The influence of auxin on respiration of the Avena coleoptile.

VARIOUS GROSS effects of auxins on plant growth are easily demonstrated and are well known. The exact mechanism, however, by which these changes are initiated still is obscure. A close connection between growth and respiration is almost axiomatic, and Bonner in 1936 demonstrated that such a close relationship existed in the Avena coleoptile. This logically suggests a study of the effect of auxin on respiration, as a means of elucidating the fundamental mechanism of auxin action. A number of approaches to such a study might be made, for example: 1. to demonstrate an "in vivo" auxin stimulation of the total respiration. 2. to demonstrate an "in vivo" auxin stimulation of a specific portion of the respiratory cycle. 3. to conclusively demonstrate, "in vitro," auxin stimulation of a single chemical reaction. Previous work in this laboratory has employed the latter two approaches. It was found that two specific cell-free enzyme systems of the Avena coleoptile (alcohol and malic dehydrogenases) were stimulated by pre-treatment of intact tissue with auxin (Berger and Avery, 1943c). However, attempts to show an "in vitro" auxin stimulation of dehydrogenase activity, using inidoleacetic acid as the auxin, were unsuccessful (Berger and Avery, 1943b). The first approach was used by Commoner and Thimann (1941), who showed that under certain conditions, indoleacetic acid stimulates the oxygen uptake of living segments of Avena coleoptile tissue. Inasmuch as we were able to duplicate this result, and no other confirmation of their report has appeared, it seems desirable to publish the results of the present work, which has had to be discontinued. Commoner and Thimann also reported that the auxin stimulation was greatly magnified by the presence of malate (or fumarate), a result which we have been unable to duplicate. MATERIALS AND METHODS. -Hulled seeds of Avena sativa, variety Victory, were soaked in distilled water for 2 hours, then set out on moist filter paper 'Received for publication March 30, 1946. This paper reports work carried out at Connecticut College. in 14 cm. Petri dishes, and grown in the dark, withl periodic red light, at 250 C. and 80-90 per cent relative humidity for 55-57 hours. After removal of the coleoptile tip and its inclosed foliage leaf, the coleoptile cylinders were cut into three segments, each about 4 mm. long, and soaked for 1.4-16 hours in various solutions, in the dark at 25$C. Thirtv segments from ten coleoptiles were then placed in fresh solutions in each respirometer flask. Respiration was measured in Warburg manometers, shaken at a constant rate of 60 times per minute. The systems were first allowed to equilibrate for 30-60 minutes in the respirometer water-bath, which was maintained at 30? C. in diffuse daylight. Exceptions to this general procedure will be noted later where they are made. The final volume of reaction-mixture in each flask was 2.5 ml. and the final concentrations of reagents, unless otherwise specified, were: potassium 1-malate, pH 7.0, 0.001 molar; indoleacetic acid (IAA), 10 mg. /liter or 5.7 X 10-5 molar; and sucrose 1 per cent. These are the same concentrations employed by Commoner and Thimann. The center well of each flask contained 0.1 ml. of 10 per cent potassium hydroxide. In general, experiments were set up in such a way as to duplicate as nearly as possible the conditions used by Commoner and Thimann. EXPERIMENTAL RESULTS. -Influence of auxin and malate on respiration. -To test for possible respiratory stimulation by added indoleacetic acid or malate, segments were "starved," i.e., depleted of excess substrate and auxin by soaking them for 18 hours in distilled water. The respiratory responses of these segments in fresh solutions are shown in table I and figure 1. It may be seen from the results of all three experiments of table 1 that addition of malate to the control causes only a very slight increase in total oxygen uptake, but that the addition of auxin to the control gives increases of 28-40 per cent. The increase in respiration occurs almost immediately (fig. 1) and is stable for at, least three hours. On the other hand, separate experiments show that the addition of auxin to segments respiring in water (no sucrose) generally causes no significant change in the rate of respiration (confirming Com-