Reactive oxygen species are involved in pollen tube initiation in kiwifruit

The role of reactive oxygen species (ROS) during pollen tube growth has been well established, but its involvement in the early germination stage is poorly understood. ROS production has been reported in germinating tobacco pollen, but evidence for a clear correlation between ROS and germination success remains elusive. Here, we show that ROS are involved in germination and pollen tube formation in kiwifruit. Using labelling with dihydrofluorescein diacetate (H2FDA) and nitroblue tetrazolium (NBT), endogenous ROS were detected immediately following pollen rehydration and during the lag phase preceding pollen tube emergence. Furthermore, extracellular H2O2 was found to accumulate, beginning a few minutes after pollen suspension in liquid medium. ROS production was essential for kiwifruit pollen performance, since in the presence of compounds acting as superoxide dismutase/catalase mimic (Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H,23H-porphin, Mn-TMPP) or as NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), ROS levels were reduced and pollen tube emergence was severely or completely inhibited. Moreover, ROS production was substantially decreased in the absence of calcium, and by chromium and bisphenol A, which inhibit germination in kiwifruit. Peroxidase activity was cytochemically revealed after rehydration and during germination. In parallel, superoxide dismutase enzymes, particularly the Cu/Zn-dependent subtype - which function as superoxide radical scavengers - were detected by immunoblotting and by an in-gel activity assay in kiwifruit pollen, suggesting that ROS levels may be tightly regulated. Timing of ROS appearance, early localisation at the germination aperture and strict requirement for germination clearly suggest an important role for ROS in pollen grain activation and pollen tube initiation.