Cytochrome P450 enzymes of the maize seedling
--Monika Frey, Ralf Kliem, Heinz Saedler and Alfons Gierl

Cytochrome P450 enzymes (P450s) are heme-containing enzymes that are most commonly integrated in microsomal membranes. Reactions catalyzed by P450s are characterized by the requirement of NADPH as a co-substrate and the photo-reversible inhibition by CO. NADPH is the substrate of the reductase that represents an integral part of the multi-enzyme complex. The ratio of reductase and P450 may be different at developmental stages and the reductase moiety does not influence the specificity of the reaction, rather substrate and reaction specificity is conferred by the P450 part of the enzyme complex. Furthermore, the reductase is exchangeable even between distantly related species. In plants the importance of P450s as key enzymes in the synthesis of secondary metabolites (e.g. gibberellins, terpens, flavonoids) has been recognized for a long time, but only recently have P450 genes been isolated molecularly (e.g. Bozak et al., Proc. Natl. Acad. Sci. USA 87:3904-3908, 1990).

It has been speculated that plants have evolved highly specific P450-linked secondary pathways to produce defense-related compounds, while in turn animals invented P450-linked systems to detoxify ingested phytoalexins or xenobiotics. The seedling has been one major source for the demonstration of P450 enzyme activity in plants. It has been shown for various species including maize that P450s are transiently expressed in the seedling. Since the seedling is a fragile structure that has to be especially protected for the successful establishment of the plant, part of these seedling specific P450 enzymes might be defense related. An example for such a seedling specific P450 enzyme having impact on defense is given in maize. A P450 N-monooxygenase participates in the synthesis of DIMBOA, a secondary metabolite belonging to the graminean specific class of benzoxazine-ones conferring general resistance to the plant (Niemeyer, Phytochemistry 27:3349-3358, 1988, for review).

We have isolated four cDNA clones representing P450 genes highly expressed in the seedling. Comparison of the amino acid sequence of the four maize P450 clones with the available plant enzyme sequences demonstrates that all plant enzymes are quite related. According to the criteria established for the animal enzymes, the maize genes belong to the same family as the ripening related avocado enzyme Cyp71A. Within this family three of the maize genes build a gene subfamily. The differences within the four maize enzymes are big enough to account for different enzymatic functions. Even for the P450s of animals that have been investigated molecularly for a long time, not much is known about involvement of protein domains in substrate recognition and reaction specificity. Comparison of the amino acid sequences of the four maize members of the Cyp71 family revealed several regions of intrafamiliar conservation that will be tested for their significance in the catalyzed reaction by site directed mutagenesis.

Making use of the recombinant inbred maize population (Burr and Burr, TIG 7:55-60, 1991) the P450 genes were mapped within a four map unit cluster on the short arm of chromosome 4 (4S023 to 4S027). Therefore, as in animals, P450 genes of families and subfamilies are clustered in maize and might have evolved via gene duplication. In contrast to the situation in animals where several large introns disrupt the gene, the structure of the isolated maize P450 genes is simple. One small intron is present close to the dioxygen binding site in all four genes and an additional intron is found in two of them. The number of introns is therefore not conserved within the gene family while intron conservation is a common feature in animal P450 gene families.

Northern analysis revealed that all four genes have a similar expression pattern: they are most highly expressed in the shoot where the maximum is reached seven days after imbibition, while in the root a distinct maximum is displayed at day three. Fourteen days after imbibition only a low level of the transcripts is discovered in the seedling and minor amounts of the transcript are found in the leaves of the mature plant. No transcript at all is detectable in the kernel. Between different maize lines the relative amount of the four P450 genes might vary. A hint for the function of the isolated maize P450 enzymes might come from their distribution in the maize seedling. In the shoot there is a shift of the major hybridization signal from the coleoptile to the outer leaves. However, the youngest, smallest leaves and the apical meristem display only background hybridization throughout the span of P450 gene expression. Transcript is detected at the base of all developed leaves and at the tip of the outer leaves but hybridization throughout the leaf blade is demonstrated only for the coleoptile and the two outer leaves. These organs build a kind of shield for the seedling but have no function for the major plant and are even degraded. The parenchymatic cells of the first internode and the compressed nodule complex that is the site for the generation of secondary roots of maize were highly decorated with silver grains after in situ hybridization. In the primary root the hybridization is restricted to the region of cell division and here to the cortex and the pith of the pro-vascular tissue. Due to this expression pattern it seems unlikely that the maize P450 genes of the Cyp71 family are involved in hormone synthesis or in the synthesis of cell wall related compounds, but it might be that they have an implication in defense mechanisms. The enzyme function will be tested by heterologous expression and the function of the genes will also be assayed by 'reverse genetics'. 

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