A multidisciplinary approach to the study of the plasma membrane of Zea mays pollen during controlled dehydration

A multidisciplinary approach (freeze-fracture, nuclear magnetic resonance, differential scanning calorimetry, isoelectric focusing and fluorochromatic reaction test) has been used to follow the behaviour of Zea mays pollen during dehydration - and to estimate its quality. At anthesis, the water content of maize pollen is 57–58% and the vegetative plasma membrane is continous and well structured with a very low density of intramembraneous particles on the extraplasmic fracture face. Maize pollen grains can withstand the drying process until a water content of 28% is reached, at which point 60–80% of the individuals show a negative reaction in the fluorochromatic test. At this water content, there is no more crystallizable water and thus metabolism decreases, leading to oxidative damage and the formation of gelphase microdomains in the plasma membrane. Consequently, the plasma-membrane permeability is modified. At 15–13% water content, all pollen grains show a negative fluorochromatic reaction, and gel-phase microdomains are more numerous but membranes still have a bilayer structure. Relaxation-time experiments indicate the occurrence of water replacement at the membrane level. Thus, sugar may stabilize the membrane structure at water contents as low as 3%. During the dehydration process, pollen walls act as elastic structures and remain closely applied to the protoplast. The combination of wall deformation and water replacement would permit pollen survival until oxidative damage occurs in the dehydrated grain.Abbreviations EF extraplasmic fracture face - FCR fluorochromatic reaction - IMP intramembraneous particle - NMR nuclear magnetic resonance - PF protoplasmic fracture face - T₂ relaxation time     

Quality of a stress-sensitive Cucurbita pepo L. pollen

The quality of Cucurbita pepo L. pollen was studied using field pollinations and the fluorochromatic-reaction test. The extreme sensitivity of this pollen to dehydration and ageing is demonstrated. Controlled stress applied to mature pollen leads to the development of seedless fruits. Molecular signals seem to be involved in the induction of this parthenocarpy. These results indicate the existence of distinct sequences involved in the completion of the fertilization program of pollen. With pollen altered by stress, the fertilization process may be stopped at different stages of its completion. We bring evidence that Cucurbita pepo plants have developed special adaptations in order to compensate for the poor viability of their pollen.Abbreviation FCR fluorochromatic reaction     

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a long (approx. 30 m) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (S_ua) is linked by plasmodesmata with the S_vn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The S_ua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.Abbreviations S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus 1=Russell and Cass (1981)     

Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts

Summary Growth of pollen tubes ofNicotiana tabacum W 38 in a defined liquid medium buffered at pH 5.9 and containing sucrose, amino-acids, boric acid, salts and an antibacterial agent was stimulated by the addition of poly(ethylene glycol) 6000 (PEG-6000) and Cu_(II) salts. In the absence of both these supplements, up to 50% of the hydrated pollen grains did not develop further, and the germinated tubes were slow-growing and abnormal, with thickened walls, kinked growth, and fragile, swollen tips containing granular cytoplasm. Addition of 10–15% (w/v) purified PEG-6000 increased germination to 80–90% and prevented the progressive bursting of pollen grains and tube tips, but growth was still slow and kinked and tips remained swollen. Addition of 30 M CuSO₄ did not stimulate germination or prevent tip bursting, but produced straight-growing tubes with smooth-sided tips resembling the tips of tubes growing through stylar tissue; the free Cu²⁺ concentration under these conditions was about 1.0 M due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 M added CuSO₄ when NH₄Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 M CuSO₄, long-term (48 h) growth of straight pollen tubes with smooth-sided tips, thin walls and long ladders of callose plugs was observed; growth occurred at 250 m/h, approximately 30–40% of the rate observed in the style. Although omission of CuSO₄ from this complete medium severely affected tube growth and callose plug deposition, it did not alter the timing of generative-nucleus division, and thus the different parameters associated with the second phase of pollen-tube growth can be uncoupled in culture. High levels of FeSO₄ (300 M) had a similar morphogenetic effect to CuSO₄, but addition of 300 M L-ascorbate or D-iso-ascorbate was required to prevent precipitation of Fe_(III) oxide and prolong the stimulation of pollen-tube growth; EDTA removed the morphogenetic effect of both CuSO₄ and FeSO₄. Further, an impure grade of PEG-4000 was contaminated with an organic morphogen that allowed continued slow growth of pollen tubes with smooth, straight-sided tips in the absence of added CuSO₄ or FeSO₄, with tube morphology unaffected by ascorbate or EDTA. However, the long-term morphogenetic effect of trace levels of CuSO₄ suggests that Cu_(II) salts play an important role in pollen-tube development in at least this species ofNicotiana.Abbreviations A₄₇₅ absorbance at 475 nm - DAPI 4,6-diamidino-2-phenylindole - EDTA ethylene-diamine N,N,N,N-tetraacetic acid - MES 2-(N-morpholino)-ethane sulphonic acid - OG ordinary grade of poly(ethylene glycol) - PEG poly(ethylene glycol) - SP Specially Purified for Biochemistry grade of poly(ethylene glycol)     

Protein synthesis of binucleate and trinucleate pollen and its relationship to tube emergence and growth

Under humid conditions, both bi- and trinucleate pollen species incorporate, on the average, very low amounts of leucine, e.g., 0.4 pmol min^-1mg pollen^-1. During germination in vitro, however, the two types of pollens greatly differ in their capacity for protein synthesis.Binucleate pollen species such as Typha, which are characterized by slow respiration in humid air and prolonged lag periods during germination in vitro, contain large amounts of monoribosomes at dehiscence. Polyribosomes are formed soon after the pollen is wetted in the germination medium, and a considerable incorporation of leucine is initiated after 10–15 min. More rapidly respiring, binucleate pollen showing a short lag period, such as Tradescantia, may already contain many polysomes at dehiscence and incorporate leucine within 2 min of germination. However, rapidly respiring, trinucleate Compositae pollen contains very limited amounts of ribosomal material and never attains any substantial level of incorporation. Cycloheximide completely inhibits both protein synthesis and tube emergence and growth in the slowly respiring, binucleate pollen species. The more rapidly respiring types are less dependent on protein synthesis, while germination of the phylogenetically advanced, trinucleate Compositae pollen proceeds completely independently. It is concluded that the level of phylogenetic advancement of the male gametophyte is characterized by its overall state of metabolic development at dehiscence rather than by the number of its generative cells.Abbreviations BSA bovine serum albumin - CHI cycloheximide - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N-tetraacetic acid - RH relative humidity - TCA trichloroacetic acid     

Water relations of growing pea epicotyl segments

The water relations of growing epicotyl segments of pea (Pisum sativum L.) were studied using the miniaturized pressure probe. For epidermal cells stationary turgor pressures of P=5 to 9 bar and half-times of water exchange of individual cells T _1/2=1 to 27 s were found. The volumetric clastic modulus () of epidermal cells varied from 12 to 200 bar and the hydraulic conductivity, Lp=0.2 to 2·10^-6 cm s^-1 bar^-1. For cortical cells P=5 to 11 bar, T _1/2=0.3 to 1 s, Lp=0.4 to 9·10^-5 cm s^-1 bar^-1 and =6 to 215 bar. The T _1/2 of cortical cells was extremely low and the Lp rather high as compared to other higher plant cells. The T _1/2-values of cortical cells were sometimes observed to change from short to substantially longer values (T _1/2=3 to 20 s). Both short and long pressure relaxations showed all the characteristics of non-artifactual curves. The change is apparently due to an increase in Lp and not , but the reason for the change in cell permeability to water is not known.In osmotic exchange experiments on peeled segments using solutions of different solutes, the half-time of osmotic water exchange for the whole segment was approximately 60 s. Water exchange occurred too quickly to be rate controlled by solute diffusion in the wall space. The data suggest that the short T _1/2-values in the cortical cells are the physiologically relevant ones for the intact tissue and that a considerable component of water transport occurs in the cell-to-cell pathway, although unstirred layer effects at the boundary between the segment and solution may influence the measured half-time. Using the theory of Molz and Boyer (1978, Plant Physiol. 62, 423–429), the gradient in water potential necessary to maintain the uptake of water for cell enlargement can be calculated from the measured diffusivities to be approximately 0.2 and 1 bar for growth rates of 1% h^-1 and 5% h^-1, respectively. Thus, although the T _1/2-values are short and Lp rather high, there may be a significant osmotic disequilibrium in the most rapidly growing tissue and as a consequence the influence of water transport on the growth rate cannot be excluded.Abbreviations P turgor pressure - T _1/2 half-time of water exchange of individual cell - Lp hydraulic conductivity - volumetric elastic modulus - t _1/2 average half-time of water exchange of tissue     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

Barley spikelet culture: An effective tool for the analysis of biochemical events in flower development

Immature detached barley spikelets were cultured in wheat spikelet medium. Fertility of cultured barley spikelets was similar to that of cultured wheat spikelets. Barley anther development within cultured spikelets was retarded relative to in planta, but viability of developing pollen, as determined by a fluorochromatic reaction, was similar in vitro and in planta. Protein synthesis by anthers in developing barley spikelets in vitro was maximal during stage 2, when pollen grains were bicellular, and declined as pollen matured. Barley spikelet culture is an effective tool for the analysis of biochemical events in flower development.Abbreviations FRC fluorochromatic reaction - WSM wheat spikelet medium     

Responses of tobacco pollen to high humidity and heat stress: viability and germinability in vitro and in vivo

Summary Responses of pollen grains of Nicotiana tabacum to high humidity (95% RH, 4 h) and temperature (38°/45° C, 4 h) stresses were investigated. Pollen grains were subjected to only RH or only temperature, or to both of these stresses. Their viability was assessed on the basis of the fluorochromatic reaction (FCR) test, and vigour was assessed on the basis of the time taken for in vitro germination as well as on the emergence of pollen tubes through the cut end of semi-vivo implanted styles. None of the stress conditions affected pollen viability and high RH or high temperature stress did not individually affect pollen vigour. However, pollen vigour was markedly affected when both the stresses were given together. Pollen grains subjected to high RH at 38° C took a longer time to germinate in vitro and the pollen tubes emerged later from the cut end of the semi-vivo styles; division of the generative cell was also delayed. Pollen grains subjected to high RH at 45° C failed to germinate in vitro, but did germinate on the stigma. Many pollen tubes subjected to this treatment showed abnormalities, and the growth of pollen tubes in the pistil was much slower than that observed in other treatments. Pollen samples subjected to all of the stress conditions were able to induce fruit and seed set. The implications of these results on the relationship between the FCR test and viability, and between viability and vigour, especially in stressed pollen, are discussed.     

Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 m of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.Abbreviations HPLC high-performance liquid chromatography - R_t retention time - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank the Australian Research Council for support, the N.S.W. Department of Agriculture for rice seed, Professor K. Mori and the Zen-Noh Corporation for authentic brassinosteroid samples, Dr. I. Hudson for statistical advice, Dr. A. Bacic and Ms. I. Bonig for helpful discussion, and J.M.S. thanks Professor R.B. Knox for laboratory facilities.     

Water status and associated processes mark critical stages in pollen development and functioning

Background

The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant – the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration – the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion – the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods – the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination – the pollen–stigma interaction phase.

Scope

This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability.

Conclusions

Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.     

Control of the developmental pathway of tobacco pollen in vitro

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.     

Pollen viability and pollen vigor

Summary Investigations were carried out to correlate pollen viability, assessed on the basis of a fluorochromatic reaction (FCR) test, with pollen vigor, assessed on the basis of the time taken for in vitro germination in pollen grains subjected to high humidity (>95% RH) and temperature (38 °C) or storage stress of Nicotiana tabacum, Agave sp., Tradescantia virginiana, and Iris sp. Both high RH and temperature, as well as storage stresses, affected pollen vigor before affecting pollen viability. The results are discussed in the light of available data on the viability and vigor of stressed pollen and of aged seeds. The need for consideration of pollen vigor, particularly in stored pollen, the inadequacy of the methods presently used, and some of the methods suitable to assess pollen vigor are elaborated.     

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     

Effects of pollen selection on progeny vigor in a Cucurbita pepo x C. texana hybrid

We examined the effects of pollen selection for rapid pollen-tube growth on progeny vigor. First, we crossed a wild gourd (Cucurbita texana) to a cultivated zucchini (Cucurbita pepo cv Black Beauty) to produce an F₁ and then an F₂ generation. Half of the F₁ seeds were produced by depositing small loads of C. texana pollen onto the stigmas of C. pepo. These small pollen loads were insufficient to produce a full complement of seeds and, consequently, both the fast- and the slow-growing pollen tubes were permitted to achieve fertilization. An F₂ generation was then produced by depositing small loads of F₁ pollen onto stigmas of F₁ plants. The F₂ seeds resulting from two generations of small pollen loads are termed the non-selected line because there was little or no selection for pollen-tube growth rate on these plants. The other half of the F₁ and F₂ seeds were produced by depositing large pollen loads (>10 000 pollen grains) onto stigmas and then allowing only the first 1% or so of the pollen tubes that entered the ovary to fertilize the ovules. We did this by excising the styles at the ovary at 12–15 h after pollination. The resulting F₂ seeds are termed the selected line because they were produced by two generations of selection for only the fastest growing pollen tubes. Small pollen loads from the F₂plants, both the selected and the non-selected lines, were then deposited onto stigmas of different C. pepo flowers, and the vigor of the resulting seeds was compared under greenhouse and field conditions. The results showed that the seeds fertilized by pollen from the selected line had greater vegetative vigor as seedlings and greater flower and fruit production as mature plants than the seeds fertilized by pollen from the non-selected line. This study demonstrates that selection for fast pollen-tube growth (selection on the microgametophyte) leads to a correlated increase in sporophyte (progeny) vigor.     

Anther opening, pollen biology and stigma receptivity in the long blooming species, Parietaria judaica L. (Urticaceae)

Background and aimsThis paper is about pollen ecophysiology, anther opening, pollen dispersal and the timing of the male and female phases in Parietaria judaica (Urticaceae).MethodsEcophysiological (effects of different relative humidities (RHs) and osmotic relationships) and cytological methods (stigmatic receptivity, pollen viability, histology and histochemistry) were used to determine pollen and pollination features during the long blooming period of this species.Key resultsPollen is dispersed by rapid uncurling of the filament and anther opening. The filament and anther lack cells with lignified wall thickenings, which are usually responsible for anther opening and ballistic pollen dispersal. Instead, dispersal is the result of the sudden movement of the filament. Pollen is of the partially hydrated type, i.e. with a water content greater than 30% at shedding, and readily loses water, and hence viability, at low RH. Pollen carbohydrate reserves differ with season. Starchless grains germinate quickly and are less subject to water loss. Flowers are protogynous, pollen release occurring only after complete cessation of the female phase within an inflorescence.ConclusionParietaria has partially hydrated pollen which differs from typical pollen of this type because of its reduced size and the absence of callose. Because of its low water content at the time of shedding it survives better at higher RH. Dispersal and pollination are adapted to pollen features.     

Phosphorylation of pollen proteins in relation to self-incompatibility in rye (Secale cereale L.)

The gametophytic two-locus self-incompatibility (SI) system in rye was investigated in view of a possible involvement of protein phosphorylation and Ca²⁺ as constituents of a signal transduction mechanism. Phosphorylation kinetics in pollen grains was found to be significantly different after in vitro treatment of pollen with either cross or self stigma proteins, with a pronounced phosphorylation activity in self-treated pollen grains. Loss of SI in self-compatible (SC) mutants was associated with a significantly decreased basic phosphorylation activity in untreated pollen grains as compared to SI genotypes. Separation of phosphorylated pollen proteins by SDS-PAGE reveals four major proteins in the MW range of 43–82 kDa which were differently phosphorylated in SI vs SC genotypes as well as in cross vs self-treated pollen grains. Application of different protein kinase inhibitors and the Ca²⁺ antagonists verapamil and La³⁺ to isolated stigmas resulted in an inhibition of the SI response in in vitro self-pollination. The role of protein kinases and Ca²⁺ as constituents of a putative SI-specific signal transduction mechanism is discussed.     

Viability and sunlight sensitivity of oak pollen and its implications for pollen-mediated gene flow

Pollen-mediated gene flow and the male reproductive success of wind-pollinated trees depend on the initial viability of the pollen and the changes that occur in its viability during transport in the atmosphere. The viability of Quercus robur pollen was determined before and during exposure to sunlight by in vitro germination and the fluorescein diacetate reaction (FCR) in 2002 and 2003, respectively. These experiments allowed us to calculate initial pollen viability and pollen sensitivity to sunlight. The germination test revealed a lower initial pollen viability (25–65%) than the FCR (53–92%). Following 9.5 h of irradiation the viability was reduced to 75–100% as determined by the in vitro germination test or to 40–70% as determined by the FCR. The actual values of initial pollen viability and pollen sensitivity to sunlight were used to define a range of values for modelling pollen dispersal using the mesoscale meteorological model METRAS. The deposition patterns of viable pollen varied by as much as a factor of 14 by changing the viability parameters in the range of the observed values. This suggests significant differences in male reproductive success. Variations in initial pollen viability have stronger effects on the gene-flow pattern than do variations in pollen sensitivity to sunlight. In particular, pollen distribution throughout the local environment is shaped by the initial pollen viability, while pollen sensitivity to sunlight mainly influences long-distance pollen dispersal.This revised version was published online in February 2005 with corrections to an incorrect sentence in the Results section. Under Pollen Sensitivity it should read: decreased faster when assessed by the germination test than by the FCR.     

Subcellular distribution of arabinogalactan proteins in pollen grains and tubes as revealed with a monoclonal antibody raised against stylar arabinogalactan proteins

Summary Monoclonal antibody PCBC3, raised against stylar extracts fromNicotians, alata flowers, was deduced from enzyme-linked immunosorbent assays and inhibition of immuno-gold labelling on tissue sections to bind specifically to carbohydrate epitopes on arabinogalactan proteins (AGPs) but not to other arabinose-containing cell wall polysaccharides. When pollen grains ofN. tabacum were hydrated in fixative, PCBC3 bound to vesicles in the vicinity of the endoplasmic reticulum but, when grains were hydrated for 20 min in culture medium before fixation, binding was restricted to the plasma membrane. The generative-cell plasma membrane was also labelled in grains ofLycopersicon peruvianum. In pollen tubes ofN. tabacum grown in liquid culture, the AGPs detected by PCBC3 were located in several regions, including the plasma membrane, tubular-vesicular structures (plasmalemmasomes) at and under the plasma membrane, and multilamellar bodies within vacuoles, features generally associated with endocytosis. Labelling was not evident in secretory vesicles or the plasma membrane at the pollen-tube tip. The AGPs detected with PCBC3 were also present in pollen-tube walls, near the interface between the inner, callosic layer and the outer, fibrillar, pectic layer. Pollen tubes ofN. tabacum grown in medium lacking added CuSO₄ produce a wall with an abnormally thickened fibrillar layer, and this layer was uniformly labelled with PCBC3. The disposition of wall AGPs thus changes in pollen tubes of different morphologies.Abbreviations AGP arabinogalactan protein - -L-Araf -L-arabinofuranose - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - PBS phosphate-buffered saline     

Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate