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 (triazoles, imidazole 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 triazole 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.