The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves

Abstract

Abscisic acid (ABA) is a plant hormone involved in seed development and germination and in responses to various environmental stresses. The last step of ABA biosynthesis involves oxidation of abscisic aldehyde, and aldehyde oxidase (EC ) is thought to catalyze this reaction. An aldehyde oxidase isoform, AOdelta, encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3, and AAO4), is the most likely candidate for the enzyme, because it can efficiently catalyze the oxidation of abscisic aldehyde to ABA. Here, we report the isolation and characterization of an ABA-deficient Arabidopsis mutant that maps at the AAO3 locus. The mutant exhibits a wilty phenotype in rosette leaves, but seed dormancy is not affected. ABA levels were significantly reduced in the mutant leaves, explaining the wilty phenotype in rosettes, whereas the level in the mutant seeds was less reduced. No AOdelta activity could be detected in the rosette leaves of the mutant. Sequence data showed that the mutant contains a G to A substitution in the AAO3 gene. The mutation causes incorrect splicing of the ninth intron of AAO3 mRNA. We thus conclude that the ABA-deficient mutant is impaired in the AAO3 gene and that the gene product, AOdelta, is an aldehyde oxidase that catalyzes the last step of ABA biosynthesis in Arabidopsis, specifically in rosette leaves. Other aldehyde oxidases may be involved in ABA biosynthesis in other organs.

Figure 1

Water loss of wild type (◊), aba3–2 (▵), aao3 (□), and aba3–2 aao3 (▴) expressed as loss of fresh weight. (Inset) The average fresh weight of the genotypes is shown. WT, wild type.

Figure 2

Germination percentage of wild type (◊), aba3–2 (▵), aao3 (□), abi3–1 (○), aba3–2 aao3 (▴), and abi3–1 aao3 (●) seeds in the light (A) and darkness (B).

Figure 3

Aldehyde oxidase activities in the rosette leaves of aao3, aba3–2, and wild-type (WT) Arabidopsis. Enzyme extracts were subjected to native PAGE, and activity bands were developed by using abscisic aldehyde (ABAld) or 1-naphthaldehyde (NAld). Each lane was loaded with 65 μg of protein.

Figure 4

Structure and expression of AAO3 and aao3. (A) Structure of the AAO3 gene and location of the mutation at the end of the ninth intron. Arrows indicate the position where primers for RT-PCR were designed. WT, wild type. (B) RT-PCR fragments obtained from cDNAs synthesized from total RNA prepared from rosette leaves using primers represented in A. (C) Illustration of RT-PCR fragments detected in wild type and aao3.

Figure 5

Expression of AAO3 mRNA and protein in the rosettes of wild type (WT) and the aao3 mutant. (A) Total RNA was extracted from rosettes of 2-month-old plants. Ten micrograms of RNA was separated on 1.5% (wt/vol) agarose gels containing 5% (vol/vol) formaldehyde. Specific probes for AAO3 and AP2 were used for hybridization. (B) Immunoblotting with protein extracts from the rosette leaves of wild-type and aao3 plants. Crude enzyme extracts (60 μg protein) of rosettes were subjected to native PAGE followed by immunoblot analysis using anti-AAO3 antibodies. Arrow indicates the position of AOδ.

Figure 6

Complementation of aao3 mutant. (A) Activity of AOδ in wild type (WT), aao3, and aao3 complemented with wild-type AAO3 gene (c-aao3) was developed by using abscisic aldehyde as a substrate. Each lane was loaded with 60 μg of protein. (B) Plants of wild type, aao3, and c-aao3 were cut from roots and left for 1 h at room conditions.