In vivo 1H NMR methods to study dynamics of chloroplast water and thylakoid membrane lipids in leaves and in photosynthetic microorganisms
Pagadala, Shanthi - \ 2017
Wageningen University. Promotor(en): H. van Amerongen, co-promotor(en): H. van As. - Wageningen : Wageningen University - ISBN 9789463431569 - 130
cell membranes - membranes - chloroplasts - thylakoids - photosynthesis - in vivo experimentation - stress conditions - stress - proteins - lipids - mobility - dynamics - celmembranen - membranen - chloroplasten - thylakoïden - fotosynthese - in vivo experimenten - stress omstandigheden - stress - eiwitten - lipiden - mobiliteit - dynamica
Dynamics of thylakoid membranes and mobility of pigment-protein complexes therein are essential for survival of photosynthetic organisms under changing environmental conditions. The published approaches to probe mobility of the thylakoid membrane lipids and protein complexes are either dependent on the use of external labels or are used only for in vitro studies. Here, we present non-invasive 1H NMR methods (DOSY and DRCOSY) to study dynamics of water in chloroplasts, lipids in oil bodies and in thylakoid membranes and pigment-protein complexes under complete in vivo conditions in leaf disks of F. benjamina and A. platanoides and in suspensions of the green alga Chlamydomonas reinhardtii and blue-green alga Synechocystissp.PCC 6803.
In leaf disks of Ficus benjamina and Acer platanoides, water in chloroplasts could be clearly discriminated from other pools. Both water in chloroplasts, and water in vacuoles of palisade and spongy cells showed resonances in the high field part of the spectra (with respect to pure water), in contrast to what has been reported in literature. Subepidermal cells (present only in F. benjamina but not in A. platanoides) may act as a water storage, buffer pool during drought. This pool prevented the fast loss of water from the chloroplasts. Nutrient stress and excess salt stress resulted in accumulated lipid bodies and in striking differences in the dynamics and spectra/composition of the different components. T2 values of the different components are compared with those observed in suspensions of Synechocystissp.PCC 6803. The differences in membrane composition (ratio of the different membrane lipids) were clearly observed in the DANS of the oil bodies and the (thylakoid) membranes, but the diffusion coefficients were quite comparable. Also the DANS of the component that is assigned to the pigment-protein complexes are quite different, reflecting the differed composition. The diffusion coefficients of this component in isolated spinach thylakoids and in C. reinhardtii are very comparable, but about a factor of 10 lower with respect to that of Synechocystis at short diffusion times. The dynamics of these complexes in these systems are thus quite different.
Utilization of complete chloroplast genomes for phylogenetic studies
Ramlee, Shairul Izan Binti - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Rene Smulders; Theo Borm. - Wageningen : Wageningen University - ISBN 9789462579354 - 186
phylogenetics - genomes - chloroplasts - models - solanum - orchidaceae - phylogenomics - dna sequencing - fylogenetica - genomen - chloroplasten - modellen - solanum - orchidaceae - phylogenomica - dna-sequencing
Chloroplast DNA sequence polymorphisms are a primary source of data in many plant phylogenetic studies. The chloroplast genome is relatively conserved in its evolution making it an ideal molecule to retain phylogenetic signals. The chloroplast genome is also largely, but not completely, free from other evolutionary processes such as gene duplication, concerted evolution, pseudogene formation and genome rearrangements. The conservation of the chloroplast genome sequence allows designing primers targeting regions conserved well beyond species boundaries, and amplification of these targets.
The small size together with their high copy number in leaf cells greatly facilitates chloroplast genome sequencing. In this thesis, chloroplast phylogenomics was conducted using complete chloroplast DNA genomes obtained by a newly developed method of de novo assembly. The method was not only cost-effective but also has the potential to extract a wealth of useful information of thousands of chloroplast genomes from Whole Genome Shotgun (WGS) data. We used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. This pipeline includes steps aimed at optimizing assemblies and filling gaps that are left due to coverage variation in the WGS dataset. The pipeline enabled successful de novo assembly across a range of nuclear genome sizes, from Solanum lycopersicon (tomato, 0.9 Gb) to Paphiopedilum heryanum (slipper orchid, 35 Gb).
The pipeline is suitable for studying structural variation in the chloroplast genome, as opposed to the common procedure of read mapping against a reference genome. To support the putative rearrangements, a flexible assembly quality comparison tool was created that combines and visualizes read mapping and alignment results in a two-dimensional plot. We have evaluated the ability of this tool using the de novo assemblies of S. lycopersicon and P. henryanum chloroplasts. The results show that not only we can immediately select the best of two options, but also determine the location of specific artefacts.
In order to explore and evaluate the utility of complete chloroplast phylogenomics, tomato and Paphiopedilum spp were used to conduct phylogenetic inferences based on the complete chloroplast genome. In total 84 tomato chloroplast genomes within the section Lycopersicon were assembled and phylogenetic trees produced. The analyses revealed that next to the chloroplast regions and spacers traditionally used for phylogenetics, additional regions of protein coding and non-coding DNA may be exploited for intraspecific phylogenetic studies. In particular, more than 50% of all phylogenetically relevant information could be included by just using four genes (ycf1, ndhF, ndhA, and ndhH), of which 34% in ycf1 alone. The topology of the phylogenetic tree inferred from ycf1 was the same as that of trees based on all other protein coding genes, although with lower bootstrap values. The phylogenetic analyses based on 32 complete Paphiopedilum spp. chloroplast genomes confirmed the division of the genus into three subgenera Parvisepalum, Brachypetalum and Paphiopedilum. The division of five sections of subgenus Paphiopedilum was also recovered. The de novo assemblies revealed several structural rearrangements including gene loss and inversion. In addition, the chloroplast genome of Paphiopedilum has experienced extreme IR expansion that has included part of or the entire SSC region, resulting in larger IR regions than commonly observed among monocots.
In conclusion, WGS data offer opportunities to generate partial or entire chloroplast genomes for phylogenetic studies. Species discrimination can be achieved already with partial data (subsets of genes), but evolutionarily young lineages may require more informative characters. Therefore, it is expected that many complete chloroplast genomes will be produced in the years to come. While generating these genomes, the urge for de novo assembly of chloroplast genomes rather than mapping against reference genomes is adamant in order to also uncover structural rearrangements in chloroplast genome.
The role of lipids in the global organization of thylakoid membranes of higher plants
Krumova, S.K.B. - \ 2009
Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): G. Garab. - [S.l.] : S.n. - ISBN 9789085852926 - 132
planten - thylakoïden - membranen - lipiden - chloroplasten - fotosynthese - plants - thylakoids - membranes - lipids - chloroplasts - photosynthesis
The first steps of photosynthesis (capturing of light and conversion of light energy into chemical energy) occur in the thylakoid membrane of the chloroplasts. It consists for 75% of membrane proteins and 25% of lipid molecules. In this thesis the global properties of the lipids in the thylakoid membrane are studied with a variety of spectroscopic techniques. Special attention is paid to the lipid packing, phase behavior and membrane permeability, as well as the role of the lipids in structural rearrangements and the overall organization of the membrane. The presented results reveal heterogeneity in the packing of the bulk lipid molecules and the participation of the lipids in both bilayer and non-bilayer structures. It is demonstrated that the lipid mixture has an active role in the lateral arrangement of the protein complexes, the formation of macrodomains and their thermal stability and indirectly in the excitation energy trapping by the photosynthetic complexes. This work provides a basis for further future investigations of the properties of the thylakoid lipid matrix and of the lipid-protein interactions in thylakoid membranes and their relevance for different functions of the membrane.
Primary photosynthetic processes: from supercomplex to leaf
Broess, K. - \ 2009
Wageningen University. Promotor(en): Herbert van Amerongen. - [S.l.] : S.n. - ISBN 9789085852988 - 124
fotosynthese - fluorescentie - fluorescentiemicroscopie - spectroscopie - membranen - chloroplasten - fotosysteem ii - planten - photosynthesis - fluorescence - fluorescence microscopy - spectroscopy - membranes - chloroplasts - photosystem ii - plants
This thesis describes fluorescence spectroscopy experiments on photosynthetic complexes that cover the primary photosynthetic processes, from the absorption of light by photosynthetic pigments to a charge separation (CS) in the reaction center (RC). Fluorescence spectroscopy is a useful tool in photosynthetic particles, because the latter are densely packed with fluorescence pigments like chlorophylls (Chl). The fluorescence of each pigment is affected by its environment and provide information about structure and dynamics of the photosynthetic complexes. In this thesis time-resolved fluorescence of Chl molecules is used for studying the ultrafast kinetics in membrane particles of photosystem II (PSII) (chapter 2, 3 and 4). In chapter 5 fluorescence lifetime imaging microscopy (FLIM) of is applied to study entire chloroplasts, either in the leaf or in isolated chloroplast form. The advantage of FLIM is that the interactions of the fluorescence pigments in both photosystems can be spatially resolved up to a resolution of 0.5 x 0.5 x 2 µm to indentify and quantify photosynthetic processes in their natural environment.
Excitation energy transfer and charge separation in PSII membranes (chapter 2,3 and 4)
In this thesis time-resolved fluorescence measurements of PSII containing membranes, the so called BBY particles, are performed in low-light conditions with open reaction centers. The BBY particles do not contain photosystem I (PSI) or stroma lamellae, but do support electron transfer and carry out oxygen evolution with high activity and are comparable with the grana in vivo. The fluorescence decay kinetics of the BBY particles are faster than observed in previous studies and also faster than observed for PSII in chloroplasts and thylakoid preparations. The average lifetime is 150 ps, which, together with previous annihilation experiments on light-harvesting complex II (LHCII) suggests that excitation migration from the antenna complexes contributes significantly to the overall charge separation time. This is in disagreement with the commonly applied exciton / radical-pair-equilibrium (ERPE) model that assumes that excitation energy diffusion through the antenna to the RC is much faster than the overall charge-separation time.
A simple coarse-grained method is proposed, based on the supramolecular organization of PSII and LHCII in grana membranes (C2S2M2). The proposed modelling procedure for BBY particles is only approximate and many different combinations of excitation migration time and the charge separation time can explain the observed fluorescence kinetics. However it is clear that charge transfer should be rather fast and is accompanied with a large drop in free energy.
In chapter 3, the fluorescence kinetics of BBY particles with open RCs are compared after preferential excitation at 420 and 484 nm, which causes a difference in the initial excited-state populations of the inner and outer antenna system. The fluorescence decay is somewhat slower upon preferential excitation of chlorophyll (Chl) b, which is exclusively present in the outer antenna. Using the coarse-grained model it was possible to fit the 420 and 484 nm results simultaneously with a two-step electron transfer model and four parameters: the hopping rate between the protein-pigment complexes, the CS rate, the drop in free energy upon primary charge separation and a secondary charge separation rate. The conclusion is that the average migration time contributes ~25% to the overall trapping time. The hopping time obtained in chapter 3 is significantly faster than might be expected based on studies on trimeric and aggregated LHCII and it is concluded that excitation energy transfer in PSII follows specific pathways that require an optimized organization of the antenna complexes with respect to each other. Analysis of the composition of the BBY particles indicates that the size of the light-harvesting system in PSII is smaller than commonly found for PSII in chloroplasts and explains why the fluorescence lifetimes are smaller for the BBY’s.
In chapter 4, four different PSII supercomplex preparations were studied. The main difference between these supercomplexes concerns the size of the outer antenna. The average lifetime of the supercomplexes becomes longer upon increasing the antenna size. The results indicate that the rate constants obtained from the coarse-grained method for BBY preparations, which is based on the supercomplex composition C2S2M2, should be slightly faster (~10%) as predicted in chapter 3. The observation that the average lifetime of the supercomplexes is relatively slow compared to what one might expect based on the measurements on BBY particles, and this will require further future studies.
Photosynthesis in plant leaves (Chapter 5)
With the use of femtosecond two-photon excitation TPE at 860 nm it appears to be possible to measure fluorescence lifetimes throughout the entire leaves of Arabidopsis thaliana and Alocasia wentii. It turns out that the excitation intensity can be kept sufficiently low to avoid artifacts due to singlet-singlet and singlet-triplet annihilation, while the reaction centers can be kept in the open state during the measurements. The average fluorescence lifetimes obtained for individual chloroplasts of Arabidopsis thaliana and Alocasia wentii in the open and closed state, are approximately ~250 ps and ~1.5 ns, respectively. The maximum fluorescence state correspond to a state in which all reaction centers are closed. The kinetics are very similar to those obtained for chloroplasts in vitro with the FLIM setup and to in vivo results reported in literature. No variations between chloroplasts are observed when scanning throughout the leaves of Arabidopsis thaliana and Alocasia wentii. Within individual chloroplasts some variation is detected for the relative contributions of PSI and PSII to the fluorescence. The results open up the possibility to use FLIM for the in vivo study of the primary processes of photosynthesis at the level of single chloroplasts under all kinds of (stress) conditions.
This thesis gives new insight of the kinetic processes in PSII membranes. With the use of a coarse-grained method that provides an easy way to incorporate existing knowledge and models for individual complexes, valuable conclusions can be drawn about the excitation energy transfer and the CS which hopefully contributes to an improvement of the knowledge about PSII functioning. In general it was shown that a large drop in free energy is needed in PSII membranes for all simulations with the coarse-grained method.
The presented results on the kinetics of chloroplasts obtained in vitro and in vitro are very similar and verify that conclusions drawn from isolated chloroplasts can be extrapolated to photosynthetic processes in their natural environment.
The electrophysiology of chloroplast protein import : the involvement of an anion channel in protein translocation across the inner membrane
Wijngaard, P.W.J. van den - \ 1999
Agricultural University. Promotor(en): W.J. Vredenberg; J.F.H. Snel. - S.l. : S.n. - ISBN 9789058081186 - 100
elektrofysiologie - chloroplasten - membranen - eiwittransport - precursors - eiwitten - onderzoek - electrophysiology - chloroplasts - membranes - protein transport - precursors - proteins - research
The electrophysiological response of the chloroplast envelope during protein import is investigated by using single channel recordings of the chloroplast envelope. An anion channel localised in the inner envelope membrane is identified. The channel has a single channel conductance of 50 pS (chapter 2 and 5). It is found that this channel is inactivated by precursor protein. The precursor induced inactivation of the channel is dependent on the presence of ATP and a functional transit sequence. A deletion mutant of preferredoxin (preFd), that is defective in in vitro import and initial binding, is unable to inactivate the channel (chapter 2). From this it is concluded that the 50 pS anion channel is involved in protein import. The channel is therefore termed P rotein I mport R elated A nion C hannel (PIRAC).
Inactivation of PIRAC is shown to require the translocation of precursor protein across the outer membrane and association of the precursor with Tic. In the presence of GTP preFd is unable to inactivate PIRAC (chapter 3). A deletion mutant of preFd (Δ15-25-preFd) is shown to inactivate PIRAC (chapter 3). This deletion mutant can compete with the wild-type precursor for import, but is not imported into the stroma. Furthermore evidence has been presented before that Δ15-25-preFd does interact with Tic. It is therefore concluded that translocation of the precursor across the inner envelope membrane is not required for PIRAC inactivation.
Blocking PIRAC activity with the anion channel blocker DIDS leads to a decrease in import efficiency of isolated pea chloroplasts (chapter 3). This indicates that normal functioning of the channel is required for protein translocation.
It is shown that the precursor protein induces a long-lived closed state of PIRAC, which is not observed in the absence of precursor (chapter 4). The mean duration of this inactive state is found to be independent of the overall precursor length. In the absence of precursor protein PIRAC is observed to have two distinct open and three distinct closed states (chapter 4). These states can be distinguished on the basis of there mean durations. It is found that the addition of precursor protein decreases the mean duration of one of the open states and two of the closed states (chapter 4). The only states of PIRAC that are unaffected by the addition of precursor are of very short duration (below 1 ms). The precursor induced decrease of the mean duration of PIRAC states is found to be concentration dependent. This leads to the conclusion that there is a direct interaction between the translocating precursor and PIRAC, leading to the switching of the channel to the long-lived inactive state.
It is observed that addition of antibodies to Tic110 at the stromal side of an excised inside out patch irreversibly inactivated PIRAC activity (chapter 5). Tic110 is a known component of the translocon of the inner membrane of the chloroplast envelope. It is an integral membrane protein with a large hydrophilic loop facing the stroma. It is therefore concluded that PIRAC is associated with the translocon of the inner membrane of the chloroplast envelope.
The electrical potential as a gauge of photosynthetic performance in plant chloroplasts : a patch-clamp study
Voorthuysen, T. van - \ 1997
Agricultural University. Promotor(en): W.J. Vredenberg; J.H.F. Snel. - S.l. : Van Voorthuysen - ISBN 9789054856696 - 155
fotosynthese - chloroplasten - elektromagnetisch veld - elektrodynamica - elektromagnetisme - photosynthesis - chloroplasts - electromagnetic field - electrodynamics - electromagnetism
The earliest events in the energisation of the photosynthetic membrane upon light capture are the formation of a transmembrane electrical potential (AV) and a transmembrane proton gradient (ΔpH). In this thesis ΔΨis employed for the study of the bioenergetics of chloroplast photosynthesis and its regulation by ΔpH in the shade adapted plant peperomia ( Peperomia metallic a) and the high-light adapted plant spinach ( Spinacia oleracea ). Electrochromism (P515) was used and a patch-clamp method was developed yielding two complementing tools for the detection of ΔΨ. The patch-clamp method enables the detection of relatively large light- induced currents (photocurrents) or potentials (photopotentials) of a single P. metallica chloroplast. An electrical equivalent scheme is introduced incorporating amongst others the thylakoid membrane resistance and capacitance and an access resistance which, at least partly, is supposed to be associated with low (lateral) conductance phases of thylakoid lamellae. The light-induced electrical responses reflect the operation of the photosynthetic current-generators and the way generated current is distributed throughout the chloroplast conductance network. Simultaneous measurements of light- and current-induced responses allow the separation of electrogenic events from changes in chloroplast conductances. A kinetically well defined slow secondary phase (R1 /Q) could be distilled from the flash- induced photocurrent/-potential which is related to the turnover of the cyt. b 6 f complex (Q-cycle). Generally, the rise of R1/Q was sigmoidal. This biphasic rise is modelled by a consecutive reaction scheme with two relaxation times of 13 and 28 ms which likely reflect the oxidation of plastoquinol and reduction of plastoquinone at the lumen and stroma membrane/water interface of the b 6 f complex, respectively. A P515 fraction (Rl /RC f ) of about 20 % is inadequately stabilised in dark-adapted spinach chloroplasts and decays rapidly with a relaxation time of 1 - 2 ms. A fast dissipation of ΔΨas generated by photosystern (PS) II is suggested to cause R1/RC f . It is hypothesised that adequate charge stabilisation depends on efficient energy coupling between PS II and the cyt. b 6 f complex which is only guaranteed in superclusters composed of both protein complexes. Energisation causes a suppression of about 50 % of PS II-dependent charge separation which is dark reversible with a relaxation time of about 20 s and is likely induced by the low lumenal pH created by lightdriven proton pumping. The results are best explained by a reaction center quenching model in which a fraction of PS II centers exhibits a rapid charge recombination. Flash-induced changes in chloroplast conductances are first demonstrated. The seal conductance decreases transiently upon a brief flash with a minimum of 0.3 - 5 % at 50 - 200 ms after the flash and a slow relaxation in 1 - 10 s. It is proposed that an important part of the conductance changes is intimately associated with changes in the lateral conductances of thylakoids, in particular those of the narrow spaced grana thylakoids.
Phytochrome and greening in etioplasts
Kraak, H.L. - \ 1986
Landbouwhogeschool Wageningen. Promotor(en): W.J. Vredenberg; R.E. Kendrick. - Wageningen : Kraak - 111
chloroplasten - fytochroom - plantenpigmenten - chloroplasts - phytochrome - plant pigments
This thesis is concerned with the role played by phytochrome (P) in the development of etioplasts into chloroplasts.
Previously dark-grown maize seedlings are not as sensitive as pea seedlings to very low fluences of red light (R) with regard to induction of rapid chlorophyll (Chl) accumulation in white light (WL), but a very low fluence response (VLFR) has been established in this plant species as well. Much higher fluences of a second R pre-irradiation are required to give an additional effect (low fluence response or LFR). When the effect of far-red light (FR) as such is accounted for, the effects of both a first and a second R pre- irradiation are 60-80% reversible by FR in maize seedlings. In high irradiance WL, the lag phase of Chl accumulation is of considerably longer duration. This indicates that photodestruction of Chl plays a role in the occurrence of a lag phase in Chl accumulation. R has a relatively large effect in high irradiance WL (Chapter 3).
Phytochrome (P) was measured spectrophotometrically for the first time in purified etioplast preparations obtained in complete darkness from dark-grown seedlings (D etioplasts) (Chapter 4). The P content of etioplast preparations from R pre-irradiated seedlings marginally exceeded that of D etioplasts. While the total P content of maize leaves, as measured in homogenates, decreased after R irradiation as a result of Pfr dark destruction, the P content of etioplasts from similar seedlings remained constant.
Attempts to demonstrate a physiological effect of etioplast- associated P were not successful. Preliminary studies on ultrastructural development of etioplasts (Chapter 5) showed that the invitro development during 1 h WL did not completely parallel development insitu . An effect of invivo R pre-irradiation on prolamellar body transformation, which was evident insitu , was not observed invitro . Insitu , formation of incipient grana in WL was stimulated by R pre-irradiation, however, isolated etioplasts proved incapable of forming incipient grana.
In the dark, following a short irradiation, regeneration of phototransformable protochlorophyll(ide) (PChl(ide)) was observed in isolated etioplasts (Chapters 6 and 7). However, regeneration kinetics differed from those invivo and no effect of invivo R pre-irradiation could be demonstrated. Invivo , the rate of PChl(ide) regeneration was increased by Pfr (Chapter 6).
Wavelength shifts of the 77K fluorescence emission maxima of newly formed chlorophyll(ide) (Chl(ide)) after a short irradiation were studied in leaves and isolated etioplasts. Derivative spectroscopy and curve fitting were applied to study kinetics of these shifts (Chapter 7). The first shift, a red shift, was slower in isolated etioplasts than in leaves. No effect of R pre-irradiation was observed on the rate of this shift. The subsequent blue shift, the so-called Shibata shift, was more rapid, but less complete in isolated etioplasts than in leaves. Whereas in leaves the rate of the Shibata shift was increased by Pfr, this was hardly, if at all, detectable in isolated etioplasts. The amount of phototransformable PChl(ide) decreased and the rate of the Shibata shift increased during storage of isolated etioplasts at 4 °C in darkness. Newly formed Chl(ide) proved unstable in isolated etioplasts.
The above results point to a decisive influence of the cytoplasm on the development of etioplasts in WL. In this respect, polypeptides of Chl-protein complexes synthesized in the cytoplasm may play an important role. However, a direct influence of etioplast-associated P in the development of etioplasts into chloroplasts, e.g. on permeability of the etioplast envelope, can not be excluded. Evidence for such an effect is found in the observation that the potentiating effect of a R pre-irradiation with regard to rapid Chl accumulation in WL is still partially reversible by FR after a dark period of 24 h. While Pfr in bulk P had already disappeared due to dark destruction after 4 h of darkness, the amount of P associated with etioplasts appeared not to decrease (see above). It is attractive to attribute at least that part of R potentiation which shows a long-term reversibility by FR, to apparently relatively stable etioplast-associated Pfr.
The results are discussed in relation to the phytochrome transport model of Raven and Spruit (Chapter 8). It is concluded that, though they do not provide a direct support for the model, they are not in disagreement with it. The transport model still appears to give an attractive explanation for a number of P responses, such as the VLFR and the Zea P paradox.
Electrochromic effects in relation to energy transduction and energy coupling in chloroplast membranes
Peters, R.L.A. - \ 1986
Landbouwhogeschool Wageningen. Promotor(en): W.J. Vredenberg. - Wageningen : Peters - 84
fotosynthese - plastiden - chloroplasten - membranen - bio-energetica - elektrische eigenschappen - energie - overdracht - photosynthesis - plastids - chloroplasts - membranes - bioenergetics - electrical properties - energy - transfer
A study was made on the kinetics of the flash-induced P515 electrochromic bandshift signal in spinach leaves and isolated chloroplasts. It was found that part of the signal (i.e. the slow component, also called reaction 2), normally present in dark-adapted membranes is absent from the signal under conditions where the membrane is energized preceding the actinic light flash. This energization can be brought about either by light-driven electron transport or by reverse electron flow caused by ATP hydrolysis. The activation of the chloroplast ATPase was found to be dependent on endogeneous factors determining ΔGATP such as of the adenylate kinase. The acceleration of the overall decay of the flash-induced P515 response found after short periods of illumination was found to be independent of the H+- permeability of the membrane as has been suggested by others. It is shown that this acceleration can be fully explained by the suppression of the reaction 2 component as a consequence of membrane energization by an activated ATPase. The occurrence of reaction 2 appeared to be dependent on the functional integrity of the membrane and a possible correlation between factors determining the structural organization of the thylakoid and the kinetics of the P515 response was found. It is concluded that the reaction 2 component of the P515 response is the reflection of an intramembranal electrical event, presumably associated with the liberation and subsequent stabilization of protons in inner-membrane domains. It appears that this stabilizing ability is lost upon the addition of a lipophilic protonophore.
Regulation of photosynthetic electron flow in isolated chloroplasts by bicarbonate, formate and herbicides
Snel, J.F.H. - \ 1985
Landbouwhogeschool Wageningen. Promotor(en): W.J. Vredenberg, co-promotor(en): J.J.S. van Rensen. - Wageningen : Snel - 123
chloroplasten - fotosynthese - plastiden - synthese - fotochemie - chloroplasts - photosynthesis - plastids - synthesis - photochemistry
This thesis describes some efforts that were made to gain a better understanding of the processes involved in the regulation of photosynthetic electron flow by bicarbonate, formate and herbicides in chloroplasts. In the past decade a large amount of research has been devoted to get insight into the mechanism of herbicide action on electron flow at the acceptor side of photosystem II. This thesis will deal mainly with studies on the regulation of electron flow at the acceptor side of photosystem II by bicarbonate and formate. Some details of the mechanism of this regulation as well as its integration in the overall process of photosynthesis were investigated.In Chapter 2 experiments are described that were aimed to provide a more quantitative description of the mutual interaction between herbicides, bicarbonate and their binding environment at the acceptor side of photosystem II. This interaction was studied by measuring the effects of bicarbonate and herbicides on electron flow in CO 2 -depleted chloroplasts. The kinetics of the reactivation of the inhibited Hill reaction in CO 2 -depleted chloroplasts by dark-incubation with bicarbonate suggest that the binding of bicarbonate involves a reaction with (pseudo) first order kinetics. It is shown that in the presence of the herbicides i -dinoseb and DCMU the reactivation of the Hill reaction in CO 2 -depleted chloroplasts by bicarbonate is retarded. It is shown that any competitive inhibitor of the binding of bicarbonate can be expected to effectively retard the binding of bicarbonate (a theoretical treatment is given in Appendix 1). Although i -dinoseb appeared to be an apparently competitive inhibitor of the stimulation of the Hill reaction by bicarbonate under equilibrium conditions, i -dinoseb and bicarbonate probably do not compete for a common binding site. This was inferred from the kinetics of the binding of i -dinoseb. These kinetics were shown to be to fast to be explained by simple competition. Therefore it is concluded that the binding of i -dinoseb affects the binding of bicarbonate in an allosteric way.In Chapter 3 a new procedure for CO 2 -depletion of chloroplasts is presented. This method yields CO 2 -depleted chloroplasts in which the Hill reaction can be almost completely reactivated by bicarbonate at pH 6.5. It is further shown that formate is a competitive inhibitor of the reactivation of the Hill reaction by bicarbonate. The true reactivation constant K r was calculated to be 78 μM bicarbonate at pH 6.5. Under the same conditions the inhibitor constant K i was calculated to be 2 mM formate. Experiments performed at bicarbonate and formate concentrations lower than K r resp. K i show that under these conditions electron flow proceeds at high rates. These observations suggest that, contrary to hitherto presented concepts, binding of bicarbonate to the regulatory site at the acceptor side of PSII is not a requirement for electron flow at the acceptor side of PS II. The results presented in Chapter 3 further suggest that formate is a potent inhibitor of electron flow at the acceptor side of PSII. The inhibitory action of formate is counteracted in a competitive way by bicarbonate.It is proposed in Chapter 3 that photorespiration may affect photosynthetic electron flow. Under certain conditions formate is produced during photorespiration. As photorespiratory formate production is dependent on the oxygen concentration, a negative feedback loop from photosynthetically produced oxygen to formate may exist under these conditions. This negative feedback loop can be powerful mechanism for the regulation of electron flow in vivo .In Chapter 4 the effects of formate and bicarbonate on the Hill reaction are further studied and characterized, especially during prolonged illumination. Formate is shown to stimulate the Hill reaction in non-depleted chloroplasts. It is therefore proposed that formate acts as an uncoupling agent. In the presence of an uncoupler formate was found not to stimulate electron flow. Instead only inhibiton of electron flow by formate was observed. The extent of this inhibition was not constant in time but appeared to increase during prolonged illumination The inhibition of electron flow by formate could be partially prevented by including bicarbonate in the reaction medium, but in the light bicarbonate appeared to be a less efficient counteracting agent than in the dark. It is estimated that the inhibitor constant K i of formate may be more than a magnitude lower in the light than in the dark, implying that formate affects steady state electron flow at sub mM concentrations.In Chapter 5 an alternative method is presented which can effectively be used to study the effects of formate and bicarbonate on electron flow in intact chloroplasts. This method is based on measurement of the magnitude of the flash-induced transmembrane potential using the P 515 response as a linear indicator of the transmembrane potential difference. In CO 2 -depleted chloroplasts the overall rate-limiting step of electron flow was found to posess a half-time of about 200-250 ms in the absence of bicarbonate. This half-time was much smaller in the presence of bicarbonate. Moreover gramicidin insensitive absorbance changes in the 500-550 nm region were found to be affected by CO 2 -depletion and readdition of bicarbonate. These absorbance changes are speculated to be associated, with protolytic reactions at the acceptor side of PSII.Chapter 6 describes effects of formate and bicarbonate on electron flow in isolated intact chloroplasts. Formate was found to inhibit PGA dependent oxygen evolution, but this inhibition could not be correlated with inhibition of electron flow at the acceptor side of PSII. Inhibition of electron flow was observed when isolated intact chloroplasts were incubated with formate at low pH. This inhibition of electron flow was abolished by addition of bicarbonate. The treatment with formate at low pH however appeared to be detrimental to carbon metabolism; PGA and CO 2 -dependent oxygen evolution were found to be irreversibly inhibited.A summarizing discussion is given in Chapter 7, in which the physiological significance of regulation of electron flow by formate and bicarbonate is discussed in relation to carbon metabolism.
Electrical events associated with primary photosynthetic reactions in chloroplast membranes
Schapendonk, A.H.C.M. - \ 1980
Landbouwhogeschool Wageningen. Promotor(en): W.J. Vredenberg, co-promotor(en): R. Kraayenhof. - Wageningen : Pudoc - ISBN 9789022007563 - 85
bio-energetica - chloroplasten - elektrische eigenschappen - membranen - fotosynthese - synthese - fotochemie - bioenergetics - chloroplasts - electrical properties - membranes - photosynthesis - synthesis - photochemistry
A study was made of electric phenomena in isolated photosynthetically-active chloroplast membranes upon energization. Energization of chloroplast thylakoid membranes gives rise to a charge separation across the membrane and an associated transmembrane electric field. The kinetics of this field were monitored as potential changes by a micro-electrode inserted in a single chloroplast and they were compared with the absorbance change of an intrinsic pigment (P515) proposed to be a reflection of a transmembrane electric field. The observed discrepancy between the flash-induced PSIS response and the electric response was caused by conformational changes in the membranal core that brought about absorbance changes of P515 in addition to its reponsiveness to the transmembrane potential. Most of the conclusions drawn hitherto from P515 kinetics, both after flash excitation and during prolonged illumination, have to be regarded with caution because they commonly are based on assumptions that are invalid under normal physiological conditions. Under certain conditions, if appropriate corrections are made, the PSIS kinetics are indeed a reflection of the transmembrane potential. In that case the results appear to be in good agreement with results obtained with the electrophysiological approach.The component of the P515 absorbance change, that is ascribed to conformational changes in the membranal core, may provide a useful tool for studies of fast structural changes in relation to energy coupling, without affecting the membrane integrity.
Licht en leven
Vredenberg, W.J. - \ 1979
Wageningen : Landbouwhogeschool - 19
membranen - fotosynthese - chloroplasten - membranes - photosynthesis - chloroplasts
Rede 29-11-79 Wageningen
|Changes in membrane potential associated with cyclic and non - cyclic electron transport in photochemical system 1 in Nitella translucens
Vredenberg, W.J. - \ 1971
Wageningen : [s.n.] (Publikatie / Centre for plant physiological research no. 101) - 8
plasmamembranen - celmembranen - chara - charophyta - chloroplasten - gegevens verzamelen - schatting - licht - meting - fotoperiode - fotoperiodiciteit - registreren - schaduw - elektrostatica - cell membranes - chara - charophyta - chloroplasts - data collection - estimation - light - measurement - photoperiod - photoperiodism - plasma membranes - recording - shade - electrostatics
|Light - induced changes in membrane potential of algal cells associated with photosynthetic electron transport
Vredenburg, W.J. - \ 1969
Wageningen : [s.n.] (Publication / Centre for plant physiological research no. 88) - 8
plasmamembranen - algen - celmembranen - chloroplasten - licht - fotoperiode - fotoperiodiciteit - schaduw - elektrostatica - algae - cell membranes - chloroplasts - light - photoperiod - photoperiodism - plasma membranes - shade - electrostatics