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.     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.     

Modeling pollen tube growth: Feeling the pressure to deliver testifiable predictions

The frequency and amplitude of oscillatory pollen tube growth can be altered by changing the osmotic value of the surrounding medium. This has motivated the proposition that the periodic change in growth velocity is caused by changes in turgor pressure. Using mathematical modeling we recently demonstrated that the oscillatory pollen tube growth does not require turgor to change but that this behavior can be explained with a mechanism that relies on changes in the mechanical properties of the cell wall which in turn are caused by temporal variations in the secretion of cell wall precursors. The model also explains why turgor and growth rate are correlated for oscillatory growth with long growth cycles while they seem uncorrelated for oscillatory growth with short growth cycles. The predictions made by the model are testifiable by experimental data and therefore represent an important step towards understanding the dynamics of the growth behavior in walled cells.     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde     

NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco

The function of the ARF-GEF family has drawn great attention recently, especially GNOM and GNL1, owing to their important role in plant development. A homolog of GBF was identified in Nicotiana tabacum, named NtGNL1, which is ubiquitously expressed throughout the tobacco life cycle. In NtGNL1 RNAi plants, irregular orientation of cell division and asynchronous cell development during early embryogenesis disrupted the symmetry of the developing embryo. In addition, root growth in transgenic lines was significantly slower than that in wild-type plants, although the structure of the root tip was largely intact. Pollen germination and pollen tube growth were also inhibited in the transgenic lines, and the tip of the pollen tube presented various aberrant morphologies in one of the transgenic lines. The phenotypes of different NtGNL1 RNAi transgenic lines suggest that the NtGNL1 is likely to be involved not only in embryogenesis and postembryonic development, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development.     

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.     

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.     

Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis

Summary The present study provides the first quantitative analysis on the distribution of organelles in pollen tubes ofNicotiana tabacum L. Organelles were studied on living pollen tubes by means of fluorescence confocal laser scanning microscopy and on cryo-fixed, freeze-substituted and serially sectioned material by electron microscopy. In the tip a 300 nm to 400 nm thick wall was secreted that proximately gradually separated into a wall with an opaque inner side and a more translucent, layered outer side. Tubular endoplasmic reticulum was particularly abundant in the tip of the tube, surrounding the region where secretory vesicles (SV) accumulated. Mitochondria were randomly distributed throughout the cytoplasm, no accumulations were present. Dictyosomes, however, showed an increased abundance at 25–30 m behind the tip. The accumulation of coated pits (CP) in a zone 6–15 m behind the tip identifies this zone as the major site of endocytosis: 50% of all CP occur in this zone. Quantification of exo- and endocytosis showed that only part of the membrane material of the SV can be retrieved after exocytosis. The typical zonation in endocytotic activity may serve to maintain a difference in membrane protein composition between the tip and the tube.     

Inhibition of RNA and protein synthesis in pollen tube development of Pinus bungeana by actinomycin D and cycloheximide

* The effects of actinomycin D and cycloheximide on RNA and protein synthesis were investigated during pollen tube development of Pinus bungeana. * RNA and protein contents, protein expression patterns, cell wall components and ultrastructural changes of pollen tubes were studied using spectrophotometry, SDS-PAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and transmission electron microscopy (TEM). * Pollen grains germinated in the presence of actinomycin D, but tube elongation and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen germination and protein synthesis, induced abnormal tube morphology, and retarded the tube growth rate. SDS-PAGE analysis showed that protein expression patterns changed distinctly, with some proteins being specific for each phase. FTIR microspectroscopy established significant changes in the chemical composition of pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of organelles involved in the secretory system. * These results suggested RNA necessary for pollen germination and early tube growth were present already in the pollen grains before germination, while the initiation of germination and the maintenance of pollen tube elongation depended on continuous protein synthesis.     

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein

Background and Aims

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation.

Methods

Two heterozygous mutant lines of arabidopsis (sia2-1+/– and qrt1 × sia2-2+/–) were investigated. sia2-2+/– was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy.

Key Results

Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary.

Conclusions

This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.     

巴旦杏授粉试验及花粉管生长的荧光显微观察

对巴旦杏4个主栽品种的授粉试验结果表明：自花授粉试验中“鹰嘴”、“双果”坐果率分别为19．74％和4．29％,“纸皮”和“麻壳”坐果率为零;自然授粉状态下4个主栽品种坐果率在9．37％－20．13％之间;人工异花授粉试验中4个品种9个授粉组合坐果率绝大多数都在40％以上,其中混合花粉授粉试验结果最好;人工养蜂条件下的坐果率明显高于无蜂自然授粉的坐果率,昆虫传粉可达到人工混合花粉授粉的效果。授粉试验还表现出巴旦杏雌蕊柱头的有效授粉时间为开花后花期的前4d,授粉越早,越有利于坐果。荧光显微观察表明,巴旦杏花粉管最早约于授粉第7天通过花柱基部到达子房室,第8天前后到达胚珠。     

Cellulose and callose of the pollen tube wall of Camellia japonica

By chemical analyses in addition to IR spectroscopy and staining reactions, cellulose and a β-1,3-glucan (callose) were demonstrated to occur in the     

Consumption of lipid granules and formation of vacuoles in the pollen tube ofTradescantia reflexa

Summary The ultrastructure in pollen tubes ofTradescantia reflexa was studied with special attention to the consumption of lipid granules and the formation of vacuoles. Electron dense, thin vesicles (EDTV) were developed from the small spherical vesicles. ER-associated lipid granules attached to both surfaces of the EDTV. Then, the EDTV became bottle-shaped, surrounding a part of cytoplasm. In the compartment surrounded by the membranes of EDTV, lipid granules were decomposed and finally disappeared. Protuberances were formed at the terminals of the expanded membranes of the EDTV and at the delimiting membranes of the compartment. These protuberances were pinched off from the membranes to form vesicles. These vesicles were transported from the site of origin to destinations in the pollen grain where they fused with each other to form vacuoles.Abbreviation EDTV electron dense, thin vesicle(s)     

Apical F‐actin‐regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall

In tip‐confined growing pollen tubes, delivery of newly synthesized cell wall materials to the rapidly expanding apical surface requires spatial organization and temporal regulation of the apical F‐actin filament and exocytosis. In this study, we demonstrate that apical F‐actin is essential for the rigidity and construction of the pollen tube cell wall by regulating exocytosis of Nicotiana tabacum pectin methylesterase (NtPPME1). Wortmannin disrupts the spatial organization of apical F‐actin in the pollen tube tip and inhibits polar targeting of NtPPME1, which subsequently alters the rigidity and pectic composition of the pollen tube cell wall, finally causing growth arrest of the pollen tube. In addition to mechanistically linking cell wall construction and apical F‐actin, wortmannin can be used as a useful tool for studying endomembrane trafficking and cytoskeletal organization in pollen tubes.     

LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.