||>年報 2002 (Vol.5)>Summary（生体機能開発研究分野）|
|［ 細胞複製 | 寒冷シグナル応答 | 生体機能開発 ］|
Kawamura, Y. and M. Uemura (2002)
Changes in the plasma membrane from Arabidopsis thaliana within 1 week of cold acclimation.
In: Plant Cold Hardiness: Gene Regulation and Genetic Engineering, P.H. Li and E.T. Palva, eds., Plenum Press, New York, pp.181-194.
In the present study, we identified six groups of plasma membrane proteins that are changed during cold acclimation of Arabidopsis. Among those
proteins, dehydrins and the proteins related to CO2 fixation, proteolysis or osmotic stress increased within only 1 day of cold acclimation and then
continued to gradually increase over a period of 7 days of cold acclimation. On the other hand, the lipid composition of plasma membrane does not change
substantially, at least after 1 day of cold acclimation. These results indicate that changes in the plasma membrane proteins occur more rapidly than those
in the lipid composition during the process of cold acclimation. Some cold?induced plasma membrane proteins may be related to changes in the incidence of
freeze-induced lesions associated with the plasma membrane in the early stage of cold acclimation.
Normal-speed (32 frames/sec) and high-speed (up to 4,000 frames/sec) video cameras attached with a cryomicroscope were utilized to analyze the freezing
process of protoplasts isolated from Arabidopsis leaves and Jerusalem artichoke tubers.
With protoplasts isolated from leaves of non-acclimated Arabidopsis seedlings, intracellular freezing was observed even when protoplasts were frozen to
-10oC at rates of 0.5 to 1.0oC/min, and the frequency increased significantly at 2.5 to 10oC/min. After cold acclimation for 1 day at 2oC, the
intracellular freezing did not occur at cooling rates of 1.0oC or slower. Cold acclimation for 2 to 7 days further resulted in a shift of the temperature
range over which intracellular freezing occurred to lower temperatures. High-speed video microscopy revealed that intracellular freezing was initiated,
with no exceptions, at places near the plasma membrane. However, it was not possible with techniques used in the present study to determine whether ice
formation initially occurs inside or outside of the cell. Nevertheless, these studies demonstrated that videomicroscopy combined with cryostage provides
an insight to understand the effect of cold acclimation on the freezing process of plant protoplasts.
In vitro-grown apical shoot tips of Gentiana were successfully cryopreserved by vitrification (V) and encapsulation-vitrification (EV)
protocols. We optimized and compared the V and EV protocols. Although both protocols resulted in a relatively high survival rate, the EV protocol seemed to
have several distinct advantages. First, the survival rate of shoot tips cryopreserved by the EV protocol was much greater than that by the V protocol
under optimized conditions. Second, the range of optimum treatment period by vitrification solution (PVS2 solution) was considerably wider with the EV
protocol than with the V protocol. Third, after rewarming from liquid nitrogen temperature, the cryopreserved shoot tips showed more vigorous growth for
the EV protocol than the V protocol. With the optimum EV protocol, we succeeded to cryopreserve shoot tips from 10 different lines of Gentiana and
uniformly obtained high survival rate. Thus, the EV protocol appears to be promising for cryopreservation of a wide range of plant germplasm.