Jim K. Heald

Non-uniform distribution and seasonal variation of endogenous indol-3yl-acetic acid in the cambial region of Pinus contorta Dougl.

Endogenous, free indol-3yl-acetic acid (IAA) levels were measured in the main stem in the 10-year-old cambial zone, in the adjoining differentiating xylem, and in the adjoining mature xylem of 15–20-year-old Pinus contorta Dougl. by single-ion-current monitoring, combined gas chromatography — mass spectrometry, on several dates from early spring to early winter. Microscopy was used to determine the state of cambial activity on each harvest date. The IAA levels were found to be nearly constant at 1 μg g-1 DW in the cambial zone from March to July, then to increase to near 2 μg g-1 DW during the remainder of the growth season. No clear correlation was evident between number of fusiform cells per radial file and IAA content in the cambial zone. By contrast, the IAA content in differentiating xylem was higher than that in the adjoining meristematic zone on all harvest dates and also exhibited marked seasonal variation, peaking near 16 μg g-1 DW in mid summer, and declining to 1 μg g-1 DW in autumn. In mature xylem, IAA levels were very low and showed negligible variation. The fresh weight to dry weight ratio of differentiating xylem was greater than that of the cambial zone, and greater in the cambial zone than in mature xylem.

Identification and measurement of indoleacetic and abscisic acids in the cambial region of Picea sitchensis (Bong.) Carr. by combined gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were identified by combined gas chromatography-mass spectrometry (GCMS) in fractions obtained by diffusion and extraction from bark peelings of Sitka spruce. A procedure is described for the quantitative analysis of IAA and ABA levels in the same extract using the GCMS technique of single-ion current monitoring. This procedure was used to measure the diffusible, free, and bound fractions of IAA and ABA in the cambial region of Sitka spruce throughout one year; the range in concentration for these fractions was 0.06–0.30, 0.46–3.85, and 0.04–0.20 μg/g oven-dry weight, respectively, for IAA, and 0–0.08, 0.03–2.21, and 0.13–0.66 μg/g oven-dry weight, respectively, for ABA. Movement in the cambial region was found to be polar for endogenous IAA and nonpolar for endogenous ABA. Recoveries of [14C]IAA internal standards showed that 73–99.5% of the IAA was lost during purification, and that there could be up to 5-fold differences in recovery between purifications, indicating that IAA loss shold be measured in quantitative analyses.

Mass spectrometric measurement of zeatin glycoside levels in Vinca rosea L. crown gall tissue.

The range of zeatin glycosides found in crown gall tissue of Vinca rosea L. has been quantified using a mass spectrometric isotope dilution procedure. Problems in the quantitative analysis of cytokinins in plant extracts are discussed.

Selected ion monitoring/isotope dilution mass spectrometric determination of 1-aminocyclopropane-1-carboxylic acid levels in ripening tomato fruit

A new method is described for the quantitation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plants. [2,2,3,3,-2H4]ACC has been synthesized and used as an internal standard for selected ion monitoring/isotope dilution quantitation of this compound in ripening tomato fruit. These data are compared with those derived from the widely used indirect oxidative ACC assay (which underestimated the ACC levels by between two- and fourfold). The greater accuracy, sensitivity (100X), and specificity of the mass spectrometric method will be of considerable benefit to those interested in factors which control ACC and ultimately ethylene levels since it is believed that ACC synthesis and its oxidative metabolism to ethylene are the key points at which ethylene biosynthesis is regulated.

A metabolomic study in oats (Avena sativa) highlights a drought tolerance mechanism based upon salicylate signalling pathways and the modulation of carbon, antioxidant and photo-oxidative metabolism

Although a wealth of information is available on the induction of one or several drought-related responses in different species, little is known of how their timing, modulation and crucially integration influence drought tolerance. Based upon metabolomic changes in oat (Avena sativa L.), we have defined key processes involved in drought tolerance. During a time course of increasing water deficit, metabolites from leaf samples were profiled using direct infusion-electrospray mass spectroscopy (DI-ESI-MS) and high-performance liquid chromatography (HPLC) ESI-MS/MS and analysed using principal component analysis (PCA) and discriminant function analysis (DFA). The involvement of metabolite pathways was confirmed through targeted assays of key metabolites and physiological experiments. We demonstrate an early accumulation of salicylic acid (SA) influencing stomatal opening, photorespiration and antioxidant defences before any change in the relative water content. These changes are likely to maintain plant water status, with any photoinhibitory effect being counteracted by an efficient antioxidant capacity, thereby representing an integrated mechanism of drought tolerance in oats. We also discuss these changes in relation to those engaged at later points, consequence of the different water status in susceptible and resistant genotypes.

Dihydrozeatin metabolism in radish seedlings

Abstract (±)-[8- 14 C]Dihydrozeatin was fed to derooted radish seedlings. After three days the plants were harvested and the cytokinin metabolites purified

Cytokinin metabolism and the modulation of cytokinin activity in radish

Abstract The metabolism of zeatin, N 6 -(Δ 2 -isopentenyl) adenine, dihydrozeatin, zeatin- O -glucoside and dihydrozeatin- O -glucoside has been studied using derooted radish seedlings. The metabolites were identified by UV and GC/MS. The patterns of metabolism are compared and provide evidence that the O -glucosyl conjugates may be storage forms of the cytokinins.

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5′-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with 15N- and 2H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N6-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.

A quantitative analysis of cytokinin using single-ion-current-monitoring

SummaryThe levels of the cytokinin 6-(o-hydroxybenzylamino)-9-β-D-ribofuranosylpurine (o-OH BAP riboside) have been measured in attached leaves of poplar (Populus x robusta Schneid) using the technique of single-ion current monitoring (SICM). The use of 6-(p-hydroxybenzylamino)-9-β-D-ribofuranosylpurine (p-OH BAP riboside) as an internal standard has enabled quantitative measurements of recovery to be made.

Gibberellins in Picea sitchensis carriere: Seasonal variation and partial characterization

SummaryThe gibberellin content of needles of Picea sitchensis was investigated during a whole vegetative cycle. During the period of active growth the major gibberellin activity was present in a less polar fraction (Fraction B) whilst during the winter a more polar fraction (Fraction A) was predominant. A similar pattern of activity was observed in buds of the same species. The pattern of change of these fractions is discussed and Fraction B is partially characterized as a gibberellin isomeric with GA9.