Journal of Experimental Botany, Vol. 52, No. 355, pp. 251-255,
February 2001
© 2001 Oxford University Press
Original Papers |
Fusicoccin- and IAA-induced elongation growth share the same pattern of K+ dependence
Institut für Allgemeine Botanik der Universität, Ohnhorststr. 18, D-22609 Hamburg, Germany
Received 14 June 2000; Accepted 22 August 2000
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Abstract |
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The dependence of growth induced by the fungal toxin fusicoccin (FC) on the K+ content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca2+ was included in the bathing medium, no FC-induced growth occurred in the absence of K+, whereas a strong response was detected in presence of K+. The optimal K+ concentration was in the range of 1–10 mM. With the exception of Rb+, none of the other alkali ions (Na+, Li+, Cs+) could replace for K+ in sustaining FC-induced growth. The potassium channel blocker tetraethylammonium (TEA) reversibly inhibited FC-induced growth. As shown earlier for auxin-induced growth, no strict potassium dependence of FC-triggered elongation was observed in Ca2+-free media. However, TEA abolished this apparently K+ independent FC-induced growth. It is concluded that FC-induced growth, like auxin-induced growth, requires K+ uptake through K+ channels.
Key words: Fusicoccin, auxin, channel blocker, potassium channel, elongation growth.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Auxin-induced growth, if measured in solutions containing sufficient amounts of calcium ions, strictly depends on the presence of K+ ions in the medium. This finding (Claussen et al., 1997
) suggests that an influx of K+ ions serves as a charge compensation for the protons exported by the H+-ATPase, which is required for an apoplasmic acidification. Additionally, K+ uptake through K+ channels may contribute to the osmotic regulation essential for the generation of turgor pressure, which is the driving force of growth (Thiel et al., 1996; Tester, 1996).
Recent molecular and electrophysiological investigations underline the physiological significance of K+ uptake by K+ inward channels in auxin action. It has been shown that auxin induces the expression of ZmK1, a K+ channel gene in maize coleoptiles (Philippar et al., 1999), leading to an increase in channel density in the plasma membrane (Thiel and Weise, 1999). These effects occur on time scales consistent with those of the growth response.
A non-auxin substance that triggers growth by stimulating H+ efflux driven by the plasma membrane ATPase (Marrè, 1979, Palmgren, 1998) is the fungal toxin fusicoccin (FC). By increasing the electrochemical gradient across the plasma membrane, fusicoccin treatment also results in an increased potassium influx (Marrè et al., 1974). This charge compensation is a prerequisite for FC-induced apoplasmic acidification which, by loosening the cell wall, causes FC-induced growth. This acid growth theory (Hager et al., 1971) has been also proposed for auxin-induced growth. If correct, the importance of charge compensation to the cell wall acidification and, consequently, growth should be similar in auxin- and fusicoccin-induced elongation. However, there are reports that auxin- and fusicoccin-induced growth differ vastly in their dependencies on the ionic composition of the apoplast. In particular, it has been claimed that FC-induced growth, but not auxin-induced elongation, depends on potassium (Terry and Jones, 1981; Kutschera and Schopfer, 1985a, b), in apparent contrast to what would be predicted by the acid growth theory of auxin action.
Recently, much of the confusion on the ionic dependencies of auxin-induced growth has been eliminated (Claussen et al., 1997). These authors found that auxin-induced growth does in fact strongly depend on K+, but only if Ca2+ or other divalent cations are included in the bathing medium. Auxin-induced growth did occur in K+-free bathing media or in distilled water in the absence of divalent cations. This explained earlier results reporting no apparent K+ dependence (Terry and Jones, 1981). However, they also found that the apparently K+-independent growth required the activity of K+ channels, since it was entirely abolished by TEA and other K+ channel blockers. This has led to the idea that K+ ions, bound in the Donnan space, can feed the potassium channels for some hours even in the absence of K+ in the bathing medium. The effect of Ca2+ has been proposed to reflect an inhibition of the K+ channel, shifting its concentration dependence to higher K+ concentrations (Thiel et al., 1996). Alternatively, Ca2+ ions may act by replacing K+ ions from the Donnan space, thereby depleting the apoplastic calcium stores (Claussen et al., 1997).
The FC side of the problem requires a critical reanalysis in the light of the new auxin data. In the present paper the following questions are addressed. (1) How does FC-induced growth depend on K+ ions in the bathing medium? (2) Can other alkali cations replace K+? (3) Do potassium channel blockers act on FC-induced growth in the way they block auxin-induced elongation? (4) Do Ca2+ ions affect the K+ dependence as in auxin-induced growth?
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Coleoptile preparation
Caryopses of maize (Zea mays L. cv. Garant, Saaten-Union, Hannover, Germany) were soaked in running tap water and grown as previously described (Lüthen et al., 1990
). Coleoptiles were harvested and abraded with SiC powder. Efficiency of abrasion was routinely monitored by staining with Neutral Red for abraded cells and with Evans Blue for killed cells as described before (Lüthen et al., 1990; Claussen et al., 1997). For this study, coleoptiles with 40% abraded cells were used. There were no detectable dead cells except at the cut surfaces.
Growth measurements
Rapid growth responses were detected in a multichannel auxanometer (Lüthen and Böttger, 1992). Briefly, growth of a column of four coleoptiles was measured by means of positional angular transducers and recorded with a computer via a custom AD-converter card. The set-up allowed six independent growth recordings. Each measuring cuvette contained 4 ml of aerated incubation buffer. Tracings shown in this paper reflect typical results of at least five independent experiments.
Incubation media and chemicals
The ionic composition of the incubation media is shown in Table 1. Deviations from the concentrations listed there are indicated in the text. During the experiments, additional salts were added as indicated. It was possible to exchange the buffer solutions rapidly by means of a pump without disturbing the measurements.
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Fusicoccin was purchased from Sigma and added from a 0.1 mM stock solution prepared in isopropanol to yield the desired final concentration. The maximal isopropanol concentration of 0.5% did not affect growth (not shown). Indole-3-actic acid (IAA) stocking solution were prepared from IAA-potassium salt (Merck). The maximal resulting contamination with K+ ions (5 µM) is below the threshold of the K+ dependence of growth induced by IAA (Claussen et al., 1997
, Fig. 2) by at least two orders of magnitude.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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K+ dependence of fusicoccin-induced growth in media containing Ca2+
Figure 1
shows the action of FC in +K/+Ca and -K/+Ca buffers. The time-courses of IAA- and FC-induced growth were different in detail, in particular in FC-induced growth the lag phase was much shorter than in auxin-induced elongation. It is evident that FC-induced growth only occurred when K+ was included to the medium (+K/+Ca buffer, Fig. 1A). FC did not induce growth in a -K/+Ca buffer (Fig. 1B), but subsequent addition of 10 mM K+ (yielding a +K/+Ca buffer) instantly triggered a growth response. This shows that in Ca2+-containing buffers FC-induced growth is strictly dependent on the presence of K+. The dependence of FC-induced growth on the K+ concentration is shown in Fig. 2
. As it has been already shown for auxin-induced growth (Fig. 2 in Claussen et al., 1997) K+ concentrations in a range of 1–50 mM were optimal. Supraoptimal concentrations were inhibitory, probably because of the excessive osmolarity of the incubation medium.
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). In +K/+Ca buffer an FC-induced growth burst occurs upon addition of 1 µM FC (
). (B) When 1 µM FC is added to coleoptiles incubated in –K/+Ca buffer, no FC-induced growth is detected. Subsequent addition of 10 mM KCl (yielding +K/+Ca buffer) at the second arrow induces a growth response (
Ionic specificity
Figure 3 shows the activity of other monovalent cations to substitute for K+. Plants were first treated with FC in a -K/+Ca buffer. No growth occurred. Monovalent cations (10 mM) were added and the resulting growth responses were recorded. The data show that Rb+, a well-known analogue of K+ and a substrate for K+ channels, had a similar effect as K+ (Fig. 3A). In some individual experiments it was even more effective than K+. The other tested alkali ions (Na+, Li+, and Cs+; Fig. 3B) were inactive. The sequence of activity was K+=Rb+>>Na+, Li+, Cs+, similar to that reported for auxin-induced growth (Claussen et al., 1997) and closely matching the selectivity patterns of plant K+ channels (Hedrich and Dietrich, 1996, and references cited therein).
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). The controls indicate growth rates of coleoptile segments with FC in –K/+Ca buffer (
) and in the absence of FC (
), Li+ (
) and Cs+ (
Effect of Ca2+ on K+ dependence
As mentioned above, auxin-induced growth occurs in K+-free solutions of low ionic strength, like distilled water (Terry and Jones, 1981). Addition of divalent cations like Ca2+ completely abolishes this response, making the auxin response strictly dependent on the presence of K+ ions (Claussen et al., 1997). Figure 4 shows the influence of Ca2+ on the K+ dependence of FC-induced growth (Fig. 4A–C) in direct comparison to IAA-induced growth (Fig. 4D–F). However, FC-induced growth (Fig. 4A), like auxin-induced elongation (Fig. 4D) occurred in the absence of external K+ in a -K/-Ca buffer, but not in -K/+Ca buffer (Fig. 4B, E). In a +K/+Ca buffer a full growth response was observed (Fig. 4C, F). For both FC and IAA, the presence of Ca2+ made the growth response K+-dependent. However, in the presence of both K+ and Ca2+, the auxin-induced growth response was much less pronounced than FC-induced growth (Fig. 4C, F).
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Activity of potassium channel blockers
Figure 5 shows the action of 30 mM TEA on growth induced by FC (Fig. 5A) and IAA (Fig. 5B), measured in a -K/-Ca buffer. It is evident that TEA completely abolished growth in both cases. Similar results were also obtained in +K/+Ca buffers containing 1 mM K+ (results not shown). The blockade of both FC- and IAA-induced growth by the channel inhibitor was completely reversible, since removal of TEA from the solution restored the increased growth rates. Similar effects were obtained with other channel blockers (Cs+, Ba2+; data not shown).
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Comparison of the ionic dependencies of FC- and auxin-induced growth
Both auxin and FC-triggered elongation (1) require the presence of K+ ions in media containing Ca2+, (2) share an ionic specificity for monovalent cations that resembles K+ selectivity of K+ channels, and (3) are abolished by K+ channel blockers.
Thus, as with auxin-induced growth, FC-induced elongation appears to be linked to the activity of K+ inward channels.
Auxin- and FC-induced growth occur in K+-free media lacking calcium. However, in both cases growth is completely abolished by TEA, suggesting the requirement for K+ channel activity for the growth process (Fig. 5). This suggests that this apparently K+-independent growth can be fed by K+ ions bound to negative surface charges in the Donnan space. The question whether calcium acts by exchanging for K+ in the Donnan space or by channel modulation will be addressed in another publication. An indirect action of extracellular calcium on potassium channels by modulating the intracellular calcium levels appears improbable, since recent experiments with caged Ca2+ did not show any effect of K+ channel activity (Thiel and Weise, 1999).
In the presence of Ca2+ and K+ the growth rates induced by fusicoccin exceed those triggered by auxin. This is consistent with the fact that FC-induced acidification surpasses IAA-induced acidification (Kutschera and Schopfer, 1985b; Lüthen et al., 1990) and tentatively supports the acid growth theory of both, IAA and FC action. The detailed mechanisms through which FC and IAA bring about cell wall acidification and growth are surely very different. This is reflected by the slight differences between the time-courses of the responses, and by the known fact that FC is unable to trigger ZmK1 expression (Philippar et al., 1999).
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG), grant BO 537.16-1.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 40 428 16 254. E-mail: h.luthen{at}botanik.uni\|[hyphen]\|hamburg.de
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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