In this work, we use a recombinant Plum pox virus (PPV, Sharka) encoding green fluorescent protein (GFP) to study their effect on antioxidant enzymes and protein expression at the subcellular level in pea plants, cv. Alaska. PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13-15 days post-inoculation (dpi). At 15 dpi, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the GFP. In the same areas, an accumulation of reactive oxygen species (ROS) was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 dpi, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide (H2O2) levels was observed, that correlated with a decrease in the enzymatic mechanisms involved in its elimination [ascorbate peroxidase (APX) and peroxidase (POX)] in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 dpi) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level.
Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly with photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PS II, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PS II stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased ROS generation in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.
This work was supported by the projects 05571/PI/07 (Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia), and 23BIO2005-04-6444 (Comunidad Autónoma de la Región de Murcia). MJCM thanks the Spanish Ministry of Science and Education for her FPI research fellowship
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