THE COMPARATIVE CELL CYCLE AND METABOLIC EFFECTS OF CHEMICAL TREATMENTS ON ROOT TIP MERISTEMS. I. IOXYNIL

In this study we investigated the cell cycle response of Vicia faba and Pisum sativum root tip meristems to ioxynil treatments at two concentrations, (10-4M and 10-"M). After 24 h of treatment at 10-4M concentration, 02 uptake and ATP concentrations were significantly reduced. The mitotic index was reduced and the cell cycle population position was shifted to indicate that previously inhibited cells reformed their nuclei and became tetraploid. Prolonged treatment at this concentration resulted in cell death. Treatment with ioxynil at 10-6M reduced the rate of entry into mitosis. Abnormal mitotic figures in all stages were observed, and the ploidy level of mitotically inhibited cells was doubled. These observations indicated that at 1076M concentration ioxynil acts as a preprophase inhibitor, that is, it does not act directly on the mitotic apparatus but does affect processes on which mitosis depends. THE CELL CYCLE is defined as that period of time from the formation of a cell by the division of its "mother-cell" until that cell itself divides to form two new cells. This period includes four phasesGi, pre-DNA synthesis phase; S, DNA synthesis; G2, pre-mitotic phase; and M, mitosis. Cells progress through the cell cycle phases in sequence and are arrested under natural conditions only in Gi and G2. Gi and G2 are particularly important phases, because it is then that the metabolic events (eg., RNA and protein synthesis and energy accumulation) occur which are needed for the initiation of S and M. The natural or artificially induced inhibition of any of these events causes the cycle to be disrupted and the cell cycle will become arrested (Van't Hof and Kovacs, 1972; Rost, 1977). The application of chemicals such as herbicides will cause cell cycle disruption. D'Amato (1960) described three categories of "antimitotic" substances which can affect the cell cycle: (1) Preprophase inhibitors inhibit entry of cells into mitosis and DNA synthesis and ultimately cause cell cycle arrest. Compounds such as 2,4-dichlorophenoxy acetic acid (2,4-D) (D'Amato, 1960) and DNA synthesis inhibitors like 5-fluorodeoxyuridine (Kovacs and Van't Hof, 1970) are examples. (2) Spindle inhibitors like colchicine or trifluralin (Hess and Bayer, 1974) inhibit spindle formation and cause aberrant mitotic figures. (3) Cytokinesis inhibitors such as caffeine inhibit the 1 Received for publication 3 November 1976; revision accepted 8 February 1977. 2This project was supported, in part, by NSF Grant NO. BMS72-02275A02 awarded to C. R. Stocking, Dept. of Botany, Univ. of Calif., Davis 95616. Thanks are extended to Karol Paterson for technical assistance on portions of this study. cell plate from developing and produce binucleate cells (Kihlman, 1966). loxynil (4-hydroxy-3,5-diiodobenzonitrile) is a contact herbicide used to control dicotyledonous weeds which are resistant to 2,4-D (Ashton and Crafts, 1973). This study was designed to investigate the effects of ioxynil, a reported oxidative phosphorylation uncoupler (Kerr and Wain, 1964; Foy and Penner, 1965; Paton and Smith, 1967), on the cell cycle of Pisum sativum root tip meristems. MATERIALS AND METHODS-General procedures and mitotic index analysis-Peas (Pisum sativum) were germinated in the dark at 19 C on wet vermiculite for 5-7 days. Seedings were suspended in 1/2 strength Hoagland's solution on stainless steel screens and aerated by bubbling in air from a small diaphragm pump. Chemicals tested were usually dissolved in 1 ml of 95% ethanol, diluted into 1/2 strength Hoagland's solution, and shaken over night. loxynil was tested at 10-4 and 10-6 M concentrations. For mitotic index analyses at least four 1-cm root tips were sampled at each sample time. Roots were fixed in absolute ethanol: acetic acid (3: 1) for at least 30 min. Root tips were rinsed in distilled water, hydrolyzed in 1N HCI at 60 C for 10 min, then rinsed again and placed into Schiff reagent for at least 20 min in the dark (Rost and Bayer, 1976). One mm tips were then squashed on a microscope slide and made permanent by placing them on dry ice and later mounting them n Euparal. Four slides (1,000 cells each) were scored for the number of normal and abnormal mitotic figures. Cytospectrophotometry-The cell cycle position of nuclei based on their relative amount of DNA