The Role of Reactive Oxygen Species in the In Vivo Germination and Growth of Petunia (Petunia hybrida E. Vilm.) Male Gametophyte in the Progamic Phase of Fertilization

Abstract

The potential role of reactive oxygen species (ROS) is studied in the male gametophytes of petunia (Petunia hybrida E. Vilm.) grown in vivo with a focus on its germination, growth support in the progamic stage of fertilization, and the function of the mechanism underlying S-RNase-based self-incompatibility. Exogenous treatment with H2O2 influences the in vivo germination and polar growth of pollen tubes (PTs), which manifests as the acceleration or inhibition of these processes depending on its concentration, time interval after pollination, and pollination variant. The H2O2 treatment of the stigma somewhat stimulates the PT elongation in the late stages of self-incompatible pollination (4–8 h) versus the strong PT inhibition observed during the first hour of germination. A different pattern is observable in cross-compatible pollination: the H2O2 treatment of pistils inhibits PT growth during the overall pollination at all tested concentrations. Treatment of pistils with the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) strongly inhibited the growth of PTs in both pollination variants. In addition, DCF-DA staining confirms that ROS are formed in pollen, PTs, stigma of nonpollinated pistil, and the pistil itself in all pollination variants. The PT growth during the function of the self-incompatibility mechanism is arrested at high ROS concentrations, which is presumably associated with the SI-induced programmed cell death. Our results demonstrate that ROS are a necessary component of pollen, PTs, exudate, and stigma cells and contribute to successful reproduction. This study provides a deeper insight into the ROS functions during the PT growth in an in vivo system.