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First published online February 5, 2004; 10.1104/pp.103.031294 Plant Physiology 134:735-747 (2004) © 2004 American Society of Plant Biologists Two Distinct Signaling Pathways Participate in Auxin-Induced Swelling of Pea Epidermal ProtoplastsInstitute for Environmental Science, Rokkasho-mura, Aomori 039–3212, Japan (M.Y.); Botanical Gardens, Graduate School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka 576–0004, Japan (K.H., M.I.); and Horticulture Research International, Warwick CV35 9EF, United Kingdom (R.M.N.)
Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca2+. In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca2+. The swelling by either pathway required extracellular K+ and Cl–. The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca2+ requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.
Since its discovery by Went (1928  ), auxin has been studied extensively as a major plant hormone, and yet controversies remain over many of the basic issues of its physiology (Iino, 2001). This is even true for the simple classical response, the stimulation of elongation growth observed in segments of coleoptiles and stems (Lüthen et al., 1999).
A large body of evidence indicates that auxin controls ion transport through the plasma membrane. It has been shown that auxin affects either the activity or the abundance of plasma membrane-located H+-ATPase (Senn and Goldsmith, 1988
ABP1 (auxin-binding protein 1) has been studied most extensively as a candidate auxin receptor for growth responses (Jones, 1994
Early studies by Cocking et al. indicated that protoplasts isolated from roots, leaves, and coleoptiles swell and eventually burst when treated with auxin (Cocking, 1962
To understand the growth of coleoptiles and stems at the organ level, we must resolve which cells perceive auxin and which cells respond with cell wall loosening. In these organs, the epidermis is under tension, and the extensibility of epidermal cell walls limits their growth (Kutschera, 1987
The protoplast swelling response offers a single-cell system with which the overall effects of auxin on plasma membrane ion transporters can be studied quantitatively. In the present study, we have employed protoplasts of pea (Pisum sativum) stem epidermis (Long and Iino, 2001
Epidermal peels of the rapidly elongating stem zone of red light-grown pea seedlings were used to isolate protoplasts. In our routine procedure, the final protoplast preparation was obtained within 3 h after the start of peeling. The standard protoplast bathing medium was composed of 0.5 M sorbitol, 10 mM KCl, 1 mM CaCl2, 20 mM Glc, and 10 mM MES-Tris (pH 6.0). In all experiments, the freshly isolated protoplasts were immediately incubated under red light on the sample stage of an inverted microscope, and, after 30 min of adaptation, they were subjected to photographic recording and experimental treatment. The purity of epidermal protoplasts was examined under a fluorescence microscope by counting parenchyma protoplasts that showed red fluorescent chloroplasts under blue light excitation. The protoplasts of guard cells would also have been included in this count. Independent preparations indicated that about 6% of the protoplasts derived from parenchyma or guard cells (the mean from six preparations was 6.1% with SD of 0.6%; about 200 protoplasts were counted for each preparation). The protoplasts showing fluorescent chloroplasts generally had a much higher density of organelles as compared with epidermal protoplasts. Although fluorescence examination of protoplasts was not possible with the inverted microscope used for actual analysis, the nonepidermal protoplasts could easily be identified in the photographic images. The protoplasts suspected to be of nonepidermal origin were not used for analysis.
The time course data shown in Figure 1 demonstrated that protoplasts swell in response to IAA and NAA. In all cases, the protoplasts maintained a nearly constant volume before auxin application. Also, those not treated with auxin retained constant volume in the subsequent period of 120 min (Fig. 1A; see also Long and Iino, 2001
Protoplast volume during the first 10 min after auxin application was not determined because this period was required before all the protoplasts settled to the bottom after mixing and could be focused for photography. With either IAA or NAA, a high rate of swelling was already detected in the first 5-min interval of measurements (Fig. 1). Therefore, the lag time for the response appeared to be 10 min or shorter. The swelling continued for the measurement period of 2 h. Generally, the rate of swelling decreased after the highest rate was established within the first 1 h. Next, we obtained dose response curves by determining the swelling response 90 min after auxin application. For either IAA (Fig. 2A) or NAA (Fig. 2B), a bell-shaped curve was identified with a threshold at 10–8.5 to 10–8 M. However, the shape of the curves was different between IAA and NAA. The curve for IAA spanned a wider concentration range, with the peak occurring at 10–5 M (IAA) or 10–6 M (NAA). A shoulder was found on the response curve for IAA (at about 10–6 M) but not for NAA. These results indicated that the response to IAA is biphasic. This feature was not apparent with NAA.
Antibodies prepared against maize ABP1 were used to examine the possible participation of ABP1 in the swelling response. Freshly isolated protoplasts were bathed in a medium containing antibodies, and the responses to IAA (Fig. 3A, solid circles) and NAA (Fig. 3B, solid circles) were examined as above. At representative concentrations, controls were obtained by using pre-immune IgG in place of the antibodies (white circles). The dose response curves in Figure 2 are reproduced for comparison.
The swelling response at 10–5 M IAA was totally inhibited by the anti-ABP1 antibodies; the response at lower concentrations (10–8–10–6 M) was not affected (Fig. 3A). The resulting dose response curve was bell shaped, showing a maximal response at 10–6 M. In the case of NAA, the response was almost totally inhibited at all concentrations (Fig. 3B). The control measurements obtained using pre-immune IgG were similar to those obtained without IgG. Figure 4 shows time courses obtained in the presence of ABP1 antibodies at two representative concentrations of IAA (10–6.5 and 10–5 M). Control time courses were obtained by using pre-immune IgG. At 10–6.5 M IAA, the time course of antibody-treated protoplasts was similar to the control time course. At 10–5 M IAA, antibody-treated protoplasts showed no swelling throughout the 2-h period after IAA application, whereas the control protoplasts showed a normal response.
These results indicated that ABP1 located on the outer surface of the plasma membrane mediated the response to NAA at all concentrations, whereas it mediated the response to IAA only at high IAA concentrations. The response at low concentrations of IAA might be caused by the ABP1 located inside the protoplast. However, this possibility was regarded as very unlikely because no comparable response was found with NAA, for which ABP1 has a high affinity (note also that all the responses demonstrated to be ABP1 mediated could be induced effectively by NAA). It seemed likely that a distinct receptor protein mediated the response not inhibited by anti-ABP1 antibodies. As evident from Figure 3A, the ABP1-independent response to IAA formed a bell-shaped optimum curve (solid circles). The ABP1-dependent response, evaluated in Figure 3A by subtracting the ABP1-independent response from the response found without the antibody treatment (dotted line and white circles), also appeared to form a bell-shaped optimal curve with a peak activity at higher IAA concentration. Comparison of this curve with the one for NAA (dotted line and white circles in Fig. 3B) indicated that the ABP1-dependent response is more sensitive to NAA than IAA. The difference in sensitivity was estimated to be between 1 and 2 orders of magnitude. Antipeptide D16 antibodies, shown previously to have an auxin agonist activity in ABP1-mediated responses, were also found to induce an auxin-like swelling response on pea epidermal protoplasts (Fig. 5). The effective concentration range appeared to be as narrow as that for NAA, in agreement with the result that the response to NAA is mediated principally by ABP1.
It was anticipated that the swelling response was caused by an enhanced influx of ions (most probably of K+ and Cl–), rather than a reduced efflux of ions or a metabolic release of osmotic substances (Iino et al., 2001 At either concentration, IAA could not induce any detectable swelling response in the protoplasts bathed in K+-free medium (Fig. 6, A and B) or Cl–-free medium (Fig. 6, C and D). When the same protoplasts were washed with and suspended in the standard medium containing both ions, they showed a clear swelling response to the second application of IAA (Fig. 6; the data after the second vertical dashed line). These results demonstrated that the ABP1-dependent and -independent responses both require the presence of extracellular K+ and Cl–. Therefore, it could be concluded that, in either response, protoplasts swell by taking up K+ and Cl– from the bathing medium. The results also indicated that K+ cannot be taken up when Cl– is absent or vice versa.
Although the Ca2+ present in the bathing medium does not contribute to the swelling response as osmotic ions, as evident from the results shown above, the external Ca2+ might be taken up as a second messenger. This possibility was investigated next. As shown in Figure 7A, protoplasts washed with and suspended in a Ca2+-free medium did not swell in response to 10–6.5 M IAA. The same protoplasts showed a clear swelling response when resuspended in the Ca2+-containing standard medium and treated again with IAA (the data after the second vertical dashed line). The results demonstrated that the ABP1-independent response requires the presence of extracellular Ca2+.
In sharp contrast, the protoplasts treated with IAA at a concentration of 10–5 M showed a clear swelling response in the Ca2+-free medium (Fig. 7B, black circles). The same protoplasts washed with and suspended in the Ca2+-containing medium showed another rapid response that was comparable with the initial response in the Ca2+-free medium. The second swelling response observed in the Ca2+-containing medium might be seen to be too large to be regarded as a response that continued after the swelling observed in the Ca2+-free medium (compare with the time course shown in Fig. 1E). In response, it might be suggested that the ABP1-mediated response partially depended on external Ca2+ and that the Ca2+-dependent portion became inducible after resuspension of the protoplasts in the Ca2+-containing medium. However, when the protoplasts incubated in the Ca2+-containing medium from the beginning were treated similarly, these protoplasts showed a swelling response to the second application of IAA that was comparable with the response to the first application (Fig. 7B, white circles). Therefore, it appeared that, although the protoplasts were made free of IAA only about 10 min after the first treatment period, they become fully responsive to the second IAA treatment. The results shown in Figure 7B also indicated that the response to the first IAA application recorded without Ca2+ was similar to that recorded with Ca2+. Taken together, these results indicated that external Ca2+ did not make any appreciable contribution to the ABP1-mediated response. Similar experiments were carried out with NAA (10–6 M) and D16 antibodies (10–8.5 M). As shown for 10–5 M IAA, the protoplasts suspended in the Ca2+-free medium showed a clear swelling response to NAA (Fig. 7C) and D16 antibodies (Fig. 7D). As also found with 10–5 M IAA, the same protoplasts showed another comparable response after resuspension in the Ca2+-containing medium. The results substantiated the conclusion that the ABP1-mediated response does not depend on external Ca2+.
To compare the results described above with the growth response, we measured dose response curves for both IAA and NAA from pea internode segments. The effects of apoplastic Ca2+ on the dose response relationship were also investigated. Experiments were designed so as to obtain the growth data under comparable experimental conditions. The segments (5 mm long) were isolated from the third internode of red light-grown seedlings and incubated for 2 h, without applied auxin, to deplete endogenous auxin. They were subsequently incubated for 2 h with auxin to determine auxin-induced growth. The incubation took place under red light. Dose response curves were obtained using an incubation medium containing 10 mM KCl, 1 mM CaCl2, 10 mM Suc, and 5 mM MES-KOH (pH 6.0). The curve for IAA revealed two peaks with a shallow trough (Fig. 8A). On the other hand, the curve for NAA was bell shaped with a single peak (Fig. 8C). It also was noted that the effective concentration range was much wider with IAA than NAA.
When IAA-induced growth was determined after 4 h of treatment (instead of 2 h), a bell-shaped dose response curve with only a single major peak (at about 10–5 M) was generated, although the curve for IAA was still wider than that for NAA (data not shown). Therefore, the timing of growth measurement appeared to be important to resolve the two peaks. To obtain dose response curves with reduced levels of apoplastic Ca2+, segments were incubated in a medium containing BAPTA, a membrane-impermeant Ca2+ chelator (CaCl2 was omitted from the medium); BAPTA was present in both the pre-incubation and treatment mediums. When segments were treated with 1 mM BAPTA, the dose response curve for IAA (Fig. 8B) or NAA (Fig. 8D) was similar to that obtained without BAPTA. At 10 mM BAPTA, the growth was substantially reduced over the entire range of auxin concentrations (Fig. 8, B and D). A clear difference was noted, however, between IAA and NAA. For NAA, the dose-dependent increase in growth occurred in the same concentration range as that found in the Ca2+-containing medium. For IAA, the dose-dependent increase occurred at concentrations higher than that in the Ca2+-containing medium. In fact, no IAA-induced increase in growth could be detected in the range from 10–8 to 10–7 M, in which the segments incubated in the Ca2+-containing medium showed a sharp increase in growth. Therefore, in contrast to the case for NAA, the response to low concentrations of IAA was selectively reduced when the apoplastic Ca2+ level was reduced by BAPTA. When segments were treated with an intermediate concentration (3 mM) of BAPTA, the response at low concentrations of IAA was not eliminated but was reduced more than that at high concentrations (Fig. 8B). As a result, the peak of response at low concentrations became less apparent. These results obtained by measuring growth supported the idea that both the ABP1-dependent and -independent signaling pathways contribute to the growth response, as will be discussed below.
We have demonstrated that protoplasts of pea stem epidermis undergo auxin-induced swelling (Fig. 1). This result provides the clearest evidence that epidermal cells respond directly to auxin. Furthermore, we have demonstrated that two distinct signaling pathways participate in the protoplast swelling response. The auxin receptor in one pathway is ABP1 located on the outer surface of the plasma membrane (Figs. 3 and 4D). In this pathway, NAA is more effective than IAA, as deduced from the results shown in Figure 3, and external Ca2+ is not required (Fig. 7, B–D). The auxin receptor for the other pathway is most probably not ABP1 (see "Results"). In sharp contrast to the ABP1 pathway, this pathway (non-ABP1 pathway) is insensitive to NAA (Figs. 3 and 4B) and depends strictly on external Ca2+ (Fig. 7A). The swelling induced by either pathway results from uptake of K+ and Cl– (Fig. 6).
Using the protoplasts isolated from maize coleoptiles and Arabidopsis hypocotyls, Steffens et al. (2001
The use of anti-ABP1 antibodies has linked ABP1 located on the outer surface of the plasma membrane to the following responses: plasma membrane hyperpolarization (Leblanc et al., 1999a
In previous studies, anti-ABP1 antibodies were shown to inhibit responses in membrane potential, H+ efflux, and protoplast volume (Barbier-Brygoo et al., 1991
Our finding that the non-ABP1 pathway is insensitive to NAA might provide a clue to the nature of the second auxin receptor. The auxin uptake carrier located in the plasma membrane was reported to have a much reduced affinity for NAA (Delbarre et al., 1996
Calcium ions have been implicated as second messengers in various plant signal transduction processes (White, 2000
Our results might provide an explanation for the contradictory results regarding whether or not apoplastic Ca2+ is required for the auxin-induced medium acidification by coleoptile segments. Cohen and Nadler (1976
As mentioned above, the binding affinity of ABP1 is at least 10 times higher for NAA than IAA. However, the dose response curves for growth have indicated that segments of coleoptiles or stems are at least 10 times more sensitive to IAA (e.g. Shinkle and Briggs, 1984 In fact, both the two signaling systems might be responsible for the auxin-induced growth of segments. The data shown in Figure 8 provide correlative evidence for this hypothesis. The dose response curve for IAA-induced growth showed two peaks (Fig. 8A), in agreement with the participation of ABP1-indepdendent and -dependent responses that have different dose requirements. On the other hand, the dose response curve for NAA-induced growth showed only one peak (Fig. 8C), in agreement with the participation of only the ABP1-dependent response. The growth response to IAA at low doses depended more critically on the presence of apoplastic Ca2+ than the response at high doses (Fig. 8B); the growth response to NAA showed no such dose-dependent difference (Fig. 8D). These results agree with the different Ca2+ requirements of the two pathways. The growth response to high IAA concentrations was also inhibited by a high concentration of BAPTA (10 mM). Therefore, the IAA-dose dependent difference in Ca2+ requirement was not as sharp as that for the protoplast swelling response (Fig. 7). Reducing apoplastic Ca2+ probably caused other less specific effects on growth.
Dose response curves for IAA-induced segment growth have been reported by a number of workers. Two distinct peaks (Fig. 8A) have not been reported previously, but biphasic dose response curves have been described. Working with segments of dark-grown oat coleoptiles, Shinkle and Briggs (1984
When the level of apoplastic Ca2+ was reduced extensively by treatment with a high-concentration BAPTA (10 mM), the dose response curve for segment growth became very similar between IAA and NAA (Fig. 8, B and D). In view of the explanations given above, this dose response curve may be attributed almost solely to the ABP1 pathway. It is interesting to note that, in this dose response curve, the response stayed more or less constant over a wide range of concentrations (i.e. the curve was not bell shaped). The mechanism underlying the descending arm of ABP1-mediated response, being distinct from that for the ascending arm, might involve apoplastic Ca2+. Our results may be related to those of Leblanc et al. (1999b
Based on the discussion above, we hypothesize that the two signaling pathways uncovered in pea epidermal protoplasts participate in the auxin-induced growth of pea internodes. In the frame of the acid growth theory, the activation of plasma membrane H+-ATPase by the ABP1 pathway (Rück et al., 1993
It might be argued that the non-ABP1 pathway, which is more sensitive to IAA than the ABP1 pathway, is more critically involved in the control of internode growth by constitutive concentrations of endogenous auxin. Many of the results reported by Jones et al. (Jones et al., 1998
Although ABP1 has been shown to function on the outer surface of the plasma membrane, there is some evidence that the site of receptor action for the control of growth is intracellular (Claussen et al., 1996
Plant Material
Seedlings of peas (Pisum sativum L. cv Alaska) were raised as described by Haga and Iino (1997
Epidermal peels were obtained from the upper part (approximately 15 mm long) of the third internode. Maximal and uniform elongation took place in this part (Haga and Iino, 1997
After the enzyme treatment, the mixture was filtered through a nylon mesh and centrifuged at 110g for 10 min. The pellet was suspended in a 2-mL bathing medium that contained 0.5 M sorbitol, 10 mM KCl, 1 mM CaCl2, 20 mM Glc, and 10 mM MES (adjusted to pH 6.0 with Tris). The suspension was loaded on an 18% (v/v) Percoll (Sigma, St. Louis) solution also containing the bathing medium components and centrifuged for 5 min at 110g. The protoplasts collected from the interface between the Percoll solution and the loaded medium were washed twice by suspending them in a 5-mL bathing medium and centrifuging them at 110g for 5 min. The protoplast pellet was suspended in a small amount of the bathing medium to obtain the final preparation. All steps of protoplast preparation were carried out in the growth room and under the red light condition used to raise seedlings. More details not described here can be found in Long and Iino (2001
A suspension (typically 225 µL) of freshly prepared protoplasts was added to an all-side clear quartz cuvette (base area 10 x 10 mm, height 45 mm). The cuvette was set on the sample stage of an inverted microscope (IMT-1, Olympus, Tokyo), and the protoplasts were incubated at 25°C ± 0.5°C under red light (50 µmol m–2 s–1); for details of the system, see Wang and Iino (1997
A 25-µL solution of IAA or NAA, made up in the bathing medium, was added to a 225-µL protoplast suspension. This addition was generally made after a period of incubation on the microscope stage. After a gentle agitation, the suspension was set again on the microscope stage to investigate the effect of auxin on the protoplast volume.
The antibodies used in the present study were those described by Venis et al. (1992 To investigate the effect of anti-ABP1 antibodies on auxin-induced swelling (Figs. 3 and 4), a 25-µL solution of the antibodies was added to a 200-µL suspension of protoplasts (to allow 25 µL for auxin solution). The final antibody concentration indicated was the concentration after addition of the auxin solution. To investigate the effect of D16 antibodies on the protoplast volume (Figs. 5 and 7D), a 25-µL solution of the antibodies was added to the protoplast suspension (225 µL).
The bathing medium components were modified as follows to investigate the roles for ions. The K+-free medium was prepared by using 10 mM TEA-Cl in place of 10 mM KCl. The Cl–-free medium was prepared by using 12 mM iminodiacetic acid, 10 mM KOH, and 1 mM Ca(OH)2 in place of 10 mM KCl and 1 mM CaCl2. To prepare the Ca2+-free medium, CaCl2 was omitted from and 1 mM EGTA was added to the bathing medium. The modified medium was used from the step of suspending the protoplast pellet after enzyme treatment. The protoplasts treated with auxin in a modified medium were resuspended in the standard medium and treated again with auxin. The change in mediums was achieved by mixing the protoplast suspension with the standard medium (about 5 mL), centrifuging the mixture for 5 min at 110g, and resuspending the pellet in a 225-µL standard medium.
Protoplast volume was determined as described previously (Wang and Iino, 1997
Internode segments (5 mm long) were excided from the upper part of the third internode (approximately 5–10 mm below the hook) with a pair of razor blades attached to an acrylic holder. The segments were first incubated for 2 h in a solution containing 10 mM KCl, 1 mM CaCl2, 10 mM Suc, and 5 mM MES (adjusted to pH 6.0 with KOH) and then for 2 h in a solution containing IAA and these components. Incubation was carried out at 25°C under red light (2.5–2.7 µmol m–2 s–1). For incubation, multiwell polystyrene containers (Nalge Nunc, Rochester, NY) were used. Each well (16-mm i.d.) contained a 0.4-mL incubation medium and one segment. The containers were shaken at 70 rpm during incubation. When the segments were treated with BAPTA, CaCl2 was omitted from and BAPTA was added to the mediums for both pre-incubation and auxin treatment. Segments were photographed on technical pan film (Eastman-Kodak, Rochester, NY) just before the onset of incubation in IAA-containing medium and at the end of 2 h of incubation. The negative film was expanded by means of a slide projector. The segment length, traced on paper, was determined using a computer-interfaced digitizer. Growth increment during the 2-h treatment period was calculated as the percentage of the initial length (the length at the onset of treatment). No light other than the red light (2.5–3.0 µmol m–2 s–1) was used to handle seedlings, to excise seedlings, and to obtain photographs. Received August 4, 2003; returned for revision September 8, 2003; accepted November 7, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.031294. * Corresponding author; e-mail iino{at}sci.osaka-cu.ac.jp; fax 81–72–891–7199.
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ABSTRACT
RESULTS
), 3 (
), and 10 (
) mM. The dotted lines reproduce the curves in A and C. Segments were treated with IAA or NAA for 2 h after 2-h pre-incubation without auxin. The ordinate indicates the increment in length, calculated as the percent of the initial length. Each point is the mean from eight to 16 segments (vertical bar = ±SE). The SE is not seen when smaller than the symbol size. The initial lengths in millimeters (mean ± SD) of all analyzed segments were 5.131 ± 0.086 (with Ca2+), 5.191 ± 0.065 (1 mM BAPTA), 5.164 ± 0.086 (3 mM BAPTA), and 5.122 ± 0.068 (10 mM BAPTA).
