Neophaseic acid catabolism in the 9'-hydroxylation pathway of abscisic acid in Arabidopsis thaliana

Abscisic acid (ABA) hydroxylation is an important pathway for ABA inactivation and homeostasis mainte-nance. Here, we discover a new downstream catabolite of neophaseic acid (neoPA) in the ABA 9'-hydroxyl pathway and identify it as epi-neodihydrophaseic acid (epi-neoDPA) by comparing its accurate mass, retention time, and MSn spectra with those of our chemically synthesized epi-neoDPA. Analyses of Arabi-dopsis seed germination and ABA-related gene expression reveal that neoPA rather than epi-neoDPA pos-sesses ABA-like hormonal activity. In vitro enzyme activity tests of prokaryotic recombinant protein reveal that NeoPAR1 (neoPA reductase 1) identified from Arabidopsis converts neoPA into epi-neoDPA. Site -directed mutation at Tyr163 in the conserved motif of NeoPAR1 abolishes the catalytic activity of NeoPAR1. Accelerated seed germination was observed in NeoPAR1 knockdown and knockout mutants, whereas retarded seed germination and the accumulation of epi-neoDPA and ABA were observed in Neo-PAR1 overexpression lines, suggesting that NeoPAR1 is involved in seed germination and maintenance of ABA homeostasis.