We describe lacerata (lcr) mutants of Arabidopsis, which display various developmental abnormalities, including postgenital organ fusions, and report cloning of the LCR gene by using the maize transposon Enhancer͞Suppressor-mutator (En͞Spm). The pleiotropic mutant phenotype could be rescued by genetic complementation of lcr mutants with the wild-type LCR gene. The LCR gene encodes a cytochrome P450 monooxygenase, CYP86A8, which catalyzes -hydroxylation of fatty acids ranging from C12 to C18:1, as demonstrated by expression of the gene in yeast. Although palmitic and oleic acids were efficient substrates for LCR, 9,10-epoxystearate was not metabolized. Taken together with previous studies, our findings indicate that LCR-dependent -hydroxylation of fatty acids could be implicated in the biosynthesis of cutin in the epidermis and in preventing postgenital organ fusions. Strikingly, the same pathway seems to control trichome differentiation, the establishment of apical dominance, and senescence in plants.T he epidermis of plants is a composite tissue that comprises several cell types. Some of these, such as stoma cells, trichomes, and papilla cells, can be easily distinguished from the predominating pavement cells by their characteristic morphological features. Other cell types are not readily distinguishable, although they apparently perform specific functions. One example of this is given by epidermal cells on the adaxial side of carpels, which exhibit a unique contact response during elaboration of the pistil and are able to adhere and redifferentiate into parenchymatous cells. Another unique feature of these epidermal cells is their ability to adhere to the growing pollen tube and guide it to the embryo sac (1, 2). In contrast to animals, where selectively established cell adhesions are common and play an enormous role in development (3, 4), examples of regular cell adhesions in higher plants are rare, and indeed may be restricted to the processes cited above. In particular mutants in several plant species fusions of organs occur during development of the shoot, in a process that resembles the regular fusion of carpels. It is not yet known whether the same molecular mechanisms underlie all instances of cell fusions. By comparison with the epidermis cells of fused carpels, epidermis cells at sutures in fusion mutants do not alter their normal anticlinal plane of division and do not redifferentiate in response to the adhesion. Cell differentiation, however, is affected in at least two fusion mutants. The epidermis of crinkly4 (cr4) maize plants contains enlarged, occasionally spherical, cells, which can divide periclinally to give rise to multilayered sectors (5). In the fiddlehead ( fdh) mutant of Arabidopsis, the epidermis of rosette leaves displays a 2-fold reduction in the number of trichomes (6). These findings indicate a link between the altered cell differentiation in the epidermis and the fusion of organs in the mutants.By using transposon tagging, FDH and CR4, two genes that result in organ fusions when m...

Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn2 -induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a Agtll cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identifi'ed in this incomplete sequence, which was used as a probe for the isolation ofa 1.7-kilobase clone in a AgtlO library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (Mr = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most similarity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family.

Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Succharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisk strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced ironkarbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1 : 1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data.

Fig. 5: Conservation of functional domains in CPRs from plant, animal, yeast and B. megaterium (the accession numbers of the sequences are given in the text). Residues printed in bold are conserved in other members of the FNR family.The numbering refers to positions in the sequence. 201Brought to you by |

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14a-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14a-methyl-D 8 -sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14a-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.Sterols are ubiquitous among most eukaryotic organisms. Bulk sterols, such as cholesterol in animals, ergosterol in yeast (Saccharomyces cerevisiae), and sitosterol in plants, serve to regulate membrane fluidity and permeability and indirectly affect the activity and distribution of integral membrane proteins, including enzymes, ion channels, and signal transduction components (Hartmann, 1998). These sterols also serve as precursors for bioactive molecules, such as mammalian steroid hormones, plant brassinosteroid (BR) hormones, and insect ecdysteroids to control developmental processes (Clouse, 2000). BRs are plant hormones that have important roles in plant development, including cell elongation, division, vascular differentiation, senescence, and stress responses (Clouse and Sasse, 1998). Plant sterols are also substrates for the synthesis of a wide range of secondary metabolites (Hartmann, 1998). Recently, the implication of sitosterol glucoside in the synthesis of cellodextrins has been proposed (Peng et al., 2002), and a link between sterol biosynthesis and cellulose production has been further documented (Schrick et al., 2004a).Plant sterols derive from cycloartenol via a series of reactions, including methylation, reduction, isomerization, and desaturation. The molecular genetic and biochemical analyses using Arabidopsis (Arabidopsis thaliana) mutants for genes encoding sterol biosynthe...

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have been investigated. Microsomes from transformed yeast catalysed trans-cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p-coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism-based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.Cytochromes P450 form a large superfamily of several hundreds to several thousands of hemoproteins, involved in oxygen activation and oxygen tranfer into lipophilic molecules. They all share some sequence identity related to their common catalytic properties, i.e. heme and oxygen binding, electron transfer and oxygen activation [l]. Differences in their primary sequences usually reflect variations in specificity for substrates oxygenated. These differences may concern more than 80% of the amino acid sequence. However, the modification of a single amino acid residue can be sufficient to completely alter the substrate specificity of the enzyme 121.Full-length amino acid sequences presently available for plant P450s share less than 30% identity. This almost certainly implies great differences in the structures of their substrate binding sites. However, no relation between the structure of the enzymes and their catalytic activities has yet been established. CYP73 is the first DNA sequence coding for a plant P450 with an identified physiological function to have been isolated 131. It catalyses the 4-hydroxylation of trunscinnamic acid into p-coumaric acid. This hydroxylation is ductase (4-hydroxylating) (EC 1.14.13.11).the second reaction in the phenylpropanoid pathway. Cinnamate 4-hydroxylase (CA4H) is thus an obligatory step for the biosynthesis of lignin, a major component of the earth's total biomass. It is involved in the formation of most of the phenylpropanoid derivatives, essential for plant development, pigmentation and defense against both ultraviolet light and pathogens. Recent data [4-61 suggest that CA4H plays a central role in the regulation of the phenylpropanoid pathway. We, therefore, decided to establish more precisely which molecules are transformed by the enzyme or interfere with its catalytic activity. The determination of a population of substrates was also intended to provide information for analysis of the structurelfunction relationship of this plant P450. We devised an optimized system for the expression of CYP73 in Saccharomyces cerevisae [7]. This system provided yeast microsomes for which the only detectable P450 was CYP73. It routine...

The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.