Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii

Abstract

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

Comparison of endogenous cytokinins, ABA and metabolites during female cone bud differentiation in low and high cone-producing genotypes of lodgepole pine

“…Our results for lodgepole pine are in Sample collection started on August 1, mean ± SE, n = 3. Asterisk indicates a significant difference (P \ 0.05) between genotypes at the individual time point accord with those of Morris et al (1990) for Douglas-fir, in which concentrations of Z-type cytokinins were also higher than iP-type cytokinins in female cone buds and vegetative buds, but not male cone buds. Z-type cytokinins are derived from iP-type compounds and not vice versa (Kakimoto 2003;Sakakibara 2006).…”

“…Cytokinins have been shown to play an important role during the reproductive process (Imbault et al 1988;Morris et al 1990;Corbesier et al 2003;Wakushima 2004). Recent studies with Arabidopsis thaliana indicate that cytokinin receptors may have different affinities for particular cytokinins, such as zeatin (Z)-type and isopentenyl (iP)-type cytokinins (Spíchal et al 2004;Romanov et al 2006).…”

The effects of plant growth regulators on growth, yield, and phenolic profile of lentil plants

“…One frequently used, gibberellic acid (GA 3 ), increases stem length, the number of flower per plant and induces fruit setting (Azuma et al, 1997). Cytokinins have been shown to participate in the regulation of numerous aspects of plant development including initiation of buds, flowering, abscission and yield by enhancing the cell expansion (Morris et al, 1990). Kinetin is a cytokinin type PGR with primary role to extend plant life by promoting cell division and cell enlargement.…”

Isolation of two cytokinin metabolites from the rhizosphere of Norway spruce seedlings (Picea abies L. Karst.)

“…The occurrence of a similar molecule was also reported for buds of Douglas fir by Morris et al . [18] . Doumas et al determined the CK content in xylem sap and roots tips of Douglas fir using immunoaffinity and radioimmunoassay [5] .…”