A continuous growth apparatus was used to measure the effect of cytokinin on auxin-induced elongation. The soybean hypocotyl segments elicited a biphasic response to auxin that appeared to be two overlapping responses. The first response, which began 12 min after auxin addition, was not inhibited by cytokinin, even after long preincubation in cytokinin, but the second response to auxin, which began about 30 min after auxin addition, was completely inhibited by cytokinin. Such overlapping reactions are shown, depending on the amount of overlap, to yield a variety of summation reactions, many of which resemble rate-time curves that have been previously reported. We have shown that the transient first phase of auxin-induced elongation is very similar to acid-activated growth, while the second phase is long lasting and very likely identical to the long-term response to auxin, as extensively studied in Avena, soybean, and other elongating cells.

The experiments characterize the inhibition by kinetin of auxin‐promoted elongation in excised hypocotyl sections of 3‐day soybean seedlings (Glycine max cv. Hawkeye 63). It was found that concentrations of kinetin above 4.2 μM did not further inhibit auxin‐promoted elongation. Kinetin is as potent an inhibitor of elongation as actinomycin D or cycloheximide. Tissue incubated for 3 or 5 h in the absence of auxin or cytokinin would, upon addition of auxin, exhibit a new growth rate similar to that of tissue grown in auxin for the entire incubation period. Similarly, tissue grown for 3 and 5 h in the presence of auxin would revert to the control rate of elongation upon addition of kinetin. A 10 to 30 min preincubation in kinetin yielded the tissue incapable, for the ensuing 6 h, of increasing its rate of elongation in response to auxin. Zeatin and isopentenyladenine were more potent than kinetin and benzyladenine in the inhibition of elongation. Levels of ethylene produced in the presence of auxin plus cytokinin indicated that it was not involved in this auxin‐cytokinin interaction. Kinetin by itself did not promote elongation; nor did it enhance auxin‐promoted elongation at low auxin concentrations.

Additional evidence for two separable responses to auxin is pre-sented. The averge of 24 control experiments indicated lag times of 12.4 and 35.4 min, and maxium rates of 0.57 and 0.54 mm-hr-', for the first and second response, respectively. The auxin analog 4-azido-2-chlorophenoxyacetic add increased the lag time of the second response (but not the first), resulting in the temporal separation of the two responses. Plots of elongation rates against time, taken from the literature, alowed the characterization of the two responses in monocotyls and dicotyls. Study of published rate-time elongation curves showed that the maimum rate of the first response is frequently greater than the maximum rate of the second response; however, the maximum rate of the second response has not yet been shown to exceed the maximum rate of the first response.The short lag time between auxin application and increased elongation rate was first described by Yamaki (27) and Kohler (9). The possible implications of this work received considerable attention when Evans and Ray (3), using a unique growth apparatus that continuously measured and recorded growth, continued the earlier work of Ray and Ruesink (22). The rapid response of stem, hypocotyl, and coleoptile cells to auxin has since been cited frequently as evidence that auxin-induced cell elongation is not mediated by gene activation (21, and references therein), as first suggested by Skoog and co-workers (23,24), and later proposed for elongating cells by Nooden and Thimann (11-13) and Key and co-workers (6-8).The cytokinin, isopentenyladenine, inhibited auxin-induced elongation in long term (6-8 hr) experiments (26, and references therein). This naturally occurring hormone, however, did not inhibit the rapid response of elongating soybean hypocotyl cells to auxin. The study of the auxin-cytokinin interaction in elongating cells has produced evidence that there are separable responses to auxin (25). This possibility has been previously con- ' This research was supported by grants from the National Science Foundation (GB-36586, BMS72-02496) (6,8,21), it was the important conclusion from this work (25) that neither is it disproven by experiments which describe the fast response to auxin.The experiments described herein further characterize the separable responses to auxin, and present additional evidence that two elongation reactions to auxin do indeed occur in soybean hypocotyl cells. MATERIALS AND METHODSSoybean seedlings (Glycine max L. Merr. var. Wayne) were germinated in the dark and the elongating segment of the hypocotyl was excised as described (26), except that all procedures were performed under green light (460-590 nm) at 30 C.Hypocotyl extension was measured continuously with a linear transducer. The apparatus was modified after that reported by Green and Cummins (4). To facilitate clamping of the segment in the growth chamber, a 2-cm section, which included the 1-cm elongating section directly below the hypocotyl hook plus a centimeter of tissue basal to it, w...

The first and second responses to auxin react differently to the inhibition of protein synthesis by cycloheximide. It was determined that the protein with the shortest half-life, among the several necessary for the first response, is different from its counterpart among the several necessary for the second response. Specifically, the protein half-lives are 28 minutes and 11 minutes for the first and second responses, respectively. "Growth-limiting proteins" have been frequently discussed in connection with auxin activity (e.g., refs. 3 and 19 and refs. therein). There is no doubt that such growth-limiting proteins exist in all living systems. For example, in growing higher plant cells which respond to auxin there are protein components in the cell wall, and there are enzymes involved in wall assembly. It is possible that without these proteins the cells will not grow; therefore, they are potentially growth-limiting proteins. If consideration is limited to only those processes directly involved in wall formation (e.g. in a direct line between amino acids and sugars, and the cell wall) and loosening, there are likely dozens of potentially growth-limiting proteins. This, although somewhat oversimplified, is why experiments which use protein synthesis inhibitors have not been very enlightening in determining the mode of action of auxin; even though a protein is required for growth, it may have nothing to do with auxin action (10,12,22). This is not to say that these general inhibitors have been without use in the study of plant hormone action. For example, the RNA inhibitor 5-fluorouracil was essential to the determination by Key and co-workers (9, 11) that ribosomal RNA synthesis was not essential to auxin activity. Similarly, the use of actinomycin D and cycloheximide was vital to experiments which have shown abscisic acid to be active at the level of translation in cotton seed germination (6).Analogously, the study of growth-limiting proteins in auxin action can provide useful information. Even though there may be many essential (potentially limiting) proteins in auxin-induced growth, experiments which use a protein synthesis inhibitor, such as cycloheximide, will measure only the half-life of one protein-that protein, among the several essential to auxin-induced growth, which has the shortest half-life.These experiments were designed to compare the half-lives of those proteins for the two responses to auxin (23,25 for the first and second responses, respectively). This result indicates that separable responses to auxin are the result of different cellular activities. MATERIALS AND METHODSSoybean seedlings (Glycine max L. Merr. var. Wayne) were germinated in the dark, and the elongating segment of the hypocotyl was excised as described (24) except that all procedures were performed under green light (460-590 nm) at 30 C.Hypocotyl extension was continuously measured with a linear transducer. The apparatus was modified after that reported by Green and Cummins (7).To facilitate clamping of the segment in t...

T-2 toxin, a mycotoxin produced by Fusarium tricinctum, inhibited elongation of excised hypocotyl sections of Glycine max var. Hawkeye 63. Auxin-promoted elongation was inhibited more severely than was control elongation, and a 1 hour preincubation of 5 AM toxin prevented the induction of a faster rate of elongation by auxin. While the inhibition of elongation by cytokinin was similar to that of the toxin, the mode of action of the two compounds appeared to be different, i.e. their effects on elongation were additive, and only kinetin promoted radial enlargement. Toxin treatment did not diminish cytokinin-induced radial enlargement. The properties of the plasma membrane, as measured by electrolyte leakage, were not affected by the toxin.Fusarium tricinctum (Cda.) Synd. and Hans. produces a potent phytotoxin, 4, 15-diacetoxy-8-(3-methylbutyryloxy)-12, 13-epoxy-A9-trichothene-3-ol (T-2 toxin, see Bamburg and Strong, ref. 2). This toxin is one of over twenty 12,13-epoxytrichothene toxins that affect both plants and animals (2, 3,11 (3). To determine whether the alterations in growth might be related to hormone-mediated control of cell growth, we have analyzed these effects of T-2 toxin on growth in the excised soybean hypocotyl system (10, 16, 17) using the fast growth apparatus, modified from the original design of Evans and Ray (4). 1 cm) of water-saturated vermiculite. For the first 48 hr, the dishes were covered with Saran wrap with 12 razor slits for air circulation. The Saran wrap was then removed, the dish was shaken gently, and the seedlings were watered and allowed to grow 24 hr more. Growth conditions: darkness, 30 C, 80% relative humidity. MATERIALS AND METHODS SeedsThe toxin from Fusarium tricinctum, strain T-2, was the generous gift of E. B. Smalley (Department of Plant Pathology, University of Wisconsin, Madison). It was prepared according to the methods of Marasas et al. (11) and references therein. Analysis and molecular weight determination have shown the crystalline (sheaves of white needles) product to be pure. Gas and paper chromatography of the toxin showed a single peak.For 8-hr incubations, 1 cm hypocotyl sections containing elongating cells (0.5-1.5 cm from cotyledon) were removed with razor blades and stored on ice for not longer than 2 hr in 1% (w/v) sucrose, at which time they were washed with distilled water. Twenty sections were incubated in 25 ml Erlenmeyer flasks with vented metal caps in 4 ml of basal medium containing 5 mM KHYPO4, pH 6.0, 30 mm sucrose, and a bacteriostat (80 ,uM chloramphenicol) at 30 C in a reciprocating shaking water bath. When present, 45 !M auxin (2,4-D) was used. Toxin concentrations are reported for each experiment. After 8 hr the tissue sections were washed with distilled water, blotted dry, and the increases in weight and length were determined. There were two replicates of each treatment in all experiments, and all experiments were repeated twice. Data are presented as the mean + (t for 0.01 X SD).For short term experiments, an apparatus similar ...