Three populations of seedlings of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) were subjected to recurrent drought cycles. Pressure-volume curves were developed to derive the osmotic and pressure potentials as functions of the symplastic water over a wide range of relative water content. The drought cycles induced large reversible changes in the relative water content of the apoplasm and in the bulk modulus of elasticity. These responses have not been reported previously in any species of higher plant. Our data suggest that turgor regulation in Douglas-fir is mediated by structural and biochemical factors associated with the cell wall, rather than by osmotic adjustment.

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

Following planting, western hemlock (Tsuga heterophylla (Raf.) Sarg.) seedlings experience water stress and declining xylem pressure potential (Psi(x)). Low Psi(x) can result in xylem cavitation and embolism formation, causing a decline in hydraulic conductance. This study focused on the relationship between Psi(x), xylem cavitation and transpiration (E) of newly planted seedlings. Leaf specific hydraulic conductance (k(AB)) declined from 0.56 to 0.09 mmol m(-2) s(-1) MPa(-1) over a 9-day period. Stomatal conductance (g(s)) declined from 143.5 to 39.15 mmol m(-2) s(-1) over the same period without an associated change in environmental conditions. A vulnerability profile indicated a 30% loss in hydraulic conductivity when seedlings experienced a Psi(x) between -2.5 and -3.0 MPa. A Psi(x) of -4.0 MPa led to a complete loss of conductivity. We conclude that following planting, western hemlock seedlings often experience Psi(x) values that are low enough to cause xylem cavitation and a decline in k(AB).

The use of spectrofluorimeter coupled to a reverse phase high performance liquid chromatography column permits selective detection of indole-3-acetic acid at the low picogram level. The value of the technique is demonstrated by the analysis of endogenous IAA in elongating shoots, xylem sap and callus of Douglas-fir. The data are also used to illustrate a procedure whereby the accuracy of chromatographic analyses can be verified within definable probability limits.

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