The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy

Abstract The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H 2 O 2 ) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H 2 O 2 and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H 2 O 2 in leaves. In this study, the M strain of Cucumber mosaic virus (M‐CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock‐inoculated leaves, H 2 O 2 accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H 2 O 2 accumulation was always higher than in healthy leaves. Subcellular H 2 O 2 localization observed using CLSM showed that H 2 O 2 in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H 2 O 2 was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H 2 O 2 and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.