Oxygen radicals and cytoplasm zoning in growing lily pollen tubes.

KEY MESSAGE: Differential modulation of ROS content of the microenvironment (O ¯/MnTMPP/OH·) affects growth speed and morphology in lily pollen tubes. Oxygen radicals influence ionic zoning: membrane potential and pH gradients. Recently, redox-regulation of tip growth has been extensively studied, but differential sensitivity of growing cells to particular ROS and their subcellular localization is still unclear. Here, we used specific dyes to provide mapping of H2O2 and O·2¯ in short and long pollen tubes. We found apical accumulation of H2O2 and H2O2-producing organelles in the shank that were not colocalized with O·2¯-producing mitochondria. Differential modulation of ROS content of the germination medium affected both growth speed and pollen tube morphology. Oxygen radicals affected ionic zoning: membrane potential and pH gradients. OH· caused depolarization all along the tube while O·2¯ provoked hyperpolarization and cytoplasm alkalinization. O·2¯accelerated growth and reduced tube diameter, indicating that this ROS can be considered as pollen tube growth stimulator. Serious structural disturbances were observed upon exposure to OH· and ROS quencher MnTMPP: pollen tube growth slowed down and ballooned tips formed in both cases, but OH· affected membrane transport and organelle distribution as well. OH·, thus, can be considered as a negative regulator of pollen tube growth. Pollen tubes, in turn, are able to reduce OH· concentration, which was assessed by electron paramagnetic resonance spectroscopy (EPR).

Dynamics of Pollen Activation and the Role of H+-ATPase in Pollen Germination in Blue Spruce (Picea pungens).

Pollen is a highly specialized structure for sexual plant reproduction. Early stages of pollen germination require the transition from dormant state to active metabolism. In particular, an important role during this early phase of angiosperm pollen germination is played by H+-ATPase. Very little is known about pollen activation in gymnosperm species, and information on the involvement of H+-ATPase is lacking. We tracked four indicators characterizing the physiological state of pollen: membrane potential, intracellular pH, anion efflux and oxygen uptake, in order to monitor the dynamics of activation in Picea pungens. Based on pH dynamics during activation, we assumed the important role of H+-ATPase in spruce pollen germination. Indeed, germination was severely suppressed by P-type ATPase inhibitor orthovanadate. In spruce pollen tubes, a pronounced pH gradient with a maximum in the apical zone was found, which was different from the pollen tubes of flowering plants. Using orthovanadate and fusicoccin, we found that the proton pump is largely responsible for maintaining the gradient. Immunolocalization of the enzyme in pollen tubes showed that the distribution of H+-ATPase generally coincides with the shape of the pH gradient: its maximum accumulation is observed in the apical zone.

Redox-regulation of ion homeostasis in growing lily pollen tubes.

The pollen tube is characterized by cytoplasm compartmentalization typical for cells with polar growth. This concept includes "ion zoning", i.e. gradient distribution of ionic currents across the plasma membrane and free inorganic ions in the cytoplasm. One of the putative mechanisms for maintaining "ion zoning" is indicated by the sensitivity of the ion transport systems to reactive oxygen species (ROS). Here we test the possibility of redox regulation of ionic gradients and membrane potential (MP) gradient in growing pollen tubes using quantitative fluorescence microscopy. ROS quencher MnTMPP and exogenic H2O2 cause different alterations of intracellular Ca2+ gradient, pH gradient and MP gradient during short-term exposure. MnTMPP significantly shifts the gradients of Ca2+ and MP at low concentrations while high concentration cause growth alterations (ballooned tips) and cytoplasm acidification. H2O2 at 0,5 and 1 mM affects ion homeostasis as well (MP, Ca2+, pH) but doesn't decrease viability or alter shape of the tubes. Here we present original quantitative data on the interconnection between ROS and ion transport during tip growth.