Action of Auxin on Cell Elongation* David L

Proceedings of the National Academy of Sciecnce8 Vol. 65, No. 1, pp. 184-191, January 1970 Action of Auxin on Cell Elongation* David L. Rayle,t Michael L. Evans, and Rainer Hertelt MSU/AEC PLANT RESEARCH LABORATORY, MICHIGAN STATE UNIVERSITY, EAST LANSING, AND DEPARTMENT OF BIOLOGY, 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-indoleacetic acid) on the elongation of segments from corn coleoptiles 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 concentrations 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 observed 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 coleoptile 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 seeds (Zea mays L., Golden Bantam 8 row and Bear Hybrid WF 9 X 38) were obtained from Vaughan's Seed 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 BOTANY: 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 segments 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 measurements, 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 following 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 relatively high concentrations) is due to the time required for the uptake of IAA into the tissue 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 hormone (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 response 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 promotion 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 A 0 b..GY 4m 0 z 0 60 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.

Load more