Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein a subunit (Galpha) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca(2+)](cyt)) and Ca(2+) channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Galpha null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Galpha overexpression lines (wGalpha and cGalpha) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca(2+)](cyt) was lower in gpa1-1 and gpa1-2 and higher in wGalpha and cGalpha. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca(2+)-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba(2+) > Ca(2+) > Mg(2+) > Sr(2+) > Mn(2+). The activity of the Ca(2+)-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGalpha and cGalpha. The results suggest that Galpha may participate in pollen germination through modulation of the hyperpolarization-activated Ca(2+) channel in the PM of pollen cells.     

In vitro pollen germination in Hypericum perforatum L. and Hypericum rumeliacum Boiss

In vitro pollen germination and pollen tube growth investigations are valuable tools used in identification of the effects of environmental factors and genotypic differences on pollen viability, pollen germination and tube elongation. In this study pollen viability, in vitro pollen germination capacity, abnormality ratios and tube length in germinated pollens of Hypericum perforatum L. and H. rumeliacum Boiss. were investigated. Both of these species has spheroid-shaped and tricolporate pollen grains. The diameters of Hypericum perforatum and H. rumeliacum pollens were found as 24 +/- 3 microm and 19 +/- 2 microm, respectively. Pollen viability of H. perforatum and H. rumeliacum was found as 83% and 72%, respectively. The germination percentages were found as 12.85% for H. perforatum and 64.42% for H. rumeliacurm. Tube lengths in germinated pollens of both taxa were measured approximately as 95.25 +/- 38 microm in H. perforatum and 165.92 +/- 53 microm in H. rumeliacium 4 h after inoculation. In germinated pollen grains of H. perlbratum and H. rumeliacumn abnormality percentages were determined as 13.23% and 43.97%, respectively. In germinated pollens of these two species, highly significant (P < 0.00001) differences in in vitro germination percents and abnormality percents were observed. Abnormalities such as swollen tube tip, branched tube, spiralled tube and excessive tube formation were observed in pollen tubes. The results of this study showed that there were obvious differences in pollen germinability between these two species growing under the same environmental conditions.     

Dynamic changes in the osmotic water permeability of protoplast plasma membrane

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.     

Characterization of whole-cell k<Superscript>+</Superscript> currents across the plasma membrane of pollen grain and tube protoplasts of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Outward and inward currents, mainly carried by K⁺, were detected in protoplasts of pollen grains (PG) and pollen tubes (PT) of Lilium longiflorum Thunb. by using the whole-cell configuration of the patch-clamp technique. The outward K⁺ current (I_{K+ out}) was similar in both protoplast types, while the inward K⁺ current (I_{K+ in}) was higher in pollen tube protoplasts. In PT but not in PG protoplasts, inward K⁺ currents were already detectable at negative membrane voltages usually monitored in lily pollen. I_{K+ in} consisted of a slow and a fast current component, as revealed by fitting a sum of two exponential functions to the time-dependent current. The contribution of the fast component to the total inward current was higher in PT than in PG protoplasts, which was even more evident at acidic pH of the external medium. Therefore, based on the measured characteristics, the I_{K+ in} of PT protoplasts may contribute to the endogenous K⁺ currents surrounding a growing pollen tube. Abbreviations: BS, bath solution; BTP, bis-Tris-propane; MES, 2-N-morpholinoethane sulfonic acid; V_act, activation voltage; V_M, membrane voltage; E_rev, reversal potential; I_{K+ in}, inward K⁺ current; I_{K+ out}, outward K⁺ current; PG, pollen grain; PT, pollen tube; PM, pipette medium     

Identification and characterization of stretch-activated ion channels in pollen protoplasts

Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.     

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.     

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.     

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.     

Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes

The pollen tube is an excellent single-cell model system for studying cellular processes in plant cell biology. This protocol describes a detailed step-by-step procedure with optimized conditions for introducing various fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains by means of biolistics for their transient expression and subsequent analysis in germinating pollen tubes. The whole experiment consists of four major stages: coating gold microcarriers with DNA constructs, preparation of pollen grains, transformation of plasmid DNA into pollen grains by particle delivery system and germination of bombarded pollen grains in optimized germination media to obtain pollen tubes for protein trafficking, protein localization, drug treatment and organelle dynamics analysis. This protocol takes about 4-12 h from pollen preparation to protein detection.     

Aquaporin as a membrane transporter of hydrogen peroxide in plant response to stresses

Hydrogen peroxide (H₂O₂) is a reactive oxygen species that signals between cells, and H₂O₂ signaling is essential for diverse cellular processes, including stress response, defense against pathogens, and the regulation of programmed cell death in plants. Although plasma membrane intrinsic proteins (PIPs) have been known to transport H₂O₂ across cell membranes, the permeability of each family member of PIPs toward H₂O₂ has not yet been determined in most plant species. In a recent study, we showed that certain isoforms of Arabidopsis thaliana AtPIPs, including AtPIP2;2, AtPIP2;4, AtPIP2;5, and AtPIP2;7, are permeable for H₂O₂ in yeast cells. Since the expression of PIPs is differently modulated in Arabidopsis by abiotic stress or H₂O₂ treatment, it is important to investigate the integrated regulation of aquaporin expression and their physiological significance in H₂O₂ transport and plant response to diverse abiotic stresses.     

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P₂ effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P₂-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P₂. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P₂ in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P₂ in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P₂ functions to coordinate cytoskeletal dynamics and secretion.

The apical plasma membrane of pollen tubes contains nanodomains where the regulatory phospholipid PtdIns(4,5)P₂ exerts a stabilizing effect on the actin cytoskeleton.     

Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.     

Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate

Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.