Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H2O2 and the hydroxyl radical ((•)OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H2O2, (•)OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H, 23H-porphin, or a spin-trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, which eliminates (•)OH. The inhibiting concentrations of exogenous H2O2 and (•)OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo-osmotic shock. (•)OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H2O2, as well as partial removal of endogenous (•)OH, over-tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H2O2-inducible inter-polymer cross-linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross-links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. (•)OH is involved in local loosening of the intine in the germination pore region, while H2O2 is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.

The antioxidant properties of the polymer matrix of exine, the outer layer of pollen grain wall, were studied. The main component of this matrix is sporopollenin, a unique biopolymer resistant to mechanical and chemical damage. Samples of isolated exine purified from soluble compounds were studied with EPR using a stable nitroxyl radical TEMPO and a spin trap DMPO. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduced TEMPO and eliminated the hydroxyl radical. Also, fluorimetric analysis demonstrated that exine decomposed H2O2, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1 M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4 M NaOH, 170°C) of ether bonds, a large amount of hydroxycinnamic acids was released, and the exine almost completely lost its antioxidant capacity. The obtained results point to the ability of the extracellular polymer matrix of the exine to eliminate free radicals and H2O2 during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.

The paper is addressed to the accumulation of sporopollenin components in the microspore wall, sporopollenin polymerization dynamics and possible participation of reactive oxygen species (ROS) in this process. Fluorescent and transmission electron microscopy (TEM) was used. It has been revealed that phenylpropanoid components of sporopollenin accumulate in the microspore wall at the middle and late tetrad stages. At the late tetrad stage they completely cover the microspore surface and accumulate abundantly in aperture areas. In accordance with this, numerous thick sporopollenin lamellae emerge in aperture areas; the lamellae are electron dense and acetolysis-resistant. The exine in non-aperture areas includes acetolysis-resistant sporopollenin, as well as washout components. These particular parts of the wall are intensively stained with fluorescent dye MitoSOX Red, which detects the presence of ROS. The staining disappeared after the treatment of the microspore with superoxide dismutase, demonstrating the presence of superoxide in the exine. Superoxide easily converts to hydrogen peroxide, which can cause oxidative polymerization of sporopollenin components, forming a chemically stable biopolymer. The obtained data favor the hypothesis of ROS involvement in the formation of sporopollenin.

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 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.

The formation of reactive oxygen species in pollen at the early germination stage, which precedes the formation of the pollen tube, was studied. During this period, pollen grain is being hydrated, abruptly increasing its volume, and it passes from the resting state to active metabolism. Fluorescent methods have made it possible to reveal reactive oxygen species in the cytoplasm and inner layer of the pollen wall, intine. The cytoplasmic reactive oxygen species were mostly found in mitochondria, while extracellular ones were localized in aperture zones of intine, as well as in the solution surrounding pollen grains in vitro. The content of extracellular reactive oxygen species decreased after superoxide dismutase (100 units per ml) and diphenylene iodonium (100 µM), which indicates NADPH oxidase as one of possible producent of them. In conditions of suppression of extracellular reactive oxygen species production (100 µM diphenilene iodonium) or their promoted removal (after addition of 10 to 100 µM ascorbic acid), the number of germinating pollen grains increased. This effect disappeared after further increase in the concentration of the listed reagents. The result is evidence of the significance of processes of generation/removal of extracellular reactive oxygen species for pollen germination.

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.

Although there are quite a few papers dealing with dynamical processes in semibounded media containing a set of inhomogeneities of various nature, these process are nowadays far from being described completely. Since the stress-strain states of such mechanical systems depend on many parameters, the traditional analytic and numerical methods for their analysis are inefficient even for a small number of imperfections, and some of them cannot be applied at large vibration frequencies and in large domains. In this connection, it is of interest to study this class of problems in a new setting and develop new numerical-analytic methods for solving these problems. It is especially important to develop methods for studying the resonance properties of mechanical systems.In the present paper, we consider a set of imperfections of the simplest type, namely, plane rigid inclusions lying in planes parallel to the interfaces in a multilayered semibounded medium. In this case, attention is mainly paid to the analysis of singular sets of the determinants for the symbols of the kernels of systems of integral equations generated by the boundary value problems in the class under study.

The main trends in the analysis of dynamic problems for discontinuous media are briefly outlined. An efficient method is proposed to solve such problems for semibounded layered media. Functional matrix relations in a new form are derived for the basic dynamic characteristics of the problem

A high aluminum concentration (0.052%) in steel 12Kh1MF favors intensive decomposition of the solid solution with formation of a large quantity of carbides (VC, M23C6), additional precipitation of aluminum nitride AIN (with > 0.01% N), more uneven distribution of dispersed particles and formation of zones free of precipitates along boundaries, and also affects the dislocation arrays. This structure is unstable and leads to brittle intercrystalline fracture; the long-term strength of steel 12Kh1MF decreases considerably in this case.

Corundum and silicate stringers lower the high-temperature ductility of nickel in torsion tests. Evenly distributed corundum and globular silicon dioxide (≤0.2%) have a negligible effect on this characteristic.