Two independent routes have been proposed for the biosynthesis of the plant hormone, abscisic acid (ABA). In higher plants, crucial evidence for the C_<40> pathway (indirect pathway or carotenoid pathway) has come from the cloning of a series of 9-cis-epoxycarotenoid dioxygenases (NCEDs). In the case of fungi, α-ionylideneethanol (4) and γ-ionylideneethanol (5) have been isolated from Cercospora rosicola and C. cruenta, respectively and proved to be biosynthetic precursors of ABA. These C_<15> alcohols were structurally related to the direct cyclization of farnesyl diphosphate (FDP, 3) derivatives, thus inferring the C_<15> pathway (direct pathway). However, the fungal C_<15> pathway is still controversial without any further evidences. As new fungal biosynthetic intermediates of abscisic acid (ABA), (R)-(2Z,4E)-α-ionylideneacetaldehyde (14) and (R)-(2Z,4E)-γ-ionylideneacetaldehyde (15) were isolated from young mycelia of Cercospora rosicola and C. cruenta, respectively. Absolute configuration of natural alcohols (4, 5), aldehydes (14, 15), and acids (16, 17) were all determined as (R) by HPLC with Chiral Cell OJ (Daicel) in accordance with the stereochemistry of the natural product, (S)-ABA. Under ^<18>O_2 atmosphere, an oxygen atom of the endogenous aldehyde 14 or 15 was exclusively labeled. Similarly, three ^<18>O atoms were incorporated into the ABA molecule recovered from prolonged incubation; selectively labeled were one of the carboxyl oxygen atoms and the two on the ring portion of ABA. Feeding experiments with [1-^<13>C]glucose proved the exclusive operation of the mevalonate pathway for the formation of both ABA and β-carotene. Putative C_<15> alcohol intermediates (6-12 and their 2-trans-isomers and diphosphate esters) were not substantially incorporated into ABA in the feeding experiments with [2-^<14>C]-labeled substrates in C. cruenta. Scrutiny on GC-EI-MS only detected a trace amount of new alcohol 13, which was proved to be a catabolite of 15 via 5 by feeding experiments with [1,2-^<13>C_2]5. The partial conversion of 15 into 5 seems to have diminished the significance of 5 as a key biosynthetic intermediate to support the fungal C_<15> pathway. These results suggested that aldehyde 14 and 15 are key ABA biosynthetic intermediates formed by the oxidative cleavage of carotenoid precursors. It is premature at this stage to conclude the fungal C_<40> pathway. However, we expect that more direct evidences will be delivered by the unequivocal identification of putative C_<40> precursors such as 18 and by the molecular cloning of the putative biosynthetic gene by the PCR technique based on the DNA sequence similarity among a series of known dioxygenases including plants NCEDs. These works are now in progress.

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