osmoregulation by oat coleoptile protoplasts

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

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,, 1 O mM KCI, 1 O mM 2-(morpholino)ethanesulfonic acid/lJ-bis- Itris(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. Swell- ing with or without IAA was inhibited by osmotic shock and was completely reversed by 0.1 mM NaN,. Sodium orthovanadate, ap- plied 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 conse- quence 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 com- mittee for Plant Molecular Integration and Function.

* Corresponding author; e-mail [email protected]; fax 1-206-543-3262.

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 auxin- induced 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 capaci- ties 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 (Pan- toja 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

Abbreviations: ABP1, auxin-binding protein 1; BTP, 1,3-bis- [tris(hydroxymethyl)methylamino]propane; 2,3-D, 2,3-dichloro- phenoxyacetic acid; IBA, indole-3-butyric acid; K-IDA, potassium iminodiacetic acid; mosmol, milliosmolal; NAA, naphthalene acetic acid; PM H+-ATPase, plasma membrane proton ATPase; TEA- C1, tetraethylammonium chloride.

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1008 Keller and Van Volkenburgh Plant Physiol. Vol. ;I 1 O, 1996

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). Pro- tòplast 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 METHODS

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 (<0.01 pmol mp2 s-’). Coleoptiles of 25 to 40 mm were harvested after 90 h. The enclosed leaf was extracted, the epidermis was peeled away with fine jewelers’ forceps, and 1-cm segments be- ginning 3 mm below the tip were prepared using a double- bladed cutter.

Prototoplast Preparation

Peeled coleoptile segments (30-50) were digested for 1.75 h in a 13 X 100 mm test tube containing 1 mL of a solution containing 3.75% (w/v) cellulase RS (Yakult Honsha Co., Tokyo, Japan), 3.75% Cellulysin (Calbiochem), 0.1% Pectol- yase Y-23 (Seishin Pharmaceutical Co., Tokyo, Japan), 0.2% BSA (Sigma), 2.23% Gamborg’s 8-5 medium (no hormones) (GIBCO), 1.75% Ficoll (Sigma), 2 mM CaCl,, and 10 mM Mes/KOH, pH 5.5 (300 mosmol). In one experiment the osmolality of the digestion medium was first 425 mosmol with added mannitol for 1.75 h, then made 300 mosmol for 10 min through the addition of 1 mL of a solution containing 3.5% Ficoll, 1 mM CaCl,, 10 mM KC1, 10 mM Mes/BTP, pH 6.5, and 190 mosmol with mannitol. The incompletely digested coleoptile segments were then removed, and the digestate containing protoplasts was overlaid with 6 mL of a rinse solution containing 1 mM CaCl,, 1.5% Ficoll, 10 mM Mes/BTP, pH 6.5, KC1 (O, 1, or 10 mM), and 290 mM mannitol (or Glc) (sufficient to bring the osmolality to between 300 and 310 mosmol as determined by a vapor- pressure osmometer [Wescor, Logan, UT, model 5100 C]). The rinse layer was then overlaid with 1 mL of a bath solution identical to the rime minus the Ficoll. The KC1 in both the rinse and the bath was replaced with 10 mM K+ salt of the impermeant anion K-IDA (pH 6.5) (Raschke and Schnabl, 1978) in some experiments. The gradient was then centrifuged at low speed for 1 to 2 min in a tabletop

centrifuge. Two hundred microliters of the protoplast in- terphase between the rinse and bath solutions were then removed and added to 1.5 mL of fresh bath solution in a narrow glass vial. The isolated protoplasts, which settle to the bottom, were essentially free of debris, wiih greater than 80% displaying unambiguous cytoplasmic streaming. They were then allowed to rest for 30 to 45 min.

Protoplast Swelling Experiments

Because the size of individual Avena coleoptile protoplasts within a sample varies considerably, it was neces- sary to follow individual protoplasts over time to determine if they increased in volume. Accordingly, a slide well system was designed: four wells were established on a 75 X 50 mm glass slide supporting a 7-mm-thick wafer of Syl- gard (Dow Corning, Midland, MI) into which four evenly spaced holes (9 mm wide) had been bored. The Sylgard formed a water-tight seal with the glass slide. The slide was lightly etched at each slide well with a grid pattern to assist in protoplast identification. Four hundred microliters of bath solution were added to each well (with or without 10 p~ IAA). In some experiments, 0.1 mM sodium azide or 0.5 mM sodium orthovanadate was included in the bath. Van- adate-containing solutions were brought to pH 6.5 with HC1, boiled, and allowed to cool, and the pfI was re- checked before addition of IAA from stock. In some experiments, the 10 mM KCl was replaced by TEA-C1.

To begin an experiment, 20 pL of rested protoplasts were added to each well and the wells were closed with glass coverslips. Approximately 20% of the enclosed volume of the chamber was air to prevent anoxia of the protoplasts over the course to the experiment. The slide was then tipped slightly to allow the meniscus in each well to contact the coverslip and establish a continuous water column between the slide and the coverslip. The slide was then placed on the stage of a microscope equipped with a camera lucida, a stage micrometer, and a 35-mm carnera. Pro- toplasts were allowed 10 min to settle to the bottom of the slide wells. Between 10 and 20 min after introducing protoplasts, photographs were made of two fields of protoplasts in each well. The micrometer coordinates of each field were recorded, and grid landmarks were recorded using the camera lucida. At subsequent time points (40 min, 1.75 h, 3 h, 4.5 h, 6 h, and 10 h) the same fields were rephotographed during a 6-min window. The microscope lamp was turned off between time points and otherwise the protoplasts were illuminated by the room lighting. Diam- eters of individual protoplasts were measured from pro- jected slide images and their volume was computed as ( 4 / 3 ) ~ ( d / 2 ) ~ , where d is the diameter. Some protoplasts lysed in the course of a11 experiments, so only measurements of protoplasts appearing intact after 10 h were included in the results. Results are expressed as percent of the volume at the earliest time point (10-20 min). Each experiment was repeated three times with similar results, and the data were pooled. Figures present experiments as they were performed, except for those comparing treatments of 10 mM KC1 to O mM KC1 (with either mannitol or Glc, with or without IAA). The 10 mM KCI treatments were

Osmoregulation by Oat Coleoptile Protoplasts 1009

all performed on separate days from treatments using O mM KCI.

Elongation Experiments

Peeled sections (12 per treatment) were placed immedi- ately after preparation into 60-mm-diameter Petri dishes each containing 15 mL of test solutions. Peeled Avena coleoptile segments have been shown to elongate in a manner similar to unpeeled segments in response to auxin (Cle- land, 1991). Solutions contained 10 mM KCI and 0.5 mM Mes/BTP, pH 6.5, with and without 10 PM IAA. In two treatments, 0.5 mM vanadate (preparation described above) was included in the medium. Incubation was at 23"C, under room light, with gentle agitation (50 rpm) provided by a rotary shaker (model G-2, New Brunswick Scientific, New Brunswick, NJ). At each time point, the sections were briefly removed from the solutions and blotted dry and their length was measured using a microscope fitted with an eyepiece micrometer. The experiment was performed three times with similar results and the data were pooled.

RESULTS

Protoplasts prepared from the apical region of peeled Avena coleoptiles were found to swell steadily (about 4% h-') over the course of 6 h (Fig. 1). A slightly faster swelling rate occurred if IAA was included in the bath medium. The IAA-treated protoplasts were significantly larger at 1.75 and 3 h. Protoplast swelling was completely prevented by 0.1 mM sodium azide.

When the KC1 concentration in the bath was increased from 1 mM (Fig. 1) to 10 mM (Fig. 2A, O, O), swelling without IAA appeared to increase slightly to about 5% h-', although the difference was significant only at 3 h. Auxin- induced swelling (the amount of swelling in the presence of IAA in excess of swelling without IAA) was greater in 10

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Figure 1 . Effect of IAA and azide on the long-term swelling of Avena coleoptile protoplasts. Aliquots of protoplasts were released into slide wells containing 1 mM KCI, 1 mM CaCI,, 10 mM Mes/BTP, pH 6.5, and 290 mM mannitol. The hath also contained 10 FM IAA in one treatment (O) and 0.1 mM N a N , in another (O). The volume of individual protoplasts was computed from the diameter of individual protoplasts in photographic images taken at the indicated times. Error hars represent 95% confidence limits. See Table I for n of each treatment.

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Figure 2. Effect of KCI concentration and Glc on the swelling of Avena coleoptile protoplasts. Protoplasts were incubated in 1 O mM (O, O) or O mM (O, W) KCI, 1 mM CaCI,, 1 O mM Mes/BTP, pH 6.5, and 290 mM mannitol (A) or Glc (6). Ten micromolar IAA was included in two treatments (O, H). Protoplast volume was monitored as in Figure 1. Error bars represent 95% confidence limits. See Table I for n of each treatment.

mM KCI than in 1 mM KCl. In the absence of KCI (Fig. 2B, O, W), swelling without IAA decreased to less than 3% h-' and auxin-induced swelling was no longer evident. To- gether these results indicate that swelling without IAA is partially dependent on the presence of KCI and that auxin- induced swelling is entirely dependent on KCI.

That protoplasts swell in the absence of KCl (without IAA) implies that solutes may also be produced internally and/or that there is uptake of some other component of the medium, probably the major osmoticum, mannitol. When mannitol was replaced with more metabolizable Glc (Fig. 2B), swelling (without IAA) increased significantly. In spite of a 29-fold higher leve1 of Glc over KC1, however, swelling (without IAA) was still enhanced by the inclusion of 10 mM KCI in the bath. Auxin-induced swelling, however, was no longer evident when Glc was provided.

The optimal concentration of IAA for auxin-induced swelling of Avena coleoptile cortex protoplasts incubated in mannitol and 10 mM KC1 appears to lie near 10 p~ because neither 1 nor 100 ~ L M IAA significantly increased swelling (Fig. 3). Besides IAA, two other auxins (IBA and 2,4-D) were also found to increase swelling significantly in mannitol and 10 mM KC1 (Fig. 4A). Increased swelling by the auxin NAA was not significant. Weak acids with no auxin properties (acetic, benzoic, and 2,3-D) did not effect swelling (Fig. 48).

1010 Keller and Van Volkenburgh Plant Physiol. Vol. 11 O, 1996

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Figure 3. Effect of IAA concentration on the swelling of Avena coleoptile protoplasts. Protoplasts were incubated in 10 mM KCI, 1 m M CaCI,, 1 O mM MedBTP, pH 6.5, and 290 mM mannitol with the indicated concentration of IAA. Protoplast volume was monitored as in Figure 1 . Error bars represent 95% confidence limits, and n was 128, 106, 108, and 160 in O, 1; 10, and 100 p~ IAA, respectively.

Since uptake of externa1 solutes, including Kt, C1-, and sugars, is energized by the PM Hf-ATPase (Sanders, 1990), poisoning of the H+-ATPase should block the uptake of both KC1 and mannitol, as well as cell enlargement and protoplast swelling dependent on the uptake of these osmotica. Figure 5 shows the effect of a vanadate-specific

1 control NAA IBA 2,4-D

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Figure 4. Effect of other auxins and nonauxin weak acids on the swelling of Avena coleoptile protoplasts. Protoplasts were incubated in 1 O mM KCI, 1 mM CaCI,, 1 O mM Mes/BTP, pH 6.5, and 290 mM mannitol with or without 1 O p~ of either one of three known auxins (NAA, IBA, 2,4-D) (A) or one of three nonauxin weak acids (6). Protoplast vol:in:e was monitored as in Figure 1 . Error bars represent 95% confidence limits, and n ranged from 75 to 94.

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Figure 5. Effect of vanadate on the elongation of coleoptile segments. Peeled segments 1 cm long from 3 mm below the apex of 4-d-old Avena coleoptiles were gently agitated in a solution containing 10 mM KCI and 0.5 mM Mes/BTP, pH 6.5 (O, O). Ten micromolar IAA was included in two treatments (O, m), as was 0.5 mM sodium orthovanadate (O, m). Length was measured at the indicated times and converted to percent of initial length. Error bars represent 95% confidence limits and are included only where they are larger than symbol size. For each treatment, n = 36.

inhibitor of the PM H+-ATPase of higher plants (Sze, 1985) on the elongation (with or without IAA) of peeled coleoptile segments incubated in 10 mM KC1 and 0.5 mM Mes/ BTP, pH 6.5. By 1.5 h, partia1 inhibition by 0.5 mM sodium orthovanadate (this concentration was found to be optimal in a separate preliminary experiment) of elongation in the absence of IAA was evident, whereas auxin-induced elongation appears to have been completely prevented by vanadate. Between 1.5 and 6 h, elongation in vanadate was less than 2.5%. By contrast, when protoplasts were treated with vanadate (Fig. 6A, O, W), inhibition of swelling was not dramatic. Partia1 inhibition of swelling (without IAA) was evident only after 3 h, and auxin-induced swelling was still evident in vanadate. Reduction of the C1- in the medium did not increase vanadate inhibition of swelling (without IAA) or of auxin-induced swelling (Fig. 6B). Oinission of Gamborg's 8-5 medium from the protoplast preparation solution, to reduce availability of phosphate, also failed to increase effectiveness of vanadate (data not shown). How- ever, when we prepared protoplasts from peeled coleoptile segments that had been preincubated for 1.5 h in 0.5 mM vanadate, subsequent swelling of protoplasts in vanadate (with or without IAA) was substantially reduced. Figure 6C also shows that 1.5 h of preincubation alcine minus vanadate did not significantly affect swelling (wií h or without IAA).

In Figure 6B (O, O), the concentration of C1- was lowered from 12.0 to 2.0 by replacement of 10 mM KC1 by K-IDA, yet swelling was not inhibited. This suggests that the KCl dependence of swelling evident in Figure 2 is a K+ and not a C1- dependence. In a separate experiment, protoplasts incubated in the complete absence of C1-, but with 10 mM K+, were found to increase in volume 23.7 5 4.1% without IAA and 29.6 5 3.7% with IAA after 4..5 h, con- firming the independence of protoplast swelling from the presence of C1- in the medium. On the other hand, the K f

Osmoregulation by Oat Coleoptile Protoplasts 101 1

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Figure 6. Effect of vanadate on the swelling of Avena coleoptile protoplasts. Protoplasts were incubated in 10 mM KCI, 1 mM CaCI,, 10 mM Mes/BTP, p H 6.5, and 290 mM mannitol (O, U). Ten micromolar IAA (O, W) and 0.5 mM sodium orthovanadate (O, W) were each included in two treatments. In B, the KCI was replaced by 10 mM K-IDA (pH 6.5). In C, protoplasts were prepared from coleoptile segments that were pretreated for 1.5 h in 1 0 mM KCI, 0.5 mM Mes/BTP, p H 6.5 (O, O) or the same medium augmented with 0.5 mM sodium orthovanadate (O, 4). Protoplast volume was monitored as in Figure 1. Error bars represent 95% confidence limits. See Table I for n of each treatment.

dependence of swelling was confirmed when K+ was replaced by an impermeant cation and known Kt channel blocker, tetraethylammonium (Hille, 1992) (Fig. 7).

Protoplasts are generally prepared under only slightly plasmolyzing conditions (Ruesink, 1980; Evans and Bravo, 1983) because an excess of osmotic pressure has been reported to impair metabolism (Ruesink, 1978). The protoplasts in the experiments described here were prepared and treated in the solutions in which the osmolalities (300-

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Figure 7. Effect of TEA-CI on the swelling of Avena coleoptile protoplasts. Protoplasts were incubated in either l O mM KCI (O, O) or l 0 mM TEA-CI (O, W), 1 mM CaCI,, 10 mM Mes/BTP, p H 6.5, and 290 mM mannitol (O, O) and with added 10 FM IAA (O, 4). Protoplast volume was monitored as in Figure 1. Error bars represent 95% confidence limits. See Table I for n of each treatment.

310 mosmol) were maintained close to that measured for the expressed cell sap of frozen, thawed coleoptile segments (278 mosmol). Osmotic shock.(lO min in 500 mM mannitol), however, has previously been shown to elimi- nate subsequent auxin-induced growth and proton efflux from Avena coleoptile segments (Rubinstein, 1977). We tested the effect of increasing t h e osmolality of t h e protoplast preparation medium to 425 mosmol. After 1.75 h the osmolality was lowered to 300 mosmol, and 10 min later the protoplasts were isolated as in the experiments described above. Figure 8 shows that this treatment results in a slightly slower rate of swelling (without IAA) (compare Figs. 2A, 6A, and 7), and auxin-induced swelling was ab- sent.

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Figure 8. Effect of osmotic shock on the swelling of Avena coleoptile protoplasts. Protoplasts were osmotically shocked by preparation in tissue-digesting medium initially at 425 mosmol with added mannitol. After 1.75 h this was diluted to 300 mosmol, and 10 min later the protoplasts were isolated as in other experiments. The protoplasts were incubated in 10 mM KCI, 1 mM CaCI,, 1 O mM Mes/BTP, pH 6.5, and 290 mM mannitol (O) with 1 0 WM IAA (O). Protoplast volume was monitored as in Figure 1. Error bars represent 95% confidence limits. See Table I for n of each treatment.

1012 Keller and Van Volkenburgh Plant Physiol. Vol. 110, 1996

Figure 9 shows a sample of protoplasts photographed at12 min, 3 h, 6 h, and 10 h following release in 10 mM KC1,1 mM CaCl2/ 10 mM Mes/BTP, pH 6.5, 10 /XM IAA, andmannitol to 300 mosmol. In all experimental treatments,some protoplasts lysed. Measurements from these proto-plasts were not included. In the course of most treatmentssome other protoplasts became nonspherical, and thustheir volumes became indeterminable, but these proto-plasts continued to exhibit cytoplasmic streaming and togrow larger and therefore were included in the results tothe point at which they became unmeasureable. The num-ber of protoplasts («) contributing to the means shown inthe figures declined at later time points due to increasingnumbers of nonspherical protoplasts. At 4.5 h in mosttreatments, >90% of protoplasts were still round, but by 10h <50% were still round in some experiments. Table Ishows the n (and the percent of initial values) for eachtreatment in the experiments described above. The num-bers of nonspherical protoplasts varied among experimen-tal replicates, which limits generalizations, but protoplaststreated with azide (Fig. 1) did not become nonspherical,whereas Glc accelerated the phenomenon (Fig. 2B).

DISCUSSION

More than 30 years ago, Cocking (1961, 1962) reportedthat protoplasts prepared from roots of Li/copersicon escu-lentum rapidly swelled by increasing their rate of vacuola-tion until they burst if incubated in a range of IAA concen-trations. Similar short-term bursting responses to auxin(i.e. less than 30 min) have also been reported for proto-plasts from the roots of Allium cepa (Pilet, 1971,1981) and Z.mays (Pilet, 1984). Protoplasts from Avena coleoptiles, how-ever, were reported by Ruesink and Thimann (1965) not to

show any tendency to burst if incubated in 0.5 M mannitoland auxin, whether buffered or unbuffered at pH 6.5. Halland Cocking (1974) later reported that rapid auxin-inducedbursting of Avena coleoptile protoplasts occurred at pH 4.3in 0.29 M mannitol. The latter researchers found that in 10JU.M IAA virtually all protoplasts lysed within 10 min, leav-ing only a few nonvacuolated protoplasts and freevacuoles.

In our experiments, long-term observation of proto-plasts began 10 min after protoplasts were released intoslide wells containing solutions with or without auxin.Auxin-induced swelling and or bursting of protoplasts isunlikely to have occurred before observations began,however, because similar numbers of intact protoplastssettled to the bottom of slide wells with or without IAA,the average diameter of protoplasts with or without IAAwas essentially the same as at the ini t ia l time point (datanot shown), and protoplasts with or without IAA were>95% clearly vacuolated. Some protoplasts were ob-served to lyse in the course of all of our experimentaltreatments, but differences in the frequency of lysis werenot apparent.

Protoplast swelling can result only from uptake of exter-nal solutes or generation of solutes from internal reserves.Azide prevents cyanide-sensitive ATP production and,therefore, the blockage of swelling by 0.1 mM sodium azideindicates that solute increase is ATP dependent. The partialdependence of swelling (without IAA) and the completedependence of auxin-induced swelling in the presence ofK+ (Figs. 1, 2A, and 7) is evidence that Aiwna coleoptileprotoplasts can increase their osmolality through uptake ofK ' . It is possible that the K ' dependence of Aveiin coleop-tile protoplast swelling might represent a broader depen-

Figure 9. Avena coleoptile protoplast swelling.Here, a sample of protoplasts is seen at theindicated times following release into 10 mMKCI, 1 mM CaCI2, 10 mM Mes/BTP, pH 6.5, 290mM mannitol, and 10 JXM IAA. In all experimen-tal treatments, some protoplasts were observedto lyse (broad arrows). Measurements from thesewere not included in results. Other protoplastsbecame nonspherical (narrow arrows), and thustheir volume became indeterminable, but theseprotoplasts continued to exhibit cytoplasmicstreaming and grow larger and were thereforeincluded in the results to the point at which theybecame unmeasureable.

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Table I . Number of protoplasts (n) measured at each time point in Figures 1, 2, 4, 5, and 6 The number of protoplasts (n) contributing to the means shown in each protoplast swelling experiment tended to fall in most experiments

because some protoplasts became nonspherical (see Fig. 7). This table shows the n (and the percent of initial) for each treatment at each time point ( h m i n ) for each protoplast in each swelling experiment.

Experiment

Figure 1 - IAA +IAA +azide

Figure 2A 10 mM KCI, -IAA

O mM KCI, -IAA 1 0 mM KCI, + IAA

0 mM KCI, +IAA Figure 2B

10 mM KCI, -IAA

0 m M KCI, -IAA 0 mM KCI, +IAA

10 mM KCI, +IAA

Figure 6A -Vanadate, -IAA -Vanadate, +IAA +Vanadate, -IAA +Vanadate, +IAA

-Vanadate, -IAA -Vanadate, +IAA +Vanadate, -IAA +Vanadate, +IAA

-Vanadate, -IAA -Vanadate, +IAA +Vanadate, +lAA +Vanadate, +IAA

Figure 66

Figure 6C

Figure 7 10 mM KCI, -IAA

10 mM TEA-CI, -IAA 10 mM KCI, +IAA

10 mM TEA-CI, +IAA Figure 8 - IAA + IAA

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107 (1 00) 105 (100) 121 (100) 121 (100)

107 (1 00) 105 (100) 85 (1 00) 83 (100)

86 (1 00) 82 (1 00) 97 (1 00) 95 (1 00)

11 5 (1 00) 11 3 ( I 00) 133 (100) 120 (1 00)

103 (100) 105 (100) 1 17 (1 00) 111 (100)

114 (100) 109 (1 00) 106 (1 00) 108 (1 00)

82 (1 00) 75 (1 00)

193 (100) 197 (100) 129 (100)

106 (99.1) 104 (99.0) 120 (99.2) 11 9 (98.3)

107 (1 00) 105 (100) 83 (97.6) 81 (97.6)

86 (1 00) 82 (100) 95 (97.9) 95 (100)

114(99.1) 112 (99.1) 133 (1 00) 120 ( I 00)

103 (1 00) 105 (1 00) 1 15 (98.3) 1 1 1 (100)

114 (100) 109 (1 00) 105 (99.1 108 (1 00)

82 (100) 75 (1 00)

193 (100) 197 (1 00) 129 (1 00)

104 (97.2) 105 (100) 121 (100) 11 4 (94.2)

107 (100) 104 (99.0) 82 (96.5) 82 (98.8)

85 (98.8) 82 (100) 96 (99.0) 94 (98.9)

1 1 2 (97.4) 1 1 1 (98.2) 128 (96.2) 11 8 (98.3)

103 (100) 105 (100) 116 (99.1) 110 (99.1)

113 (99.1) 1 09 (1 00) 104 (98.1) 108 (1 00)

82 (100) 75 (100)

193 (100) 197 (1 00) 129 (1 00)

103 (96.3) 101 (96.2) 11 8 (97.5) 11 7 (94.2)

101 (94.4) 1 00 (95.2)

77 (90.6) 80 (96.4)

82 (95.3) 81 (98.8) 95 (97.9) 93 (97.9)

110 (95.7) 108 (95.6) 125 (96.2) 11 3 (94.2)

101 (98.1) 105 (100) 116 (99.1) 107 (96.4)

111 (97.4) 1 09 (1 00) 105 (99.1) 108 ( I 00)

80 (1 00)

192 (99.5) 196 (99.5) 129 (1 00)

101 (94.4) 102 (97.1) 11 6 (95.9) 114 (94.2)

91 (85.0) 89 (84.8) 74 (87.1) 71 (85.5)

81 (94.2) 77 (93.9) 94 (96.9) 87 (91.6)

102 (88.7) 101 (89.4) 120 (90.2) 108 (90.0)

97 (94.2) 104 (99.0) 11 5 (98.3) 106 (95.5)

11 2 (98.2) 108 (99.1) 105 (99.1) 108 (1 00)

77 (93.9) 74 (98.74) 73 (97.3)

190 (98.4) 183 (92.9) 129 (1 00)

87 (81.3) 95 (90.5)

106 (87.6) 96 (79.3)

82 (76.6) 75 (71.4) 67 (78.8) 60 (72.3)

73 (84.9) 74 (90.2) 85 (87.6) 81 (85.3)

91 (79.1) 84 (74.3)

111 (83.5) 98 (81.7)

93 (90.3) 96 (91.4)

1 09 (93.2) 1 O1 (91 .O)

11 2 (98.2) 109 (100) 104 (98.1) 108 (1 00)

70 (85.4) 67 (89.3)

167 (86.5) 140 (71.1) 129 (1 00)

73 (68.2) 85 (81 .O) 85 (70.2) 57 (47.1)

51 (47.7) 48 (45.7) 37 (43.5) 27 (32.5)

44 (51.2) 61 (74.4) 71 (73.2) 67 (70.5)

51 (44.3) 71 (62.8) 88 (66.2) 84 (70.0)

73 (70.9) 85 (80.9)

11 3 (96.6) 93 (83.8)

1 1 3 (99.1) 107 (98.2) 97 (91.5) 99 (91.7)

35 (42.7) 44 (58.7)

dence on monovalent cations, because Stevenson and Cle- land (1981) have shown that NaCl supports auxin-induced growth of Avena coleoptile segments equally as well as KCI. In a preliminary experiment, however, swelling of protoplasts after 4.5 h of incubation in a medium in which the 10 mM KC1 was replaced by NaCl was no greater (with or without IAA) than that of controls with O mM NaCl or KC1.

Rubinstein and Light (1973) and Rubinstein (1974) found that auxin induces the uptake of "6C1- into oat coleoptile cells, but Haschke and Lüttge (1975a, 1975b) found that although CI- did not accumulate in oat coleoptiles, malate increased and was stoichiometrically correlated with potassium uptake. Our results showing that swelling (with or without IAA) is unaffected by a lowering of C1- concentration (Fig. 6B) indicate that CI- is not required as a counterion for absorbed K f and implies that malate may accumulate in swelling protoplasts as well. The complete dependence of auxin-in-

duced swelling on the presence of K f implies that malate production is not directly increased by auxin but requires an influx of Kf to occur.

Substantial swelling (without IAA) occurs in the absence of KCl (Fig. 2A) and must result from generation of interna1 solutes (e.g. through the breakdown of starch or possibly from CO, fixation [Yamaki, 19541) or from uptake of other medium components, most likely mannitol. Auxin does stimulate uptake of Glc (Baker and Ray, 1965), and manni- to1 has been shown to be taken up by Z. mays and Hordeum vulgare roots (Cram, 1984) and by Z. mays coleoptile cells (Hohl and Schopfer, 1991). Absorbed mannitol, however, is only slowly metabolized in Zea and Hordeum root cells (Cram, 1984). When mannitol was replaced by more absorbable (Reinhold and Kaplan, 1984) and more cataboliz- able Glc, swelling (without IAA) increased and auxin-induced swelling was no longer evident (Fig. 2B). Absorbed Glc can be expected to be catabolized to other osmotically active solutes, increasing the respiratory ATP concentra-


tion and perhaps increasing the activity of the PM H+- ATPase and the driving force for the uptake of solutes.

The effect of vanadate on auxin-induced swelling (Fig. 6A) was tested to confirm a role for the PM H+-ATPase in swelling. We found that vanadate most effectively inhibited swelling (with or without IAA) when protoplasts were prepared from tissues preincubated in vanadate (Fig. 6C). Inhibition of swelling by vanadate was less apparent when protoplasts were treated after isolation (Fig. 6, A and B). This suggests that uptake of vanadate is inhibited by protoplast preparation. Vanadate inhibition of the higher- plant H+-ATPase in vivo has sometimes been found to be ineffective (Schwartz et al., 1991). Vanadate appears to work from the cytoplasmic side of the membrane (Cantley et al., 1978). The effect of lowering the C1- concentration in the bath was tested (Fig. 6B), because limiting the concentration of permeant anions in the bath has been found to increase vanadate inhibition of stomatal opening in epidermal peels (Schwartz et al., 1991) and blue-light-stimulated swelling of Vicia faba guard cell protoplasts (Amodeo et al., 1992), possibly by increasing vanadate uptake via a nonspecific anion carrier (Schwartz et al., 1991). We also tested the possibility that phosphate nutrition during protoplast preparation was reducing the effectiveness of vanadate, because Kuroda et al. (1980) found that vanadate inhibition of growth and membrane potential of Neurospora requires prior phosphate starvation. Perhaps protoplast preparation disrupts the activity of either the nonspecific anion carrier (Schwartz et al., 1991) or a more specific phosphate carrier (Kuroda et al., 1980) responsible for vanadate uptake into intact cells. Alternatively, the function of the uptake carrier could be hampered indirectly. Severa1 reports suggest that the membrane potential of protoplasts are at least partially depolarized (Assmann et al., 1985; Pantoja and Willmer, 1986; Ketchum et al., 1989; Barbier-Brygoo et al., 1991). The uptake carrier could be voltage sensitive, or, if the carrier represents a H+/vanadate symport, a decrease in the proton motive force might lessen activity.

Avena protoplasts can increase in volume more rapidly than do growing coleoptile cells (compare Figs. 2A and 5) (note that Avena coleoptile segments grow almost entirely in length [Ray and Ruesink, 19631, so increases in length are directly proportional to increases in volume). In protoplasts, osmoregulation should be rate limiting for swelling. The rate at which coleoptile segments elongate, however, is limited by cell-wall loosening, which in turn is determined by the apoplast acidity mediated by the PM H+-ATPase (Lüthen et al., 1990; Rayle and Cleland, 1992). Any inhibition of the rate-limiting activity of PM H+-ATPase will slow coleoptile growth by inhibiting wall loosening. Tur- gor must be maintained to drive growth, but osmoregulation can be expected gradually to become rate limiting only in the absence of absorbable solutes (Stevenson and Cle- land, 1981). Under optimal conditions, however, coleoptile segments can grow at rates approaching 5% h-' for many hours (Kefford and Bonner, 1961), so perhaps induction of increased osmoregulation by auxin serves to prevent osmoregulation from becoming rate limiting for growth. Pro- toplast osmoregulation, unlike wall loosening, may be lim-

ited by something other than the activity of the PM H+- ATPase. If so, protoplast swelling would be expected to be less sensitive to vanadate poisoning of the PM H+-ATPase and may explain why inhibition of swelling (with or without IAA) by vanadate was found to be incomplete even when the protoplasts were prepared from coleoptile segments preincubated in vanadate (Fig. 6C).

The fact that swelling (without IAA) is sensitive to K+ (Fig. 7) , even in the presence of 290 mM Glc (Fig. 2B), implies that the inward conductance of K+, piresumably through K+ channels, is at least partially rate limiting in osmoregulation. Although direct measurement of PM H+- ATPase activity (Lohse and Hedrich, 1992; Riick et al., 1993) indicates that electrogenic (presumably H+) current is stimulated by auxin, the complete dependence of auxin- induced swelling on K+ (Figs. 1, 2, and 7 ) implies that the rate-limiting K+ conductance is also increased by IAA. Blatt and Thiel (1994) have also recently reported evidence that auxins modulate the activity of K+ channels. They found a bimodal effect of IAA and NAA on an inward- rectifying K+ conductance in guard cells of V. faba stomates; between 0.1 and 10 p~ the current was augmented, whereas above 30 PM the effect was inhibitory. Although 100 p~ IAA was found to be supraoptimal for induction of protoplast swelling (Fig. 3), it did not appear to inhibit swelling, as might be expected if it also acts to inhibit K+ uptake in this system.

Blatt's group (Thiel et al., 1993) has also reported evi- dente for a role for the putative auxin receptor ABPl (Jones, 1994) in the regulation of the inward-rectifying K+ conductance of V. faba guard cells. Treatment with a syn- thetic peptide corresponding to the C-terminal domain of ABPl was found to inactivate this channel. The ABPl protein from corn has been found to bind more strongly to NAA than to other auxins (Lobler and Klampt, 1985). We found, however, that 10 p~ NAA was a relatively ineffective inducer of protoplast swelling (Fig. 4). Although this result would tend to suggest that ABPl is not involved in the auxin-induced swelling response, we have recently described another auxin response, this time by intact tissues of Avena coleoptile cortex, in which NAA is more effective than other auxins (Keller and Van Volkenburgh, 1996). We found that a C1- conductance, evtdent as a depolarization of the membrane potential that was sensitive to the extracellular C1- concentration, is rapidly activated by 10 p~ NAA and not by other auxins, although 100 p~ IAA activated a similar conductance. We also found a poor correlation with average induction of the C1- conductance by different concentrations of NAA and IAA and growth induction of excised coleoptile tissue by the same concentrations. The relative effectiveness of different auxins at 10 p~ in inducing protoplast swelling (i.e. IAA 2

2,4-D 2 IBA 2 NAA) (Figs. 2 and 4) is the same as that for induction of coleoptile segment elongation (Keller, 1994). This suggests that the increased osmoregulation and cell-wall loosening are linked to the same auxin- receptor-signal transduction mechanism, which is appar- ently separate from that which activates increased C1- conductance.


Inhibition of swelling (without IAA) a n d elimination of auxin-induced swelling by osmotic shock during protoplast preparation (Fig. 8) could result from either profound inhibition of PM H+-ATPase activity or inhibition of the activity of the inward conductance of K+. This result un- derscores the need to ensure that the method of protoplast preparation does not disrupt the physiological response to be studied in, for example, patch-clamp experiments rely- ing on protoplasts.

In a11 experiments some protoplasts became nonspherical. Nonspherical protoplasts d id not result from adher- ence to the bottom of the slide well, because they retained their shape if swirled up off the bottom. Nonspherical protoplasts could, however, have been the result of a t least t w o developments. First, rapid cell-wall regeneration could differentially constrict regions of the expanding plasma membrane. The rate of cell-wall regeneration depends on the source of the protoplasts, with regeneration by protoplasts isolated from rapidly growing tissues or cell cultures commencing most rapidly (Franz and Blaschek, 1985). Al- ternatively, anchoring of the plasma membrane to cytoskeletal elements could also hold back regions. Destruction of cytoskeletal elements by laser microsurgery or with the actin disrupter cytochalasin B has been found to cause nonspherical protoplasts from Hibisctls rosa-sinensis to become round (Hahne a n d Hoffmann, 1984). In preliminary experiments, the possibility w a s tested that nonspherical protoplasts resulted from cytoskeletal anchoring of the plasma membrane by including 10 pg/mL cytochalasin B in the bath solution. Cytochalasin B rapidly arrested cytoplasmic streaming a n d disrupted cytoplasmic strands. Swelling appeared to be unaffected, bu t no protoplasts became nonspherical. If, however, chytochalasin B was added a t 10 h, once a substantial portion of the protoplasts h a d become nonspherical, streaming w a s arrested a n d strands disappeared, bu t the protoplasts d id not become round. This suggests that it is the formation of new cell wall a n d not cytoskeletal anchoring of the plasma membrane that results i n the development of nonspherical protoplasts a n d that cell-wall regeneration requires cytoplasmic streaming a n d / o r a n intact cytoskeleton.

ACKNOWLEDGMENT

We are grateful to Dr. Robert E. Cleland for a critica1 reading of a draft of this report.

Received August 2, 1995; accepted November 29, 1995. Copyright Clearance Center: 0032-0889/96/110/1007/10.

LITERATURE ClTED

Amodeo G, Srivastava A, Zeiger E (1992) Vanadate inhibits blue light-stimulated swelling of Vicia guard cell protoplasts. Plant Physiol 100: 1567-1570

Assmann SM, Simoncini L, Schroeder JI (1985) Blue light stimulates electrogenic ion pumping in guard cell protoplasts of Vicia faba. Nature 318: 285-287

Baker DB, Ray PM (1965) Direct and indirect effects of auxin on cell wall synthesis in oat coleoptile tissue. Plant Physiol 4 0 345-352

Barbier-Brygoo H, Ephritikhine G, Klambt D, Maurel C, Palme K, Schell J, Guern J (1991) Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts. Plant J 1:

Blakeley SD, Thomas B, Hall JL, Vince-Prue D (1983) Regulation of swelling of etiolated-wheat-leaf protoplasts by phytochrome and gibberellic acid. Planta 158: 416421

Blatt MR, Theil G (1993) Hormonal control of ion channel gating. Annu Rev Plant Physiol Plant Mo1 Biol 44: 543-567

Blatt MR, Theil G (1994) K+ channels of stomatal guard cells: bimodal control of K+ inward-rectifier evoked by auxin. Plant J

Bossen ME, Dassen HHAS, Kendrick RE, Vredenberg WJ (1988) The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts. Planta 1 7 4 94-100

Bossen ME, Tretyn A, Kendrick RE, Vredenberg WJ (1991) Com- parison between swelling of etiolated wheat (Tritium aestivunl L.) protoplasts induced by phytochrome and d-naphthaleneacetic acid, benzylaminopurine, gibberellic acid, abscisic acid and acetylcholine. J Plant Physiol 137: 706-710

Cantley LC, Resh MD, Guidotti G (1978) Vanadate inhibits the red cell (Na+, K+) ATPase from the cytoplasmic side. Nature

Cleland RE (1991) The outer epidermis of Avena and maize coleoptiles is not a unique target for auxin in elongation growth. Plant Physiol 186 75-80

Cleland RE, Prins HBA, Harper JR, Higinbotham N (1977) Rapid hormone-induced hyperpolarization of the oat coleoptile trans- membrane potential. Plant Physiol 59: 395-397

Cocking EC (1961) Properties of isolated plant protoplasts. Nature

Cocking EC (1962) Action of growth substances, chelating agents and antibiotics on isolated root protoplasts. Nature 193: 998-999

Cram WJ (1984) Mannitol transport and suitability as an osmoticum in root cells. Physiol Plant 61: 396-404

Evans DA, Bravo JE (1983) Protoplast isolation and culture. In DA Evans, WR Sharp, PV Ammirato, Y Yamada, eds, Handbook of Plant Cell Culture. Macmillan, New York, pp 125-137

Franz G, Blaschek W (1985) Wall regeneration in protoplasts of higher plants. In PE Pilet, ed, The Physiological Properties of Plant Protoplasts. Springer-Verlag, Berlin, pp 171-183

Hahne G, Hoffmann F (1984) The effect of laser microsurgery on cytoplasmic streaming in isolated protoplasts. Eur J Cell Biol 33:

Hall MD, Cocking EC (1974) The response of isolated Avena coleoptile protoplasts to indole-3-acetic acid. Protoplasma 79:

Haschke H-P, Liittge U (1975a) Stoichiometric correlation of malate accumulation with auxin-dependent K+-H+ exchange and growth in Avena coleoptile segments. Plant Physiol 56:

Haschke H-P, Liittge U (1975b) Interactions between IAA, potassium, and malate accumulation, and growth in Avena coleoptile segments. Z Pflanzenphysiol 76: 450-455

Hille B (1992) Ionic Channels of Excitable Membranes, Ed 2. Sinauer Associates, Sunderland, MA

Hohl M, Schopfer P (1991) Water relations of growing maize coleoptiles. Comparison between mannitol and polyethylene glycol 6000 as externa1 osmotica for adjusting turgor pressure. Plant Physiol 95: 716-722

Jones AM (1994) Auxin-binding proteins. Annu Rev Plant Physiol Plant Mo1 Biol 4 5 393420

Kefford NP, Bonner J (1961) Steady state growth of Avena coleoptile sections in high auxin concentrations. Plant Physiol 36:

Keller CP (1994) Characterization of the electrical response of Avem sativa L. coleoptile cortex cells to auxin treatment. PhD thesis, University of Washington, Seattle

Keller CP, Van Volkenburgh E (1996) The electrical response of Avetia coleoptile cortex to auxins: evidence in vivo for activation of a C1- conductance. Planta 198: 404412

83-93

5: 55-68

272 552-554

191: 780-782

175-1 79

225-234

696-698

323-325


Ketchum KA, Shrier A, Poole RJ (1989) Characterization of potassium-dependent currents in protoplasts of corn suspension cells. Plant Physiol 89: 1184-1192

Kim HY, Cote GGJ, Crain RC (1993) Potassium channels in Sa- manea saman protoplasts controlled by phytochrome and the biological clock. Science 260: 960-962

Kuroda H, Warncke J, Sanders D, Hansen U-P, Allen KE, Bow- man BJ (1980) Effects of vanadate on the electrogenic proton pump in Neuuospoua. In RM Spanswick, WJ Lucas, J Dainty, ed, Plant Membrane Transport: Current Conceptual Issues. Elsevier, Amsterdam, pp 507-508

Lobler M, Klampt D (1985) Auxin-binding protein from coleoptile membranes of corn (Zea mnys L.). I. Purification by immunolog- ical methods and characterization. J Biol Chem 260: 9848-9853

Lohse G, Hedrich R (1992) Characterization of the plasma-membrane H+-ATPase from Vicia faba guard cells. Modulation by extracellular factors and seasonal changes. Planta 188: 206-214

Liithen H, Bigdon M, Bottger M (1990) Reexamination of the acid growth theory of auxin action. Plant Physiol 93: 931-939

Marten I, Lohse G, Hedrich R (1991) Plant growth hormones control voltage-dependent activity of anion channels in plasma membrane of guard cells. Nature 353: 758-762

Pantoja O, Willmer CM (1986) Pressure effects on membrane potentials of mesophyll cell protoplasts and epidermal cell protoplasts of Commelina conirrzunis L. J Exp Bot 37: 315-320

Pilet PE (1971) Effet de quelque auxines sur les protoplasts raci- naires. CR Acad Sci (Paris) 273: 2253-2256

Pilet PE (1981) Root protoplasts: some physiological properties. In H Goring, E Paul, eds, Pflanzliche Gewebekultur. Humbolt Uni- versitat, Berlin, pp 35-42

Pilet PE (1984) Auxin effect on protoplasts from gravireacting maize roots. Z Pflanzenphysiol 113: 373-376

Raschke K, Schnabl H (1978) Availability of chloride affects the balance between potassium chloride and potassium malate in guard cells of Vicia faba L. Plant Physiol 6 2 84-87

Ray PM, Ruesink AW (1963) Osmotic behavior of oat coleoptile tissue in relation to growth. J Gen Physiol 47: 83-101

Rayle DL, Cleland RE (1992) The acid growth theory of auxin- induced cell elongation is alive and well. Plant Physiol 99: 1271-1274

Reinhold L, Kaplan A (1984) Membrane transport of sugars and amino acids. Annu Rev Plant Physiol 35: 45-83

Rubinstein B (1974) Effect of pH and auxin on chloride uptake into Avena coleoptile cells. Plant Physiol 5 4 835-839

Rubinstein B (1977) Osmotic shock inhibits auxin-stimulated acid- ification and growth. Plant Physiol 59: 369-371

Rubinstein B, Light EN (1973) Indoleacetic-acid-enhanced chloride uptake into coleoptile cells. Planta 110: 43-56

Rubinstein B, Tattar TA (1980) Regulation of amino acid uptake into oat mesophyll cells: a comparison between protoplasts and leaf segments. J Exp Bot 31: 269-279

Riick A, Palme K, Venis MA, Napier RM, Felle HH (1.993) Patch- clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts. Plant J 4: 41-46

Ruesink A (1978) Leucine uptake and incorporation by Convolvu- lus tissue culture cells and protoplasts under severe osmotic stress. Physiol Plant 44: 48-56

Ruesink A (1980) Protoplasts of plant cells. Methods Ilnzymol 69:

Ruesink AW, Thimann KV (1965) Protoplasts from the Avenn coleoptile. Proc Natl Acad Sci USA 54: 56-64

Sanders D (1990) Kinetic modeling of plant and funga1 membrane transport systems. Annu Rev Plant Physiol Plant Mo1 Biol 41:

Schwartz A, Illan N, Assmann SM (1991) Vanadate inhibition of stomatal opening in epidermal peels of Commelinu communis. Planta 183: 590-596

Senn AP, Goldsmith MHM (1988) Regulation of electrogenic proton pumping by auxin and fusicoccin as related to the growth of Avena coleoptiles. Plant Physiol 88: 131-138

Stevenson TT, Cleland RE (1981) Osmoregulation in the Avena coleoptile in relation to auxin and growth. Plant Physiol 67: 749-753

Sze H (1985) H+-translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol 3 6 175-208

Theil G, Blatt MR, Fricker MD, White IR, Millner P (1993) Modulation of K+ channels in Vicia stomatal guard cells by peptide homologs to the auxin-binding protein C terminus. Proc Natl Acad Sci USA 9 0 11493-11497

Vreugdenhil D, Harkes PAA, Libbenga KR (1980) Auxin-binding by particulate fractions from tobacco leaf protoplasts. Planta 150:

Webb MS, Williams JP (1984) Changes in the lipid artd fatty acid composition of Vicia faba mesophyll protoplasts induced By isolation. Plant Cell Physiol 2 5 1541-1550

Weist SC, Steponkus PL (1978) Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing tem- peratures. Plant Physiol 62: 699-705

Wolfe J, Dowgert MF, Steponkus PL (1986) Mechanical study of the deformation and rupture of the plasma membranes of protoplasts during osmotic expansion. J Membr Biol 93: 63-74

Yamaki T (1954) Effect of IAA upon oxygen uptake, C02 fixation and elongation of Avena coleoptile cylinders in the clarkness. Sci Pap Coll Gen Educ Univ Tokyo 4: 127-154

Zeiger E, Hepler PK (1977) Light and stomatal function: blue light stimulates swelling of guard cell protoplasts. Science

Zhou S, Jones AM, Scott TK (1990) Phytochrome-mecliated swelling of etiolated leaf protoplasts and its possible biological sig- nificance. Plant Cell Rep 9: 435-438

69-84

77-107

9-12

196: 887-889

osmoregulation by oat coleoptile protoplasts

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