Effect of Ethylene and Gibberellic Acid on Auxin Synthesis in Plant Tissues

Treatment of plant tissues with gibberellic acid leads to increased levels of auxin and stem elongation in several plant species (7,13). Pretreatment of plant tissues with ethylene is known to decrease levels of diffusible auxin (5, 10, 20) as well as cell elongation (3,4). Recent evidence has indicated that a significant influence of gibberellin on auxin levels and growth processes is ithrough its effect on auxin synthesis in plant tissues (8,17). The possibility that ethylene may also regulate auxin levels in plant tissues by affecting the synthesis of auxin is suggested by the literature (4, 5, 10, 11, 20). Further support of this proposal is suggested by papers (1, 10, 20) which show ethylene to have no direct effect on the polar transport of auxin. Another indication that ethylene, in addition to gibberellin, may be involved in the regulation of auxin synthesis, lies in the fact that gibberellin and ethylene have opposing effects on the elongation of hypocotyls of lettuce seedlings (14). The possibility that ethylene affects the formation of auxin from tryptophan was investigated and the results of the experiments are presented in this paper. Pea seedlings (Pisum sativum L. varieties Alaska and Little Marvel) were grown under 14 hours per day of light at a temperature of 23 ? 20 during the day and 200 at night. The seedlings were grown in a medium of Perlite and vermiculite (1:1) saturated with half-strength Hoagland's nutrient medium. Eighteen hours before harvesting the tissue for tryptophan and auxin studies, the plants were placed in plastic or glass chambers and ethylene (25 l/l1) was added by means of a hypodermic syringe. Seedlings receiving gibberellic acid were treated in the apical region with 0.1 ml of 10 !&M gibberellic acid in 0.05 % Tween-20. Assays for tryptophan-1lC conversion and IAA"C destruction by cell free preparations of the apical tissue were conducted as described in an earlier report (17), except that Penicillin G at a final concentration of 0.25 mM served as the antibiotic during a 3 hour incubation period. L-Tryptophan-1-'4C (0.1 ,uc) was supplied with the enzyme preparation at a final concentration of 0.2 mm and a specific activity of 0.125 mc/mmole. Indoleacetic acid-1-14C (0.07 Ac) was included in other incubation flasks at a final concentration of 1.5 pM and a specific activity of 12 mc/mmole. This is a level of auxin within the concentration range of diffusible auxin present in Coleus stem tissue as reported earlier (12). The conversion of tryptophan to auxin by,cell free preparations of Coleus tissue was s1tudied by linctubating 1 ml of heated (900) or unheated enzyme preparation with 1 ml of tryptophan (1.54 mg/ml) and 0.1 ml of 10 mm Penicillin G. The tryptophan had been purified previously with peroxide-free ether by refluxing for 24 hours in a Soxhlet extractor. A portion of -the auxin formed during the 2 hours of incubation in the dark, was trapped in 13 agarose blocks (1 %), each of a volume of 8 jI, which had been included in each incubation flask. The auxin content of the blocks was then determined using the Avena curvature bioassay. Diffusible auxin from the apical bud regions of the stems was collected iin agarose blocks over a 2 hour period during which the tissue was exposed to diffuse light. Where comparisons are made between diffusible auxin levels and rates of tryptophan-1-14C conversion with the release of 14CO9, the enzyme preparations were made from the same apical btud regions at the end of the 2 hour diffusion period. In each experimental condition the diffusible auxin was collected from the apical bud regions of 10 Coleus plants, 12 Alaska pea seedlings, and 16 dwarf pea seedlings. Standard errors are included in table I and the t-test (15) was employed in evaluating significant differences between treatments. Studies -of tryptophan-1-l4C conversion with the release of 14CO2 were repeated a minimum of 5 itimes with each type of plant ti'ssue. Auxin determinations were repeated 3 times. The possibility that the tryptophan conversion observed in these experiments is due to bacterial 1 Supported in part by Grants No. GB-3870 and No. GB-6722 from the National Science Foundation. 2 Publication No. 193, Department of Biology, Wayne State University. 8 Present address: Department of Biology, Eastern Nazarene College, Quincy, Massachusetts.