Exogenously applied indole-3-acetic acid (IAA) strongly pro- moted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress ...
Plant Physiol. (1993) 102: 717-724
Magnitude and Kinetics of Stem Elongation lnduced by Exogenous Indole-3-Acetic Acid in lntact Light-Crown Pea Seedlings' Tao Yang, David M. Law, and Peter J. Davies*
Section of Plant Biology, Plant Science Building, Cornell University, Ithaca, New York 14853
1987). Nonetheless, severa1 recent studies have directly related endogenous auxin contents of elongating tissues to growth (e.g. bean stem [Bialek et al., 19831; lupin hypocotyl [Ortuíío et al., 19901). In particular, Law and Davies (1990) found that there was a close correlation between the endogenous leve1 of IAA and stem growth in a range of genetic lines of pea (Pisum sativum L.) differing in height, including slender phenotypes, which are ultratall regardless of their GAI content. Thus, this line of evidence contends that auxin is also involved in regulating the growth of intact plant stems. The major argument against such a role for auxin is that auxin applications to intact plants fail to elicit an appreciable growth response (Hanson and Trewavas, 1982). More recently, the growth response of intact plants to auxin application has been reexamined in dark-grown pea epicotyls(Hall et al., 1985) and in hypocotyls of sunflower, marrow (Tamimi and Fim, 1985), and watermelon (Carrington and Esnard, 1988). It was consistently found in a11 of these studies that exogenous auxin did strongly promote stem elongation, but that the promoting effect was short-lived (3-5 h), and auxin subsequently became inhibitory. The lack of a prolonged growth promotion by applied auxin seems to support the contention of Hanson and Trewavas (1982) that the effect of applied auxin on intact stem growth is too insignificant to account for any role of auxin in regulating stem growth. An altemative view proposes that endogenous auxin may be active, but its supply in intact plants is not a limiting factor for stem growth (Tamimi and Fim, 1985). It should be noted, however, that previous studies on the effect of applied auxin on the growth of intact stems were performed almost exclusively with dark-grown plants. In view of many differences in developmental morphology and physiology between darkgrown and light-grown tissues, it seems invalid to extend the implications of the growth response to applied auxin in darkgrown plants to the situation in light-grown plants. The difference in stem height between tall and dwarf pea varieties is relatively small in etiolated seedlings, but it becomes striking if they are grown in the light, with stem growth in the dwarf being much more reduced than that of the tall (Muir, 1972; Ross and Reid, 1989). Furthermore, auxin levels are often lower in apices and elongating stems of light-grown dwarf plants than in their tall counterparts (Law and Davies, 1990). Therefore, should applied auxin
Exogenously applied indole-3-acetic acid (IAA) strongly promoted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress No. 9) and tal1 (cv Alaska) peas (Pisum safivum l.), with the relative promotion being far greater in dwarf plants. In dwarf seedlings, solutions of IAA (between 10-4 and 10-3M), when continuously applied to the uppermost two internodes via a cotton wick, increased whole-stem growth by at least 6-fold over the first 24 h. The magnitude of growth promotion correlated with the applied IAA concentration from 10-6 to 10-3 M, particularly over the first 6 h of application. IAA applied only to the apical bud or the uppermost internode of the seedling stimulated a biphasic growth response in the uppermost internode and the immediately lower internode, with the response in the latter being greatly delayed. This demonstrates that exogenous IAA effedively promotes growth as it i s transported through intact stems. IAA withdrawal and reapplication at various times enabled the separation of the initial growth response (ICR) and prolonged growth response (PCR) induced by auxin. The ICR was inducible by at least 1 order of magnitude lower IAA concentrations than the PCR, suggesting that the process underlying the ICR i s more sensitive to auxin indudion. In contrast to the magnitude of the IAA effect in dwarf seedlings, applied IAA only doubled the growth in tal1 seedlings. These results suggest that endogenous IAA i s more growth limiting in dwarf plants than in tal1 plants, and that auxin promotes stem elongation in the intact plant probably by the same mechanism of adion as in isolated stem segments. However, since dwarf plants to which IAA was applied failed to reach the growth rate of tal1 plants, auxin cannot be the only limiting factor for stem growth in peas.
Auxin often strongly stimulates elongation in isolated stem segments, and much progress has been made in understanding the mode of auxin action in this system (Cleland, 1987; Rayle and Cleland, 1992). However, owing to a lack of direct evidence, it remains uncertain whether the results obtained from studies on isolated stem segments are applicable to the intact stem, and whether endogenous auxin directly participates in regulating stem elongation in intact plants. In fact, in an increasing number of species, GA has been shown to play an essential role in the control of vegetative stem elongation in intact plants, with GAI as the prime effector (Reid, This work was supported in part by Hatch funds from the College of Agriculture and Life Sciences at Comell University, and a CurtisKnudson summer fellowship from Comell to T.Y. * Correspondingauthor; fax 1-607-255-5407.
Abbreviations: IGR, initial growth response; PGR, prolonged growth response. 71 7
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effectively promote growth in intact plants, the effect ought to be clearly manifested in a light-grown dwarf pea. We have developed a precisely controlled system for auxin application to intact plants. This, along with a high-resolution growth recording system (Behringer et al., 1990), allowed us to characterize the stem elongation responses of intact lightgrown dwarf and tall pea seedlings to exogenously applied IAA. We have already shown that IAA can indeed provide a long-term stimulation of stem elongation in intact pea plants (Behringer et al., 1992). Herein, we report on the comparative responses of dwarf and tall plants and the kinetics of IAAinduced growth in the whole plant stem. MATERIALS AND METHODS Plant Material
Seeds of Pisum sativum L. cv Progress No. 9 (Agway, Syracuse, NY) and cv Alaska (Burpee, Warminster, PA) were sown individually in moist vermiculite in 100-mL plastic pots and grown and treated at 23°C under continuous light (30 ^mol m~2 s~' at plant level) from cool-white fluorescent lamps (General Electric). Thirteen to 15-d-old dwarf Progress No. 9 and tall Alaska seedlings that exhibited healthy growth and uniformity were selected for experiments. At this stage, the uppermost internode (sixth, counting the cotyledons as zero) of both varieties was about 20 to 30% fully expanded, or between 2 to 3 mm long in the dwarf and around 15 mm long in the tall plant. Auxin Application
To ensure precise control of dose-application and treatment duration, we developed a method for applying aqueous auxin solutions to intact stems. Solutions of IAA (Sigma) were freshly prepared in 1 mM Na2HPO4-citrate (pH 6.0) with 0.2% Tween-20. Control plants received the same solution without IAA. Treatment of a seedling was accomplished by a flow of solution through thin (approximately 0.1 mm), absorbent cotton strings that were evenly wrapped around the uppermost one or two internodes, depending upon the experiment type (Fig. 1). The solution was delivered at a constant rate of 0.15 mL min"1 by a computerized pump (Cole-Farmer Instrumental Co., Chicago, IL) through plastic tubing to the upper end of the cotton wick. After flowing around the wrapped region of the apical shoot, the solution was diverted laterally off the plant into a waste container by the extending cotton wick beneath the site of application, so as to avoid contact between the solution and any other part of the plant or soil. The addition of Tween-20 as a surfactant in all treatment solutions greatly facilitated the flow-through of solution along the cotton wick, and presumably also aided in the uptake of auxin through the cuticle around the stem surface. In this way, a seedling could be exposed to a constant strength of treatment solution throughout a prolonged period. Interchange between IAA and control solutions was completed within 30 s using computer-driven valves, which allowed withdrawal and readdition of a treatment solution with great precision.
Figure 1. A 14-d-old dwarf cv Progress No. 9 pea seedling illustrating the application method and stem-growth measurement. The closest stipule at the second node below the apical bud was removed to show the apical bud and uppermost internode. A continual flow of treatment solution was supplied to the seedling from the top via a cotton wick, which then wrapped around the uppermost one or two internodes (depending on the experiment type), and was then diverted away from the plant to waste by the extending cotton wick beneath the site of application (down-pointing arrows). Whole-stem elongation was recorded by a transducer (T) attached above the uppermost internode (horizontal arrow, Tl). Extension in the stem below the uppermost internode was simultaneously measured in some experiments by a co-attached transducer located on the second node below the apical bud (horizontal arrow, T2). Bar = 10 mm.
Growth Measurement
Stem elongation of intact seedlings was recorded using position transducers that were interfaced with a microcomputer for data acquisition and analysis, details of which have been previously described (Behringer et al., 1990). In brief, a very fine barb at the end of the transducer arm was pressed into the uppermost node (just below the apical bud) to record growth in the whole stem. In some experiments, a second transducer was attached to the penultimate node to measure stem growth below the uppermost internode (Fig. 1). Electric signals from the transducers were amplified, converted into digital form, and logged by a microcomputer. Stem growth was monitored over various time periods at 1-min intervals, from which the growth rate was computed with smoothing
IAA-lnduced Stem Elongation in lntact Light-Grown Peas
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on the running average of 10 data points. The weight imposed by wicking on the upper stem did not appear to affect seedling growth. After attachment to the apparatus, seedlings were allowed to equilibrate for about 1 h, during which a flow of buffer solution without IAA was applied. A11 treatments were performed at least five times. To exhibit the distinct features of the kinetic response, we did not average the experiment replicates and used a typical response curve for figure presentation. RESULTS Characteristics of IAA-lnduced Stem Elongation Response in Dwarf cv Progress No. 9 Seedlings
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Figure 2. Characteristic dose-response curves of elongation growth induced by IAA continuously applied around the two uppermost internodes of dwarf cv Progress No. 9 seedlings. Treatments started at time zero. A, Cumulative whole-stem elongation; B, elongation
rate.
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Dwarf seedlings treated with buffer solution elongated at a nearly constant rate of about 1 pm/min. Continuous application of IAA (10-4 M or higher concentrations) around the two uppermost intemodes promoted the elongation in the whole stem by at least 6-fold over the first 20 h (Fig. 2A; Table I). However, the induced growth in the whole stem was found to be distributed only in the two uppermost internodes, because little or no growth could be recorded by a transducer attached below these intemodes. Following the application of 2.5 X 10-4 M IAA to this entire growing stem region, there was about an 18-min lag before the stem elongation rate increased sharply (Fig. 28; Table I). The growth rose about 10 times that of the control seedling to an initial peak (IGR) approximately 50 min after the start of treatment. This was followed by a drop in the growth rate, usually to a minimum around 75 min, before a second increase to a steady-state PGR at 2 to 3 h after the application. The PGR was subsequently maintained for a period of more than 20 h at a rate about 6-fold higher than that of control seedlings (Fig. 2B). The IGR and PGR observed here in the
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Table 1. Effect of IAA on stem elongation in Iight-grown dwarf cv Progress No 9 pea seedlings Buffered solution (1 mM Na2HPO4-citrate,pH 6.0, 0.2% Tween-20)with or without IAA was continuously applied to the intact stem by a flow of solution around the two uppermost internodes Whole stem elongation response at selected time points after start of t h e treatment is shown. Data are means k SE (number of replicates shown in parentheses) NS, Not specific IAA Concentration (M) Parameter
Rate at O h (pmlmin) Latent period (min) Maximum rate (pmlmin) Time until maximum rate (min) Rate during treatment (pm/min) At 3 h At 6 h At 9 h At 12 h At 15 h At 20 h
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10-6
10-5
5 x 10+
2 5 x 10-~
(8)
(5)
(6)
(5)
(6)
1.48 f 0.19 NS NS NS
1.21 & 0.18 28 f 3
3.53 f 0.21 106 f 5
1.35 2 0.20 26 f 2 5.99 f 0.51 100 f 6
1.39 f 0.34 23 f 2 8.31 f 0.69 74 f 5
1.27 f 0.26 18f 1 10.36 f 0.91 49 f 2
1.57 f 0.16 17f 1 11.42 f 0.53 43 f 1
1.22k0.17 1.24 f 0.25 1.31 f 0.28 1.02 f 0.19 0.66 f 0.17 0.56 f 0.17
2.12f0.16 1.05 f 0.13 0.66 f 0.09 0.91 f 0.14 0.74 f 0.13 0.65 f 0.09
2.73f0.65 2.16 f 0.34 1.20 k 0.25 1.32 f 0.16 1.04 f 0.14 0.89 f 0.16
6.65f0.28 5.17 f 0.41 4.46 f 0.54 3.38 f 0.51 2.21 k 0.35 1.55 f 0.49
7.91 f 0 . 5 0 7 . 0 7 f 0.43 6.01 f 0.34 5.55 f 0.37 5.52 f 0.42 4.06 f 0.26
8.28f0.61 7.50 f 0.25 6.09 k 0.34 5.10 f 0.39 4.24 f 0.45 2.89 +- 0.41
0.48k0.05 1.14 k 0.09
0.59f0.03 1.20 k 0.06
1.04fO.09 1.89 f 0.25
1.96f0.17 4.30 f 0.29
2.49f0.17 7.03 f 0.34
3.08 f 0.09 7.16 f 0.49
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(6)
Total elongation (mm) By 6 h By 20 h
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whole stem clearly resemble those of the IAA-induced growth response in isolated stem segments (Cleland, 1987). The promotion of stem growth tended to be progressively dampened after seedlings had been continuously treated for more than 24 h, and there was little increase after 48 h of treatment. However, if IAA was withdrawn at this time, and 3 to 4 d were allowed to elapse for the formation of new stem tissue, reapplication of IAA to the apical bud or newly formed internode again substantially promoted stem elongation as recorded above the new uppermost internode (results not shown). IAA Dose-Response Kinetics
IAA at concentrations below 10-6 M had no effect on stem growth. Within the range from 10-6 to 10-3 M, a higher IAA concentration correlated with a stronger growth promotion over the first 6 h of treatment, a shorter lag time, and a higher maximum elongation rate (Fig. 2B; Table I). Seedlings showed only a single, small burst of growth at about 2 h following exposure to 10-6 M IAA. This was followed by a decline to the control rate by 4 h, so the long-term growth was not clearly enhanced. Increasing IAA concentration from 10-6 to 10-5 M only increased the magnitude of the IGR and appeared largely ineffective in initiating or maintaining the PGR, since .the response was only marginal after the initial peak (Table I). Higher concentrations of IAA (above 5 X l O P 5 M) effectively elicited both the IGR and PGR. IAA at 10-3 M most strongly stimulated growth over the first 6 h of treatment, but tended to be less optimal than 2.5 X 10-4 M thereafter, and over a long term (20 h), there was no significant difference in the effect between the two concentrations (Fig. 2A; Table I). However, we observed that in some replicates 10-3 M IAA was more effective than 2.5 X 10T4M IAA throughout this long-term treatment. Thus, it appears that 10-3 M IAA was approaching a concentration saturating to the growth response, and the duration of its large effect was dependent upon the response capacity of the stem, which varied somewhat with individual plants. Growth Kinetics following IAA Application and Withdrawal
Withdrawal of IAA (2.5 X 10-4 M) after the PGR was stabilized (e.g. 5 h from the start of treatment) resulted in a rapid decline in the stem growth rate following a characteristic lag period of about 25 min (Fig. 3A). The half-time for the growth-rate decline to the leve1 of the endogenous growth was 50 to 55 min (including the lag period). Reapplication of IAA 5 h after the time of IAA withdrawal produced a kinetic growth response similar to that induced by the first IAA treatment (Fig. 3A). When IAA was reapplied only 30 to 60 min after IAA withdrawal, the stem growth rate, although rapidly declining at the time of reapplication, was again increased with a typical latent period (Fig. 3C). It was found that the magnitude of the PGR to reapplied IAA was relatively constant, regardless of the timing of IAA reapplication after the previous withdrawal, whereas the IGR to IAA reapplied within 1 h after the withdrawal was hardly visible. The magnitude of the IGR was increasingly greater as the
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Whole-stem elongation kinetics in dwarf Progress No. 9 seedlings following IAA (2.5 X 10-4 M) application and withdrawal. Treatment solutions were continuously applied to the two uppermost internodes of the seedling. Downward-pointed arrow!; indicate start of IAA application, and upward-pointed arrows indicate time of withdrawal of IAA treatment, which was replaced by treatment with control solution. Each set of experiments was performed on a new seedling. A, IAA was withdrawn after 5 h of treatment and reapplied 5.5 h after the withdrawal for another 5-h trealment; B, IAA was withdrawn after 0.5 h of treatment and reapplied 6.5 h following the withdrawal for a further 1 h; C , IAA was withtdrawn after 5 h of treatment and reapplied 1 h after the withdrawal for a further 2 h. See Figure 2B for the control rate. Figure 3.
reapplication was further delayed beyond 1 h, approaching a maximum a1 about 6 h (compare Fig. 3C with 3A). The typical growth rate minimum, following the initial IGR peak but before the PGR stabilized, was occasionally not seen following either the first IAA application or a reapplication (e.g. the response to the reapplication in Fig. 3A). As prolposed by Vanderhoef and Stahl (1975), the lack of such ii rate minimum is probably due to an increased overlap between the IGR and PGR. An IAA treatment as short as 10 to 30 min could fully elicit the IGR and initiate the PGR, but it failed to maintain the latter ( e g Fig. 3B). Increasing the duration of IAA treatment only extended the maintenance of the F'GR and did not appear to affect the IGR (Fig. 3B). Withdrawal of IAA after a
IAA-lnduced Stem Elongation in lntact Light-Grown Peas
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in the uppermost intemode. The lag time for a growth increase in the uppermost intemode that directly received applied IAA coincided with that of the whole stem response, whereas it was much longer in the lower intemode (60-80 min). However, the growth responses of the two elongating intemodes exhibited a similar kinetic pattem, with the characteristic IGR and PGR independently produced in each intemode (Fig. 4). The multiphasic growth response of the whole stem to IAA applied to the uppermost intemode thus represents a temporal and spatial summation of the unsynchronized growth responses in the two elongating intemodes, which were exposed at different times to the IAA being basipetally transported after application.
TIME (hour)
Distribution of elongation induced by applied IAA in the stem of dwarf cv Progress No. 9 seedlings. IAA at 10-4 M was continuously applied around the uppermost internode, starting at time zero. Curves represent the growth rate of (a) the whole stem (solid line), (b) the uppermost internode (light dots), (c) the stem below the uppermost internode (dark dots). Whole-stem elongation of control seedlings is depicted in Figure 2. Figure 4.
short treatment (30-60 min) was followed by a much longer lag, before the decline of stem growth rate (Fig. 3B), than that after a prolonged treatment (Fig. 3A), suggesting that newly treated stems are much more sensitive to growth induction by IAA than those previously treated by auxin for a prolonged period.
Effect of IAA on Stem Growth in Tall cv Alaska Seedlings
Tall, control seedlings elongated at an average rate of 8 pm/min with large rhythmic fluctuations (Fig. 5B). The period of time for these growth oscillations was around 90 to 120 min. This contrasts with a lack of any regular growth fluctuations in dwarf cv Progress No. 9 seedlings (Fig. 2B). IAA applied around the uppermost intemode of the tal1 seedling enhanced the growth rate of the whole stem by as much as 3-fold during the first 6 h of continuous treatment, with the total growth reaching nearly twice that of the control by 15 h (Fig. 5; Table 11). The early pattem of the whole stem growth following IAA application to the uppermost intemode was characterized by multiple peaks in the growth rate (Fig.
Crowth Distribution following IAA Application to the Apical Bud or Uppermost lnternode
When IAA was applied only to the apical bud or to the uppermost internode, it produced a more complex response kinetics than did application of IAA to the uppermost two intemodes; the IGR comprised two prominent peaks (Fig. 4). However, the initial lag and overall magnitude of promotion of the whole stem growth was similar between application to the uppermost one or two internodes (Fig. 2 versus Fig. 4). The only difference between application to the apical bud and uppermost intemode was that the lag time following apical bud application was extended to 25 to 30 min (data not shown). To determine the relative distribution of the induced growth in the two uppermost internodes following IAA application only to the uppermost intemode, growth in the whole stem and the stem below the uppermost intemode was simultaneously recorded in the same seedling (see Fig. 1). Net growth in the uppermost intemode was determined by subtracting growth in the stem below it from the whole stem growth. The growth in the intemode just below the uppermost internode was, in fact, fully represented by that recorded in the entire stem below the uppermost internode, given that the remaining stem below the uppermost two internodes did not show any extension in response to IAA treatment, presumably because it had reached maturity. As shown in Figure 4, the IAA-induced growth was confined mainly to the internode immediately below the uppermost intemode, with a relatively small amount of growth occurring
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Figure 5. Characteristic elongation response in t h e whole stem of light-grown tal1 Alaska seedlings to 1O-4 M IAA continuously applied
around t h e uppermost internode. Treatment started at time zero. A, Cumulative whole-stem elongation; B, elongation kinetics.
Yang et al.
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Table II. Comparison of the stem elongation response of lightgrown ta// (cv Alaska) and dwarf (cv Progress No. 9) pea seedlings to exogenous IAA Buffered solution with or without IAA (10-4 M) was continuously applied around the uppermost internode of the seedling. Values indicate whole stem eloneation (mm). Mean k SE of five redicates. Dwarf
Treatment Period
Control
Tal1 +IAA
Control
+IAA
h
6 15
0.48k0.07 2.54 k 0.19 3.45 k 0 . 2 4 7.22 k 0.29 0 . 9 7 f 0 . 1 1 6 . 2 8 f 0 . 2 1 8 . 1 7 k 1 . 0 8 15.04k0.91
5B). As in the dwarf seedlings, these early peaks represent the overlapping effect of IAA-induced growth responses in individual elongating intemodes, which occurred with successive delays as the applied IAA was transported down the stem. However, the induced growth was found to be distributed throughout the uppermost three intemodes in tal1 seedlings (data not shown) instead of in the uppermost two internodes, as in dwarf seedlings, which explains why the overlapping responses in the former gave rise to more peaks during the early treatment period than in dwarf plants (Fig. 5B). The IAA-induced growth stabilized after approximately 6 h of treatment, attaining a rate of about 15 pm/min, twice the steady-state rate induced by IAA in dwarf seedlings. The steady promotion by IAA treatment clearly masked the regular growth oscillations that were typical in control seedlings, but this plateau usually began to decline slowly 12 h after the start of treatment (see Fig. 58). DISCUSSION
This study unequivocally demonstrates that exogenous auxin strongly stimulates stem elongation in intact lightgrown pea seedlings over a long term. We found that the effect was particularly pronounced in dwarf seedlings, in which stem growth was promoted by at least 6-fold by a continuous IAA treatment throughout 24 h. Our finding of a prolonged growth promotion by auxin in intact plants can in large measure be ascribed to the use of light-grown plants, as well as to the method of application, namely, continuous delivery of aqueous auxin solution via a cotton wick to the apical stem parts. I
Comparison of IAA Effects on Light- and Dark-Grown Plants
In intact etiolated seedlings, it has been shown previously that applied auxin effectively promotes stem growth for only a few hours and is subsequently growth inhibiting, accompanied by immense swelling in the actively elongating stem (eg. Sargent et al., 1974; Hall et al., 1985). However, we found that elongation in light-grown seedlings was not inhibited by continuously applied aqueous .IAA, even at a concentration as high as 10-3 M over the first 48 h of treatment. This lack of stem-growth inhibition by high concentrations of auxin in intact, light-grown plants is consistent with the early findings from studies on isolated, light-grown stem
Plant Physiol. Vol. 102, 1993
segments (Galston and Kaur, 1961; Burg and Burg, 1968). Thus, it appears that light-grown stem tissue is far less sensitive to growth inhibition by auxin than is dark-grown tissue. Characteristics of IAA Action in lntact Stems
Auxin-depleted, isolated pea stem segments are capable of large growth responses to added exogenous IAA (Penny et al., 1972; Pamsh and Davies, 1975). Interestingly, we found that intact stems of dwarf cv Progress No. 9 pea, possessing a low content of endogenous IAA compared with tal1 cv Alaska (Law and Davies, 1990; R.H. Hamilton, perjonal communication), are remarkably similar to isolated stem segments in their response to exogenous IAA, with respect to both dose-response (Brian and Hemming, 1955; Galston and Kaur, 1961) antd short-term kinetics (Barkley and Leopold, 1972; Penny et al., 1972). This may suggest that elongation induced by exogenous IAA in intact stems involves the same mechanism of mxin action as in isolated stem segments. Based on sktdies on isolated stem segments, it has been proposed that the two phases of growth response induced by auxin (IGR ancl PGR) may arise from different mechanisms: the IGR is effected by a turgor-driven burst of cell extension following auxin-stimulated wall loosening, which might be mediated by wall acidification, whereas the PGR represents a late spectrum of auxin action involving steady regeneIation of the capacity of the walls to undergo wall loosening (Vanderhoef and Dute, 1981; Rayle and Cleland, 1992). ‘These notions may he corroborated by our results showing the pattems of the IGR and PGR in intact stems follclwing addition and withdrawal of IAA at various time points (Fig. 3). The variable magnitude of the IGR, as regulated by the time between TAA withdrawal and reapplication, may provide a measure of the amount of extensible tissue within the stem readily available for immediate elongation induction by IAA. It seems íhat the IGR is a spontaneous process once it is initiated, whereas the PGR is heavily dependent on the continued presence of IAA for maintenance. Despite current uncertainty on the biochemical nature underlying the two responses, IAA dose-response kinetics in intact sterrts recorded in our study (e.g: Fig. 2B; Table I) suggest that the K;R is far mole sensitive than the PGR to auxin induction and can be separated from the latter by a much lower inductive concentration threshold. IAA-induced growth in the intact stem was steadily maintained for a prolonged period (>20 h) by a contiriuous treatment (Fig. 2). The withdrawal of IAA resulted in a rapid decline in the stem-growth rate after a lag of about 25 min (Fig. 3A). This confinns that the action of exogenous I A 4 in intact stems is intrinsically transient. The transient action of IAA might be occasioned by the ephemeral nature of IAAinducible mRNAs and proteins, since the combined half-life of IAA-induced mRNA (30 min) and growth-limiting proteins (20 min) estimated in maize coleoptile segments (Edelimann and Schopfer, 1989) is consistent with that of IAA-induced growth response (50-55 min) as noted in our study. The responsiveness of the intact stem to applied IAA decreased consistently with time after more than 24 h of continuous treatment. However, the responsiveness could be
IAA-lnduced Stem Elongation in lntact Light-Grown Peas largely restored in the seedling after a long intermption of the IAA treatment, during which a new intemode was formed, since the reapplication of IAA to the newly formed intemode became effective in promoting stem growth. It seems that the eventual lack of growth response to continuously applied IAA may be due to a shortage of extensible cells within the stem after the preexisting tissue has been fully extended. This suggests that the cellular basis of auxininduced elongation in intact stems conforms to that in isolated stem segments, i.e. mainly through an increase in cell elongation rather than cell division (Evans, 1984). Auxin as a Regulator of Stem Crowth in lntad Plants
Johnson and Moms (1989) have demonstrated that IAA applied to the apical bud is transported basipetally in lightgrown pea seedlings at a slow rate (about 10 mm/h). Internally transported IAA from an apical exogenous source is clearly effective in promoting the growth of elongating internodes (Fig. 4). Following the application of IAA to the stem apical region, each successively lower, growing intemode showed an increased lag in the growth response compared with that in the uppermost intemode (Figs. 4 and 5), indicating that there was a basipetal distribution of growth increase in the elongating stem region. Thus, it appears that the growth response correlates with the profile of the polar IAA transport as reported by Johnson and Moms (1989). Apically applied auxin is mainly transported in parenchyma cells associated with the vascular tissue in intact stems (Moms and Johnson, 1985). However, the primary target cells of auxin for growth are probably located in the outer-stem tissues, i.e. the epidennis and cortex (Rayle and Cleland, 1992). According to Sinchez-Bravo et al. (1991), exogenous IAA transported in inner-stem tissues can efficiently diffuse to the outer, responsive cells to elicit a growth response. If we assume that exogenous and endogenous IAA behave similarly in intact stems in terms of transport and action, our results suggest that endogenous IAA is actively involved in promoting stem elongation as it polarly travels through the tissues of the elongating region. The endogenous growth rate of tall seedlings was 7 to 8 times higher than that of dwarf seedlings. However, exogenous IAA promoted growth by at least 6-fold for more than 24 h in dwarf seedlings, whereas it only enhanced growth in tall seedlings by less than 1-fold and for a shorter duration (e.g. Figs. 2 and 4 versus Fig. 5). This suggests that endogenous IAA is much more limiting to growth in dwarf seedlings than in tall seedlings, and that IAA is an important regulator of stem growth in the intact plant. This assertion is supported by the close correlation between the endogenous IAA content and stem height in various pea genotypes differing in height (Law and Davies, 1990). Thus, the far greater relative promotion of growth by applied IAA in dwarf than in tall seedlings may be a consequence of the fact that the stems of tall plants have been much more extended by their higher levels of endogenous auxin than those of dwarf plants. However, it should be noted that the absolute magnitude of this promotion was larger in tall seedlings, and that stem growth in dwarf seedlings could hardly be promoted by exogenous IAA to the level of endogenous growth in tall
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seedlings (Table 11). This indicates that stem elongation in intact plants is not regulated solely by auxin. Compared with s t e m in dwarf peas, stems of tall peas exhibit a much greater number of cells (Reid et al., 1983), which has been attributed largely to an effect of their higher GAI content (Reid, 1987). As noted in our study, auxin-induced growth in the intact stem appears to be essentially confined to an increase in cell elongation in the subtending intemodes; therefore, it is conceivable that tall plant s t e m that are endowed with a much higher number of cells showed a greater absolute elongation potential in response to exogenous IAA than their dwarf counterparts. Taken together, it appears that stem growth in peas is regulated at least by both GA and auxin. Work is currently in progress to examine the possible interaction between the two hormones in this process. ACKNOWLEDCMENTS
D.M.L. dedicates this paper to Professor Robert H. Hamilton on the anniversary of his retirement, with appreciationfor his important contibutions to the field of plant physiology, and with gratitude for his roles as exemplary doctoral advisor, teacher, and valued friend. The application technique successfully employed in this work was developed from ideas generated in this laboratory and in conversations between D.M.L. and R.H. Hamilton. We thank Dr. Friedrich J. Behringer for initial construction of the growth-recordingapparatus and for help with growth measurement, and we thank Sandra G. Brown for typing the manuscript. Received December 14, 1992; accepted March 10,1993. Copyright Clearance Center: 0032-0889/93/102/0717/08. LITERATURE CITED
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