Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

The identification of a pollen-specific LEA-like protein in Lilium longiflorum

A novel pollen‐specific LEA‐like protein, LP28, was detected in Lilium longiflorum using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N‐terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen‐specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.     

The influence of relative humidity on the swelling of pollen grains in vitro

The volume of hydrated pollen grains of Petunia hybrida L. during swelling in germination medium increased three times. The volume of desiccated pollen grains increased only two times after transfer to the same medium. This difference in swelling ability is attributed to different rigidities of the pollen grain wall,ccaused by the different hydration states. The relationship between pollen grain swelling and germination metabolism with regard to relative humidity is discussed.Abbreviation RH relative humidity     

Uptake and metabolism of flavonols during in-vitro germination of Petunia hybrida (L.) pollen

Flavonol-deficient petunia pollen [conditionally male fertile (CMF) pollen] is unable to germinate but application of nanomolar concentrations of flavonol aglycones completely restores function (Mo et al. 1992). In this study a chemically synthesized radioactive flavonol, [4′-O-¹⁴C]kaempferide, was used as a model compound to study the metabolism of flavonols during the first few hours of pollen germination. [4′-O-¹⁴C] Kaempferide was as efficient at inducing CMF pollen germination as kaempferol and quercetin, the aglycone form of the endogenous flavonols in petunia pollen. Analysis by high-performance liquid chromatography (HPLC) of extracts from both in-vitro-germinated pollen and the germination medium showed that more than 95% of the applied radioactivity was recovered as three kaempferide 3-O-glycosides and unmetabolized kaempferide; no flavonol catabolites were detected. Only HPLC fractions that contained the aglycone, or produced it upon acid hydrolysis, could induce CMF pollen germination in vitro. Structurally diverse flavonols could be classified according to how efficiently the aglycone was internalized and glycosylated during pollen germination. The ability of an individual flavonol to restore germination correlated with the total uptake of flavonols but not with the amount of glycoside formed in the pollen. Thus this study reinforces the conclusion that flavonol aglycones are the active compound for inducing pollen germination. Received: 4 November 1996/Accepted: 4 December 1996     

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.     

Staining for viability testing,germination and maturation of Sambucus nigra L. pollen in vitro

Freshly released pollen of black elderberry (Sambucus nigra L.) was incubated under various culture conditions until germination was achieved. Optimal conditions for germination were determined and used for maturation of unicellular microspores in vitro. Staining with 5-diphenyltetrazolium bromide, propidium iodide and iodine potassium iodide was used to assess pollen viability, nuclear phase and maturation, respectively. The germination rate was highest when fresh pollen was agitated at 40 rpm in Petri dishes containing a liquid medium consisting of Brewbaker and Kwack salts, 15% (w/v) sucrose, 500 mg/l MES sodium salt, at pH 5.0; germination reached nearly 70% after only 1 h in culture. Under these conditions, and with addition of 200 mg/l glutamine, 260 mg/l cytidine and 500 mg/l uridine, uninucleate microspores developed into mature pollen at a 12% germination rate. Our report is the first demonstration of maturation of S. nigra microspores in vitro.     

The interrelationship between the accumulation of lipids,protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.To whom correspondence should be addressed     

The Germination of Vinca rosea L. Pollen Grains and the Growth of the Pollen Tubes in vitro

The effect of medium concentration, pollen grain concentration, pH of the media, light and temperature on the germination of Vin ca rosea pollen grains, and the growth of their pollen tubes in vitro have been studied. The pollen grains germinate best at a sucrose concentration between 14.2% and 30%; when the pollen grain concentration exceeds 800 per 0.0234 ml; at near neutral pH (6.5); in darkness and at a temperature close to 30°. Moreover buffering ions affect the growth of the pollen tubes. Pollen grains remain viable in a wide range of temperatures, and the wall of the pollen grain is capable of withstanding severe osmotic imbalance. Low temperature induces spherical swellings at the tips of the pollen tubes, followed by accumulation of a hyaline plug.     

Localization of Phosphorus and Cation Reserves in Lilium longiflorum Pollen

Transmission electron microscopy of pollen from Lilium longiflorum Thunb. reveals electron-dense inclusions in storage body organelles ubiquitous in the cytosol. In ungerminated pollen, these inclusions are rounded in appearance and appressed to the inner surface of the smooth membrane of the storage body. During pollen germination, these inclusions become less rounded, smaller, and enclosed in storage bodies that have developed crenated membranes. Energy dispersive x-ray analysis reveals high levels of P, Mg, K, and Ca in the inclusions relative to other regions of the cytosol in which elemental signals can be obtained. The elemental composition and the degradation of inclusions during germination are offered as evidence for storage of phytin in these structures which are thus analogous to phytin storage globoids of seed tissues.     

In-vitro culture of tobacco pollen: gene expression and protein synthesis

Summary Immature pollen grains of Nicotiana tabacum L., cv Petit Havana were isolated at the mid-binucleate stage and cultured in vitro. During the first 66 h of in-vitro culture the pollen developed the same ability to set seed and germinate as pollen matured in vivo. No fertile pollen was produced when protein synthesis was inhibited temporarily at an early stage of development. In the case of inhibition at day 2 of development a delay in the total time necessary for maturation was observed that was equal to the length of time the inhibitor was applied. Hybridization experiments with a pollen-specific cDNA probe showed that the pattern of gene expression in vitro was similar to that in vivo. However, the model system differs from natural pollen development with respect to the dehydration period, which is absent in the model system, and the synthesis of proteins. Protein synthesis of in-vitro cultured pollen differed significantly from that of pollen developing in vivo, even though pollen maturation in vitro proceeded in the same time as in vivo, and led to fully matured fertile pollen. Pollen development in vitro is thus an ideal model system for studying gene expression in relation to fertility and for experimental manipulation of microsporogenesis.     

Fructokinase and hexokinase from pollen grains of bell pepper (Capsicum annuum L.): possible role in pollen germination under conditions of high temperature and CO2 enrichment

The processes of pollen grain development and germination depend on the uptake and metabolism of pollen sugars. In pepper (Capsicum annuum L.), initial sugar metabolism includes sucrose hydrolysis by invertase and subsequent phosphorylation of glucose and fructose by hexose kinases. The main objective of this study was to investigate changes in fructokinase (EC 2.7.1.4) and hexokinase (EC.2.7.1.1) activities in pepper flowers during their development, and to study the possible roles of these enzymes in determining pollen germination capacity under high temperature and under CO(2) enrichment, previously shown to modify sugar concentrations in pepper pollen (Aloni et al., 2001 Physiologia Plantarum 112: 505-512). Fructokinase (FK) activity was predominant in pepper pollen, and increased during pollen maturation. Pollen hexokinase (HK) activity was low and did not change throughout pollen development. High-temperature treatment (day/night, 32/26 degrees C) of pepper plants reduced the percentage of pollen that germinated compared with that under normal temperatures (26/22 degrees C), and concomitantly reduced the activity of FK in mature pollen. High temperature also reduced FK and HK activity in the anther. Under high ambient CO(2) (800 micro l l(-1)) pollen FK activity was enhanced. The results suggest that pollen and anther FK may play a role in the regulation of pollen germination, possibly by providing fructose-6-phosphate for glycolysis, or through conversion to UDP-glucose (UDPG) to support the biosynthesis of cell wall material for pollen tube growth. High temperature stress and CO(2) enrichment may influence pollen germination capacity by affecting these pathways.     

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.     

The size and germinability of Scots pine pollen in different temperatures in vitro

The effect of genotype, the origin of genotype, and germination temperature on Scots pine pollen grain size, hydration rate, germinability, and tube growth was studied in vitro. The mean sizes of dry and germinated pollen grains varied among pollen genotypes in different ways, thus the hydration rate varied among genotypes. Pollen from Scots pine that originates in northern Finland hydrated more than pollen from a population in southern Finland. Germination temperature had no effect on the hydration rate. Germinability and tube growth rate of northern genotypes were higher at 20 °C than at 15 °C. Differences among southern genotypes were not significant. At 15 °C, the germinability and pollen tube growth rate of northern genotypes were lower than southern genotypes. At 20 °C, the differences were not significant. It appears that germination and growth of pollen from northern populations are enhanced at higher temperatures whereas pollen from southern populations is unaffected.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

In situ molecular identification of the Ntf4 MAPK expression sites in maturing and germinating pollen

BACKGROUND INFORMATION: MAPKs (mitogen-activated protein kinases) are involved in the transduction of different signals in eukaryotes. They regulate different processes, such as differentiation, proliferation and stress response. MAPKs act through the phosphorylation cascade, being the last element that phosphorylates the final effector of the cell response. They are activated when their threonine and tyrosine residues are phosphorylated. Ntf4, a MAPK with a molecular mass of 45 kDa, has been reported to be expressed in pollen and seeds. Biochemical studies have indicated that the expression and the activation of Ntf4 is regulated during pollen maturation, although an increase of the activation is observed when the pollen is hydrated, just at the beginning of the germination. However, nothing is known about its subcellular localization. RESULTS: In the present study, the in situ expression and subcellular localization of Ntf4 have been analysed during the tobacco pollen developmental pathway. Cryosections, freeze-substitution and cryo-embedding in Lowicryl K4M were used as processing techniques for subsequent immunofluorescence, immunogold labelling and in situ hybridization assays. During pollen maturation, Ntf4 showed an increase in expression, as demonstrated by in situ hybridization, and specific subcellular distributions. We found that the protein was expressed from mid bicellular pollen stage until the pollen was mature. In germinating pollen, the protein increased after the initiation of germination. Translocation of the protein to the nucleus was found at specific stages; the presence of Ntf4 in the nucleus was found in the last stage of the pollen maturation and in germinating pollen. Double immunofluorescence and immunogold labelling with anti-Ntf4 (AbC4) and anti-P-MAPK (phosphorylated MAPK) antibodies revealed the co-localization of both epitopes in the nucleus at late developmental stages. CONCLUSIONS: The temporal and spatial pattern of the expression sites of Ntf4 has been characterized during pollen development, indicating that Ntf4 is a 'late gene' that is upregulated during maturation and germination, with a possible role in the gametophytic function. The translocation of the Ntf4 protein from the cytoplasm to the nucleus at late pollen developmental stages, and its co-localization with the P-MAPK epitope in several nuclear sites, indicates a relationship between the Ntf4 nuclear translocation and its active state.     

Ultrastructure and Germination Percentage of Crocus biflorus Miller subsp. biflorus (Iridaceae) Pollen

Pollen of Crocus biflorus Miller subsp. biflorus from natural habitats of Tusculum (Frascati, near Rome, Italy) has been studied in order to compare its structure and physiology to pollen of other Crocus species belonging to the Crocus sativus group. Mature pollen grains are rounded, 60 μm in diameter, in-aperturate (but with surface incisions where exine is lacking). DAPI staining reveals a spindle-shaped generative nucleus which is intensely fluorescent, and vegetative nucleus which is less fluorescent, and is elongated with numerous lobes. At anthesis the pollen is bicellular, but about 2% of tricellular grains occur among the pollen grains released from the anthers as well as on both naturally or handpollinated stigmas. Pollen germination is low in vitro, but higher in vivo. The pollen tubes are of normal shape. An electron-dense surface coat is sometimes visible on the exine, which in many cases, is detached from the exine. The vegetative cytoplasm is very rich in glycolipid bodies surrounded by endoplasmic reticulum. The generative cell has a lobed cell wall and is surrounded by the vegetative nucleus.     

Visualisation of transglutaminase-mediated cross-linking activity in germinating pollen by laser confocal microscopy

Abstract

Transglutaminases (TGs) are a multigenic family of calcium-dependent protein cross-linking enzymes, which are present in animal and plant cells. We have previously reported the presence of TGs in the cytosol and, more recently, in the cell wall of Malus domestica pollen, where it may be involved in pollen germination and pollen–stylar interactions. In this report we describe a simple method for the in situ visualisation of TG activity in germinating pollen. The method is based on the incorporation, mediated by pollen TG, of a fluorescently labelled exogenous diamine substrate of TG (fluorescein-cadaverine) into endogenous pollen substrates. Following the in situ TG activity reaction, the presence of cross-linked pollen proteins was visualised in fixed specimens of germinated pollen by laser confocal microscopy. Our data indicate the presence of TG cross-linking activity mainly at the apical part of the pollen tube, in the region proximal to the grain, and in the pollen grain itself. In planta, the products of this activity may provide strength to the pollen tube migrating through the style.     

In vitro pollen maturation and successful seed production in detached spikelet cultures in wheat (Triticum aestivum L.)

Summary Two spring wheat genotypes (cv Orofen and Chinese Spring) were compared for their in vitro pollen maturation capacity in detached spikelet cultures in a defined solid medium. Under these in vitro conditions Chinese Spring produced normal trinucleate pollen in 66.8% and Orofen in only 37.5%. In both cultivars the pollen maturation process from the middle uninucleate stage took approximately 3 days longer in vitro than in vivo. The pollen maturation time depended on the microspore developmental stage at the time that the culturing started. The viability, germination capacity, and fertilizing ability of the in vitro matured pollen also differed between the two genotypes. The seed set achieved in vitro (averagely 12.8%) offers promise for the practical application of this method for producing controlled or selected offspring.     

Hermodactylus tuberosus L. (Iridaceae) pollen organisation before and after anther dehiscence

ABSTRACT

The morphology, cytology and viability of Hermodactylus tuberosus L. (Iridaceae) pollen were examined from the first mitosis until maturation and after anther opening. During maturation, the pollen coat becomes modified, and the vegetative cell cytoplasm accumulates several types of reserve substances. In the vegetative cell cytoplasm, starch is quickly utilised whereas lipid inclusions of different dimensions, shape and composition occur during pollen maturation. Pollen from opened anthers have a thin pollen coat; the cytoplasm has mostly lipid reserves, and many small vesicles and vacuoles. It is similar in size or larger than pollen located inside the anther, and its viability does not decrease until one day after anther dehiscence. Large osmiophilic bodies, different from those of the vegetative cell cytoplasm, are present in the generative cell cytoplasm starting from the first stage of pollen development. The poorly developed pollen coat in pollen from opened anthers suggests that it plays a minor role in attracting insects for pollination. The size and structural and ultrastructural features of mature pollen indicate that it does not undergo dehydration and possesses sufficient vigour for immediate germination.