I. P. Yermakov

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (Si) and the swelling coefficient (Kcw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pHi = f (Si), the Gregors equation was employed. It was shown that the Gregors model fits well the experimental data. The total number of the cation exchange (Stcat) and the anion exchange (Stan) groups were determined in the root cell walls. The number of the functional group of each type (ΔSj) was estimated, and the corresponding values of pKaj were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferichs equation [pKaj = f (CK+)] were determined. It was found that the swelling of root cell walls changes with pH, CK+ and strongly depends on plant species. Within the experimental pH and CK+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (ΔSj, pKaj, Kcw), the equation {pKaj = f (CK+)} and the dependencies {Kcw = f (CK+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.

A new approach to the investigation on the tonogenic groups of root cell walls

Acid-base properties of wheat, lupin, pea root cell walls were investigated. The roots of etiolated and green plants of different age were analysed by the potentiometric method. The ion exchange capacity of root cell walls (Si) was estimated at various pH values (pHi 2 to pHi 12) and constant ion strength of the solution (10 mM). To analyse polysigmoid curves pHi =f (Si), Gregor’s equation was used. It was shown that Gregor’s model fits fairly well the experimental data. The total quantities of cation-exchange (St cat) and anion-exchange (St an) groups were determined in the root cell walls. It was shown that the quantity of anion exchange groups is varied through a small range (60–185 µmol/g dry wt.) in plant species tested, and that the St cat differs widely from 550 to 1300 µmol/g dry wt. For leguininous plants the quantity of acidic groups (fixed anions) is nearly twice as large as that for cereals. It was found that in seedlings as well as in plants, there are 3 cation-exchange groups and one anion-exchange group in root cell walls. The quantity of functional groups of each type (Sj) was estimated, and the corresponding values of nj and pKa j were calculated. It can be assumed that the groups with the pKa 1 ≈ 3.2 are amine groups, the ones with PKa 2 ≈ 5 are groups of galacturonic acid, the ones with pKa ≈ 7.5 are the carboxyl groups of the second species, and the ones with pKa 4 ≈ 40 are the phenolic groups. The values of dissociation constants (pKa j) and Sj indicate that the root cell walls of wheat, lupin and pea are identical in qualitative structure of ionogenic groups but vary in the quantity of each ionogenic group. It was demonstrated that the summarized quantity of carboxyl groups (S2 + S3) should be connected directly with the pH gradient in the extracellar space at the membrane surface. The gradient arises from ion-exchange reactions between cations of an outer medium and protons of the ionized carboxyl groups of the cell walls. The results suggest that, St cat and St an allow the quantitative estimation of ion exchange properties of the cell walls. The resulting parameters (Sj, pKa j and nj) allow prediction of changes in an ionic composition of a medium that bathes the cell membrane, during the first step of mineral nutrition uptake.

Generation of Reactive Oxygen Species During Pollen Grain Germination

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.

Ion Exchange Properties of the Root Cell Walls Isolated from the Halophyte Plants (Suaeda altissima L.) Grown Under Conditions of Different Salinity

Ion exchange properties of root cell walls of 50- to 60-day-old plants of Suaeda altissima L., grown in nutrient medium in the presence of different NaCl concentration (0.3, 250, 750 mM) were investigated. For all growth conditions, the ion exchange capacity of the cell walls was estimated at various pH values and at different salt concentrations. The total amount of cation and anion exchange groups in the cell walls was determined. It is shown that four ionogenic groups in the polymeric structure of the halophyte cell walls are always observed independently of growth conditions: three are cation exchangeable, and one exchanges anions. The amount of the functional groups of each type is estimated, and the corresponding values of pKaj are calculated. It is shown that acidic properties are enhanced for all types of cation exchangeable groups by increasing electrolyte concentration (NaCl) in the nutrient solution. At the level of ‘optimal salinity’ (250 mM NaCl) the amount of polygalacturonic acid groups in cell walls is approx. 1.5 times greater than for the plants grown at either low (0.3 mM NaCl) or high salinity (750 mM NaCl). Ion exchange capacities of the root cell walls of the halophytes grown in different salt concentration are discussed in terms of S.altissima adaptation to salt stress.

Ni2+ effects on Nicotiana tabacum L. pollen germination and pollen tube growth

To investigate the mechanisms of Ni2+ effects on initiation and maintenance of polar cell growth, we used a well-studied model system—germination of angiosperm pollen grains. In liquid medium tobacco pollen grain forms a long tube, where the growth is restricted to the very tip. Ni2+ did not prevent the formation of pollen tube initials, but inhibited their subsequent growth with IC50xa0=xa0550xa0μM. 1xa0mM Ni2+ completely blocked the polar growth, but all pollen grains remained viable, their respiration was slightly affected and ROS production did not increase. Addition of Ni2+ after the onset of germination had a bidirectional effect on the tubes development: there was a considerable amount of extra-long tubes, which appeared to be rapidly growing, but the growth of many tubes was impaired. Studying the localization of possible targets of Ni2+ influence, we found that they may occur both in the wall and in the cytoplasm, as confirmed by specific staining. Ni2+ disturbed the segregation of transport vesicles in the tips of these tubes and significantly reduced the relative content of calcium in the aperture area of pollen grains, as measured by X-ray microanalysis. These factors are considered being critical for normal polar cell growth. Ni2+ also causes the deposition of callose in the tips of the tube initials and the pollen tubes that had stopped their growth. We can assume that Ni2+-induced disruption of calcium homeostasis can lead to vesicle traffic impairment and abnormal callose deposition and, consequently, block the polar growth.

Membrane potential changes during pollen germination and tube growth

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.

Are the carboxyl groups of pectin polymers the only metal-binding sites in plant cell walls?

Background and AimsAre the carboxyl groups of pectin polymers the only metal-binding sites in plant cell walls? To answer this question we investigated Cu2+ and Ni2+ -exchange capacities of isolated root cell walls of seven plant species. As the ionization degree of carboxyl groups and consequently their ability to bind metal ions is dependent upon the solution pH it is possible to reveal the predominant sites of metal binding at a certain pH.MethodsAmounts of different ionogenic groups in the isolated cell walls was determined by potentiometric titration, Cu2+ and Ni2+ binding capacities – by measuring the amount of adsorbed metal ions after incubation in 1u2009mM CuCl2 or NiCl2 at different pHs.ResultsCarboxyl groups of polygalacturonic acid (PGA) are the only Cu2+ and Ni2+ binding sites at pHu2009<u20095. At рНu2009≥u20095 the ratio between the amount of the metal bound and the amount of PGA carboxyl groups is >1 in some plant species, indicating the involvement of carboxyl groups of hydroxycinnamic acids (HCA) in Cu2+ and Ni2+ binding. HCA carboxyl groups in cell walls of grasses bind 40–85xa0% of total cell wall Cu2+ and Ni2+ at рНu2009≥u20095, but in dicotyledons their contribution does not exceed 45xa0% and varies widely between species.ConclusionCarboxyl groups of both polygalacturonic and hydroxycinnamic acids provide Cu2+ and Ni2+ adsorption sites in root cell walls. Their relative content depends on plant species, and ability to bind Cu2+ and Ni2+ – on the solution pH.

Reactive oxygen species are involved in regulation of pollen wall cytomechanics

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.

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.

Transmembrane transport of K+ and Cl− during pollen grain activation in vivo and in vitro

We studied the possibility of K+ and Cl− efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl− efflux; potassium channel inhibitor TEA (tetraethylammonium chloride) partially reduced K+ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba2+ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K+ and Cl− release. An important role in these processes is played by NPPB-, TEA- and Ba2+-sensitive plasmalemma ion channels.