Hydrogen peroxide in tobacco stigma exudate affects pollen proteome and membrane potential in pollen tubes.

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 H2 O2 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 H2 O2 treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H2 O2 . 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 H2 O2 -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 H2 O2 and sensitivity of Ca2+ - and K+ -conducting channels to this ROS. Thus, H2 O2 is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen.

Bipolar pollen germination in blue spruce (Picea pungens).

Direct growth of a pollen tube is an effective mechanism of sperm delivery characteristic for the majority of seed plants. In most cases, only one tube grows from one grain to perform the delivery function; meanwhile in Picea the appearance of two tubes from a single pollen grain is quite common during in vitro germination. Here, we describe the phenomenon of bipolar germination and test two hypotheses on its nature and possible role in gametophyte functioning. The hypothesis on "trophic" function of multiple tubes provoked by poor nutrition discussed in literature was not confirmed by in vitro growth tests; bipolar germination strongly decreased with lowering sucrose availability. The highest proportion of bipolar germination occurred in optimal conditions. We then assumed that bipolar germination occurs because turgor pressure is a non-directional force and effective systems of cell wall mechanical regulation are lacking. In hypertonic medium, bipolar germination was sufficiently lower than in isotonic medium, which was consistent with prediction of the «mechanical¼ hypothesis. Scanning electron microscopy and fluorescence microscopy analysis of pollen morphology and cell wall dynamics during both types of germination showed that the appearance of a single tube or bipolar germination depends on the extension of exine rupture. Cell wall softening by short-term ·OH treatment sufficiently decreased the percent of bipolar germination without affecting total germination efficiency. We concluded that mechanical properties of the cell wall and turgor pressure could shift the balance towards one of the germination patterns.

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts.

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.

[PLASMALEMMAL ION TRANSPORT IN POLLEN TUBES IS REGULATED BY HYDROGEN PEROXIDE].

Pollen tube growth is a key step in the life cycle of seed plants, which defines the success of sexual reproduction. One of the most important contributions to this process is made by ion transport through plasmalemma, which is tightly coordinated in time and space. Different classes of signaling molecules are involved in the regulation of transmembrane ion transport including reactive oxygen species as it has been recently demonstrated. Here, using subprotoplasts isolated from pollen tubes, we have demonstrated a connection between hydrogen peroxide, on one side, and two groups of targets on the plasma membrane, on the other side: nifedipine-sensitive Ca(2+)-permeable channels and transport systems controlling membrane potential. H2O2 interaction with these targets causes the increase in cytoplasmic Ca2+ concentration and plasmalemma hyperpolarization. One of the consequences of target modification was acceleration of cell wall regeneration.