M. Luisa Pérez-Bueno 1 Jaber Rahoutei 1 Carlota Sajnani 1 Isabel García-Luque 2 Matilde Barón 1 1 Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Granada, Spain 2 Department of Plant Biology, Centro de Investigaciones Biológicas CSIC, Madrid, Spain Proteomic analysis of the oxygen-evolving complex of photosystem II under biotec stress: Studies on Nicotiana benthamiana infected with tobamoviruses We have previously shown that tobamovirus infection induces an inhibition of photo- system II electron transport, disturbing the oxygen-evolving complex (OEC). In the infected plants, the OEC polypeptide pattern was modified when compared to healthy plants, the levels of the PsbP and PsbQ extrinsic proteins being lowered to different extents. In this work we have further investigated by two-dimensional polyacrylamide gel electrophoresis (2-DE) the changes on the OEC protein pattern of thylakoid mem- branes isolated from Nicotiana benthamiana Domin plants infected with the Spanish strain of pepper mild mottle virus. When the thylakoid membranes from healthy plants were analyzed for the presence of PsbO and PsbP proteins by 2-DE (pI range 4–7) and further immunoassayed by using specific-antisera against these two proteins, it was observed that four polypeptides cross-reacted with each antiserum. These data, along with the N-terminal amino acid sequence determined for the eight polypeptides, indi- cate that the N. benthamiana PsbO and PsbP proteins correspond to protein families. In the silver-stained 2-DE gels of thylakoid membranes isolated at different days post- inoculation from virus-infected plants, it was observed that the content of PsbP poly- peptides decreased dramatically with respect to those of PsbO, during the progress of the infection. Interestingly, there was a differential decrease of the different PsbP pro- teins, indicative of a distinct regulation of their expression. Keywords: Biotic stress / Chloroplast proteomic / Oxygen-evolving complex / Photosystem II / Plant virus PRO 0655 1 Introduction In the last two decades many investigations have described PSII, the photosynthetic complex of the thyla- koid membrane responsible for photosynthetic oxygen evolution, as a target of many abiotic stress conditions such as heavy metals, photoinhibition, drought, ozone, high and low temperature, etc. [1–3]. More recently some authors have shown that infection by pathogens, such as virus and fungi, disturbs the PSII photochemistry and induces photoprotective mechanisms in order to preserve the integrity of this complex in the host plant [4–16]. Thus, PSII is a target within the photosynthetic apparatus for both biotic and abiotic stress conditions. In addition, pathogen infection and some abiotic stress factors induce similar lesions on the PSII electron transport and generate identical defense response to avoid photoinhibi- tion [8]. A common response pattern against both stress situations consists of the enhancement of nonphoto- chemical-quenching related to energy dissipation before being trapped by the PSII reaction center [2]. We have previously analyzed the effect of tobamoviral infection in Nicotiana benthamiana plants. Our experi- ments have demonstrated that the infection induces a reduction of PSII electron transport originated on distur- bances on the polypeptide composition of the oxygen- evolving complex (OEC). The levels of both PsbP and PsbQ proteins from the OEC were reduced in the infected plants when compared with the levels in the healthy con- trol plants. Consequently these losses might affect the oxygen evolution rates. The infected plants also showed a reduction in the efficiency of light capture in PSII by photoprotective thermal dissipation of excess excitation energy [14, 15]. Thermoluminiscence (TL) assays for test- ing the redox cycling of the S-states in the OEC revealed Correspondence: Dr. Matilde Barón, Department of Biochemis- try, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), C/ Profesor Albareda 1, E-18008 Granada, Spain E-mail: mbaron@eez.csic.es Fax: 134-958-121011 Abbreviations: aa, amino acid; d.p.i., days postinoculation; OEC, oxygen-evolving complex; PMMoV-S, Spanish strain of pepper mild mottle virus; PSII, photosystem II; PsbO, PsbP and PsbQ, extrinsic OEC proteins of 33, 24 and 16 kDa, respectively 418 Proteomics 2004, 4, 418–425  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200300655