Leaf movement imaging and analysis
A. thaliana seeds were sown in-vitro individually, with an
approximately 1 cm gap in between each seed. To promote hypocotyl
elongation plants were grown under the same condition as described above
in individual 5 cm tall cardboard boxes for 10 - 12 days. Individual
seedlings were cut out in 1 cm2 blocks of agar with a
scalpel and transferred to vertical standing 25-well 10
cm2 culture dishes (Sterilin, UK) in constant white
light (100 µmol m-2 s-2). Circadian
rhythms of leaf movement were recorded using time-lapse image capture
from an array of video cameras controlled by
MetamorphTM software (Molecular Devices LLS, USA).
Images were captured every 20 minutes for seven days. Leaf position data
were extracted from images using MetamorphTM software.
Rhythmic traces were analysed using Biological Rhythms Analysis Software
System (BRASS) software package which obtains period estimates and
relative amplitude error (RAE) values using a Fast-Fourier Transform
non-linear least squares (FFT-NLS) method. The first 24 hours of data
were excluded from analysis, and movements were considered rhythmic if
they had a period between 15 and 35 hours.