Francis Durst

Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid ω-hydroxylation in development

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 Enhancer/Suppressor-mutator (En/Spm). 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 ω-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 ω-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.

Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase.

Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns. Purification was monitored using carbon monoxide and type I substrate binding properties of the enzyme. The protein, purified to electrophoretic homogeneity, showed an Mr of about 57,000 on SDS-PAGE. Polyclonal antibodies raised against this protein selectively reacted with a 57-kDa polypeptide on Western blots of induced Jerusalem artichoke microsomes. The antibody selectively and strongly inhibited CA4H activity from several plant species.

Wounding-induced cinnamic acid hydroxylase in Jerusalem artichoke tuber

Abstract Cinnamic acid hydroxylase (CAH), which catalyses transformation of trans-cinnamic acid into p-hydroxy-cinnamic acid, is a multi-enzyme system localized on the endoplasmic reticulum. Electrons are transferred from NADPH, the preferential electron donor for the system, to cytochrome P-450 via NADPH-cytochrome P-450 reductase, an enzyme regulated by the NADPH-NADP+ ratio. The induction and subsequent changes of CAH activity during ageing are accounted for by the variations in reductase and cytochrome P-450 content. The content of cytochrome b5, already present in the dormant tuber, is markedly enhanced by wounding; its participation in electron transport from NADH to the hydroxylase is discussed.

Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (insecta, orthoptera)

It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.

Arabidopsis cyp51 Mutant Shows Postembryonic Seedling Lethality Associated with Lack of Membrane Integrity

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14α-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 14α-methyl-Δ8-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 14α-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

ω-hydroxylation of Z9-octadecenoic, Z9,10-epoxystearic and 9,10-dihydroxystearic acids by microsomal cytochrome P450 systems from Vicia sativa

A microsomal fraction from etiolated Vicia sativa seedlings incubated aerobically with [1-14C]oleic acid (Z9-octadecenoic acid) or [1-14C]9,10-epoxystearic acid or [1-14C]9,10-dihydroxystearic acid catalyzed the NADPH-dependent formation of hydroxylated metabolites. The chemical structure of compounds formed from oleic, 9,10-epoxystearic or 9,10-dihydroxystearic acids was established by gas chromatography/mass spectra analysis to be 18-hydroxyoleic acid, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, respectively. The reactions required O2 and NADPH and were inhibited by carbon monoxide. As expected for monooxygenase reactions involving cytochrome P450, inhibition could be partially reversed by light and all three reactions were inhibited by antibodies raised against NADPH-cytochrome P450 reductase from Jerusalem artichoke. The omega-hydroxylation of the three substrates was enhanced in microsomes from clofibrate induced seedlings.

Regulation of the Cinnamate 4-Hydroxylase (CYP73A1) in Jerusalem Artichoke Tubers in Response to Wounding and Chemical Treatments

trans-Cinnamate 4-hydroxylase (C4H) is a plant-specific cytochrome (P450) that is encoded by the gene CYP73A and catalyzes the second step of the multibranched phenylpropanoid pathway. Increases in C4H activity in response to physical and chemical stresses have been well documented, but the mechanism of these increases has never been studied in detail. This paper reports on the regulatory mechanism controlling C4H activity in Jerusalem artichoke (Helianthus tuberosus) tubers in response to wounding and chemical treatments. We compared induction of C4H and other P450-catalyzed activities. C4H was moderately induced by chemicals relative to other P450s. Increases in enzyme activity, C4H protein, and transcripts were quantified and compared in tuber tissue 48 h after wounding and chemical treatments. Our data suggest that induction of the enzyme activity results primarily from gene activation. Time-course experiments were performed after wounding and aminopyrine treatment. Compared with wounded tissues, aminopyrine triggered an additional and delayed peak of transcript accumulation. The timing of the induced changes in activity, protein, and transcripts confirms that C4H induction results primarily from an increase in CYP73A1 mRNA, in both wounded and aminopyrine-treated tissues. However, posttranscriptional mechanisms might also contribute to the regulation of C4H activity.

Autocatalytic inactivation of plant cytochrome P-450 enzymes: selective inactivation of cinnamic acid 4-hydroxylase from Helianthus tuberosus by 1-aminobenzotriazole.

Abstract Incubation of either microsomes or slices of Helianthus tuberosus (Jerusalem artichoke) with 1-aminobenzotriazole results in time-dependent autocatalytic inactivation of cinnamic acid 4-hydroxylase, a cytochrome P-450 monooxygenase found only in plants. The inactivation is characterized by a pseudo-first-order rate constant between 4.3 and 16.5 × 10−3 s−1. Cytochrome b5 and NADPH cytochrome c (P-450) reductase are not affected. Lauric acid hydroxylation, a second type of cytochrome P-450 catalyzed reaction, is only weakly inhibited in microsomal incubations. Both lauric acid hydroxylation and cytochrome P-450 levels are decreased in tuber slices by 1-aminobenzotriazole, but at a much slower rate than cinnamic acid hydroxylation. The results suggest that 1-aminobenzotriazole is a selective (perhaps specific) suicide substrate for cinnamic acid 4-hydroxylase in this plant tissue.

Induction by manganese, ethanol, phenobarbital, and herbicides of microsomal cytochrome P-450 in higher plant tissues.

Abstract Cytochrome P -450 content and, to a lesser extent the activity of tr -cinnamic acid 4-hydroxylase, are induced in ageing Jerusalem artichoke ( Helianthus tuberosus L.) tuber cells by manganese ions, ethanol, phenobarbital, and herbicides. Manganese, ethanol, and phenobarbital induced cytochrome P -450 and also modified the time-course of its appearance. In contrast the herbicides tested stimulated the cytochrome P -450 content without modifying its time-course. The extent of induction was enhanced when the aging medium was supplemented with iron.

Multiple forms of NADPH-cytochrome P450 reductase in higher plants

We report on the presence of multiple forms of NADPH-cyt P450 reductase in microsomes from higher plants. This contrasts with the animal cyt P450 monooxygenases, where the numerous cyt P450 isoforms are reduced by a single form of reductase. Three NADPH-cyt c reductases have been resolved from Jerusalem artichoke tuber microsomes by chromatography on Reactive Red Agarose and Concanavalin A-Sepharose. Their molecular weights, determined by sodium dodecylsulfate-gel electrophoresis, are 80,000, 82,000 and 84,000. The three proteins share common epitopes and are dependent upon FMN for catalytic activity. They are highly selective for NADPH as electron donor, and allowed effective reconstitution of trans-cinnamic acid and 3,9-dihydroxypterocarpan 6a-hydroxylase activities with purified cyt P450 fractions from Helianthus tuberosus and Glycine max, respectively. As such, they appear as true isoenzyme forms of NADPH-cyt P-450 reductase.