Effect of Colchicine on the Growth and Gravitropic Response via Ethylene Production in Arabidopsis Roots

Abstract

Inhibitory effect of colchicine on growth and gravitropic responses in Arabidopsis root was explored to find whether there was an involvement of ethylene production. It has been known that cytoskeleton components are implicated in sedimentation of statoliths to respond to gravitropism and growth. The root growth was inhibited by 25% and 40% over control for 8 hr treatment of colchicine at a concentration of $10^{-5}M$ and $10^{-7}M$, respectively. The roots treated with colchicine at the concentration of $10^{-7}M$ showed the same pattern as control in 3 hr, however, gravitropic response was decreased in the next 5 hr. The colchicine treatment at the concentration of $10^{-5}M$ inhibited the gravitropic response resulting in $60^{\circ}$ of curvature. In order to better understand the role of colchicine, the production of ethylene was measured with and without the treatment of colchicine. Colchicine increased the ethylene production by 20% when compared to control via the activation of ACC oxidase and ACC synthase activity. These results suggest that the inhibition of the growth and gravitropic responses of Arabidopsis roots by the treatment of colchicine could be attributed to the rearrangement of microtubule, and increase of ethylene production.

Fig. 1. Effect of colchicine on root growth in Arabidopsis seedlings for 8 hr. Colchicine was applied to the root using an agar plate. After a vernalization for 1 day at 4°C, seeds were grown for 6 days under diurnal rhythm such as day and night was 16 hr and 8 hr, respectively. These seedlings were transferred the agar plate containing several concentrations of colchicine such as 10^-7 M and 10^-5 M, and placed in vertical position. The growth was measured for 8 hr using a camera as described in Material and Methods. Symbols are mean values ± SE from 10 independent experiments.

Fig. 2. Effect of colchicine on the gravitropic response in Arabidopsis seedlings for 8 hr. After a vernalization for 1 day at 4°C, seeds were grown for 6 days under diurnal rhythm such as day and night was 16 hr and 8 hr, respectively. These seedlings were transferred the agar plate containing several concentrations of colchicine such as 10^-7 M and 10^-5 M, and placed in horizontal position. The growth was measured for 8 hr using a camera as described in Material and Methods. Symbols are mean values ± SE from 10 independent experiments.

Fig. 3. Effect of colchicine on in vitro ACS and in vitro ACO activities. To measure in vitro ACS activity, one hundred of roots were incubated for 4 hr in solution containing 10^-7 M and 10^-5 M colchicine. These root segments were ground in liquid nitrogen, and added to 250 mM phosphate buffer, and ethylene production was measured as described in Material and Methods. To measure in vitro ACO activity, one hundred of roots were incubated with colchicine for 4 hr. These root segments were ground in liquid nitrogen, and added to an extraction buffer. After centrifugation, the supernatant was transferred to a new vial containing incubation buffer. Ethylene production was measured as described in Material and Methods to detect ACO activity. Symbols are mean values ± SE from 10 independent experiments.

Table 1. Effect of colchicine on ethylene production in root segments of Arabidopsis for 8 hr. After a vernalization for 1 day at 4°C, seeds were grown for 6 days under diurnal rhythm such as day and night was 16 hr and 8 hr, respectively. Root segments (10 ㎜) were incubated for 8 hr in solution containing 10^-7 M and 10^-5 M colchicine. At every 2 hr, 1 ㎖ of gas sample was withdrawn from the vials for measuring ethylene production

Table 2. Effect of colchicine on root growth in Arabidopsis seedlings for 4 hr and 8 hr. After a vernalization for 1 day at 4°C, seeds were grown for 6 days under diurnal rhythm such as day and night was 16 hr and 8 hr, respectively. These seedlings were transferred the agar plate containing 10^-4 M cobalt ion and/or 10^-5 M colchicine, and placed in vertical position. The growth was measured for 4 hr and 8 hr using a camera as described in Material and Methods