CTK and abscisic acid (ABA) quantification
CTK and ABA were quantified using the following enzyme-linked
immunosorbent assay protocol (Yang et al. 2001). Briefly, samples were
homogenised in liquid nitrogen and extracted in cold 80% (v/v) methanol
with butylated hydroxytoluene (1 mM) overnight at 4°C. The extracts were
collected after centrifugation at 10000 × g (4°C) for 20 min,
passed through a C18 Sep-Pak cartridge (Waters, Milford, MA, USA), and
dried in N2. The residues were dissolved in
phosphate-buffered saline (0.01 M, pH 7.4) to determine the CTK and ABA
levels. Microtitration plates were coated with synthetic CTK or
ABA-ovalbumin conjugates in NaHCO3 buffer (50 mM, pH
9.6) and left overnight at 37°C. Ovalbumin solution (10 mg/mL) was added
to each well to block nonspecific binding. After incubation for 30 min
at 37°C, standard CTK and ABA, samples, and antibodies were added and
incubated for an additional 45 min at 37°C. Antibodies against CTK and
ABA were obtained as described by Weiler et al. (1981). Then,
horseradish peroxidase-labelled goat anti-rabbit immunoglobulin was
added to each well, and samples were incubated for 1 h at 37°C. Finally,
the buffered enzyme substrate (orthophenylenediamine) was added, the
enzyme reaction was carried out in the dark at 37°C for 15 min, and the
reaction was then terminated using 3 M
H2SO4. The absorbance was recorded at
490 nm. Calculations of the enzyme immunoassay data were performed as
described by Weiler et al. (1981).