The generation of polarity in the cells of eukaryotes in most cases requires an external asymmetrical input. This signal may originate from a random event such as the point of penetration of an egg by a sperm or from asymmetries present in the maternal environment as, for example, in megasporogenesis in flowering plants, or from an asymmetry in the physical environment usually involving either light or gravity. The latter affords the best opportunities for experimental manipulation and single cell systems in algae, mosses and ferns allow both cell biological and genetical investigation of the mechanisms establishing polarity. These simple systems have already shown that the generation of polarity involves two distinguishable processes, axis alignment and axis orientation, but a detailed understanding of the mechanisms by which the environmental inputs are transduced to set up a polar axis is still lacking. Non‐motile organisms must also be able to modify cell polarity in response to changed environmental inputs, and here too, lower plant systems afford the best prospect for understanding the mechanisms responsible.
Defining polarity in biological terms
Polarity is central to the development of animals and plants both in their evolution and for the production of individual complexity. This paper will focus particularly on the generation and modification of polarity at the level of individual cells. Polarity at this level is at the core of the development of complex multicellular organisms, since the division of a polar cell generates non‐equivalent daughter cells, allowing subsequent differential development.
The term ‘polarity’ is often used loosely and needs more rigorous definition. Recent studies of polarity which will be reviewed here have led to the need for further clarification of terminology related to polar axes. I myself have previously used the terms ‘axis polarity’, ‘axis alignment’ and ‘axis orientation’ inconsistently, and dictionary definitions suggest that the terms, ‘polarity’, ‘alignment’ and ‘orientation’ may be interchangeable in common usage. However, here these three terms will be defined differentially to allow more critical analysis of systems in which the generation of polarity may be studied. Polarity has recently been defined in a biological context, as ‘persistent asymmetrical and ordered distribution of structures along an axis’ (Cove et al., 1999) and this definition is not at variance with its general usage and applies equally to polarity at the level of the organelle, cell, organ or whole organism. Thus, most flowering plants have, at the level of the whole organism, a single major polar axis with roots below the soil and shoots and leaves above while a zygomorphic flower has two. I now wish to define more precisely, two further properties of a polar axis, axis alignment and axis orientation. Axis alignment does not specify the direction of polarity, only the relationship between the axis and some external reference. The direction in which the ‘asymmetrical and ordered distribution of structures’ occurs will be defined as the orientation of the axis. Thus two polar objects may both be oriented to an external gradient, for example, gravity, but one may be more closely aligned than the other. On the other hand the two may be equally aligned, but oriented in opposite directions (see the legend to Fig. 1 for the use of these terms).
Is a polar input needed to achieve cell polarity?
Although in many systems, the setting up of a polar axis requires an asymmetrical input, some polar biological structures develop autonomously. Asymmetrical virus capsids such as those of the T even bacteriophages, have a highly polar structure that is achieved by assembly of the capsid components without a requirement for an external polar input (Mosig and Eiserling, 1988). Organelles such as ribosomes may also achieve polarity without an external input.
There is evidence that, in some systems, a polar axis may be generated in a cell without an external polar input. It has been proposed that the differentiation of one daughter cell into a pre‐spore, and the other into a spore mother cell, at the onset of sporulation in the spore‐forming bacterium, Bacillus subtilis, is achieved independently of external inputs (Errington, 1992; Jacobs and Shapiro, 1998). There are also examples where eukaryote cells appear to be able to develop a polar axis in the absence of any external input. The polarity of the micro‐gametophyte (pollen grain) of flowering plants is related to the proximal‐distal axis of the microspore from which it develops (Twell et al., 1998). Although this relationship may vary between species, for a particular species it is constant, indicating that the polarity of the pollen grain owes its origin to the topology of microspore tetrad formation.
At the 32‐cell stage of asexual development of the green alga, Volvox carteri, 16 cells undergo the first of three crucial asymmetrical cell divisions that result in each producing one large, asexual propagule, the gonidium, and numerous small cells that differentiate into somatic cells (Kirk, 1997). The 32‐cell embryo is already a polar structure and the 16 cells that undergo an asymmetrical division are at the anterior of the embryo. The generation of the embryo's anterio‐posterior axis does not appear to require an external input. The alignment and orientation of the polar division is stereotypical and related to the topology of the embryo. The polar axis is such that the gonidia‐forming cells are produced to the outside of the embryo. Subsequently in development, inversion occurs so that the gonidia come to lie in the interior of the mature plant. Numerous mutants affecting this pattern of development, and the related patterns of sexual development, have been isolated and the establishment of a transposon‐tagging system now allows their molecular basis to be investigated (Miller et al., 1993). Thus the glsA gene, loss of function of which leads to the normally‐asymmetrical division becoming symmetrical, has now been isolated and sequenced (Miller and Kirk, 1999). Although it can be argued that this gene is involved in polar axis interpretation rather than polar axis generation (see below), it is interesting to find that the GlsA protein product is found to be associated with the mitotic spindle and Miller and Kirk propose that it may play a role in altering the plane of cell division.
Isolated protoplasts of the moss Ceratodon purpureus regenerate into cell filaments by polar outgrowth. Although the initial alignment of the regeneration axis can be polarized by a directional light input (Cove et al., 1996), protoplasts of this species are able to regenerate in darkness. In these conditions, there is only a very weak alignment to the gravity vector, suggesting that some internal cue is sufficient to allow polar development. Spores of the fern, Ceratopteris richardii, normally align their germination axis to gravity, but recently have been successfully germinated in microgravity in a space craft in orbit around the earth (S Roux, unpublished data), again indicating that an external cue may not be essential for the acquisition of polarity.
External signals are usually required to establish a polar axis
The development of a polar axis often requires an input provided by a biological signal or by the physical environment, particularly a light gradient or gravity stimulus. In many animal systems, including the nematode, Caenorhabditis elegans, and amphibia such as Rana, the point of sperm entry into the egg provides the stimulus that breaks symmetry. The point of sperm entry is also the default stimulus for setting up the polar axis in the developing zygote of the fucoid alga, Pelvetia compressa (Kropf et al., 1999), although, if the zygote develops in a light gradient, the axis may be re‐specified with a different alignment. It appears that the sperm entry point is in many systems random, so a chance biological signal is responsible for the initiation of the programme leading to the establishment of the polar axis.
The determination of the alignment of the dorso‐ventral axis in the embryo of the fruit fly, Drosophila melanogaster, is another example where it appears that a chance biological event generates polarity. Early in maturation, the oocyte nucleus migrates from its original median position to an anterior position. The asymmetry leading to the establishment of the dorso‐ventral axis is achieved by the chance position occupied by the oocyte nucleus after migration. Transcription in the oocyte nucleus of the gurken gene, leads to the localized production of a secreted molecule, which signals to the maternal cells nearest to it, setting in train the mechanisms that specify the dorsal side of the embryo (Klinger and Tautz, 1999).
The biological input may, however, often be more difficult to pinpoint, the origins of asymmetry remaining obscure. Even for the most studied animal system, Drosophila melanogaster, detail of how the anterio‐posterior axis of the embryo is generated is still lacking. The establishment of the axis involves the setting up of an asymmetrical distribution of mRNAs in the oocyte (Klinger and Tautz, 1999). These mRNAs are transcribed in the nuclei of maternal tissue surrounding the oocyte and are then transported into the oocyte where they come to be localized. The product of the nannos gene, for example, is localized at the end of the oocyte destined to become the animal's posterior. Several lines of evidence indicate that the required polar transport is dependent on pre‐existing polarized microtubules. If this is the case, the mechanism responsible for establishing the polarity of the cytoskeleton needs in turn, to be explained. It is probable that ultimately the programme for setting up the anterio‐posterior axis owes its origin to the maternal anatomy. This example illustrates a problem facing developmental geneticists. The characterization of nannos mutants established that the nannos gene product was necessary for setting up the anterio‐posterior axis. Similar work, also showed that products of other genes, such as bicoid, were also required. However, none of these gene products can be said to be involved in the origin of the anterio‐posterior polarity, but are instead required for axis elaboration. In many systems, the origin may prove impossible to describe with precision since it may be that vital asymmetry is simply passed down the maternal line from mother to daughter. Although less well understood than Drosophila, the origins of polarity in the megaspore of flowering plants may be similar, although the occurrence of somatic embryogenesis indicates that plants may have more capacity for self organization. The review for the June issue (Souter and Lindsey, 2000) deals specifically with polarity in flowering plant embryogenesis and so this topic will not be reviewed here.
The mechanisms leading to the acquisition of a consistently‐oriented third polar axis (in addition to anterio‐posterior and dorso‐ventral axes) pose a greater conceptual problem. Such third axis polarity, which leads to chirality, is found in plants. Although the direction of phyllotaxis appears to be random in many plants, there is some evidence for a genotypic component of consistent phyllotactic bias in some species of Tifolieae (Rijven, 1968). In species with twining stems, the chirality of the twine is in most cases consistent (Hart, 1990). It seems unlikely that consistent chirality can be achieved by external inputs and so some intrinsic property of the biological system may be responsible. The characterization of genes that mutate to reverse chirality would be revealing. Similar genes have already been characterized in mammals, including man, where mutant phenotypes showing a reversal of the normal arrangement of organs along the left‐right axis are known. Such an arrangement, in which for example, the heart is on the right rather than the left, is called situs inversus. Two mouse mutations that lead to a situs inversus phenotype have been characterized (Yokoyama et al., 1993; Supp et al., 1998). All inv/inv homozygous mice have a situs inversus phenotype, whereas 50% of iv/iv mice are situs inversus and 50% are normal. In both cases alignment along the left‐right axis is nearly normal, but in inv/inv individuals, the orientation of the left‐right polar axis has been reversed, while it appears that homozygosity for the iv mutation leads to an inability to orient the axis. The sequence of the iv gene suggests that it encodes a dynein heavy chain, while that of the inv gene shows homology with ankyrins (Mochizuko et al., 1998). Thus in this case, evidence is again emerging for a role of the cytoskeleton in determining the orientation of a polar axis, and this suggests that similar studies of chirality in plants will be worthwhile.
The different developmental demands of polarity in relation to the external environment
Although polarity is universal in multicellular organisms, allowing differential cell function and organ formation, the requirements for the generation of polarity will depend on whether the developing organism is motile or non‐motile. A motile organism can adjust its orientation to the physical world. In motile organisms, a polar axis is of adaptive value, with organs of sensory perception and analysis concentrated in the part of the body that encounters the environment first, conventionally its anterior. For motile terrestrial animals, locomotory organs are ventral where they can interact with the ground. However, there is no need for a motile organism to develop oriented to the gravity vector, since it can adjust its orientation subsequently. Thus, although the alignment of the dorso‐ventral axis in Drosophila appears to rely on chance, it is only necessary to set this axis up at right angles to the anterio‐posterior axis; alignment within the perpendicular plane being unimportant.
Plants being non‐motile, have a more rigorous need for environmental inputs, but in many cases the need is not for a polar axis to be aligned and oriented to the physical environment at the time of its establishment, but to be amenable to modification in response to environmental inputs. Thus the embryo within a seed will not require its polar axis to be aligned to light or gravity at the time of its establishment, but it is essential that it should be responsive to these inputs during germination. Only where no further movement will occur following polar development, is there likely to be a requirement for direct monitoring of the external environment during polar axis generation, and even here the axis may be initially established without environmental inputs and alignment modified subsequently in response to light or gravity.
Environmental inputs and the generation of cell polarity
The relationship between experimental material and physical environment is usually easy to manipulate and so experimental studies of the effects of light and gravity on the generation of cell polarity have been particularly rewarding. The relative direction of either a light source or the gravity vector can be changed, and light quality can be manipulated both in intensity and wavelength. With access to orbiting spacecraft, gravitational forces less than that on earth have become available. A number of different single‐celled plant systems are amenable to study.
Zygotes of brown algae, Fucus distichus and Pelvetia compressa, have been particularly amenable to cell biological study (Fowler and Quatrano, 1995). Fucus eggs are about 80 μm in diameter, Pelvetia eggs somewhat larger. Fertilization activates zygote development leading to the polar outgrowth of a rhizoid. The first cell division generates a cell plate that is perpendicular to the long axis of the developing zygote, separating a rhizoid cell which divides to generate the holdfast, from the cell which gives rise to the thallus. As outlined above, the point of sperm entry is a sufficient cue to set up the polar axis (Kropf et al., 1999). The alignment of polar development can, however, be changed if zygotes, once they have settled on the substratum, are exposed to a directional light gradient. The rhizoid will now emerge on the shaded side of the zygote. Re‐orientation of zygotes with respect to a light gradient at varying times, shows that the alignment of the polar axis remains plastic until axis fixation occurs, in Fucus at about 12 h post‐fertilization. Once the axis becomes fixed, further changes in light direction do not affect axis alignment (Quatrano and Shaw, 1997). There is evidence that the cell wall plays an integral role in axis fixation (Kropf et al., 1988) and it is proposed that fixation occurs as a result of cross linking between the actin cytoskeleton and the cell wall (Quatrano and Shaw, 1997). The earliest observable evidence of ordered asymmetry in the polar development of the zygote is the localization of F‐actin at the future site of rhizoid outgrowth. In Pelvetia, localization resulting from sperm entry is observed within 3 h of fertilization, but is revised following exposure to a light gradient, and reversal of this gradient before axis fixation results in the repositioning of the localized F‐actin in less than an hour (Alessa and Kropf, 1999). The mechanisms bringing about the formation of the F‐actin patch remain unknown. It has been suggested that sperm proteins, introduced upon fertilization, may be the instigators of the recruitment of F‐actin (Kropf et al., 1999), but even if this is the mechanism by which sperm entry cues polar axis alignment, the response to a light stimulus must involve intrinsic mechanisms.
Details of the mode of transduction of a light stimulus are emerging. Jaffe established that polarization of the germination axis was induced by blue light, but not by red, and that the photoreceptors concerned were located in or near to the cell membrane (Jaffe, 1958). Recently, all trans retinal has been isolated from zygotes of Pelvetia fastigiata, and it has therefore been proposed that the photoreceptor is a rhodopsin‐like protein. (Robinson et al., 1998). Rhodopsin is associated with the control of cyclic GMP (cGMP) levels in other systems, and so a role for a rhodopsin‐like protein as the photoreceptor for axis generation is supported by the finding (Robinson and Miller, 1997) that the establishment of a cGMP gradient may be one of the earliest events in the generation of polarity. Blue light irradiation leads to increases in cGMP levels, inhibitors of cGMP formation block the development of polarity, bathing in an excess of a cGMP analogue reduces polarization, and allowing zygotes to germinate in darkness in the presence of a gradient of a cGMP analogue leads to a tendency for rhizoid emergence to the low side of the concentration gradient. It is likely that the localization of Ca2+ channels that leads to a Ca2+ influx in the region of future rhizoid outgrowth, is an early event in axis determination (Shaw and Quatrano, 1996). In this work, evidence for the localization of Ca2+ channels was provided by the use of fluorescently‐labelled dihydropyridine (DHP), which is known to bind to calcium channels in mammalian cells. The same work, showed that the localization of DHP receptors was disrupted by cytochalasin B, a drug that interferes with actin function, and the establishment of a calcium gradient is therefore likely to be downstream of F‐actin localization.
The fern, Ceratopteris richardii, provides another system for the experimental investigation of the way in which the physical environment determines the polarity of an individual isolated cell. Ceratopteris spores, like fucoid zygotes, divide unequally to produce a small rhizoid cell and a larger cell that develops into the thallus. The alignment of the germination axis can be cued by gravity, but spores only become sensitive to gravity after germination has been triggered by light. Repositioning experiments, in which spores are rotated 180° with respect to the gravity vector at varying times following the initiation of germination, show that spores remain sensitive to the changes in the direction of the gravity vector up to about 10–12 h into the germination programme after which time, the axis is fixed for most spores (Edwards and Roux, 1994). Within 1 h of the initiation of germination by light, a polar calcium current can be detected which is aligned to the gravity vector, such that calcium moves into the cell on its lower side, and out of the cell from its upper side. When treated with the calcium blocking drug, nifedipine, the calcium current is reduced and the spore is no longer sensitive to the gravity vector. Nifedipine also leads to a partial inhibition of polar axis formation (Chatterjee et al., 2000), and so there is evidence that, as in Fucus and Pelvetia, an early event in polar axis formation is the establishment of an internal Ca2+ gradient.
The first morphological indication of polarity in Ceratopteris spores is the downward migration of the spore nucleus, and the position of the nucleus predicts the subsequent direction of rhizoid outgrowth. Nuclear movement occurs in material germinated on a klinostat, but the final position of the nucleus still predicts the position of rhizoid outgrowth, suggesting that the events are causally related. Light also affects the determination of axis polarity but only weakly, the rhizoid tending to develop away for the direction of a light source (Edwards and Roux, 1998). Protoplasts isolated from Ceratopteris gametophytes regenerate by polar outgrowth producing a rhizoid in a manner similar to spore germination. The alignment of the regeneration axis is sensitive to light and to gravity, but in contrast to their effects on spore germination, light is now much the stronger morphogenetic influence. Regeneration in a light gradient results in over 80% of regenerating protoplast producing a rhizoid on their shaded side. Cell biological and genetic studies of Ceratopteris spore germination and protoplast regeneration are at an early stage, but the system should allow progress to be made in the understanding of how light and gravity generate cell polarity.
Regeneration of moss protoplasts shows many similarities to the germination of fucoid zygotes and the regeneration of Ceratopteris protoplasts. Isolated protoplasts of the mosses, Physcomitrella patens and Ceratodon purpureus regenerate by polar outgrowth (Stumm et al., 1975; Cove et al., 1996). In Physcomitrella, protoplast regeneration only occurs at high light intensities (Jenkins and Cove, 1983a), but Ceratodon protoplasts are able to regenerate in darkness. Ceratodon protoplasts regenerated in darkness show a weak alignment of their regeneration axes to the gravity vector. Because there is no absolute requirement for light during protoplast regeneration in Ceratodon, it is possible to investigate its role in axis formation in more detail. Light quality has an effect on the overall response of populations of regenerating protoplasts and their behaviour can best be interpreted by considering axis alignment and axis orientation separately (Cove et al., 1996). In red light, the axes of most regenerating protoplasts are closely aligned to the direction of the light source, whereas in blue light, axis alignment shows much more variation. Axis orientation in red light is, however, much poorer than in blue light. In red light, only about two‐thirds of protoplasts orient outgrowth towards the light source, the remaining one‐third growing away, while in blue light almost all protoplasts orient towards the source of light. Reorientation experiments, similar to those carried out for Fucus, show that there is a period before which axis fixation occurs, during which a change of light direction results in a revision of the polar axis. These experiments allow the independent investigation of the effect of changing the direction of the light input on axis alignment and orientation. Results are consistent with axis alignment being fixed before axis polarity. There is thus a period during which the alignment of the axis with respect to the light direction is unaffected by changing the direction of the light source, but during which the orientation of the axis is still labile and so is disturbed by the change. As a result of these observations, it has been proposed that the mechanisms for the determination of axis alignment and orientation are at least partially independent, perhaps involving different morphogen gradients (Cove et al., 1996). There is evidence that the detection of light direction is by way of phytochrome (D Cove, unpublished data), but no cell biological investigations, similar to those carried out in Fucus and Pelvetia, have yet been made of the role of the cytoskeleton in axis development or of the cell wall in axis fixation.
The recognition that during the regeneration of Ceratodon protoplasts, axis alignment and orientation may involve different processes, was due both to a biological feature of the system and also to what may seem a minor difference in experimental design. Ceratodon protoplasts do not regenerate synchronously; there is about 30 h difference between the most rapidly regenerating protoplasts and those that are slowest. This allows the kinetics of repositioning to be studied in detail, albeit by the use of computer model fitting (Cove et al., 1996). In addition, Ceratodon experiments involving the repositioning of protoplasts with respect to light, rotated material by 90° relative to the direction to the light source. Fucus and Pelvetia zygotes show more synchronous development and, in repositioning experiments, 180° rotation has been used. As a result, the type of analysis that revealed the complexities of axis determination in Ceratodon protoplasts has not yet been carried out in Fucus or Pelvetia.
Environmental inputs and the modification of cell polarity
As discussed above, it is to be expected that plants, lacking motility at the level of both the whole organism and the individual cell, will often show an ability to modify polarity in response to environmental inputs. At the multicellular level, tropic responses of the shoots and roots of flowering plants have been extensively studied. The modification of the polarity of individual cells in response to light and gravity is however, as with the generation of cell polarity, often more easily studied in non‐flowering plant species.
The protonemal stage of moss gametophyte development provides particularly good material for the investigation of the effects of light and gravity on the modification of cell polarity. Protonemal filaments extend by the growth and division of their apical cells, and the response of individual living cells to environmental signals can readily be observed using time‐lapse video microscopy. In Physcomitrella patens, there are two types of protonemal filaments, chloronema and caulonema. Chloronemal filaments, which are produced following spore germination or tissue regeneration, only grow in the light. Chloronemal filaments developing from germinating spores are sensitive to light direction, growing towards a low intensity light source, at right angles to the direction of a high intensity red light source and away from a high intensity blue light source (Jenkins and Cove, 1983b). Caulonemal filaments are produced from chloronema and grow much more rapidly. The direction of extension of their apical cell is sensitive both to directional light inputs and to the gravity vector, but sensitivity to gravity is only shown by cells growing in darkness (Cove and Knight, 1987). As with chloronemal apical cells, the response of caulonemal apical cells to light depends on wavelength and intensity. No effect of gravity on the apical cells of chloronemal filaments has been observed, but since chloronemal cell division and growth only occur in the light, this is not unexpected.
Mutants of Physcomitrella abnormal in the response of their caulonemal apical cells to light or gravity have been isolated, but no mutant showing an altered response to both light and gravity has been isolated, indicating that the responses must be to a large extent independent. Physcomitrella mutants that are unable to align their caulonemal apical cells to light ( ptr mutants) comprise at least three complementation groups. ptr mutants produce gametophores that are also insensitive to light direction, showing that the gene products concerned are involved in response to light at both the level of the individual cell and the multicellular shoot (Cove and Knight, 1987). The response of the chloronemal apical cells of ptr mutants to directional light has been investigated, and surprisingly, they show a normal response to both low and high light intensities, but the change from a low‐light to a high‐light response occurs at lower intensities (Jenkins and Cove, 1983c). No explanation has been suggested for this pleiotropic phenotype.
Physcomitrella mutants having caulonemal apical cells that are insensitive to gravity (gtr mutants) have also been obtained and comprise at least two complementation groups. All gtr mutants so far obtained, produce gametophores that retain the wild‐type gravity response, showing that in contrast to light, the response to gravity of the individual cell and of the multicellular shoot must be at least partially independent. In addition to mutants insensitive to gravity, there are mutants of Physcomitrella (designated gtrC) that show a reversal in the orientation of their gravity response while retaining the wild type's ability to align to the gravity vector (Knight and Cove, 1991a). These mutants therefore have caulonemal filaments that grow downwards rather than away from gravity.
Protonemal filaments of Ceratodonpurpureus do not show clear differentiation into chloronemal and caulonemal cell types and studies of the response to light and gravity have concentrated on the apical cells of filaments on older cultures. The responses of these are similar to those of caulonemal apical cells of Physcomitrella. Plastid sedimentation in a sub‐apical zone of the Ceratodon apical cell is clearly observable and it is proposed that this is part of the gravi‐sensing apparatus (Sack, 1993). Mutants of Ceratodon with altered responses to light and gravity have also been isolated, although little genetic analysis has yet been carried out on them. Ceratodon ptr mutants fall into two classes (Lamparter et al., 1996). Some have their ability to respond to directional light restored by growth in the presence of biliverdin, a precursor of the phytochrome chromophore. These mutants are likely to be deficient in the biosynthesis of phytochrome, and provide evidence that light direction must be detected by way of phytochrome, as was proposed earlier for Physcomitrium turbinatum by Nebel (Nebel, 1968). This class of Ceratodon ptr mutant show other pleiotropic effects including reduced chlorophyll synthesis (Lamparter et al., 1997), and a continuing response to gravity when grown in light. It is therefore proposed that the response of apical cells to gravity in the wild type is actively turned off in light, detected by way of phytochrome. The second class of Ceratodonptr mutants, show no other pleiotropic effects, and the products of the genes concerned are therefore likely to be acting downstream of light detection, to bring about the regulated polar extension of the apical cell. The identification and molecular characterization of these genes should therefore be a high priority.
Mutants of Ceratodon having protonemal apical cells that respond abnormally to gravity (gtr) include completely insensitive mutants and mutants which respond to gravity more slowly than the wild type (Fig. 1). Ceratodon mutants resembling Physcomitrella gtrC mutants, in which the orientation of the gravity response is reversed, have also been isolated. This latter class of mutant have consequently been designated wrong way response (wwr) mutants (Wagner et al., 1997). Wild‐type strains of both Physcomitrella and Ceratodon also show reversal in the orientation of their response to gravity Knight and Cove, 1991b; Young and Sack, 1992). Reversal occurs immediately upon repositioning cultures with respect to gravity and during cell division. wwr Mutants show similar reversals of the orientation of their responses, mirroring the wild type, i.e. growth is down when the wild type shows upward growth, and up when the wild type grows downwards (Wagner et al., 1997). Plastid sedimentation in wwr mutants is also similar to that in the wild type, indicating that the wild‐type wwr gene must play a role in establishing axis orientation in response to gravity but not axis alignment.
To understand how a polar axis of an individual cell is generated or its polarity is modified in response to the physical environment, requires the study of systems that are accessible to both cell biological techniques and genetic analysis using molecular and genomic tools. Mutant isolation in the fucoid algae does not seem to be a near prospect, but the amenability of Fucus and Pelvetia to cell biological studies including imaging and micro‐injection will ensure that their continued study will contribute to the knowledge of polarity generation. Fern spore germination and moss protonemal growth may, however, be able to provide the necessary combination of accessibility to both cell biology and genetic analysis. To exploit these resources further, cell biological study of mosses and ferns need to be extended. Future genetic studies in both mosses and ferns must include the molecular characterization of genes involved in axis development. Recently, the aphototropic phenotype of a Ceratodon mutant blocked in phytochrome chromophore synthesis, has been repaired by the micro‐injection of appropriate cDNA (Brücker et al., 2000). This micro‐injection procedure has considerable potential in the analysis of cell polarity and promises to add to the repertoire of techniques available to study gene function in mosses. Molecular genetic studies of mosses are progressing rapidly. With the establishment that homologous recombination occurs with high efficiency between transforming DNA and genomic sequences (Kammerer and Cove, 1996; Schaefer and Zryd, 1997), the techniques of gene knockout and allele replacement are now available in Physcomitrella (see, for example, Strepp et al., 1998), allowing the detailed study of gene function. However, no method of gene tagging is yet available and so it has not yet been possible to isolate and characterize the genes involved in the responses to light and gravity that have already been identified by forward mutation. If such a technique becomes available, it should be possible to make rapid progress in understanding the mechanisms needed for polar axis development at the level of the individual cell.
I am grateful to Drs Ian Hope and Ralph Quatrano, as well as my students, for stimulating me to think through my ideas more clearly and to Drs Steve Miller, Ralph Quatrano and Stan Roux for their very helpful comments on parts of the manuscript.
Alessa L, Kropf DL.
Brücker G, Zeidler M, Kohchi T, Hartmann E, Lamparter T.
Chatterjee A, Marshall Porterfield D, Smith PS, Roux SJ.
Cove DJ, Hope IA, Quatrano RS.
Cove DJ, Knight CD.
Cove DJ, Quatrano RS, Hartmann E.
Edwards ES, Roux SJ.
Edwards ES, Roux SJ.
Fowler J, Quatrano RS.
Jacobs C, Shapiro L.
Jenkins GI, Cove DJ.
Jenkins GI, Cove DJ.
Jenkins GI, Cove DJ.
Kammerer W, Cove DJ.
Klinger M, Tautz D.
Knight CD, Cove DJ.
Knight CD, Cove DJ.
Kropf DL, Bisgrove SR, Hable WE.
Kropf DL, Kloareg B, Quatrano RS.
Lamparter T, Esch H, Cove D, Hartmann E.
Lamparter T, Esch H, Cove D, Hughes J, Hartmann E.
Miller SM, Kirk DL.
Miller SM, Schmitt R, Kirk DL.
Mochizuki T, Saijoh Y, Tsuchiya K et al.
Mosig G, Eiserling F.
Quatrano RS, Shaw SL.
Robinson KR, Lorenzi R, Ceccarelli N, Gualtieri P.
Robinson KR, Miller BJ.
Schaefer DG, Zrÿd J‐P.
Shaw SL, Quatrano RS.
Souter M, Lindsey K.
Strepp R, Scholz S, Kruse S, Speth V, Reski R.
Stumm I, Meyer Y, Abel WO.
Supp DM, Brueckner M, Potter SS.
Twell D, Park SK, Lalanne E.
Wagner TA, Cove DJ, Sack FD.
Yokoyama T, Copeland NG, Jenkins NA, Montgomery CA, Elder FFB, Overbeek PA.