The biosynthesis of abscisic acid

This work is the first investigation into the biosynthesis of abscisic acid. Green tomato fruit were injected with a solution of either the potassium salt of (±)-[2- 14 c] mevalonic acid in O.05M phosphate buffer, pH 7.3, containing 10% ethanol, or {±)-[2- 14 C] mevalonic acid in ethanol. Radioactivity was detected in material which co-chromatographed with authentic abscisic acid and the authentic derivatives of abscisic acid. This material exhibited the highly characteristic spectral properties of (+)-abscisic acid. Similar results were obtained when (t)-[2- l4 c] mevalonic acid was supplied to avocado fruit preparations. The results of this work have been published in the Biochemical Journal, Vol.ll2, p.547-548 (1969). Many analogues of abscisic acid have been synthesised at Shell Research Limited, Woodstock Agricultural Research Centre, and their growth inhibitory activity assessed. The strong inhibitory activity of (+)-5-(l',2'~epoxy-2' ,6',6'-trimethylcyclohexyl)-3- methylpenta-cis-2-trans-4-dienoic acid (epoxide) on plant growth was of particular interest since the compound has the same carbon skeleton and similar substitution to abscisic acid. To discover whether this compound was active per se or because it was converted to abscisic acid the (±)-[2- 14 C] labelled material was synthesised and injected, as an ethanol solution, into tomato fruit. Epoxide was converted to abscisic acid in 1.8% yield (3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid was derived from the precursor. The 2-trans isomer was not converted to abscisic acid. Neither epoxide nor its 2-trans isomer were converted to abscisic acid in boiled tomato fruit. Treatment of epoxide with dilute mineral acid produced the 1',2'-dihydroxy derivative. The 1',2'-dihydroxy derivative was not converted to abscisic acid in tomato fruit. Wright and Hiron (1969) reported that the abscisic acid concentration in wheat leaves increased 40-fold during the first 4 hours of wilting. It was found that this increase was caused by synthesis of abscisic acid rather than by its release from a conjugate or a precursor. This was shown by the increased incorporation of [2- 3 H] mevalonic acid into abscisic acid by wilted wheat shoots in comparison with turgid shoots which had absorbed equal amounts of [2- 3 H] mevalonic acid. When (+)-[2-14C] labelled epoxide was supplied, larger amounts of labelled material were again incorporated into abscisic acid by wilted plants. The incorporation of [14C] epoxide into abscisic acid shows that the carbon skeleton of the epoxide becomes the carbon skeleton of abscisic acid. The fate of the epoxy oxygen was also investigated with wilting wheat because any [18o] containing abscisic acid formed during the experiment would not be diluted by a large pool of endogenous [16o] material. (+)- [1’2’-18o] epoxide was converted to abscisic acid, the oxygen of the l',2'-epoxy group became the tertiary hydroxyl of abscisic acid and the conversion was quantitative. For the [18ᴏ] to be retained the C-l'-oxygen bond must remain intact and epimerization at C-l' is not possible. It follows that only one enantiomer of the epoxide is converted into abscisic acid: that one in which the I' ,2'-epoxy group is on the same side of the six-membered ring as the hydroxyl group in abscisic acid. The results of this work have been published in the Biochemical Journal, VOl.119, p.727-734 (1970). The 1',4'-diols of synthetic abscisic acid were found to inhibit the growth of a number of plants (private communication, Mr. R. Leach and Dr M. Anderson) although they were less potent than abscisic acid. The demonstration of the conversion of the epoxide into (+)-abscisic acid which entails the introduction of a 4'-keto-group, and which by analogy with similar oxidations (Murphy and West, 1969) probably takes place by a hydroxylation, suggested that plants were able to oxidise the 4'-hydroxy group in the l',4'-diols of abscisic acid to a 4' ketone. The abscisic acid produced would then account for the observed growth inhibitory activity of the l',4'-diols. (±)-[2- 14 C]- cis-1’, -l' ,4' and trans-l',4' diols of abscisic acid are both converted to abscisic acid by wilted and turgid wheat shoots. The abscisic acid recovered showed a preponderance of the unnatural (-) enantiomer. The unreacted cis-1’, 4'-diol which was recovered from the tissues also showed an excess of the (-) enantiomer, unreacted trans-I' ,4'-diol which was recovered from the tissue lacked optical activity and is presumed to be a racemic mixture.