strains of Staphylococcus aureus with efflux pump. The minimum inhibitory concentrations (MICs) for norfloxacin, tetracycline and erythromycin were determined by the microdilution broth method, in the absence and presence of the extract at a sub-inhibitory concentration (MIC/4). The extracts and isolated compounds showed no significant antimicrobial activity, but they changed the antibiotic activity, decreasing bacterial resistance by 2 to 4x. Using a checkerboard method, it was also possible to observe the synergistic effect (ΣFIC ≤ 0.5) between Sp-1 and the antibiotics erythromycin and norfloxacin. The results indicate that the seaweed Sargassum polyceratium and pheophytin are potential sources of an antibiotic adjuvant that modulates bacterial resistance, acting as a putative efflux pump inhibitor.

Switching sides-Reengineered primary charge separation in the bacterial photosynthetic reaction center.

We report 90% yield of electron transfer (ET) from the singlet excited state P* of the primary electron-donor P (a bacteriochlorophyll dimer) to the B-side bacteriopheophytin (HB) in the bacterial photosynthetic reaction center (RC). Starting from a platform Rhodobacter sphaeroides RC bearing several amino acid changes, an Arg in place of the native Leu at L185-positioned over one face of HB and only ∼4 Šfrom the 4 central nitrogens of the HB macrocycle-is the key additional mutation providing 90% yield of P+HB- This all but matches the near-unity yield of A-side P+HA- charge separation in the native RC. The 90% yield of ET to HB derives from (minimally) 3 P* populations with distinct means of P* decay. In an ∼40% population, P* decays in ∼4 ps via a 2-step process involving a short-lived P+BB- intermediate, analogous to initial charge separation on the A side of wild-type RCs. In an ∼50% population, P* → P+HB- conversion takes place in ∼20 ps by a superexchange mechanism mediated by BB An ∼10% population of P* decays in ∼150 ps largely by internal conversion. These results address the long-standing dichotomy of A- versus B-side initial charge separation in native RCs and have implications for the mechanism(s) and timescale of initial ET that are required to achieve a near-quantitative yield of unidirectional charge separation.

Application of Bioactive Thermal Proteome Profiling to Decipher the Mechanism of Action of the Lipid Lowering 132-Hydroxy-pheophytin Isolated from a Marine Cyanobacteria.

The acceleration of the process of understanding the pharmacological application of new marine bioactive compounds requires identifying the compound protein targets leading the molecular mechanisms in a living cell. The thermal proteome profiling (TPP) methodology does not fulfill the requirements for its application to any bioactive compound lacking chemical and functional characterization. Here, we present a modified method that we called bTPP for bioactive thermal proteome profiling that guarantees target specificity from a soluble subproteome. We showed that the precipitation of the microsomal fraction before the thermal shift assay is crucial to accurately calculate the melting points of the protein targets. As a probe of concept, the protein targets of 132-hydroxy-pheophytin, a compound previously isolated from a marine cyanobacteria for its lipid reducing activity, were analyzed on the hepatic cell line HepG2. Our improved method identified 9 protein targets out of 2500 proteins, including 3 targets (isocitrate dehydrogenase, aldehyde dehydrogenase, phosphoserine aminotransferase) that could be related to obesity and diabetes, as they are involved in the regulation of insulin sensitivity and energy metabolism. This study demonstrated that the bTPP method can accelerate the field of biodiscovery, revealing protein targets involved in mechanisms of action (MOA) connected with future applications of bioactive compounds.

The in vitro synergistic denaturation effect of heat and surfactant on photosystem I isolated from Arthrospira Platensis.

Photosystem I (PSI) generates the most negative redox potential found in nature, and the performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermal stability of PSI. Thus, understanding thermal denaturation is an important prerequisite for the use of PSI at elevated temperatures. To assess the thermal stability of surfactant-solubilized PSI from cyanobacteria Arthrospira Platensis, the synergistic denaturation effect of heat and surfactant was studied. At room temperature, surfactant n-dodecyl-ß-D-maltoside solubilized PSI trimer gradually disassembles into PSI monomers and free pigments over long time. In the solubilizing process of PSI particles, surfactant can uncouple pigments of PSI, and the high concentration of surfactant causes the pigment to uncouple more; after the surfactant-solubilizing process, the uncoupling is relatively slow. During the heating process, changes were monitored by transmittance T800nm, ellipticity θ686nm and θ222nm, upon slow heating (1.5 °C per minute) of samples in Tris buffer (20 mM, pH 7.8) from 20 to 95 °C. The thermal denaturation of surfactant-solubilized PSI is a much more complicated process, which includes the uncoupling of pigments by surfactants, the disappearance of surrounding surfactants, and the unfolding of PSI α-helices. During the heating process, the uncoupling chlorophyll a (Chla) and converted pheophytin (Pheo) can form excitons of Chla-Pheo. The secondary structure α-helix of PSI proteins is stable up to 87-92 °C in the low-concentration surfactant solubilized PSI, and high-concentration surfactant and pigments uncoupling can accelerate the α-helical unfolding.

Characterization of chlorophyll f synthase heterologously produced in Synechococcus sp. PCC 7002.

In diverse terrestrial cyanobacteria, Far-Red Light Photoacclimation (FaRLiP) promotes extensive remodeling of the photosynthetic apparatus, including photosystems (PS)I and PSII and the cores of phycobilisomes, and is accompanied by the concomitant biosynthesis of chlorophyll (Chl) d and Chl f. Chl f synthase, encoded by chlF, is a highly divergent paralog of psbA; heterologous expression of chlF from Chlorogloeopsis fritscii PCC 9212 led to the light-dependent production of Chl f in Synechococcus sp. PCC 7002 (Ho et al., Science 353, aaf9178 (2016)). In the studies reported here, expression of the chlF gene from Fischerella thermalis PCC 7521 in the heterologous system led to enhanced synthesis of Chl f. N-terminally [His]10-tagged ChlF7521 was purified and identified by immunoblotting and tryptic-peptide mass fingerprinting. As predicted from its sequence similarity to PsbA, ChlF bound Chl a and pheophytin a at a ratio of ~ 3-4:1, bound ß-carotene and zeaxanthin, and was inhibited in vivo by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Cross-linking studies and the absence of copurifying proteins indicated that ChlF forms homodimers. Flash photolysis of ChlF produced a Chl a triplet that decayed with a lifetime (1/e) of ~ 817 µs and that could be attributed to intersystem crossing by EPR spectroscopy at 90 K. When the chlF7521 gene was expressed in a strain in which the psbD1 and psbD2 genes had been deleted, significantly more Chl f was produced, and Chl f levels could be further enhanced by specific growth-light conditions. Chl f synthesized in Synechococcus sp. PCC 7002 was inserted into trimeric PSI complexes.

Mg-dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii.

The STAY-GREEN (SGR) gene encodes Mg-dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion-mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (Fv /Fm ) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light-harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that ChlamydomonasSGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgr1 sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the ChlamydomonasSGR participates in PSII formation despite having the same catalytic property.

FERONIA mutation induces high levels of chloroplast-localized Arabidopsides which are involved in root growth.

The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT-ICR)-MS approach was used for initial screening of wild-type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non-wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole-time-of-flight (Q-TOF) MS by direct injection and LC-MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild-type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild-type and fer mutant leaves. More significantly, five micron high-spatial resolution MALDI-MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.

Chlorophyll-Mediated Changes in the Redox Status of Pancreatic Cancer Cells Are Associated with Its Anticancer Effects.

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10-125 µmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.

Modelling of the cathodic and anodic photocurrents from Rhodobacter sphaeroides reaction centres immobilized on titanium dioxide.

As one of a number of new technologies for the harnessing of solar energy, there is interest in the development of photoelectrochemical cells based on reaction centres (RCs) from photosynthetic organisms such as the bacterium Rhodobacter (Rba.) sphaeroides. The cell architecture explored in this report is similar to that of a dye-sensitized solar cell but with delivery of electrons to a mesoporous layer of TiO2 by natural pigment-protein complexes rather than an artificial dye. Rba. sphaeroides RCs were bound to the deposited TiO2 via an engineered extramembrane peptide tag. Using TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) as an electrolyte, these biohybrid photoactive electrodes produced an output that was the net product of cathodic and anodic photocurrents. To explain the observed photocurrents, a kinetic model is proposed that includes (1) an anodic current attributed to injection of electrons from the triplet state of the RC primary electron donor (PT) to the TiO2 conduction band, (2) a cathodic current attributed to reduction of the photooxidized RC primary electron donor (P+) by surface states of the TiO2 and (3) transient cathodic and anodic current spikes due to oxidation/reduction of TMPD/TMPD+ at the conductive glass (FTO) substrate. This model explains the origin of the photocurrent spikes that appear in this system after turning illumination on or off, the reason for the appearance of net positive or negative stable photocurrents depending on experimental conditions, and the overall efficiency of the constructed cell. The model may be a used as a guide for improvement of the photocurrent efficiency of the presented system as well as, after appropriate adjustments, other biohybrid photoelectrodes.

Suppressor of Overexpression of CO 1 Negatively Regulates Dark-Induced Leaf Degreening and Senescence by Directly Repressing Pheophytinase and Other Senescence-Associated Genes in Arabidopsis.

Although the biochemical pathway of chlorophyll (Chl) degradation has been largely elucidated, how Chl is rapidly yet coordinately degraded during leaf senescence remains elusive. Pheophytinase (PPH) is the enzyme for catalyzing the removal of the phytol group from pheophytin a, and PPH expression is significantly induced during leaf senescence. To elucidate the transcriptional regulation of PPH, we used a yeast (Saccharomyces cerevisiae) one-hybrid system to screen for its trans-regulators. SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a key flowering pathway integrator, was initially identified as one of the putative trans-regulators of PPH After dark treatment, leaves of an SOC1 knockdown mutant (soc1-6) showed an accelerated yellowing phenotype, whereas those of SOC1-overexpressing lines exhibited a partial stay-green phenotype. SOC1 and PPH expression showed a negative correlation during leaf senescence. Substantially, SOC1 protein could bind specifically to the CArG box of the PPH promoter in vitro and in vivo, and overexpression of SOC1 significantly inhibited the transcriptional activity of the PPH promoter in Arabidopsis (Arabidopsis thaliana) protoplasts. Importantly, soc1-6 pph-1 (a PPH knockout mutant) double mutant displayed a stay-green phenotype similar to that of pph-1 during dark treatment. These results demonstrated that SOC1 inhibits Chl degradation via negatively regulating PPH expression. In addition, measurement of the Chl content and the maximum photochemical efficiency of photosystem II of soc1-6 and SOC1-OE leaves after dark treatment suggested that SOC1 also negatively regulates the general senescence process. Seven SENESCENCE-ASSOCIATED GENES (SAGs) were thereafter identified as its potential target genes, and NONYELLOWING1 and SAG113 were experimentally confirmed. Together, we reveal that SOC1 represses dark-induced leaf Chl degradation and senescence in general in Arabidopsis.

Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center.

This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical line shape function for the P870 band is calculated numerically. On the basis of the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (∼50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian, providing new insight into the mutant's complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P(+)QA(-) state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870 band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P(+) is addressed by persistent HB spectra burned within the B band in chemically oxidized samples.