Roles for NO and ROS signalling in pollen germination and pollen-tube elongation in Cupressus arizonica

Roles for nitric oxide (NO) and reactive oxygen species (ROS) during pollen-tube growth have been well established in angiosperms, but there remains lack of information regarding their potential signalling roles in pollen tubes in gymnosperms. Here, the pollen-tube elongation of Arizona cypress (Cupressus arizonica Greene) was investigated. Nitric oxide, ROS, and actin were detected using their respective fluorescent probes. Both NO and ROS were observed in the nuclei of generative cells and pollen-tube cells, and in the cytoplasm in the tip region. An intracellular NO content in the pollen cells was lowered using an NO scavenger or an NO-synthase inhibitor. Similarly, an endogenous ROS content in the pollen cells was lowered using an NAD(P)H oxidase inhibitor. These treatments reduced pollen germination and pollen-tube growth, and induced severe morphological abnormalities. Inhibition of NO and ROS accumulation also severely disrupted the actin cytoskeleton in the pollen tubes. These data indicate that NO and ROS had signalling roles in pollen germination and pollen-tube formation in cypress.