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...

Suberin composition of various plants including Arabidopsis (Arabidopsis thaliana) has shown the presence of very long chain fatty acid derivatives C20 in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis, this subfamily is represented by CYP86B1 and CYP86B2, which share about 45% identity with CYP86A1, a fatty acid v-hydroxylase implicated in root suberin monomer synthesis. Here, we show that CYP86B1 is located to the endoplasmic reticulum and is highly expressed in roots. Indeed, CYP86B1 promoter-driven b-glucuronidase expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knockouts and two weak ones were RNA interference-silenced lines. These root aliphatic plant hydroxylase lines had a root and a seed coat aliphatic polyester composition in which C22-and C24-hydroxyacids and a,vdicarboxylic acids were strongly reduced. However, these changes did not affect seed coat permeability and ion content in leaves. The presumed precursors, C22 and C24 fatty acids, accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a very long chain fatty acid hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development.

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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.

The mixed function oxidase trans-cinnamic acid 4-hydroxylase, cytochrome P450, cytochrome b5, and NADPH-cytochrome c (P450) reductase were measured in microsomes from aging artichoke tuber slices exposed to (24,25), and trans-cinnamic acid (1, 18) are hydroxylated, whereas p-chloro-N-methylaniline (27) was N-demethylated in Cyt P-450-dependent reactions. Involvment of Cyt P-450 is also suggested in the N-dealkylation of the herbicide Monuron2 (10).

In plants, hydroxy-fatty acid production is mainly the result of enzymatic reactions catalyzed by cytochrome P450 dependent fatty acid hydroxylases. One can distinguish x-hydroxylases that catalyze the hydroxylation of the terminal methyl of aliphatics acids (x position) and sub-terminal or in-chain hydroxylases that oxidize carbons in the chain (x-n position). Since both types of enzymes were discovered about three decades ago, the majority of investigations have focused on the CYP94 and CYP86 families, which mediate x-hydroxylations. The activities of x-hydroxylases in cutin synthesis have been clearly established, but the studies of LCR (LACERATA) and att1 (aberrant induction of type three genes), which are the first Arabidopsis thaliana mutants with alterations in coding sequences of CYP86A8 and CYP86A2, show that these types of x-hydroxylases can be involved in many aspects of plant development. The existence of different x-hydroxylases in plants with distinct regulation patterns suggests that these enzymes mediate diverse biological processes. Much less information concerning in-chain hydroxylases is available despite the fact that they were initially reported along with x-hydroxylases. This lack of information might be explained by the very few examples of sub-terminal hydroxyfatty acids described in plants. We present here the best characterized fatty acid hydroxylases and we discuss their possible roles in plant defense and development, fatty acid catabolism, plant reproduction and detoxification.

A full length cDNA encoding a new cytochrome P450‐dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10‐epoxystearic acid was converted into one major metabolite identified by GC/MS as 18‐hydroxy‐9,10‐epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 ± 0.2 µm and Vmax,app = 27 ± 1 nmol·min−1·nmol−1 P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10‐epoxy‐octadecan‐1,18‐dioic acid. The diacid was also produced in microsomal incubations of 18‐hydroxy‐9,10‐epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10‐epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the ω‐hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.

We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the -1 and -2 positions. This work was performed after recoding and heterologous expression of a fulllength cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K m(app) of 8 M and V max(app) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed.