We have developed a simple method for extracting and purifying (+)-abscisic acid (ABA) and eight ABA metabolites--phaseic acid (PA), dihydrophaseic acid (DPA), neophaseic acid (neoPA), ABA-glucose ester (ABAGE), 7'-hydroxy-ABA (7'-OH-ABA), 9'-hydroxy-ABA (9'-OH-ABA), ABAaldehyde, and ABAalcohol--before analysis by a novel technique for these substances, ultra-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS). The procedure includes addition of deuterium-labelled standards, extraction with methanol-water-acetic acid (10:89:1, v/v), simple purification by Oasis((R)) HLB cartridges, rapid chromatographic separation by UPLC, and sensitive, accurate quantification by MS/MS in multiple reaction monitoring modes. The detection limits of the technique ranged between 0.1 and 1 pmol for ABAGE and ABA acids in negative ion mode, and 0.01-0.50 pmol for ABAGE, ABAaldehyde, ABAalcohol and the methylated acids in positive ion mode. The fast liquid chromatographic separation and analysis of ABA and its eight measured derivatives by UPLC-ESI-MS/MS provide rapid, accurate and robust quantification of most of the substances, and the low detection limits allow small amounts of tissue (1-5mg) to be used in quantitative analysis. To demonstrate the potential of the technique, we isolated ABA and its metabolites from control and water-stressed tobacco leaf tissues then analysed them by UPLC-ESI-MS/MS. Only ABA, PA, DPA, neoPA, and ABAGE were detected in the samples. PA was the most abundant analyte (ca. 1000 pmol/g f.w.) in both the control and water-stressed tissues, followed by ABAGE and DPA, which were both present at levels ca. 5-fold lower. ABA levels were at least 100-fold lower than PA concentrations, but they increased following the water stress treatment, while ABAGE, PA, and DPA levels decreased. Overall, the technique offers substantial improvements over previously described methods, enabling the detailed, direct study of diverse ABA metabolites in small amounts of plant tissue.

Laboratory of Growth Regulators Palacký University and Institute of Experimental Botany ASCR CZ 783 71 Olomouc Czech Republic

References provided by Crossref.org

Domestication has altered the ABA and gibberellin profiles in developing pea seeds

Phytochromes mediate germination inhibition under red, far-red, and white light in Aethionema arabicum

Distinct hormonal and morphological control of dormancy and germination in Chenopodium album dimorphic seeds

High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing

The Effects of Exogenous Salicylic Acid on Endogenous Phytohormone Status in Hordeum vulgare L. under Salt Stress

A stable isotope dilution method for a highly accurate analysis of karrikins

Lycopene β-cyclase expression influences plant physiology, development, and metabolism in tobacco plants

Timing-dependent effects of salicylic acid treatment on phytohormonal changes, ROS regulation, and antioxidant defense in salinized barley (Hordeum vulgare L.)

The Response of Maize to Inoculation with Arthrobacter sp. and Bacillus sp. in Phosphorus-Deficient, Salinity-Affected Soil

Root-shoot communication in tomato plants: cytokinin as a signal molecule modulating leaf photosynthetic activity

The biochemistry underpinning industrial seed technology and mechanical processing of sugar beet

Aethionema arabicum: a novel model plant to study the light control of seed germination

The NAC Transcription Factor SlNAP2 Regulates Leaf Senescence and Fruit Yield in Tomato

Plant Hormonomics: Multiple Phytohormone Profiling by Targeted Metabolomics

Gibberellin-Abscisic Acid Balances during Arbuscular Mycorrhiza Formation in Tomato

The LSM1-7 Complex Differentially Regulates Arabidopsis Tolerance to Abiotic Stress Conditions by Promoting Selective mRNA Decapping

Hormonal and epigenetic regulation during embryogenic tissue habituation in Cucurbita pepo L

Spatio-temporal changes in endogenous abscisic acid contents during etiolated growth and photomorphogenesis in tomato seedlings

Quo vadis plant hormone analysis?

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression