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).