Features of ionogenic group composition in polymeric matrix of lily pollen wall

The composition of ionogenic groups and ion-exchange capacity were studied in the polymeric matrix of cell walls isolated from the pollen grain and tissues of vegetative organs (leaves and stems) of Lilium longiflorum Thunb. The ion-exchange capacity was evaluated at different pH values and ionic strength of 100 mM. In the two-layered pollen wall and the somatic cell walls four types of ionogenic groups were found: amino groups, two carboxyl groups (represented by residues of uronic and hydroxycinnamic acids), and phenolic OH-groups. The groups of all four types are present in the intine, whereas the exine contains one type of anion-exchange and two types of cation-exchange groups. The contents of each type group and their ionization constants were determined. The qualitative and quantitative compositions of structural polymers of the pollen intine and somatic cell walls are significantly different. It is suggested that hydroxycinnamic acids should be involved in cross-linking of polysaccharide chains in both the intine and somatic cell primary walls, and such cross-links play a crucial role in the structural organization and integrity of the pollen grain wall.     

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.     

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.     

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.     

Characterization of hexose kinases from camellia and lily pollen grains

Extracts from Camellia japonica, Lilium longiflorum and L. lancifolium pollen grains showed a far higher kinase activity with fructose than with glucose. Fructokinase (EC 2.7.1.4) and hexokinase (EC 2.7.1.1) preparations were obtained by partial fractionation of the extracts by DEAE-cellulose chromatography; the former was practically inactive with glucose. All had a pH optimum at 7.0–8.0 and required Mg¹⁺ ions for activity with optima at 0.5-1 m M and 1-2 m M for fructokinase and hexokinase activities, respectively. Fructokinases had K_m of 0.2-0.4 m M for fructose and similar affinities for ATP and UTP, and were inhibited by fructose above 1 m M . Hexokinases had a higher affinity for glucose than for fructose and a lower affinity for UTP compared to ATP. In camellia pollen most of hexose kinase activities were found to be due to fructokinase. These results are discussed in relation to stimulation of camellia pollen tube growth by oligosaccharides susceptible to invertase (EC 3.2.1.26).     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

Protection of pollen germination from adverse temperatures: a possible role for proline

Abstract After germination, pollen grains of Lilium longiflorum became very sensitive to short periods of heat stress as shown by the greatly reduced germination percentages upon subsequent incubation at the optimal temperature. Addition of proline to the incubation medium made pollen more resistant to heat. It was demonstrated that in a short time a large amount of proline was taken up by the cell. Germination and metabolic functions were completely or partially protected from heat damage by proline treatment. As well, it was shown that proline treatment at least partially protected pollen grains from cold stress. These results suggest that the high proline concentrations found in pollen of many species may confer resistance to germinating pollen grains at unfavourable temperatures thereby enhancing the chances of successful fertilization.     

Boric acid stimulates the plasma membrane H+-ATPase of ungerminated lily pollen grains

The stimulation of the plasma membrane (PM) H⁺-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H⁺ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H⁺ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H⁺-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (V_m) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H⁺-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on V_m may be reduced by additional stimulation of a K⁺ inward current of opposite direction to the H⁺-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H⁺-ATPase. A boron-increased PM H⁺-ATPase activity in turn may stimulate germination and growth of pollen tubes.     

Qualitative changes in protein synthesis in germinating pollen of Lilium longiflorum after a heat shock

Abstract Germination and protein synthesis of lily pollen subjected to a short heat shock after imbibition are strongly inhibited. The proteins synthesized after the heat shock were analysed by polyacrylamide gel electrophoresis. The patterns obtained from heat-treated pollen are strikingly different from those of control samples. The difference is nearly completely climinated by a high concentration of proline in the incubation medium. This proline effect correlates with the protection of pollen germination from high temperature by the amino acid.     

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.     

Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway

Summary. An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth stopped. Brefeldin A (1 μM) and cytochalasin D (1 μM), which block the production and transport of secretory vesicles, respectively, inhibited the acPase secretion. The Ca²⁺ channel blocker gadolinium (100 μM Gd³⁺) also inhibited acPase secretion and tube growth, whereas 3 mM caffeine, another Ca²⁺ uptake inhibitor, stimulated the acPase release, while tube growth was inhibited. The Yariv reagent (β-D-glucosyl)₃ Yariv phenylglycoside stopped tube growth by binding to arabinogalactan proteins of the tube tip cell wall but did not affect acPase secretion. A strong correlation between tube growth and acPase release was detected. The secreted acPase activity had a pH optimum at pH 5.5, a K _M of 0.4 mM for p-nitrophenyl phosphate, and was inhibited by zinc, molybdate, phosphate, and fluoride ions, but not by tartrate. In electrophoresis gels the main acPase activity was detected at 32 kDa. The conspicuous correlation between activity of the secretory pathway and acPase secretion during tube elongation strongly indicates an important role of the acPase during pollen tube growth and the secreted acPase activity may serve as a useful marker enzyme assay for secretory activity in pollen tubes Received July 25, 2001 Accepted January 15, 2002     

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     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

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.     

Factors influencing T-DNA transferring into pollen of lily in vitro

Direct pollen transformation method improves the classical transformation procedures because some tissue culture steps and subsequent regeneration can be avoided. A critical step in the development of Agrobacterium-mediated transformation is the establishment of optimum conditions for T-DNA delivery into tissue. The pollen grains of David lily (Lilium davidii Duchartre) are transformable by Agrobacterium during their germination, and extremely high GUS expression frequency of pollen had been achieved (92.7 ± 2.7%), but not for the ungerminated pollen. The culture medium, Agrobacterium cell density, duration of co-cultivation, and the combination of bacterial strains and plasmids should be optimized to get the highest transformation frequency. Thus, a method for pollen monocotyledonous species reproductive tissues transformation by Agrobacterium in monocots has been successfully developed. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 475–480 The text was submitted by the authors in English.     

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model – protoplasts obtained from lily pollen grains at the early germination stage – to reveal the effect of H₂O₂ on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS‐sensitive currents on the pollen grain plasma membrane: the hyperpolarisation‐activated calcium current, which is strongly enhanced by H₂O₂, and the outward potassium current, which is modestly enhanced by H₂O₂. We used low concentrations of H₂O₂ that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.     

Calcium in electron-dense globoids during pollen grain maturation in Chlorophytum elatum R.Br.

The localization, elemental composition and quantitative changes in insoluble, electron-dense globoids observed in the course of pollen grain development in Chlorophytum elatum R.Br. have been investigated. During pollen maturation, small electron-dense globoids were only found within larger electron-light vesicles in the cytoplasm of the vegetative cell. The number and diameter of the electron-dense globoids increased during pollen grain maturation, and decreased after pollen germination. By energy-dispersive X-ray microanalysis, the presence of calcium, magnesium and phosphorus was detected in these globoids. The results are discussed in the light of previous findings with regard to phytate metabolism and the role of calcium in pollen germination. Received: 28 December 1996 / Accepted: 7 May 1997     

Proteolytic activity in the maize pollen wall

A new protease from maize ( Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS-PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn₂₊. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.     

The conserved nature and taxonomic utility of pollen morphology in Cantua (Polemoniaceae)

Pollen exine morphology of nine of the ten species of Cantua (Polemoniaceae) is examined using light and scanning electron microscopy. A total of 28 specimens of C. bicolor, C. buxifolia, C. candelilla, C. cuzcoensis, C. flexuosa, C. pyrifolia, C. quercifolia, C. volcanica, and an as yet undescribed species (Cantua sp. nov.) are examined using either fresh or herbarium material. Pollen grains are found to be spheroidal, pantoporate, and quite large; mean diameter varies from 62 to 87?μm. Mean number of pores varies from 4.5 to 21.2 and mean pore size varies from 4.86 to 12.40?μm. Pollen grains of all species have insulate semitectate sexines. This feature distinguishes the pollen of Cantua species from the remainder of the Polemoniaceae. Insulae are evenly distributed over the surface of the pollen grain, with the exception of C. flexuosa and occasionally C. buxifolia, where insulae are more sparsely and haphazardly distributed. The majority of the species examined have irregularly shaped tectal insulae, with the exception of the large rounded insulae in C. quercifolia and the elongated narrow insulae in C. volcanica. Cantua quercifolia and C. volcanica have supratectal verrucae, a possible synapomorphy. In comparison to close relatives, the pollen grains of Cantua are evolutionarily conserved, and show little variation among species.     

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