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Tao Yang† & Peter J. Davies. ∗. Section of Plant Biology, Cornell University, Ithaca, NY 14853, USA (. ∗ corresponding author; †current address: Center for ...
Plant Growth Regulation 27: 157–160, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L. Tao Yang† & Peter J. Davies∗

Section of Plant Biology, Cornell University, Ithaca, NY 14853, USA (∗ corresponding author; † current address: Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, MD 20742, USA) Received 9 October 1998; accepted 8 January 1999

Key words: indole-3-acetic acid, indole-3-butyric acid, pea, Pisum sativum, stem elongation

Abstract While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants. Abbreviations: IAA – indole-3-acetic acid; IBA – indole-3-butyric acid

1. Introduction Although IAA is widely considered to be the principal natural auxin, there is now ample evidence for the occurrence of other auxins in plants. Most notably, IBA has been repeatedly identified in peas by GC-MS [e.g., 11], and may occur in the shoot apex at a level higher than that of IAA [9]. Even in leaf tissue, the total IBA content can reach 75% of that of IAA [12]. Despite our increased awareness of its natural occurrence, the role of IBA in plants remains unclear, other than its speculative involvement in root formation [9, 14]. Exogenous IAA strongly stimulates stem elongation in intact pea plants [15, 16]; moreover, IAA from the apical source actively induces the growth of the

subtending internodes while being transported basipetally in the intact stem [15]. These findings provide important evidence in establishing the involvement of IAA in the control of stem growth in intact plants. Given the significant level of endogenous IBA in a shoot apex [9], the potential contribution to stem growth by IBA as an additional auxin in the intact plant cannot be discounted. This study was thus prompted to determine the characteristics of IBA action on stem growth in intact plants in comparison to IAA. The results hereby obtained in a light-grown dwarf le pea may validate IBA as an auxin active in promoting stem elongation in intact plants.

158 2. Materials and methods The dwarf le pea plants (cv. Progress No. 9) used in this study were light-grown (at 30 µmol m−2 s−1 ) in moist vermiculite in 100 mL plastic pots and selected for experiments about 14 days after sowing [15]. At this stage the sixth internode of the plants was about 20–30% expanded (2–3 mm long). IBA (Sigma Chemical Co., St. Louis, MO, USA) was applied to the intact plants by pumping an 0.25 mM aqueous solution (buffered at pH 6 with 1 mM Na2 HPO4 -citrate containing 0.2% Tween 20) at 0.15 mL min−1 down a thin (ca. 0.1 mm) cotton wick [15]. This was wrapped round the upper stem several times with about one turn every 2–3 mm of stem, prior to being diverted off laterally to waste. No liquid ran down the exterior of the stem. Control plants were treated similarly but without IBA in the solution. Stem elongation of intact seedlings was recorded using position transducers interfaced with a microcomputer for data analysis as described in Behringer et al. [3]. A very fine barb on the end of a balanced thin-wire transducer arm was pressed into the side of the stem at the node of the insertion of the stipules enclosing the apical bud, such that the transducer arm rose as the stem grew [15]. Data was taken at minute intervals with a resolution of better than 1µ. Solution changes were accomplished with computer controlled valves. Unless specified otherwise, all experiments were repeated six times.

Table 1. Effect of IBA on stem elongation in light-grown dwarf Progress No.9 pea seedlings. Buffered solution (1 mM Na2 HPO4 -citrate, pH 6.0, 0.2% Tween-20) with or without 0.25 mM IBA was continuously applied to the intact stem by a flow of solution through a cotton wick around the two uppermost internodes. Whole stem elongation response at selected time points after start of the treatment is shown. The results for IAA (0.25 mM) were extracted from [15] for comparison. Data expressed as mean ± SE (n = 6). ∗ Not applicable Parameter Rate at 0 h (µm/min) Latent period (min) Maximum rate (µm/min) Time until maximum rate (min) Rate during treatment (µm/min) At 3 h At 9 h At 15 h At 20 h Total elongation (mm) By 6 h By 20 h

Control

IAA

1.48 ± 0.19 1.27 ± 0.26 18 ± 1 ∗ 10.36 ± 0.91 ∗ 46 ± 2



IBA 1.28 ± 0.35 39 ± 3 10.01 ± 1.10 83 ± 5

1.22 ± 0.17 1.31 ± 0.28 0.66 ± 0.17 0.56 ± 0.17

7.91 ± 0.50 6.01 ± 0.34 5.52 ± 0.42 4.06 ± 0.26

8.04 ± 0.78 5.79 ± 0.47 4.65 ± 0.32 4.21 ± 0.19

0.48 ± 0.05 1.14 ± 0.09

2.49 ± 0.17 7.03 ± 0.34

2.43 ± 0.19 6.64 ± 0.31

3. Results The two uppermost internodes below the apical bud were used in these experiments. These comprise the extending stem region in dwarf le pea seedlings [15]. IBA (0.25 mM) continuously applied around these two uppermost internodes promoted stem growth at a magnitude comparable to that induced by IAA over a 20 h period (Table 1). The biphasic growth response, with overlapping initial and prolonged growth responses, inducible by IAA, appeared to be typical also of the IBA effect. However, the growth response to IBA was delayed, so that the lag period and the time required to attain the maximum growth rate were nearly double those needed to observe a similar response to IAA (Table 1; Figures 1 and 2). Withdrawal of IBA after treatment for 5 h, during the prolonged growth response phase, resulted in a rapid decline in growth rate, as in the case for IAA,

Figure 1. Whole-stem elongation kinetics in dwarf Progress No. 9 seedlings following IBA (2.5 × 10−4 M) application (as described in Table 1) (downward-pointed arrow) and withdrawal (upward-pointed arrow). IBA treatment started at time zero and was withdrawn after 5 h. Note the occurrence of a plateau of moderate growth stimulation (area marked “P”) after the phase of rapid decline in growth rate following IBA withdrawal, but before the rate slowly decreased to the control level.

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Figure 2. Whole-stem elongation kinetics in dwarf Progress No. 9 seedlings following IAA (2.5 × 10−4 M) application (downward-pointed arrow) and withdrawal (upward-pointed arrow). IAA treatment started at time zero and was withdrawn after 5 h. (Redrawn after data in [15]).

Figure 3. Distribution of elongation induced by applied IBA in the upper internodes of the whole stem of dwarf Progress No. 9 seedlings. 10−4 M IBA was continuously applied around the uppermost internode, starting at time zero. Curves represent the growth rate of (a) the whole stem (heavy solid line), (b) the uppermost internode (light dashed line), and (c) the stem below the uppermost internode (heavy dashed line).

4. Discussion

but the lag before the decline was considerably longer (45 min average vs 25 min for IAA) (Figures 1 and 2). While the growth rate declined steadily to or below the baseline rate in the control plants following the removal of IAA [15], in the case of IBA the growth rate only declined by about 70%, and was subsequently remained at that rate for about 2 h in a plateau (the “P” area in Figure 1) before it slowly decreased to the control level. With this characteristic pattern of rate decline, the half-time for a complete loss of the IBA-induced response following removal of the treatment was at least twice as long as that in the case of IAA. To determine whether IBA applied to the stem apex can be transported downward and elicit growth in a subtending internode, it was applied only to the uppermost internode while the growth of the lower internode was recorded. As shown in Figure 3, a sustained increase in elongation was detectable in the lower internode to which IBA was not directly applied; although the response was consistently delayed (by about 50 min) as compared to the plant similarly treated with IAA [15], the intensity of growth stimulation produced was generally comparable between transported IBA and IAA.

These results demonstrate that exogenous IBA, albeit with a delayed effect, is as effective as applied IAA in promoting elongation in intact pea plants, regardless of whether IBA is applied directly around, or transported through, the expanding internodes. It has been shown that IBA application can moderately increase the level of IAA in pea stems, probably through a conversion of IBA to IAA; however, this activity only becomes significant about 24 h after the treatment [9]. Thus, the strong effect of applied IBA on stem growth recorded in our study is probably due to the IBA action per se. Given the in vivo transport capacity for IBA in the stem (deduced from our study) and its high endogenous content in the apical shoot as in peas [9], IBA may therefore serve as an important form of auxin in contributing to stem elongation in intact plants. The existing evidence for the pattern of IBA transport appears to be mixed. Early studies with excised stem segments repeatedly suggested that IBA can be basipetally transported, but at a markedly slower rate than IAA [e.g. 7]. However, no prominent difference between the transport of IBA and IAA was found in Arabidopsis [8]. The results in the present study, which show a clear growth response in the lower internode following IBA application to the upper internode, though later than with IAA, imply that IBA is trans-

160 ported but probably at a slower rate than IAA, at least down through the node region in peas. The long lag of the IBA effect on growth in the intact plant coincides with that found in excised stem segments [10]. Interestingly, there is also a long lag prior to the complete loss of the IBA effect following removal of the treatment. Thus, the delay in the IBA effect, as compared to that with IAA, may be due to the slowness of IBA action, metabolism and/or transport per se rather than a general difference in the uptake process. The rapid initial decline in growth rate after the removal of IBA confirms that the IBA action is inherently transient as is that of IAA. Within a few hours of their application to pea or other dicot stem tissues, exogenous IAA and IBA are largely metabolized to other compounds, primarily by a process of conjugation [6]. While IAA is almost exclusively conjugated into IAA-aspartate [1, 5], IBA may be metabolized into multiple conjugates, with IBA-aspartate being predominant [2, 9, 13, 14]. The biological activity of auxin conjugates appears to be related to the ability to release free auxin through hydrolysis within the tissue, which is very poor in the case of IAA-aspartate [4], whereas IBA-conjugates can be readily hydrolyzed [13, 14]. Considering this difference in hydrolysis between IAA- and IBAconjugates, it is thus possible that the residual but sustained growth stimulation long after removal of the treatment (the “P” area in Figure 1) is due to some free IBA released from the accumulated IBA-conjugates. In contrast, IAA removal was always followed by a steady decline in growth rate to the base level [15], which may reflect the inert nature of IAA-aspartate to release free auxin for a residual growth stimulation typically seen in the IBA case. Given that a large proportion of auxin exists in its conjugate state in plants [6], the ability of IBA-conjugates to release free IBA may further solidify the role of IBA as a physiologically important auxin.

Acknowledgements This research was supported in part by Hatch funds from the College of Agriculture and Life Sciences at Cornell University, and by Curtis-Kudson and graduate school fellowships from Cornell to TY.

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