Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytokinins from vegetative, female, and male buds of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The purified cytokinins were separated by reverse phase high performance liquid chromatography and analyzed by radioimmunoassay. Confirmation of cytokinin identities was by gas chromatography-mass spectrometry. Immediately prior to bud burst, all bud types contained three major cytokinins: isopentenyladenosine, zeatin riboside, and a hexose conjugate of zeatin nboside (not zeatin nboside 0-glucoside). Zeatin-type cytokinins were present in relatively high concentration in vegetative and female buds. In male buds, however, relatively high levels of isopentenyladenosine were found together with low levels of zeatin-type cytokinins. (4, 1 1), and tZ, zeatin glucoside, and tZR were shown to be present in the leaves of Podocarpus henkelii (21). In a study on Pinus radiata buds, tZR was identified by GC-MS. A glycoside of tZR was also present in which the glycoside moiety was believed to be attached to the ribosyl moiety ( 19).As part of a study of the role of cytokinins in bud development, we have examined the cytokinin content of female, male, and vegetative buds from Douglas fir. In this paper we describe the nature of the major cytokinins present in these bud types, using immunoaffinity chromatography for isolation, HPLC and RIA for the quantitation, and GC-MS for identification.
MATERIALS AND METHODS
Bud CollectionSix Douglas-fir trees (Pseudotsuga menziesii Mirb. Franco) approximately 15 years old were selected from two seed orchards near Corvallis, OR. Buds were collected from the upper third of the crown during April and May, 2 weeks prior to bud break. Male, female, and vegetative buds were harvested from each of the six trees separately, frozen, and stored in liquid nitrogen until extraction.
ExtractionBuds (1-3 g fresh weight) were homogenized (Polytron, Brinkmann Instruments) in a mixture of methanol and ammonium acetate buffer (40 mM, pH 7.0), (80/20 v/v), which contained Dieca (0.2 mg/mL) and butylated hydroxytoluene (0.5 mg/mL) as antioxidants. Aliquots of [3H]iPA trialcohol (1-2 pmol; 25,000 dpm) were added before extractions as an internal standard. The homogenate was stirred for 30 min at 4°C and centrifuged (1,000g, 30 min). The supernatant was 4