Plant Physiol. (1998) 117: 1473-1486

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Ken Haga and Moritoshi Iino^*

Botanical Gardens, Faculty of Science, Osaka City University, Kisaichi, Katano-shi, Osaka 576, Japan

	ABSTRACT

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Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied ³H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.

	INTRODUCTION

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There can be no doubt that the auxin IAA is a key substance in the control of plant growth, and yet many aspects of its physiological functions are the subject of controversy. Trewavas (1981) pointed out that the classical concept that growth is controlled by changes in auxin concentration is not valid and underscored the alternative idea that tissue "sensitivity" to auxin plays an important role (Hanson and Trewavas, 1982). This argument has stimulated many authors to further discussion (Trewavas and Cleland, 1983; Firn, 1986; Guern, 1987; Davies, 1995). Recent studies of pea (Pisum sativum) seedlings, however, have shown that growth has a positive correlation with the level of endogenous IAA (Law and Davies, 1990; McKay et al., 1994).

To clarify the causal relationship between IAA levels and growth, demonstration beyond a correlative relationship is needed. One approach is to investigate the growth changes induced by experimental manipulation of auxin levels, preferably using intact plants. After many years of uncertainty (Cleland, 1977; Thimann, 1977), it has been demonstrated in maize (Zea mays L.) coleoptiles (Baskin et al., 1986; Iino, 1995), pea internodes (Hall et al., 1985; Yang et al., 1993, 1996), and watermelon hypocotyls (Carrington and Esnard, 1988) that growth of intact plants is stimulated by externally supplied IAA. Therefore, the level of auxin appears to limit growth rate in many growing tissues, supporting the idea that growth is controlled by changes in the level of auxin.

Our understanding of the relationship between IAA and growth has been complicated, however, by the observation that growth stimulation by exogenous IAA occurs for only a short time even if supply is continuous (Hall et al., 1985; Carrington and Esnard, 1988). Detailed analyses of this short-term growth stimulation in maize coleoptiles (Iino, 1996) and pea internodes (Haga and Iino, 1997) have indicated that the level of auxin in the target tissue is regulated in such a way that the initially enhanced level declines after a few hours. The responsiveness (or the capacity to respond) to IAA also declines in pea internodes in response to an increase in IAA level (Haga and Iino, 1997). Thus, the causal relationship between the IAA level and the growth rate is complex. It does not appear to be a simple choice between regulation by the level of IAA versus regulation by IAA sensitivity or responsiveness.

It has long been considered that the auxin supplied from the tip of grass coleoptiles or the apical part of dicotyledonous seedling shoots determines the growth of coleoptiles or stems (Went and Thimann, 1937). Firn and coworkers (Tamimi and Firn, 1985; Parsons et al., 1988a, 1988b), however, concluded that this apical control of growth is not valid. Their major arguments were that auxin transport inhibitors cannot inhibit growth in a predictable way and that growth inhibition by decapitation cannot be explained by auxin depletion but, rather, by a wounding effect. McKay et al. (1994), however, were able to observe that auxin transport inhibitors reduce the growth of intact pea internodes. No further study has discussed the critical questions raised by Firn and coworkers (Tamimi and Firn, 1985; Parsons et al., 1988a, 1988b) in the original studies.

Another controversial issue concerns the concept of "regeneration of the physiological tip" (Went and Thimann, 1937). The growth of oat coleoptiles inhibited by decapitation recovers spontaneously, and this has been considered to result from regeneration of auxin-producing activity in an upper part of the coleoptile stump (Dolk, 1926; Thimann and Bonner, 1933). Evidence for this idea was provided by measurements of auxin by the Avena curvature test (e.g. van Overbeek, 1941) and later by physicochemical measurements of IAA in maize coleoptiles (Iino and Carr, 1982b). Growth recovers in isolated, nontip segments of oat and maize coleoptiles (Anker, 1973; Evans and Schmitt, 1975), and the production of IAA is also initiated in such segments (Iino and Carr, 1982b). However, Vesper and Evans (1978) provided a view different from the classical one. They interpreted the growth recovery as being due to an increase in auxin sensitivity. Hatfield and LaMotte (1984) used this sensitivity view to explain their results in decapitated maize coleoptiles. Parsons et al. (1988a) concluded that the growth inhibition by decapitation results from wounding and therefore argued that the growth recovery cannot be explained by regeneration of the auxin-producing activity.

The issues described above are central to our understanding of the physiological roles of auxin, and the conflicting results and ideas must be clarified before molecular mechanisms of auxin action are truly understood. Therefore, we conducted simple but detailed experiments with seedlings of maize and pea. The major approaches we used were to monitor growth of an elongating zone of maize coleoptiles and pea internodes after decapitation or apical application of the auxin-transport-inhibitor NPA and to investigate the IAA dose-response relationship in NPA-treated or decapitated plants. A mathematical model was introduced to evaluate response elements that underlie the changes in the dose-response relationship.

	MATERIALS AND METHODS

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Plant Materials

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Growth Measurement and Analysis

The time courses of growth in maize coleoptiles and pea internodes were expressed as increments in zone length (Iino, 1996) and the natural logarithm of zone length (Haga and Iino, 1997), respectively. The growth in intact plants was linear with these expressions (Haga and Iino, 1997). The increments were determined for individual plants, and the means and SEs were calculated from a set of plants. The growth rate of a zone during a given time interval was calculated as the slope of the increase in length (maize) or the natural logarithm of length (pea). The rates were determined for individual plants, and the mean and the SE were calculated from a set of plants.

Preparation of Lanolin Mixtures of IAA and NPA

We occasionally found that the effective IAA concentrations differed when different batches of IAA/lanolin were used, necessitating the use of the same IAA/lanolin preparations in a series of experiments. IAA powder does not always dissolve totally in lanolin, resulting in uncontrolled effectiveness. Since the analysis of dose-response relationships was one of the major aspects of the present study, more thorough mixing of IAA and lanolin was carried out, in addition to the use of the same preparations. The procedure used to obtain IAA/lanolin was: (a) add weighed powders of IAA to a weighed amount of lanolin in a glass container, (b) melt the mixture by placing the container in hot water for 1 min while stirring with a glass rod, (c) stir the mixture rigorously for an additional 10 min in air and repeatedly crush visible IAA particles, if any, with the glass rod, and (d) repeat the melting and stirring steps several times. The IAA/lanolin mixtures at concentrations from 0.1 to 10 mg g¹ were prepared as described above; those at lower concentrations were prepared by mixing a portion of IAA/lanolin (10 times higher concentration) with lanolin and repeating the melting and stirring procedure several times.

IAA and NPA Treatments and Decapitation

Application of [³H]IAA and Measurement of Radioactivity

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The [³H]IAA/lanolin mixture was applied to a specified region above the NPA application site. After a scheduled time, zone 3 was excised, split vertically into two pieces, and added to a vial containing 80% methanol (250 µL). In maize the leaf inside the coleoptile segment was removed. Segments from three plants were used for each measurement. The vial was allowed to stand for 12 h in the dark at room temperature, and 10 mL of scintillation cocktail (Clearsol 1, Nacalai Tesque, Kyoto, Japan) was added. Radioactivity was determined with a liquid-scintillation counter (model LS6200, Beckman).

Application of a Mathematical Model to IAA Dose-Response Data

At the steady state the growth rate, R, is given by:

(1)

where [IAA] is the concentration of IAA available for the binding reaction, [X]₀ is the total concentration of X (free plus bound forms), K is the dissociation constant (i.e. k₂/k₁), and a is the proportionality constant. When IAA is supplied externally, [IAA] is the sum of the concentration of endogenous IAA ([IAA]_en) and that of exogenous IAA ([IAA]_ex):

(2)

We assume that [IAA]_ex is linearly related to x, the applied concentration (Assumption 2):

(3)

where b is the proportionality constant. In view of Equations 2 and 3, Equation 1 becomes:

(4)

where

(5)

(6)

and

(7)

It is assumed that [IAA]_en is not affected by applied IAA, at least in the ascending part of the dose-response curve (Assumption 3). With this assumption, c (Eq. 6) can be regarded as a constant.

In Equation 4, the parameter A determines the growth rate at saturating IAA doses, and a change in A without changes in K and c results in proportional changes in growth rate at all IAA doses. The receptor concentration [X]₀ is an element affecting A (Eq. 5). The parameter K determines the position of the dose-response curve in the log dose axis, and a change in K without changes in A and c results in its parallel shift along the log dose axis. When b is not modified, a change in K parallels the change in K (Eq. 7). The parameter c is related to [IAA]_en. The value of c parallels [IAA]_en when b is not altered (Eq. 6).

To obtain IAA dose-response data, we applied IAA to the surface of a monitored zone. With this application method, IAA would enter the tissue and reach to near the site of its action by diffusion. Therefore, it is probable that [IAA]_ex is a linear function of x (i.e. Assumption 2). More critical is the assumption that [IAA]_en is not affected by applied IAA (Assumption 3). Endogenous IAA is transported to the vicinity of its action through a carrier-mediated transport system that resides in the plasma membrane (Lomax et al., 1995). Therefore, exogenous IAA would compete with endogenous IAA for transport and, as the concentration of exogenous IAA increased, [IAA]_en would decrease. However, for the following reasons, it is possible that this decrease in [IAA]_en is relatively insignificant at the critical IAA doses that form the ascending part of the dose-response curve. First, since the maximal growth rate achieved in maize (Iino, 1996) and pea (Haga and Iino, 1997) by IAA application was the same whether IAA was applied directly or apically, it appears that the saturation was not imposed by the transport of IAA; i.e. the transport system has the capacity to translocate IAA to the site of action, exceeding the saturation level. Second, since endogenous IAA is expected to reach near the site of action largely by diffusion (see above), the competition of exogenous IAA with endogenous IAA in the transport system would be relatively weak at the critical IAA doses.

Dose-response data were fitted to Equation 4 by the least-squares method with the aid of a computer program (Iino, 1987). The computed values of the parameters were used to evaluate the response elements underlying the changes in the dose-response relationship.

	RESULTS

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IAA Dose-Response Relationship in Intact Plants

View larger version (25K): [in this window] [in a new window]   Figure 1. Relationships between IAA dose (applied concentrations) and response (growth rate) in intact maize coleoptiles (A) and pea third internodes (B). Coleoptiles and internodes were marked with India ink at 5-mm intervals. After 1 h, IAA/lanolin was applied to the two opposite sides of zone 3 as vertical stripes (about 1 mm wide), and the length of this zone was measured at 1-h intervals (see illustrations). From increments in length (maize) or the natural logarithm of length (pea), the growth rate at each 1-h interval was calculated. The data shown are those obtained at the three intervals indicated. Each point and vertical bar represent the mean and ± SE, respectively, from 22 to 24 (A) or 27 to 32 (B) plants. Lines are the best-fit curves of the function: R = A(c + x)/[K + (c + x)], where R is the growth rate (in millimeters per hour [A] or per hour [B]), x is the applied IAA concentration (in milligrams per gram), and A, K, and c are the constants. The computed values of the constants are indicated in each panel. The SDs of the experimentally determined growth rates from the growth rates on the best-fit curve: A, 0.011 (1-2 h), 0.018 (2-3 h), and 0.0047 (3-4 h); B, 0.00098 (1-2 h), 0.0023 (2-3 h), and 0.0022 (3-4 h).

In maize the growth rate remained enhanced for at least several hours beginning about 1 h after IAA application (Iino, 1996). In pea the growth rate became maximal 1 to 3 h after IAA application, but decreased sharply afterwards; the extent of this decrease was greater at higher concentrations (Haga and Iino, 1997). The data in Figure 1 are in essential agreement with those reported previously, although the effective range of IAA concentrations shifted to lower concentrations. This difference might be due to the use of different IAA preparations (see ``Materials and Methods'').

Mathematical models have been presented to describe the relationship between IAA dose and the growth response (McRae et al., 1953; Nissen, 1985; Weyers et al., 1987; Fitzsimons, 1989). In "Materials and Methods," a mathematical model (Eq. 4) was formulated by extending the model based on a reversible binding reaction between IAA and its receptor (Weyers et al., 1987). We considered two basic characteristics of the dose-response curve: responsiveness and sensitivity to IAA. The responsiveness is represented by the growth rate at saturating IAA doses, and the sensitivity is represented by the position of the ascending part of the dose-response curve along the log dose axis. In addition, the growth rate measured without applied IAA is considered to be due to endogenous IAA. In Equation 4, the parameters A and K determine the responsiveness and the sensitivity, respectively. (Note that the sensitivity becomes greater as K becomes smaller.) The parameter c, together with A and K, determines the growth rate without applied IAA (x = 0 in Eq. 4) and represents the concentration of endogenous IAA (Eq. 6).

The lines in Figure 1 were obtained by fitting the data to Equation 4. In maize the computed values of the three parameters were very similar in the first two periods (1-2 h and 2-3 h) and deviated somewhat in the third period (3-4 h; Fig. 1A). In pea the values were relatively similar in the first two periods and deviated considerably in the third (Fig. 1B). The time-dependent change in the dose-response relationship after IAA application may be caused by an element that varies in an IAA dose-dependent manner (Haga and Iino, 1997). If so, the model is no longer applicable to the dose-response data obtained in later periods. Because of this possibility, we did not assign a physiological significance to the parameter values obtained in the period from 3 to 4 h.

Effects of NPA Treatment

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View larger version (24K): [in this window] [in a new window]   Figure 2. Effects of applied NPA on the growth of zone 3 in maize coleoptiles (A) and pea internodes (B). Coleoptiles and internodes were marked as described in Figure 1. After 1 h a ring (about 1 mm wide) of NPA/lanolin was applied to the region just above the top of zone 2 (A) or the top of zone 1 (B) (see illustrations). Following the NPA application, the increment in length (A) or the natural logarithm (Ln) of length (B) of zone 3 was monitored. The concentrations of NPA were: 0 (), 0.01 (), 0.1 (), 1 (), and 10 () mg g1. The means ± SE from eight plants are shown.

The maize coleoptiles treated with NPA at low concentrations (0.01 and 0.1 mg g¹) exhibited unique time courses: growth once suppressed by NPA recovered gradually beginning at about 5 h (Fig. 2A). This recovery is the first indication of an increase in auxin sensitivity induced by a decrease in endogenous auxin level. No such growth recovery was apparent in pea (Fig. 2B).

The results obtained from the experiment designed to determine whether, as expected, the NPA treatment inhibits the basipetal transport of IAA are summarized in Figure 3. In these experiments [³H]IAA/lanolin was applied above the position to which NPA had been applied, and the radioactivity content in zone 3 was measured after an interval (see the legend to Fig. 3 for details). The data demonstrated that NPA indeed inhibits the transport of IAA. The dependence of the inhibition of [³H]IAA transport on NPA concentration should be compared with that of the growth inhibition achieved in a later period that is close to the time of radioactivity determination (5 h for maize and 7 h for pea after NPA application). Apparently, the growth data (Fig. 2) were in reasonable agreement with the [³H]IAA transport data (Fig. 3). The data for pea, however, suggested that NPA might have a somewhat greater effect on [³H]IAA transport than on growth.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effects of NPA on transport of [3H]IAA in maize coleoptiles (A) and pea internodes (B). A ring of NPA/lanolin or plain lanolin as the control was applied as described in Figure 2. After 1 h (A) or 2.5 h (B), [3H]IAA/lanolin (1.63 MBq g1) was applied as a ring (about 0.8 mm wide) to the position 2 mm below the top (A) or as a spot (about 3 mm2) to the surface just above the upper node of the third internode (B) (see illustrations). Zone 3 was excised 4 h (A) or 4.5 h (B) after [3H]IAA application and its radioactivity was determined. The means ± SE were obtained from three measurements; three plants (zone segments) were used in each measurement.

If the growth inhibition by NPA treatment is caused by a decrease in endogenous auxin level, then the inhibited growth should be restored by an external supply of IAA. This was investigated by applying IAA to the monitored zone 1 or 4 h after NPA application. As shown by the representative time courses in Figure 4, growth was substantially stimulated by applied IAA in both maize and pea. The dose-response data shown in Figure 5 indicated that in both maize and pea the maximally enhanced growth rate was very close to that achieved in intact plants. These results indicate that NPA treatment inhibited growth by reducing the level of endogenous auxin and that the responsiveness to IAA was not affected by NPA treatment.

View larger version (25K): [in this window] [in a new window]   Figure 4. Effects of applied IAA on the growth of zone 3 in NPA-treated maize coleoptiles (A) and pea internodes (B). After application of NPA/lanolin (1 mg g1 [A] or 10 mg g1 [B]; Fig. 2), the growth of zone 3 was monitored for length (A) and the natural logarithm (Ln) of length (B). At the time indicated (1 or 4 h), zone 3 was treated with IAA/lanolin (Fig. 1) at the following concentrations: 0 (), 0.01 (), 0.1 (), and 1 () mg g1. The means ± SE from eight plants are shown.

View larger version (30K): [in this window] [in a new window]   Figure 5. IAA dose-response relationships in NPA-treated maize coleoptiles (A) and pea internodes (B). Growth rates in the periods 1 to 2 h and 2 to 3 h after IAA application were determined from time-course data, some of which are shown in Figure 4, with IAA application 1 h () or 4 h () after NPA application. Each point and vertical bar represent the mean and ± SE, respectively, from eight plants. Dashed and solid lines are the best-fit curves (Fig. 1) that coincided with the parameter values indicated. The SDs (Fig. 1) were: A, 0.015 (1-2 h, ), 0.024 (1-2 h, ), 0.017 (2-3 h, ), 0.017 (2-3 h, ); B, 0.0017 (1-2 h, ), 0.0050 (1-2 h, ), 0.0035 (2-3 h, ), 0.0061 (2-3 h, ). Dotted lines reproduce the curves in Figure 1 at the corresponding periods after IAA application.

It was noted, however, that the growth rate in maize was more effectively enhanced at low concentrations when IAA was applied at 4 h rather than at 1 h (Fig. 5A). This difference was observed for the measurement periods of 1 to 2 h and 2 to 3 h. The results indicated that IAA sensitivity was enhanced as the time of IAA application was delayed from 1 to 4 h after NPA application. In pea no such sensitivity change was apparent (Fig. 5B).

Also shown in Figure 5 are the best-fit curves of Equation 4 and the computed values of the parameters. In both maize and pea the value of c was considerably smaller in NPA-treated plants (Fig. 5) than in nontreated plants (Fig. 1). In both maize and pea the four sets of data provided similar values of A (Fig. 5), as is also evident from the similar saturation levels of the curves. These values of A were also similar to those in intact plants (Fig. 1). These analytical results, in agreement with the conclusions given above, indicate that the endogenous level of auxin is reduced by NPA treatment and that the responsiveness to IAA was not affected by NPA treatment.

The value of K was subject to modification in maize (Fig. 5A). It was similar to that in nontreated coleoptiles when IAA was applied at 1 h but was smaller when IAA was applied at 4 h. The change in K was reflected in a nearly parallel shift of the best-fit curve along the log dose axis. These analytical results support the conclusion that IAA sensitivity was enhanced as the time after NPA application elapsed and indicated that the sensitivity was enhanced by a factor of 3 to 5 (see the two K values in Figure 5A, top and bottom).

In pea the values of K obtained by applying IAA 1 and 4 h after NPA application were similar (Fig. 5B), indicating that IAA sensitivity was held constant. This was also evident from the similarity in position of the best-fit curves (Fig. 5B, dashed versus solid line). However, these K values were somewhat greater than those from nontreated plants (Fig. 1B). This means that the internode became less sensitive to IAA rapidly after NPA treatment. We do not conclude, however, that this relatively small difference in K represents the true change in sensitivity, because it is possible that one or more of the assumptions introduced to formulate Equation 4 are not exactly valid for pea.

Effects of Decapitation

View larger version (23K): [in this window] [in a new window]   Figure 6. Effects of decapitation on the growth of zone 3 in maize coleoptiles (A) and pea internodes (B). Coleoptiles and internodes were marked as described in Figure 1 and, after 1 h, subjected to decapitation. Maize coleoptiles were decapitated 2 mm from the top (a) or at the base of zone 1 (b), and pea internodes were decapitated at the top (a), middle (b), or base (c) of zone 1 (see illustrations). Following decapitation, the growth of zone 3 was monitored for length (A) and the natural logarithm (Ln) of the length (B). , Nondecapitated controls. The means ± SE from six plants are shown.

The growth response to applied IAA was investigated. For maize IAA application was done 1 h after decapitation. (The IAA-growth relationship in the recovery phase is characterized in "Growth Recovery in Decapitated Maize Coleoptiles.") For pea IAA application was either 1 or 4 h after decapitation. As shown by the representative time courses in Figure 7, growth was stimulated by applied IAA in all cases. Figure 8 shows the dose-response data 1 to 2 h after IAA application. The maximal growth rate at saturating IAA doses was somewhat lower than that achieved in intact plants in all cases. The results indicated that the growth inhibited by decapitation could be restored by applied IAA, although the responsiveness to IAA was a little reduced by decapitation. The results agree in principle with the view that the growth of coleoptiles and internodes depends on the endogenous auxin supplied from the apical region.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effects of applied IAA on the growth of zone 3 in decapitated maize coleoptiles (A) and pea internodes (B). The growth of zone 3 was monitored after decapitation for length (A) and the natural logarithm of length (B); maize coleoptiles were decapitated at 2 mm and pea internodes were decapitated at the base of zone 1 (Fig. 6). At the time indicated (1 or 4 h), zone 3 was treated with IAA/lanolin (Fig. 1) at the following concentrations: 0 (), 0.01 (), 0.1 (), and 1 () mg g1. The means ± SE from eight plants are shown.

View larger version (21K): [in this window] [in a new window]   Figure 8. IAA dose-response relationships in decapitated maize coleoptiles (A) and pea internodes (B). Growth rates of zone 3 in the period 1 to 2 h after IAA application were determined from time-course data, some of which are shown in Figure 7; IAA application was 1 h () or 4 h () after decapitation. Each point and vertical bar represent the mean and ± SE, respectively, from eight plants. Dashed and solid lines are the best-fit curves (Fig. 1) that coincided with the parameter values indicated. The SDs (Fig. 1) were: A, 0.013; B, 0.0018 (), 0.0029 (). Dotted lines reproduce the curves in Figure 1 at the corresponding times after IAA application.

The best-fit curves of Equation 4 are shown in Figure 8 together with the parameter values. As expected, the values of c in decapitated plants were smaller than those in intact plants. The reduced responsiveness to IAA was reflected in the slightly smaller values of A in decapitated plants (Fig. 8; compare with Fig. 1). Decapitated maize showed a somewhat greater value of K (reduced IAA sensitivity) compared with intact maize. In pea the value of K from either set of data (Fig. 8B) was greater than that in intact plants (Fig. 1B, 1-2 h), and this increase in K (see the values for dashed lines) was greater than that found with NPA treatment (Fig. 5B, 1-2 h). Perhaps a part of the sensitivity decrease was a decapitation-specific response.

The data shown in Figure 8B suggested that the sensitivity to IAA might be enhanced slightly in pea internodes as the time after decapitation elapsed (open versus closed circles or dashed versus solid lines). The sensitivity enhancement was, however, much less apparent than that detected in NPA-treated maize coleoptiles (Fig. 5A). In pea the growth inhibition induced by the decapitation at the top of zone 1 (Fig. 6B) was clearly smaller than that induced by applying NPA to the comparable position (Fig. 2B). This apparent disagreement will be considered in ``''.

The experimental and analytical results described above indicated that the reduced supply of auxin is the primary cause of the growth inhibition induced by decapitation, although some reduction of the responsiveness and the sensitivity to IAA may contribute to the inhibition.

Growth Recovery in Decapitated Maize Coleoptiles

View larger version (22K): [in this window] [in a new window]   Figure 9. Effects of applied NPA and IAA on the growth of zone 3 in decapitated maize coleoptiles. A, Effects of NPA in decapitated coleoptiles. Coleoptiles were decapitated at 2 mm. After 1 h a ring of NPA/lanolin (1 mg g1) was applied to the region just above the top of zone 2 () or to the middle of zone 2 (). Controls () were treated with plain lanolin; the data obtained by applying lanolin to the two positions were combined because they were similar. The means ± SE from 6 plants (12 plants for controls) are shown. B, Effects of IAA in decapitated and NPA-treated coleoptiles. One hour after decapitation, a ring of NPA/lanolin (1 mg g1) was applied to the middle of zone 2. Zone 3 was treated 3 h after NPA application with IAA/lanolin at the following concentrations: 0 (), 0.01 (), 0.1 (), and 1 () mg g1. The means ± SE from 7 to 11 plants are shown.

The IAA dose-response data for the growth in decapitated and NPA-treated maize coleoptiles are shown in Figure 10. The data and the best-fit curve (solid line) indicated that the sensitivity to IAA was higher than that detected in decapitated coleoptiles by applying IAA 1 h after decapitation (compare with the dashed line in Fig. 10). Therefore, as shown with NPA treatment (Fig. 5A), the sensitivity appeared to increase as the time of IAA application was delayed from 1 to 4 h after decapitation. The increase in sensitivity was about 4-fold.

View larger version (19K): [in this window] [in a new window]   Figure 10. IAA dose-response relationship in decapitated and NPA-treated maize coleoptiles. Growth rates in the period 1 to 2 h after IAA application were determined from time-course data, some of which are shown in Figure 9B. Each point and vertical bar represent the mean and ± SE, respectively, from 7 to 11 plants. The solid line is the best-fit curve (Fig. 1) that coincided with the parameter values indicated; the SD (Fig. 1) was 0.0102. The dotted and the dashed lines reproduce the curves in Figures 1A and 8A, respectively, at the corresponding period after IAA application.

	DISCUSSION

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Our results substantiated the classical concept that growth is regulated by changes in the concentration of endogenous auxin. It has also become clear that in certain cases modifying the auxin concentration leads, after a period of time, to a modified relationship between the auxin concentration and the growth rate. These issues are discussed separately below. We also discuss the mechanism of the spontaneous growth recovery observed in decapitated coleoptiles.

Auxin as a Growth-Limiting Factor

Our conclusion is in direct conflict with that of Firn and coworkers (Tamimi and Firn, 1985; Parsons et al., 1988b) obtained from similar experiments using the ring application of auxin-transport inhibitors. Parsons et al. (1988b) used a morphactin as the transport inhibitor in experiments with maize coleoptiles. Tamimi and Firn (1985) used a morphactin and NPA in experiments with sunflower and marrow hypocotyls. They generally observed that a ring of inhibitor/lanolin placed in an upper position in intact plants did not cause a significant inhibition of growth, whereas the same treatment inhibited transport of [³H]NAA. When NPA was used, its concentration in lanolin was about 0.03 mg g¹. Our data indicate that both the transport of [³H]IAA (Fig. 3) and the growth in intact plants (Fig. 2) would be partially but significantly inhibited at this concentration of NPA. It remains to be investigated whether the different results are due to different materials or to different experimental conditions (e.g. NAA versus IAA).

Decapitation is another effective way to suppress the growth of maize coleoptiles and pea internodes (Fig. 6). The short-term change in the IAA dose-response relationship after decapitation was generally similar to that observed after NPA treatment (Fig. 8, open circles). In maize coleoptiles decapitation at 2 and 5 mm resulted in similar growth inhibition (Fig. 6A), in agreement with the data indicating that the apical few millimeters is the major site of IAA production (Briggs, 1963; Iino, 1982, 1991; Iino and Carr, 1982a). These results, supplementing those from NPA treatment, support the view that coleoptile and internode growth depends on auxin supplied from the apical region. This conclusion disagrees with that of Parsons et al. (1988a), who inferred that the inhibition of maize coleoptile growth by decapitation is due to wounding. We observed that relatively small decreases occur in the responsiveness and the sensitivity to IAA in decapitated maize coleoptiles and pea internodes. These responses, which were not found with NPA treatment, were probably due to wounding. We conclude that these responses contribute to the growth inhibition caused by decapitation but are not the primary cause of the inhibition.

Decapitation at an apical position of the internode was less effective at inhibiting growth than NPA application at the same position in pea (Fig. 6B versus Fig. 2B). This apparent disagreement should be addressed by further careful investigation. Scott and Briggs (1960) showed that the apical bud of pea seedlings is the major source of auxin in the internodes. However, the site of auxin production may not be confined to the apical bud, extending down to an upper region of the top elongating internode, and the supply of auxin from this region to the lower regions may be inhibited by NPA that has moved below the application site by slow diffusion (Thomson et al., 1973). This possibility can also explain why the inhibitory effect of NPA on [³H]IAA transport (Fig. 3B) was somewhat greater than the effect on growth (Fig. 2B).

A little growth activity remained after treatment with high concentrations of NPA (Fig. 2). The same treatments could not totally suppress IAA transport (Fig. 3). It is probable that the small growth activity retained after NPA treatment depends on endogenous auxin. Therefore, the results support the notion of "without auxin, no growth" (Went and Thimann, 1937). The total dependence of growth on auxin is also the basis of our model (see ``Materials and Methods'').

The dose-response data obtained by treating isolated tissue segments with IAA solutions have often, but not always, shown that the effective range of IAA dose is as wide as 4 orders of magnitude. It has therefore been argued that auxin cannot effectively modulate growth through changes in its concentration (Trewavas, 1981; Hanson and Trewavas, 1982). The dose-response curves obtained by incubating segments in IAA solutions, however, may not parallel the true relationship between the concentration at the site of action and growth rate (Guern, 1987; Davies, 1995). In fact, the effective IAA dose spans only 2 orders of magnitude in all of the dose-response curves obtained here (Figs. 1, 5, 8, and 10), and the data could be described by Equation 4 or a simple hyperbolic function. Together with the results showing that the auxin concentration in intact maize coleoptiles or pea internodes is maintained at a level that is about one-half saturation (Iino, 1996; Haga and Iino, 1997; Fig. 1), it is suggested that endogenous auxin can effectively modulate the growth rate through changes in its concentration.

IAA-Dependent Regulation of the IAA Concentration-Response Relationship

The results of present study indicate that auxin sensitivity is enhanced in maize coleoptiles when the auxin level is reduced for a certain period. This sensitivity enhancement, detectable as a shift of the dose-response curve to lower doses, was observed when a period of 4 h was allowed between NPA and IAA application (Fig. 5A). Time courses of growth that follow NPA application have indicated that the initially inhibited growth recovers gradually when the NPA concentration is low (Fig. 2A). Perhaps the sensitivity is enhanced even when the decrease in auxin level is partial, and the observed growth recovery represents enhanced sensitivity. No such growth recovery was evident at high concentrations, probably because the level of auxin became too low for the expression of a significant growth enhancement. Straightforward demonstration of the sensitivity response in decapitated coleoptiles was difficult because it was masked by the restored growth activity. However, when the growth recovery was inhibited with NPA treatment, the enhanced IAA sensitivity could be shown even in decapitated coleoptiles (Fig. 10).

The dose-response data shown in Figures 5A and 10 indicate that the sensitivity increases by a factor of 3-5. We have not analyzed the kinetics of the sensitivity change in detail. No sensitivity enhancement was evident when IAA was applied 1 h after NPA treatment (Fig. 5A) or decapitation (Fig. 8A). Therefore, a period longer than 1 h is necessary for the response. It remains to be investigated whether the 4-h period in which the sensitivity enhancement was found is optimal for the response. It is expected that the enhanced sensitivity is reduced again after IAA application. We have not investigated this expected decrease in sensitivity. The data shown in Figure 5A indicate that the sensitivity remains enhanced as long as 3 h after IAA application.

As discussed above, plants do not exhibit a fixed relationship between auxin concentration and growth rate; the relationship is subject to regulation by auxin itself. Two modes of regulation have been identified: responsiveness regulation and sensitivity regulation, which are initiated when the auxin level in intact plants is enhanced and reduced, respectively. It has also become apparent that the contribution of each mechanism differs from plant to plant or organ to organ. The responsiveness regulation was found in pea internodes but not definitively in maize coleoptiles (Iino, 1996; Haga and Iino, 1997); the sensitivity regulation was found in maize coleoptiles but not definitively in pea internodes (present study). The mechanisms for changes in the concentration-response relationship probably reside within the target tissue itself. However, the tissue responsible for the detection of the change in auxin level can be other than the target tissue.

The sensitivity enhancement detected in maize coleoptiles supports the sensitivity concept of Vesper and Evans (1978). However, our interpretation of the growth recovery observed in excised maize coleoptile segments, on which these authors based the concept, is different from theirs (see below).

The observed changes in the IAA concentration-response relationship occur in such a way that the growth rate returns to the rate before the change. Therefore, in agreement with the view of Vesper and Evans (1978), the phenomenology is analogous to the sensory adaptation recognized, for example, in photobiological responses (Iino, 1987; Wang and Iino, 1997). We have detected changes in the concentration-response relationship by experimentally manipulating the auxin concentration. In nature the responsiveness regulation as found in pea might take place when the tissue content of IAA is enhanced by infection with bacteria such as Agrobacterium tumefaciens (Zambryski et al., 1989). On the other hand, the sensitivity regulation as found in maize might take place when tissues are injured. It should also be interesting to investigate whether the sensitivity or responsiveness regulation takes place in response to the changes in IAA level induced by environmental factors such as light (Iino, 1982, 1991; Jones et al., 1991).

Mechanisms of the Growth Recovery in Decapitated Coleoptiles

Our data are best reconciled with the classical view. The most critical results are those shown in Figure 9, which indicate that the growth recovery was greatly impaired when the coleoptile stump was ringed with NPA at a position above the monitored zone and that the inhibited growth could be restored by applying IAA. The most reasonable explanation of the data is that growth recovery was suppressed by NPA because the supply of auxin from the regenerated source was inhibited.

Nevertheless, we found that IAA sensitivity was enhanced in decapitated coleoptiles. This sensitivity increase was detected by applying IAA 4 h after decapitation and measuring growth rates 1 to 2 h later (Fig. 10). Therefore, the timing of sensitivity increase in decapitated coleoptiles approximately matched that of growth recovery (Fig. 6A). We conclude that IAA sensitivity was enhanced in decapitated coleoptiles, although the principal cause of the growth recovery was the regenerated auxin-producing activity, and that, during the early period that followed the regeneration of the auxin-producing activity, IAA sensitivity was also enhanced. In an early phase of growth recovery, the growth rate temporarily exceeded the rate in intact plants (Fig. 6). This can be explained in terms of the sensitivity increase that initially accompanies the auxin supply from the regenerated source.

Although the growth recovery was found in decapitated coleoptiles, it was not found in NPA-treated coleoptiles. This implies that the regeneration of auxin-producing activity is not a simple consequence of auxin depletion. Wounding may also be required for the initiation of the regeneration.

Spontaneous growth recovery after decapitation has not been reported for stems of dicotyledons. The data shown in Figure 6B indicated that growth cannot recover in decapitated pea internodes. Therefore, the regeneration of the auxin-producing activity seems to be a unique property of grass coleoptiles. Auxin is a signal for apical dominance, and depletion of auxin from the stem (at least in its upper part) after decapitation results in outgrowth of lateral buds (Tamas, 1995). Perhaps auxin-producing activity is not regenerated in the decapitated stem to allow this mechanism of lateral bud outgrowth.

Further Implications of the Model Describing the Dose-Response Relationship

In spite of the uncertainty described, the model offers a framework for the molecular studies of the IAA concentration-response relationship. The increase in sensitivity that follows NPA treatment or decapitation in maize results from a reduction of K. Such a reduction results most likely from a reduction of the dissociation constant K (Eq. 7). On the other hand, the reduction of the responsiveness to IAA that follows IAA application in intact pea (Haga and Iino, 1997) results from a reduction of A. The receptor concentration is an element determining A (Eq. 5).

Mutants showing a reduced sensitivity to auxin are available (Mirza and Maher, 1985; Ephritikhine et al., 1987; Estelle and Somerville, 1987). A tobacco line with an enhanced sensitivity to auxin can be generated by transforming the plant with a fragment of Agrobacterium rhizogenes T-DNA (Maurel et al., 1991). Our results provide a physiological basis for such genetic changes. On the other hand, analyses of these mutants and transformed plants may illuminate the molecular mechanisms of the auxin-dependent control of auxin sensitivity. It is of special interest to determine whether such genetic changes result from altered K, as indicated by the model.

	FOOTNOTES

   Received March 10, 1998; accepted May 18, 1998.

	ABBREVIATIONS

Abbreviation: NPA, N-1-naphthylphthalamic acid.

	LITERATURE  CITED

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