The polar growth of the pollen tube is a key stage in the life cycle of seed plants, which is critical for successful sexual reproduction. One of the most important components of this process is ion transport across the cell membrane coordinated in time and space. Different classes of signal molecules, including reactive oxygen species, as has been found recently, participate in regulation of ion transmembrane transport. In this study, based on the model system of subprotoplasts isolated from pollen tubes, we showed that hydrogen peroxide can regulate two targets located on the plasma membrane: nifedipine-sensitive Ca2+ channels and ion transport, both of which control the membrane potential. The interaction of hydrogen peroxide with these targets resulted in an increase in an intracellular Ca2+ concentration and hyperpolarization of the plasma membrane. Faster regeneration of the cell wall was a consequence of elevation of the Ca2+ intracellular concentration.

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.

Cell walls were isolated from roots of six plant species to study their ion-exchange capacity for nickel ions (S Ni) at Ni2+ concentration of 10−3 M. The S Ni values varied depending on the plant species from 50 to 150 μmol Ni2+ per gram dry wt; the sorption capacity increased in a row: Poaceae < Chenopodiaceae < Fabaceae. At pH 5 the sorption capacity of cell walls for nickel ions was determined by the presence of carboxyl groups of polygalacturonic acid in the polymeric cell-wall matrix. In all cases the ion-exchange capacity of cell walls was higher at pH 8 than at pH 5, indicating that Ni2+ binds also to a carboxyl group different from that of polygalacturonic acid. Irrespective of plant species, the presence of EDTA in the solution diminished drastically the absorption capacity of cell walls for Ni2+. It is concluded that the presence of 10−3 M EDTA weakens the defense properties of cell walls. The sequestration of Ni2+ in the cell wall can be considered as an effective means of plant cell defense against elevated concentrations of nickel ions in the external medium.

Исследовали ионообменные свойства клеточной стенки кустистого лишайника Cladonia rangiferina (L.) F. H. Wigg. Для выделения клеточных стенок использовали живые части подециев, а также “молодые части”, которые представляли собой четыре верхних междоузлия подециев, и “старые части” (с 4-го по 8-е междоузлие). Получены зависимости ионообменной способности клеточной стенки от рН в диапазоне изменения рН 2–12 и постоянной ионной силе раствора 10 мМ. Установлено, что в трехмерной структуре клеточных стенок C. rangiferina содержались три типа ионогенных групп, которые определяют ионообменные свойства оболочек. Это аминогруппы с рКа 3, карбоксильные группы с рКа 7 и фенольные ОН-группы с рКа 10. Определены количество групп каждого типа и константы их ионизации и показано, что в клеточных стенках молодых частей количество аминогрупп и карбоксильных групп выше по сравнению со старыми частями подециев (в 1.5 и 2.0 раза соответственно). Установлено, что с возрастом изменялось содержание азота и доля деацетилированных аминогрупп в клеточных стенках от 34% (молодые части подециев) до 40% (старые части подециев). Показано, что у C. rangiferina N-ацетилглюкозамин и глюкозамин не являются основными мономерами полимеров клеточных стенок, так как и в слоевищах, и в изолированных из него клеточных стенках содержание общего азота составляло менее 1%.

С использованием флуоресцентных методов изучали участие Cl- в поляризации цитоплазмы пыльцевой трубки и регуляции мембранного потенциала в процессе прорастания in vitro пыльцевого зерна Nicotiana tabacum. Выход Cl- из клеток блокировали с помощью ингибитора анионных каналов (5-nitro-2-(3-phenylpropylamino)benzoic acid) или путем добавления в среду инкубации Cl-. Использовали концентрации этого ингибитора (40 мкМ) и внеклеточного Cl-, полностью подавляющие прорастание пыльцевого зерна (200 мМ Cl-) и рост трубки (100 мМ Cl-). Выявлен выход анионов из пыльцевого зерна в первые минуты гидратации, который сохранялся в присутствии 200 мМ Cl-. Ингибитор полностью блокировал этот процесс, что указывает на важную роль чувствительных к нему анионных каналов в трансмембранном переносе Cl- на начальном этапе активации. В присутствии 100 мМ Cl- происходила гиперполяризация мембраны пыльцевой трубки, однако экзогенный Cl- не влиял на компартментацию и движение органелл в трубке. Ингибитор вызывал деполяризацию плазматической мембраны в пыльцевом зерне и в трубке, нарушал полярную организацию цитоплазмы и движение органелл. Таким образом, активность чувствительных к указанному ингибитору хлоридных каналов необходима для регуляции потенциала на плазматической мембране и для поддержания функциональной компартментации цитоплазмы, которая обеспечивает полярный рост.

Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54–0.91 and total nitrogen in the range 1.0–4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.

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.

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.

Изучали образование активных форм кислорода в пыльце на ранней стадии прорастания, подготавливающей образование пыльцевой трубки. В это время пыльцевое зерно гидратируется, резко увеличивает свой объем и переходит из состояния покоя к активному метаболизму. Использование флуоресцентных методов позволило обнаружить активные формы кислорода в цитоплазме и внутреннем слое оболочки пыльцевого зерна – интине. Цитоплазматические активные формы кислорода обнаружены, главным образом, в митохондриях, а внеклеточные – в апертурных зонах интины, а также в растворе, окружающем пыльцевые зерна in vitro. Содержание внеклеточных активных форм кислорода снижалось под действием супероксиддисмутазы (100 ед/мл) и дифенилениодониума (100 мкМ), что указывает на НАДФ·Н-оксидазу в качестве одного из возможных их продуцентов. В условиях подавления продукции внеклеточных активных форм кислорода (100 мкМ дифенилениодониума) или усиленной их ликвидации (при добавлении 10–100 мкМ аскорбиновой кислоты) число проросших пыльцевых зерен возрастало. Этот эффект исчезал при дальнейшем увеличении концентрации указанных реагентов. Результаты свидетельствуют о значимости процессов генерации/ликвидации внеклеточных активных форм кислорода для прорастания пыльцевого зерна.

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.

The diffusion within the polymeric matrix of the cell wall is a process that determines the possibility and the rate of ion penetration into cell. In this work, we quantitatively determined the diffusion of Methylene Blue cation within the cell walls isolated from the crustose lichen Cladonia rangiferina (L.) F. H. Wigg. and its possible contribution to the processes of absorption into the lichen thalli. The swelling coefficient of the cell wall matrix and the diffusion coefficient of the organic cation within the cell walls were determined. Our results show that lichen cell walls are characterized by a higher crosslinking degree and a smaller diffusion coefficient than plants.

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.