Aislamiento y caracterización de mutantes Rhodospirillum rubrum con alteraciones en el centro fotosintético de reacción

Tesis Univ. Complutense de Madrid.Fac. de Ciencias BiológicasTRUEProQuestpu

Aislamiento y caracterización de mutantes Rhodospirillum rubrum con alteraciones en el centro fotosintético de reacción

Tesis Univ. Complutense de Madrid.Fac. de Ciencias BiológicasTRUEProQuestpu

Editorial: Molecular basis of the response of photosynthetic apparatus to light and temperature stress

2 Pags. This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science.- © 2017 Picorel, Alfonso and Velitchkova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).Work in the laboratory of RP and MA was supported by Grant AGL2014-55300-R from Ministry of Economy and Competitiveness (MINECO) of Spain. Work in MV laboratory was supported by Swiss National Science Foundation under BSRP (IZEBZO-143169/1).Peer reviewe

Pigment Content of D1-D2-Cytochrome b559 Reaction Center Preparations after Removal of CP47 Contamination: An Immunological Study

5 pages, figures, and tables statistics.Isolated D1 -D2-cytochrome b559 photosystem I1 reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction centers prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose- 12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any crossreaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D 1 -D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-D1 -D2-Cyt b559 complex and that native photosystem I1 reaction centers contain 4 core chlorophylls per 2 pheophytins.Peer reviewe

Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions

The definitive version is available at www.blackwell-synergy.comToxic Cu(II) effect on Cytochrome b559 under aerobic photoinhibitory conditions was examined in two different PSII membrane preparations active in oxygen evolution. The preparations differ in the content of Cytochrome b559 redox potential forms. Difference absorption spectra showed that the presence of Cu(II) induced the oxidation of the high-potential form of Cytochrome b559 in the dark. Addition of hydroquinone reduced the total oxidised high-potential form of Cytochrome b559 present in Cu(II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of Cytochrome b559 during photoinhibitory treatment showed slower kinetics of Cu(II) effect on Cytochrome b559 as compared to the rapid loss of oxygen evolution activity in the same conditions. This result indicates that Cytochrome b559 is affected after PSII centers are photoinhibited. The high-potential form was more sensitive to toxic Cu(II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of Cytochrome b559 was completely lost, however the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. Results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of Cytochrome b559, respectively.M. Bernal was recipient of an I3P Programme fellowship from Consejo Superior de Investigaciones Científicas. This work was supported by the Dirección General de Investigación (Grant BMC2002-00031) to R.P. and Gobierno de Aragón (Grant P015/2001) to I.Y., and it has been done within GC DGA 2002 Program of Gobierno de Aragón.Peer reviewe

El tráfico de cobre en el cloroplasto de plantas superiores: los transportadores de membrana

Introducción: El cobre (Cu) es un elemento esencial en la mayoría de organismos, aunque también puede ser tóxico a elevadas concentraciones (Festa y Thiele, 2011). En las plantas, el Cu participa en procesos fisiológicos como la fotosíntesis, la respiración mitocondrial, la percepción del etileno, el metabolismo de la pared celular, la protección frente al estrés oxidativo, la síntesis del cofactor de molibdeno y la regulación del ciclo circadiano (Puig y col., 2007; Mendel, 2013; Perea-García y col., 2010). Esto es debido a su habilidad para encontrarse en dos estados de oxidación diferentes in vivo, Cu+ y Cu2+. Por esta razón las plantas han desarrollado mecanismos para mantener la homeostasis, que permiten un eficiente uso del metal, a pesar del potencial daño que puede constituir para la célula (Ellingsen y col., 2007). En ella intervienen, transportadores de Cu de alta afinidad, metalochaperonas y ATPasas de tipo P (Ravet y Pilon, 2013). Muchos de los componentes de esta red han sido identificados y caracterizados durante los últimos años. Sin embargo, es necesaria más investigación en este campo para comprender completamente estos mecanismos. Por todo ello, el objetivo de esta Tesis Doctoral es el estudio de la homeostasis de Cu en plantas de soja (Glycine (G.) max), concretamente se ha realizado la caracterización de PAA2 (P-type ATPase in Arabidopsis 2), la ATPasa de tipo P1B encargada de transportar el Cu a través de la membrana tilaicoidal (Abdel-Ghany y col., 2005), y el estudio de la chaperona CCS (Copper chaperone for the Cu/Zn superoxide dismutase), encargada de la entrega del Cu a la Cu/Zn superóxido dismutasa (Pilon y col., 2011). Desarrollo: 1. Caracterización de PAA2. En la especie G. max, existen dos genes PAA2 (Bernal, 2006). Dichos genes, GmPAA2-1 y GmPAA2-2, tienen alta identidad entre ellos y se ha detectado un mecanismo de procesamiento alternativo en el gen GmPAA2-1, lo que podría conducir a la producción de una proteína completa (GmPAA2-1) y una proteína truncada (GmPAA2-1T). A su vez, el gen GmPAA2-2 retiene un hipotético intrón que produciría un codón de STOP prematuro y, por tanto, una proteína truncada. Utilizando la técnica de 5’ RACE, se vio que este gen tiene diferentes inicios de la transcripción según las condiciones nutricionales de Cu in vivo. Se ha realizado una caracterización de las proteínas GmPAA2-1 y GmPAA2-2 mediante la complementación funcional de los mutantes de S. cerevisiae ccc2, donde parecía que las proteínas GmPAA2-1, GmPAA2-1T o GmPAA2-2 no son capaces de complementar el fenotipo mutante. A su vez, en las levaduras silvestres, tampoco se observó un fenotipo apreciable por hipersensibilidad a cobre a consecuencia de la expresión de las proteínas de soja. Posteriormente se realizó la complementación del mutante paa1-1 de A. thaliana, que carece del transportador de Cu de la envuelta del cloroplasto (Shikanai y col., 2003), y se observó que las proteínas GmPAA2-1 y GmPAA2-2 no complementan el mutante paa1-1, indicando que las proteínas PAA1 y PAA2 tienen funciones que no se solapan. A continuación se realizó la complementación funcional del mutante paa2-1 de A. thaliana (Abdel-Ghany y col., 2005) con las proteínas GmPAA2-1 y GmPAA2-2. En este caso, GmPAA2-2 parece no complementar el fenotipo mutante paa2-1, en cambio, GmPAA2-1 es capaz de complementar el mutante paa2-1, lo que indica que la proteína de G. max es capaz de realizar la función de transporte de Cu+ a través de la membrana tilacoidal como en el caso de AtPAA2. Además, se ha estudiado la localización de GmPAA2-1 y GmPAA2-2 mediante expresión transitoria y estable de las proteínas fusionadas a YFP. Los datos indican que ambas proteínas tienen una localización cloroplástica. También se estudió la expresión de los genes mediante la técnica de qRT-PCR, y se vio que la expresión del gen GmPAA2-1 aumenta significativamente en algunas de las hojas según el aporte de Cu en la planta. En cambio, el gen GmPAA2-2 no parece mostrar una regulación por Cu. La localización in vivo de las diferentes proteínas GmPAA2 de soja se estudió mediante western blot con anticuerpos específicos. Los resultados mostraron bandas que podrían corresponder con las proteínas GmPAA2-1 y GmPAA2-1T, aunque no se observó la proteína GmPAA2-2. A pesar de ello, serán necesarios más estudios para conocer la función y caracterizar las nuevas posibles proteínas adicionales de soja. 2. Caracterización de la chaperona CCS de soja. La proteína GmCCS recombinante se sobreexpresó en E. coli y se purificó por cromatografía IMAC-Co2+ mayoritariamente en forma de apoproteína. Mediante estudios de interacción con su ligando por calorimetría isotérmica de titulación, se observó que la proteína GmCCS parece tener dos sitios de unión a Cu+ independientes con diferente afinidad y capacidad calorífica. La apoproteína GmCCS se encuentra en forma monomérica y dimérica, siendo más abundante la forma monomérica como indicaron los experimentos de cromatografía de exclusión molecular. La adición de Cu+ parece tener un efecto en el estado oligomérico, favoreciendo la dimerización de la proteína. Conclusiones: 1. La duplicación génica y los eventos de procesamiento alternativo pueden constituir un escenario favorable para la especiación, adaptación y evolución de las especies. En la especie G. max se han encontrado dos genes PAA2, con procesamiento alternativo en al menos uno de ellos, que podrían producir proteínas con funciones diferentes. Esto podría traducirse en una característica diferencial y, posiblemente, favorable para las plantas de esta especie. 2. Homólogos del gen PAA2 se han encontrado en todas las especies analizadas, lo que sugiere que su función es de gran importancia para las plantas. En soja, la proteína GmPAA2-1 parece realizar la función de transporte de Cu+ a través del tilacoide de manera similar a la proteína AtPAA2. A su vez, GmPAA2-1 tiene una función específica que no se superpone con la de PAA1 a pesar de compartir una gran similitud estructural con ésta. 3. La función de las proteínas adicionales GmPAA2 que podrían existir en la especie G. max es desconocida todavía. Es posible que realicen funciones alternativas al transporte de Cu+ a través de la membrana tilacoidal, pudiendo representar una característica positiva para las plantas que cuentan con ellas. Sin embargo, es necesaria más investigación para determinar estas cuestiones. 4. El mecanismo de actuación de la proteína CCS, así como su función de transferencia de Cu a su diana, Cu/ZnSOD, todavía cuentan con múltiples cuestiones por resolver. La proteína GmCCS parece encontrarse en dos estados oligoméricos diferentes (monómero y dímero), viéndose el dímero favorecido por la presencia de su ligando, Cu+. Esto podría tener una implicación en la interacción con la Cu/ZnSOD, mayoritariamente dimérica, en la que la transferencia del Cu podría producirse desde el sitio de unión a metal en el dominio III de GmCCS, con menor afinidad y mayor capacidad calorífica que el sitio de unión a metal en el dominio I

Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas Reinhardtii

31 Pags. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkIt is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction centers (RCs) is pheophytin a (Pheo a) within the D1 protein (PheoD1), while PheoD2 (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the last two decades assigned the Qy-states of PheoD1 and PheoD2 bands near 678–684 nm and 668–672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986–998; Cox et al. J. Phys. Chem. B 2009, 113, 12364–12374] of the electronic structure of the PSII RC reversed the location of the active and inactive Pheos, suggesting that the mean site energy of PheoD1 is near 672 nm, whereas PheoD2 (~677.5 nm) and ChlD1 (~680 nm) have the lowest energies (i.e., the PheoD2-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Qy absorption maxima at 676–680 nm [Germano et al. Biochem. 2001, 40, 11472–11482; Germano et al. Biophys. J. 2004, 86, 1664–1672]. To provide more insight into the site energies of both PheoD1 and PheoD2 (including the corresponding Qx transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch PheoD1 is genetically replaced with chlorophyll a (Chl a). We show that the Qx-/Qy-region site-energies of PheoD1 and PheoD2 are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803–8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.Partial support to B.N. (involved in calculations) was provided by the NSF EPSCoR Grant. V.Z. (involved in writing the manuscript) acknowledges support by NSERC. R.T.S., R.P., and M.S. were involved in the design and preparation of D2-mutant and RCs. They acknowledge support from USDOE, Photosynthetic Antennae Research Center (R.T.S.), MICIN (Grant AGL2008-00377) in Spain (R.P.), and the U.S. Department of Energy’s Photosynthetic Systems Program within the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences under NREL Contract #DE-AC36-08-GO28308 (M.S.).Peer reviewe

The CP43 Proximal Antenna Complex of Higher Plant Photosystem II Revisited: Modeling and Hole Burning Study. I

The final version is available at: http://pubs.acs.org/journal/jpcbfkThe CP43 core antenna complex of photosystem II is known to possess two quasi-degenerate “red”-trap states (Jankowiak, R. et al. J. Phys. Chem. B 2000, 104, 11805). It has been suggested recently ( Zazubovich, V.; Jankowiak, R. J. Lumin. 2007, 127, 245) that the site distribution functions of the red states (A and B) are uncorrelated and that narrow holes are burned in the subpopulations of chlorophylls (Chls) from states A and B that are the lowest-energy Chl in their complex and previously thought not to transfer energy. This model of uncorrelated excitation energy transfer (EET) between the quasidegenerate bands is expanded by taking into account both electron−phonon and vibrational coupling. The model is applied to fit simultaneously absorption, emission, zero-phonon action, and transient hole burned (HB) spectra obtained for the CP43 complex with minimized contribution from aggregation. It is demonstrated that the above listed spectra can be well-fitted using the uncorrelated EET model, providing strong evidence for the existence of efficient energy transfer between the two lowest energy states, A and B (either from A to B or from B to A), in CP43. Possible candidate Chls for the low-energy A and B states are discussed, providing a link between CP43 structure and spectroscopy. Finally, we propose that persistent holes originate from regular NPHB accompanied by the redistribution of oscillator strength due to excitonic interactions, rather than photoconversion involving Chl−protein hydrogen bonding, as suggested before (Hughes J. L. et al. Biochemistry 2006, 45, 12345). In the accompanying paper ( Reppert, M.; Zazubovich, V.; Dang, N. C.; Seibert, M.; Jankowiak, R. J. Phys. Chem. B 2008, 9934), it is demonstrated that the model discussed in this manuscript is consistent with excitonic calculations, which also provide very good fits to both transient and persistent HB spectra obtained under non-line-narrowing conditions.This work was supported by the start-up funding at the Department of Chemistry, Kansas State University (RJ, NCD, MR and BN), and in part by the U.S. Department of Energy (DOE) EPSCoR grant (RJ), Energy Biosciences Program, Basic Energy Sciences, DOE (MS and NCD) and BFU2005-07422-CO2-01; Spain (RP). VZ acknowledges support by NSERC.Peer reviewe

Spin-label EPR study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation

El pdf del artículo es la versión post-print.Fatty acid desaturation effect on the lipid fluidity in thylakoid membranes isolated from the STR7 mutant was investigated by electron paramagnetic resonance (EPR) using spin label probes. The spectra of both 5- and 16-n-doxylstearic acid probes were measured as a function of the temperature between 10-305 K and compared to those of the wild type. This complete thermal evolution provides a wider picture of the dynamics. The spectra of the 5-n-doxylstearic acid probe were identical in both STR7 mutant and wild type thylakoids as well as their temperature evolution. However, differences were found with the 16-n-doxylstearic acid probe at temperatures between 230-305 K. The differences in the thermal evolution of the EPR spectra can be interpreted as a 5-10 K shift toward higher temperatures of the probe motional rates in the STR7 mutant as compared with that in the wild type. At temperatures below 230 K no differences were observed. The results indicated that the lipid motion in the outermost region of the thylakoids is the same in the STR7 mutant than in the wild type while the fluidity in the inner region of the STR7 mutant membrane decreases. Our data point out a picture of the STR7 thylakoid membrane in which the lipid motion is slower most probably as a consequence of fatty acid desaturation deficiency.M.A. and I.G.-R. were recipients of a contract and a fellowship, respectively, from the Ministerio de Educación y Cultura of Spain. This work was supported by the Dirección General de Investigación Científica y Técnica (Grant PB98-1632) and by the Diputación General de Aragón (Project P17/98).Peer reviewe