Here we give a critical analysis of the opinion of Andreev (2011) on membrane potential distribution along the pollen tube plasmalemma. He assumes that a lateral gradient of dipole potential exists, but suggests a lateral gradient of transmembrane potential impossible. We demonstrate by concrete examples that the argumentation of the initiator of discussion is based on inaccurate citation of our experimental data (Breygina et al., 2009) and incomplete analysis of previously published articles. Speaking about transmembrane potential, he doesn't consider numerous facts demonstrating the uneven distribution of transmembrane ion fluxes and ion-transport proteins in the pollen tube plasmalemma, as well as data obtained by modeling of transmembrane potential distribution in objects of different shape. In addition, the assumption on the uneven distribution of dipole potential doesn't have an experimental basis neither in studies of the pollen tube, nor in the practice of using fluorescent voltage-sensitive dyes DiBAC4(3) and Di-4-ANEPPS. We are expecting the author to obtain experimental data in support of his position.

The basic feature of polar growing cells is uneven distribution of organelles, forming distinct cytoplasmic zones. It is closely related to the cytosolic free ion gradients and transmembrane ion fluxes, which may cause uneven membrane potential distribution along the cell surface. Participation of Ca(2+). H(+) and K(+) in pollen tube growth has been proved in numerous studies Data on inorganic anions contribution to the growth process are scarce and controversial. In somatic plant cells anion channels have vital functions, including membrane voltage and turgor pressure regulation. In this chapter we give an overview of recent findings in ionic regulation of pollen germination and give evidence for the important role of anion channels in this process. The use of inhibitory analysis combined with fluorescent methods has allowed us to observe both temporal and spatial changes of membrane potential and reveal the involvement of anion channels in the regulation of this value Our data on the key role of anion channels in structural and functional compartmentalization of the polarized pollen tube cytoplasm are considered. A contribution of mitochondrial anion channels to the pollen tube growth regulation is also discussed.

The influence of anion channel blockers NPPB and DIDS on pollen tube growth and its mitochondria functioning was studied by means of fluorescence microscopy and flow cytometry. NPPB (40 μM) blocked pollen tube growth completely, but didn’t change its diameter. DIDS (20–80 μM) caused pollen tube swelling and bursting, suggesting that DIDS-sensitive channels take part in the regulation of pollen tube osmotic balance. The osmotic effect of low DIDS concentration (20 μM) wasn’t accompanied by changes in the tube growth rate. The mapping of plasma membrane potential of pollen tubes using Di-4-ANEPPS revealed the involvement of NPPB-sensitive but not DIDS-sensitive anion channels in the maintenance of the longitudinal membrane potential gradient along the tube surface. The study of isolated pollen mitochondria showed that DIDS increased their capacity to take up potential-dependent dye DiOC5(3), i.e. caused hyperpolarization of mitochondrial membranes. At the same time DIDS influenced on intramitochondrial ROS content and ROS release from mitochondria. Thus, NPPB and DIDS in different ways influenced on plasma membrane potential distribution along pollen tube, on its osmotic balance, and on mitochondria functioning. This set of data suggests that pollen tube growth is dependent on activity of anion channels that differ in localization and functions.

Changes in the composition of ionogenic groups of the polymeric matrix of the cell walls of lily ( Lilium longiflorum Thunb.) pollen grains were studied during its activation at the early stages of pollen germination. In the cell walls isolated from nonactivated and activated pollen grains, four types of ionogenic groups were identified: amino groups, carboxylic groups of uronic acids, phenolic OH-groups. and groups with pK a 7–8. During the early stages of germination, ionization constants of each type groups remained unchanged, but the quantitative composition of ionogenic groups in the intine changed. In this matrix, a decrease in the content of phenolic groups and demethylated carboxylic groups of uronic acids was detected. It is supposed that, at early stages of germination, the intine loses some part of acid pectins and some phenolic compounds.

Using methods of quantitative fluorescent microscopy, we studied membrane potential changes during pollen germination and in growing pollen tubes. Two voltage-sensitive dyes were used, i.e., DiBAC4(3), to determine the mean membrane potential values in pollen grains and isolated protoplasts, and Di-4-ANEPPS, to map the membrane potential distribution on the surfaces of the pollen protoplast and pollen tube. We have shown that the activation of the tobacco pollen grain is accompanied by the hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (−108 mV) with respect to the pollen grains (−23 mV) from which they were isolated. We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H+-ATPase inhibitor sodium orthovanadate (1 mM) or its activator fusicoccin (1 μM), the longitudinal voltage gradient was modified, but did not disappear. Anion channel blocker NPPB (40 μM) fully discarded the gradient in pollen tubes. The obtained results indicate the hyperpolarization of the plasma membrane during pollen germination and uneven potential distribution on the pollen grain and tube surfaces. An inhibitory analysis of the distribution of the potential in the tube has revealed the involvement of the plasma membrane H+-ATPase and anion channels in the regulation of its value.

The involvement of Cl− in cytoplasm polarization in the pollen tube and membrane potential control during pollen germination in vitro was studied by fluorescence techniques in Nicotiana tabacum. Cl− release from cells was blocked by the anion channel inhibitor nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or by the addition of Cl− to the incubation medium. The concentrations of the inhibitor (40 μM) and extracellular Cl− completely inhibiting pollen germination (200 mM) and pollen tube growth (100 mM) were used. The release of anions from the pollen grain has been revealed in the first minutes of hydration also in the presence of 200 mM Cl−. The inhibitor blocked this process completely, which points to the significance of the NPPB-sensitive anion channels in the transmembrane Cl− transport at the early activation stage. The pollen tube membrane was hyperpolarized in the presence of 100 mM Cl−; however, exogenous Cl− had no effect on the compartmentalization and organelle movement in the tube. The inhibitor depolarized the plasma membrane in the pollen grain and tube and affected the polar organization of the cytoplasm and organelle movement. Thus, activity of NPPB-sensitive chloride channels was required to regulate the potential on the plasma membrane and to maintain the functional compartmentalization of the cytoplasm, which provides for the polar growth.

Ion-exchange characteristics of the cell walls isolated from different zones of the foliose lichen Peltigera aphthosa (L.) Willd were determined. Four types of ionogenic groups were revealed in the thallus cell walls of P. aphthosa , namely amino groups, carboxylic groups of uronic acids, carboxylic groups of phenolic acids, and phenolic OH groups. They may participate in the ion-exchange reactions with the ions of the environment. The amount of ionogenic groups in P. aphthosa cell walls was found to depend on the zone and age of the thallus.

Proteins diffusing from tobacco pollen grains into external medium, being inactivated by low temperature (0°C), were shown to stimulate pollen germination in vitro. Fractionation of these proteins by affinity chromatography using α-D-methylmannopyranoside (MMP) immobilized on agarose resulted in the isolation of lectins stimulating germination. The mol wts of these lectins were estimated by SDS-PAGE as 58, 69, and 74 kD. A stimulatory effect of these lectins was determined by their specific interaction with carbohydrate determinants because a competitive sugar (0.3 M MMP) suppressed completely lectin effect on germination. Polyvalent lectins capable of erythrocyte agglutination were also found among diffused proteins. These lectins are glycoproteins with Glu/Man carbohydrate determinants. MMP did not affect their capability of agglutination. This finding permits a conclusion that pollen grain wall contains lectins differing in their carbohydrate specificity.

We studied the effects of short-term (5–10 min) treatment of Nicotiana tabacum L. pollen grains with concanavalin A (ConA) on their activation (changes in the membrane potential and intracellular pH) and germination in vitro. ConA (10–1000 μg/ml) induced plasma membrane hyperpolarization in the vegetative cell and enhanced pollen grain germination. These effects depended on ConA concentration and were interrelated: the value of the membrane potential was negatively correlated with the number of pollen grains germinated for 1 h of their incubation (r = –0.96). In addition, ConA (100 μg/ml) increased the intracellular pH value by 0.3 unit. All these effects of ConA are determined by its specific interaction with carbohydrate determinants because a competitive sugar methyl-α-mannopyranoside (0.1 M) completely blocked ConA effects. The data obtained presume that the specific receptors are present on the surface of pollen grains, evidently on their plasma membrane, and their interaction with lectins has a functional significance for pollen grain activation and germination.

The regulatory role of intracellular pH changes and of transmembrane Cl – transport in the activation of Nicotiana tabacum L. pollen grains at a stage preceding in vitro germination was studied. The acidification of the cytosol with propionic acid hindered the germination of pollen grains, whereas its alkalization by fusicoccin-stimulated H + -ATPase activity of plasma membranes sharply increased the germination frequency with respect to control values. The activation of pollen grains was accompanied by the Cl – efflux. The blockage of Cl – efflux with 1 mM ethacrynic acid significantly decreased the intracellular pH and fully inhibited germination. The results allow assumption that the intracellular pH rise and Cl – efflux are prerequisites for pollen grain activation.

Pollen hydration and germination on the "wet" stigma of Nicotiana tabacum L. were studied by SEM and TEM to reveal the role of the stigma in selecting the germinative pore, and in establishing the axis of polarity in the pollen grain. Pollinated stigmas were fixed with glutaraldehyde or osmium tetroxide vapour, or processed with rapid freeze fixation and freeze substitution. Fixation was performed in 5, 15 or 30 min and 3.5 h after pollination. The tube easily emerged from either pore, this process not depending on the pollen grain orientation relative to the stigma. The orientation of pollen tubes remained random till their length becomes longer than the pollen grain diameter. The TEM analysis of ultrastructural changes in poral regions during pollen hydration and germination showed that the germinative pore was positioned just near the generative cell and vegetative nucleus. Within the first 5 min after pollination a new layer of the electron-lucent wall adjacent to the plasma membrane was formed in the region of a future germinative pore. Following 15 min, marked changes were revealed in the cytoplasm region, close to the germinative pore. Minute dictyosome vesicles were accumulated near the plasma membrane. Small mitochondria and short ER cisternae were distal to a zone of secretory vesicles. The data suggest that the axis of polarity in the germinating pollen grain is predetermined by a spatial organization of the vegetative cell.