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Whither chemotropism and pollen tube guidance?
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Whither chemotropism and pollen tube guidance? W. Mary Lush Pollen tubes follow a well-defined path to deliver male gametes to female gametes, but the mechanisms they use to locate this path are poorly understood. The major hypothesis is (and long has been) that pollen tubes are guided by chemical gradients and/or physical structures. Recently, parallels have been drawn between chemical mechanisms of guidance in pollen tubes and other cells, such as axons. These comparisons highlight a problem with the current models for pollen tube guidance, namely the distance over which chemical guidance is proposed to occur. Based on this new perspective, some models are either invalid or pollen tubes are uniquely responsive to chemical guidance cues.
P
ollen grains are delivered to the receptive surface of the stigma, which is remote from the ovules (Fig. 1). The male gametes, which are not motile, reach the female gametes via a tube that grows out of the pollen grains, and into and through the female tissues of the flower until it reaches the micropyle of an ovule. What guides the growth of pollen tubes along this path? According to early hypotheses, pollen tubes are guided by a continuous gradient in a single, target-derived chemical1,2. A more common view is that chemical cues guide pollen tubes during growth on the stigma and in the ovary, but that the route through the style is determined by physical structures1. Recently, parallels have been drawn between pollen tubes, migrating cells3 and growing axons4,5. This has led to a more complex view of guidance that involves multiple cues that act in overlapping spatial and temporal frames, and include chemical attractants and repellents, and physical guidance4. Guidance of pollen tubes and other cells Ð general considerations
By definition, chemotropism is a growth response to a chemical stimulus, the direction of growth being determined by the direction of the chemical gradient in the attractant or the repellent (Fig. 2). Chemical guidance can occur only when the external environment permits propagation of the cue at concentrations that vary within the response range of pollen, and when pollen is competent to respond to the cue. There are thus many chemicals, including nutrients, that are indirectly involved in pollen tube guidance (for effects on pollen-tube growth rate see Refs 6,7). Candidates for the role of chemical guidance cue should meet the criteria routinely applied to address the general question of whether a substance (including a gene product), plays a role in a particular process8:
free, as in the examples already given, or bound to a surface, as proposed for the stylar canal3. Competent pollen tubes will respond to a chemical gradient by altering the polarity of tip growth, provided that the absolute concentration of the cue is above the threshold for detection (but below saturation), and that there is a detectable difference in concentration across the tip (Fig. 2). It is proposed that pollen tubes are guided over distances of 50–100 mm by single chemical cues3,11, whereas the experimentally determined limit of guidance in other systems is usually ,1 mm (Ref. 9). There are theoretical reasons for chemical guidance distances being short, one of which is that there is a limit to the number of cumulative increases in the concentration of the guidance cue that can occur before the responding system is saturated (Fig. 2). What, theoretically, is the maximum path length over which pollen tubes can be guided by a chemical cue? Between 1.2 and 9.3 mm is the answer generated by applying
• The substance should be present at the correct time and place, and in the correct quantities. • If the source of the substance is excised surgically, or by mutation or by down-regulation of genes, the response should cease. • The response should be restored by the substitution of the purified Stigma chemical for the natural source or Pollen by the replacement of a funcPlug tube tional copy of the gene. Pollen Sperm Style tube • The response should be reprocells ducible in isolation from other Vegetative tissues (i.e. it should act directly nucleus upon the responding system). Extending • The hypothesis is strengthened if tip Transmitting it has generality. tract Ovary • The response should be specific Ovule Trends in Plant Science to one substance or its close analogs. Fig. 1. General structure of the flower. The pistil is Where chemotropism is proposed, made up of three parts, the receptive surface or two additional constraints apply: stigma, the ovary, which houses the ovules and, • There must be an external, direcseparating the two, the style. The stigma surface tional gradient in the putative can be ‘dry’, or ‘wet’ (covered by either a hydroguidance substance. philic or hydrophobic secretion). Pollen tubes grow • The distance over which a single through the central part of the style, which can be a hollow, fluid-filled canal, or consist of parallel files chemotropic agent acts should be of longitudinally oriented cells, called transmitting no more than 10 mm for bound tract cells. The ovary can contain a single ovule or cues and less for diffusible cues. up to thousands of ovules. The male pollen grains In most working models of chemiare usually desiccated when they make contact with cal attraction, it is proposed that a the stigma. Each grain hydrates, and germinates to chemical gradient arises by the difproduce a tube that grows into the intercellular fusion of the chemical from its orispaces of the stigma and through the style (inset). 9 gin at the target tissue . An example The tip of the growing tube contains cytoplasm, two of this model in pollination biology male gametes (sperm cells), and a vegetative nuis pollen tube attraction by the secrecleus, which is thought to regulate most aspects of tube growth. As the tube elongates by depositing tions of the embryo sac10. However, new cell wall material at the tip (‘tip growth’), the gradients could arise also by the older parts of the tube are cut off by cross walls, graded expression or processing of the called plugs. Fertilization occurs when a pollen tube chemical by tissues along the pathenters an ovule at the micropyle, and the tip releases way, as proposed for a transmitting the sperm cells. tract-specific glycoprotein (TTS pro11 tein) . The guiding chemical can be
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Response system Guidance region saturated
Cue below detection level
(a)
Guidance range 5 Ð10 mm DC, d
(b)
Style
Growth stimulant
(c)
Growth attractant Trends in Plant Science
Fig. 2. Schematic models of pollen tube guidance by a chemical attractant. (a) Pollen tubes grow randomly in regions in which attractant concentrations are below the detection threshold of pollen tubes. When the tubes, by chance, enter a region with a detectable gradient in concentration, they respond by growing up the gradient and towards the source of the cue. They might eventually reach regions of high concentration in which receptors for the chemical are saturated and in which they resume random growth. Pollen tubes follow a relatively straight path through the guidance region, because if the growth axis (shown by arrows) deviates from the direction of the chemical gradient, a difference in concentration (DC) becomes detectable across the tip. The steeper the gradient or the wider the tip, the smaller the deviation that must occur before the gradient is detected. The sensing interval, indicated as d, is the minimum distance (in the direction of the cue) over which differences in concentration can be detected, and is less than or equal to the diameter of the pollen tube. In some in vitro assays of putative chemo-attractants, the growth pattern of pollen tubes emerging from a cut style have been observed10,23. If the chemical is a growth stimulant (b), the elongation rate of pollen tubes that grow by chance into effective concentrations of the chemical is accelerated, but the direction of growth is unaltered. If the chemical is an attractant (c), pollen tubes that by chance enter the guidance range of the chemical are directed towards higher concentrations of the chemical, but their rate of growth does not change.
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the rules of compounding (Table 1). Because this estimate is based on parameters of the responding system, it applies to all stable chemical gradients, regardless of their origin and means of propagation. Estimates of the maximum guidance range of bound cues for axons [,10 mm (Ref. 12)], are consistent with the above estimate. However, the guidance range of diffusible cues is likely to be much shorter because of the temporal and spatial realities of diffusion, which limit the maximum distance over which chemical morphogens influence development of animal cells to ~1 mm (Ref. 13). The growth cone of axons is slightly larger than the tip of most pollen tubes, and numerical models of axon guidance suggest that the maximum guidance range for unbound cues is probably ,2.5 mm (Table 2), which is consistent with experimental results9. The maximum guidance range increases with the increasing size of the sensor (Table 1). The type of sensor envisaged for pollen tubes (and axons), compares concentrations on either side of the tip (or growth cone), and this method of gradient sensing is called spatial sensing. However, guidance ranges for small cells, such as bacteria, would be short if cells depended on spatial sensing. Instead, motile cells sense gradients temporally by comparing the concentration at time (T) = 0 with the concentration at T = 1. The maximum effective size of temporal sensors is thus the distance the cell moves during the time between measurements. Spatial and temporal mechanisms are not mutually exclusive, and pollen tubes could also take advantage of temporal sensing. Although pollen tubes are not motile, they have features in common with migrating cells. The apical region of pollen tubes is the only living part of the tube and its growth is in some ways like that of a migrating cell, which ‘leaves a trail of extracellular matrix…behind’14. Furthermore, tip growth might have more in common with
amoeboid growth than with the turgor-driven growth usually associated with plant cells15. Guidance on the stigma
Pollen tubes emerging from grains in oily exudates on the wet stigmas of Nicotiana alata, grow from a hydrophobic environment towards and into the aqueous environment of the stigma (Fig. 3). Tube growth towards an aqueous environment is reproducible in cultures in the absence of any style-specific components16–18 (Fig. 3). In vitro guidance of pollen tubes surrounded by oil occurs with high fidelity (97% of pollen tubes grow towards the aqueous phase16), and ends when pollen tubes enter the aqueous phase. The trajectory of pollen tube growth is determined by the aperture of emergence, the angle of emergence and, to a lesser extent, by subsequent reorientation of the growing tip (Fig. 3), suggesting that pollen continuously monitors a guidance cue. Internal cue gradients in the pollen, resulting from possible uptake by the pollen, will be minimized by the rapid cytoplasmic streaming characteristic of pollen grains and tubes. Models and commentary
The response time (6 h) and distance (up to 75 mm) for pollen tube growth towards a source of water are consistent with guidance by a diffusible chemical cue (Table 2). The cue acting in vitro and on the stigma is probably a gradient of water across the pollen grain or tube. The gradient is steepest for grains at the interface between oil and water, but there must also be a gradient of water as a solute within the oil between stigma (or aqueous medium) and pollen. This is maintained by the activity of pollen as a sink and the localized nature of the source. However, proving conclusively that water is the cue is difficult16, because pollen tubes readily develop abnormal morphologies when culture conditions are modified, and in this particular case, pollen will not hydrate or grow if the putative cue is ‘excised’. The hypothesis might have generality in that pollen on dry stigmas germinates in a hydrophobic environment created by the pollen coat19, and thus water could be a guidance cue. On the other hand, water cannot account for directional pollen tube growth on stigmas with hydrophilic exudates, because the supply of water to pollen is presumably not directional. A conceptual difficulty in considering water as a guidance molecule is that the classical receptor models of responding systems are unlikely to apply when water is the signal. It is possible that the degree of hydration of the cell wall, or of membrane components at the pollen tube tip, affects the position of vesicle fusion and hence the direction of tip growth. Another explanation for guidance on the stigma is that specific lipids in the pollen– stigma environment are the guidance cue or are
trends in plant science perspectives
Table 1. Theoretical maximum guidance ranges of chemical attractants: based on an ideal gradient chosen to maximize the potential guidance distancea Gradient across pollen tube
1% 2%
Maximum guidance distance, mm
Narrow sensor
Wide sensor
4600 2300
9300 4700
a
This data is based on the compound interest formula. The minimum [(min)] and maximum [(max)] concentrations of the guidance cue are assumed to differ by a factor of 104. Reducing the factor to 103, changes the guidance range by a factor of 0.75. The sensing interval [represented as d in Fig. 2] is assumed to be 5 mm (narrow sensor) or 10 mm (wide sensor). The length of the pathway (l) over which a pollen tube can be guided was calculated according to the relation (max) 5 (min) 3 Rl/d , where l 5 d{log[(max)/(min)]/logR} and R is the fractional change in concentration detected by pollen tubes.
The N. alata homologue of TTS protein, galactose-rich style glycoprotein (GaRSGP) has also been purified24. Although GaRSGP and TTS protein have 97% identity (at the amino acid level), the method of extraction of GaRSGP indicates that it is associated with the cell walls of transmitting tract cells. There is no apparent gradient in its degree of glycosylation within the style and, in a different in vitro assay to that used for TTS protein, it does not attract pollen tubes. Recently, ‘N. alata TTS’ was shown to have similar properties to TTS protein (A. Cheung, pers. commun.). However, it remains to be established whether GaRSGP and N. alata TTS are the same molecule, and why work in different laboratories has given contrasting results. Chemical guidance is also proposed for the long styles of Lilium species, in which pollen tubes grow down the fluid-filled lumen of the hollow style, in contact with epidermal cells or other pollen tubes3. A component(s) of lily styles, when extracted and bound to artificial membranes, causes the tips of pollen tubes to adhere to the membrane. Adhesion to the surface increases the growth rate of pollen tubes. It has been proposed that a gradient in the adhesion factor from the top to the bottom of styles could be a guidance cue. Work to characterize the adhesive chemical is in progress, but no gradient has been detected. Models and commentary
actively involved in generation of the guidance gradient4,16,18. Lipids or derivatives of lipids guide the movement of some bacteria20 and algae21 in aqueous environments, and cause root hairs to resume tip growth during root nodule formation22. However, for a lipid to be the guidance cue requires not just heterogeneity in the lipid phase around pollen grains (which could occur if pollen is a source or sink of the cue), but that this heterogeneity is directional with respect to the aqueous phase. It is difficult to imagine how directional heterogeneity can arise in an environment in which the reservoir of the cue (the oil phase) surrounds the responding system. Guidance within the style
The TTS protein, which occurs in the intercellular spaces of the solid transmitting tract (Fig. 3) of N. tabacum, is a candidate for the role of stylar guidance cue11,23. This hypothesis is based on observations using isolated pollen tubes grown in the presence of TTS protein, which also acts as a growth stimulant in the assay. To date, there are no data on the concentration of TTS protein within the style, but it is suggested that, because TTS protein is less highly glycosylated at the stigma end of the style than at the ovary end, a difference in glycosylation could be the guidance cue11.
The hypothesis that gradients in bound or free chemicals guide pollen tubes through the style is far from proven. Pollen tubes from mid-style pollinations of solid and hollow styles grow both up and down the style, and tubes will also grow through inverted sections of styles1,25,26. Tubes grow out of cut styles, where there should be some detectable concentration of the putative attractant, and into growth media entirely lacking the attractant (Fig. 3). Indeed, the direction of pollen tube growth through the style is determined by the direction of growth at the point of entry, which is more consistent with growth through the style being constrained by its architecture rather than by chemical guidance1. If it could be proven that pollen tubes are chemically guided through the 50–100 mm long styles of Nicotiana and lilies, the phenomenon would be of considerable interest because it is at least an order of magnitude longer than expected (Table 1). The key information required for such proof includes quantitative analyses of the growth of isolated pollen tubes in the presence of defined gradients of the putative attractants. Both TTS protein and the Lilium adhesion molecule stimulate pollen tube growth in the in vitro assays, and without such quantitative data it is difficult to distinguish between differential growth in response to variation in the concentration of a growth stimulant, and guided growth in response to gradients in an attractant (Fig. 2).
Table 2. Theoretical maximum guidance ranges of chemical attractants: based on diffusion from a point source of the guidance cuea Gradient across pollen tube
Maximum guidance distance, mm
Rapid diffusion 1% 2%
Slow diffusion
2500 (0.50 d)b 2400 (3.6 d) 1200 (0.05 d) 1200 (1.0 d)
a
This data is based on diffusion from a point source that secretes the guidance chemical at a constant rate. The model was developed for axon guidance9. The context for axon guidance is analogous to that for pollen tubes. Sensor width is assumed to be 10 mm, and maximum guidance distance changes with width of the sensor as shown in Table 1. Two rates of diffusion are considered. ‘Rapid diffusion’ (diffusion constant D 5 1026 cm2/s) represents an upper limit to the diffusion rate, and is unlikely to be exceeded because it is in the same order as the diffusion of small molecules in water. ‘Slow diffusion’ (D 5 1027 cm2/s) is based on the expected rate of diffusion of netrin-1 (75 kDa) in cytoplasm (D 5 4.0 3 1027 cm2/s), with an allowance for diffusion being further slowed by the structure of the extracellular matrix. Because gradients in the cue change with time, the maximum guidance distance also changes with time. b Brackets show time [days (d) after the start of secretion] at which the maximum guidance distance applies. The maximum guidance range is shorter at other times.
Guidance within the ovary
In Arabidopsis, pollen tubes enter the ovary through transmitting tissue in the centre of a septum that bisects the ovary, and then diverge from the transmitting tract to enter one of the locules5. Pollen tubes in the locule grow directly to ovules that are close to the placenta (~20 mm), and along longitudinal furrows in the funicle of more distant ovules4,27,28. Pollen tubes are not guided to the micropyle with absolute fidelity (Fig. 3), but the accuracy of guidance has not been quantified. Usually only one pollen tube approaches each ovule, which must have a fully-developed embryo sac5,29,30. Low rates of fertilization are associated with the failure of pollen tubes to adhere to surfaces within the locule31. No chemical cue has been identified. October 1999, Vol. 4, No. 10
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Fig. 3. Pollen tube growth on the stigma, and in the style and the ovary. (a) and (b) The stigma. Nicotiana alata stigmas are papillate and covered with a secreted oil. (a) A pollen tube in oil is growing towards the aqueous environment of the stigma (scale bar 5 20 mm). (b) The growth of pollen tubes in oil towards an aqueous environment is reproduced in cultures of N. alata pollen, in which refined olive oil is substituted for the natural exudate, and the aqueous phase is a pollen tube growth medium (scale bar 5 20 mm). (c) Transmitting tissues of the solid style of N. alata (in cross section; scale bar 5 2 mm). Pollen tubes grow through the parallel channels created by the spaces between longitudinal files of transmitting tract cells. (d) Pollen tubes grow out of the cut end of a Rhododendron style (hollow) and into an artificial growth medium (scale bar 5 500 mm). Photograph Philip Taylor. (e) and (f) The ovary and the ovule. (e) Part of one locule of an Arabidopsis ovary showing multiple ovules (scale bar 5 100 mm). (f) Arabidopsis pollen tubes in the locules tend to follow grooves in the surfaces of funicles and ovules, and ultimately enter the embryo sac via the micropyle (scale bar 5 20 mm). Photographs (e) and (f) John Alvarez. Abbreviations: Pap, papillar cell; PG, pollen grain; PT, pollen tube; TTC, transmitting tract cell; St, style; Fun, funicle; M, micropyle; Ov, ovule.
In vitro experiments with Torenia fournieri10, provide evidence that the embryo sac secretes a diffusible chemical guidance cue. In these experiments, pollen tubes that by chance approach to within ~75 mm of an intact embryo sac are usually attracted to the micropylar end of the sac. Meandering pollen tubes are more likely to reach an ovule than those that grow straight. This last observation is interesting, because a meandering pollen tube is, potentially, a more sensitive gradient detector than a straight one (see below). The guidance chemical has not been identified. Models and commentary Fig. 4. Pollen tubes of Nicotiana alata. (a) In favourable in vitro (and in vivo) environments tubes follow the relatively straight growth trajectories depicted in Fig. 2. However, in unfavourable environments pollen tubes and tips can swell (b), or growth trajectories can become convoluted (c). Scale bar 5 20 mm.
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No guidance cues have been identified in the ovary, although calcium has been proposed on several occasions2. Chemical guidance by bound or diffusible cues within the ovary is feasible in that the distances involved, at least
trends in plant science perspectives
Conclusion and speculation
For most of this century, biologists have postulated that pollen tubes are guided by chemical cues, but none of the postulated attractants or repellents has been conclusively identified. Does chemical guidance of pollen tubes exist? The critical framework used here is based on conventional criteria and on estimates of the maximum guidance range of chemical cues. None of the guidance systems reviewed has been shown to meet all these criteria. It is important to consider whether the criteria are appropriate and adequate (and if not, to propose new sets of rules). This is because within the confines of these criteria, long-distance chemical guidance can only be rescued by major changes in the assumptions about the ability of pollen tubes to perceive gradients, or in the way in which we view the gradient itself. The assumptions made about pollen tube growth were: • Pollen tubes respond to a 10 000-fold variation in concentration of the guidance chemical. Expansion of this variation to
(a) T2
(c)
(b)
T1 Trends in Plant Science
in Arabidopsis, are within the maximum guidance range (Tables 1 and 2). But how do pollen tubes identify the closest ovule in multi-ovule ovaries, and why does only one pollen tube follow a particular path? Individual pollen tubes would follow independent paths if those paths were defined by an attractant secreted by an embryo sac, and a repellent secreted by the pollen tube, particularly if the attractant dissipates rapidly once the pollen tube reaches the sac4,28. However, repulsion between pollen tubes has not been demonstrated, and the model requires an attractant that forms a stable gradient for the receptive life of ovules (probably several days), but disperses rapidly if the source is removed. Slowly diffusing (or bound) molecules are likely to meet the first criterion, but rapidly diffusing molecules are required to meet the second (Table 2). None of the current models address the problem of guidance in the presence of multiple sources of the cue, such as would occur in multi-ovule ovaries (Fig. 3). The concentration of a putative chemical cue at any point in the ovary is the sum of the amounts derived from all sources, and consequently the steepest parts of concentration gradients might not lead to sources of the cue32. By analogy with yeast, pollen tubes could increase the accuracy of locating an ovule in the presence of multiple sources by destroying the cue32. However, modelling is needed to determine whether the rate of diffusion of the destructive agent would be fast enough relative to the growth rate of pollen tubes for this mechanism to be effective. The destruction of cues by pollen tube secretions is a potential means of preventing more than one pollen tube following a particular path.
(d)
Fig. 5. Spatial and temporal sensing by pollen tubes. Pollen tubes are shown entering a region in which there is a gradient in the concentration of a guidance molecule. Pollen tube tips respond to the gradient by elongating in the directions shown by the arrows. In (a) the concentration difference across the tip is too small to be detected and the tube’s direction of growth is unchanged. In (b) the swollen tip of the tube is now wide enough to detect differences in concentration and the tip grows towards high concentrations of the chemical. The general point made in (a) and (b) is that the wider the sensor, the greater the sensitivity to gradient detection. In (c) a hypothetical, meandering pollen tube is shown using temporal sensing to detect the gradient. Concentration measured at time T1 is perceptibly lower than the concentration at T2. The effective width of the sensor is the distance (parallel to the direction of the gradient) that the tip moves between measurements. The tip responds by directing growth towards the high concentration, and prolonging this movement relative to movements towards the low concentration. Growth oscillations that previously occurred randomly are now biased so that net growth is towards the high concentration. In (d) a pollen tube is chemically constrained to follow a narrow band of an attractant. If the tube deviates from this path, it detects a decrease in the attractant concentration, which results in the direction of growth changing and the tube returning to the highest concentration.
higher orders of magnitude would increase the guidance range and has implications for the number of receptors or their affinity for the substrate9. • The fractional change in concentration that pollen tubes can detect is 1%. This assumption is based on the accepted limits to sensitivity in other systems, and is in close agreement with the theoretical limit to sensitivity (the point at which a gradient cannot be distinguished from ‘noise’ associated with the random movement of molecules33). • Pollen tubes measure the fractional change in concentration over intervals of ,10 mm, the diameter of the tube. The maximum range of pollen tube guidance will increase if the sensing interval is wider than 10 mm (Table 1). Pollen tubes grown in vivo34 and in vitro (Fig. 4) have a range of morphologies and growth trajectories. The sensing interval is, potentially, increased by tip swelling (spatial sensing) and by meandering trajectories (temporal sensing) of the tube (Fig. 5). Both swelling (from which pollen tubes can recover35) and meandering trajectories occur more frequently in suboptimal growth conditions. Temporal sensing by chemotactic organisms along gradients
of bound or free chemicals results in distinctive growth paths called ‘biased random walks’, in which cells move transiently in all directions, but predominantly along the gradient20,36. These sorts of trajectories cannot occur where pollen tubes are physically constrained, as they are in solid styles, but growth paths in hollow styles and in ovaries should be examined in more detail. Nevertheless, it is difficult to imagine how the maximum guidance range can be increased sufficiently to accommodate some models of long-distance guidance. Perhaps a more promising model for long-distance chemical guidance is that pollen tubes, like axons37, are held on a path by gradients in attractants (or repellents) at right-angles to the direction of growth (Fig. 5). Future progress in pollen tube guidance must be based on realistic expectations of both the guidance system (pistil) and the response system (pollen). We must establish acceptable critical frameworks, and ways of quantifying pollen tube responses. We should also broaden our perspective to encompass the possibility that growth is sometimes random, and examine non-chemotropic explanations for pollen tube guidance. Adhesion, for example, could be a pre-requisite for sensing physical October 1999, Vol. 4, No. 10
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trends in plant science perspectives guidance cues [fungal hyphae can sense changes in surface topography of 0.4 mm (Ref. 38)]. Acknowledgements
I thank John Alvarez, Franz Grieser, John Golz, Geoffrey Goodhill, Maria Herrero, Chris O’Brien, Julie and Jeremy PickettHeaps, Jens Sommer-Knudsen, Philip Taylor for their contributions and the Australian Research Council for funding. References 1 Heslop-Harrison, J. (1987) Pollen germination and pollen-tube growth, Int. Rev. Cytol. 107, 1–78 2 Mascarenhas, J.P. (1993) Molecular mechanisms of pollen tube growth and differentiation, Plant Cell 5, 1303–1314 3 Jauh, G.Y. et al. (1997) Adhesion of lily pollen tubes on an artificial matrix, Sex. Plant Reprod. 10, 173–180 4 Wilhelmi, L.K. and Preuss, D. (1997) Blazing new trails. Pollen tube guidance in flowering plants, Plant Physiol. 113, 307–312 5 Hülskamp, M., Schneitz, K. and Pruitt, R. (1995) Genetic evidence for a long-range activity that directs pollen tube guidance in Arabidopsis, Plant Cell 7, 57–64 6 McCormick, S. (1998) Self-incompatibility and other pollen–pistil interactions, Curr. Opin. Plant Biol. 1, 18–25 7 Herrero, M. and Hormaza, J.I. (1996) Pistil strategies controlling pollen tube growth, Sex. Plant Reprod. 8, 343–347 8 Jacobs, W.P. (1959) What substance normally controls a given biological process? I. Formulation of some rules, Dev. Biol. 1, 527–533 9 Goodhill, G.J. (1997) Diffusion in axon guidance, Eur. J. Neurosci. 9, 1414–1421 10 Higashiyama, T. et al. (1998) Guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri, Plant Cell 10, 2019–2031 11 Wu, H-M., Wang, H. and Cheung, A.Y. (1995) A pollen tube growth stimulatory glycoprotein is deglycosylated by pollen tubes and displays a glycosylation gradient in the flower, Cell 82, 395–403 12 Goodhill, G.J. (1998) Mathematical guidance for axons, Trends Neurosci. 21, 226–231
13 Crick, F. (1970) Diffusion in embryogenesis, Nature 225, 420–422 14 Jauh, G.Y. and Lord, E.M. (1995) Movement of the tube cell in the lily style in the absence of the pollen grain and spent pollen tube, Sex. Plant Reprod. 8, 168–172 15 Pickett-Heaps, J.D. and Klein, A.G. (1998) Tip growth in plant cells may be amoeboid and not generated by turgor pressure, Proc. R. Soc. London Ser. B 265, 1453–1459 16 Lush, W.M., Grieser, F. and Wolters-Arts, M. (1998) Directional guidance of Nicotiana alata pollen tubes in vitro and on the stigma, Plant Physiol. 118, 733–741 17 Lush, W.M., Grieser, F. and Spurck, T. Does water direct the initial growth of pollen tubes towards the stigma of solanaceous plants? in Pollen and Spores: Morphology and Biology (Harley, M.M., Morton, C.M. and Blackmore, S., eds), Royal Botanic Gardens, Kew, UK (in press) 18 Wolters-Arts, M., Lush, W.M. and Mariani, C. (1998) Lipids are required for directional pollen tube growth, Nature 392, 819–821 19 Dickinson, H. (1995) Dry stigmas, water and self-incompatibility in Brassica, Sex. Plant Reprod. 8, 1–10 20 Kearns, D.B. and Shimkets, L.J. (1998) Chemotaxis in a gliding bacterium, Proc. Natl. Acad. Sci. U. S. A. 95, 11957–11962 21 Boland, W. (1995) The chemistry of gamete attraction: chemical structures, biosynthesis, and (a)biotic degradation of algal pheromones, Proc. Natl. Acad Sci. U. S. A. 92, 37–43 22 De Ruijter, N.C.A. et al. (1998) Lipochitooligosaccharides re-initiate root hair tip growth in Vicia sativa with high calcium and spectrin-like antigen at the tip, Plant J. 13, 341–350 23 Cheung, A.Y., Wang, H. and Wu, H-M. (1995) A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their growth, Cell 82, 383–393 24 Sommer-Knudsen, J. (1998) Re-evaluation of the role of a transmitting tract-specific glycoprotein on pollen tube growth, Plant J. 13, 529–535 25 Iwanami, Y. (1959) Physiological studies of pollen, J. Yokohama Munic. Univ. 116 (C34-Biol. 13), 1–137
26 Mulcahy, G.B. and Mulcahy, D.L. (1987) Induced pollen directionality, Am. J. Bot. 74, 1458–1459 27 Bowman, J.L. (1994) Arabidopsis: An Atlas of Morphology and Development, Springer-Verlag 28 Smyth, D. (1997) Plant development: attractive ovules, Curr. Biol. 7, R64–R66 29 Ray, S., Park, S-S. and Ray, A. (1997) Pollen tube guidance by the female gametophyte, Development 124, 2489–2498 30 Hauser, B.A., Villanueva, J.M. and Gasser, C.S. (1998) Arabidopsis TS01 regulates directional processes in cells during floral organogenesis, Genetics 150, 411–423 31 Wilhelmi, L.K. and Preuss, D. (1996) Selfsterility in Arabidopsis due to defective pollen tube guidance, Science 274, 1535–1537 32 Barkai, N., Rose, M.D. and Wingreen, N.S. (1998) Protease helps yeast find mating partners, Nature 396, 422–423 33 Goodhill, G.J. and Urbach, J.S. Theoretical analysis of gradient detection by growth cones, J. Neurobiol. (in press) 34 Williams, E.G., Knox, R.B. and Rouse, J.L. (1982) Pollination sub-systems distinguished by pollen tube arrest after incompatible interspecific crosses in Rhododendron (Ericaceae), J. Cell Sci. 53, 255–277 35 Lush, W.M. and Clarke, A.E. (1997) Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility, Sex. Plant Reprod. 10, 27–35 36 Manson, M.D. (1992) Bacterial motility and chemotaxis, Adv. Microb. Physiol. 33, 277–346 37 Tessier-Lavigne, M. and Goodman, C.S. (1996) The molecular biology of axon guidance, Science 274, 1123–1133 38 Corrêa, A., Staples, R.C. and Hoch, H.C. (1996) Inhibition of thigmostimulated cell differentiation with RGD-peptides in uromyces germlings, Protoplasma 194, 91–102
W. Mary Lush is at the School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia (fax 161 3 9347 1071; e-mail [email protected]).
Trends in Plant Science Perspectives Perspectives articles are your opportunity to express personal views on topics of interest to plant scientists. The range of appropriate subject matter covers the breadth of plant biology: historical, philosophical or political aspects; potential links between hitherto disparate fields; or simply a new view on a subject of current importance. Specific issues might include: the allocation of funding; commercial exploitation of data; the gene sequencing projects; genetic engineering; the impact of the Internet; education issues; or public perception of science. Current developments in other disciplines may also be relevant. Although Perspective articles are more opinionated in tone, they remain subject to stringent peer-review to ensure clarity and accuracy. The decision to publish rests with the Editor. If you wish to write for the Perspectives section, please contact the Editor before embarking on your manuscript, with a 200-word proposal and a list of key recent references ([email protected]).
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Whither chemotropism and pollen tube guidance? W. Mary Lush Pollen tubes follow a well-defined path to deliver male gametes to female gametes, but the mechanisms they use to locate this path are poorly understood. The major hypothesis is (and long has been) that pollen tubes are guided by chemical gradients and/or physical structures. Recently, parallels have been drawn between chemical mechanisms of guidance in pollen tubes and other cells, such as axons. These comparisons highlight a problem with the current models for pollen tube guidance, namely the distance over which chemical guidance is proposed to occur. Based on this new perspective, some models are either invalid or pollen tubes are uniquely responsive to chemical guidance cues.
P
ollen grains are delivered to the receptive surface of the stigma, which is remote from the ovules (Fig. 1). The male gametes, which are not motile, reach the female gametes via a tube that grows out of the pollen grains, and into and through the female tissues of the flower until it reaches the micropyle of an ovule. What guides the growth of pollen tubes along this path? According to early hypotheses, pollen tubes are guided by a continuous gradient in a single, target-derived chemical1,2. A more common view is that chemical cues guide pollen tubes during growth on the stigma and in the ovary, but that the route through the style is determined by physical structures1. Recently, parallels have been drawn between pollen tubes, migrating cells3 and growing axons4,5. This has led to a more complex view of guidance that involves multiple cues that act in overlapping spatial and temporal frames, and include chemical attractants and repellents, and physical guidance4. Guidance of pollen tubes and other cells Ð general considerations
By definition, chemotropism is a growth response to a chemical stimulus, the direction of growth being determined by the direction of the chemical gradient in the attractant or the repellent (Fig. 2). Chemical guidance can occur only when the external environment permits propagation of the cue at concentrations that vary within the response range of pollen, and when pollen is competent to respond to the cue. There are thus many chemicals, including nutrients, that are indirectly involved in pollen tube guidance (for effects on pollen-tube growth rate see Refs 6,7). Candidates for the role of chemical guidance cue should meet the criteria routinely applied to address the general question of whether a substance (including a gene product), plays a role in a particular process8:
free, as in the examples already given, or bound to a surface, as proposed for the stylar canal3. Competent pollen tubes will respond to a chemical gradient by altering the polarity of tip growth, provided that the absolute concentration of the cue is above the threshold for detection (but below saturation), and that there is a detectable difference in concentration across the tip (Fig. 2). It is proposed that pollen tubes are guided over distances of 50–100 mm by single chemical cues3,11, whereas the experimentally determined limit of guidance in other systems is usually ,1 mm (Ref. 9). There are theoretical reasons for chemical guidance distances being short, one of which is that there is a limit to the number of cumulative increases in the concentration of the guidance cue that can occur before the responding system is saturated (Fig. 2). What, theoretically, is the maximum path length over which pollen tubes can be guided by a chemical cue? Between 1.2 and 9.3 mm is the answer generated by applying
• The substance should be present at the correct time and place, and in the correct quantities. • If the source of the substance is excised surgically, or by mutation or by down-regulation of genes, the response should cease. • The response should be restored by the substitution of the purified Stigma chemical for the natural source or Pollen by the replacement of a funcPlug tube tional copy of the gene. Pollen Sperm Style tube • The response should be reprocells ducible in isolation from other Vegetative tissues (i.e. it should act directly nucleus upon the responding system). Extending • The hypothesis is strengthened if tip Transmitting it has generality. tract Ovary • The response should be specific Ovule Trends in Plant Science to one substance or its close analogs. Fig. 1. General structure of the flower. The pistil is Where chemotropism is proposed, made up of three parts, the receptive surface or two additional constraints apply: stigma, the ovary, which houses the ovules and, • There must be an external, direcseparating the two, the style. The stigma surface tional gradient in the putative can be ‘dry’, or ‘wet’ (covered by either a hydroguidance substance. philic or hydrophobic secretion). Pollen tubes grow • The distance over which a single through the central part of the style, which can be a hollow, fluid-filled canal, or consist of parallel files chemotropic agent acts should be of longitudinally oriented cells, called transmitting no more than 10 mm for bound tract cells. The ovary can contain a single ovule or cues and less for diffusible cues. up to thousands of ovules. The male pollen grains In most working models of chemiare usually desiccated when they make contact with cal attraction, it is proposed that a the stigma. Each grain hydrates, and germinates to chemical gradient arises by the difproduce a tube that grows into the intercellular fusion of the chemical from its orispaces of the stigma and through the style (inset). 9 gin at the target tissue . An example The tip of the growing tube contains cytoplasm, two of this model in pollination biology male gametes (sperm cells), and a vegetative nuis pollen tube attraction by the secrecleus, which is thought to regulate most aspects of tube growth. As the tube elongates by depositing tions of the embryo sac10. However, new cell wall material at the tip (‘tip growth’), the gradients could arise also by the older parts of the tube are cut off by cross walls, graded expression or processing of the called plugs. Fertilization occurs when a pollen tube chemical by tissues along the pathenters an ovule at the micropyle, and the tip releases way, as proposed for a transmitting the sperm cells. tract-specific glycoprotein (TTS pro11 tein) . The guiding chemical can be
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Response system Guidance region saturated
Cue below detection level
(a)
Guidance range 5 Ð10 mm DC, d
(b)
Style
Growth stimulant
(c)
Growth attractant Trends in Plant Science
Fig. 2. Schematic models of pollen tube guidance by a chemical attractant. (a) Pollen tubes grow randomly in regions in which attractant concentrations are below the detection threshold of pollen tubes. When the tubes, by chance, enter a region with a detectable gradient in concentration, they respond by growing up the gradient and towards the source of the cue. They might eventually reach regions of high concentration in which receptors for the chemical are saturated and in which they resume random growth. Pollen tubes follow a relatively straight path through the guidance region, because if the growth axis (shown by arrows) deviates from the direction of the chemical gradient, a difference in concentration (DC) becomes detectable across the tip. The steeper the gradient or the wider the tip, the smaller the deviation that must occur before the gradient is detected. The sensing interval, indicated as d, is the minimum distance (in the direction of the cue) over which differences in concentration can be detected, and is less than or equal to the diameter of the pollen tube. In some in vitro assays of putative chemo-attractants, the growth pattern of pollen tubes emerging from a cut style have been observed10,23. If the chemical is a growth stimulant (b), the elongation rate of pollen tubes that grow by chance into effective concentrations of the chemical is accelerated, but the direction of growth is unaltered. If the chemical is an attractant (c), pollen tubes that by chance enter the guidance range of the chemical are directed towards higher concentrations of the chemical, but their rate of growth does not change.
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the rules of compounding (Table 1). Because this estimate is based on parameters of the responding system, it applies to all stable chemical gradients, regardless of their origin and means of propagation. Estimates of the maximum guidance range of bound cues for axons [,10 mm (Ref. 12)], are consistent with the above estimate. However, the guidance range of diffusible cues is likely to be much shorter because of the temporal and spatial realities of diffusion, which limit the maximum distance over which chemical morphogens influence development of animal cells to ~1 mm (Ref. 13). The growth cone of axons is slightly larger than the tip of most pollen tubes, and numerical models of axon guidance suggest that the maximum guidance range for unbound cues is probably ,2.5 mm (Table 2), which is consistent with experimental results9. The maximum guidance range increases with the increasing size of the sensor (Table 1). The type of sensor envisaged for pollen tubes (and axons), compares concentrations on either side of the tip (or growth cone), and this method of gradient sensing is called spatial sensing. However, guidance ranges for small cells, such as bacteria, would be short if cells depended on spatial sensing. Instead, motile cells sense gradients temporally by comparing the concentration at time (T) = 0 with the concentration at T = 1. The maximum effective size of temporal sensors is thus the distance the cell moves during the time between measurements. Spatial and temporal mechanisms are not mutually exclusive, and pollen tubes could also take advantage of temporal sensing. Although pollen tubes are not motile, they have features in common with migrating cells. The apical region of pollen tubes is the only living part of the tube and its growth is in some ways like that of a migrating cell, which ‘leaves a trail of extracellular matrix…behind’14. Furthermore, tip growth might have more in common with
amoeboid growth than with the turgor-driven growth usually associated with plant cells15. Guidance on the stigma
Pollen tubes emerging from grains in oily exudates on the wet stigmas of Nicotiana alata, grow from a hydrophobic environment towards and into the aqueous environment of the stigma (Fig. 3). Tube growth towards an aqueous environment is reproducible in cultures in the absence of any style-specific components16–18 (Fig. 3). In vitro guidance of pollen tubes surrounded by oil occurs with high fidelity (97% of pollen tubes grow towards the aqueous phase16), and ends when pollen tubes enter the aqueous phase. The trajectory of pollen tube growth is determined by the aperture of emergence, the angle of emergence and, to a lesser extent, by subsequent reorientation of the growing tip (Fig. 3), suggesting that pollen continuously monitors a guidance cue. Internal cue gradients in the pollen, resulting from possible uptake by the pollen, will be minimized by the rapid cytoplasmic streaming characteristic of pollen grains and tubes. Models and commentary
The response time (6 h) and distance (up to 75 mm) for pollen tube growth towards a source of water are consistent with guidance by a diffusible chemical cue (Table 2). The cue acting in vitro and on the stigma is probably a gradient of water across the pollen grain or tube. The gradient is steepest for grains at the interface between oil and water, but there must also be a gradient of water as a solute within the oil between stigma (or aqueous medium) and pollen. This is maintained by the activity of pollen as a sink and the localized nature of the source. However, proving conclusively that water is the cue is difficult16, because pollen tubes readily develop abnormal morphologies when culture conditions are modified, and in this particular case, pollen will not hydrate or grow if the putative cue is ‘excised’. The hypothesis might have generality in that pollen on dry stigmas germinates in a hydrophobic environment created by the pollen coat19, and thus water could be a guidance cue. On the other hand, water cannot account for directional pollen tube growth on stigmas with hydrophilic exudates, because the supply of water to pollen is presumably not directional. A conceptual difficulty in considering water as a guidance molecule is that the classical receptor models of responding systems are unlikely to apply when water is the signal. It is possible that the degree of hydration of the cell wall, or of membrane components at the pollen tube tip, affects the position of vesicle fusion and hence the direction of tip growth. Another explanation for guidance on the stigma is that specific lipids in the pollen– stigma environment are the guidance cue or are
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Table 1. Theoretical maximum guidance ranges of chemical attractants: based on an ideal gradient chosen to maximize the potential guidance distancea Gradient across pollen tube
1% 2%
Maximum guidance distance, mm
Narrow sensor
Wide sensor
4600 2300
9300 4700
a
This data is based on the compound interest formula. The minimum [(min)] and maximum [(max)] concentrations of the guidance cue are assumed to differ by a factor of 104. Reducing the factor to 103, changes the guidance range by a factor of 0.75. The sensing interval [represented as d in Fig. 2] is assumed to be 5 mm (narrow sensor) or 10 mm (wide sensor). The length of the pathway (l) over which a pollen tube can be guided was calculated according to the relation (max) 5 (min) 3 Rl/d , where l 5 d{log[(max)/(min)]/logR} and R is the fractional change in concentration detected by pollen tubes.
The N. alata homologue of TTS protein, galactose-rich style glycoprotein (GaRSGP) has also been purified24. Although GaRSGP and TTS protein have 97% identity (at the amino acid level), the method of extraction of GaRSGP indicates that it is associated with the cell walls of transmitting tract cells. There is no apparent gradient in its degree of glycosylation within the style and, in a different in vitro assay to that used for TTS protein, it does not attract pollen tubes. Recently, ‘N. alata TTS’ was shown to have similar properties to TTS protein (A. Cheung, pers. commun.). However, it remains to be established whether GaRSGP and N. alata TTS are the same molecule, and why work in different laboratories has given contrasting results. Chemical guidance is also proposed for the long styles of Lilium species, in which pollen tubes grow down the fluid-filled lumen of the hollow style, in contact with epidermal cells or other pollen tubes3. A component(s) of lily styles, when extracted and bound to artificial membranes, causes the tips of pollen tubes to adhere to the membrane. Adhesion to the surface increases the growth rate of pollen tubes. It has been proposed that a gradient in the adhesion factor from the top to the bottom of styles could be a guidance cue. Work to characterize the adhesive chemical is in progress, but no gradient has been detected. Models and commentary
actively involved in generation of the guidance gradient4,16,18. Lipids or derivatives of lipids guide the movement of some bacteria20 and algae21 in aqueous environments, and cause root hairs to resume tip growth during root nodule formation22. However, for a lipid to be the guidance cue requires not just heterogeneity in the lipid phase around pollen grains (which could occur if pollen is a source or sink of the cue), but that this heterogeneity is directional with respect to the aqueous phase. It is difficult to imagine how directional heterogeneity can arise in an environment in which the reservoir of the cue (the oil phase) surrounds the responding system. Guidance within the style
The TTS protein, which occurs in the intercellular spaces of the solid transmitting tract (Fig. 3) of N. tabacum, is a candidate for the role of stylar guidance cue11,23. This hypothesis is based on observations using isolated pollen tubes grown in the presence of TTS protein, which also acts as a growth stimulant in the assay. To date, there are no data on the concentration of TTS protein within the style, but it is suggested that, because TTS protein is less highly glycosylated at the stigma end of the style than at the ovary end, a difference in glycosylation could be the guidance cue11.
The hypothesis that gradients in bound or free chemicals guide pollen tubes through the style is far from proven. Pollen tubes from mid-style pollinations of solid and hollow styles grow both up and down the style, and tubes will also grow through inverted sections of styles1,25,26. Tubes grow out of cut styles, where there should be some detectable concentration of the putative attractant, and into growth media entirely lacking the attractant (Fig. 3). Indeed, the direction of pollen tube growth through the style is determined by the direction of growth at the point of entry, which is more consistent with growth through the style being constrained by its architecture rather than by chemical guidance1. If it could be proven that pollen tubes are chemically guided through the 50–100 mm long styles of Nicotiana and lilies, the phenomenon would be of considerable interest because it is at least an order of magnitude longer than expected (Table 1). The key information required for such proof includes quantitative analyses of the growth of isolated pollen tubes in the presence of defined gradients of the putative attractants. Both TTS protein and the Lilium adhesion molecule stimulate pollen tube growth in the in vitro assays, and without such quantitative data it is difficult to distinguish between differential growth in response to variation in the concentration of a growth stimulant, and guided growth in response to gradients in an attractant (Fig. 2).
Table 2. Theoretical maximum guidance ranges of chemical attractants: based on diffusion from a point source of the guidance cuea Gradient across pollen tube
Maximum guidance distance, mm
Rapid diffusion 1% 2%
Slow diffusion
2500 (0.50 d)b 2400 (3.6 d) 1200 (0.05 d) 1200 (1.0 d)
a
This data is based on diffusion from a point source that secretes the guidance chemical at a constant rate. The model was developed for axon guidance9. The context for axon guidance is analogous to that for pollen tubes. Sensor width is assumed to be 10 mm, and maximum guidance distance changes with width of the sensor as shown in Table 1. Two rates of diffusion are considered. ‘Rapid diffusion’ (diffusion constant D 5 1026 cm2/s) represents an upper limit to the diffusion rate, and is unlikely to be exceeded because it is in the same order as the diffusion of small molecules in water. ‘Slow diffusion’ (D 5 1027 cm2/s) is based on the expected rate of diffusion of netrin-1 (75 kDa) in cytoplasm (D 5 4.0 3 1027 cm2/s), with an allowance for diffusion being further slowed by the structure of the extracellular matrix. Because gradients in the cue change with time, the maximum guidance distance also changes with time. b Brackets show time [days (d) after the start of secretion] at which the maximum guidance distance applies. The maximum guidance range is shorter at other times.
Guidance within the ovary
In Arabidopsis, pollen tubes enter the ovary through transmitting tissue in the centre of a septum that bisects the ovary, and then diverge from the transmitting tract to enter one of the locules5. Pollen tubes in the locule grow directly to ovules that are close to the placenta (~20 mm), and along longitudinal furrows in the funicle of more distant ovules4,27,28. Pollen tubes are not guided to the micropyle with absolute fidelity (Fig. 3), but the accuracy of guidance has not been quantified. Usually only one pollen tube approaches each ovule, which must have a fully-developed embryo sac5,29,30. Low rates of fertilization are associated with the failure of pollen tubes to adhere to surfaces within the locule31. No chemical cue has been identified. October 1999, Vol. 4, No. 10
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Fig. 3. Pollen tube growth on the stigma, and in the style and the ovary. (a) and (b) The stigma. Nicotiana alata stigmas are papillate and covered with a secreted oil. (a) A pollen tube in oil is growing towards the aqueous environment of the stigma (scale bar 5 20 mm). (b) The growth of pollen tubes in oil towards an aqueous environment is reproduced in cultures of N. alata pollen, in which refined olive oil is substituted for the natural exudate, and the aqueous phase is a pollen tube growth medium (scale bar 5 20 mm). (c) Transmitting tissues of the solid style of N. alata (in cross section; scale bar 5 2 mm). Pollen tubes grow through the parallel channels created by the spaces between longitudinal files of transmitting tract cells. (d) Pollen tubes grow out of the cut end of a Rhododendron style (hollow) and into an artificial growth medium (scale bar 5 500 mm). Photograph Philip Taylor. (e) and (f) The ovary and the ovule. (e) Part of one locule of an Arabidopsis ovary showing multiple ovules (scale bar 5 100 mm). (f) Arabidopsis pollen tubes in the locules tend to follow grooves in the surfaces of funicles and ovules, and ultimately enter the embryo sac via the micropyle (scale bar 5 20 mm). Photographs (e) and (f) John Alvarez. Abbreviations: Pap, papillar cell; PG, pollen grain; PT, pollen tube; TTC, transmitting tract cell; St, style; Fun, funicle; M, micropyle; Ov, ovule.
In vitro experiments with Torenia fournieri10, provide evidence that the embryo sac secretes a diffusible chemical guidance cue. In these experiments, pollen tubes that by chance approach to within ~75 mm of an intact embryo sac are usually attracted to the micropylar end of the sac. Meandering pollen tubes are more likely to reach an ovule than those that grow straight. This last observation is interesting, because a meandering pollen tube is, potentially, a more sensitive gradient detector than a straight one (see below). The guidance chemical has not been identified. Models and commentary Fig. 4. Pollen tubes of Nicotiana alata. (a) In favourable in vitro (and in vivo) environments tubes follow the relatively straight growth trajectories depicted in Fig. 2. However, in unfavourable environments pollen tubes and tips can swell (b), or growth trajectories can become convoluted (c). Scale bar 5 20 mm.
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No guidance cues have been identified in the ovary, although calcium has been proposed on several occasions2. Chemical guidance by bound or diffusible cues within the ovary is feasible in that the distances involved, at least
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Conclusion and speculation
For most of this century, biologists have postulated that pollen tubes are guided by chemical cues, but none of the postulated attractants or repellents has been conclusively identified. Does chemical guidance of pollen tubes exist? The critical framework used here is based on conventional criteria and on estimates of the maximum guidance range of chemical cues. None of the guidance systems reviewed has been shown to meet all these criteria. It is important to consider whether the criteria are appropriate and adequate (and if not, to propose new sets of rules). This is because within the confines of these criteria, long-distance chemical guidance can only be rescued by major changes in the assumptions about the ability of pollen tubes to perceive gradients, or in the way in which we view the gradient itself. The assumptions made about pollen tube growth were: • Pollen tubes respond to a 10 000-fold variation in concentration of the guidance chemical. Expansion of this variation to
(a) T2
(c)
(b)
T1 Trends in Plant Science
in Arabidopsis, are within the maximum guidance range (Tables 1 and 2). But how do pollen tubes identify the closest ovule in multi-ovule ovaries, and why does only one pollen tube follow a particular path? Individual pollen tubes would follow independent paths if those paths were defined by an attractant secreted by an embryo sac, and a repellent secreted by the pollen tube, particularly if the attractant dissipates rapidly once the pollen tube reaches the sac4,28. However, repulsion between pollen tubes has not been demonstrated, and the model requires an attractant that forms a stable gradient for the receptive life of ovules (probably several days), but disperses rapidly if the source is removed. Slowly diffusing (or bound) molecules are likely to meet the first criterion, but rapidly diffusing molecules are required to meet the second (Table 2). None of the current models address the problem of guidance in the presence of multiple sources of the cue, such as would occur in multi-ovule ovaries (Fig. 3). The concentration of a putative chemical cue at any point in the ovary is the sum of the amounts derived from all sources, and consequently the steepest parts of concentration gradients might not lead to sources of the cue32. By analogy with yeast, pollen tubes could increase the accuracy of locating an ovule in the presence of multiple sources by destroying the cue32. However, modelling is needed to determine whether the rate of diffusion of the destructive agent would be fast enough relative to the growth rate of pollen tubes for this mechanism to be effective. The destruction of cues by pollen tube secretions is a potential means of preventing more than one pollen tube following a particular path.
(d)
Fig. 5. Spatial and temporal sensing by pollen tubes. Pollen tubes are shown entering a region in which there is a gradient in the concentration of a guidance molecule. Pollen tube tips respond to the gradient by elongating in the directions shown by the arrows. In (a) the concentration difference across the tip is too small to be detected and the tube’s direction of growth is unchanged. In (b) the swollen tip of the tube is now wide enough to detect differences in concentration and the tip grows towards high concentrations of the chemical. The general point made in (a) and (b) is that the wider the sensor, the greater the sensitivity to gradient detection. In (c) a hypothetical, meandering pollen tube is shown using temporal sensing to detect the gradient. Concentration measured at time T1 is perceptibly lower than the concentration at T2. The effective width of the sensor is the distance (parallel to the direction of the gradient) that the tip moves between measurements. The tip responds by directing growth towards the high concentration, and prolonging this movement relative to movements towards the low concentration. Growth oscillations that previously occurred randomly are now biased so that net growth is towards the high concentration. In (d) a pollen tube is chemically constrained to follow a narrow band of an attractant. If the tube deviates from this path, it detects a decrease in the attractant concentration, which results in the direction of growth changing and the tube returning to the highest concentration.
higher orders of magnitude would increase the guidance range and has implications for the number of receptors or their affinity for the substrate9. • The fractional change in concentration that pollen tubes can detect is 1%. This assumption is based on the accepted limits to sensitivity in other systems, and is in close agreement with the theoretical limit to sensitivity (the point at which a gradient cannot be distinguished from ‘noise’ associated with the random movement of molecules33). • Pollen tubes measure the fractional change in concentration over intervals of ,10 mm, the diameter of the tube. The maximum range of pollen tube guidance will increase if the sensing interval is wider than 10 mm (Table 1). Pollen tubes grown in vivo34 and in vitro (Fig. 4) have a range of morphologies and growth trajectories. The sensing interval is, potentially, increased by tip swelling (spatial sensing) and by meandering trajectories (temporal sensing) of the tube (Fig. 5). Both swelling (from which pollen tubes can recover35) and meandering trajectories occur more frequently in suboptimal growth conditions. Temporal sensing by chemotactic organisms along gradients
of bound or free chemicals results in distinctive growth paths called ‘biased random walks’, in which cells move transiently in all directions, but predominantly along the gradient20,36. These sorts of trajectories cannot occur where pollen tubes are physically constrained, as they are in solid styles, but growth paths in hollow styles and in ovaries should be examined in more detail. Nevertheless, it is difficult to imagine how the maximum guidance range can be increased sufficiently to accommodate some models of long-distance guidance. Perhaps a more promising model for long-distance chemical guidance is that pollen tubes, like axons37, are held on a path by gradients in attractants (or repellents) at right-angles to the direction of growth (Fig. 5). Future progress in pollen tube guidance must be based on realistic expectations of both the guidance system (pistil) and the response system (pollen). We must establish acceptable critical frameworks, and ways of quantifying pollen tube responses. We should also broaden our perspective to encompass the possibility that growth is sometimes random, and examine non-chemotropic explanations for pollen tube guidance. Adhesion, for example, could be a pre-requisite for sensing physical October 1999, Vol. 4, No. 10
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trends in plant science perspectives guidance cues [fungal hyphae can sense changes in surface topography of 0.4 mm (Ref. 38)]. Acknowledgements
I thank John Alvarez, Franz Grieser, John Golz, Geoffrey Goodhill, Maria Herrero, Chris O’Brien, Julie and Jeremy PickettHeaps, Jens Sommer-Knudsen, Philip Taylor for their contributions and the Australian Research Council for funding. References 1 Heslop-Harrison, J. (1987) Pollen germination and pollen-tube growth, Int. Rev. Cytol. 107, 1–78 2 Mascarenhas, J.P. (1993) Molecular mechanisms of pollen tube growth and differentiation, Plant Cell 5, 1303–1314 3 Jauh, G.Y. et al. (1997) Adhesion of lily pollen tubes on an artificial matrix, Sex. Plant Reprod. 10, 173–180 4 Wilhelmi, L.K. and Preuss, D. (1997) Blazing new trails. Pollen tube guidance in flowering plants, Plant Physiol. 113, 307–312 5 Hülskamp, M., Schneitz, K. and Pruitt, R. (1995) Genetic evidence for a long-range activity that directs pollen tube guidance in Arabidopsis, Plant Cell 7, 57–64 6 McCormick, S. (1998) Self-incompatibility and other pollen–pistil interactions, Curr. Opin. Plant Biol. 1, 18–25 7 Herrero, M. and Hormaza, J.I. (1996) Pistil strategies controlling pollen tube growth, Sex. Plant Reprod. 8, 343–347 8 Jacobs, W.P. (1959) What substance normally controls a given biological process? I. Formulation of some rules, Dev. Biol. 1, 527–533 9 Goodhill, G.J. (1997) Diffusion in axon guidance, Eur. J. Neurosci. 9, 1414–1421 10 Higashiyama, T. et al. (1998) Guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri, Plant Cell 10, 2019–2031 11 Wu, H-M., Wang, H. and Cheung, A.Y. (1995) A pollen tube growth stimulatory glycoprotein is deglycosylated by pollen tubes and displays a glycosylation gradient in the flower, Cell 82, 395–403 12 Goodhill, G.J. (1998) Mathematical guidance for axons, Trends Neurosci. 21, 226–231
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W. Mary Lush is at the School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia (fax 161 3 9347 1071; e-mail [email protected]).
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