Induction of Coleoptile Elongation by Carbon Dioxide 1 Received for publication

The ability of C02 to induce elongation of Avena sativa coleoptile segments was examined with the use of a high resolution growth-recording device. CO-,saturated water causes an 8to 16-fold promotion in the rate of elongation within 1 minute. This elongation is insensitive to a variety of metabolic inhibitors that suppress auxin-induced elongation, and the C02 effect cannot be prevented by pretreatment with these inhibitors. Buffers of pH 3 to 4 also stimulate elongation quickly, and it seems that at least a major part of the action of CO2 depends upon its ability to reduce pH. The rate of elongation of auxin-promoted segments can be further enhanced by treatment with C02 but not vice versa. The response to C02 can be inhibited by mannitol at osmotic concentrations that inhibit normal growth, by calcium, and by brief pretreatment with heavy water (D20). The elongation rate that results from C02 treatment is sensitive to temperature, but the induction by C02 itself appears to be almost temperature-independent. Elongation following treatment with C02 may be a physical flow phenomenon, essentially independent of immediate biochemical participation, which occurs when wall polymer interactions that normally restrict strain in the cell wal are weakened or broken by CO2 in a manner that in effect substitutes for the role of metabolism in normal auxin-inducible cell enlargement. The literature contains a number of reports on the promotion of elongation, cell enlargement, or water uptake by CO, (3, 7-9, 19, 29, 35, 36) and by acid pH (1, 2, 16, 18, 22, 27, 34, 35). Reinhold and Glinka (29) provided evidence that induction of water uptake by CO2 in sunflower hypocotyl tissue involves a lowering of turgor stress. The brevity of the CO, treatment required (10 sec) and the fact that the effect can be obtained at 0 C led them to suggest that the CO2 effect was a physical one. They speculated that the lowering of turgor stress by CO2 might be due to cell wall loosening resulting from alterations in cell wall structure induced by low pH. The purpose of the present work was to characterize the 1 Supported by National Science Foundation grants to P. M. Ray, M. L. Evans, and K. V. Thimann. Much of the material presented here is drawn from the Ph.D. thesis of M. L. Evans (5). 2Present address: Faculty of Botany, Ohio State University, Columbus, Ohio 43210. 8Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305. ' Present address: Department of Botany, The Hebrew University of Jerusalem, Israel. elongation response of coleoptiles to CO2 and to examine the mechanism involved, using a high-resolution growth-recording device. MATERIAS AND METHODS Plant Material. The experiments were done with 8-mm segments taken beginning 3 mm below the tip of 3-day-old etiolated oat coleoptiles (Avena sativa L., var. Victory). Oats were grown as described elsewhere (6). Growth Measurements. The growth-recording device used is these experiments is described in detail elsewhere (6). Briefly, 13 hollow coleoptile segments are strung on a thread and rest on a supporting platform at the bottom. A piece of red Pyrex glass capillary tubing weighing about 90 mg is also strung on the thread so that it rests upon the uppermost coleptile segment and serves as a weight. The thread assembly is then inserted into a tubular glass chamber 14 ml in volume, and the chamber is clamped into a vertical position and filled with a particular growth medium. A small arc lamp is used to cast a sharp shadow of the glass weight onto a narrow vertical slit in a baffle placed about 1 m from the chamber. Immediately behind the slit a long piece of photographic paper is drawn horizontally by a kymograph drum turning at a rate of 3.08 mm/min. This arrangement allows continuous shadowgraphic recording of the elongation of the entire column of coleoptile segments by recording the vertical displacement of the shadow of the glass weight as the latter is pushed upward along the thread by the growing segments below. All curves shown in this work are direct tracings of original shadowgraphic records with time scales indicated. Since the magnification differs slightly for each record, a marker representing a 1-mm increase in length of the column of segments is shown at the end of each curve. Unless otherwise noted, the growth medium surrounding the coleoptile segments was continuously gassed with oxygen. Solutions could be drained and replenished within 15 sec through the appropriate outlet and connecting funnel. All experiments were done under dim red light and at 26 C, except as noted. Treatment with CO2. CO2 treatment was given by emptying the chamber and refilling it with a solution of C02-saturated water. The C02-saturated water remained in the chamber for 3 min. During this period, gassing of the solution was with CO, instead of oxygen. After 3 min, oxygenation of the C02-saturated water within the chamber was begun. CO2 treatment was given in this manner instead of continuously, since a continuous supply of CO2 is not necessary for the achievement of the full stimulatory effect, and complications due to anaerobiosis and to irreversible damage to membranes may thus be avoided (29). CO2 concentrations below saturation were made by mixing CO2-saturated water with the appropriate amount of distilled water and immediately adding the mixture to the growth-measuring chamber. During the 3 min that such solutions were in the chamber there was no gassing. 335 www.plantphysiol.org on June 29, 2017 Published by Downloaded from Copyright © 1971 American Society of Plant Biologists. All rights reserved. EVANS, RAY, AND REINHOLD In some experiments the segments were pretreated with 10-3 M KCN at pH 6.5. This solution was prepared by adding a small amount of HCI to 10-3 M KCN in 103 M phosphate buffer (initial pH, 6.5) so that the final pH remained at 6.5. The HC1 was added to the KCN solution just prior to using the solution in the growth-measuring chamber. CO2-saturated 10M KCN was prepared by diluting 102 M KCN (pH adjusted to 6.5) with CO2-saturated water. In experiments in which it was desired to follow the time course of expansion of segments whose turgor had been reduced by evaporating water from them, the evaporation was accomplished by draining the chamber and forcing a stream of air around the segments. When the segments had shrunk the desired amount, the chamber was filled with water, and their expansion was recorded. In order to measure the temperature dependence of the CO. response, a constant temperature bath with a cooling unit was used. By suitable methods (5) cold water could be circulated through the growth-measuring chamber when desired. A copper-constantan thermocouple was used to measure temperature. Temperature changes in the chamber were completed within 1