Xylem cavitation and loss of hydraulic conductance in western hemlock following planting.

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).

Influence of drought stress and low irradiance on plant water relations and structural constituents in needles of Pinus ponderosa seedlings.

The influence of low light on tolerance to prolonged drought was tested on unshaded and shaded seedlings of ponderosa pine (Pinus ponderosa var. scopulorum Dougl. ex Laws.). Unshaded seedlings of P. ponderosa var. ponderosa were also drought stressed to compare varietal responses to drought. The maximum irradiance received by shaded seedlings was 10% of full light. Seedlings were progressively drought stressed until predawn water potentials (Psi(x)) were -5.0 MPa. Relative water content (RWC) and the reciprocal of Psi(x) were analyzed by means of an unusual application of the pressure-volume relationship for determination of RWC of the apoplast (RWC(a)), osmotic potential at full turgor (Psi(oft)), and ratio of fully turgid weight to dry weight. Major varietal differences in drought response were in RWC(a) and needle cellulose content. The shaded seedlings showed tissue damage at relative water contents < 60%, and were killed by water deficits from which unshaded seedlings recovered. Correspondingly, shaded plants had significantly higher cell volume/cell mass ratio, Psi(oft), less cellulose in needle tissue, and lower RWC(a) than unshaded plants. These differences suggest that low irradiance restricts drought adaptation in ponderosa pine.

Transient gene expression of microprojectile-introduced DNA in Douglas-fir cotyledons.

Plasmid DNA containing the reporter gene uidA encoding ß-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was introduced on high-velocity microprojectiles into cultured cotyledons of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. Transient gene expression was measured by counting the number of distinct loci of GUS activity per cotyledon. Contrary to published results on angiosperms, repeated bombardments did not increase expression in Douglas-fir. Expression varied significantly among cotyledons from different seedlings. The amount of time between DNA delivery and treatment of cotyledons with auxins and cytokinins strongly affected GUS expression. The optimal cytokinin pretreatment produced an average of 20 loci per cotyledon. In several experiments, more than 95% of the treated cotyledons exhibited at least some transient expression. Expression remained constant up to three days following DNA delivery into cotyledons.

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons.

The effects of high-concentration, 2-h liquid pulses of N(6)-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 µM BA, and 100 and 200 µM TD. Shoots developed best from buds induced with 300, 400, and 800 µM BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 µM BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.

Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii.

Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytoklnins 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 riboside (not zeatin riboside O-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.

Differences in Proteins Synthesized in Needles of Unshaded and Shaded Pinus ponderosa var Scopulorum Seedlings during Prolonged Drought.

Proteins were radiolabeled and extracted from needles of Pinus ponderosa var scopulorum (Dougl. ex Laws.) seedlings progressively drought-stressed for about 1 month. A set of novel, low molecular weight proteins was detected in fluorographs of two-dimensional gels when relative water content of needles fell below 70%. Their synthesis was undetectable in the fully recovered seedlings within 48 hours after rewatering. In similarly stressed seedlings that were shaded to 10% full light, the low molecular weight polypeptides were not detected or appeared at very low levels. The shaded seedlings, in which drought tolerance was reduced, did not recover upon termination of the drought. The results suggest that protein synthesis induced by water deficit in drought-tolerant seedlings may contribute to resisting the effects of cellular dehydration.

Distribution of endogenous indole-3-acetic Acid and compression wood formation in reoriented branches of douglas-fir.

Five-year-old segments of intact 7-year-old branches of Douglas-fir (Pseudotsuga meziesii [Mirb.] Franco) were reoriented to determine the relation between indole-3-acetic acid (IAA) and the formation of compression wood. Eight branches per treatment were either left at their original angle (mean of 69 degrees , the control), or bent proximal to the segment to reorient it up or down 30 degrees . Differentiating xylem tissue from the upper and lower sides of each segment was collected and extracted separately for IAA analysis by in-line fluorescence detection of free IAA and IAA methyl ester after sequential C(16) reversed-phase high performance liquid chromatography. The IAA methyl ester was confirmed by gas chromatography-mass spectroscopy. Compression wood formed on the upper side of branches reoriented up and on the lower side of controls or branches reoriented down. IAA was present in all samples. The difference in IAA concentration between upper and lower sides was either not correlated, or negatively correlated in segments reoriented down, with both the occurrence of compression wood and the rate of new tracheid production. Mean concentrations for whole branch segments were not affected by the treatments, regardless of whether IAA concentrations were expressed on a surface area, weight, or cell basis.

Evidence of cytokinin bases, ribosides and glucosides in roots of Douglas-fir, Pseudotsuga menziesii.

Concentrations of cytokinin-like compounds were measured in root tips of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) collected in spring. Cytokinin bases and ribosides were estimated by radioimmunoassay after purification by immunoaffinity (zeatin riboside and isopentenyladenosine antibodies coupled to cellulose gel) and high performance liquid chromatography. Cytokinin glucosides were similarly analyzed in root extracts treated with beta-glucosidase and octadecyl-silica column chromatography in conjunction with immunoaffinity purification. The identity of predominant cytokinins obtained after enzymatic degradation was confirmed by gas chromatography-mass spectrometry. Each of the observed cytokinin bases and ribosides seemed to have a corresponding hexose conjugate form. Total quantity of cytokinin glucosides was twice that of cytokinin bases and ribosides, and the major compound was isopentenyladenosine hexose conjugate.

Seasonal changes in levels of cytokinin-like compounds from Douglas-fir xylem extrudate.

High-performance liquid chromatography, immunochromatography, and radioimmunoassay were used to identify cytokinin-like bases and glycosides in xylem sap of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). Isopentenyladenosine-type (isopentenyladenine and isopentenyladenosine) and zeatin-riboside type (zeatin, zeatin riboside, and dihydrozeatin riboside) cytokinins were detected during springtime. A glucosyl conjugate of zeatin riboside was also present in small amounts. Levels of cytokinin-like compounds varied throughout the spring but were generally highest in late April to early May.

Alteration of Cell-Wall Water Content and Elasticity in Douglas-Fir during Periods of Water Deficit.

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.

Analysis of picogram quantities of indole-3-acetic acid by high performance liquid chromatography-fluorescence procedures.

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.

Indole-3-acetic Acid in Douglas Fir: Analysis by Gas-Liquid Chromatography and Mass Spectrometry.

We sought evidence for the occurrence and seasonal variation of indole-3-acetic acid (IAA) in shoots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco).Collections obtained in December and June were extracted with methanol and diethyl ether. Extracts were purified by solvent partitioning and with Sephadex LH-20. Qualitative and quantitative information was acquired by gas-liquid chromatography of methyl, trimethylsilyl, or both derivatives of plant extract components. Analysis was performed with polar (XE-60) and moderately polar (Hi-Eff-8-BP) stationary phases. Results from three collections demonstrated that IAA does occur in Douglas fir and that amounts vary seasonally. Mass analysis of the proposed endogenous IAA peak from two representative extracts supported gas-liquid chromatography data and established the presence of IAA in Douglas fir.

Trace enrichment of cytokinins from Douglas-fir xylem extrudate.

Quantitative trace-enrichment of cytokinins from a plant source (xylem sap from the Douglasfir, Pseudotsuga menziesii (Mirb.) Franco) has been achieved by adsorption onto octadecyl-silica columns followed by elution with ethanol. Adsorption is rapid and efficient and allows complete recovery of cytokinins at the nanomolar level. Douglas-fir sap contains at least four compounds having cytokinin activity, one of which co-elutes with zeatin and another with ribosylzeatin.

Spring shoot growth in douglas-fir may be initiated by gibberellins exported from the roots.

Trials conducted under controlled environments demonstrated that the delay of bud activity of Douglas-fir (Pseudotsuga menziesii) seedlings occasioned by low temperature of the soil could be eliminated by application of gibberellic acid. Analyses of field-grown plants showed a parallel increase in bud activity, level of gibberellin-like compounds in xylem sap, and soil temperature during February and March.

Polar transport related to mobilization of plant constituents.

Indole-3-acetic acid (IAA), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), sucrose, and mannitol, were tested for polar transport through 5-mm hypocotyl segments of bean (Phaseolus vulgaris var. Pinto) seedlings. All 4 compounds were transported in a polar direction. Autoradiographs of segments through which (14)C-labeled compounds were transported indicated areas of (14)C concentration near the morphological base of segments. There was a direct correlation of IAA transport with the ability to initiate roots as well as with the degree of (14)C accumulation in the morphological base of segments. Unlabeled IAA in lanolin applied to segments appeared to reduce transport of IAA-(14)C through those segments when measured by the accumulation of (14)C in a receiver-block of agar, caused an increase in (14)C accumulation in the region to which the unlabeled IAA was applied, and also caused a decrease in accumulation of (14)C at the morphological base of upright segments. Histological studies showed that IAA accelerated cell division and the formation of root primordia particularly at the basal ends, and that these responses sometimes occurred at the expense of cell proliferation at the apical ends of segments. The data presented support the hypothesis that polar movement of IAA, and other endogenous and exogenous substances, in isolated stem segments was controlled by mobilization and utilization of plant constituents at the growth centers.