7.3@ Cell cycle inhibitors
The
moss Physcomitrella patens has an
amazing ability for regeneration, whose leaf cells readily change to protonemal
apical cells with excision signals without any exogenous phytohormones,
resulting in regeneration of new whole plants. In particular, given that a part of a leaf is excised from a
gametophore, leaf cells facing to the dissected plane start to behave similarly
to a protonemal apical cell and readily reenter cell cycle in a few days. To
investigate early events during these processes in excised leaves, we use
chemical inhibitors for cell cycle studies, roscovitine and aphidicolin.
Procedure
1. Inoculate
protonemata tissues blended by a polytron homogenizer, glass beads or motor and
pestle.
2. Cultivate
3 to 4 weeks on BCDAT solid medium (until growth of gametophores with
approximately 10 leaves or more) at 25˚C under continuous light.
3. Collect
gametophores, of which rhizoids and protonema tissues should be removed, within
1 hour with forceps.
4. Put the
collected gametophores in 50 ml tube containing 10 ml of BCDAT liquid medium or
sterilized water.
5. Blend with
a polytron homogenizer or glass beads to cut leaves apart from the
gametophores. (By the treatment, the number of leaf cells facing to the excised
planes in a leaf is also increased.)
6. Wash with
BCDAT liquid medium twice.
7. Suspend in
5~10 ml BCDAT liquid medium.
8. A part of
suspension (1~2 ml) is inoculated in a 35 mm dish or a well of 24-well plate.
9. Add
roscovitine (final conc. 100 µM) or aphidicolin (final conc. 30 µM)
and incubate 25˚C for 48 hours at under continuous white light conditions.
10.
Harvest the leaves.
Note
E Roscovitine
inhibits the CDK activity, resulting in the G1 arrest of the P.patens leaf cells after treatment with
a polytron homogenizer.
E Aphidicolin
inhibits DNA polymerase a
and d,
resulting in the G1/S phase arrest. At 48 hours or more after the treatment,
you may see leaves with leaf cells changed to protonemal apical cells, despite
of the cell cycle arrest by aphidicolin.
Reference
Chemical
inhibitors: a tool for plant cell cycle studies.
Planchais
S, Glab N, Inzé D, Bergounioux C. FEBS Lett. (2000) 476: 78-83.