Proceedings of the National Academy of Sciecnce8 Vol. 65, No. 1, pp. 184-191, January 1970

Action of on Elongation* David L. Rayle,t Michael L. Evans, and Rainer Hertelt

MSU/AEC PLANT RESEARCH LABORATORY, MICHIGAN STATE UNIVERSITY, EAST LANSING, AND DEPARTMENT OF , KALAMAZOO COLLEGE, KALAMAZOO, MICHIGAN Communicated by Anton Lang, December 1, 1969 Abstract. The early time course (0-30 min) of the action of auxin (3-- acetic acid) on the elongation of segments from corn was studied, using a high-resolution continuous recording technique. Two different effects of auxin were observed: (1) After addition of low auxin concentrations (2 X 10-v M) to 4-mm sections, a very rapid (2-3 min) enhancement of elongation was found. Similar early responses were seen following the addition of low con- centrations of the methyl ester of indoleacetic acid. (2) Following a large step- up in the auxin level (10-s, 10-4, or 10-3 Al), a rapid transient decrease was ob- served one to three minutes after the addition of indoleacetic acid. It lasted 10-15 minutes at which time the steady rate of auxin-promoted elongation be- came evident. Similar kinetic patterns of auxin effects other than on elongation and the im- plications of the findings on hypotheses of the primary action of auxin are dis- cussed.

When an auxin such as 3-indoleacetic acid (IAA) is added to auxin-depleted sections, the rate of elongation increases strikingly but only after a lag period of 10 to 15 minutes.1' 2 Other auxin effects, however, can be seen without such a long delay. For example, effects on protoplasmic streaming,3 electrical potentials, and growth inhibition in roots4 are evident within one to two minutes after auxin application. In view of these differences we have further investigated the early time course of IAA-controlled elongation. We will present evidence that during the first few minutes after application, auxin can cause either a rapid promotion or a transient inhibition of elongation. These findings may have some bearing on the validity of some hypotheses of the primary action of auxin. Materials and Methods. Plant material: Corn (Zea mays L., Golden Bantam 8 row and Bear Hybrid WF 9 X 38) were obtained from Vaughan's Co., Chicago, Illinois, and Bear Hybrid Corn Co. The seeds were sown and coleoptile segments prepared as described previously.,' 6 Oat (Avena sativa L. cv. Victory) and pea (Pisum sativum L. cv. Alaska) seeds were sown and segments prepared as described in references 2 and 7. Chemicals: IAA was obtained from Nutritional Biochemical Corp., Cleveland, Ohio. The methyl ester of IAA was synthesized by methylation with diazomethane. The product was recrystallized twice from benzene-petroleum ether. Confirmation of synthesis was achieved by comparison of the product with an authentic sample using thin-layer chromatography and by hydrolysis (1.0 N KOH) to IAA. 184 Downloaded by guest on September 28, 2021 VOL. 65, 1970 : RAYLE ET AL. 185

Measurements of elongation: Elongation of coleoptiles was measured by the high-resolution continuous recording technique of Evans and Ray.2 A vertical column of coleoptile segments is positioned within a specially constructed glass chamber which is then filled with the desired growth medium. A small weight is placed on the uppermost coleoptile segment, and a shadow of the weight is cast by an arc lamp onto a vertical slit in a piece of cardboard. The vertical displacement of the weight by the growing seg- ments causes the shadow of the weight to move up the slit. This movement is contin- uously recorded on a piece of photographic paper moving horizontally behind the slit. The growth curves shown are direct tracings of such shadowgraph records with magnifi- cation factors and time scales as indicated. Elongation measurements of pea segments were made using the same technique except that the column of segments was held in a vertical position by a plastic tube slightly larger in diameter than the pea stem segments themselves. The plastic tube was provided with openings to allow rapid access of the growth media to the segments. A large slit was cut in the upper portion of the tube to allow light to pass through and cast a shadow of the weight resting on the pea segments within. The plastic tube holding the pea segments and the weight were positioned within a glass chamber similar to that used with coleoptiles but somewhat larger. Details can be found in references 7 and 8. All media were buffered to pH 6.3 using 10- M phosphate buffer. Elongation rate: In some experiments, in addition to the direct growth measure- ments, we have made calculations of the elongation rate. The elongation rate was determined by placing a sheet of graph paper over the original tracings, and for each consecutive 24-second point the difference on the ordinate (= length) between the fol- lowing and previous 24-second points was determined. The values so obtained were transformed (arithmetically, using the calibration on the original tracing) to microns per minute, plotted, and curves fitted through the points. Results. Fast, stimulatory action on elongation: It has been shown pre- viously2 that under standard experimental conditions the length of the lag period before onset of auxin-stimulated elongation is nearly constant (about 12 min) over concentrations of IAA ranging from 5 X 10-6 to 10-3 M. This finding seems to indicate that no significant portion of the observed lag (at these rela- tively high concentrations) is due to the time required for the uptake of IAA into the in the sense that a certain threshold amount of auxin has to be reached in the cell before an observable reaction starts. Since uptake does not saturate, but is approximately linear with outside concentration in this range, a threshold level should be reached much earlier with a high concentration of the (e.g., 10-3 M) than with a low concentration (e.g., 5 X 10-6 M). Since the observed lag is thus not due to the time required for uptake of IAA, Evans and Ray2 proposed that there is a sequence of time-consuming reactions leading to the elongation, each one requiring a certain time and these times adding up to the lag period. This explanation, however, now appears improbable in view of the two cases presented below where a positive auxin response is initiated within two to three minutes after hormone application. In agreement with a finding by Polevoy,9 the elongation of coleoptile sections was shown to take place after a lag of only two or three minutes when certain concentrations of the methyl ester of IAA were applied (Fig. 1). Since it is thought that the ester promotes growth only after conversion to IAA, the re- sponse represents a fast effect of IAA itself. Interestingly, coleoptile segments respond to high concentrations (i.e., 10-5 M) of the ester with a lag or inhibition period similar to that seen with high IAA concentrations (see below). Downloaded by guest on September 28, 2021 186 BOTANY: RA YLE ET AL. PROC. N. A. S.

/Imm FIG. 1.-Time course of growth promo- tion by IAA and by the methyl ester of IAA z Growth medium from o phosphate(MIA). buffer to the solutionchangedindicated i IAA (5x10-M) at the arrow. Coleoptile segments were z from Golden Bantam corn. Note the rela- oi /tively short latent period in response to MIA. The vertical bar by each curve in MIAt(2o M) this and subsequent figures represents 1.0 mm of elongation, for that particular record, for the entire row of segments.

10 0 10 20 30 40 50 60 TIME, MINUTES

Another fast, stimulatory effect (1-2 min) can be seen if one applies low IAA concentrations (2 X 10-7 M) to 4-mm coleoptile sections (Fig. 2). The short lag obtained with 4-mm segments can be contrasted with the longer lag seen with 8-mm segments. In the latter case at least part of the lag is thought to be due to the time required for uptake when low concentrations of IAA are used. The uptake of auxin by short segments is more rapid than by longer ones.10 This

200 B

N 100_ * 4I~1AA

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tR4mm A M 2lilto^,i L j~~~~~t 50 t j 5uIO'M I U 1

b AA 1t00 4mmeoptile segmens2Xole BatMcorn IB)Datafrom Fig. 2A plotted as growth rate vs. time. See Mat

0 10 20 30 40 0 1 0 3 TIME. MINUTES TIME, MINUTES

FIG. 2.-(A) Time course of the growth response to low concentrations of IAA in 4- and 8-mm coleoptile segments of WF hybrid corn. Growth medium changed from phosphate buffer to the concentration of IAA indicated at the arrow. Similar results were obtained using coleoptile segments of Golden Bantam corn. (B) Data from Fig. 2A plotted as growth rate vs. time. See Materials and Methods. Downloaded by guest on September 28, 2021 VOL. 65, 1970 BOTANY: RAYLE ET AL. 187

may be a partial explanation for the fast reaction seen with short segments and the relatively slow effect seen with longer segments. We would like to point out that the short lag times seen with the methyl ester of IAA and with low IAA concentrations are not under strict experimental con- trol. The above effects cannot be demonstrated in every trial (roughly 50%). However, the effects have been repeated many times and are quite real. At the present time we are unable to explain why the effects are not perfectly predict- able. Fast inhibitory action on elongation: When a large step-up in the concentra- tion of IAA is given to coleoptile sections growing in a buffered solution, an in- crease in the elongation rate is observed after 10-15 minutes. However, fur- ther examination of the response shows an earlier effect of IAA. This early effect is a reduction of the growth rate below the basal one. The effect is rapid, transient (i.e., overcome after 10-15 min by the promotion of elongation), and occurs in the three species of plants tested (Fig. 3; also see ref. 8).

FIG. 3.-Effect of high, nonphysio- logical concentrations of IAA on the elongation rate of three plant species. Growth medium changed from phos- phate buffer to the solution indicated at OA/ each arrow. In curve C the solution changes from buffer to buffer (first arrow), zo and the two solution changes from 10-3 I /| M IAA to 10-3 M IAA (second and third z A " S arrows) are included to show that changes I- c Iaa in elongation rate are not artifacts of e H i manipulations made during solution OM /Ol changes. In curve D the concentration RN of IAA indicated at the arrow was at- BBUFFER (Gold.lU.) tained by adding a small amount of 10-3 M IAA to a known volume of buffer in /2I10M 1AA the chamber.

-20 -10 0 10 30 TIME, MINUTES

Since the initial rate of elongation in buffer alone is presumably due to residual IAA, one might expect that a large step-up in IAA concentration given to sections already growing at a low rate in low concentrations of exogenously supplied IAA would cause a transient inhibition prior to establishing a more rapid elongation rate. This is indeed the case, as shown in Figure 4. The transient inhibition caused by a step-up in IAA concentration appears to be a specific auxin response and not a trivial effect of increasing the molarity of the solution or altering its oxygen content. The inhibition appears whether one changes solutions by emptying the chamber and refilling it with the appropriate new solution, or simply by the addition of highly concentrated stock solutions to Downloaded by guest on September 28, 2021 188 BOTANY: RA YLE ET AL. PROC. N. A. S.

A I

z 0 I iOf t~~~~~~~~ 5xIO-TCM ,AA S~~~~~~~~~~~~' 10__.3M 1AA 107?M 1AA 0 10 20 30 40 50 60 TIME, MINUTES

100 B 1AA+BA .'50:--;-;. -:v E * FIG. 4.-(A) Effect of high concentra- tions of IAA and benzoic acid (BA) on the 0 elongation rate of coleoptile segments pre- 0 ' 20 30 40 50 viously treated with low concentrations of C IAA. Lower curve: growth medium z changed from M to 10- M o IAA at the second1O-7 arrow.IAAUpper curve: AL loo 1 growth medium changed from 5 X 10-7 M 0 163M IAA1AA to 5 X 10-7 M IAA plus 10-3 M w I benzoic acid at the arrow. WF hybrid 4 *::L 4 I *..:.corn. 50. (B) Data of Fig. 4a plotted as growth rate vs. time. See Materials and Methods.

0 30 40 50 60 TIME, MINUTES

the original medium. The addition of high levels of benzoic acid did not inhibit the growth rate (Fig. 4). Other (1-naphthaleneacetic acid and 2,4-dichlo- rophenoxyacetic acid) gave results similar to those obtained with IAA. Since IAA step-ups act first by inhibiting elongation (transient inhibition) and subsequently by promoting elongation, one might expect that sequential addi- tions of auxin will prolong the IAA-induced lag by lengthening the transient in- hibition. In Figure 5 we present evidence that tenfold step-ups in IAA con- centration can indeed lengthen the lag (here 24 min vs. 12 min for the control). Similar extensions of the lag could be demonstrated in oats and peas. Discussion. The results presented here show that, under certain conditions, there are two distinct and rapid effects of auxin on the elongation of coleoptile segments. Following a large step-up in auxin concentration, a growth inhibition is realized within one to three minutes. This inhibition is transient and after 10 to 15 minutes a steady rate of auxin-enhanced elongation is established. Fol- lowing application of a low auxin concentration to short sections, a rapid (1-3 min) enhancement of elongation is initiated and maintained without an apparent Downloaded by guest on September 28, 2021 VOL. 65, 1970 BOTANY: RAYLE ET AL. 189

FIG. 5.-Effect of sequential in- creases in hormone concentration on the z length of the latent period in the re- 0 t sponse of coleoptile segments of Golden !a0-6M Bantam corn to IAA. Lower curve: - mt BAA growth medium changed to the indi- 0 IO6M cated concentration of IAA at each W IAA AA arrow. Upper curve: growth medium t 10-3M changed three times in succession with .10-4M BAA no change in concentration of IAA. lo- BAA BAA [AA

_ -10 0 10 20 30 TIME, MINUTES

transient inhibition. The failure to observe an equally fast enhancement at low concentrations when long sections are used may be due in part to the slower up- take rate and subsequent equilibration in the tissue. The rapid growth stimula- tion caused by the IAA-methyl ester at 2 X 10-6 M, previously reported and here confirmed, appears to be similar to the effect of low IAA concentrations on short sections. Assume the methyl ester is an active auxin only after conversion to IAA. The ester enters very rapidly and IAA is gradually liberated.7 This would be equivalent to a small step-up in the concentration of IAA. However, the faster penetration of the ester eliminates the uptake problem seen with 8-mm segments and low IAA concentrations. The rapid stimulation of growth seen under some circumstances implies that there are no time-consuming events between the primary recognition of the hor- mone and the enhancement of elongation. This idea is supported by observa- tions of several other fast stimulatory effects reported elsewhere. For example, if one assumes the Cholodny-Went theory to be correct, the appearance of an asymmetric auxin flow leads to asymmetric elongation without a significant lag period." Another well-documented and rapid stimulatory effect of auxin (20 sec) is the promotion of protoplasmic streaming.3 The transient inhibition of growth which is observed after a step-up in auxin concentration also implies that time-consuming events need not take place after auxin recognition to initiate a growth response-in this case an inhibition. It should perhaps be noted that high concentrations (i.e., 10-5 M) of the methyl ester also serve to induce the transient inhibition. This in our opinion simply implies that the hydrolysis of the ester is rapid enough to induce a paralysis similar to that achieved when high levels of auxin are applied externally. One should also note that a transient inhibition can be induced with low concentra- tions of the ester if high concentrations of free auxin are added externally at the same time. As far as tested, the transient inhibition of growth seems to be an auxin-specific reaction. It has several parallels in other auxin controlled processes. The rate of deplasmolysis in epidermal cells of Rhoeo is initially inhibited by external auxin before it is accelerated with respect to the controls.12 Auxin also first inhibits Downloaded by guest on September 28, 2021 190 BOTANY: RAYLE ET AL. PROC. N. A. S.

(0-10 min) and then promotes the uptake of glutamic acid.-3 A similar effect of auxin on electrical potentials in coleoptiles has been observed. Following asym- metric application of auxin the electric potential first drops on the treated side, but rises after 10-15 minutes to a stable, positive value.'4 Furthermore, high concentrations of various auxins inhibit protoplasmic streaming in Avena coleoptiles' and in Tradescantia hair cells.'5 The rapid in- hibition of elongation by auxinl6 may be due to a similar mechanism, espe- cially since in some cases recovery could be observed.4 There are at least two different ways to rationalize the transient inhibition observed with high IAA concentrations. One would be to assume that growth stimulation is brought about by an interaction of auxin with a recognition site X. At high concentrations there is also an interaction with another site Y. Inter- action with Y leads to growth inhibition (Y may be simply a modified form of X), but the inhibitory action of Y is expended with time. A second model would again assume that a growth stimulation can be brought about by an interaction of auxin with site X. When a large step-up occurs in auxin concentration, the binding of auxin to site X would cause an overexcitation and paralysis. The end of the transient inhibition might then be due to an adaptation that brings the auxin-induced excitation back to moderate levels where elongation is enhanced. Any model of primary auxin action has to account not only for the known early promoting effects of auxin on elongation, protoplasmic streaming, plasmoly- sis, uptake of certain amino acids, auxin transport, and electrical potential changes but it must also allow for the observed transient inhibitions caused by a large step-up in auxin concentration. It is difficult to visualize how the gene- activation hypothesis'7-2' can meet these requirements. We would suggest that the primary interaction of auxin is with the plasma membrane, perhaps through sites that are also common to auxin transport (see also refs. 22 and 23). If auxin does act on the membrane, the early kinetics of auxin action here reported may suggest some interesting features of membrane function.

* Supported in part by Atomic Energy Commission contract AT-(11-1)-1338, by Grant-in- Aid 66-12-6 from the Sloan Foundation, by a National Science Foundation Postdoctoral Fellowship to D. L. R., and by grant GB 14469 from the National Science Foundation. t Present address: Department of Botany, University of Washington, Seattle, Washington 98105. t Present address: Institut fur Genetik, Universitht Freiburg, D-78 Freiburg i. Br., Schhnz- lestr. 11, Germany. ' Ray, P. M., and A. W. Ruesinck, Develop. Biol., 4, 377 (1962). 2 Evans, M. L., and P. M. Ray, J. Gen. Physiol., 53, 1 (1969). 3 Thimann, K. V., and B. M. Sweeney, J. Gen. Physiol., 21, 123 (1937). 4List, A., Jr., Planta, 87, 1 (1969). 6 Evans, M. L., and R. Hokanson, Planta (Berl.), 85, 85 (1969). 6 Hertel, R., M. L. Evans, A. C. Leopold, and H. M. Sell, Planta (Berl.), 85, 238 (1969). 7 Evans, M. L., and D. L. Rayle, Plant Physiol., in press. 8 Barkley, G. M., and M. L. Evans, Plant Physiol., in press. 9 Polevoy, V. V., Wiss. Z. Univ. Rostock, Math.-Naturwiss. Reihe, 16, 477 (1967). 1O Hertel, R., and M. L. Evans, unpublished data. "Filner, B., R. Hertel, C. L. Steele, and V. Fan, unpublished data. 12 Guttenberg, H. von, and A. Beythien, Planta (Berl.), 40, 36 (1951). 13 Hertel, R., unpublished data. 14 Woodcock, O., and M. B. Wilkins, unpublished data. Downloaded by guest on September 28, 2021 VOL. 65, 1970 BOTANY: RAYLE ET AL. 191

"Kelso, J. M., and J. S. Turner, Austr. J. Biol. Sci., 8, 19 (1955). 16 Scott, B. I. H., Austr. J. Biol. Sci., 10, 164 (1957). 17 Briquet, M. V., J. R. DeCallonne, R. R. Lambert, and A. L. Wiaux, Physiol. Plknt, 20, 337 (1967). 18 Masuda, Y., and E. Tanimoto, Plant Cell Physiol., 8, 459 (1967). 19 Masuda, Y., E. Tanimoto, and S. Wada, Physiol. Plant., 20, 713 (1967). 'I Trewavas, A., J. Arch. Biochem. Biophys., 123, 324 (1968). 21 Trewavas, A. J., Phytochem., 7, 673 (1968). 22 Hertel, R., and R. Flory, Planta (Berl.), 82, 123 (1968). 23 Rayle, D. L., R. Ouitrakul, and R. Hertel, Planta (Berl.), 87, 49 (1969). Downloaded by guest on September 28, 2021