Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

Sucrose metabolism during the growth of Camellia japonica pollen

The free sugar in the mature pollen grains of Camellia japonica is almost all sucrose and the sucrose content decreases rapidly during pollen growth. The activity of soluble invertase increases during culturing and a high constant activity was found at the later stages of pollen tube growth. By contrast, the level of sucrose synthetase activity remains constant during pollen growth and that of wall-bound invertase activity is very low. Cycloheximide has little effect on the activity of these enzymes. Exogenous sucrose or glucose was simultaneously incorporated into the pollen grains when they absorbed water and swelled. The free sugar levels in growing pollen depend on the nature of the exogenous sugar. The sugar metabolism in the pollen at the stage of germination differs from that during tube growth, the latter being particularly influenced by exogenous sugar.     

Breaking strengths of pollen grain walls

50% breaking point pressures of pollen grain walls of eleven species were determined. The breaking point pressures of most pollen grain walls are equivalent to those reported in the literature for other types of living cell walls such as bacterial spore coats, algal cell walls, mold sporophyte cells, and dicot suspension culture cells. The strongest pollen grain walls are two or three orders of magnitude stronger, however. Pollen grain walls are proportionately very thick in comparison to other types of cell walls. It is this thickness, not the construction or physical properties of the pollen grain wall, that most probably accounts for their strength.     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Seed set,pollen morphology and pollen surface composition response to heat stress in field pea

Pea (Pisum sativum L.) is a major legume crop grown in a semi‐arid climate in Western Canada, where heat stress affects pollination, seed set and yield. Seed set and pod growth characteristics, along with in vitro percentage pollen germination, pollen tube growth and pollen surface composition, were measured in two pea cultivars (CDC Golden and CDC Sage) subjected to five maximum temperature regimes ranging from 24 to 36 °C. Heat stress reduced percentage pollen germination, pollen tube length, pod length, seed number per pod, and the seed–ovule ratio. Percentage pollen germination of CDC Sage was greater than CDC Golden at 36 °C. No visible morphological differences in pollen grains or the pollen surface were observed between the heat and control‐treated pea. However, pollen wall (intine) thickness increased due to heat stress. Mid‐infrared attenuated total reflectance (MIR‐ATR) spectra revealed that the chemical composition (lipid, proteins and carbohydrates) of each cultivar's pollen grains responded differently to heat stress. The lipid region of the pollen coat and exine of CDC Sage was more stable compared with CDC Golden at 36 °C. Secondary derivatives of ATR spectra indicated the presence of two lipid types, with different amounts present in pollen grains from each cultivar.     

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.     

Lipid synthesis in germinating Tradescantia pollen

Synthesis of neutral and polar lipids in pollen of Tradescantia paludosa during germination and tube growth was studied by the incorporation of acetate-[1-¹⁴C] into lipids in the presence and absence of inhibitors of RNA and protein synthesis. The proteins required for the synthesis of both neutral lipids and phospholipids are not made de novo during germination but are already present in the mature ungerminated pollen grain and they are functionally stable during the first 2 hr of pollen growth.     

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.     

Lectins of the <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> pollen grain walls stimulating in vitro pollen germination

Proteins diffusing from tobacco pollen grains into external medium, being inactivated by low temperature (0°C), were shown to stimulate pollen germination in vitro. Fractionation of these proteins by affinity chromatography using α-D-methylmannopyranoside (MMP) immobilized on agarose resulted in the isolation of lectins stimulating germination. The mol wts of these lectins were estimated by SDS-PAGE as 58, 69, and 74 kD. A stimulatory effect of these lectins was determined by their specific interaction with carbohydrate determinants because a competitive sugar (0.3 M MMP) suppressed completely lectin effect on germination. Polyvalent lectins capable of erythrocyte agglutination were also found among diffused proteins. These lectins are glycoproteins with Glu/Man carbohydrate determinants. MMP did not affect their capability of agglutination. This finding permits a conclusion that pollen grain wall contains lectins differing in their carbohydrate specificity.     

Wall architecture with high porosity is established at the tip and maintained in growing pollen tubes of Nicotiana tabacum

A major question in pollen tube growth in planta remains: do the pollen tube walls form a barrier to interaction with the environment? Using cryo‐FESEM, we directly assessed the 3D construction and porosity of tobacco pollen tube walls. Fractured mature primary walls showed a 40–50 nm spaced lattice of continuous fibers interconnected by short rods in the primary wall. These observations agree with TEM observations of sectioned walls. In the secondary callose wall, for which no structure is visible using TEM, cryo‐FESEM also revealed a 50 nm lattice consisting of longer fibers, approximately 10–15 nm wide, with rod‐like, thinner interconnections at angles of approximately 90° with the longer fibers. Such architecture may reflect functional needs with respect to porosity and mechanical strength. The wall does not form a mechanical barrier to interaction with the environment and is gained at low cost. Cryo‐FESEM additionally revealed another special feature of the wall: the tubes were tiled with scales or rings that were highly conspicuous after pectin extraction with EDTA. These rings cause the typical banding patterns of pectin that are commonly seen in pollen tubes during oscillatory growth, as confirmed by staining with toluidine blue as well as by DIC microscopy. Growth analysis by VEC‐LM showed that the ring‐ or scale‐like structures of the primary wall consist of material deposited prior to the growth pulses. The alternating band pattern seen in the callose wall is probably imposed by constrictions resulting from the rings of the primary wall.     

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Cell-wall proteins in pollen and roots of Lilium longiflorum: extraction and partial characterization

Cell-wall proteins of pollen grains, in-vitro-germinated pollen and young roots of Lilium longiflorum were studied by gel electrophoresis and amino-acid analysis. The proteins were removed from extensively purified walls by successive saline and alkali extractions. The major part including the hydroxyproline-containing proteins is covalently bound to the wall. Clear differences were observed between the proteins, especially the glycoproteins, of the pollen grain and the pollen tube. During elongation of the tubes some proteins decrease in quantity and many new proteins appear. The amount of protein in the cell walls is much lower in roots than in pollen and the root cell walls also contain fewer glycoproteins.     

Phosphatases from pollen of Brassica campestris and Lilium regale

Soluble and wall-bound acid phosphatases isolated from rape seed pollen showed similar properties except for the pH optimum curve which was elevated for the cell wall enzyme. About 50 % of the phosphatase activity of washed pollen wall preparations could be solubilized with Triton X-100, compared with only ca 20% for the corresponding preparation from lily pollen. A comparison of the wall-bound acid phosphatase of rape seed and lily pollen showed a marked difference in specificity towards fructose-6-phosphate and glucose-6-phosphate. A Mg²⁺-dependent alkaline pyrophosphatase was obtained from rape seed pollen but this activity could not be detected in cell wall preparations.     

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.     

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.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

Amorphophallus: New insights into pollen morphology and the chemical nature of the pollen wall

In pollen characters, Amorphophallus is one of the most diverse genera in the Araceae. The present work is a critical survey of contradicting reports on the impact of acetolysis treatment on Amorphophallus pollen, on the chemical nature of the outer pollen wall layer and of electron-dense (dark) granules found within it. Furthermore, we wanted to clarify the pollen polarity and to test conclusions based on different preparation techniques. Pollen morphology of 25 species is investigated by light microscopy, scanning electron microscopy and transmission electron microscopy. Our results show that Amorphophallus pollen is not resistant to acetolysis treatment. The use of different transmission electron microscopy staining methods proved the polysaccharide nature of the outer pollen wall layer and of the granules within it. Moreover, an additional thin surface layer was found in all investigated species. Microspores in early and late tetrad stages show that the less convex side of the microspore is the proximal face and the more convex side the distal face. The extrusion of pollen in strands is illustrated for the first time by light microscopy and scanning electron microscopy. Furthermore, observations of pollen in water showed that in some of the investigated species the pollen wall is shed immediately before pollen tube formation.