RESUMO
Reactive oxygen species (ROS) participate in signaling events that regulate ion channel activity and gene expression. However, excess ROS exert adverse effects that stem from their interaction with macromolecules. Thus, the assessment of the effects of salinity on ROS changes are central to understanding how plants respond and cope with this stress. ROS determination in salt-stressed plants poses specific challenges. On the one hand, salinity comprises osmotic and ion-specific effects which may, in turn, have different effects on ROS production. On the other hand, changes in ROS production may happen when tissues from salinized plants are subject to water potential (Ψ) changes when incubated in non-isosmotic solutions. This chapter provides detailed accounts of methods for ROS detection in tissues from salt-stressed plants and includes suggestions for avoiding artifacts when dealing with such tissues.
Assuntos
Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Salinidade , Tolerância ao Sal/fisiologia , Estresse Fisiológico , Citoplasma/metabolismo , Peróxido de Hidrogênio/metabolismo , Íons/metabolismo , Plântula/química , Plântula/metabolismo , Cloreto de Sódio/química , EspectrofotometriaRESUMO
The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.