Fusicoccin- and IAA-induced elongation growth share the same pattern of K q dependence

Transcription

1 Journal of Experimental Botany, Vol. 52, No. 355, pp. 251±255, February 2001 Fusicoccin- and IAA-induced elongation growth share the same pattern of K q dependence Katrin Tode and Hartwig LuÈ then 1 Institut fuè r Allgemeine Botanik der UniversitaÈ t, Ohnhorststr. 18, D Hamburg, Germany Received 14 June 2000; Accepted 22 August 2000 Abstract The dependence of growth induced by the fungal toxin fusicoccin FC) on the K q content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca 2q was included in the bathing medium, no FC-induced growth occurred in the absence of K q, whereas a strong response was detected in presence of K q. The optimal K q concentration was in the range of 1±10 mm. With the exception of Rbq, none of the other alkali ions Na q, Li q, Cs q ) could replace for K q 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 Ca 2q -free media. However, TEA abolished this apparently K q independent FC-induced growth. It is concluded that FC-induced growth, like auxininduced growth, requires K q uptake through K q channels. Key words: Fusicoccin, auxin, channel blocker, potassium channel, elongation growth. Introduction Auxin-induced growth, if measured in solutions containing suf cient amounts of calcium ions, strictly depends on the presence of K q ions in the medium. This nding Claussen et al., 1997) suggests that an in ux of K q ions serves as a charge compensation for the protons exported by the H q -ATPase, which is required for an apoplasmic acidi cation. Additionally, K q uptake through K q 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 signi cance of K q uptake by K q inward channels in auxin action. It has been shown that auxin induces the expression of ZmK1, a K q 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 q ef ux driven by the plasma membrane ATPase MarreÁ, 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 in ux MarreÁ et al., 1974). This charge compensation is a prerequisite for FC-induced apoplasmic acidi cation 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 acidi cation and, consequently, growth should be similar in auxinand 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 auxininduced growth does in fact strongly depend on K q, but only if Ca 2q or other divalent cations are included in 1 To whom correspondence should be addressed. Fax: q h.luthen@botanik.uni-hamburg.de ß Society for Experimental Biology 2001

2 252 Tode and LuÈ then the bathing medium. Auxin-induced growth did occur in K q -free bathing media or in distilled water in the absence of divalent cations. This explained earlier results reporting no apparent K q dependence Terry and Jones, 1981). However, they also found that the apparently K q -independent growth required the activity of K q channels, since it was entirely abolished by TEA and other K q channel blockers. This has led to the idea that K q ions, bound in the Donnan space, can feed the potassium channels for some hours even in the absence of K q in the bathing medium. The effect of Ca 2q has been proposed to re ect an inhibition of the K q channel, shifting its concentration dependence to higher K q concentrations Thiel et al., 1996). Alternatively, Ca 2q ions may act by replacing K q 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 q ions in the bathing medium? 2) Can other alkali cations replace K q? 3) Do potassium channel blockers act on FCinduced growth in the way they block auxin-induced elongation? 4) Do Ca 2q ions affect the K q dependence as in auxin-induced growth? Materials and methods 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 LuÈthen et al., 1990). Coleoptiles were harvested and abraded with SiC powder. Ef ciency of abrasion was routinely monitored by staining with Neutral Red for abraded cells and with Evans Blue for killed cells as described before LuÈthen 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 LuÈthen and BoÈttger, 1992). Brie y, growth of a column of four coleoptiles was measured by means of positional angular transducers and recorded with a computer via a custom ADconverter card. The set-up allowed six independent growth recordings. Each measuring cuvette contained 4 ml of aerated incubation buffer. Tracings shown in this paper re ect typical results of at least ve 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. Fusicoccin was purchased from Sigma and added from a 0.1 mm stock solution prepared in isopropanol to yield the desired nal 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 q ions 5 mm) is below the threshold of the K q dependence of growth induced by IAA Claussen et al., 1997, Fig. 2) by at least two orders of magnitude. Results and discussion K q dependence of fusicoccin-induced growth in media containing Ca 2q Figure 1 shows the action of FC in qkuqca and KuqCa buffers. The time-courses of IAA- and FCinduced 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 FCinduced growth only occurred when K q was included to the medium qkuqca buffer, Fig. 1A). FC did not induce growth in a KuqCa buffer Fig. 1B), but subsequent addition of 10 mm K q yielding a qkuqca buffer) instantly triggered a growth response. This shows that in Ca 2q -containing buffers FC-induced growth is strictly dependent on the presence of K q. The dependence of FC-induced growth on the K q concentration is shown in Fig. 2. As it has been already shown for auxin-induced growth Fig. 2 in Claussen et al., 1997) K q concentrations in a range of 1±50 mm were optimal. Supraoptimal concentrations were inhibitory, probably because of the excessive osmolarity of the incubation medium. Ionic specificity Figure 3 shows the activity of other monovalent cations to substitute for K q. Plants were rst treated with FC in a KuqCa buffer. No growth occurred. Monovalent cations 10 mm) were added and the resulting growth responses were recorded. The data show that Rb q, a wellknown analogue of K q and a substrate for K q channels, Table 1. Composition of the various buffers Buffer designation Composition Ca 2q concentration K q concentration KuqCa 5 mm MES; add Ca OH) 2 to ph mm 0 qkuqca qcau K buffer; add 10 mm KCl 1.25 mm 10 mm Ku Ca 1 mm MES; add NaOH to ph Effective Na q concentration 0.6 mm qku Ca Cau K buffer; add 10 mm KCl 0 10 mm

3 Potassium and elongation growth in maize 253 Fig. 1. Fusicoccin-induced growth in the presence of Ca 2q ions. A) In KuqCa buffer, no FC-induced growth is detected j). In qkuqca buffer an FC-induced growth burst occurs upon addition of 1 mm FC h). B) When 1 mm FC is added to coleoptiles incubated in ±KuqCa buffer, no FC-induced growth is detected. Subsequent addition of 10 mm KCl yielding qkuqca buffer) at the second arrow induces a growth response h). Addition of KCl in the absence of FC does not induce any growth effect j). Fig. 2. Dependence of FC-induced growth on K q concentration in a qkuqca buffer. Means of steady-state growth of four independent experiments at each concentration are shown, FC concentration was 1 mm. More explanations in the text. had a similar effect as K q Fig. 3A). In some individual experiments it was even more effective than K q. The other tested alkali ions Na q, Li q, and Cs q ; Fig. 3B) were inactive. The sequence of activity was K q ˆ Rb q ))Na q,li q,cs q, similar to that reported for auxin-induced growth Claussen et al., 1997) and Fig. 3. Effect of various alkali cations on fusicoccin-induced growth. Coleoptiles were treated with 1 mm FCin±KuqCa buffer rst arrow). After an incubation time of 30 min, at the time of the second arrow, 10 mm of the indicated alkali chloride were added. A) Activity of the K q channel substrates K q m) and Rb q %). The controls indicate growth rates of coleoptile segments with FC in ±KuqCa buffer \) and in the absence of FC h). B) Effects of the alkali ions Na q k), Li q n) and Cs q j), which do not permeate K q inward channels. Obviously only Rb q can substitute for K q in maintaining the growth response, while Na q,li q and Cs q cannot. closely matching the selectivity patterns of plant K q channels Hedrich and Dietrich, 1996, and references cited therein). Effect of Ca 2q on K q dependence As mentioned above, auxin-induced growth occurs in K q -free solutions of low ionic strength, like distilled water Terry and Jones, 1981). Addition of divalent cations like Ca 2q completely abolishes this response, making the auxin response strictly dependent on the presence of K q ions Claussen et al., 1997). Figure 4 shows the in uence of Ca 2q on the K q 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 q in a Ku Ca buffer, but not in KuqCa buffer Fig. 4B, E). In a qkuqca buffer a full growth response was observed Fig. 4C, F). For both FC and IAA, the presence of Ca 2q made the growth response K q -dependent. However, in the presence of both K q and Ca 2q, the auxin-induced growth response was much less pronounced than FC-induced growth Fig. 4C, F). 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

4 254 Tode and LuÈ then Fig. 4. In uence of Ca 2q ions on K q dependence of FC-induced growth. A) Ku Ca buffer: In the absence of any K q in the incubation medium, 1 mm FC, added at the arrow, induces a full response. B) FC is ineffective when added in a KuqCa buffer. C) In a qkuqca buffer FC induces a full growth response. D), E) and F) show experiments equivalent to A), B) and C), respectively, in which growth was induced by addition of 5 mm IAA instead of 1 mm FC. in a Ku Ca buffer. It is evident that TEA completely abolished growth in both cases. Similar results were also obtained in qkuqca buffers containing 1 mm K q results not shown). The blockade of both FC- and IAAinduced 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 q,ba 2q ; data not shown). Conclusions Comparison of the ionic dependencies of FC- and auxin-induced growth Both auxin and FC-triggered elongation 1) require the presence of K q ions in media containing Ca 2q, 2) share an ionic speci city for monovalent cations that resembles K q selectivity of K q channels, and 3) are abolished by K q channel blockers. Thus, as with auxin-induced growth, FC-induced elongation appears to be linked to the activity of K q inward channels. Auxin- and FC-induced growth occur in K q -free media lacking calcium. However, in both cases growth is completely abolished by TEA, suggesting the requirement for K q channel activity for the growth process Fig. 5). This suggests that this apparently K q -independent growth can be fed by K q ions bound to negative surface charges in the Donnan space. The question whether calcium acts by exchanging for K q 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 Ca 2q did not show any effect of K q channel activity Thiel and Weise, 1999). In the presence of Ca 2q and K q the growth rates induced by fusicoccin exceed those triggered by auxin. This is consistent with the fact that FC-induced acidi cation surpasses IAA-induced acidi cation Kutschera and Schopfer, 1985b; LuÈ then 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 acidi cation and growth are

5 Fig. 5. Effect of 30 mm TEA on growth induced by FC A) and IAA B). The experiment was carried out in Ku Ca buffer. Auxin or fusicoccin were added at the rst arrow 1 mm FC to A), 5 mm IAA to B)). Growth was completely and instantaneously blocked by the addition of 30 mm TEA at the time of the second arrow. The effect was reversible, as indicated by the instant recovery after removal of TEA third arrow) by exchanging the buffer to a TEA-free solution and re-supplying the indicated concentrations of IAA or FC, respectively. For clarity, data points prior to the addition and removal of TEA were omitted in two curves. surely very different. This is re ected 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). Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft DFG), grant BO References Potassium and elongation growth in maize 255 Claussen M, LuÈthen H, Blatt M, BoÈttger M Auxininduced growth and its linkage to potassium channels. Planta 201, 227±234. Hager A, Menzel H, Krauss A Versuche und Hypothese zur PrimaÈrwirkung des Auxins beim Streckungswachstum. Planta 100, 47±75. Hedrich R, Dietrich P Plant K q channels: similarity and diversity. Botanica Acta 109, 94±101. Kutschera U, Schopfer P. 1985a. Evidence against the acid-growth theory of auxin action. Planta 163, 483±493. Kutschera U, Schopfer P. 1985b. Evidence for the acid-growth theory of fusicoccin action. Planta 126, 494±499. LuÈthen H, Bigdon M, BoÈttger M Re-examination of the acid growth theory of auxin action. Plant Physiology 93, 931±939. LuÈthen H, BoÈttger M A high-tech low-cost auxanometer for high-resolution determination of elongation rates in six simultaneous experimental setups. Mitteilungen des Institut fuèr Allgemeine Botanik Hamburg 24, 13±22. MarreÁ E Fusicoccin: a tool in plant physiology. Annual Review of Plant Physiology 30, 273±288. MarreÁ E, Lado P, Rasi Caldogno F, Colombo R, MarreÁ E Evidence for the coupling of proton extrusion to K q uptake in pea internode segments treated with fusicoccin or auxin. Plant Science Letters 3, 365±379. Palmgren MG Proton gradients and plant growth. Role of plasma membrane H q -ATPase. Advances in Botanical Research 28, 1±70. Philippar K, Fuchs I, LuÈthen H, Hoth S, Bauer CS, Haga K, Thiel G, Ljung K, Sandberg G, BoÈttger M, Becker D, Hedrich R Auxin-induced K q -channel expression represents an essential step in coleoptile growth and gravitropism. Proceedings of the National Academy of Sciences, USA 96, 12186± Terry ME, Jones RL Effect of salt on auxin-induced acidi cation and growth by pea internode sections. Plant Physiology 68, 59±64. Tester M Functions of ion channels in plant cells. In: Smallwood M, Knox P, Bowles D, eds. Membranes: specialised functions in plant cells. Oxford: Bios Scienti c Publishers, 231±245. Thiel G, BruÈdern A, Gradmann D Small inward rectifying K q channels in coleoptiles. Inhibition by external Ca 2q and function in cell elongation. Journal of Membrane Biology 149, 9±20. Thiel G, Weise R Auxin augments conductance of K q inward recti er in maize coleoptile protoplasts. Planta 208, 38±45.