No abstract

ROS are known to be accumulated in stigmas of different species and can possibly perform different functions important for plant reproduction. Here we tested the assumption that one of their functions is to control membrane potential and provoke synthesis of unique proteins in germinating pollen. We used spectrofluorometry and spectrophotometry to detect H2O2 in stigma exudate, quantitative fluorescent microscopy of pollen tubes and flow cytometry of pollen protoplasts to reveal effects on membrane potential, and a label‐free quantification approach to study pollen proteome changes after H2O2 treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H2O2. This effect is abolished by catalase treatment and the ROS quencher, MnTMPP. Inhibitory analysis indicates probable participation of Ca2+‐ and K+‐conducting channels in the observed hyperpolarization. For a deeper understanding of pollen response, we analysed proteome alterations in H2O2‐treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Observed hyperpolarization and proteome alterations agree well with previously reported stimulation of pollen germination by H2O2 and sensitivity of Ca2+‐ and K+‐conducting channels to this ROS. Thus, H2O2 is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen.

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 H 2 O 2 and O • 2 ¯ in short and long pollen tubes. We found apical accumulation of H 2 O 2 and H 2 O 2 -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 along with H 2 O 2 . Serious structural disturbances were observed upon exposure to OH• and H 2 O 2 and O • 2 ¯ quencher MnTMPP: pollen tube growth slowed down and ballooned tips formed in both cases, but in the presence of OH• membrane transport and organelle distribution was affected as well. OH•, thus, can be considered as a negative influence on pollen tubes which, presumably, have mechanisms for leveling it. The assumption was confirmed by EPR spectroscopy: pollen tubes actively reduce OH• content in the incubation medium.Recent studies of pollen tube growth have been attributed to internal and external signals affecting cytoplasm structural and functional as well as non-uniformly distributed ion currents. A number of data indicate the great importance of redox-regulation: ROS are produced on stigmas of various plants species during the pollination period

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.

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 H 2 O 2 and O • 2¯ in short and long pollen tubes. We found apical accumulation of H 2 O 2 and H 2 O 2 -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 along with H 2 O 2 . Serious structural disturbances were observed upon exposure to OH• and H 2 O 2 and O • 2¯ quencher MnTMPP: pollen tube growth slowed down and ballooned tips formed in both cases, but in the presence of OH• membrane transport and organelle distribution was affected as well. OH•, thus, can be considered as a negative influence on pollen tubes which, presumably, have mechanisms for leveling it. The assumption was confirmed by EPR spectroscopy: pollen tubes actively reduce OH• content in the incubation medium.