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See detailThe tropical sea anemone Aiptasia pallida as a lab model for the study of coral bleaching
Ladrière, Ophélie ULg; Roberty, Stéphane ULg; Baudesson, Charlotte et al

Poster (2008)

Bleaching is still among major events threatening coral reefs. New tools have to be developped to better understand the mechanisms leading to this pathology : we studied the use of the hermatypic anemone ... [more ▼]

Bleaching is still among major events threatening coral reefs. New tools have to be developped to better understand the mechanisms leading to this pathology : we studied the use of the hermatypic anemone Aiptasia pallida as experimental model for coral bleaching. Aiptasia appears as a good candidate as it is easy to maintain in aquarium and subjected to bleaching like corals. Both morphological and physiological approaches were performed to investigate the ultrastructure of the anemone tissues (TEM) and the zooxanthellae photophysiology (chlorophyll a fluorescence, respiration and pigmentation). Experiments under light and dark stress reveal that anemone tissues ultrastructure can be differently affected. In darkness, the ectoderm activity is reoriented to capture prey by increasing cnidocyte density. In contrast, intense light affects especially the gastroderm : intercellular spaces increase, the expulsion of intact algae in the gastric cavity and the degradation of zooxanthellae inside vacuoles seem to reduce the zooxanthellae density, chloroplast thylakoids lose their parallel arrangement. The analysis of the fluorescence induction curve appears as a powerful tool to analyse the physiological events series previous to bleaching. Although no significant zooxanthellae density reduction was observed, the decrease of pigments concentrations indicates that light or dark stresses induce anemone bleaching. Under strong light intensity, A. pallida zooxanthellae show an increased proportion of PSII QB non reducing, leading to partial photoinhibition. This phenomenon favours the ROS production that damages cellular structures of host and zooxanthellae. In darkness, there is no photosynthesis; anemones have therefore to find other feeding sources, as suggested by the ultrastructural approach. As the present results confirm some of those obtained on scleractinians, A. pallida can be regarded as a good model for coral bleaching studies and has numerous advantages for experimentation. [less ▲]

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See detailGrowth, fluorescence, photosynthetic O-2 production and pigment content of salt adapted cultures of Arthrospira (Spirulina) platensis
Ben Dhiab, R.; Ben Ouada, H.; Boussetta, H. et al

in Journal of Applied Phycology (2007), 19(4), 293-301

The effect of salt concentration (NaCl) on growth, fluorescence, photosynthetic activities and pigment content of the cyanobacterium Arthrospira platensis has been investigated over 15 days. It has been ... [more ▼]

The effect of salt concentration (NaCl) on growth, fluorescence, photosynthetic activities and pigment content of the cyanobacterium Arthrospira platensis has been investigated over 15 days. It has been observed that high NaCl concentration induces an increase of the growth, photosynthetic efficiency (alpha), phycobilin/chlorophyll ratio and a slight decrease of dark respiration and compensation points. Moreover, high NaCl concentration enhances photosystem II (PSII) activity compared to photosystem I (PSI). Results show that the phycobilin-PSII energy transfer compared to the chlorophyll-PSII (F-695,F-600/F-695,F-440) increases. However, data obtained about the maximal efficiency of PSII photochemistry are controversial. Indeed, the Fv/Fm ratio decreases in salt adapted cultures, while at the same time the trapping flux per PSII reaction center (TR0/RC) and the probability of electron transport beyond QA (Psi(0)) remain unchanged at the level of the donor and the acceptor sites of PSII. This effect can be attributed to the interference of phycobilin fluorescence with Chl a when performing polyphasic transient measurements. [less ▲]

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See detailThe light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana
Tzvetkova-Chevolleau, T.; Franck, Fabrice ULg; Alawady, A. E. et al

in Plant Journal (The) (2007), 50(5), 795-809

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants ... [more ▼]

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress. [less ▲]

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See detailChronic ozone exposure affects leaf senescence of adult beech trees: a chlorophyll fluorescence approach
Gielen, B.; Low, M.; Deckmyn, G. et al

in Journal of Experimental Botany (2007), 58(4), 785-795

Accelerated leaf senescence is one of the harmful effects of elevated tropospheric ozone concentrations ([O-3]) on plants. The number of studies dealing with mature forest trees is scarce however ... [more ▼]

Accelerated leaf senescence is one of the harmful effects of elevated tropospheric ozone concentrations ([O-3]) on plants. The number of studies dealing with mature forest trees is scarce however. Therefore, five 66-year-old beech trees (Fagus sylvatica L.) have been exposed to twice-ambient (2xambient) [O-3] levels by means of a free-air canopy O-3 exposure system. During the sixth year of exposure, the hypothesis of accelerated leaf senescence in 2xambient [O-3] compared with ambient [O-3] trees was tested for both sun and shade leaves. Chlorophyll (chl) fluorescence was used to assess the photosynthetic quantum yield, and chl fluorescence images were processed to compare functional leaf homogeneity and the proportion of O-3-injured leaf area (stipples) under ambient and 2xambient [O-3] regimes. Based on the analysis of chl fluorescence images, sun leaves of both ambient and 2xambient [O-3] trees had apparently developed typical necrotic O-3 stipples during high O-3 episodes in summer, while accelerated senescence was only observed with sun leaves of 2xambient [O-3] trees. This latter effect was indicated along with a faster decrease of photosynthetic quantum yield, but without evidence of changes in non-photochemical quenching. Overall, treatment effects were small and varied among trees. Therefore, compared with ambient [O-3], the consequence of the observed O-3-induced accelerated leaf senescence for the carbon budget is likely limited. [less ▲]

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See detailThe photosynthetic electron transport network: occurrence and function of cyclic and side pathways
Franck, Fabrice ULg

Conference (2006, September 07)

The current knowledge on cyclic and side pathways of the thylakoidal electron transport system will be discussed. Cyclic or pseudo-cyclic pathways include the PSI-driven cyclic electron transport, the ... [more ▼]

The current knowledge on cyclic and side pathways of the thylakoidal electron transport system will be discussed. Cyclic or pseudo-cyclic pathways include the PSI-driven cyclic electron transport, the cytochrome b6/f plastoquinone cycle and the oxygen-dependent water/water cycle. Side pathways altogether make up the chlororespiratory pathway, including the non-photochemical, NAD(P)H-dependent plastoquinone reduction by chloroplastic dehydrogenases and plastoquinol oxidation by a putative chloroplastic oxidase. The state of knowledge concerning the components, the importance and function of these pathways will be discussed. Results obtained in Liège using the unicellular green alga Chlamydomonas reinhardtii or higher plant chloroplasts will be presented. The importance of specific pathways in the context of mitochondria-chloroplast metabolic interactions and global cellular bio-energetics will be highlighted. Some of the methodological aspects in this field will be shortly described, in particular the use of fluorescence and absorbance spectroscopy. In some micro-algae, the electron transport chain can also use protons as terminal electron acceptors under anaerobic conditions, thereby allowing hydrogen evolution in the light. The potential of this fascinating reaction in perspective of the photobiological production of ‘bio-hydrogen’ will be briefly discussed. [less ▲]

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See detailND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme
Cardol, Pierre ULg; Lapaille, Marie ULg; Minet, P. et al

in Eukaryotic Cell (2006), 5(9), 1460-1467

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants ... [more ▼]

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed. [less ▲]

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See detailDelayed chlorophyll accumulation and pigment photodestruction in the epicotyls of dark-grown pea (Pisum sativum)
Boddi, B.; Loudeche, R.; Franck, Fabrice ULg

in Physiologia Plantarum (2005), 125(3), 365-372

A comparison was performed of the tetrapyrrole transformations that occur upon irradiation of epicotyl or leaves of dark-grown Pisum sativum L. (var. Zsuzsi, Hungary). High performance liquid ... [more ▼]

A comparison was performed of the tetrapyrrole transformations that occur upon irradiation of epicotyl or leaves of dark-grown Pisum sativum L. (var. Zsuzsi, Hungary). High performance liquid chromatography analysis after continuous or flash-irradiation showed that the biosynthetic pathway from protochlorophyllide (Pchlide) to chlorophyll (Chl) a was markedly slowed down at the step of the reduction of geranylgeranyl(gg)-Chl to dihydrogeranylgeranyl (dhgg)-Chl in epicotyls, whereas phytyl-Chl was synthesized in leaves subjected to the same light treatments. Quantitative pigment analysis during continuous irradiations of different intensities also showed that significant Pchlide photodestruction occurred in epicotyls even under weak light. When both Pchlide and chlorophyllide and/or chlorophylls were present in epicotyls, Pchlide photodestruction was faster under 630-nm light than under 670-nm light, which indicates that this process is most efficiently promoted by Pchlide excitation. Pre-incubation of epicotyl segments with 10 mM ascorbate partly alleviated pigment photodestruction in white light. It is concluded that formation of photoactive Pchlide-Pchlide oxidoreductase complexes is important to prevent fast pigment photooxidation after Pchlide accumulation in the dark. [less ▲]

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See detailChanges in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves
Franck, Fabrice ULg; Dewez, D.; Popovic, R.

in Photochemistry & Photobiology (2005), 81(2, Mar-Apr), 431-436

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves ... [more ▼]

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F-O (basal level of fluorescence yield at transient O) and F-M (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F-O to F-M, a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F-M to FT (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII. [less ▲]

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See detailProtochlorophyllide phototransformation in the bundle sheath cells of Zea mays
Dewez, D.; Franck, Fabrice ULg; Popovic, R. et al

in Journal of Photochemistry and Photobiology B : Biology (2004), 75(1-2), 73-80

The protochlorophyllide transformation process was investigated by using comparative analysis of 77 K fluorescence spectral changes occurring in isolated bundle sheath (BS) cells of etiolated Zea mays ... [more ▼]

The protochlorophyllide transformation process was investigated by using comparative analysis of 77 K fluorescence spectral changes occurring in isolated bundle sheath (BS) cells of etiolated Zea mays leaves after being exposed to a 200 ms saturating flash. Deconvolution analysis of the fluorescence spectra showed essential differences in the ratio of protochlorophyll(ides) and chlorophyll(ides) spectral forms indicating for BS cells to have a characteristic pathway of protochlorophyllide transformation. Bundle sheath cells showed a high ratio between non-photoactive protochlorophyll(ide)-F632 and photoactive protochlorophyllide-F655. In those cells, the 200 ins flash triggered a preferential formation of chlorophyll(ide)-F675 which remained stable in the dark for at least 90 min. Isolated BS cells showed an accumulation of chlorophyll(ide)-F675 resulting in the formation of inactive photosystem II. However for mesophyll cells of intact leaves, it was found to have a high ratio between photoactive and non-photoactive protochlorophyll(ide), showing the succession of chlorophyll(ide) forms usually known in C-3 plants. Protochlorophyllide phototransformation pathway in BS cells related to early stages of plastid differentiation triggered by light may indicate specific conditions for PSII assembly process leading to inactive PSII forms. (C) 2004 Elsevier B.V. All rights reserved. [less ▲]

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See detailPhotosynthesis and state transitions in mitochondrial mutants of Chlamydomonas reinhardtii affected in respiration
Cardol, Pierre ULg; Gloire, Geoffrey ULg; Havaux, M. et al

in Plant Physiology (2003), 133(4), 2010-2020

Photosynthetic activities were analyzed in Chlamydomonas reinhardtii mitochondrial mutants affected in different complexes (I, III, IV, I + III, and I + IV) of the respiratory chain. Oxygen evolution ... [more ▼]

Photosynthetic activities were analyzed in Chlamydomonas reinhardtii mitochondrial mutants affected in different complexes (I, III, IV, I + III, and I + IV) of the respiratory chain. Oxygen evolution curves showed a positive relationship between the apparent yield of photosynthetic linear electron transport and the number of active proton-pumping sites in mitochondria. Although no significant alterations of the quantitative relationships between major photosynthetic complexes were found in the mutants, 77 K fluorescence spectra showed a preferential excitation of photosystem I (PSI) compared with wild type, which was indicative of a shift toward state 2. This effect was correlated with high levels of phosphorylation of light-harvesting complex II polypeptides, indicating the preferential association of light-harvesting complex II with PSI. The transition to state 1 occurred in untreated wild-type cells exposed to PSI light or in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells exposed to white light. In mutants of the cytochrome pathway and in double mutants, this transition was only observed in white light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. This suggests higher rates of non-photochemical plastoquinone reduction through the chlororespiratory pathway, which was confirmed by measurements of the complementary area above the fluorescence induction curve in dark-adapted cells. Photo-acoustic measurements of energy storage by PSI showed a stimulation of PSI-driven cyclic electron flow in the most affected mutants. The present results demonstrate that in C. reinhardtii mutants, permanent defects in the mitochondrial electron transport chain stabilize state 2, which favors cyclic over linear electron transport in the chloroplast. [less ▲]

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See detailProtochlorophyllide reduction: Mechanisms and evolution
Schoefs, B.; Franck, Fabrice ULg

in Photochemistry and Photobiology (2003), 78(6), 543-557

Protochlorophyllide (Pchlide) reductases are key enzymes in the process of chlorophyll biosynthesis. In this review, current knowledge on the molecular organization, substrate specificity and assembly of ... [more ▼]

Protochlorophyllide (Pchlide) reductases are key enzymes in the process of chlorophyll biosynthesis. In this review, current knowledge on the molecular organization, substrate specificity and assembly of the light-dependent reduced nicotinamide adenine dinucleotide phosphate:Pchlide oxidoreductases are discussed. Characteristics of light-independent enzymes are also described briefly, and the possible reasons for the selection of light-dependent enzymes during the course of evolution are discussed. [less ▲]

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See detailEarly reactions of light-induced protochlorophyllide and chlorophyllide transformations analyzed in vivo at room temperature with a diode array spectrofluorometer
Boddi, B.; Popovic, R.; Franck, Fabrice ULg

in Journal of Photochemistry and Photobiology B : Biology (2003), 69(1), 31-39

The steps of protochlorophyllide (Pchlide) photoreduction and subsequent chlorophyllide (Chlide) transformations which occur in the seconds to minutes time-scale were studied using a diode array ... [more ▼]

The steps of protochlorophyllide (Pchlide) photoreduction and subsequent chlorophyllide (Chlide) transformations which occur in the seconds to minutes time-scale were studied using a diode array spectrofluorometer in dark-grown barley leaves. The intensity of the excitation light was varied between 3 and 2500 mumol m(-2) s(-1) and a series of fluorescence spectra were recorded at room temperature in the seconds and minutes time scales. In certain experiments, 77-K emission spectra were measured with the same equipment. The high quality of the spectra allowed us to run spectral resolution studies which proved the occurrence, at room temperature, of multiple Pchlide and Chlide forms found previously in 77-K spectra. The comparison of the 77-K and room-temperature spectra showed that the fluorescence yields of the nonphotoactive 633-nm Pchlide form and of the Chlide product emitting at 678 nm were temperature independent. The fluorescence intensity of aggregated NADPH-pigment-POR complexes (photoactive 656-nm Pchlide and 693-nm Chlide forms) were strongly increased at 77 K, while that of the NADP(+)-Chlide-POR (684-686-nm Chlide form) was much less affected by temperature. Information was obtained also about the dynamics of the transformation of pigment forms in the light at different photon densities. At low light intensities, the phototransformation of the 642-644-nm Pchlide form was faster than that of the 654-656-nm form. The relative amplitudes of Gaussian components related to different Chlide forms found after exposure to a constant amount of photons strongly depended on the light intensity used. Strong quenching of all Chlide components occurred upon prolonged exposure to high intensity light. These effects are discussed by considering the interconversion processes between different forms of the pigment-protein complexes, their relative fluorescence yields and energy migration processes. (C) 2002 Elsevier Science B.V. All rights reserved. [less ▲]

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See detailResolution of the Photosystem I and Photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature
Franck, Fabrice ULg; Juneau, P.; Popovic, R.

in Biochimica & Biophysica Acta (2002), 1556(2-3), 239-246

Green leaves illuminated with photosynthetically active light emit red fluorescence, whose time-dependent intensity variations reflect photosynthetic electron transport (the Kautsky effect). Usually ... [more ▼]

Green leaves illuminated with photosynthetically active light emit red fluorescence, whose time-dependent intensity variations reflect photosynthetic electron transport (the Kautsky effect). Usually, fluorescence variations are discussed by considering only the contribution of PSII-associated chlorophyll a, although it is known that the fluorescence of PSI-associated chlorophyll a also contributes to the total fluorescence [Aust. J. Plant Physiol. 22 (1995) 13 1]. Because the fluorescence emitted by each photosystem cannot be measured separately by selecting the emission wavelength in in vivo conditions, the contribution of PSI to total fluorescence at room temperature is still in ambiguity. By using a diode array detector, we measured fluorescence emission spectra corresponding to the minimal (F-O) and maximal (F-M) fluorescence states. We showed that the different shapes of these spectra were mainly due to a higher contribution of PSI chlorophylls in the F-O spectrum. By exciting PSI preferentially, we recorded a reference PSI emission spectrum in the near far-red region. From the F-O and F-M spectra and from this PSI reference spectrum, we derived specific PSI and PSII emission spectra in both the F-O and F-M states. This enables to estimate true value of the relative variable fluorescence of PSII, which was underestimated in previous works. Accurate separation of PSI-PSII fluorescence emission spectra will also enable further investigations of the distribution of excitation energy between PSI and PSII under in vivo conditions. (C) 2002 Elsevier Science B.V. All rights reserved. [less ▲]

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See detailRegulation of Etioplast Pigment-Protein Complexes, Inner Membrane Architecture, and Protochlorophyllide a Chemical Heterogeneity by Light-Dependent Nadph:Protochlorophyllide Oxidoreductases a and B
Franck, Fabrice ULg; Sperling, U.; Frick, G. et al

in Plant Physiology (2000), 124(4), 1678-96

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of ... [more ▼]

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB. [less ▲]

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See detailLocalization of Nadph-Protochlorophyllide Reductase in Plastids of Barley at Different Greening Stages
Barthelemy, X.; Bouvier, G.; Radunz, A. et al

in Photosynthesis Research (2000), 64(1), 63-76

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley ... [more ▼]

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids. [less ▲]

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