RESPIRATION AND CELL DIVISION IN PLANTS. I. OXYGEN CONSUMPTION AND CELL DIVISION IN THE LEAVES OF LIGUSTRUM LUCIDUM AND HEDERA HELIX

THE PRESENT paper is an attempt to further the knowledge of the relationship of oxygen consumption to cell division with data obtained by use of leaves. Here the number of cell divisions considered is much greater than in fertilized eggs of invertebrates or of algae, which in the past have served as materials for such studies. Some authors have found no relationship, while others have claimed that cell division involves oxidative processes dependent on oxygen consumption. Lyon (1904) working with Arbacia, Warburg (1908) with Echinus, Gray (1931) with Echinus, Whitaker (1932b) with Cumingia and (1933a) with Chaetupterus, did not find any relationship between nuclear division and respiration. They concluded that respiration is a function of the cytoplasm and is independent of nuclear syntheses. Mathews (1907) working with Echinus, Vles (1922) with Paracentrotus, Whitaker (1932a, c) with Fucus and Nereis and (1933a) with Arbacia, found a direct relationship between nuclear division and respiration. They concluded that nuclear division increases the rate of oxygen consumption and is, therefore, dependent on oxidative processes in the cell. More recently, Clowes and Krahl (1937, 1940), Krahl and Clowes (1937, 1940), and Krahl et al. (1941) have tested the action of phenols, cyanides, carbon monoxide, and other respiratory inhibitors on cell division using sea urchin eggs. They reported "that fertilized eggs of Arbacia punctulata have a small or negligible rate of glycolysis, both aerobic and anaerobic, so that these eggs depend almost exclusively on oxygen consumption for the energy required to support division" (Clowes and Krahl, 1940), and that when respiration was inhibited below a certain value, 30 to 40 per cent of the controls, a reversible block to cell division was produced. MATERIALS AND METHODS.-Young leaves from five different plants of both Ligustrum lucidum and Hedera helix were used in this study. Five leaves were collected each hour in both species, using leaves of uniform size from the second pair on a growing shoot; three of these were used for the study of mitotic frequency, two in the respirometer. The Warburg microrespirometer technic, with modified vessels designed by Pomerat and Zarrow (1937), was used for determination of oxygen consumption. The vessels, including a side-arm chamber for KOH, have a capacity of approximately 30 cc. Although the vessels were larger than neces1 Received for publication July 2, 1945. Aided by a grant from the University Research Fund of the University of Alabama. sary, they were found to be convenient for this type of work. The large vessel opening permitted the insertion of a leaf without contamination by the lubricating cement, and the extensive floor allowed the placement of a leaf on a dry surface, with the petiole in the water which remained around the side of the vessel. Five manometers were used, one of which was the thermobarometer. The experiments were conducted from dusk to daylight and in the dark in order to exclude the process of photosynthesis. The leaves used in the respirometer were weighed, and the volumes were determined by submersion. Following this they were blotted dry and put in the vessels with 1 cc. of distilled water. The leaves were allowed eight to ten minutes for temperature adaptation. The temperature was maintained at 250C. ? 0.050. Readings were taken for fifty-minute periods of each hour, the remaining time being necessary for the adaptation of the new leaves and for the preparation of the vessels for a new run. Although preliminary trials revealed that a leaf could be used for four runs without any great variation in results, due to the time that the leaf has been removed from the plant, each leaf was used for only two runs. This procedure gave lhourly readings for two leaves which had been in the vessels one hour and two leaves for two hours. The procedure used in determining the amount of oxygen consumption is given by Dixon (1943). The data on oxygen consumption are given per gram of wet leaf tissue. On the leaves used for the study of mitotic activity, an area, between specific veins, was located in the lower half of the leaves for the selection of the material. A millimeter disc of tissue was removed from each leaf and placed in Carnoy's solution (Speese, 1939). In the preparation of the slides, the tissue was placed in an HCl-alcohol solution for five minutes, or until the tissue became translucent, was transferred back into Carnoy's for two minutes (Baldwin, 1939), and put into aceto-carmine for an hour. After staining, each disc was placed on a slide in a small drop of aceto-carmine, and, by means of a specially constructed apparatus with a plunger, the discs were gently smeared so that they retained their circular shape. The slides were then heated and sealed with a paraffin-gum-acacia mixture. The number of mitoses was determined by a sampling method. The stages of mitosis used in these experiments were from mid-prophase to mid-telophase. Ten areas were counted and the average ascertained. The smears were then projected and