Endogenous ROS, including those produced by NADPH oxidase, are required for spruce pollen germination and regulate membrane potential in pollen tubes; [Formula: see text] and H O 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 HO in pollen tube apex. Colocalization with mitochondria-derived [Formula: see text] showed that HO is produced in mitochondria and amyloplasts in addition to its apical gradient in the cytosol. The necessity of intracellular ROS and, particularly, [Formula: see text] 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.

No abstract

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model - protoplasts obtained from lily pollen grains at the early germination stage - to reveal the effect of H2 O2 on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS-sensitive currents on the pollen grain plasma membrane: the hyperpolarisation-activated calcium current, which is strongly enhanced by H2 O2 , and the outward potassium current, which is modestly enhanced by H2 O2 . We used low concentrations of H2 O2 that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.