Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside

Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth. They are secreted at pollen tube tips in Lilium longiflorum. Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species. In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L. longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum. With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips. Only A. cherimola showed evidence of AGPs at the pollen tube tip as does lily. The Yariv reagent causes arrest of tube growth in both A. cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species. We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion. Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium. After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site. During this recovery, esterified pectins colocalized with AGPs. Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.     

Composition of the cell walls of Nicotiana alata Link et Otto pollen tubes

Cell walls isolated from pollen of Nicotiana alata germinated in vitro contain glucose and arabinose as the predominant monosaccharides. Methylation analysis and cytochemical studies are consistent with the major polysaccharides being a (13)--D-glucan (callose) and an arabinan together with small amounts of cellulose. The cell walls contain 2.8% uronic acids. Alcian blue stains the pollen-tube walls intensely at the tip, indicating that acidic polysaccharides are concentrated in the tip. Synthetic aniline-blue fluorochrome is specific primarily for (13)--D-glucans and stains the pollen-tube walls, except at the tip. Protein (1.5%), containing hydroxyproline (2.4%), is present in the cell wall.     

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity

 A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421–1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes. Received: 23 February 1999 / Accepted: 22 April 1999     

Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h⁻¹ in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location. Received: 1 December 1998 / Accepted: 21 January 1999     

Pectin and the role of the physical properties of the cell wall in pollen tube growth of<Emphasis Type="Italic"> Solanum chacoense</Emphasis>

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

Ultrastructure of cell wall and plugs of tobacco pollen tubes after chemical extraction of polysaccharides

Tobacco pollen tubes grown in vitro and from pollinated tobacco styles were treated by chemical solvents to remove one or more of the following polysaccharides from the tube walls: pectin (ethylenediamine tetraacetic acid); hemicellulose (alkali); callose (alkali; potassium hypochlorite); cellulose (cuprammonium); and all polysaccharides with exception of cellulose (H₂O₂/glacial acetic acid). Both the inner tube wall, which we had regarded as the secondary wall, and the plugs contained, in addition to callose, microfibrils of cellulose and non-cellulosic microfibrils that had pectin-like properties. When using the expressions callosic or callose layer and callose plugs in reference to pollen tubes, one should realize that they do not imply the exclusive presence of callose in the inner tube wall layer and its localized thickenings.Extended version of a contribution (poster) presented at the International Symposium Advances in Plant Cytoembryology in Lublin, Poland, in June 1980 Dedicated to Professor J. Straub (Köln-Vogelsang) on his 70th birthday in 1981     

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.     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

Effects of anoxia on pollen tube growth and tube wall formation of Impatiens glandulifera

Summary When pollen of Impatiens glandulifera was cultured in aerated liquid medium for 1 h, 70% of the pollen grains germinated; these attained an average tube length of 1 mm. Subsequently, these aerobic growth conditions were changed to anaerobic by substituting a nitrogen inlet for the air inlet. As a result, the pollen tubes stopped elongating and burst. The ultrastructural changes which occurred upon inducing anoxia were studied with samples taken at 0 s, 45 s, and 4 min after changing the gas. Anoxia caused rapid and considerable changes in the ultrastructure of the dictyosome vesicles involved in cell wall formation. There was an increase in the osmiophyly of the vesicle content, and the presence of fibrillar material became apparent. Simultaneously, the fusion behavior of the dictyosome vesicles changed. Instead of the normal fusion of the dictyosome vesicles with the plasma membrane, there was a premature fusion of the vesicles with each other inside the cytoplasm that resulted in the formation of aggregates. Furthermore, the cell wall precursors that were excreted were not incorporated in their usual configuration into the growing cell wall. Instead of a smooth inner cell wall surface, irregular thickenings were formed.     

Sugar composition of pollen grain and pollen tube cell walls

The sugar composition of pollen grain and pollen tube cell walls was studied for Camellia japonica, C. sasanqua, C. sinensis, Tulipa gesneriana and Lilium longiflorum. In all species, the main components of pollen grain walls were arabinose, galactose, glucose and uronic acid. On the other hand, the pollen tube walls consisted mostly of glucose. The pollen tube wall of C. japonica was fractionated into hemicellulose, α-cellulose and pectic substance fractions in yields of 61, 19 and 3 %, respectively. The hemicellulose fraction was composed essentially of glucose. The sugar composition of the pollen tube wall was not influenced by the nature of exogenously supplied sugars. Rapid growth of the pollen tube seemed to correlate with the synthesis of hemicellulosic glucan.     

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     

Changes in the composition of ionogenic groups of the wall of the lily pollen grain during germination

Changes in the composition of ionogenic groups of the polymeric matrix of the cell walls of lily (Lilium longiflorum Thunb.) pollen grains were studied during its activation at the early stages of pollen germination. In the cell walls isolated from nonactivated and activated pollen grains, four types of ionogenic groups were identified: amino groups, carboxylic groups of uronic acids, phenolic OH-groups. and groups with pK_a 7–8. During the early stages of germination, ionization constants of each type groups remained unchanged, but the quantitative composition of ionogenic groups in the intine changed. In this matrix, a decrease in the content of phenolic groups and demethylated carboxylic groups of uronic acids was detected. It is supposed that, at early stages of germination, the intine loses some part of acid pectins and some phenolic compounds.     

Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.     

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     

Ultrastructural immunolocalization of periodic pectin depositions in the cell wall ofNicotiana tabacum pollen tubes

Summary The monoclonal antibody (MAb) JIM5, marking acidic pectins, was used to localize ultrastructurally pectin molecules in the pollen tube wall ofNicotiana tabacum. Longitudinal sections of LR-White embedded pollen tubes were exposed to antibody treatment; accumulations of pectins were identified by counting the density of the gold particles representing the pectin epitopes along the pollen tube wall. Significant accumulations of gold grains were marked and the distances between them were measured. In many pollen tubes a more or less regular distribution of the accumulations was observed along the tube indicating a periodical deposition of pectin. The distances between the accumulations were 4–6 m. Most of the label was found in the inner part of the outer layer of the bilayered cell wall. These findings correspond to and confirm the earlier observation by our group reporting ring-shaped periodical deposits in pollen tubes after immunofluorescence labelling with the MAb JIM5 under the confocal laser scanning microscope.Abbreviations Ab antibody - MAb monoclonal antibody     

The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth

The effects of anti-calmodulin (CaM) serum, the CaM antagonist W7-agarose, the Ca²⁺ chelator ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and exogenous pure CaM on pollen germination and tube growth of Hippeastrum rutilum Herb were studied. Pollen germination and tube growth were inhibited or completely stopped by anti-CaM serum in a dose-dependent manner, while the same amount of preimmune serum had no effect on either process. Pollen germination and tube growth were also inhibited or completely stopped by the CaM antagonist W7-agarose and the Ca²⁺ chelator EGTA. The addition of exogenous pure CaM enhanced pollen germination and tube growth, whereas the same amount of bovine serum albumin had no effect. The inhibitory effects caused by anti-CaM serum, W7-agarose and EGTA-washing could be reversed completely by the addition of exogenous pure CaM. These results indicate that extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes, and may provide a novel view for understanding the control of pollen germination and tube growth. Received: 12 December 1996 / Accepted: 15 January 1997