Dynamic changes in concentrations of auxin, cytokinin, ABA and selected metabolites in multiple genotypes of Douglas-fir (Pseudotsuga menziesii) during a growing season.

Changes in concentrations of several endogenous phytohormones and metabolites were analyzed in the long shoots of nine genotypes of coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) at five developmental stages: (1) closed buds, (2) flushing buds, (3) rapidly elongating shoots, (4) growing shoots and (5) near full-length shoots during one growing season. When averaged across genotypes, indole-3-acetic acid (IAA) concentration was high at stages 1 and 3. The only pattern that correlated with cone productivity was the one that was unique to IAA, in which high concentrations at stages 3 and 4 were found in all genotypes with high female cone productivity. Concentrations of isopentenyl adenosine (iPA) decreased and zeatin riboside (ZR) concentrations increased as the buds initiated and differentiated; ZR was 30 and 28 ng g(-1) dry weight (DW) at stages 1 and 4, respectively, before increasing to 166 ng g(-1) DW at stage 5. Isopentenyl adenosine peaked at 92 ng g(-1) DW at stage 2 and declined to low concentrations at stages 4 and 5. Zeatin-O-glucoside was 30 ng g(-1) DW at stage 1, declined at stages 2 and 3 and increased at stages 4 and 5. High abscisic acid (ABA) concentrations were positively correlated with rapid shoot elongation (stages 1 and 2), but as growth slowed and terminated, ABA concentrations decreased. Abscisic acid was 7 microg g(-1) DW at stage 1, increased to 13 microg g(-1) DW at stage 2 and then declined. The glucosyl ester (GE) of ABA decreased rapidly in early summer, and increased inversely with an increase in ABA. Between stages 1 and 2, ABA-GE decreased from 10 to 0.2 microg g(-1) DW and then increased. Of the ABA catabolites studied, 7'-hydroxy-ABA was about 2 microg g(-1) DW at stage 1, declined at stages 2 and 3 and increased at stages 4 and 5; phaseic acid concentrations were low at all stages, whereas dihydrophaseic acid was detected only at stages 4 and 5.

Measurement of plant hormones by liquid chromatography-mass spectrometry.

Abscisic acid (ABA) plays a number of key roles in the growth, development, and stress response of plants. For example, it is vital to a plant's response to drought stress, and is the signalling molecule responsible for closure of the stomata in order to promote water conservation. The hormone is rapidly turned over in plant tissue, mainly by oxidation or conjugation. Accurate and sensitive quantification of ABA and its metabolites has made a significant contribution to the knowledge of the role of this hormone, and also of its relationship to the induction of numerous ABA-induced genes in plants. High-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become an essential technique for the analysis and quantification of these compounds.

Phytohormones and their metabolites during long shoot development in Douglas-fir following cone induction by gibberellin injection.

Changes in plant hormones and metabolites in long-shoot stems of interior Douglas-fir (Pseudotsuga menziesii var. glauca (Beissn.) Franco) during cone induction by gibberellic acid (GA) treatment were analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode. A mixture of GA(4) and GA(7), including small amounts of GA(3) and GA(1), was stem-injected into each tree in amounts of 0, 4, 40 or 400 mg. One week after injection, concentrations of GA(4), GA(7) and GA(3) were elevated in all GA-treated samples. The ratio of GA(4) to GA(7) decreased significantly at Week 3. Absolute concentrations of all gibberellins declined sharply at Week 3 after GA application. After 5 weeks, GA(1) and GA(4) were below detection limits in all samples, and GA(7) and GA(3) were found only in the samples from trees treated with 40 or 400 mg of GA. Endogenous indole-3-acetic acid (IAA) concentrations increased following GA injection, and peaked at Week 2 or Week 3 in the trees treated with 40 or 400 mg GA, respectively. Injection of 400 mg of GA brought about a twofold increase in IAA concentration compared with control values. Injection of 40 and 400 mg of GA caused significant increases in stem dry mass in Week 5. Seed orchard data revealed that injection of either 40 or 400 mg GA enhanced female cone formation, whereas male cone formation was enhanced only by 400 mg GA. Slight decreases in concentrations of abscisic acid (ABA) and isopentenyl adenosine were observed after GA application. No significant changes were detected in the concentrations of ABA metabolites except for a slight decrease in the concentration of 7'-hydroxy ABA. The concentration of ABA declined during the growing season and the concentration of ABA glucose ester increased correspondingly.

Increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates.

Spot size reduction and increased detection sensitivity in matrix-assisted laser desorption/ionisation (MALDI) of small molecules are accomplished by using an inexpensive and removable hydrophobic coating for MALDI targets, based on 3M Scotch Gard surface treatment. Several variations in sample preparation were explored, such as surface coating technique, identity of the matrix, solvent composition, and the type of metal support plate used. These were investigated on both uncoated and coated surfaces and their impact on spot size, crystal coverage, and sensitivity is presented here. Additionally, crystallisation behaviour obtained on coated plates is compared with that on uncoated plates using scanning electron microscope analysis. To demonstrate the potential of the new coating technique, erythromycin A and valinomycin are studied to determine the increase in detection sensitivity of coated plates in comparison to uncoated plates, and to reveal the suitability of the plates for application in combined high-performance liquid chromatography/MALDI (HPLC/MALDI), where widely varying solvent compositions and droplet volumes are observed. It is shown that enhancements in detection sensitivities correlate very well with the achieved spot size reduction. The versatility of the coated plates is also exhibited by the ease of removing the surface layer, after which the plates can be rigorously cleaned without worry about damaging the hydrophobic surface, followed by a quick reapplication of new hydrophobic coating material. This makes the non-polar coating superior to more expensive commercial hydrophobic-coated targets, which are much more delicate to clean. Furthermore, cleaning and reapplication eliminate potential carry-over effects and the easy application procedure also makes the fabrication of inexpensive, disposable MALDI targets readily possible.

Automated coupling of capillary-HPLC to matrix-assisted laser desorption/ionization mass spectrometry for the analysis of small molecules utilizing a reactive matrix.

This study describes the application of a novel, reactive matrix for the mass spectral analysis of steroids by capillary-high performance liquid chromatography (capillary-HPLC) coupled to matrix-assisted laser desorption/ionization (MALDI). The mass spectral analysis of steroids was accomplished after fully automated peak deposition of chromatographic peaks onto MALDI targets. The seven corticosteroids used as test compounds were: triamcinolone, prednisone, cortisone, fludrocortisone, dexamethasone, deoxycorticosterone, and budesonide. They were separated using a PepMap C(18) (3 microm particle size, 100 A pore width) column at five different concentration levels of 25, 15, 7.5, 2.5 and 1 ng/microL, and the peaks were detected at a wavelength of 237 nm. The column effluent was mixed with 2,4-dinitrophenylhydrazine (DNPH) directly following the UV detector. The chromatographic peaks were then deposited onto the MALDI target with a robotic micro-fraction collector triggered by the UV detector signals. A special hydrophobic surface coating allowed the deposition of up to 4 microL (up to 90 % of the chromatographic peak volume) onto one sample spot. The compounds were then identified by MALDI mass spectrometry. Depending on the nature of the analyte, radical cations ([M](+.)) and sodium adduct ions ([M+Na](+)) of the steroids as well as protonated steroid-dinitrophenylhydrazone derivatives ([M(D)+H](+)) were detected in positive ion mode. The detection limits were between 0.5 and 15 ng injected with capillary-HPLC-MALDI-TOF-MS and between 0.3 and 3 ng on target with MALDI-TOF when deposited manually.