Reactive Oxygen Species in the Elongation Zone of Maize Leaves Are Necessary for Leaf Extension

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Abstract
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 (H2O2) presence in the apoplast. 2′,7′-Dichlorofluorescein fluorescence and apoplastic H2O2 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 H2O2 but did not inhibit O⋅ 2 − production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H2O2 (50 or 500 μm) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H2O2 is necessary for leaf elongation, but it could not be distinguished whether H2O2, or other ROS, are the actual active agents.