Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress—New Tools for Reactive Oxygen Species Research in Seed Biology

The intracellular homeostasis of reactive oxygen species (ROS) and especially of superoxide anion and hydrogen peroxide participate in signaling cascades which dictate developmental processes and reactions to stresses. ROS are also biological molecules that play important roles in seed dormancy and germination. Because of their rapid reactivity, short half-life and low concentration, ROS are difficult to measure directly with high accuracy and precision. In presented work tissue printing method with image analysis and dual excitation flow cytometry (FCM) were developed for rapid detection and localization of O(2)(•−) and H(2)O(2) in different part of seed. Tissue printing and FCM detection of ROS showed that germination of wild oat seeds was associated with the accumulation of O(2)(•−) and H(2)O(2) in embryo (coleorhiza, radicle and scutellum), aleurone layer and coat. To verify if printing and FCM signals were specified, the detection of O(2)(•−) and H(2)O(2) in seeds incubated in presence of O(2)(•−) generation inhibitor (DPI) or H(2)O(2) scavenger (CAT) were examined. All results were a high level of agreement among the level of ROS derived from presented procedures with the ones created from spectrophotometric measured data. In view of the data obtained, tissue printing with image analysis and FCM are recommended as a simple and fast methods, which could help researchers to detection and level determination of ROS in the external and inner parts of the seeds.