Detecting ROS Production by Pollen Grains and Tubes

Reactive oxygen species (ROS) are signaling agents that actively participate in pollen germination. Pollen produces ROS both during the initial stages of germination and at later stages, including polar growth. ROS are distributed unevenly within the pollen tube, forming a gradient. Some ROS are released into the extracellular space, while others accumulate intracellularly, including in organelles. The main experimental difficulty lies in distinguishing between hydrogen peroxide, superoxide radicals, and other ROS. In this chapter, we describe methods for detecting ROS production in pollen: microscopic, spectrophotometric, and EPR-based. Fluorescence microscopy is accessible and easy to use, but it has limited accuracy and sensitivity. EPR spectroscopy is a relatively uncommon method, primarily used in physics and biophysics. However, it produces excellent results and allows for the quantification of total ROS production and superoxide radical production in liquid medium. For hydrogen peroxide detection, we recommend highly specific approaches: a non-oxidative dye PFBSF for pollen grains and tubes, and a spectrophotometric method FOX-1 for extracellular H₂O₂.