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 89-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 89-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 89-hydroxylation of (1)-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 (1)-ABA to yield phaseic acid, the isomerized form of 89-hydroxy-ABA. The microsomes from the insect cells exhibited very strong activity of 89-hydroxylation of (1)-ABA (K m ¼ 1.3 mM and k cat ¼ 15 min ÿ1 ). The solubilized CYP707A3 protein bound (1)-ABA with the binding constant K s ¼ 3.5 mM, but did not bind (ÿ)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 89-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 89-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 89-hydroxylation of (1)-ABA.

Plant cytochromes P450 (P450s) catalyze a wide variety of monooxygenation/hydroxylation reactions in primary and secondary metabolism. The number of P450 genes in plant genomes is estimated to be up to 1% of total gene annotations of each plant species. This implies that diversification within P450 gene superfamilies has led to the emergence of new metabolic pathways throughout land plant evolution. The conserved P450 families contribute to chemical defense mechanisms under terrestrial conditions and several are involved in hormone biosynthesis and catabolism. Species-specific P450 families are essential for the biosynthetic pathways of species-specialized metabolites. Future genome-wide analyses of P450 gene clusters and coexpression networks should help both in identifying the functions of many orphan P450s and in understanding the evolution of this versatile group of enzymes.

D22-Unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with b-sitosterol to produce stigmasterol (CYP710A1, K m ¼ 1.0 mM and kinetic constant [k cat ] ¼ 0.53 min ÿ1 ; CYP710A11, K m ¼ 3.7 mM and k cat ¼ 10 min ÿ1 ). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6-to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and b-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:b-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the D 22 -unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.

We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis tha/iana using a C4H cDNA from mung bean as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-' nmol-' P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PALl and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PALZ and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression.

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