The role of reactive oxygen species in pollen germination in Picea pungens (blue spruce)

Endogenous ROS, including those produced by NADPH oxidase, are required for spruce pollen germination and regulate membrane potential in pollen tubes; $${\mathbf{O}}_{{\mathbf{2}}}^{{{\mathbf{ \cdot - }}}}$$ and H 2 O 2 are unevenly distributed along the tube. Recently, the key role of reactive oxygen species (ROS) in plant reproduction has been decisively demonstrated for angiosperms. This paper is dedicated to the involvement of ROS in pollen germination of gymnosperms, which remained largely unknown. We found that ROS are secreted from pollen grains of blue spruce during the early stage of activation. The localization of different ROS in pollen tube initials and pollen tubes demonstrated the accumulation of H2O2 in pollen tube apex. Colocalization with mitochondria-derived $${\text{O}}_{2}^{ \cdot - }$$ showed that H2O2 is produced in mitochondria and amyloplasts in addition to its apical gradient in the cytosol. The necessity of intracellular ROS and, particularly, $${\text{O}}_{2}^{ \cdot - }$$ for pollen germination was demonstrated using different antioxidants. ·OH and extracellular ROS, on the contrary, were found to be not necessary for germination. Exogenous hydrogen peroxide did not affect the germination efficiency but accelerated pollen tube growth in a concentration-dependent manner. The optical measurements of membrane potential showed that in spruce pollen tubes there is a gradient which is controlled by H+-ATPase, potassium- and calcium-permeable channels, anion channels and ROS, as demonstrated by inhibitory analysis. An important role of NADPH oxidase in the regulation of ROS balance in particular, and in germination in general, has been demonstrated by inhibiting the enzyme, which leads to the reduction in ROS release, depolarization of pollen tube plasma membrane, and blocking of pollen germination.