Signalling pathways in pollen germination and tube growth

Summary. Signalling is an integral component in the establishment and maintenance of cellular identity. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among these, pollen tubes (PTs) are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca²⁺, calmodulin, phosphoinositides, protein kinases, cyclic AMP, and GTPases. These constitute a large and complex web of signalling networks that intersect at various levels such as the control of vesicle targeting and fusion and the physical state of the actin cytoskeleton. Here we discuss some of the most recent advances made in PT signal transduction cascades and their implications for our future research. For reasons of space, emphasis was given to signalling mechanisms that control PT reorientation, so naturally many other relevant works have not been cited. Correspondence and reprints: Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

Membrane potential changes during pollen germination and tube growth

Using methods of quantitative fluorescent microscopy, we studied membrane potential changes during pollen germination and in growing pollen tubes. Two voltage-sensitive dyes were used, i.e., DiBAC₄(3), to determine the mean membrane potential values in pollen grains and isolated protoplasts, and Di-4-ANEPPS, to map the membrane potential distribution on the surfaces of the pollen protoplast and pollen tube. We have shown that the activation of the tobacco pollen grain is accompanied by the hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (−108 mV) with respect to the pollen grains (−23 mV) from which they were isolated. We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H⁺-ATPase inhibitor sodium orthovanadate (1 mM) or its activator fusicoccin (1 μM), the longitudinal voltage gradient was modified, but did not disappear. Anion channel blocker NPPB (40 μM) fully discarded the gradient in pollen tubes. The obtained results indicate the hyperpolarization of the plasma membrane during pollen germination and uneven potential distribution on the pollen grain and tube surfaces. An inhibitory analysis of the distribution of the potential in the tube has revealed the involvement of the plasma membrane H⁺-ATPase and anion channels in the regulation of its value.     

Genetic variation for in vitro sesame pollen germination and tube growth

  In vitro pollen germination and tube length studies are valuable in elucidating mechanisms (germination capacity and rate, tube growth rate) possibly associated with genetic differences in male transmission. On each of two collection dates, the percentage germination and tube length of the binucleate pollen grains from five diverse sesame (Sesamum indicum L.) genotypes were determined at eight times (30, 60, 90, 120, 150, 180, 240, 300 min) after inoculation on a semisolid medium containing 10% (100 g l^-1) sucrose (C₁₂H₂₂O₁₁), 0.4% (4 g l^-1) purified agar (Fisher Lot 914409), 0.1% (1 g l^-1) calcium nitrate [Ca(NO₃)₂ ⋅ 4H₂O] and 0.01% (100 mg l^-1) boric acid (H₃BO₃). Before heating, the pH of the medium was adjusted to 7.0 with a 0.1 N potassium hydroxide (KOH) solution. Over the five genotypes, 5% germination was found 30 min after inoculation and a maximum of 37% germination 120 min after inoculation with no significant changes thereafter. As indicated by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which germination was initiated and maximum germination attained. Over all five genotypes, the tube length was 91 μm 30 min after inoculation, reaching a maximum of 1000 μm 300 min after inoculation. As shown by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which tube length was observed and the maximum tube length was attained. Little or no relationship between percent germination and tube length was observed among the genotypes. For both percent germination and tube length, the statistical significance of collection date and its interactions with genotype and time after inoculation indicated that environment in the form of collection date was also an influencing factor. These results indicated that genetic differences among genotypes were present for in vitro germination capacity, germination rate and tube growth rate and that these factors singly or in combination could alter male transmission of genetic elements. Received: 5 February 1997 / Accepted: 23 June 1997     

Cdi gene is required for pollen germination and tube growth in Arabidopsis

Pollen germination and tube growth are of essential importance to sexual reproduction of flowering plants. Several biological processes including cell wall biosynthesis and modification are known to be involved in pollen tube growth, though the underlying molecular mechanisms remain largely to be investigated. Here we report the identification and functional characterization of the Arabidopsis gene Cdi, which encodes a putative nucleotide-diphospho-sugar transferase. Cdi is preferentially expressed in pollen grains and pollen tubes. Transient expression of Cdi:GFP fusion protein showed that CDI is localized in the cytosol. Mutation in Cdi impaired pollen germination and pollen tube growth thus leading to disrupted male transmission. These results suggest that Cdi is an essential gene required for pollen germination and tube growth.     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains containing either theWx,wx,Su ₁,Su ₁,Sh ₂orsh ₂alleles were stored for 0, 1, 2, 3, 4 or 5 days at 2 °C. After each storage period, a portion of each genotype was cultured on a 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid medium, while another portion was placed on receptive silks, the number of kernels produced being a measure of fertilization ability. Regardless of the allele present in the pollen grain, 1 day of storage greatly increased the germination percentage and significantly increased pollen tube length. After 4 days of storage, there was noin vitro germination but some fertilization ability was found. The experiment was designed so that comparisons free from genetic background effects could be made between alleles at each locus. Significant differences at each storage period and a differential response to storage were obtained at some loci for germination percentage, ruptured percentage, pollen tube length and fertilization ability. A relationship between dominance of the allele and response to storage was detected only for fertilization ability. Since alleles at these loci affect the biochemical composition of pollen grains containing them, the results suggest that differences inin vitro germination characteristics and fertilization ability may be associated with biochemical composition.Journal Series Paper No. 3950, Florida Agricultural Experiment Station.     

Reversible protein tyrosine phosphorylation affects pollen germination and pollen tube growth via the actin cytoskeleton

Summary. Phenylarsine oxide (PAO) and genistein are two well-known specific inhibitors of tyrosine phosphatases and kinases, respectively, that have been used in the functional analysis of the status of protein phosphotyrosine in different cell types. Our experiments showed that both PAO and genistein arrested pollen germination and pollen tube growth and led to the malformation of the pollen tubes, although genistein had a lesser effect. The malformations of the pollen tubes caused by PAO and genistein were, however, quite different. In addition, it was found that the rate of pollen germination and tube growth recovered to a certain extent when phalloidin was present during PAO treatment, but not when it was present during genistein treatment. Furthermore, PAO treatment also had a great effect on the dynamic organization of filamentous actin in the pollen grain and pollen tube, while genistein only caused reorganization of actin at the turning point of the pollen tube. Our results suggest that reversible protein tyrosine phosphorylation is a crucial step in pollen germination and pollen tube growth, but that tyrosine kinases and phosphatases may have different effects which may function through the reorganization of the actin cytoskeleton. Correspondence and reprints: Key Laboratory of Cell Proliferation and Regulation Biology of the Ministry of Education, College of Life Science, Beijing Normal University, Beijing 100875, People’s Republic of China.     

Flavonols stimulate development, germination, and tube growth of tobacco pollen

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 μm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.     

Cyclic-amp control of some oxido-reductases during pine pollen germination and tube growth

Cyclic-AMP markedly increased the activities of peroxidase, malate dehydrogenase and succinate dehydrogenase but not glucose-6-phosphate dehydrogenase. Using inhibitors of protein and RNA synthesis, it was found that a part of enzyme activity increase caused by cyclic-AMP required fresh protein synthesis. The question of specificity of enzyme induction by cyclic-AMP has been examined.     

Tomato pollen respiration in relation to in vitro germination and pollen tube growth under favourable and stress-inducing temperatures

Tomato pollen germination, pollen tube growth and respiratory activity were recorded during incubation in a liquid medium for 7 h over a temperature range of 15–35°C. Although the initial rate of respiration was highest at 30°C, both at 30°C and 35°C respiration decreased after the first hour of incubation due to high temperature impairment of germination and pollen tube growth. The total per cent germination of pollen over the 7-h period was maximal at 15°C whereas pollen tube length was maximal at 25°C. Although the production of CO₂ measured at hourly intervals throughout the incubation period did not correlate to a statistically significant level with either the per cent pollen germination or the length of the pollen tubes alone, nevertheless from 2 h after the start of incubation, it closely correlated with the values for germination × pollen tube length, indicating that the respiratory activity of tomato pollen at a given time is a function of both the per cent germination and the pollen tube growth. We suggest therefore that the rate of respiration might be preferable to a simple germination test for the assessment of pollen germination ability since it expresses not only the pollen germination potential but also the growth vigour of the pollen tubes. In addition, where in vitro tests are designed to assess pollen germination–temperature interactions, they should employ a long incubation period (e.g. 7 h) to permit differences in sensitivity to temperature to be observed.     

Ion dynamics and its possible role during in vitro pollen germination and tube growth

Summary One of the most interesting aspects of plant fertilization is the growth and orientation of the pollen tube from the stigma to the ovary. Considerable research has been carried out in this field but little is yet known about the mechanisms involved in the growth process. Recent research has been focused on the regulation of molecular events in order to discover the specific genes involved in tube growth. Important results in the biochemical and physiological aspects of molecular regulation have been reported. The following review attempts to cover these aspects. It is primarily based on talks presented by the authors at the 13th International Congress on Sexual Plant Reproduction and is mainly addressed to non-experts in the fields of electrophysiology and ion signalling. We aim to present a general overview of electrical currents, ion dynamics, and ion transporters in pollen, and their possible role during pollen tube germination and growth. Together with results on other tip-growing cells, a general model of pollen tube germination and growth as a self-organizing process is proposed.     

Pollen tube growth following compatible and incompatible intraspecific pollinations in Petunia hybrida

The observation that both compatible and incompatible pollen tubes grow at identical speeds on the stigma in many plants with gametophytically controlled self-incompatibility (SI) systems has, in Petunia, been extended to cover all other facets of pollen behaviour on this tissue. On entry into the stylar transmitting tissue both types of tubes accelerate, but the compatible achieve a higher terminal velocity than do the incompatible, which eventually slow and stop. Grafting experiments show that the top 1 mm of the stylar tissue can play an important rôle in determining the future development of the pollen tube. Following mixed pollinations, proportionally too many compatible pollen tubes reach the ovary than would be expected from the results of pure compatible and incompatible pollinations indicating that incompatible pollen in some way helps prime the style for growth of compatible pollen tubes. This data is considered in terms of recent structural studies of these tissues, and related to the pollination conditions pertaining to Petunia populations in the field.Abbreviation SI self-incompatibility     

The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth

Summary The involvement of exogenous calcium ions in the regulation of pollen tube formation has been investigated in Haemanthus albiflos L. and Oenothera biennis L. by following the changes that occur in pollen germination, tube growth, and ⁴⁵⁺Ca²⁺ uptake and distribution upon application of Verapamil (an inhibitor of calcium channels), lanthanum (a Ca²⁺ substitute), and ruthenium red (believed to raise the intracellular calcium level). It was found that exogenous Ca²⁺ takes part in the formation of the calcium gradient present in germinating pollen grains and growing pollen tubes. Ca²⁺ ions enter the cells through calcium channels. Raising or reducing ⁴⁵Ca²⁺ uptake causes disturbances in the germination of the pollen grains and in the growth of the pollen tubes.     

The pollen receptor kinase LePRK2 mediates growth-promoting signals and positively regulates pollen germination and tube growth

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.     

Sodium thioglycollate enhances pollen germination and pollen tube elongation in cruciferous species

Sodium thioglycollate is a reducing agent used in microbiological growth media to enhance the growth of anerobic, microaerophilic, and facultative organisms, and in eukaryotic tissue extraction buffers to inhibit damaging oxidative reactions. Sodium thioglycollate was added to a semi-solid pollen germination medium to evaluate its effects on in vitro pollen germination and pollen tube elongation, based on the assumption that conditions within stylar tissues are less aerobic than in ambient conditions. We observed significant increases in the percent germination and pollen tube elongation of both crop and weedy mustard family species, on a medium containing 2.2 mM sodium thioglycollate. This suggests that sodium thioglycollate may be a useful amendment to semi-solid media and to solutions that are used to study pollen vigor, physiology, or gene expression, and to bioassay sensitivities of different species or genotypes to diverse physical and chemical factors.     

Proline metabolism in pollen: degradation of proline during germination and early tube growth

Proline degradation inPetunia pollen germinated in vitro was studied in cultures supplemented with [¹⁴C]proline labeled at different positions. Despite its abundance in the cell, this amino acid is only a minor substrate for respiration. Proline is partially converted via the citrate cycle into metabolites which can be traced in the ethanol-insoluble fraction of the cellular constituents. The fact that this conversion is much more extensive than the use of proline in respiration indicates that, in germinating pollen, the citrate cycle serves mainly for synthetic purposes. There are no indications that proline carbon is used for feeding of other amino-acid pools used for protein synthesis.     

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.     

Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth

As a highly reduced organism, pollen performs specialized functions to generate and carry sperm into the ovule by its polarily growing pollen tube. Yet the molecular genetic basis of these functions is poorly understood. Here, we identified 322 unique proteins, most of which were not reported previously to be in pollen, from mature pollen of Oryza sativa L. ssp japonica using a proteomic approach, 23% of them having more than one isoform. Functional classification reveals that an overrepresentation of the proteins was related to signal transduction (10%), wall remodeling and metabolism (11%), and protein synthesis, assembly and degradation (14%), as well as carbohydrate and energy metabolism (25%). Further, 11% of the identified proteins are functionally unknown and do not contain any conserved domain associated with known activities. These analyses also identified 5 novel proteins by de novo sequencing and revealed several important proteins, mainly involved in signal transduction (such as protein kinases, receptor kinase-interacting proteins, guanosine 5'-diphosphate dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPase), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). The study is the first close investigation, to our knowledge, of protein complement in mature pollen, and presents useful molecular information at the protein level to further understand the mechanisms underlying pollen germination and tube growth.