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Effect of 2 , 4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue 1
| Content Provider | Semantic Scholar |
|---|---|
| Author | Murr, D. P. Yang, Shang Fa |
| Abstract | Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-'4C was also inhibited in the presence of 2, 4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2, 4-dinitrophenol. In the absence of 2, 4-dinitrophenol, indoleacetic acid-induced ethylene parallels the free indoleacetic acid level within the tissue. However, in the presence of 2,4-dinitrophenol the rate of ethylene production did not correlate with the free indoleacetic acid level. These results indicate that both indoleacetic acid-induced ethylene production and indoleacetic acid conjugation require a continuous supply of ATP, the formation of which was inhibited by 2, 4-dinitrophenol. Auxins are known to stimulate ethylene production in a wide variety of plant tissue (1, 4, 6-8, 11-13). In excised tissue from legume seedlings, kinetin alone slightly stimulates ethylene production, but a remarkable synergistic effect of kinetin on IAA-induced ethylene production has been observed (4, 6, 8). It has been shown that IAA-induced ethylene production parallels the free IAA level, which, in turn, is regulated by the rate of IAA metabolism (4, 7, 8). When tissue is incubated with IAA, most of the IAA entering the tissue is rapidly converted to IAAsp2 which has little or no auxin activity in the pea test (1) or in inducing ethylene production in mung bean (8). Kinetin markedly suppressed the conjugation of IAA and therefore increased the free IAA level and ethylene production rate of the tissue (8). The presence of IAA has been found to stimulate synthesis of a new enzyme protein responsible for IAA 1 This work was supported by the National Science Foundation Grant GB-33907X. 2 Abbreviations: IAAsp: indole-3-acetylaspartic acid; DNP: 2. 4-dinitrophenol. conjugation (16), which requires oxygen and probably proceeds via formation of indoleacetyl-CoA (18). Using a model system employing octanoate thiokinase from liver mitochondria, Zenk (18) found that ATP is required for the synthesis of indoleacetyl-CoA from IAA and CoA; the oxygen is required for ATP synthesis. Compounds such as DNP and other haloand nitrophenols are known to uncouple oxidative phosphorylation which leads to a depletion of ATP in the tissue. However, these agents do not uncouple substrate level phosphorylation (9). In fruit tissue DNP has been shown to inhibit ethylene production and respiration (5, 15) as well as the conversion of methionine to ethylene (Murr and Yang, in preparation). The inhibition caused by DNP could be reversed partially by ATP (5). These results suggest that at least one step in the synthesis of ethylene required ATP (5, 15; Murr and Yang, in preparation). The present paper will describe the effect of DNP on IAAinduced ethylene production in relation to IAA conjugation by mung bean hypocotyl segments. MATERIALS AND METHODS Plant Materials and Chemicals. Seeds of mung bean (Phaseolus mungo L.) purchased from a local market were sorted and surface-sterilized with 0.02% sodium hypochlorite solution for 10 min. After thorough washing, the seeds were imbibed and aerated for 8 hr and then were grown in vermiculite for 4 days, in darkness, at 25 C. Under dim green light, 2-cm long segments were cut from hypocotyls at a point 1 cm below the hook. Twenty segments were incubated in 5 ml of incubation medium containing 50 mm potassium phosphate buffer (pH 6) and 2% sucrose in a 50-ml Erlenmeyer flask. Where indicated. 0.15 ,umole of IAA-2-14C (2 ttCi), 0.25 ,Lmole of DNP, and 0.2 I-mole of kinetin was included. A plastic center well containing 0.2 ml of 40% KOH was hung in the flask to absorb the CO2 evolved. The flask was sealed with a rubber serum cap and incubated in a shaker at 25 C, in darkness. Determination of Ethylene. At the end of the incubation, a 1-ml gas sample was withdrawn from the flask with a hypodermic syringe, and ethylene was assayed with a gas chromatograph equipped with an alumina column and a flame-ionization detector. Analysis of IAA and IAAsp. Aliquots of incubation medium before and after incubation were assayed for total radioactivity with a liquid scintillation counter. The segments were washed with 10 /LM unlabeled IAA solution and then with water. The segments were then homogenized and extracted, first with 95% ethvl alcohol and then with 80% ethyl alcohol. The combined extracts were concentrated under reduced pressure to a final volume of 2 ml. An aliquot of the concentrated extract was 182 www.plantphysiol.org on October 15, 2017 Published by Downloaded from Copyright © 1974 American Society of Plant Biologists. All rights reserved. DNP ON IAA-INDUCED ETHYLENE PRODUCTION chromatographed on paper, along with unlabeled IAA and IAAsp, using 1-butanol-3% ammonia (1:1, v/v) as developing solvent. After the chromatograms dried, they were scanned for radioactivity and the radioactivity was estimated from the peak area as previously described (8). |
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| Content Type | Text |
| Resource Type | Article |
