Plant Physiol. (1991) 96, 669-674 Received for publication December 27, 1990 0032-0889/91 /96/0669/06/$01 .00/0 Accepted March 11, 1991

Review Multiple Forms of Plant Cytochromes P-4501

Robert P. Donaldson* and Douglas G. Luster Department of Biological Sciences, The George Washington University, Washington, D.C. 20052 (R.P.D.), and U.S. Department of Agriculture-Agricultural Research Service, Foreign Disease-Weed Science Research, Ft. Detrick, Frederick, Maryland 21702 (D.G.L.)

ABSTRACT produce only small quantities of end products (e.g. gibberel- lins), and thus the level of activity of each Cyt P-450 enzyme Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases may be quite low. Also, the products of Cyt P-450 in plants are implicated in the metabolism of sterols, terpenes, gibberellins, are often hydrophobic. Many specific Cyt P-450 are expressed isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 only under certain circumstances (e.g. pathogenesis) and may are involved in the detoxification of herbicides (chlorotoluron, only be expressed in a few cell types (e.g. epidermal glands of primsulfuron, and diclofop) includes photoreversible CO inhibition mints). The Cyt P-450 hemoproteins are membrane bound of the reactions, and a requirement for 02 and NADPH. Several in eukaryotes and are often inactivated by proteolysis and cytochromes P-450, Mr 45,000 to 65,000, have been isolated, oxygen. For all these reasons, Cyt P-450 have been difficult including hydroxylases of cinnamic acid, 3,9-dihydroxypterocar- to isolate and characterize. However, recently several investi- pan, and digitoxin. In some cases the purified cytochrome P-450 gators have succeeded in purifying some distinct Cyt P-450. has been successfully reconstituted with NADPH:cytochrome P- Immunological techniques have been used to characterize 450 reductase (Mr 72,000-84,000 protein). This reductase ap- pears to be a nonspecific electron donor to different forms of some Cyt P-450 in plants and a cDNA for a putative plant cytochrome P-450. Immunological techniques and specific inhib- Cyt P-450 has been sequenced. itors (, derivatives) are being used to charac- A comprehensive review of plant Cyt P-450 was published terize plant cytochromes P-450 and the NADPH:cytochrome P- in 1980 (33). In this review we will summarize selected recent 450 reductase. Specific cytochromes P-450 are induced by findings which exemplify the current understanding of plant wounding or pathogens, others are expressed in specific cell Cyt P-450. These findings will be compared with what is types. Plant cytochromes P-450 are found in various subcellular known about Cyt P-450 in animal tissues. In the process we locations, including endoplasmic reticulum, plasma membranes, will examine some of the questions which remain regarding glyoxysomes, and perhaps mitochondria. A cytochrome P-450 plant Cyt P-450 structural features and functions. demethylase from avocado has recently been sequenced and found to have a hydrophobic N terminus similar to the membrane anchor of cytochromes P-450 from other organisms. The exist- Cyt P-450 ARE CHARACTERIZED BY SPECTROSCOPY ence of cytochromes P-450 in different subcellular locations AND SPECIFIC INHIBITION suggests that there are many genes for cytochromes P-450 in Cyt P-450 are hemoproteins in the range of Mr 50,000 in plants which have yet to be identified and classified. which the heme is available for 02 or CO binding. The binding of CO to a reduced Cyt P-450 heme moiety shifts the Soret absorption maximum to 450 nm and blocks further redox activity. The extinction coefficient of the hemoprotein-CO adduct is about 91 mM-' (25). Exposure to light (450 nm Cyt P-450 are membrane-bound heme-containing proteins action maximum) causes the CO to be displaced from the which have been implicated in a variety of oxidative reactions heme; thus CO binding is photoreversible. For this reason, in plant tissues. A wide range of reactions are mediated by Cyt P-450 enzymes exhibit photoreversible inhibition by CO. specific forms of these proteins. Cyt P-450-linked enzymes The 450 nm CO-hemoprotein adduct is often accompanied have been implicated in biosynthetic pathways leading to the by a peak at 420 nm, and solubilization with detergents and/ synthesis oflignin phenolics, membrane sterols, phytoalexins, or prolonged exposure to 02 may increase the 420 nm peak and terpenoids (33). It has been postulated that plants have at the expense of the 450 nm peak (25). For this reason the evolved highly specific Cyt P-450-linked secondary pathways CO-hemoprotein adduct with the 420 nm peak, termed P- to produce defense-related phytoalexins, while animals have 420, is often considered to be a degradation product of Cyt evolved parallel less specific, Cyt P-450-linked systems to P-450. detoxify ingested phytoalexins and other xenobiotics (11, 22). In addition to the nonspecific inhibition by CO, certain Cyt Cyt P-450 have been an elusive group of enzymes in plants. P-450 activities are selectively inhibited by various imidazole, Many ofthe pathways in which Cyt P-450 enzymes participate pyrimidine, and derivatives. Inhibitor binding also can be determined spectrophotometrically by changes in dif- Supported by National Science Foundation grant DMB 8716740. ference spectra. A typical Cyt P-450 spectrum of any mem- 669 670 DONALDSON AND LUSTER Plant Physiol. Vol. 96, 1991 brane preparation almost certainly represents a mixture of namic acid hydroxylase Cyt P-450 (15). Also, a Cyt P-450 several Cyt P-450 enzymes. The selective inhibitors can be digitoxin 12-f hydroxylase has been isolated from cell cultures used to distinguish different types of Cyt P-450 as discussed of foxglove (Digitalis lanata) and reconstituted with the below. reductase (26). A wide variety of oxidative and reductive mechanisms have Such reconstitution experiments demonstrate that the re- been elucidated for Cyt P-450-linked reactions in animals, ductase is necessary for the activity of each specific reaction, bacteria, and fungi. However, the majority of Cyt P-450- and that the substrate specificity of the reaction is conferred linked reactions studied to date in plant systems are hydrox- by the Cyt P-450. Inhibition of both lauric acid hydroxylase ylations or oxidative demethylations. In the most common and cinnamic acid hydroxylase by the anti-NADPH:Cyt P- Cyt P-450 oxygenase reactions two electrons from Cyt P-450 450 reductase antibodies (2) also suggests that, as in animal are donated to 02 and then one oxygen atom combines systems, the different Cyt P-450 are serviced by the same (hydroxylates) with the substrate (RH) and the second oxygen reductase and that the specificity for the various reactions is atom forms water: due to the individual Cyt P-450 and not the reductase (see Fig. 1). NADPH + H + R-H -- NADP+ + + 0 2+ HO R-OH During the course of castor bean germination, Cyt P-450 Oxidative demethylations (and dealkylations) involve the develops in the ER of the endosperm at a different stage than same hydroxylation mechanism followed by the elimination the NADPH:Cyt P-450 reductase (1). The total amount of of the hydroxylated methyl (alkyl) group as the aldehyde. ER Cyt P-450, measured spectrally, is greatest at 4 d of Cyt P-450 is reduced with cytosolic NADPH by germination, whereas the NADPH:Cyt P-450 reductase NADPH:Cyt P-450 reductase, a membrane-bound flavopro- achieves its highest activity at 2 d. A possible explanation is tein (Fig. 1). Thus electron flow is from cytosolic reductant that specific Cyt P-450 enzymes which are active at 2 d require to 02- In some mammalian systems an iron-sulfur protein a greater rate of electron flux. (e.g. adrendoxin or putaredoxin) participates in this reduc- tion. Such iron-sulfur proteins have not been reported in NADH:Cyt b5 Reductase and Cyt b5 plants. However there may be undiscovered proteins present Cyt P-450-dependent processes are often stimulated by the in plants involved in the delivery of electrons to Cyt P-450. addition of NADH (13). This may be because, as in animal systems, Cyt P-450 located in the ER can receive electrons Cyt P-450 INTERACT WITH OTHER PROTEINS from Cyt b5 in addition to the electrons donated by the NADPH:Cyt P-450 Reductase NADPH:Cyt P-450 reductase (Fig. 1). Cyt b5 in plants and animals obtains electrons in vivo from NADH:Cyt b5 reduc- NADPH:Cyt P-450 reductase is an integral membrane fla- tase (12). NADH:Cyt b5 reductase is an integral membrane voprotein, measured in vitro as NADPH:Cyt c reductase, flavoprotein, often measured in vitro as NADH:ferricyanide which has been purified to homogeneity from a variety of reductase (although some NADH:ferricyanide reductase ac- plant tissues. Most recently, antibodies raised against an Mr tivities may not represent NADH:Cyt b5 reductase). The 82,000 NADPH:Cyt P-450 reductase isolated from micro- activity measured in vitro as NADH:Cyt c reductase is thought somes ofaged Jerusalem artichoke tubers were shown to cross- to be the result of transfer of electrons from NADH to Cyt c react with Mr 82,000 microsomal proteins from maize, potato, through both Cyt b5 and its reductase. Because NADH:Cyt c avocado, bramble, tulip, and leek, but not Euglena, Ginko reductase is dependent on two proteins, it can be disrupted biloba, or animal reductase preparations (2). NADPH:Cyt P- by detergent solubilization (17) whereas the NADH:b5 (ferri- 450 reductase has also been isolated from fungal elicitor- cyanide) reductase is not. NADPH:Cyt P-450 reductase can challenged soybean cell suspension cultures (Mr 74,000) and donate electrons in vitro to Cyt c as well as to Cyt P-450 (Fig. reconstituted with a purified Cyt P-450 to produce active 3,9- 1), thus the addition of exogenous Cyt c to a Cyt P-450 dihydroxypterocarpan 6a-hydroxylase, as separated from cin- reaction can inhibit the reaction (13). It should be stressed

Fe(CN)6 Figure 1. Protein components of cytochrome P- e 450 enzyme systems in the cytosolic face of the endoplasmic reticulum. Electrons flow from cy- tosolic NADPH through NADPH:Cyt P-450 re- ductase. In some cases Cyt b5, which receives electrons from NADH:Cyt b5 reductase, may do- nate electrons to a Cyt P-450. There are many different Cyt P-450 enzymes, indicated here as x, y, and z. Some Cyt P-450 are specific hydrox- ylases as shown (R-H -* R-OH). In vitro, Cyt c or ferricyanide (Fe(CN)6) can accept electrons as indicated. PLANT CYTOCHROMES P-450 671 that such experiments reflect in vitro conditions and do not microsomes prepared by centrifugation ofpost-mitochondrial indicate participation of mitochondrial Cyt c in vivo. supernatants at 100,000g. Microsomal preparations may in- The transfer ofelectrons from Cyt b5 to Cyt P-450 has been clude membrane vesicles derived from the breakage ofvarious demonstrated by reconstitution of the Cyt and reductases membranes such as those of ER, Golgi, plasma membrane, from animal microsomes (30). There is no direct evidence for tonoplast, plastid envelopes, nucleus, and peroxisomes. The this transfer in plant systems although the stimulation ofplant composition of a microsomal fraction would depend on ho- Cyt P-450 hydroxylations by NADH (13) suggests the partic- mogenization techniques and the abundance of the different ipation of NADH:Cyt b5 reductase and Cyt b5. types ofmembranes in the source tissue. Isolation and spectral characterization of plant Cyt P-450 have usually employed Protein Kinases microsomal fractions as the starting material. The subcellular origins of the microsomal membranes have been determined Some mammalian Cyt P-450 are phosphorylated by protein in only a few cases kinases, and the phosphorylation may affect substrate binding For example, a Cyt P-450-containing microsomal fraction, and catalytic activity (6). Phosphorylation of Cyt P-450 can demonstrated to be ER, was isolated by sucrose density gra- be blocked by Cyt b5 (6), which implies that the binding of dient centrifugation from homogenates of germinating castor Cyt b5 to Cyt P-450 occludes the phosphorylation site or bean endosperm (16). This fraction was characterized by the changes its conformation. presence of NADPH:Cyt c (P-450) reductase and an enzyme of phosphoglyceride synthesis. When the fractionation was THE SUBCELLULAR LOCATIONS OF PLANT carried out in the presence of Mg2", ribosomes remained Cyt P-450 ARE DIVERSE membrane-bound and the membranes were found at a higher Topography and Insertion density than without Mg2". This density shift established the identity of these membranes as ER. Later it was shown that Although bacterial Cyt P-450 are soluble, eukaryotic Cyt Cyt P-450 cinnamate 4-hydroxylase and demethylase activi- P-450 are bound to membranes. In animal tissues Cyt P-450 ties were present in this ER fraction (32). and the attendant reductases have been found in a variety of Another study indicated that a Cyt P-450 monoterpene subcellular locations, including the ER, mitochondria, and hydroxylase was located in small vacuoles rather than the ER peroxisomes (9). In the ER the Cyt P-450 and reductases are in the cotyledons ofCatharanthus roseus (19). Yellow vesicles, exposed to the cytosol. Mammalian Cyt P-450 appears to be bearing this enzyme and NADPH:Cyt c reductase, were iso- anchored in the membrane by a 50 amino acid N-terminal lated on sucrose gradients at a density of 1.1 g/cm3. Increasing sequence, with the heme-containing, catalytic domain on the the Mg2' concentration in the gradient did not shift the cytosolic surface of the membrane (4, 31). Cyt b5 and its density ofthe hydroxylase. It was suggested that these vacuoles reductase are held in the membrane by C-terminal anchors. represented a discrete compartment for the synthesis and The orientations of the anchor sequences are related to the storage of the indole alkaloids which develop in this tissue mechanisms of insertion of the proteins in the membrane. during germination. Cyt b5 and its reductase are translated on cytoplasmic ribo- Cyt P-450 and its putative degradation product, Cyt P-420, somes and inserted after synthesis is complete, whereas Cyt have been measured in plasma membranes isolated from P-450 and NADPH:Cyt P-450 reductase are cotranslationally cauliflower by aqueous polymer two-phase partitioning (14). inserted on membrane bound ribosomes (23). The level ofCyt P-450 plus P-420 was estimated by difference Information on targeting, insertion and topography of Cyt spectroscopy to be in the range of 100 pmol/mg membrane P-450 in plant membranes is sparse. A Cyt P-450 p-chloro- protein. The amount of Cyt P-450 found in the plasma N-methylaniline demethylase isolated from avocado has an membrane fraction appeared to be greater than could be N-terminal sequence similar to that of animal Cyt P-450, accounted for by ER contamination. Nondenaturing lithium which has about 20 hydrophobic residues followed by 7 dodecylsulfate polyacrylamide gel electrophoresis of these charged residues and a cluster ofprolines (24). The hydropho- membranes yielded a protein of Mr 93,000, possibly a dimer bic sequence is thought to be responsible for membrane of a Cyt P-450, which was detected by a stain for heme. insertion and the charges are thought to prevent translocation A recent demonstration of immuno-cross-reactivity of a through the membrane. Cyt P-450 antibody with proteins in the Mr 50,000 range associated with a plant mitochondrial fraction suggests that The Functions of Cyt P-450 Probably Differ in Various Cyt P-450 may be present in these mitochondria (27). Another Subcellular Locations report indicated that Cyt P-450 might be present in the In mammalian tissues, Cyt P-450 are found in ER, mito- membranes of glyoxysomes along with Cyt b5 and its reduc- chondrial and peroxisomal membranes. The peroxisomal Cyt tase (12). This latter identification may be related to the P-450 may contribute to cholesterol synthesis in liver (9). The finding of Cyt P-450 in liver peroxisomes (9). mitochondrial forms, which have narrow substrate specifici- Analytic subcellular fractionation of plant tissues, with the ties, participate in steroidogenesis in tissues such as the use of marker enzymes, will be necessary to definitively adrenal cortex. The ER Cyt P-450, which are less substrate- localize Cyt P-450 to specific membranes in plants. Immu- specific, apparently are primarily involved in xenobiotic nohistochemical localization, using antibodies raised against detoxification. specific Cyt P-450, would be especially useful for this purpose. The Cyt P-450 from plant sources are usually obtained in Since there are many forms of Cyt P-450 having different 672 DONALDSON AND LUSTER Plant Physiol. Vol. 96, 1991 functions in plant cells, it is likely that these proteins will be Some Plant Cyt P-450 May Be Involved in Modification of found in different membranes, tissues, and organs. Herbicides OF P-450 IN PLANTS Plants have evolved detoxification reactions for elimination THERE ARE MANY FORMS Cyt of xenobiotic or allelopathic chemicals. In general the detox- In the following subsections we will review the types of ification ofxenobiotics in tolerant plants and animals involves evidence that indicate that many different Cyt P-450 enzymes conjugation to glutathione or hydroxylation of the molecule exist in plants. Cyt P-450-linked enzymes are implicated in followed by conjugation to carbohydrates, and subsequent the biosynthesis ofa variety ofofstructural, growth regulatory, vacuolar sequestration or elimination. The involvement of and protective substances in plant cells. Different Cyt P-450 plant Cyt P-450 in detoxification of herbicides has been well- have been implicated in numerous plant metabolic pathways. documented in only a few cases. Proof of involvement of Cyt Specific Cyt P-450 participate in the synthesis of substances P-450 in such detoxification reactions requires demonstration such as gibberellins, indole alkaloids, flavonoids, polyphenolic of photoreversible CO inhibition of the reaction, a require- acids, pterocarpanoids, terpenes, and sterols. Other, perhaps ment for 02 and NADPH, and, if possible, spectral evidence less specific, Cyt P-450 are responsible for the detoxifications of substrate (e.g. herbicide) binding to the Cyt. of herbicides and allelopathic substances. The different sub- Recently, such evidence has been acquired for at least three strate specificities ofCyt P-450 enzymes and the selectivity of important herbicides, chlorotoluron (21), primsulfuron (7), different types of inhibitors indicate the involvement of dif- and diclofop (20). Microsomes isolated from etiolated wheat ferent Cyt P-450 proteins. In at least one case two different seedlings hydroxylated chlorotoluron and diclofop. Diclofop- Cyt P-450 enzymes have been isolated from cultured plant methyl, an herbicide used to control grasses in cereal crops, cells. particularly wheat, is hydrolyzed to the active product, diclo- fop, in the plant. Resistant crops metabolize diclofop by Specific Plant Cyt P-450 Are Produced in Response to hydroxylation and subsequent conjugation to glucose. Diclo- Insult fop hydroxylation was shown to be inhibited by tetcyclasis, Many isoflavonoid branch pathways leading to phytoalex- an established Cyt P-450 inhibitor (20). Primsulfuron, a sul- ins are wound- and pathogen-induced. Because these path- fonurea herbicide which inhibits plant acetohydroxyacid syn- ways include Cyt P-450 linked enzymes, many ofthe individ- thase in the branched chain amino acid pathway, was found ual Cyt P-450 proteins involved in the pathways are probably to be hydroxylated in two different positions by maize seedling induced as well. For example, wound-induced, Mr 65,000 and microsomes (7). Primsulfuron, chlorotoluron and diclofop 45,000 proteins, which were cross-reactive with antibodies hydroxylations all required 02 and were inhibited by CO with raised against a Pseudomonas camphor hydroxylase Cyt P- at least some reversal of the inhibition by white light, impli- 450, were recently detected in pea epicotyl section micro- cating a Cyt P-450 in each of the hydroxylations. These Cyt somes (29). This indicated that pea microsome Cyt P-450 P-450 dependent hydroxylations indicate that herbicide de- with epitopes similar to the bacterial camphor hydroxylase toxification by Cyt P-450 ranks along with glutathione con- were induced by wounding. jugation as an important mechanism in plant xenobiotic The synthesis of the phytoalexin, glyceollin, in fungal- metabolism. infected soybean is a process which depends on several Cyt P-450 enzymes. As mentioned above, two Cyt P-450 have Individual Cyt P-450 Can Convert a Common Substrate to been purified from microsomes of cultured soybean cells Different Products exposed to a fungal elicitor extract (15). The fungal elicitor induces two specific Cyt P-450 enzymes, cinnamate 4-hy- Cyt P-450 with different reaction specificities participate in droxylase and 3,9-dihydroxypterocarpan 6a-hydroxylase. The monoterpene synthesis in epidermal glands of mints (13). In purification of these two activities appears to be the first three different species of mints the common precursor, (-)- successful resolution of distinct forms of Cyt P-450 from a limonene, is hydroxylated at distinct carbon atoms by micro- single plant tissue source. Furthermore, these workers recon- somal preparations from epidermal glands. In peppermint stituted the 3,9-dihydroxypterocarpan 6a-hydroxylase activity (Mentha piperita), limonene is hydroxylated at C-3 leading by combining the purified Mr 55,000 Cyt P-450 with purified to the formation of menthone. In spearmint (Mentha spicata) NADPH:Cyt P-450 reductase in the presence ofphospholipid. C-6 hydroxylation of limonene leads to carvone synthesis. Cinnamate 4-hydroxylase is induced by a fungal elicitor in Perillyl aldehyde is produced in Perilla following C-7 hydrox- exponential growth phase cell cultures of peanut (28), with a ylation. All three hydroxylations have the characteristics of time course similar to that observed for soybean (15). The Cyt P-450-catalyzed reactions, that is dependence on 02 and elicitor-induced hydroxylase in peanut cells is involved in the NADPH, photoreversible inhibition by CO, and stimulation synthesis of stilbene phytoalexins. This induction could be by cofactors for the NADPH:Cyt P-450 reductase, FMN or mimicked by micromolar concentrations of vanadate in the FAD. The different specificities for the hydroxylation of lim- exponential phase but not in stationary phase cells. The time onene (C-3, C-6, or C-7) may indicate that the Cyt P-450 courses for the inductions of the microsome-associated hy- hydroxylating enzymes are different proteins and originate droxylase and phenylalanine ammonia-lyase were parallel but from different genes. Also, these Cyt P-450 differ in sensitivity not correlated with increased phospholipid synthesis, indicat- to inhibition by N-substituted . The C-6 hydrox- ing a specific enrichment ofthe microsomal membranes with ylation is especially sensitive to , while C-3 and C- the hydroxylase and lyase. 6 hydroxylations are sensitive to . Ancymidol, a PLANT CYTOCHROMES P450 673 pyrimidine derivative which inhibits the Cyt P-450 kaurene ing avocado fruit (3). The N-terminal amino acid sequence oxidase of gibberellin synthesis (see below), does not inhibit predicted from the cDNA was identical to a 40 amino acid these monoterpene hydroxylations (13). sequence from a Cyt P-450 protein isolated from avocado fruit (24), and had characteristics associated with Cyt P-450 Specific Inhibitors Distinguish Plant Cyt P-450 from animal sources, such as an N-terminal hydrophobic sequence and a heme-binding domain (see "Topography and Certain triazole plant growth regulators (e.g., paclobutrazol) Insertion" above). The protein sequence predicted by the inhibit Cyt P-450 enzymes which participate in the biosyn- cDNA had less than 40% positional identity with other known thesis of sterols (27) and gibberellins (8). Recent evidence Cyt P-450 families, and it was proposed from these data that demonstrates that the previously reported inhibition of both the plant sequence belongs in a new Cyt P-450 gene family. sterol and gibberellin precursor synthesis by certain triazoles is actually specific inhibition of the individual Cyt P-450- PROSPECTS linked reactions by mixtures of enantiomers of the triazoles. Certain enantiomers of triazoles inhibit kaurene oxidase, a Isolation of individual Cyt P-450 proteins from plants and Cyt P-450 enzyme in the gibberellin pathway (5). Other production of antibodies to them should soon lead to the enantiomers specifically inhibit obtusifoliol- 14a- demethyl- identification ofthe genes and allow comparisons ofthe plant ase, recently demonstrated to be a Cyt P-450-linked reaction Cyt P-450 with the gene families from animals and bacteria. in the sterol biosynthesis pathway of higher plants (27). Eval- The different substrate and inhibitor specificities of the Cyt uation of the relative inhibition of plant gibberellin pathway P-450 enzymes discussed above, and in earlier reports, suggest versus sterol pathway Cyt P-450-linked processes by various that plants contain a broad range of Cyt P-450 enzymes, enantiomers of paclobutrazol and triadimenol indicated that probably representing many genes. Plant Cyt P-450 may be the configuration about the chiral carbon bearing a critical more diverse, with narrower substrate specificities than mam- hydroxyl group determined which pathway was inhibited (5). malian Cyt P-450, which have apparently evolved broad An inhibitor of plant sterol biosynthesis has been used to xenobiotic detoxification capacities. It will be interesting to select resistant mutant cell lines (18). The recent synthesis of see how the plant genes are related to the gene families of an azido form of paclobutrazol may provide a photoaffinity animal and prokaryotic Cyt P-450 (22). Comparisons ofplant probe which could be used to identify the specific Cyt P-450 Cyt P-450 which synthesize toxic substances to the animal responsible for kaurene oxidase, a Cyt P-450 enzyme in the Cyt P-450 which detoxify them will be especially intriguing biosynthetic pathway leading to gibberellins (10). since the substrate binding sites would be expected to have common features. In the long term, manipulation of genetic Cyt P-450 GENE FAMILIES information for Cyt P-450 substrate binding sites could pro- vide the tools for engineering plants which synthesize specified To date over 100 Cyt P-450 genes from microbial, yeast, protective substances to resist pathogens, weeds or pests. vertebrate, and, recently, plant sources have been sequenced. Many are distinguished by the types of compounds which LITERATURE CITED induce expression of the genes. The nucleotide sequences for 1. Alani AA, Luster DG, Donaldson RP (1990) Development of the genes encoding microbial, yeast and animal Cyt P450 glyoxysomal and ER membrane redox activities during castor have been compared and classified (22). The mammalian bean germination. Plant Physiol 94: 1942-1848 genes comprise 8 families, distinguished by at least 36% 2. Benveniste I, Lesot A, Hasenfratz M, Durst F (1989) Immuno- in nucleotide sequences within each chemical characterization of NADPH-cytochrome P-450 re- similarity family. ductase from Jerusalem artichoke and other higher plants. Subfamilies were defined with a 68% similarity cutoff (22). It Biochem J 259: 847-853 has been suggested that four of the gene families evolved in 3. Bozak KR, Yu H, Sirevag R, Christoffersen RE (1990) Sequence animals in response to exposure to plant derived substances analysis of ripening-related cytochrome P-450 cDNAs from (phytoalexins) during the past billion years (22). The genes avocado fruit. Proc Natl Acad Sci USA 87: 3904-3908 4. Brown CA, Black SD (1989) Membrane topology of mammalian encode P-450 hemoproteins with overlapping substrate spec- cytochromes P-450 from liver endoplasmic reticulum. J Biol ificities. The existence of P-450 with extremely similar nu- Chem 264: 4442-4449 cleotide sequences and overlapping substrate specificities 5. Burden, RS, Carter GA, Clark T, Cooke DT, Croker SJ, Deas makes the distinction between the proteins at the nucleotide AHB, Hedden P, James CS, Lenton JR (1987) Comparative activity ofthe enantiomers of tridimenol and paclobutrazol as sequence level an absolute requirement. The mammalian inhibitors of fungal growth and plant sterol and gibberellin sequences encode hemoproteins with (average) predicted biosynthesis. Pestic Sci 21: 253-267 structures consisting of46% a-helix, 12% ,8-sheet, 9% fl-turns, 6. Epstein PM, Curti M, Jansson I, Huang CK, Schenkman JB and 33% random coils. Two adjacent conserved f-a-fl units (1989) Phosphorylation of cytochrome P-450: regulation by are as a site of interaction with cytochrome b5. Arch Biochem Biophys 271: 424-433 recognized possible 7. Fonne-Pfister R, Gaudin J, Kreuz K, Ramsteiner K, Ebert E NADPH:Cyt P-450 reductase (31). Expression of the mam- (1990) Hydroxylation of primsulfuron by an inducible cyto- malian genes is under control at the transcriptional level, in chrome P450-dependent monooxygenase system from maize. response to xenobiotics, environmental conditions, and tis- Pestic Biochem Physiol 37: 165-173 sue-specific cues (22). 8. Graebe JD (1987) Gibberellin biosynthesis and control. Annu Rev Plant Physiol 38:419-465 Recently, a cDNA sequence encoding a putative plant Cyt 9. Gutierrez G, Okita R, Krisans S (1988) Demonstration of cyto- P-450 was identified by differential screening ofcDNA librar- chrome reductases in rat liver peroxisomes: biochemical and ies prepared using mRNA isolated from unripe versus ripen- immunochemical analyses. J Lipid Res 29: 613-628 674 DONALDSON AND LUSTER Plant Physiol. Vol. 96, 1991

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