A new abscisic acid catabolic pathway.

We report the discovery of a new hydroxylated abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism in Brassica napus siliques. This metabolite reveals a previously unknown catabolic pathway for ABA in which the 9'-methyl group of ABA is oxidized. Analogs of (+)-ABA deuterated at the 8'-carbon atom and at both the 8'- and 9'-carbon atoms were fed to green siliques, and extracts containing the deuterated oxidized metabolites were analyzed to determine the position of ABA hydroxylation. The results indicated that hydroxylation of ABA had occurred at the 9'-methyl group, as well as at the 7'- and 8'-methyl groups. The chromatographic characteristics and mass spectral fragmentation patterns of the new ABA metabolite were compared with those of synthetic 9'-hydroxy ABA (9'-OH ABA), in both open and cyclized forms. The new compound isolated from plant extracts was identified as the cyclized form of 9'-OH ABA, which we have named neophaseic acid (neoPA). The proton nuclear magnetic resonance spectrum of pure neoPA isolated from immature seeds of B. napus was identical to that of the authentic synthetic compound. ABA and neoPA levels were high in young seeds and lower in older seeds. The open form (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid, but not neoPA, exhibited ABA-like bioactivity in inhibiting Arabidopsis seed germination and in inducing gene expression in B. napus microspore-derived embryos. NeoPA was also detected in fruits of orange (Citrus sinensis) and tomato (Lycopersicon esculentum), in Arabidopsis, and in chickpea (Cicer arietinum), as well as in drought-stressed barley (Hordeum vulgare) and B. napus seedlings.

Rapid extraction of abscisic acid and its metabolites for liquid chromatography-tandem mass spectrometry.

We have described a simple, reliable and rapid method of extracting and partially purifying the phytohormone (+)-abscisic acid and its catabolites for liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) analysis. Lyophilized tissue samples were powdered by high-speed agitation with ceramic beads for 5 s. Metabolites were extracted from the tissue powder using acetone-water-acetic acid (80:19:1, v/v) with the addition of deuterated internal standards for quantification. Essentially all endogenous hormones were recovered by three successive tissue extractions. However we demonstrated that, with the use of internal standards, one extraction with vigorous vortexing was sufficient to obtain accurate results (recovery 65-90%). Solvents were optimized for partial purification of abscisic acid and related compounds by solid-phase extraction using Oasis HLB cartridges. The eluted metabolites were then analyzed by LC-MS-MS. To illustrate the applicability of these techniques, we analyzed the levels of abscisic acid and metabolites in seeds and valves of Brassica napus siliques at two stages of development. We detected abscisic acid, phaseic acid, 7'-hydroxyabscisic acid, dihydrophaseic acid and abscisic acid glucose ester. In both tissues, dihydrophaseic acid was the major accumulating product, reaching 97300 pmol/g dry mass in valves at 24 days after anthesis. The amount of abscisic acid in seeds was high at 24 days after anthesis (23300 pmol/g dry mass), but low in the other samples (292-447 pmol/g dry mass).