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First published online April 2, 2004; 10.1104/pp.103.037614 Plant Physiology 134:1439-1449 (2004) © 2004 American Society of Plant Biologists Arabidopsis CYP707As Encode (+)-Abscisic Acid 8'-Hydroxylase, a Key Enzyme in the Oxidative Catabolism of Abscisic Acid1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611–0011, Japan (S.S., K.S., M.M.); International Innovation Center, Kyoto University, Kyoto 606–8501, Japan (N.H.); Graduate School of Agriculture, Kyoto University, Kyoto 606–8502, Japan (C.M., H.O.); College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599–8531, Japan (D.O.)
Abscisic acid (ABA) is involved in a number of critical processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, a physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. The hydroxylation at the 8'-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 8'-hydroxylation of (+)-ABA. First, all four CYP707A cDNAs were cloned from Arabidopsis and used for the production of the recombinant proteins in insect cells using a baculovirus system. The insect cells expressing CYP707A3 efficiently metabolized (+)-ABA to yield phaseic acid, the isomerized form of 8'-hydroxy-ABA. The microsomes from the insect cells exhibited very strong activity of 8'-hydroxylation of (+)-ABA (Km = 1.3 µM and kcat = 15 min–1). The solubilized CYP707A3 protein bound (+)-ABA with the binding constant Ks = 3.5 µM, but did not bind (–)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 8'-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 8'-hydroxylase. The transcripts of the CYP707A genes increased in response to salt, osmotic, and dehydration stresses as well as ABA. These results establish that the CYP707A family plays a key role in regulating the ABA level through the 8'-hydroxylation of (+)-ABA.
The plant hormone abscisic acid (ABA) regulates many important physiological and developmental processes in plants as well as adaptive responses to environmental stresses (Zeevaart and Creelman, 1988  ). During seed development and dormancy, ABA content increases as a potential signal to organize expression of many embryo-specific genes. The ABA level is also elevated in response to various environmental stresses such as drought, high salinity, and low temperature conditions. These physiological processes controlled by ABA are primarily regulated by bioactive ABA pool size, which is thought to be maintained through fine-tuning the rates of de novo biosynthesis and catabolism. Thus, to understand the molecular mechanism that controls the ABA contents in plant tissues, the genes and enzymes in biosynthesis and catabolism of ABA must be studied in detail.
ABA is a sesquiterpene, and the first committed step in ABA biosynthesis is the oxidative cleavage of a 9-cis-epoxycarotenoid (C40) to form a xanthoxal (C15). The xanthoxal is oxidized to form abscisic aldehyde, and then further oxidized to ABA. To date, most of the genes in the ABA biosynthetic pathway have been isolated through identification and characterization of ABA deficient mutants from several plant species (Schwartz et al., 2003
In contrast to ABA biosynthesis, the genes and enzymes in ABA catabolism as well as their regulation remain largely unknown (Cutler and Krochko, 1999
Complete sequencing of Arabidopsis genome has revealed 246 full-length P450 genes distributed into 45 families (http://drnelson.utmem.edu/Arablinks.html). Arabidopsis P450s are divided into two main classes of A-type and non-A-type genes (Durst and Nielsen, 1995 ; Schuler and Werck-Reichhart, 2003). About 60% of Arabidopsis P450s are grouped as the A-type and are primarily involved in unique plant biosynthetic pathways of various secondary metabolites such as phenylpropanoids, terpenoids, glucosinolates, and phytoalexins. On the other hand, the non-A-type P450s (Fig. 2
) show similarities to P450s from other kingdoms (mammal, yeast, fungi, etc.) and often represent more fundamental enzymes involved in biosynthesis of sterols, oxygenated fatty acid, and phytohormones.
Aiming at elucidating P450s involved in essential reactions for plant growth, we have been focusing on biochemical characterization of P450 genes in the 85-clan within the non-A type P450s. The 85-clan (Fig. 2, circled), one of the clades in the non-A-type P450s, contains several P450s (CYP85, 88, and 90s) involved in brassinosteroid (BR) and gibberellin (GA) biosynthesis (Szekeres et al., 1996 ; Choe et al., 1998; Helliwell et al., 2001; Shimada et al., 2001). Recently, rice (Oryza sativa) genome has been sequenced completely, and classification of the P450 gene family in rice genome has also been completed (http://drnelson.utmem.edu/ricefams.html). In the 85-clan of rice P450s, there are no P450 genes belonging to the CYP702, 708, 716, 718, and 720 families found in Arabidopsis. In other words, it is possible that these P450s may function in metabolic pathways unique to Arabidopsis. Among the 85-clan P450s overlapping between Arabidopsis and rice, we selected CYP707A as the first target for our biochemical approach to identify the P450 function, because the CYP707A family is most closely related to CYP88As encoding ent-kaurenoic acid hydroxylase in the early steps of GA biosynthesis. The CYP707A family in Arabidopsis consists of four genes, implicating the possibility that the physiological function of this family may not be characterized by standard mutant screening due to their functional redundancy. Thus, biochemical characterization of the CYP707A family is crucial for identifying their enzymatic and physiological functions. In this study, we isolated the cDNAs of four CYP707A genes in Arabidopsis and characterized the biochemical properties of the recombinant CYP707A proteins expressed in the baculovirus system as well as the expression patterns of the CYP707A genes in vivo. The results have demonstrated that Arabidopsis CYP707As encode (+)-ABA 8'-hydroxylase.
Isolation of CYP707A1 and CYP707A3 cDNAs Several P450 genes in the 85-clan of the non-A-type P450s have already been shown to function in phytohormone biosynthesis, and therefore we expected that other 85-clan members might be involved in metabolic pathways related to plant bioactive compounds. During the course of our systematic biochemical characterization of the 85-clan P450s, we investigated Arabidopsis CYP707A family members consisting of four genes (CYP707A1, A2, A3, and A4). First, we isolated the full-length cDNAs for CYP707A1 and CYP707A3 from a cDNA library of Arabidopsis seedlings. The CYP707A1 protein sequence is 87% identical to CYP707A3, while it is only about 60% identical to CYP707A2 and CYP707A4. A BLAST search with CYP707A1 and CYP707A3 revealed that these P450s show the highest identities (35%) to Arabidopsis CYP88A3 and CYP88A4 of ent-kaurenoic acid hydroxylase in the GA biosynthetic pathway, and 30% to 35% identities to CYP85A1, CYP90A1, CYP90B1, and CYP90C1, which are involved in BR biosynthesis (Fig. 2). In rice genome, two CYP707A genes (CYP707A5 and CYP707A6) have been identified (http://drnelson.utmem.edu/biblioD.html). The scanning of the CYP707A ESTs available in the TIGR database (http://www.tigr.org/tdb/tgi/plant.shtml) also indicated that the CYP707A-like genes occur in a variety of monocotyledonous and dicotyledonous plants. These results suggested that the CYP707A family functions in a fundamental metabolism common among higher plants.
CYP707As also share about 30% identities to CYP725A1, A2, and A3 from Taxus cuspidate, which are also in the 85-clan and involved in the biosynthesis of a diterpenoid, taxol (Jennewein et al., 2001
Plant hormones such as GA and BR negatively regulate their own biosynthetic genes and positively regulate their own catabolic genes in feed-back and feed-forward mechanisms, respectively. It should be noted that ABA is inactivated through the hydroxylation of the 8'-methyl group by a P450 (Krochko et al., 1998
Heterologous Expression in Insect Cells In order to characterize the enzymatic properties of the CYP707A family, we produced the recombinant CYP707A1 and CYP707A3 proteins in insect cells using a baculovirus system. SDS-PAGE analysis showed that the new intense bands of 53 and 52 kD appeared in the microsomal fractions of the insect cells upon infection with the recombinant viruses of CYP707A1 and CYP707A3, respectively (Fig. 4A ). The microsomes were solubilized with 1% (w/v) Emulgen 913, and the resultant solubilized fractions were analyzed for P450 contents. The reduced-CO difference spectrum of the solubilized CYP707A1 fractions showed two Soret absorption peaks at 420 and 450 nm (Fig. 4B), indicating that the recombinant CYP707A1 would be easily inactivated. On the other hand, the solubilized CYP707A3 fractions showed a clear absorption peak at 450 nm (Fig. 4C). The solubilized fractions from the mock-infected cells did not give the 450-nm peak (data not shown). Thus, a part of the recombinant CYP707A1 and most of the recombinant CYP707A3 were expressed in insect cells as active forms.
ABA 8'-Hydroxylase Activity
We studied ABA 8'-hydroxylase activity using the insect cell culture by adding (+)-ABA directly into the culture medium. The insect cells expressing either CYP707A1 or CYP707A3 were incubated in the presence of 200 µM (+)-ABA for 24 h at 27°C. HPLC analyses revealed that the ABA concentration (the peak at 12.1 min) was greatly decreased in the culture medium from the cells expressing CYP707A3 (Fig. 5
). On the other hand, two new compounds, 1 and 2, were detected in the same analysis at 5.8 and 7.1 min, respectively, with a ratio of 98:2. These new peaks were identical in the retention times to authentic standards of PA (5.8 min) and 8'-OH-ABA (7.1 min), respectively. The assay for CYP707A1 also gave the similar results (Fig. 5). Compounds 1 and 2 were also detected inside the cells, but the amounts were much lower than those from the medium (data not shown). Compounds 1 and 2 were not in the assays of either the mock-infected Sf9 cells (Fig. 5) or cells expressing the Arabidopsis CYP73A5 cDNA encoding a trans-cinnamic acid 4-hydroxylase (Mizutani et al., 1997
To identify compounds 1 and 2, we carried out a large-scale expression of the recombinant CYP707A3 protein. The suspension-cultured Sf9 cells (60 mL) expressing the CYP707A3 protein were incubated with 200 µM (+)-ABA for 24 h, and compounds 1 and 2 were collected by preparative HPLC of the extract from the culture medium. Compound 1 was identified as (–)-PA by comparison of its spectral data with those of an authentic sample (data are presented in "Materials and Methods"). In this assay, 36% of the applied ABA (3.17 mg) was converted to PA (1.15 mg). Most of the formed PA was secreted into the medium, and only 4% (48 µg) of the formed PA was found within the cells. The collected material corresponding to compound 2 was a mixture of compounds 1 and 2 with a ratio 40:60 on HPLC analysis, showing that compound 2 partially isomerized to PA during isolation. The 1H NMR data of the mixture indicated the signals assignable to 8'-OH-ABA in addition to PA, and the mass spectrum of methyl ester of the mixture coincided with that of the methyl ester of PA. These data identified compound 2 as 8'-OH-ABA. PA and 8'-OH-ABA are in equilibrium with a final ratio 98:2 at 25°C (Todoroki et al., 2000 ), strongly suggesting that PA in the extracts from the reaction mixtures was formed from 8'-OH-ABA by spontaneous isomerization during extraction and preservation. This meant that CYP707A3 possesses at least the activity of (+)-ABA 8'-hydroxylase, although it was unclear whether CYP707A3 possesses the activity of the 8'-OH-ABA isomerization as well. To examine whether the other two members of the Arabidopsis CYP707A family also have the same catalytic activity, the cDNAs for CYP707A2 and CYP707A4 were isolated by RT-PCR, and the recombinant protein of either CYP707A2 or CYP707A4 was expressed in insect cells. The cells expressing CYP707A4 were able to convert (+)-ABA to PA, while the cells expressing CYP707A2 did not metabolize (+)-ABA to PA (data not shown). This was probably ascribed to the instability of the recombinant CYP707A2 protein, which was found in spectral analysis only as the inactive form with the 420-nm Soret absorption peak (data not shown).
Enzymatic properties were studied using the microsomal fractions from the insect cells expressing CYP707A3. The CYP707A3 microsomes were incubated with 100 µM (+)-ABA and 100 µM NADPH at 30°C for 10 min, and the reaction products were analyzed by HPLC (Table I). The CYP707A3 microsomes produced PA, indicating that endogenous NADPH-P450 reductase in the insect cells was able to support the electron transfer to the CYP707A3 protein to some extent. When the recombinant Arabidopsis NADPH-P450 reductase (AR1; Mizutani and Ohta, 1998
Substrate Binding
Most P450 enzymes are present in a low-spin state in the absence of substrate. Upon substrate binding, the heme electronic state is modified, and the heme iron spin state shifts from low-spin state to high-spin state. The recombinant CYP707A3 was solubilized with 1% (w/v) sodium cholate and used for substrate binding experiments with (+)-ABA and (–)-ABA at a final concentration of 100 µM. (+)-ABA produced a Type I difference spectrum with a maximum at 386 nm and a minimum at 419 nm (Fig. 6A
). The amplitude of this difference was proportional to increasing amounts of (+)-ABA, and the binding constant was determined to be the Ks value of 3.5 ± 0.1 µM (Fig. 6B). This value is closely similar to that of the mung bean cinnamate 4-hydroxylase with trans-cinnamic acid (Ks = 2.8 µM; Mizutani et al., 1993
Characteristics of the Reactions
Kinetic parameters of the CYP707A3 reaction were determined using the CYP707A3 microsomes. The Km value for (+)-ABA was estimated to be 1.3 ± 0.3 µM, indicating a high affinity of CYP707A3 for (+)-ABA. This value was comparable to the binding constant Ks determined above. Cutler et al. (2000)
Although (+)-7'-hydroxy-ABA has been shown to be produced as a minor metabolite in some plant species (Lehmann and Schwenen, 1988
In the cell assays of the CYP707A enzymes, overnight culture with ABA resulted in the detection of PA as a major product (98%) and only a very small amount of 8'-OH-ABA (2%; Fig. 5). It was not clear whether the detected PA was formed by either spontaneous isomerization of 8'-OH-ABA or CYP707A enzyme-dependent isomerization. At the final equilibrium at 25°C, the PA to 8'-OH-ABA ratio should be 98:2 (Todoroki et al., 2000
Expression Patterns of CYP707A Family Genes in Arabidopsis Firstly, we determined the expression profiles of the CYP707A genes in various tissues of Arabidopsis by RT-PCR (Fig. 8 ). The expression of the CYP707A genes was ubiquitous in various organs with different transcript accumulation levels. The PCR amplification with 31 cycles gave clear amplified bands for the CYP707A1 and CYP707A3 transcripts, while the number of PCR cycles had to be increased to 35 to obtain the signals for CYP707A2 and CYP707A4 gene expression, indicating that overall expression levels of CYP707A1 and CYP707A3 are much higher than those of CYP707A2 and CYP707A4. Flower buds and flowers were relatively abundant in the transcripts of all CYP707As compared with the other tissues, while mature siliques showed lower expression levels of CYP707As. In rosette leaves, the CYP707A3 mRNA was the major transcript, while CYP707A2 was abundantly expressed in inflorescence stems. In roots, CYP707A1 and CYP707A3 were moderately expressed, whereas weak expression of CYP707A2 and no expression of CYP707A4 were observed. These results indicated that the expression of the CYP707A genes is differently regulated in each organ.
It is known that environmental stresses such as high salinity and drought stresses induce the accumulation of ABA in various plant species. We studied how the expression of the CYP707A genes might respond to salt, osmotic, and drought stresses by semiquantitative RT-PCR (Fig. 9 ). When liquid-cultured Arabidopsis seedlings were stressed by adding 250 mM NaCl, strong induction of CYP707A1 and CYP707A4 expression and moderate increase of the CYP707A2 and CYP707A3 transcript levels were observed (Fig. 9A). The osmotic stress by 400 mM mannitol also had similar effects on the expression of the CYP707A genes (Fig. 9A). The expression patterns of the CYP707A genes in response to these stresses were quite similar to those in response to ABA. On the other hand, the expression patterns of the CYP707A genes under the drought stress were relatively complicated (Fig. 9B). CYP707A3 was rapidly induced within 30 min of the drought stress and the induced expression level was retained thereafter. The expression of CYP707A1 also increased in 30 min after the stress but reduced to a basal level at 1 h. The CYP707A1 transcript level increased again 3 h after treatment. CYP707A4 gene expression was slowly induced 3 h after treatment, while CYP707A2 expression was reduced at the early stages (30 min and 1 h) of the stress and returned to a basal level 3 h after treatment.
CYP707A Family Encodes ABA 8'-Hydroxylase
In this study, we have cloned the cDNAs of all four genes in the Arabidopsis CYP707A family, and CYP707A1, A3, and A4 have been functionally expressed in the baculovirus system. The enzymatic characterization of these recombinant P450s provides convincing evidence that the CYP707A family encodes (+)-ABA 8'-hydroxylase. First, CYP707A3 catalyzed hydroxylation at C-8' methyl group of (+)-ABA but not at C-7' position. Second, CYP707A3 bound (+)-ABA with a high affinity (Ks = 3.5 µM) but did not bind and hydroxylate (–)-ABA. Also, we demonstrated that CYP707A3 catalyzed the 8'-hydroxylation of (+)-ABA with a high affinity and efficiency (Km = 1.3 µM and kcat = 15 min–1). These enzymatic properties of CYP707A3 are quite consistent with those of ABA 8'-hydroxylase activity found in the microsomes from suspension-cultured maize cells (Krochko et al., 1998
We have also investigated whether PA was produced by either spontaneous isomerization of 8'-OH-ABA or CYP707A enzyme-dependent isomerization (Fig. 7). The isomerization of 8'-OH-ABA to PA is a cyclization reaction triggered by the intramolecular conjugated nucleophilic addition of 8'-hydroxy group oxygen into the electron-deficient enone at C-2' position. This reaction is likely promoted by deprotonation of the 8'-hydroxy group, and the reaction rate can be affected by the pH of the medium. At 25°C, the PA to 8'-OH-ABA ratio was 98:2 at the final equilibrium, and the half-life of 8'-OH-ABA was determined to be 30 h at pH 3, 4 h at pH 7, and shorter than 1 min at pH 10 (Todoroki et al., 2000
Several T-DNA-inserted knockout lines for CYP707A1, A2, and A3 genes are found at the Arabidopsis Biological Resource Center (ABRC; Alonso et al., 2003
It has been reported that ABA negatively regulates ABA accumulation by enhancing the ABA 8'-hydroxylase level (Cutler and Krochko, 1999
This raises the question regarding what signals activate the CYP707 genes in response to environmental stresses such as high salinity, osmotic, and dehydration stresses. These stresses may be direct signals for CYP707A induction. It is also possible that the ABA level elevated by the stresses may indirectly induce CYP707A gene expression. To address the question, it should be investigated how CYP707A gene expression could be induced in ABA deficient and insensitive mutants. In the ABA insensitive mutant abi1-1 seedlings, induction of CYP707A1 and CYP707A3 as well as many ABA responsive genes in response to ABA treatment was abolished (Hoth et al., 2002
The conversion of ABA to DPA is the primary inactivation pathway of ABA. 8'-OH-ABA still has biological activity similar to that of ABA in stimulating biosynthesis of a very long chain of fatty acids in Brassica napus embryos (Zou et al., 1995
Instruments Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker (Billerica, MA) ARX500 instrument (500 MHz for 1H) at 300 K, using tetramethylsilane as the internal standard. Electron impact ionization mass spectrometry (EIMS) measurements were carried out with a JEOL JMS-600H mass spectrometer set at an electron potential of 70 eV, the temperature of the direct probe being increased from 30°C to 450°C at a rate of 128°C/min. UV spectra and optical rotation were measured with a UV 2200AI instrument (Shimadzu, Kyoto) and a DIP-1000 polarimeter (JASCO, Tokyo), respectively. The other all spectrophotometric determinations were carried out at room temperature with a UV-3101 spectrophotometer (Shimadzu).
(±)-ABA and (+)-ABA were purchased from Wako Pure Chemical Industries (Osaka) and BAL Planning Co. (Ichinomiya, Aichi, Japan), respectively. (–)-ABA was prepared by optical resolution of (±)-ABA (10.5 mg) by HPLC: column, Daicel Chiralcel OD (Osaka; 250-mm length x 4.6 mm i.d.); solvent, i-propanol-n-hexane (20:80) containing 0.1% (v/v) acetic acid; flow rate, 1.0 mL/min; detection at 254 nm. (+)-ABA and (–)-ABA were eluted at a retention time of 7.5 and 8.6 min, respectively, and the latter was collected to give 4.5 mg of colorless solids. For preparation of PA,
Arabidopsis ecotype Columbia (Col) was grown under continuous light conditions at 22°C. Various organs such as buds, flowers, mature siliques, rosette leaves and inflorescence stems were obtained from nonsterilized plants grown in soil for 4 weeks. To isolate total RNA from roots, Arabidopsis seedlings were grown for 2 weeks under a sterile condition on 0.8% (w/v) agar plates containing GM medium (Valvekens et al., 1988
A cDNA library of Arabidopsis was prepared using total RNA from 7-d-old seedlings treated with 1 µM uniconazole for 3 d, using Superscript Lambda system (Invitrogen, Carlsbad, CA). A DNA fragment of the CYP707A1 gene was amplified from genomic DNA of Arabidopsis by PCR with a set of primers; 707A1N: 5'-ATGGATATCTCCGCCTTGTTTCTC-3', and 707A1C: 5'-TTCTGTCATTCTACACTTCGATCT-3'. The PCR fragments were gel-purified, labeled with an AlkPhos direct labeling system (Amersham Biosciences, Piscataway, NJ), and used for the probe to isolate the CYP707A cDNAs. The cDNA library (200,000 plaques) was screened, and nine positive clones were isolated. The pZL1 plasmids containing the cDNA inserts were excised by in vivo excision according to the manufacturer's instruction, and the partial cDNA sequences were determined. Two of nine positive clones were found to be full-length CYP707A1 cDNAs, and the other seven clones were full-length CYP707A3 cDNAs. The CYP707A1 and CYP707A3 cDNAs were completely sequenced (DDBJ accession numbers, AB122149 and AB122150, respectively).
The cDNA containing an entire open-reading frame of either CYP707A2 or CYP707A4 was amplified by RT-PCR. Nucleotide sequences of gene-specific primers were as follows; CYP707A2: 5'-GGATCCATGCAAATCTCATCTTCATCGTCTTCAAATTTC-3' (BamHI site in italic and start codon is underlined) and 5'-GTCGACTTAGGCTTAAATCGGGGTTACTCTTATTGG-3' (SalI site in italic and stop codon is underlined); CYP707A4: 5'-GGATCCATGGCTGAAATTTGGTTCTTGGTTGTACCA-3' (BamHI site in italic and start codon is underlined) and 5'-CTCGAGCTAAAGAGAATGTCGACGAAATGTAGCGGG-3' (XhoI site in italic and stop codon is underlined). First-strand cDNA was synthesized with an oligo(dT) primer and a ReverTra Ace reverse transcriptase (Toyobo, Osaka) in a 10-µL reaction mixture containing 0.6 µg of total RNA from (±)-ABA treated Arabidopsis seedlings, and the RT reactions were carried out at 42°C for 30 min, 99°C for 5 min, and chilled to 5°C for 5 min. One µL of the reaction products was used as a template for the PCR reaction in a 10-µL reaction mixture containing 2.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, 0.25 unit of KOD-plus Taq DNA polymerase (Toyobo), 2 mM of dNTP, and 0.5 µM of gene-specific primers described above. Addition of 3'-A-overhangs was done by incubation of the PCR reaction with 1 unit of Taq DNA polymerase at 72°C for 10 min. The PCR product was gel-purified and cloned into the TA cloning vector pCR2.1 using a TOPO TA cloning kit (Invitrogen). The cloned inserts were sequenced to confirm no PCR errors in the inserts.
The full-length cDNAs of CYP707A1 and CYP707A3 in pZL1 plasmid vector were excised with the restriction enzymes, SalI and NotI, and were purified by 1% (w/v) agarose gel electrophoresis. The cDNAs of CYP707A2 and CYP707A4 in pCR2.1 were digested with restriction enzymes, BamHI and SalI for CYP707A2, and BamHI and XhoI for CYP707A4, and agarose gel-purified. These cDNAs were then ligated into pFastBac1 vector (Invitrogen) digested with the same sets of the restriction enzymes. The pFastBac1-CYP707A constructs were used for the preparation of recombinant Bacmid DNA by transformation of Escherichia coli strain DH10Bac (Invitrogen). Spodoptera furugiperda 9 (Sf9) cells were maintained at 27°C as a monolayer culture in Grace's insect cell medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum. For large-scale expression, Sf9 cells were propagated as suspension cultures in Grace's insect medium containing 0.1% (w/v) Pluronic F-68 (Invitrogen), and incubated in a rotary shaker at 27°C and 150 rpm. For expression of recombinant CYP707A proteins, Sf9 cells were cultured in the above Grace's insect medium supplemented with 100 µM 5-aminolevulinic acid and 100 µM ferrous citrate to compensate for the low heme synthetic capacity of the insect cells.
The suspension cultures of Sf9 insect cells were infected with the recombinant baculovirus each containing CYP707A 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 cells and the culture medium were separately collected by centrifugation at 1,000g for 5 min and were 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 subjected to HPLC: column, YMC AQ-311 (Kyoto; ODS, 100-mm length x 6 mm i.d.); solvent, 45% (v/v) methanol in water containing 0.1% (v/v) acetic acid; flow rate, 1.0 mL/min; detection at 254 nm. Retention times of authentic samples were 5.8 min for PA, 9.6 min for 2E-ABA, and 12.1 min for ABA. The detection limit for these three compounds was 5 ng.
For preparation of microsomes, the cells (250 mL of suspension-cultured cells) expressing either CYP707A1 or CYP707A3 were washed with PBS and suspended in buffer A consisting of 20 mM potassium phosphate (pH 7.25), 20% (w/v) glycerol, 1 mM EDTA, and 1 mM dithiothreitol. The cells were sonicated, and cell debris was removed by centrifugation at 10,000g for 15 min. The supernatant was further centrifuged at 100,000g for 1 h, and the pellet was homogenized with buffer A to provide microsomal fractions. The complete reaction mixture (2 mL) contained 50 mM potassium phosphate (pH 7.25), 50 µg/mL recombinant CYP707A3 microsomes, 100 µM NADPH, and 100 µM (+)-ABA. Reactions were initiated by the addition of NADPH and carried out at 30°C for 10 min. After stopping the reactions by adding 200 µL of 1 N HCl, the reaction products were extracted four times with an equal volume of ethyl acetate. The organic layers were combined, washed with a small amount of water twice, dried over Na2SO4, filtered, and concentrated to give ethyl acetate-soluble materials. The materials were dissolved in 50 µL of methanol, and 5 µL of the sample was subjected to HPLC in the same conditions as those described in "Cell Assay." For kinetic analysis, CYP707A3 was assayed using the microsomes (25 µg/mL) and (+)-ABA at concentrations ranging from 1 to 32 µM. Reactions were initiated by the addition of NADPH and carried out at 30°C for 10 min. The kinetic constants were calculated from triplicated data sets. Km and Vmax were calculated from a double reciprocal plot of the initial velocity (v0) versus substrate concentration.
The reaction mixture (100 µL) contained 50 mM potassium phosphate (pH 7.25), 2 mg/mL recombinant CYP707A3 microsomes, 200 µM NADPH, and 200 µM (+)-ABA. Reactions were carried out at 30°C and stopped by acidification to pH 2 with 5 µL of 1 N HCl. The reaction mixtures were briefly centrifugated at 3,000g, and a portion of each reaction mixture (10 µL) was directly subjected to HPLC in the same conditions as those described in "Cell Assay."
Sf9 cells expressing CYP707A3 were cultured in 60 mL of a medium containing (+)-ABA (3.17 mg) at 27°C for 24 h in the darkness. The cell suspension was centrifuged at 1,000g for 10 min. The supernatant was acidified to pH 2 with 1 N HCl and partitioned with 30 mL of ethyl acetate four times. The organic layers were combined, washed with a small amount of water, dried over Na2SO4, filtered, and concentrated to give solids (7.9 mg). The solids dissolved in 1.2 mL of methanol were subjected to preparative HPLC in the same conditions as those described in "Cell Assay." The materials eluted at the retention times 5.8 min and 7.1 min were collected separately and concentrated to give compounds 1 (1.15 mg) and 2 (48 µg), respectively. Compound 1 (PA):
P450 was estimated from the CO-difference spectrum using an extinction coefficient (
The microsomes from the cells expressing the recombinant CYP707A3 were solubilized with 1% (w/v) sodium cholate for 1 h, and centrifugated at 100,000g for 1 h. The supernatant was used as the solubilized recombinant CYP707A3. Either (+)-ABA or (–)-ABA was added to the solubilized CYP707A3 at a final concentration of 100 µM, and type I difference spectra were recorded between 500 and 350 nm. For the reference, dimethylsulfoxide, which is the solvent for 20 mM ABA stock solution, was added at a final concentration of 1% (v/v). For spectrophotometric titration, (+)-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 (Ks) was calculated from a double reciprocal plot of absorbance difference
Total RNA from Arabidopsis plants was isolated with a MagExtractor-RNA (Toyobo). RT-PCR was performed using a ReverTra Dash RT-PCR kit (Toyobo) according to the manufacturer's instruction. First-strand cDNA was synthesized in a 10-µL reaction mixture containing 0.6 µg of total RNA with an oligo(dT) primer and a ReverTra Ace reverse transcriptase (Toyobo), and the RT reactions were carried out at 42°C for 30 min, 99°C for 5 min, and chilling to 5°C for 5 min. The reaction mixture was diluted 2 times, and 0.5 µL of aliquot was used as a template for each of the PCR amplifications. The PCR reaction was carried out in a 10-µL reaction mixture containing 2.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, 0.25 unit of KOD dash Taq DNA polymelase (Toyobo), 2 mM of dNTP, and 0.5 µM of each gene-specific primer as shown below. Nucleotide sequences of gene-specific primers were as follows; CYP707A1, 707A1-F: 5'-CTTTTTCCAATCAAAACAGAAAAGGTATGG-3' and 707A1-R: 5'-TCGCTCCAACAATTGACCAACTGTACTTGG-3'; CYP707A2, 707A2-F: 5'-CACTTTTTCATAAGTCCATGAAGGCAAGAA-3' and 707A2-R: 5'-TACGTGTAAGGTTTTGGTGCCACCTCGAAT-3'; CYP707A3, 707A3-F: 5'-TGTTCTTTGCAGCAAAACAGAGAAGATACG-3' and 707A3-R: 5'-TAGGCCCTACGATTGACCATCTGTACTTAG-3'; CYP707A4, 707A4-F: 5'-CCTACCAGGAGATGAAGAAGTTCGCCTTTG-3' and 707A4-R: 5'-CTCCTCCCTTCACTTCCCATCGGAAATTGG-3'; Actin2, act2-F: 5'-GTGAAGGCTGGATTTGCAGGA-3' and act2-R: 5'-AACCTCCGATCCAGACACTGT-3'. The RT sample was first denatured at 94°C for 2 min, and the PCR amplification was performed in following conditions; CYP707A1 and CYP707A3: 29, 31, 33, and 35 cycles of 94°C for 30 s, 54°C for 2 s, and 74°C for 45 s; CYP707A2: 31, 33, 35, and 37 cycles of 94°C for 30 s, 60°C for 2 s, and 74°C for 45 s; CYP707A4: 31, 33, 35, and 37 cycles of 94°C for 30 s, 54°C for 2 s, and 74°C for 45 s; and Actin2: 19, 21, 23, and 25 cycles of 94°C for 30 s, 54°C for 2 s, and 74°C for 45 s by using GeneAmp PCR system 9700 (Applied Biosystems, Norwalk, CT). The PCR products were analyzed with a 1% (w/v) agarose gel containing 20 ng/mL of ethydium bromide. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AB122149 and AB122150. Received December 12, 2003; returned for revision February 2, 2004; accepted February 6, 2004.
1 This work was supported by a part of the R&D Project of Industrial Science and Technology Frontier Program supported by NEDO (New Energy and Industrial Technology Development Organization), Japan (to D.O.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037614. * Corresponding author; e-mail mizutani{at}scl.kyoto-u.ac.jp; fax 81–774–38–3229.
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ABSTRACT
RESULTS
A(386–419 nm) versus the substrate concentration (inset).
-hydroxy-
–30° (c 0.056, methanol); UV 
max nm (methanol): 256 (
18,400); 1H-NMR (500 MHz, CD3OD): 
1.01 (3H, s, H-9'), 1.21 (3H, s, H-7'), 2.04 (3H, d, J = 1.2 Hz, H-6), 2.38 (1H, dd, J = 18.1 and 2.6 Hz, H-5'proR), 2.45 (1H, dd, J = 18.0 and 2.6 Hz, H-3'proS), 2.70 (1H, dd, J = 18.1 and 2.6 Hz, H-5'proS), 2.80 (1H, d, J = 18.0 Hz, H-3'proR), 3.66 (1H, d, J = 7.6 Hz, H-8'proS), 3.94 (1H, dd, J = 7.6 and 2.9 Hz, H-8'proR), 5.80 (1H, brs, H-2), 6.40 (1H, d, J = 15.9 Hz, H-5), 8.06 (1H, d, J = 15.9 Hz, H-4). Compound 1 (0.89 mg) was methylated with ethereal diazomethane to give its methyl ester. EIMS (probe) m/z (relative intensity): 294 [M]+ (24), 276 [M-H2O]+ (18), 263 (12), 244 (13), 233 (9), 217 (11), 177 (22), 167 (30), 163 (31), 154 (31), 139 (37), 135 (39), 125 (100), 122 (68). Compound 2 (a mixture of PA and 8'-OH-ABA): 1H-NMR (500 MHz, CD3OD): signals corresponding to 8'-OH-ABA; 
1.08 (3H, s, H-9'), 1.92 (3H, s, H-7'), 1.93 (3H, s, 6-H), 2.37 (1H, d, J = 17.1 Hz, H-5'proR), 2.48 (1H, d, J = 17.1 Hz, H-5'proS), 3.70 (1H, d, J = 11.0 Hz, H-8'proS), 5.83 (1H, brs, H-2), 5.90 (1H, s, H-3'), 5.93 (1H, d, J = 16.1 Hz, H-5), 7.58 (1H, d, J = 16.1 Hz, H-4), and the signal of H-8'proR overlapped with solvent signals; the other signals coincided with those of compound 1. Compound 2 (28 µg) was methylated with ethereal diazomethane to give its methyl ester. EIMS (probe) m/z (relative intensity): 294 [M]+ (20), 276 [M-H2O]+ (22), 244 (12), 233 (11), 217 (13), 177 (25), 167 (36), 163 (33), 154 (31), 139 (39), 135 (41), 125 (100), 122 (76). 
= 91 mM–1 cm–1; Omura and Sato, 1964
-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10: 231–243
