In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its 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 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species 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 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) 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 d post-inoculation) 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 to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII 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 generation of reactive oxygen species 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 describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H₂O₂ contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H₂O₂ levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H₂O₂ levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4–6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.