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Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.
Eustigmatophyte algae represent an interesting model system for the study of the regulation of the excitation energy flow due to their use of violaxanthin both as a major light-harvesting pigment and as the basis of xanthophyll cycle. Fluorescence induction kinetics was studied in an oleaginous marine alga Nannochloropsis oceanica. Nonphotochemical fluorescence quenching was analyzed in detail with respect to the state of the cellular xanthophyll pool. Two components of nonphotochemical fluorescence quenching (NPQ), both dependent on the presence of zeaxanthin, were clearly resolved, denoted as slow and fast NPQ based on kinetics of their formation. The slow component was shown to be in direct proportion to the amount of zeaxanthin, while the fast NPQ component was transiently induced in the presence of membrane potential on subsecond timescales. The applicability of these observations to other eustigmatophyte species is demonstrated by measurements of other representatives of this algal group, both marine and freshwater.
- Klíčová slova
- Chl a fluorescence, Eustigmatophyceae, Nannochloropsis, Nonphotochemical quenching, Xanthophyll cycle,
- MeSH
- fluorescence MeSH
- fotosyntéza MeSH
- mořské řasy chemie MeSH
- Publikační typ
- časopisecké články MeSH
Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiAA24) and Bacillus sp. DMS133(aiiADMS133) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiAA24) and pME7080 (aiiADMS133) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiAA24 and AiiADMS133 proteins suggested that the replacement of proline 210 from AiiAA24 to serine in AiiADMS133 caused the reduction of enzyme activity in AiiADMS133.
- Klíčová slova
- Pectobacterium carotovorum, Acyl-homoserine lactone, Molecular dynamic modeling, Quorum sensing, Virulence genes,
- MeSH
- Bacillus * genetika enzymologie MeSH
- bakteriální proteiny * genetika metabolismus MeSH
- biofilmy růst a vývoj MeSH
- karboxylesterhydrolasy * genetika metabolismus MeSH
- klonování DNA MeSH
- metaloendopeptidasy MeSH
- Pectobacterium carotovorum genetika enzymologie patogenita MeSH
- quorum sensing * MeSH
- regulace genové exprese u bakterií MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- AiiA protein, Bacillus MeSH Prohlížeč
- bakteriální proteiny * MeSH
- karboxylesterhydrolasy * MeSH
- metaloendopeptidasy MeSH
- N-acyl homoserine lactonase MeSH Prohlížeč
Water molecules (H2O) often reduce luminescence lifetimes of various luminescence probes. The change of lifetime is usually caused by dynamic luminescence quenching induced by O-H oscillators which effectively take away energy from excited molecule. The process can be described by Stern-Volmer equation. We have studied selected luminescence systems where it is possible to detect considerable changes of lifetime in presence/absence of H2O and D2O in this work for analytical purposes. We have tested both, inorganic (Ln3+) and organic compounds using three different instrumentation in order to find the largest change between τH and τD. The Ln3+ containing systems have shown considerable increase/decrease of lifetimes in the presence/absence of D2O (Eu3+: τD/τH = 34.5) whereas organic systems gave significantly lower values of τD/τH (coumarin 123 lifetime ratio, τD/τH = 1.94). The calculated LOD varied from 0.04 mol l-1 (samarium nitrate) to 6.55 mol l-1 (riboflavin).
Higher plants defend themselves from bursts of intense light via the mechanism of Non-Photochemical Quenching (NPQ). It involves the Photosystem II (PSII) antenna protein (LHCII) adopting a conformation that favors excitation quenching. In recent years several structural models have suggested that quenching proceeds via energy transfer to the optically forbidden and short-lived S 1 states of a carotenoid. It was proposed that this pathway was controlled by subtle changes in the relative orientation of a small number of pigments. However, quantum chemical calculations of S 1 properties are not trivial and therefore its energy, oscillator strength and lifetime are treated as rather loose parameters. Moreover, the models were based either on a single LHCII crystal structure or Molecular Dynamics (MD) trajectories about a single minimum. Here we try and address these limitations by parameterizing the vibronic structure and relaxation dynamics of lutein in terms of observable quantities, namely its linear absorption (LA), transient absorption (TA) and two-photon excitation (TPE) spectra. We also analyze a number of minima taken from an exhaustive meta-dynamical search of the LHCII free energy surface. We show that trivial, Coulomb-mediated energy transfer to S 1 is an unlikely quenching mechanism, with pigment movements insufficiently pronounced to switch the system between quenched and unquenched states. Modulation of S 1 energy level as a quenching switch is similarly unlikely. Moreover, the quenching predicted by previous models is possibly an artifact of quantum chemical over-estimation of S 1 oscillator strength and the real mechanism likely involves short-range interaction and/or non-trivial inter-molecular states.
- Klíčová slova
- LHCII, carotenoid, energy-dissipation, non-photochemical quenching (NPQ), photosystem (PSII), transient absorption,
- Publikační typ
- časopisecké články MeSH
Non-photochemical quenching (NPQ) is a photoprotective mechanism in light-harvesting antennae. NPQ is triggered by chloroplast thylakoid lumen acidification and is accompanied by violaxanthin de-epoxidation to zeaxanthin, which further stimulates NPQ. In the present study, we show that violaxanthin can act in the opposite direction to zeaxanthin because an increase in the concentration of violaxanthin reduced NPQ in the light-harvesting antennae of Chromera velia. The correlation overlapped with a similar relationship between violaxanthin and NPQ as observed in isolated higher plant light-harvesting complex II. The data suggest that violaxanthin in C. velia can act as an inhibitor of NPQ, indicating that violaxanthin has to be removed from the vicinity of the protein to reach maximal NPQ.
- Klíčová slova
- Chromera velia, light-harvesting antennae, nonphotochemical quenching, photoprotection, violaxanthin, zeaxanthin,
- MeSH
- Alveolata cytologie metabolismus účinky záření MeSH
- časové faktory MeSH
- chlorofyl metabolismus MeSH
- fluorescence MeSH
- fotochemické procesy * MeSH
- světlosběrné proteinové komplexy izolace a purifikace metabolismus MeSH
- xanthofyly metabolismus MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl MeSH
- světlosběrné proteinové komplexy MeSH
- violaxanthin MeSH Prohlížeč
- xanthofyly MeSH
The present paper aims to open discussion on the information content, physical mechanism(s), and measuring protocols to determine the partitioning of the absorbed light energy in oxygenic photosynthetic organisms. Revisiting these questions is incited by recent findings discovering that PSII, in addition to its open and closed state, assumes a light-adapted charge-separated state and that chlorophyll a fluorescence induction (ChlF), besides the photochemical activity of PSII, reflects the structural dynamics of its reaction center complex. Thus, the photochemical quantum yield of PSII cannot be determined from the conventional ChlF-based protocol. Consequently, the codependent quantity - the quantum yield of the so-called nonregulatory constitutive nonphotochemical quenching (npq) - loses its physical meaning. Processes beyond photochemistry and regulatory npq should be identified and characterized by multifaceted studies, including ChlF. Such investigations may shed light on the putative roles of dissipation and other energy-consuming events in the stress physiology of photosynthetic machinery.
- Klíčová slova
- Fv/Fm, chlorophyll a fluorescence, constitutive nonregulatory dissipation, nonphotochemical quenching, quantum yield, structural dynamics,
- Publikační typ
- časopisecké články MeSH
Plants harvest photons for photosynthesis using light-harvesting complexes (LHCs)-an array of chlorophyll proteins that can reversibly switch from harvesting to energy-dissipation mode to prevent over-excitation and damage of the photosynthetic apparatus. In unicellular algae and lower plants this process requires the LHCSR proteins which senses over-acidification of the lumen trough protonatable residues exposed to the thylakoid lumen to activate quenching reactions. Further activation is provided by replacement of the violaxanthin ligand with its de-epoxidized product, zeaxanthin, also induced by excess light. We have produced the ppLHCSR1 protein from Physcomitrella patens by over-expression in tobacco and purified it in either its violaxanthin- or the zeaxanthin-binding form with the aim of analyzing their spectroscopic properties at either neutral or acidic pH. Using femtosecond spectroscopy, we demonstrated that the energy dissipation is achieved by two distinct quenching mechanism which are both activated by low pH. The first is present in both ppLHCSR1-Vio and ppLHCSR1-Zea and is characterized by 30-40ps time constant. The spectrum of the quenching product is reminiscent of a carotenoid radical cation, suggesting that the pH-induced quenching mechanism is likely electron transfer from the carotenoid to the excited Chl a. In addition, a second quenching channel populating the S1 state of carotenoid via energy transfer from Chl is found exclusively in the ppLHCSR1-Zea at pH5. These results provide proof of principle that more than one quenching mechanism may operate in the LHC superfamily and also help understanding the photoprotective role of LHCSR proteins and the evolution of LHC antennae.
- Klíčová slova
- Carotenoids, Femtosecond spectroscopy, LHCSR, Non-photochemical quenching,
- MeSH
- biologické modely MeSH
- chlorofyl metabolismus MeSH
- fotosyntéza * genetika účinky záření MeSH
- geneticky modifikované rostliny genetika metabolismus účinky záření MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- mechy genetika metabolismus účinky záření MeSH
- přenos energie MeSH
- spektrální analýza MeSH
- světlosběrné proteinové komplexy genetika metabolismus účinky záření MeSH
- tabák genetika metabolismus účinky záření MeSH
- transport elektronů MeSH
- vazba proteinů MeSH
- xanthofyly metabolismus MeSH
- zeaxanthiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl MeSH
- světlosběrné proteinové komplexy MeSH
- violaxanthin MeSH Prohlížeč
- xanthofyly MeSH
- zeaxanthiny MeSH
Chl synthase (ChlG) is an important enzyme of the Chl biosynthetic pathway catalyzing attachment of phytol/geranylgeraniol tail to the chlorophyllide molecule. Here we have investigated the Flag-tagged ChlG (f.ChlG) in a complex with two different high-light inducible proteins (Hlips) HliD and HliC. The f.ChlG-Hlips complex binds a Chl a and three different carotenoids, β-carotene, zeaxanthin and myxoxanthophyll. Application of ultrafast time-resolved absorption spectroscopy performed at room and cryogenic temperatures revealed excited-state dynamics of complex-bound pigments. After excitation of Chl a in the complex, excited Chl a is efficiently quenched by a nearby carotenoid molecule via energy transfer from the Chl a Qy state to the carotenoid S1 state. The kinetic analysis of the spectroscopic data revealed that quenching occurs with a time constant of ~2ps and its efficiency is temperature independent. Even though due to its long conjugation myxoxanthophyll appears to be energetically best suited for role of Chl a quencher, based on comparative analysis and spectroscopic data we propose that β-carotene bound to Hlips acts as the quencher rather than myxoxanthophyll and zeaxanthin, which are bound at the f.ChlG and Hlips interface. The S1 state lifetime of the quencher has been determined to be 13ps at room temperature and 21ps at 77K. These results demonstrate that Hlips act as a conserved functional module that prevents photodamage of protein complexes during photosystem assembly or Chl biosynthesis.
- Klíčová slova
- Carotenoids, Cyanobacteria, Energy transfer, Femtosecond spectroscopy, High-light inducible proteins, Non-photochemical quenching,
- MeSH
- bakteriální proteiny chemie MeSH
- fotolýza MeSH
- karotenoidy farmakologie MeSH
- ligasy tvořící vazby C-O chemie MeSH
- sinice enzymologie MeSH
- světlosběrné proteinové komplexy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- bakteriální proteiny MeSH
- chlorophyll synthetase MeSH Prohlížeč
- high light-inducible protein, cyanobacteria MeSH Prohlížeč
- karotenoidy MeSH
- ligasy tvořící vazby C-O MeSH
- světlosběrné proteinové komplexy MeSH
In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light. Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport. The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics. Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.
- Klíčová slova
- cyclic electron transport, frequency analysis, nonphotochemical quenching, photosynthetic oscillation, regulation,
- MeSH
- Arabidopsis * metabolismus MeSH
- chlorofyl metabolismus MeSH
- fotosyntetická reakční centra (proteinové komplexy) * genetika MeSH
- fotosyntéza fyziologie MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- membránové proteiny metabolismus MeSH
- mutace genetika MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- světlo MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- transport elektronů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- chlorofyl MeSH
- fotosyntetická reakční centra (proteinové komplexy) * MeSH
- fotosystém II (proteinový komplex) MeSH
- membránové proteiny MeSH
- PGR5 protein, Arabidopsis MeSH Prohlížeč
- PGRL1 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku * MeSH
- světlosběrné proteinové komplexy MeSH
