ИСТИНА

The role of H2O2 in sexual plant reproduction is poorly understood. Endogenous ROS production is crucial for pollen germination and tube growth; H2O2 is also accumulated in pistil tissues awaiting pollination. These facts point towards the possible signaling role of hydrogen peroxide during pollen germination in vivo. We have made a first step to test this hypothesis by performing experiments with tobacco pollen protoplasts in vitro. We have observed a rapid increase in [Ca2+]cyt after H2O2 addition (10 µM), measured by Fluo-3 staining. Ca2+-channel inhibitor nifedipine (100 µM) abolished this effect, indicating that Ca2+ influx is carried by plasmalemmal Ca2+-channels. Since these channels can be voltage-gated, we assumed that H2O2-induced Ca2+ influx can be associated with membrane potential shifts. Therefore, we have used voltage-sensitive dye Di-4-ANEPPS to record membrane potential dynamics during H2O2 treatment. We have observed membrane hyperpolarization occurring at the same time as [Ca2+]cyt increase. These data was confirmed by measurement of absolute membrane potential values using the DiBAC4(3) staining: it has also revealed H2O2-induced hyperpolarization in a large protoplast population. The two observed membrane effects could cause various delayed physiological changes. One of the key processes determining pollen tube growth rate is cell wall assembly. We have found that H2O2–treated protoplasts were able to regenerate cell wall significantly faster than the control ones. Nifedipine removed this effect, indicating that elevation of [Ca2+]cyt is crucial for H2O2–mediated acceleration of pollen wall synthesis. Thus, rapid and delayed effects of H2O2 revealed in this study point towards the possible role of ROS in exogenous signal transduction in growing pollen tubes. The key targets for H2O2 are plasmalemmal ion-transport systems, which in turn activate the cell wall assembly. This study was supported by Russian Foundation for Basic Research (project 14-04-31431).