Experiments were designed to determine the relative radiosensitivity of the cell transition points of G1 → S and G2 → M in root meristems of several plant species. Label and mitotic indices and microspectrophotometry were used to measure the proportions of cells in each mitotic cycle stage in root meristems after protracted gamma radiation. The criterion of radiosensitivity was the dose rate needed to produce a tissue with less than 1% cells in S and none in M after 3 days of continuous exposure. The results show that DNA is the primary radiation target in proliferative root meristems and that the cycle duration stipulates the time interval of vulnerability. In each species, nonrandom reproducible cell proportions were established with 2C:4C:8C amounts of nuclear DNA after 3 days of exposure. Roots of Helianthus annuus, Crepis capillaris, and Tradescantia clone 02 had 80% cells with a 2C amount of DNA and 20% had a 4C amount of DNA. In these species the transition point of G1 → S was more radiosensitive than G2 → M. Roots of Pisum sativum and Triticum aestivum had cell proportions at 2C:4C:8C amounts of DNA in frequencies of 0.10-0.20:0.40-0.60:0.30-0.40. In these two species 0.30-0.40 cells underwent radiation-induced endoreduplication that resulted from a rapid inhibition of cell transit from G2 → M and a slower impairment of G1 → S. Cells increased from 2C to 4C and from 4C to 8C amounts of DNA during irradiation. The proportions of nuclei with 2C:4C:8C amounts of DNA were dependent in part upon the relative radiosensitivity of the G1 → S and G2 → M control points. The data show the relative radiosensitivity of the transition points from G1 → S and from G2 → M was species specific and unrelated to the cycle duration and mean nuclear DNA content of the plant species.

Earlier studies with synchronized plant cell populations suggested that radiation damage resulting ultimately in mitotic delay could be expressed either immediately or at some time after exposure. Whether damage resulted in an immediate cessation of progression through the mitotic cycle or whether cessation occurred latently was contingent on whether the protein requirements for further progression in the mitotic cycle were satisfied at the time of exposure. Experiments were designed to determine the effects of γ-rays on protein synthesis and whether a relationship existed between radiation-induced mitotic delay and changes in rate of protein synthesis of synchronous populations. The results obtained demonstrated that an elevated rate of protein synthesis following γ-irradiation is associated with mitotic delay. Elevated rates of polypeptide synthesis measured by sedimentation analysis, and protein synthesis measured autoradiographically, occurred only when irradiated populations were no longer making progress toward mitosis. The results suggested that: a) during mitotic delay an elevated rate of protein synthesis represents those proteins required for recovery; and b) that recovery occurs at the expense of further preparation for cycle progression, resulting in an impairment of division-oriented protein synthesis.

The mitotic delay of two biochemically different G1 cell populations of cultured 48-hour stationary phase (S.P.) and synchronized pea root meristems was measured after exposure to 300 R of gamma rays. The indices of delay were the transition from G1 to S and the time required for the population to divide (G1 → S → G2 → M). The criteria for biochemical differences were the time required to enter the S period and the sensitivity of DNA synthesis initiation to actinomycin D, puromycin, and cycloheximide. The population of S.P. meristems was delayed in G1 for approximately 5 hours but subsequently traversed S and G2 at a control rate; puromycin treatment prevented all cells from advancing to S for at least 12 hours and none divided for 24 hours. Actinomycin D treatment failed to stop about 10% of the G1 cells from moving to S, and a few of these eventually divided. Synchronized cells at the G1/S boundary progressed to S unimpaired but were delayed somewhere in either S or G2 and they appeared in mitosis 6-7 hours later than the controls. When irradiated and treated with an inhibitor, these cells were not prevented from advancing to S. It was concluded that defective protein synthesis was the cause of delay in all irradiated G1 cells and that the time and cycle period where it occurred varied and was dependent on whether the requisite proteins for functional completion of a given period were already formed at the moment of exposure.

Recovery after 300 R of γ-rays by G 1 and G 2 stationary-phase meristematic cells of excised pea root tips was measured as a function of the duration of an induced postirradiation stationary phase. The recovery indices measured were the initiation of DNA synthesis by G 1 cells ( 3 H-thymidine incorporation) and the entrance of G 2 cells into mitosis. Results showed that cells in each phase required 24 to 26 hours for full recovery. Recovery rate of G 1 cells was relatively constant throughout the 24-hour period, while that of G 2 cells varied, being higher in the first half and lower during the last half of the recovery period. Recovery of G 1 and G 2 cells exposed to a total of 300 R at a rate of 300 R/min or 0.19 R/min was the same, provided the stationary phase was not continued after γ-ray exposure.

Prolonged carbohydrate starvation of sterile excised pea roots induces a stationary phase characterized by the accumulation of meristematic cells in either the G 1 (90%) or G 2 (10%) periods of the mitotic cycle which is relieved by carbohydrate supply. With the presence of carbohydrate, DNA synthesis and mitosis are resumed and the cells return to a normal mitotic cycle. In these experiments three groups of roots were irradiated with 300 R or 600 R of X-rays after: (1) 24 hours in the stationary phase, (2) 48 hours in the stationary phase, or (3) 24 hours in the stationary phase followed by another 24 hours in this phase before carbohydrate provision. Three cytological measurements were made: the increase with time in the number of ${}^{3}{\rm H}\text{-thymidine-labeled}$ cells, the frequency of mitotic figures, and the appearance of ${}^{3}{\rm H}\text{-thymidine-labeled}$ cells in mitosis. Cells in the first and second groups of roots displayed a delay in the increase of ${}^{3}{\rm H}\text{-thymidine-labeled}$ cells, an initial reduction in division figures, and a delayed appearance of labeled mitotic figures. The recovery roots, however, displayed only a delayed appearance of labeled mitotic figures. Oxygen consumption measurements indicated the recovery of DNA synthesis, and the onset of cell division took place while metabolism was at a very low level. The overall results demonstrated that retardation of cell division enhanced the recovery rate of DNA synthesis and radiation-induced mitotic delay.