Pollen and pistil in the progamic phase

The progamic phase, the period of pollen tube growth through the pistil, is a period of specific interactions between the male gametophyte and the pistil. Understanding of pollen germination and pollen tube growth are relevant for the study of pollen-pistil interactions and for understanding the function of components specifically accumulated in the transmitting tissue cell walls and intercellular matrix that may interact with pollen tubes. Received: 18 January 2001 / Accepted: 19 June 2001     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.     

The cell wall of kiwifruit pollen tubes is a target for chromium toxicity: alterations to morphology, callose pattern and arabinogalactan protein distribution

Trivalent chromium has previously been found to effectively inhibit kiwifruit pollen tube emergence and elongation in vitro . In the present study, a photometric measure of increases in tube wall production during germination showed that 25 and 50 μ m CrCl₃ treatment induced a substantial reduction in levels of polysaccharides in walls over those in controls. Moreover, chromium-treated kiwifruit pollen tubes had irregular and indented cell walls. Callose, the major tube wall polysaccharide, was deposited in an anomalous punctuate pattern. Arabinogalactan proteins (AGPs), which are integral in maintaining correct tube growth and shape in kiwifruit pollen, were found to be strongly altered in their distribution after CrCl₃ treatment compared to control tube walls. Transmission electron microscopy–immunogold analysis using four monoclonal antibodies (JIM8, JIM13, JIM14 and MAC207) revealed discontinuous AGP distribution within the treated tube walls. Such clearly discernable alterations in the molecular and morphological architecture of pollen tube walls may be detrimental in vivo for the male gametophyte to accomplish its vital role in the fertilisation process.     

Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize

Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.Key words: male gametophyte, generative cell, sperm, pollen tube, tubulin, fertilization, maize     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

Effect of temperature on the progamic phase in high-mountain plants

Progamic processes are particularly temperature-sensitive and, in lowland plants, are usually drastically reduced below 10 °C and above 30 °C. Little is known about how effectively sexual processes of mountain plants function under the large temperature fluctuations at higher altitudes. The present study examines duration and thermal thresholds for progamic processes in six common plant species (Cerastium uniflorum, Gentianella germanica, Ranunculus alpestris, R. glacialis, Saxifraga bryoides, S. caesia) from different altitudinal zones in the European Alps. Whole plants were collected from natural sites shortly before anthesis and kept in a climate chamber until further processing. Flowers with receptive stigmas were hand-pollinated with allopollen and exposed to controlled temperatures between -2 and 40 °C. Pollen performance (adhesion to the stigma, germination, tube growth, fertilisation) was quantitatively analysed, using the aniline blue fluorescence method. Pollen adhesion was possible from -2 to 40 °C. Pollen germination and tube growth occurred from around 0 to 35 °C in most species. Fertilisation was observed from 5 to 30-32 °C (0-35 °C in G. germanica). The progamic phase was shortest in G. germanica (2 h at 30 °C, 12 h at 5 °C, 24 h at 0 °C), followed by R. glacialis (first fertilisation after 2 h at 30 °C, 18 h at 5 °C). In the remaining species, first fertilisation usually occurred after 4-6 h at 30 °C and after 24-30 h at 5 °C. Thus, mountain plants show remarkably flexible pollen performance over a wide temperature range and a short progamic phase, which may be essential for successful reproduction in the stochastic high-mountain climate.     

Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins

In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.     

Gametophyte–Sporophyte Interactions in the Pollen–Pistil System: 3. Hormonal Status at the Progamic Phase of Fertilization

The content of hormones, IAA, ABA, and cytokinins, as well as the rate of ethylene production in petunia (Petunia hybrida L.) pistils and their parts (stigma, style, and ovary) were determined over 8 h after compatible pollination. At the progamic phase of fertilization in the pollen–pistil system, the phytohormones were virtually absent from the ovary but were present in various proportions in stigma and style. The stigma was the main site of ethylene synthesis and contained 90% of ABA while the style contained 80% of cytokinins of their contents in the whole pollinated pistil. Stigma and style did not differ in their IAA levels. The interaction of the male gametophyte with the stigmatic tissues was accompanied by a threefold increase in the ethylene production and a 1.5-fold increase in the IAA content in the pollen–pistil system within 0–4 h. Growth of pollen tubes in the stylar tissues (4–8 h) was accompanied by a further increase in IAA content and a decrease in the ethylene production by stigmatic tissues, as well as by a decrease in the cytokinin content in the stylar tissues. The ethylene/auxin status of the stigma may be suggested to control the processes of adhesion, hydration, and germination of pollen grains during pollination, while the auxin/cytokinin status of the style controls the pollen tube growth.     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach

Temperature is a major climatic factor that limits geographical distribution of plant species, and the reproductive phase has proven to be one of the most temperature-vulnerable stages. Here, we have used peach to evaluate the effect of temperature on some processes of the progamic phase, from pollination to the arrival of pollen tubes in the ovary. Within the range of temperatures studied, 20 degrees C in the laboratory and, on average, 5.7 degrees C in the field, the results show an accelerating effect of increasing temperature on pollen germination and pollen tube growth kinetics, as well as an increase in the number of pollen tubes that reach the style base. For the last two parameters, although the range of temperature registered in the field was much lower, the results obtained in the laboratory paralleled those obtained in the field. Increasing temperatures drastically reduced stigmatic receptivity. Reduction was sequential, with stigmas first losing the capacity to sustain pollen tube penetration to the transmitting tissue, then their capacity to offer support for pollen germination and, finally, their capacity to support pollen grain adhesion. Within a species-specific range of temperature, this apparent opposite effect of temperature on the male and female side could provide plants with the plasticity to withstand changing environmental effects, ensuring a good level of fertilization.     

Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.     

Post‐pollination process in a partially self‐compatible distylous plant,Primula sieboldii (Primulaceae)

Pollen germination and pollen‐tube growth under natural conditions were observed in a population of a distylous species, Primula sieboldii, in which partial self‐compatibility has been demonstrated in some long‐styled genets. We observed post‐pollination processes microscopically in styles collected after self‐morph and inter‐morph hand pollination (with standardized pollen load on the stigmas) in four genets each from the following three ‘genet types’: self‐incompatible long‐styled (SI), partially self‐compatible long‐styled (SC) and self‐incompatible short‐styled morph genets. Irrespective of the genet type, pollen germination began within 24 h after pollination and tubes of pollen reached to the style base with 48–96 h after inter‐morph pollination. Although pollen tubes germinated after self‐pollination in the SC genets, the number of germinated pollen tubes was significantly lower than in the case of inter‐morph pollination. Few pollen tubes germinated after self‐pollination of the SI or short‐styled genets. In SC genets, the rate of pollen‐tube growth did not differ between self‐morph and inter‐morph pollination (～1.9 mm/day). Therefore, differences in self‐compatibility between SC and SI genets in P. sieboldii are likely to be attributable to differential pollen germination rates rather than to differential pollen‐tube growth rates.     

Role of ethylene in the control of gametophyte-sporophyte interactions in the course of the progamic phase of fertilization

We investigated dynamics of the content of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene production in male gametophyte development and germination in fertile (self-compatible and selfincompatible) and sterile clones of petunia. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during the microspore development simultaneously with the degeneration of both the tapetal tissues and the middle layers of the anther wall. The second peak coincided with dehydration and maturation of pollen grains. In the anther tissues of the sterile line of petunia, tenfold higher ethylene production was observed at the meiosis stage compared with that in fertile male gametophytes. This fact correlated with the degeneration of both microsporocytes and tapetal tissues. Exogenously applied ethylene (1–100 ppm) induced a degradation of the gametophytic generation at the meiosis stage. According to the obtained data, ethylene synthesis in germinating male gametophyte is provided by a 100-fold ACC accumulation in mature pollen grains. The male gametophyte germination, both in vitro, on the culture medium, and in vivo, on the stigma surface, was accompanied by an increase in ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by various levels of ACC and ethylene release. The male gametophyte germination after self-compatible pollination was accompanied by higher content of ACC as compared with the self-incompatible clone, whereas, after the self-incompatible pollination, we observed a higher level of ethylene production compared with compatible pollination. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. Inhibitor of ethylene action, 2,5-norbornadiene (NBN), blocked both the development and germination of the male gametophyte. These results suggest that ethylene is an important factor in male gametophyte development, germination, and growth at the progamic phase of fertilization.     

A role for LORELEI,a putative glycosylphosphatidylinositol‐anchored protein,in Arabidopsis thaliana double fertilization and early seed development

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre‐5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)‐anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre‐5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre‐5/lre‐5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre‐5/lre‐5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre‐5/lre‐5 seeds. The lre‐5/lre‐5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI‐like‐GPI‐anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre‐5/lre‐5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.     

Role of Wall-bound β-Glucanases in Regulating Tip-growth of Lilium longiflorum Pollen Tubes

β-Glucanases were found in the cell wall of Lilium longiflorum Thunb. pollen tubes grown in vitro . The activity of β-glucanases was, in a certain extent, decreased by nojirimycin, an inhibitor of glucosidase. Pollen germination percentage reduced dramatically when nojirimycin was applied in the culture medium. In case that nojirimycin was added at 0 or 1 h after the onset of incubation, the inhibition rate was 99.6% and 91.4%, respectively. When 3 mmol/L of nojirimycin was applied in the liquid medium at 0, 1, 1.5 and 2 h after the onset of incubation, the growth of pollen tubes was interrupted, which resulted in the morphological change of the pollen tubes such as the newly grown portion of pollen tubes being bent, curved and swollen. Tracing the growth pattern of the individual pollen tube grown in semi-solid medium by video microscopy, the authors demonstrated that pollen tube growth rate was strongly inhibited by nojirimycin at concentrations ranged from 0.003 to 3 mmol/L. Moreover, the cytoplasmic arrangement and the morphology of the pollen tubes were also affected by nojirimycin. The growth inhibition brought about by nojirimycin was reversible. These results indicated that β-glucanases, which degrade 1,3-β-glucan and/or 1,4-β-glucan or 1,3:1,4-β-glucan constructed in the cell wall, are involved in pollen germination and pollen tube growth. It provides new insight into an understanding of the contribution of β-glucanases to the cell wall extensibility and the crucial role of cell wall in regards to the regulation of pollen tube growth.     

MARIS plays important roles in Arabidopsis pollen tube and root hair growth

In flowering plants, male gametes are delivered to female gametes for double fertilization through pollen tubes.Therefore, pollen tube growth is crucial for double fertilization. Despite its importance to sexual reproduction, genetic mechanisms of pollen tube growth remain poorly understood.In this study, we characterized the receptor-like cytoplasmic protein kinase(RLCK) gene, MARIS(MRI) that plays critical roles in pollen tube growth. MRI is preferentially expressed in pollen grains, pollen tubes and roots. Mutation in MRI by a Ds insertion led to a burst of pollen tubes after pollen germination. Pollen-rescue assay by pollen and pollen tubespecific expression of MRI in the mri-4 mutant showed that loss of MRI function also severely affected root hair elongation. MRI protein interacted with the protein kinase OXIDATIVE SIGNAL INDUCIBLE1(OXI1) in the in vitro and in vivo assays, which functions in plant defence and root hair development, and was phosphorylated by OXI1 in vitro. Our results suggest that MRI plays important roles in pollen tube growth and may function in root hair elongation through interaction with OXI1.