1,8-cineole inhibits both proliferation and elongation of BY-2 cultured tobacco cells

Abstract

Volatile monoterpenes such as 1,8-cineole inhibit the growth of Brassica campestris seedlings in a dose-dependent manner, and the growth-inhibitory effects are more severe for roots than hypocotyls. The preferential inhibition of root growth may be explained if the compounds inhibit cell proliferation more severely than cell elongation because root growth requires both elongation and proliferation of the constituent cells, whereas hypocotyl growth depends exclusively on elongation of existing cells. In order to examine this possibility, BY-2 suspension-cultured tobacco (Nicotiana tabacum) cells were treated with 1,8-cineole, and the inhibitory effects on cell proliferation and on cell elongation were assessed quantitatively. Treatment with 1,8-cineole lowered both the mitotic index and elongation of the cells in a dose-dependent manner, and the half-maximal inhibitory concentration (IC₅₀) for cell elongation was lower than that for cell proliferation. Moreover, 1,8-cineole also inhibited starch synthesis, with IC₅₀ lower than that for cell proliferation. Thus, the inhibitory effects of 1,8-cineole were not specific to cell proliferation; rather, 1,8-cineole seemed inhibitory to a variety of physiological activities when it was in direct contact with target cells. Based on these results, possible mechanisms for the mode of action of 1,8-cineole and for its preferential inhibition on root growth are discussed.

Fig. 1

Effects of various concentrations of 1,8-cineole on Nicotiana tabacum seed germination and seedling growth. Germination rate (top), hypocotyl length (middle), and root length (bottom) were measured on day 10. Each data point for germination rate was calculated from the results of all 50 individuals contained in a single container, while each for hypocotyl and root length represents the average value from 30 individuals that successfully germinated. Vertical bars represent standard errors. IC₅₀ values calculated from the data are shown in respective graphs

Fig. 2

Effects of 1,8-cineole on growth of Nicotiana tabacum seedlings. a Effects on hypocotyl length and root length. N. tabacum seedlings were grown in the absence (control) or presence (cineole) of 440 μM of 1,8-cineole for 10 days, and the hypocotyl length (top) and root length (bottom) were measured. Each data point represents the average of 20 individuals that successfully germinated. Vertical bars represent standard errors. b Effects on the cell elongation and cell proliferation in the affected roots. N. tabacum seedlings were grown in the absence (control) or presence (cineole) of 440 μM 1,8-cineole for 10 days, and the sizes of matured cells (top) and mitotic index in the root apical meristem (bottom) were measured. For cell length (top), each point represents average value of 60 cells derived from more than 4 individuals. Vertical bars represent standard errors. For mitotic index, each point represents the average value from 8 (control) or 7 (cineole) individuals. Vertical bars represent standard errors. n.s.; not significant (P > 0.05), ***; P < 0.001, Student’s t-test

Fig. 3

Behavior of BY-2 protoplasts/cells during culture. Four-day-old BY-2 cells were converted to protoplasts and cultured in either 2,4-D medium A or BA-NAA medium B, and the changes in the mitotic index (●) and cell length (○) were followed during the culture. Each data point for mitotic index was determined by counting more than 1,000 cells/protoplasts. Each point for the length of cells/protoplasts represents the average value from 30 (for 2,4-D medium) or 60 (for BA-NAA medium) cells/protoplasts. Vertical bars represent standard errors

Fig. 4

Effects of 1,8-cineole and its application methods on the proliferation and elongation of BY-2 protoplasts/cells. A Schematic representation of the experimental system. 1,8-Cineole was applied to either filter paper hanging from the cap of the air-tight container (volatilization method) or the culture medium in the flask (direct-addition method). The calculated final concentrations of 1,8-cineole in the airspace (gas-phase) were 0 to 3,000 μM. In direct-addition method, the initial concentrations of 1,8-cineole in the culture medium (liquid-phase) were 50 times higher than the calculated final concentrations in the airspace (gas-phase). (B) Effects of 1,8-cineole on the proliferation of BY-2 protoplasts grown in 2,4-D medium. Various doses of 1,8-cineole were applied through either the volatilization method (left) or the direct-addition method (right), and the mitotic index was examined on d 2. Each data point was determined by counting more than 1,000 cells/protoplasts. C Effects of 1,8-cineole on the elongation of BY-2 protoplasts grown in BA-NAA medium. Various doses of 1,8-cineole were applied through either the volatilization method (left) or the direct-addition method (right), and cell length was examined on day 0 and day 7. Each point represents the average value from 200–600 cells/protoplasts. Vertical bars represent standard errors. In B and C, IC₅₀ values estimated from the data are shown in the respective graphs

Fig. 5

Effects of 1,8-cineole on the proliferation and elongation of BY-2 cells. A The effects of a range of doses of 1,8-cineole on the proliferation of BY-2 cells cultured in D-medium are shown. The mitotic index was examined on d 2. The average values from three independent experiments are shown. Vertical bars represent standard errors. B The effects of a range of doses of 1,8-cineole on elongation (top) and starch accumulation (bottom) in BY-2 cells cultured in B-medium. Cell length and starch content were examined on d 2. The average values from three independent experiments are shown. Vertical bars represent standard errors. In A and B, 1,8-cineole was applied through the direct-addition method only. The estimated final concentrations of 1,8-cineole in the airspace (gas phase) were 0 to 240 μM, while the initial concentrations in the culture medium (liquid phase) immediately after application were 25 times higher. IC₅₀ values estimated from the data are shown in respective graphs