The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism

Abstract

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

Figure 1

Catabolic pathway of ABA in plants.

Figure 2

Functional expression of CYP707A genes in yeast. (A) HPLC profiles of reaction products on incubating (+)-S-ABA with 2 μg of microsomal protein. Retention time is given in min, while the vertical axis indicates UV absorbance at 254 nm. The positions of authentic ABA and PA (with an arrow) under the same conditions are labeled. (B) Mass spectra of the reaction product from (+)-S-ABA by CYP707A1.

Figure 3

Effect of P450 inhibitors on ABA 8′-hydroxylase activity in vitro. The vertical axis shows the percentage of PA production compared to control in the absence of the inhibitor.

Figure 4

Expression of CYP707A genes among various tissues. QRT–PCR was performed using dry seed, rosette, stem, flower, silique and root. Transcript levels were normalized using 18S rRNA as an internal control. Results from triplicate samples in three independent experiments are shown with error bars.

Figure 5

Changes in the level of ABA, PA and DPA, and expression of CYP707A genes during seed imbibition. (A) Quantification of total ABA, PA and DPA levels including both the endogenous and medium leaked fraction during seed imbibition. Levels of ABA, PA and DPA were measured by GC-MS from both seeds and medium, imbibed under continuous light at designated time points. An average from five independent experiments is shown with error bars. DW indicates dry weight. (B) The level of CYP707A transcripts during seed imbibition. QRT–PCR was performed using dry and imbibed seed at the designated time points. Transcript levels were normalized using 18S rRNA as an internal control. An average from duplicate experiments is shown. (C) Induction of CYP707A transcript levels by exogenous ABA (30 μM) at 12 h after seed imbibition. An average from three independent experiments is shown with error bars.

Figure 6

Changes in ABA, PA and DPA levels, and CYP707A gene expression during dehydration and rehydration. (A) Quantification of endogenous ABA, PA and DPA levels during dehydration and rehydration. Endogenous levels of these metabolites were measured by GC-MS from 2-week-old plants subjected to dehydration (open circle). Rehydrated plants were watered after 6 h of dehydration (filled circle). An average from at least duplicate samples in two independent experiments is shown. FW indicates fresh weight. (B) Induction of the NCED3 expression during dehydration and rehydration. QRT–PCR was performed using plants subjected to dehydration (open circle) and rehydration after 6 h of dehydration (filled circle) at the designated time points. Transcript levels were normalized using 18S rRNA as an internal control. Induction of NCED3 expression is indicated as fold increase. An average from duplicate experiments is shown. (C) Induction of CYP707A gene expression during dehydration and rehydration. Induction of CYP707A gene expression is indicated as fold increase.

Figure 7

Changes in PA and DPA levels and induction of CYP707A gene expression upon ABA treatment. (A) Quantification of endogenous PA and DPA levels upon ABA treatment. (B) QRT–PCR was performed using plants subjected to 30 μM (+)-S-ABA (filled circle) or water control (open circle) at the designated time points. Transcript levels were normalized using 18S rRNA as an internal control. Induction of CYP707A gene expression is indicated as fold increase. An average from duplicate samples in two independent experiments is shown.

Figure 8

Phenotypic analysis of the cyp707a2 and cyp707a3 mutants. (A) T-DNA insertion in the CYP707A genes. The T-DNA insertion in the cyp707a2-1 mutant is located at the junction between the fifth exon and fifth intron, which disrupts the splicing site, while the cyp707a2-2 mutant contains the T-DNA at the seventh intron. The cyp707a3-1 and cyp707a3-2 mutants harbor the T-DNA at the first exon and second exon, respectively. (B) Dormancy of the cyp707a mutants. Freshly harvested seeds were sown on filter paper moistened with water. Radical emergence was scored as a criterion for germination. Triplicate experiments were performed and the averages are shown with error bars. Wild type, open circle; the cyp707a2-1 mutant, gray circle; the cyp707a2-2 mutant, filled circle; the cyp707a3-1 mutant, gray square; and the cyp707a3-2 mutant, filled square. (C) The endogenous level of ABA, PA and DPA in dry seeds of wild type and the cyp707a2-1 mutant. An average from duplicate experiments from independent samples is shown. (D) The change in the level of endogenous ABA in wild type and the cyp707a2-1 mutant during seed imbibition.