A novel
abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for
paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds.
Carotenoid composition analysis demonstrated that the defective locus affects
neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted
protein has four putative helical transmembrane domains and shows significant similarity to predicted
proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-
neoxanthin, indicating that the ABA4
protein has a direct role in
neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using
violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both
violaxanthin and
neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid
dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on
dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via
neoxanthin isomer precursors.