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Lutz A Eichacker - One of the best experts on this subject based on the ideXlab platform.
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Printed in U.S. A. In Vitro Synthesis of Chlorophyll A in the Dark Triggers Accumulation of Chlorophyll A Apoproteins in Barley Etioplasts”
2015Co-Authors: Lutz A Eichacker, Jiirgen Solls, Petra Lauterbachs, Wolfhart Riidigerl, Robert R. Kleinll, John E. MulletAbstract:An in vitro translation system using lysed Etioplasts was developed to test if the accumulation of plastid-encoded chlorophyll a apoproteins is dependent on the de novo synthesis of chlorophyll a. The P700 apopro-teins, CP47 and CP43, were not radiolabeled in pulse-chase translation assays employing lysed Etioplasts in the absence of added chlorophyll precursors. When chlorophyllide a plus phytylpyrophosphate were added to lysed etioplast translation assays in the dark, chlo-rophyll a was synthesized and radiolabeled P700 apo-proteins, CP47 and CP43, and a protein which comi-grates with D1 accumulated. Chlorophyllide a or pby-tylpyrophosphate added separately to the translation assay in darkness did not induce chlorophyll a forma-tion or chlorophyll a apoprotein accumulation. Chlo
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Determination of Chlide, ChlGG, and ChlPY in fluorescent Lil3 bands.
2015Co-Authors: Astrid Mork-jansson, Janine Arnold, Veronika Reisinger, Karol Kmiec, Ann Kristin Bue, Daniela Gargano, Clemens Furnes, Lutz A EichackerAbstract:For identification of Chlide specific fluorescence in protein complexes, isolated Etioplasts (1x108) were incubated without (lanes 1, 3) or supplemented with (lanes 2, 4) GGPP on ice for 1 min in darkness, plastids were frozen on dry ice and either maintained in the dark (lanes 1, 2) or exposed to light for 10 seconds (lanes 3, 4) (A). Plastids were lyzed, membranes were solubilized and protein complexes separated by LN-PAGE (3–12% acrylamide gradient) (A). For identification of pigments bound to fluorescent Lil3 bands, pigments were extracted from Etioplasts (B) or from gel-bands after separation by 7.5% native PAGE (C). For identification of pigments in etioplast membranes (B), acetone extraction was conducted using Etioplasts kept in darkness (B, lane 1), illuminated (B, lane 2), illuminated and incubated with GGPP (B, lane 3), or illuminated in the presence of GGPP and NADPH (B, lane 4), or FPP and NADPH (B, lane 5). Etioplasts isolated after a 10 sec in vivo illumination of etiolated plants (B, lane 6) were extracted and loaded as control. For identification of pigments bound to protein complexes (C), fluorescent Lil3 bands F3 (C, lane 1 and 2), F2 (C, lane 3), and F1 (C, lane 4) were extracted. Pigment synthesis was induced by a 10 s light exposure of Etioplasts (C, lane 1) or of etiolated plants (C, lane 2–4). HPTLC and native gels were scanned for fluorescence by laser excitation at λ = 633nm. The position of fluorescent protein complexes Cyt b6f, F3, and of pigments in the gel front (PF) of the native gel (A), and of pigments Pchlide, Chlide, Pchl, ChlGG, ChlPY, ChlF, and unidentified GGPP and FPP dependent tetrapyrrol derivatives (ChlGG* and ChlF*) after HPTLC separations (B, and C) are marked.
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Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley Etioplasts.
The Plant cell, 1999Co-Authors: Bernd Müller, Lutz A EichackerAbstract:Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated Etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in Etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated Etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b 559 . Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in Etioplasts. We therefore conclude that in Etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a –triggered accumulation of reaction center monomers.
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Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a–triggered accumulation of reaction center II in barley Etioplasts
1999Co-Authors: Bernd Müller, Lutz A EichackerAbstract:Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seed-lings and isolated Etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in Etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane pro-tein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated Etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 pre-cursor in a monomeric RCII precomplex that also included D2 and cytochrome
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Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts.
Plant physiology, 1996Co-Authors: Lutz A Eichacker, Ingrid Edhofer, Gerhard WannerAbstract:Methods for the cryopreservation of protein import and integration in pea chloroplasts and of protein import or protein synthesis in tobacco mitochondria were modified to yield enzymatically active cryopreserved Etioplasts from barley (Hordeum vulgare L.). The cryoprotectants ethylene glycol and dimethy sulfoxide were about 64 and 77% effective, respectively, for the cryopreservation of etioplast intactness. Phototransformation of protochlorophyllide a, esterification of chlorophyllide a or zinc-pheophorbide a, and stabilization of the de novo synthesized plastid-encoded chlorophyll-apoproteins P700, CP47, CP43, D2, and D1 were successfully preserved in liquid nitrogen. Cryopreservation of freshly prepared intact Etioplasts completely retained enzymatic activities for accumulation of chlorophyll a or resulted in a slightly decreased yield of zinc-pheophytin a.
Gregory A Armstrong - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis light dependent protochlorophyllide oxidoreductase a pora is essential for normal plant growth and development
Plant Molecular Biology, 2012Co-Authors: Troy Paddock, Klaus Apel, Daniel Lima, Mary E Mason, Gregory A ArmstrongAbstract:During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within Etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.
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regulation of etioplast pigment protein complexes inner membrane architecture and protochlorophyllide a chemical heterogeneity by light dependent nadph protochlorophyllide oxidoreductases a and b
Plant Physiology, 2000Co-Authors: Fabrice Franck, Ulrich Sperling, B Van Cleve, Genevieve Frick, Klaus Apel, Babette Pochert, Gregory A ArmstrongAbstract: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.
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etioplast differentiation in arabidopsis both pora and porb restore the prolamellar body and photoactive protochlorophyllide f655 to the cop1 photomorphogenic mutant
The Plant Cell, 1998Co-Authors: Ulrich Sperling, Fabrice Franck, B Van Cleve, Genevieve Frick, Klaus Apel, Gregory A ArmstrongAbstract:The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of Etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.
Bernd Müller - One of the best experts on this subject based on the ideXlab platform.
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Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley Etioplasts.
The Plant cell, 1999Co-Authors: Bernd Müller, Lutz A EichackerAbstract:Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated Etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in Etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated Etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b 559 . Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in Etioplasts. We therefore conclude that in Etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a –triggered accumulation of reaction center monomers.
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Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a–triggered accumulation of reaction center II in barley Etioplasts
1999Co-Authors: Bernd Müller, Lutz A EichackerAbstract:Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seed-lings and isolated Etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in Etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane pro-tein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated Etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 pre-cursor in a monomeric RCII precomplex that also included D2 and cytochrome
Dmitriy Shevela - One of the best experts on this subject based on the ideXlab platform.
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biogenesis of water splitting by photosystem ii during de etiolation of barley hordeum vulgare l
Plant Cell and Environment, 2016Co-Authors: Dmitriy Shevela, Janine Arnold, Veronika Reisinger, Hansmartin Berends, Karol Kmiec, Sergey Koroidov, Ann Kristin Bue, Johannes MessingerAbstract:Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of Etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de-etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll-binding protein complexes and development of water splitting via O2 production in plastids (etiochloroplasts) isolated during de-etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light-harvesting antenna [PSII-light-harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de-etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de-etiolation, etiochloroplasts revealed the same water-splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de-etiolation precedes assembly of the PSII-LHCII supercomplexes. Taken together, data show a rapid establishment of water-splitting activity during etioplast-to-chloroplast transition and emphasize that assembly of the functional water-splitting site of PSII is not the rate-limiting step in the formation of photoactive thylakoid membranes.
Klaus Apel - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis light dependent protochlorophyllide oxidoreductase a pora is essential for normal plant growth and development
Plant Molecular Biology, 2012Co-Authors: Troy Paddock, Klaus Apel, Daniel Lima, Mary E Mason, Gregory A ArmstrongAbstract:During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within Etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.
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regulation of etioplast pigment protein complexes inner membrane architecture and protochlorophyllide a chemical heterogeneity by light dependent nadph protochlorophyllide oxidoreductases a and b
Plant Physiology, 2000Co-Authors: Fabrice Franck, Ulrich Sperling, B Van Cleve, Genevieve Frick, Klaus Apel, Babette Pochert, Gregory A ArmstrongAbstract: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.
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etioplast differentiation in arabidopsis both pora and porb restore the prolamellar body and photoactive protochlorophyllide f655 to the cop1 photomorphogenic mutant
The Plant Cell, 1998Co-Authors: Ulrich Sperling, Fabrice Franck, B Van Cleve, Genevieve Frick, Klaus Apel, Gregory A ArmstrongAbstract:The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of Etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.