Arabidopsis CYP707As Encode (+)-Abscisic Acid 8′-Hydroxylase, a Key Enzyme in the Oxidative Catabolism of Abscisic Acid

Phylogenetic tree of the non-A-type P450s from Arabidopsis. The tree was built using a single gene of each subfamily, except for all four CYP707As. The 85-clan is circled, and the P450 families involved in brassinosteroid and gibberellin biosynthesis are shown in bold.

Expression patterns of CYP707As in response to ABA, BR, and GA. Arabidopsis plants (2 weeks old) cultured in GM liquid medium were treated with 1 μm of (±)-ABA (ABA), brassinolide (BL), and gibberellin (GA₃), and incubated for 6 h. Total RNA isolated from the plants was subjected to RT-PCR using gene specific primers. The Actin2 (Act2) RT-PCR was included as a constitutive control. −, control; ABA, (±)-ABA; BL, brassinolide; GA, GA₃. The number indicated on the right is the number of PCR cycles required to amplify the DNA fragment of each CYP707A transcript.

Heterologous expression of CYP707A1 and CYP707A3 by using the baculovirus-insect cell system. A, SDS-PAGE of the microsomes prepared from the cells without baculovirus infection (mock-infected) or the cells expressing CYP707A1 and CYP707A3. Fifteen micrograms of proteins were loaded per lane. Reduced CO-difference spectra of the recombinant CYP707A1 (B), CYP707A3 (C). The spectra were recorded using the solubilized fractions of the CYP707A1 and CYP707A3 microsomes.

HPLC analysis of reaction products of ABA metabolized by insect cells expressing either the CYP707A1 or CYP707A3 proteins. The suspension cultures of Sf9 insect cells were infected with the recombinant baculovirus containing each P450 cDNA on a rotary shaker (150 rpm) at 27°C for 72 h. After 72-h culture, 200 μm (+)-ABA was added to the culture medium, and the cells were further incubated at 27°C for 24 h. The culture medium was collected by centrifugation and extracted four times with an equal volume of ethyl acetate. After evaporation, the ethyl acetate extracts were resuspended in 1.2 mL of methanol, and 1 μL of the sample was analyzed by HPLC. Mock-infected, the cells without baculovirus infection; CYP73A5, Arabidopsis cinnamate 4-hydroxylase.

Substrate binding spectra of the CYP707A3 protein. The recombinant CYP707A3 microsomes were solubilized with 1.0% (w/v) sodium cholate, and the solubilized CYP707A3 was obtained by centrifugation at 100,000g. A, Type I difference spectra of CYP707A3. Either (+)-ABA or (−)-ABA was added to the solubilized CYP707A3 at a final concentration of 100 μm. B, Spectrophotometric titration of CYP707A3 with (+)-ABA. (+)-ABA was added to the solubilized CYP707A3 at concentrations ranging from 1 to 50 μm, and difference spectra were recorded between 500 and 350 nm. The spectral dissociation constant (K_s) was calculated from a double reciprocal plot of absorbance difference, ΔA_{(386–419 nm)} versus the substrate concentration (inset).

HPLC analysis of reaction products from the recombinant CYP707A3 microsomes. The recombinant CYP707A3 microsomes were incubated without (+)-ABA (A), with 200 μm (+)-ABA for 10 min (B), or for 60 min (C), and with 200 μm PA (D). Reactions were carried out at 30°C, and stopped by acidification to pH 2 with 1 n HCl. A portion of reaction mixture (10 μL) was directly injected into the YMC AQ-311 column (6 × 100 mm, ODS). The reaction mixture contained 50 mm potassium phosphate (pH 7.25), 2 mg/mL recombinant CYP707A3 microsomes, and 200 μm NADPH.

Tissue specific expression of CYP707As. Total RNA was extracted from various tissues in Arabidopsis plants (4 weeks old) except for root (2 weeks old). RT-PCR was performed using gene specific primers. The Act2 RT-PCR was included as a constitutive control. The number indicated on the right is the number of PCR cycles required to amplify the DNA fragment of each CYP707A cDNA.