Plant Physiol. (1996) 110: 1007-1016
Osmoregulation by Oat Coleoptile Protoplasts' Effect of Auxin Christopher P. Keller* and Elizabeth Van Volkenburgh
Department of Botany, Box 351 330, University of Washington, Seattle, Washington 981 95 shown that an anion channel is also modulated by auxin in the plasma membrane of V .faba guard cells (Marten et al., 1991). The use of a patch clamp has also shown that auxininduced proton efflux from Zea mays coleoptile protoplasts is abolished by antibodies directed against a putative auxin receptor, ABPl (Rück et al., 1993). Patch-clamped protoplasts of V. faba guard cells, however, were found not to contain auxin-sensitive K+ channels (Marten et al., 1991). Furthermore, stimulation of proton efflux in Z . mays coleoptile protoplasts follows auxin application after a lag of only 30 to 40 s rather than 8 to 10 min, as is the case with intact cells (Rück et al., 1993). These last results suggest that the auxin-sensitive physiology of protoplasts may differ from that of cells from which they are generated. Vreugdenhil et al. (1980) reported that when tobacco mesophyll cells were converted to protoplasts, the activity of auxin-binding proteins was completely lost, reappearing after 2 d. Conversion of cells to protoplasts is known to alter some other physiological processes. For example: protoplasts have different capacities for amino acid uptake compared to the parent cells (Rubinstein and Tatter, 1980); the membrane potential of protoplasts is often only slightly negative (Assmann et al., 1985; Ketchum et al., 1989) or completely depolarized (Pantoja and Willmer, 1986; Barbier-Brygoo et al., 1991; but see Lohse and Hedrich, 1992); and large changes in membrane lipid composition occur during protoplast isolation (Webb and Williams, 1984). In some cases, cell-wall remova1 has not appeared to alter physiological responsiveness significantly; protoplasts from the mesophyll of Triticum aestivum and Z . mays swell in response to red light (Blakeley et al., 1983; Bossen et al., 1988; Zhou et al., 1990), V. faba guard cell protoplasts swell in response to blue light (Zeiger and Hepler, 1977; Amodeo et al., 1992), and protoplasts of Samanea saman pulvini retain a circadian rhythm and also respond to light (Kim et al., 1993). Plant protoplasts behave as ideal osmometers against a wide range of externa1 osmolalities (Weist and Steponkus, 1978). Adjustment is rapid; changes in volume greater than 50% can occur in 2 min (Wolfe et al., 1986). Protoplast
The effect of auxin on the physiology of protoplasts from growing oat (Avena sativa L.) coleoptiles was investigated. Protoplasts, isolated iso-osmotically from peeled oat coleoptile segments, were found to swell steadily over many hours. lncubated in 1 mM CaCI,, 1O mM KCI, 1O mM 2-(morpholino)ethanesulfonic acid/lJ-bisItris(hydroxymethyl)methylaminolpropane, pH 6.5, and mannitol to 300 milliosmolal, protoplasts swelled 28.9% f 2.0 (standard error) after 6 h. Addition of 10 p~ indoleacetic acid (IAA) increased swelling to 41.1 YO 2 2.1 (standard error) after 6 h. Swelling (in the absence of IAA) was partially dependent on K+ in the bath medium, whereas auxin-induced swelling was entirely dependent on K+. Replacement of mannitol in the bath by Clc increased swelling (in the absence of IAA) and eliminated auxin-induced swelling. Swelling with or without IAA was inhibited by osmotic shock and was completely reversed by 0.1 mM NaN,. Sodium orthovanadate, applied at 0.5 mM, only gradually inhibited swelling under various conditions but was most effective with protoplasts prepared from tissue preincubated in vanadate. Our data are interpreted to suggest that IAA increases the conductance of the plasma membrane to K+. ~
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Current objectives in the study of auxin action include identifying rapidly initiated electrical events at the plasma membrane (Blatt and Theil, 1993) because these are likely to be triggered by early steps in the signal transduction sequence of the hormone and may be critica1 to the growth response (Rayle and Cleland, 1992). The plasma membrane of oat (Avena sativa) coleoptile cells first depolarizes then hyperpolarizes in response to auxin (IAA) (Cleland et al., 1977). The hyperpolarization phase is most likely a consequence of activation of the PM H+-ATPase (Senn and Goldsmith, 1988). Voltage clamp recordings of the guard cells of Vicia faba stomates, where auxin has also been shown to influence stomatal aperture, have suggested that K+ channel activity is also regulated by auxin (Theil et al., 1993; Blatt and Theil, 1994). The powerful patch-clamp technique, in which protoplasts are used in the place of intact cells, has confirmed stimulation of electrogenic proton efflux by auxin (Lohse and Hedrich, 1992) and has This work was supported by National Science Foundation grants Nos. MCB-9220110 and MCB-9316947 to E.V.V. During much of this work, C.P.K. was supported by the University of Washington Graduate School through the interdisciplinary committee for Plant Molecular Integration and Function. * Corresponding author; e-mail
[email protected]; fax 1-206-543-3262.
Abbreviations: ABP1, auxin-binding protein 1; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; 2,3-D, 2,3-dichlorophenoxyacetic acid; IBA, indole-3-butyric acid; K-IDA, potassium iminodiacetic acid; mosmol, milliosmolal; NAA, naphthalene acetic acid; PM H+-ATPase, plasma membrane proton ATPase; TEAC1, tetraethylammonium chloride. 1007
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swelling is thus a direct measure of increases in the osmolality of the protoplast protoplasm. During auxin-induced growth of intact Avena coleoptile tissue, the uptake of both sugar and salts, and thus osmoregulation, is stimulated (Baker and Ray, 1965; Stevenson and Cleland, 1981). Protòplast swelling would therefore appear to be an excellent subject for the study of the auxin sensitivity of protoplasts. A 30-min treatment with a-naphthaleneacetic acid has been reported to cause swelling of protoplasts prepared from T . aestivum leaf mesophyll (Bossen et al., 1991). In preparation for a patch-clamp study of the auxin responses of Avena coleoptile protoplasts, we chose to determine if such protoplasts swell in response to auxin. We wished to determine if and under what conditions protoplasts retained osmoregulatory responsiveness to auxin. As well, we wished to determine whether auxin-induced protoplast osmoregulation depended on extracellular solutes. MATERIALS A N D M E T H O D S Plant Material
Oat (Avena sativa L. cv Victory [Swedish General Seed Co., Svalof, Sweden]) seeds were soaked for 1 to 3 h in aerated tap water and then planted in moist, well-drained vermiculite. The plants were germinated and grown at 26°C under extremely dim red light (
