JXB Advance Access originally published online on December 20, 2004
Journal of Experimental Botany 2005 56(412):653-662; doi:10.1093/jxb/eri040
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RESEARCH PAPER |
Effect of the energy supply on filamentous growth and development in Physcomitrella patens
1Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, SE-750 07 Uppsala, Sweden
2Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden
* To whom correspondence should be addressed. Fax: + 46 18 471 4673. E-mail: Hans.Ronne{at}imbim.uu.se
Received 15 July 2004; Accepted 27 September 2004
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Abstract |
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The filamentous gametophyte of the moss Physcomitrella patens consists of two filament types called chloronemata and caulonemata. Chloronemal cells are photosynthetically active with numerous chloroplasts, while caulonemata help to spread the colony by radial growth. The balance between the two filament types is affected by external factors such as light and plant hormones. In the present study, caulonema formation and chloronemal branching have been monitored during high and low light conditions and in the presence of glucose, auxin, or cytokinin. These experiments were performed both in a wild-type strain and in a hxk1 knockout mutant which lacks the major hexokinase of Physcomitrella. It was found that caulonema formation is induced by high energy conditions such as high light and external glucose, while chloronemal branching is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant. The hxk1 mutation also causes buds to appear on chloronemal filaments, which is rarely seen in the wild type, and shows increased sensitivity to cytokinin and abscisic acid. Based on these findings a model is proposed in which the energy supply of the moss colony regulates the balance between chloronemal and caulonemal growth.
Key words: Abscisic acid, auxin, caulonemata, chloronemata, cytokinin, hexokinase, Physcomitrella patens, protonemata
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Introduction |
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The moss Physcomitrella patens has recently emerged as a powerful model system in plant molecular genetics (Cove et al., 1997
; Reski, 1998; Schaefer, 2002; Nishiyama et al., 2003). Similar to higher plants, growth and development in Physcomitrella is regulated both by environmental factors, such as the quantity and quality of light (Cove et al., 1978; Imaizumi et al., 2002), and by phytohormones including auxins, cytokinins, and abscisic acid (Ashton et al., 1979; Knight et al., 1995). Physcomitrella is particularly well suited for reversed genetic studies since it has a high frequency of homologous recombination, which makes gene targeting possible (Schaefer and Zrÿ;d, 1997; Schaefer, 2002).
Physcomitrella colonies that are formed from regenerating spores or protoplasts are initially made up of protonemal filaments that grow by apical cell divisions (Cove et al., 1997; Reski, 1998). There are two types of protonemal filaments, chloronemata and caulonemata (Fig. 1). The photosynthetically more active chloronemal cells have well-developed chloroplasts, while caulonemal cells have fewer and smaller chloroplasts. The first filaments that are formed in a Physcomitrella colony are chloronemal. Caulonemal filaments are formed later, and help to spread the colony by rapid radial growth. Caulonema formation is also the first step towards a new generation since it gives rise to gametophores, which are needed for sexual reproduction. Thus, some caulonemal side-branch initials differentiate into buds (Fig. 1), which subsequently develop into gametophores, also known as leafy shoots. From the base of each gametophore, root-like rhizoids protrude (Fig. 1) which are morphologically similar to caulonemal filaments. The transition from chloronema to caulonema is induced by auxin (Ashton et al., 1979), an effect which is in turn inhibited by chryptochrome-dependent blue light signalling (Imaizumi et al., 2002). However, the effects of light on caulonema formation are complex since low light conditions favour chloronema formation and retard differentiation into caulonema (Jenkins and Cove, 1983; Reski, 1998).
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A gene has recently been cloned and characterized, named PpHXK1, which encodes the major hexokinase in Physcomitrella, a soluble protein that is located in the chloroplast stroma (Olsson et al., 2003
). Hexokinase is a key enzyme in the primary carbon metabolism, but it has also been proposed to function as a hexose sensor that mediates sugar regulation of gene expression, in both plants (Jang and Sheen, 1994; Jang et al., 1997; Rolland et al., 2002) and other eukaryotes (Entian, 1980; Rolland et al., 2001). According to this hypothesis, hexokinase generates a signal that is triggered by hexoses but is independent of their further metabolization. It is not easy to distinguish experimentally between the proposed sensory and metabolic roles of hexokinase since alterations in hexose phosphorylation with resulting changes in the glycolytic flow will have complex effects on cellular metabolism (Halford et al.; 1999; Moore and Sheen, 1999; Hardie, 1999). However, evidence was recently provided that the Arabidopsis hexokinase AtHxk1 has a hexose-sensing function which is independent of its hexose phosphorylating activity (Moore et al., 2003).
The characterization of the Physcomitrella hxk1 knockout mutant unexpectedly revealed that 0.15 M glucose induces caulonema formation in light-grown colonies, and that this effect is much reduced in the hxk1 mutant (Olsson et al., 2003). This finding prompted the present investigation of how chloronemal and caulonemal growth is affected by high and low light, glucose, and plant hormones, both in the wild type and in the hxk1 mutant. It was found that caulonema formation is induced by high energy growth conditions such as intense light and glucose, and by auxin. By contrast, chloronemal branching is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant, but also by cytokinin. Based on these findings a model is proposed in which the energy supply of the moss colony regulates the balance between chloronemal and caulonemal growth.
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Plant material and growth conditions
The standard growth conditions used in this report was growth at 25 °C under constant white light in a Sanyo MLR-350 light chamber with irradiation from the sides. Light was supplied from fluorescent tubes (FL40SS W/37, Toshiba) at 30 µmol m–2 s–1 (high intensity) or 6 µmol m–2 s–1 (low intensity). Routine subculturing of Physcomitrella protonemal tissue was done on cellophane-covered 0.8% agar plates (90 mm in diameter) containing 27 ml of BCD media (1 mM MgSO4, 1.85 mM KH2PO4, 10 mM KNO3, 45 µM FeSO4, 1 mM CaCl2, 1x Hoagland's Number 2 solution) supplemented with 5 mM ammonium tartrate. In cultures used for phenotypic analysis, the ammonium tartrate was omitted from the medium.
Phenotypic analysis of protonemata
For morphological studies, small pieces of fresh protonemal tissue (approximately 2 mm in diameter) were inoculated on ammonium tartrate-free BCD plates without cellophane, with different additives as described in the figure legends. For colony growth assays, representative 26-d-old colonies were photographed using a dissecting microscope and the diameters of at least four independent colonies were measured. In the caulonemal filament induction assay, the numbers of caulonemal filaments clearly protruding from the edge of a 30-d-old colony were counted using a dissecting microscope. Moss colonies grown under these standard conditions have a closely connected edge in the wild type, and even more so in the hxk1 mutant. This made it possible to define a filament extending beyond the rim of the colony as a protruding filament. Such protruding filaments were counted for at least four colonies in each case.
For the quantification of the number of branches per chloronemal cell, a large number of filaments were analysed by light microscopy. Using a razor blade, tissue was detached from the periphery of 4-week-old colonies, and caulonema-derived chloronemal filaments of more than 10 cells in length with intact apical cells were analysed. The number of cells and the number of branches, including both side-branch initials and bud-like structures, were counted for each such caulonema-derived chloronemal filament. The number of branches was divided by the number of cells in each filament. Furthermore, the number of cells of each side branch were counted. The total number of cells in the side branches were divided by the total number of side branches. The number of buds per side branch initial was counted in the same way for both caulonema-derived and chloronema-derived chloronemal filaments. For the ABA-induced cell expansion assay, chloronemal filaments were detached from the periphery of colonies grown with or without 1 µM ABA. The lengths and widths of a large number of cells were measured in a light microscope and the length to width ratio for each cell was used to calculate the averages and standard errors of mean. The analysis was limited to chloronemal cells without branch initials, positioned neither as the first cell in a new branch nor as the apical or one of the first two subapical cells.
Determination of endogenous abscisic acid levels
In order to measure endogenous ABA levels under different conditions, protonemal tissue was cultivated for 7 d on cellophane-covered ammonium tartrate-free BCD plates. The cellophane with the tissue was then transferred to treatment plates containing 0.15 M glucose, 0.15 M mannitol or no additives, and incubated under standard conditions for 24 h. At least eight independent experiments were done for each treatment. The tissue was harvested and 200 mg of protonemal tissue from each experiment were frozen in liquid nitrogen and ground to a powder, after which 2 ml of 80% (v/v) acetone was added. After extraction at 4 °C for 24 h in the dark, the samples were centrifuged, and the resulting supernatant was diluted 50-fold and 100-fold in TBS. ABA was quantified by competitive ELISA using the Phytodetek ABA kit (Sigma) using (+)cis/trans abscisic acid (Sigma) as a standard.
Statistical analysis
The significances of the observed differences between the wild type and the hxk1 mutant were tested using a two-tailed two-sample t-test assuming unequal variances.
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Results |
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Protonemal morphology of wild-type and hxk1 mutant strains under different growth conditions
The finding that caulonemal filament formation in Physcomitrella is enhanced in the presence of externally added glucose, and that this effect is suppressed in a hxk1 knockout mutant (Olsson et al., 2003
), suggested a link between developmental regulation and the energy supply. This prompted a more detailed investigation as to how growth and development of protonemal tissue are affected by different external conditions both in the wild type and in the hxk1 mutant. Accordingly, wild-type and hxk1 mutant colonies grown in high light (30 µmol m–2 s–1) or low light (6 µmol m–2 s–1) on media containing different additives were examined. Several independent colonies from both the mutant and the wild type were examined. Figure 2 shows pictures of representative colonies. The average diameter of several colonies (with standard error of means) for each treatment is shown below the pictures. The most striking phenotype of the hxk1 mutant is its previously noted reduced number of caulonemal filaments in the presence of 0.15 M glucose (Olsson et al., 2003). However, there are several more subtle differences between the mutant and the wild type. Thus, hxk1 mutant colonies generally have a somewhat smaller diameter than the wild type, indicating a reduced growth rate. Furthermore, the periphery of the hxk1 mutant colonies are characterized by very dense growth, and the edge of the colony, which is distinct already in the wild type, is even smoother in the hxk1 mutant.
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The hxk1 mutant is hypersensitive to cytokinin
Interestingly, the hxk1 mutant also exhibits altered sensitivities to plant hormones. Thus, the average colony diameter is reduced by 34% compared with the wild type in the presence of 1 µM 6-benzylaminopurine (BAP), indicating a hypersensitivity of the mutant to this cytokinin (Fig. 2). Cytokinin treatment reduces the average size of wild-type colonies as well, but the effect is clearly more pronounced in the hxk1 mutant (Fig. 2). By contrast, no obvious difference was seen between the wild type and the hxk1 mutant in the response to the auxin 1-naphthaleneacetic acid (1-NAA). The latter treatment reduces colony size of both the wild type and the hxk1 mutant under low light, but not under high light conditions (Fig. 2).
The effect of cytokinin on the growth of the wild type as well as the hxk1 mutant was further investigated in a cytokinin dose–response assay (Fig. 3). Low concentrations of cytokinins have been reported to induce bud formation in Physcomitrella, while higher concentrations inhibit growth and induce the formation of callus-like tissue (Ashton et al., 1979). Consistent with this, severe callus formation and a slightly reduced colony diameter in the wild type were observed at cytokinin concentrations above 1 µM (Fig. 3A). The hxk1 mutant is cytokinin hypersensitive with pronounced callus formation and growth reduction evident already at 0.1 µM cytokinin (Fig. 3A). A quantification of the growth inhibition (Fig. 3B) showed that the difference between the wild type and the hxk1 mutant increases with increasing cytokinin concentrations, up to the highest concentration tested (100 µM). The hxk1 mutant was also compared with the wild type in an auxin sensitivity test. The results showed that both the wild type and the mutant are similarly affected by increasing amounts of 1-NAA (data not shown). This confirms that the hxk1 mutant responds normally to auxin while it is hypersensitive to cytokinin.
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The hxk1 mutant is hypersensitive to abscisic acid
As shown in Fig. 2, it was further found that the hxk1 mutant is hypersensitive to the plant hormone abscisic acid (ABA). Thus, a reduction in the colony diameter by 37% compared with the wild type is seen in the presence of 1 µM ABA. The effects of ABA were further investigated in a dose–response assay where the hxk1 mutant was compared with the wild type. Figure 3C shows that the inhibitory effect of ABA on wild-type colony size is dose-dependent, ranging from a minor effect at 0.1 µM to almost complete growth inhibition at 10 µM ABA. Furthermore, the hxk1 mutant shows a clear hypersensitivity to the effects of ABA on colony size. A quantification of this effect (Fig. 3D) revealed that it is most pronounced at 1 µM ABA. At 10 µM ABA no difference is seen, since the wild type is already fully growth inhibited at that point.
Light microscopy analysis of chloronemal cells grown under standard conditions (in the absence of plant hormones) revealed that the hxk1 mutant cells have a somewhat swollen appearance compared with the wild type (Fig. 3E). ABA is known to cause a change in cell shape, producing thicker and less elongated protonemal cells, known as brood cells (Goode et al., 1992). This prompted the investigation of the cell shape in the presence of ABA. It was found that both the wild type and the hxk1 mutant respond to 1 µM ABA in the medium by cell swelling, but the effect is much more pronounced in the mutant (Fig. 3E). The differences in cell shape were quantified by measuring the length-to-width ratio of a large number of chloronemal cells (Fig. 3F). This confirmed that a significant difference exists between the hxk1 mutant and the wild type, which is further emphasized in the presence of externally added ABA.
The finding that the hxk1 mutant exhibits slightly swollen cells also in the absence of ABA suggested that ABA signalling might be constitutive in the hxk1 mutant, either due to hyper-signalling or due to elevated endogenous ABA levels. Therefore a test to examine whether the mutant had altered ABA levels was performed. ABA was measured in extracts of protonemal tissue from the wild type and hxk1 mutant grown for 24 h on BCD plates containing either no additive, 0.15 M glucose or, as an osmotic control, 0.15 M mannitol. As shown in Fig. 3G, it was found that both the wild type and the hxk1 mutant show a moderate increase in endogenous ABA after growth on glucose. This effect is also triggered by the non-metabolized sugar mannitol, which suggests that it is due to the well-known role of ABA in the osmotic response (Zhu, 2002). No significant differences were seen between the wild type and the hxk1 mutant under any of the conditions tested (Fig. 3G). It was concluded that the cell-shape phenotypes of the hxk1 mutant are not mediated by changes in endogenous ABA levels.
Caulonema formation is stimulated by glucose, high light and auxin, but inhibited by cytokinin
In order to understand why externally added glucose stimulates the formation of caulonemal filaments (Olsson et al., 2003), it was next investigated how caulonemal filament formation is affected by different treatments in both the wild type and in the hxk1 mutant. Caulonema formation is known to require relatively high light intensities (Reski, 1998; Schumaker and Dietrich, 1997). It was therefore reasoned that the two effects might be mediated by a common mechanism, since sugars are the main products of photosynthesis. To test this hypothesis, it was examined how caulonema formation in the wild type as well as in the hxk1 mutant is affected by different conditions such as high or low light and externally supplied glucose, cytokinin or auxin. As expected, caulonema formation was largely absent under low light conditions, although a few caulonemal filaments were seen in the presence of glucose. For colonies grown in high light, the effects of different treatments were quantified by counting the total number of caulonemal filaments protruding beyond the rim of the colony (Fig. 4). As previously reported (Olsson et al., 2003) it was found that externally added glucose strongly enhances caulonema formation in the wild-type strain. Thus, 0.15 M glucose increases the average number of caulonemal filaments per colony 9.8-fold, from 9.8 to 97. The addition of 1 µM 1-NAA increased the number of caulonemal filaments 3.8-fold, to 37 per colony, whereas the addition of 1 µM BAP virtually abolished caulonema formation (Fig. 4). It was concluded that caulonema formation during growth in high light is stimulated by auxin and strongly inhibited by cytokinin. These results are consistent with previous findings that auxins stimulate caulonemal growth while cytokinins suppress it (Ashton et al., 1979; Reski, 1998).
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Caulonema formation is strongly reduced in the hxk1 knockout mutant
It has previously been reported that the caulonema formation that is induced by 0.15 M glucose is greatly diminished in the hxk1 mutant (Olsson et al., 2003
). This is also evident in Fig. 2. However, the present analysis revealed that caulonema formation is impaired in the hxk1 mutant under all the conditions tested, and not only in the presence of glucose, where it is more easily observed (Fig. 4). Thus, the effect of the hxk1 knockout in the absence of additives is a 5.8-fold reduction in the number of caulonemal filaments, from 9.8 to 1.7 per colony. In the presence of glucose, it reduces the number of caulonemal filaments 9.3-fold, to 10.3, and in the presence of auxin it reduces the number 6.2-fold, to 6.0 filaments per colony (Fig. 4). No caulonema formation was observed in the hxk1 mutant during growth in low light, either in the presence or in the absence of glucose. From these experiments it was concluded that the basal rate of caulonema formation is reduced several-fold in the hxk1 mutant compared with the wild type, irrespective of growth conditions and the resulting wild-type frequencies of caulonema formation. It therefore appears that the formation of caulonemal filaments is generally impaired in the hxk1 mutant.
Chloronemal branching is stimulated by cytokinin but inhibited by auxin, glucose and high light
Chloronemal filaments grow by apical cell divisions and subapical branching. As illustrated in Fig. 5A, branching frequencies can be highly variable. Furthermore, individual branches are frequently longer under conditions where branching is sparse. In order to find out how chloronemal growth is affected by different external factors, both branching frequencies (Fig. 5B) and side branch lengths (Fig. 5C) were measured in wild-type chloronema exposed to high and low light, glucose, auxin and cytokinin. It was found that high light compared with low light caused a slight but significant decrease (by 21%) in the branching frequency, while no significant difference was seen for side-branch lengths. In low light, auxin and glucose both reduced the branching frequency by 21% and 47%, respectively, while cytokinin enhanced chloronemal branching by 63%. Similar effects were seen in high light, where auxin and glucose reduced the branching frequency by 50% and 21%, respectively, and cytokinin stimulated it by 53%. The effects on side-branch lengths were less pronounced, although a slight inverse correlation with branching frequencies was seen in several cases (Fig. 5C). There was one notable exception, however. Thus, when 0.15 M glucose was added to colonies grown in low light, a 2.4-fold increase in the average number of cells per chloronemal side branch was seen. A similar but smaller effect (a 35% increase) is seen when glucose is added to colonies grown in high light. From these observations it was concluded that high light and auxin both reduce chloronemal branching, while cytokinin instead stimulates it. Glucose has a more complex effect, since it reduces the chloronemal branching frequency but increases the number of cells per chloronemal side branch.
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Chloronemal branching is enhanced in the hxk1 knockout mutant
It is possible that an increased branching frequency at the expense of branch lengths might explain why the hxk1 mutant colonies have both reduced diameters and a more dense morphology (Fig. 2). To test this hypothesis, it was examined how chloronemal branching frequencies and side branch lengths are affected by high or low light, glucose and phytohormones in the hxk1 mutant. As shown in Fig. 5B, it was found that the number of branches per chloronemal cell is enhanced in the hxk1 mutant under all conditions tested. For example, there is a 1.9-fold increase in high light and a 1.8-fold increase in low light, compared with the wild type. This is consistent with the notion that increased branching contributes to the dense colony morphology of the hxk1 mutant. The most pronounced effect is seen in the presence of auxin and low light, where the hxk1 mutation causes a 2.6-fold increase in the branching frequency. Chloronemal side-branch lengths correlate inversely with the effects on branching frequencies; i.e. branch lengths are generally reduced in the hxk1 mutant (Fig. 5C). The effect is moderate but in most cases significant. It was concluded that the effect of the hxk1 mutation resembles that of externally added glucose in that both branching frequencies and average side-branch lengths are affected, but in opposite ways. However, the effects of the hxk1 mutation are the very opposite of glucose in both cases.
The hxk1 knockout mutation promotes atypical bud formation on chloronemal filaments
An unexpected finding was that the hxk1 knockout mutant showed significant bud formation on chloronemal filaments (Fig. 5D). Such chloronemal buds are seen sporadically in the wild type, and their abundance is increased in the presence of cytokinin (Fig. 5E), which is known generally to stimulate bud formation (Reski and Abel, 1985). These wild-type chloronemal buds are, however, only seen on chloronemal filaments that are directly derived from a caulonemal filament, the normal site for bud formation. By contrast, the hxk1 mutant has a significant number of chloronemal buds already under normal growth conditions, and their abundance is not increased further in the presence of cytokinin (Fig. 5E). Furthermore, chloronemal buds in the hxk1 mutant are not restricted to caulonema-derived chloronemal filaments, but are also found on chloronema-derived filaments. It was further found that the addition of 1 µM 1-NAA completely blocked the formation of chloronemal buds, both in the wild type and in the hxk1 mutant (Fig. 5E). This is consistent with previous findings that auxin inhibits normal bud development (Ashton et al., 1979). It was concluded that the developmental program that controls bud formation is disturbed in the hxk1 mutant.
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Discussion |
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The present investigation has examined how the balance between chloronemal and caulonemal growth in Physcomitrella protonemata is affected by external conditions such as high or low light, glucose, auxin, and cytokinin has been investigated. These experiments were performed both in a wild-type strain and in a hxk1 knockout mutant which lacks the major hexokinase of Physcomitrella and therefore has a strongly reduced hexose phosphorylation (Olsson et al., 2003
). These results show that caulonema formation is stimulated by high light, glucose, and auxin, but inhibited by cytokinin, and in the hxk1 mutant (Fig. 4). Conversely, chloronemal branching is reduced in the presence of high light, glucose, and auxin, but enhanced in the presence of cytokinin, and in the hxk1 mutant (Fig. 5). The effects on caulonema formation and chloronemal branching are thus opposite for all the above treatments. This suggests that a regulated balance exists between the two filament types, and that conditions which enhance the formation of one of them generally inhibit the other. It does not necessarily mean that different factors affect this balance through the same mechanisms or that filaments of a similar type that are induced by different treatments should be considered as identical. On the contrary, caulonemata that are induced by auxin differ from those seen in the presence of glucose in that they do not reach out nearly as far from the rest of the colony (Fig. 2). Caulonemal filaments that are induced by glucose are also more heavily pigmented than those induced by auxin.
Interestingly, the balance is shifted towards caulonema formation by high light and glucose, both of which will increase the available energy and carbon supply (that 0.15 M glucose can serve as an adequate external source of carbon and energy is shown by the fact that Physcomitrella can grow in the dark on 0.15 M glucose). Conversely, low light and the hxk1 mutation both inhibit caulonemal growth and stimulate chloronemal branching, thus shifting the balance towards more chloronemata. PpHxk1 is the major glucose phosphorylating enzyme in Physcomitrella, and accounts for 80% of the enzymatic activity in protonemal tissue (Olsson et al., 2003). One would therefore expect the hxk1 knockout to significantly reduce in vivo hexose phosphorylation, thereby also reducing ATP production and creating a condition of artificial starvation. This is consistent with the finding that hxk1 mutant colonies behave similarly to colonies grown in low light, i.e. by enhancing chloronemal branching and reducing caulonemal growth. Taken together, these findings suggest that the available energy supply affects the balance between the two filament types (Fig. 6). Such regulation makes sense since the increased chloronema formation that is stimulated by low energy conditions can help to restore the energy supply due to the higher photosynthetic activity of chloronemata. Conversely, if energy is readily available, the colony can afford to form caulonemata which promotes its radial growth and also paves the way for the next stage in the life cycle, i.e. budding and leafy shoot formation. According to this model, the balance between chloronemal and caulonemal growth would therefore be homeostatically regulated to provide sufficient energy for metabolism and growth during different conditions (Fig. 6).
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An important question is how the available energy supply is sensed. In this context, it is interesting to note that the Arabidopsis Type B hexokinase AtHxk1 has been proposed to be involved in hexose sensing and signalling (Moore et al., 2003
). It could be argued that a similar function for PpHxk1 might explain the observed phenotype since an inability to sense hexoses, whether externally added or generated in vivo from photosynthesis, could also produce a condition of artificial starvation where the cell would adopt a low energy growth mode. However, this is not a likely explanation. PpHxk1 differs from AtHxk1 in that it is a Type A hexokinase that resides inside the chloroplast stroma, which makes it less likely to be involved in signal transduction (Olsson et al., 2003). More significantly, even though it has a reduced caulonema formation and an increased frequency of chloronemal branching, the hxk1 mutant still responds to changes in the energy supply (light or glucose) to the same approximate degree as the wild type. This is true both for caulonema formation (Fig. 4) and chloronemal branching (Fig. 5), and it suggests that any sensing and signalling required to elicit these responses is still intact in the hxk1 mutant.
A possible candidate for sensing the energy level is Snf1-related protein kinase 1 (SnRK1), a plant homologue of the yeast Snf1 kinase (Halford and Hardie, 1998; Hardie et al., 1998; Halford et al., 2003). The Snf1 kinase (Celenza and Carlson, 1986), which plays a key role in energy homeostasis, is inactivated by glucose in a way that requires the major yeast hexokinase Hxk2 (Entian, 1980; Johnston, 1999). This epistatic relationship causes the yeast snf1 and hxk2 mutants to have opposite phenotypes: a snf1 mutant is constitutively glucose repressed while a hxk2 mutant is constitutively derepressed (Johnston, 1999). Two Physcomitrella genes, PpSNF1a and PpSNF1b, encoding Snf1-related kinases belonging to the SnRK1 subfamily (Thelander et al., 2004) have recently been cloned and characterized. Interestingly, it was found that a snf1a snf1b double knockout mutant suffered from a constitutive high energy growth mode, which made it unable to grow in low light or darkness. The phenotypes of the double mutant are to a large extent the opposite of what was seen in the hxk1 mutant, with a striking reduction of chloronemata and an excess of caulonemata. Furthermore, the snf1a snf1b double mutant is resistant to cytokinin. The opposite phenotypes of the hxk1 and snf1a snf1b knockouts suggest that hexokinase may function upstream of the Snf1-related kinases in plants as well. However, it does not necessarily mean that PpHxk1 functions as a glucose sensor. The hxk1 mutation may simply cause a reduced metabolic flux which is sensed by SnRK1 downstream of glucose phosphorylation.
Another important question is how the observed changes in the frequencies of caulonemal and chloronemal filaments arise. Increased caulonema formation could be due either to a developmental switch from chloronema to caulonema, or to the enhanced growth of caulonemal filaments already present in the colony. Auxins are known to induce the chloronema to caulonema transition but, as noted above these caulonemal filaments differ qualitatively from those seen in the presence of glucose. Therefore it is probable that the stimulatory effect of glucose on caulonema formation is primarily due to an increase in caulonemal growth rate, although a direct effect on caulonema induction cannot be ruled out. As for the effects on chloronemal branching, it should be noted that the number of branches per chloronemal cell reflects the net contribution of two opposing factors, the growth rate and the rate at which new branches are formed, and that both are probably affected by light intensity. High light is likely to cause an increase in the apical growth rate due to enhanced photosynthesis which, given a constant branching rate, will decrease the average number of branches per cell. By contrast, light will also enhance the number of side branches per chloronemal cell through cryptochrome-mediated down-regulation of the inhibitory effects of auxins on branching (Imaizumi et al., 2002). In any event, it appears from the results in Fig. 5 that light, auxin, or cytokinin mainly cause changes in the number of branches per chloronemal cell, while glucose and the hxk1 mutant also affect the number of cells per chloronemal side branch.
The observation that auxin stimulates and cytokinin inhibits caulonema formation (Fig. 4) is consistent with previous findings by Ashton et al. (1979). In the present investigation, it was further found that the two plant hormones have opposite effects on chloronemal branching, which is instead stimulated by cytokinin and inhibited by auxin (Fig. 5B). The inhibitory effect of auxin on chloronema formation has been described (Ashton et al., 1979), but the fact that chloronemal branching is enhanced by cytokinin has, as far as we know, not been reported previously. Interestingly, it was found that this effect is completely blocked in the hxk1 mutant. Thus, the higher level of chloronemal branching that is seen in the hxk1 mutant is not further elevated in the presence of cytokinin (Fig. 5B). One possible interpretation of this result is that cytokinin acts to relieve Hxk1-dependent inhibition of chloronemal branching. Further evidence of functional interaction between Hxk1 and cytokinin signalling comes from the finding that the hxk1 mutant is hypersensitive to cytokinin (Fig. 3). Interestingly, two point mutations in the Arabidopsis hexokinase AtHxk1 also caused cytokinin hypersensitivity (Moore et al., 2003). However, these mutants were also hypersensitive to auxin, which is not the case for the hxk1 knockout mutant.
The finding that the hxk1 mutant has a significant number of chloronemal buds (Fig. 5E) provides another connection between hexokinase and cytokinin, since cytokinin treatment also induces chloronemal buds in the wild type (Fig. 5E). The effect of the hxk1 mutant is, however, more pronounced both in its magnitude and in that it also induces bud formation on chloronema-derived chloronemal filaments, something which is never seen in the wild type, not even in the presence of cytokinin. It should also be noted that cytokinin does not further increase chloronemal bud formation in the hxk1 mutant, which is instead somewhat reduced (Fig. 5E). This is analogous to the effects on chloronemal branching (see above) and suggests that cytokinin and hexokinase may affect bud formation through a common mechanism. Finally, it was noted that no chloronemal buds were formed in the presence of auxin, in either the wild type or the hxk1 mutant. The inhibitory effect of auxin on bud development (Ashton et al., 1979) is thus epistatic to the effect of the hxk1 mutation. One possible interpretation of this is that the auxin-controlled step in bud formation is downstream of the step affected by hexokinase and cytokinin.
The hxk1 mutant is also hypersensitive to ABA as shown both by growth inhibition (Fig. 3C) and the effect on cell shape (Fig. 3E). It has previously been reported that Arabidopsis mutants that are deficient in endogenous ABA production have a glucose-insensitive phenotype (Arenas-Huertero et al., 2000). Moreover, treatment of Arabidopsis seedlings with 6% glucose caused a 7-fold increase in endogenous ABA (Cheng et al., 2002). This suggested the possibility that the ABA sensitivity of the hxk1 mutant could be due to an effect on endogenous ABA levels. However, it was found that externally added glucose only had a marginal effect on endogenous ABA levels in Physcomitrella, and no significant differences were seen between the hxk1 mutant and the wild type (Fig. 3G). The small increase in ABA that is caused by glucose is most likely an osmotic effect since the non-metabolized sugar mannitol gave similar results. It thus seems that the effect of externally added glucose on ABA levels that has been reported in Arabidopsis (Cheng et al., 2002) is absent in Physcomitrella. The hypersensitivity of the hxk1 mutant to ABA is therefore more likely to reflect an effect on the detection or transduction of the ABA signal.
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This work was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS).
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Abbreviations: ABA, abscisic acid; BAP, 6-benzylaminopurine; 1-NAA, 1-naphthaleneacetic acid.
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