Bellido, D.

Boronat, A.

Eichacker, L.

Odena, M.A.

Oliveira, Eliandre de

Petrizzo, R.

Reisinger, V.

Rose Campbell, J.K.

Repositorio Institucional de la Universidad de Córdoba

70 PROTEÓMICA • NÚMERO 1 • FEBRERO 2008al., 2005). Debido a la difi cultad del trabajo con pro-teínas de membrana, el estudio de éstas se ha pasado por alto en muchos análisis proteómicos sobre la interacción con tiorredoxinas (Meyer et al., 2005). Materiales y métodosEn este trabajo hemos desarrollado un proce-dimiento para aislar proteínas de membrana que interaccionen con la tiorredoxina TrxA mediante la unión “in situ” a una tiorredoxina alterada (Tr-xAC35S), con una sola cisteína en el sitio activo, y con una secuencia adicional de seis histidinas, la cual fue añadida directamente a membranas intactas. Posteriormente al fraccionamiento y solubilización de las membranas, las potenciales proteínas diana de tiorredoxina fueron aisladas mediante cromato-grafía de afi nidad por níquel y electroforesis des-naturalizante bidimensional bajo condiciones no reductoras en la primera dimensión y reductoras en la segunda. ResultadosUtilizando este método hemos identifi cado 50 posibles proteínas diana de tiorredoxina, 38 de las cuales han sido identifi cadas como tales por primera vez para Synechocystis en este trabajo, aunque 10 de ellas ya fueron descritas como tales en trabajos previos para otros organismos. Entre las dianas cabe destacar varias subunidades ATPasa de transporta-dores y miembros de la familia AAA+ de ATPasas, e.g. FtsH. (Mata-Cabana, et al., 2007).Conclusiones En este estudio se describe por primera vez una posible regulación por la luz a través de tiorredoxi-nas de procesos tales como el transporte a través de la membrana o la proteolisis, con lo que se abren nuevas vías de estudio para profundizar en estas nuevas relaciones tanto en cianobacterias como en otros organismos.Bibliografía Cooper, C.E., Patel, R.P., Brookes, P.S., Darley-Usmar, V.M. Trends in Biochemical Sciences 2002, 27, 489-492 Mata-Cabana, A., Florencio, F.J., Lindahl, M. Proteom-ics 2007, 7, 3953-3963Meyer, Y., Reichheld, J.P., Vignols, F. Photosynthesis Research 2005, 86, 419-433Srivastava, R., Pisareva, T., Norling, B. PCC 6803. Proteomics 2005, 5, 4905-4916Tomato chromoplast proteome: challenges and perspectivesPetrizzo R.1, Reisinger V.2, Eichacker L.2, Rose Campbell J. K3, Bellido D.4, Oliveira E.4, Odena MA4 and Boronat A.1 1Department of Biochemistry and Molecular Biology, University of Barcelona, Faculty of Biology, Avda. Diagonal 645, 08028-Barcelona. 2Department für Biologie I, Ludwig-Maximilians-Universitat, München, Germany. 3Emerson Hall Department of Plant Biology, Cornell University, Ithaca, NY 14853 USA. 4Plataforma de Proteòmica, Parc Científi c de Barcelona, Campus Diagonal, Universitat de Barcelona, C/ Josep Samitier 1-5, 08028 Barcelona.IntroductionIn our work we focus on non-photosynthetic plastids (i.e Chromoplasts) which are part of the compartimentalised genetic machinery of the plant cell and originated from endosymbiosis. Chromo-plasts derive from the chloroplasts present in the green fruit (Camara et al., 1995). The chloroplast to chromoplast transition is marked by the degradation of the thylakoid membrane system and the reduction in the level of proteins associated with photosynthe-sis (Cheung et al., 1995). The role of chromoplasts in other metabolic processes relevant for fruit qual-ity (such as the production of fl avors, aromas, or PROTEÓMICA • NÚMERO 1 • FEBRERO 2008 71defense compounds) remains unexplored. To evalu-ate the metabolic role of chromoplasts during fruit ripening we have undertaken a proteomic study of this organelle. We report about different proteomics technology to characterize the functional genome of the organelle.Material and methodsFor chromoplast isolation and protein extrac-tion, approxymatively 300 g of fi rm ripe tomato fruits in a specifi c ripening stage (B+8 to B+12) were collected from wild-type, T1 (DXS), and T2 (PSY). Fruits harvesting and protein preparation were repeated from four independent batches of plants, to give four independent biological replicate. Chromoplasts from fi rm-ripe fruits have been puri-fi ed using sucrose density-gradient centrifugation. The purity of the isolated chromoplasts have been evaluated by electron microscopy but also and the use of enzymatic markers (cytosol, mitochondria, peroxisome and ER). After extraction with a phenol based protocol, proteins were labelled with CyDyes DIGE® fl uorochromes (GE Healthcare).  Equal amounts (50 µg) of transgenic (Cy3), Wt (Cy5) and the mixture (Cy2) of the individually labeled samples were loaded on the gel resulting in each 75 µg of protein from transgenic and Wt. First dimen-sion (IEF) was carried out on both 18 and 24cm pre-cast IPG strips (GE Healthcare) with a pH gradient of 3-11NL. The second dimension (SDS-PAGE) was performed using the Ettan DALTsix system. To ensure statistical signifi cance 4 biological replicates were used. After SDS-PAGE gels were scanned us-ing a TyphoonTRIO (GE Healthcare). Gel images were processed using the DeCyder Software v6.5 (GE Healthcare). After the Student’s t-test (P-values ≤ 0.05), spots of interest were picked on preparative gels containing 500 μg of a protein mixture from all samples and stained by Colloidal Coomassie. Picked spots were submitted to tryptic digestion and analyzed by nanoLC/ESI-Q-TOF. BN-PAGE was performed as described by Reisinger et al. (2006). Acquired spectra were then searched with Mascot (Matrix Science, UK) against the NCBI database and blasted against the Tomato EST database.ResultsThe total chromoplast proteome could be re-solved into 660 protein spots after the application of the fi ltering option offered by the DeCyder soft-ware, and the gels showed a similar pattern, show-ing that protein extraction procedure, labeling and 2-D PAGE are reproducible. Our preliminary results show clear differences between tomato chromoplasts from Wt and transgenic lines. A number of spots dis-playing at least 2-fold changes in relative abundance were chosen for identifi cation. Since tomato genome has not been yet fully sequenced, the obtained MS and MS/MS results were matched against all se-quence data available on public databases using the Mascot and dBEST engines. Chromoplast proteins from wt and transgenic line that showed signifi cant changes in abundance were classifi ed into different categories. The dominant group of proteins chang-ing in abundance in our study was defence/repair and ripening related.ConclusionsIn conclusion, in this study we applied subcel-lular fractionation and protein quantifi cation using differential gel electrophoresis (DIGE) to obtain a fi rst insight into tomato chromoplast proteome of wt and transgenic fruits. We know that 2D-DIGE and DeCyder image analysis is a sensitive, MS-com-patible, and reproducible technique for identifying statistically signifi cant differences in the protein expression profi les of multiple tomato samples. The differential analysis revealed the presence of sig-nifi cant differences in 62 spots; 45 of them were identifi ed by nanoLC-ESI-MS/MS. The identifi ed proteins, differentially expressed in the transgenic line and that participate in specifi c carotenoid ac-cumulation within the tomato chromoplasts but also in other plastid metabolic functions, indicating that carotenoid accumulation involves a more complex mechanism than just accumulation of the caroten-oid biosynthetic pathway enzymes. We identifi ed those spots having moderate and intense fl uores-cence intensity. Spots with lower intensity and some spots corresponding to low MW proteins were not identifi ed. This is probably due to the shift that low MW proteins labelled with CyDye do have in DIGE studies. This study not only provides a new insight into carotenoid accumulation within the chromo-plast of transgenic lines of tomato fruit, but also demonstrates the importance of subfractionation for the full understanding of the changes in protein abundance during metabolic responses.The proteins identifi ed by DIGE analysis were grouped in functional categories: 1) metabolism; 2) ionic channels, ion transporter and related; 3) 72 PROTEÓMICA • NÚMERO 1 • FEBRERO 2008defence/repair; 4) miscellaneous; 5) Unknown; 6) Not identifi ed. Membrane protein complexes were also studied using BN-PAGE. The BN-SDS PAGE technique has been proven to be a good method to resolve highly hydrophobic integral membrane proteins from chromoplast preparations. These membrane protein complexes are just now being analyzed by nanoLC/ESI-Q-TOF. With BN-PAGE we have been able to get for the fi rst time the chro-moplast membrane pattern and to identify all sub-units of the ATPase complex and 2 subunits of the NdhI complex. The last one was also confi rmed by in gel enzymatic assay, indicating a possible role of that complex in chromorespiration.ReferencesCamara B, Hugueney P, Bouvier F, Kuntz M, Moneger R (1995). Int Rev Cytol 163:175-247.Cheung AY, McNellis T, Piekos B (1993). Plant Physiol 101:1223-1229.Fraser PD, Bramley PM (2004). Prog Lipid Res 43:228 –265.Krinski NI, Johnson EL (2005).  Mol Asp Med 26: 459-516.Reisinger V, et al. (2006). Proteomics 6:suppl2:6-15Tannu NS and Hemby SE (2006). Nat Protocols 1(4):1732-42Cambios inducidos por el virus del moteado suave del pimiento (PMMoV) en el proteoma cloroplastídico de Nicotiana benthamianaPineda, M.1,2, Sajnani, C.1,2 y Barón, M.1,*1: Estación Experimental del Zaidín, CSIC. C/ Profesor Albareda, 1. C.P. 18008. Granada (España). 2: Estos autores han contribuido por igual y éstán colocados en orden alfabético. IntroducciónEl uso de la técnicas proteómicas para el aná-lisis de la respuesta de las plantas a cambios en su entorno está mereciendo un interés creciente (ver revisiones Rossignol et al., 2006; Jorrín et al., 2007). El cloroplasto ha resultado ser un efi caz sensor am-biental y en los últimos años se han registrado nota-bles avances en el conocimiento de su proteoma (ver PPDB, http://ppdb.tc.cornell.edu/subproteome.aspx; van Wijk 2004). Análisis proteómicos recientes han destacado además el papel de este orgánulo en la defensa de la planta frente al ataque por patógenos y en las rutas de señalización que éstos desencadenan (Jones et al. 2006; Zhou et al. 2006). En ensayos previos con plantas de Nicotiana benthamiana in-fectadas con PMMoV hemos detectado cambios en el proteoma cloroplastídico de la planta huésped que afectaban al sistema de oxidación/fotolisis del agua (Pérez-Bueno et al., 2004); en el presente trabajo, hemos profundizado en el impacto causado por la infección viral en la expresión proteica de este or-gánulo. Material y métodosEl cultivo de plantas, así como la infección ex-perimental, se realizaron según Pérez-Bueno et al., (2004) y las preparaciones cloroplastídicas se ais-laron de plantas control e infectadas (Reche et al., 1997) a los 14 días post-inoculación. Los cambios en el proteoma cloroplastídico de N. benthamia-na durante la infección con la cepa española de PMMoV (PMMoV-S) fueron analizados mediante electroforesis bidimensional  (rangos de pH 4-7 y 6-11; Pineda, 2007), seguida de espectrometría de masas (MALDI-TOF/TOF MS) y búsqueda en bases de datos.Resultados y conclusionesLa técnica de electroforesis bidimensional permitió la obtención de mapas proteicos de clo-roplastos de plantas control, en los que se distin-guieron  al menos 200 manchas, de las cuales un 75% aparecieron en el rango de pH 4-7. Se consi-guieron identifi car un total de 72 polipéptidos, 55 

Tomato chromoplast proteome: challenges and perspectives

Tomato chromoplast proteome: challenges and perspectives

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