SFPH proteins as therapeutic targets for a myriad of diseases.

The stomatin/prohibitin/flotillin/HflK/HflC (SPFH) domain is present in an evolutionarily conserved family of proteins that regulate a myriad of signaling pathways in archaea, bacteria and eukaryotes. The most studied SPFH proteins, prohibitins, have already been targeted by different families of small molecules to induce anticancer, cardioprotective, anti-inflammatory, antiviral, and antiosteoporotic activities. Ligands of other SPFH proteins have also been identified and shown to act as anesthetics, anti-allodynia, anticancer, and anti-inflammatory agents. These findings indicate that modulators of human or bacterial SPFH proteins can be developed to treat a wide variety of human disorders.

Arabidopsis thaliana EPOXIDE HYDROLASE1 (AtEH1) is a cytosolic epoxide hydrolase involved in the synthesis of poly-hydroxylated cutin monomers.

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization in vivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaves with constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis.

The Rise and Fall of Jasmonate Biological Activities.

Jasmonates (JAs) constitute a major class of plant regulators that coordinate responses to biotic and abiotic threats and important aspects of plant development. The core biosynthetic pathway converts linolenic acid released from plastid membrane lipids to the cyclopentenone cis-oxo-phytodienoic acid (OPDA) that is further reduced and shortened to jasmonic acid (JA) in peroxisomes. Abundant pools of OPDA esterified to plastid lipids also occur upon stress, mainly in the Arabidopsis genus. Long thought to be the bioactive hormone, JA only gains its pleiotropic hormonal properties upon conjugation into jasmonoyl-isoleucine (JA-Ile). The signaling pathway triggered when JA-Ile promotes the assembly of COI1-JAZ (Coronatine Insensitive 1-JAsmonate Zim domain) co-receptor complexes has been the focus of most recent research in the jasmonate field. In parallel, OPDA and several other JA derivatives are recognized for their separate activities and contribute to the diversity of jasmonate action in plant physiology. We summarize in this chapter the properties of different bioactive JAs and review elements known for their perception and signal transduction. Much progress has also been gained on the enzymatic processes governing JA-Ile removal. Two JA-Ile catabolic pathways, operating through ω-oxidation (cytochromes P450) or conjugate cleavage (amido hydrolases) shape signal dynamics to allow optimal control on defense. JA-Ile turnover not only participates in signal attenuation, but also impact the homeostasis of the entire JA metabolic pathway.

The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly.

The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning.

CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection.

Induced resistance to the necrotrophic pathogen Botrytis cinerea depends on jasmonate metabolism and signalling in Arabidopsis. We have presented here extensive jasmonate profiling in this pathosystem and investigated the impact of the recently reported jasmonoyl-isoleucine (JA-Ile) catabolic pathway mediated by cytochrome P450 (CYP94) enzymes. Using a series of mutant and overexpressing (OE) plant lines, we showed that CYP94B3 and CYP94C1 are integral components of the fungus-induced jasmonate metabolic pathway and control the abundance of oxidized conjugated but also some unconjugated derivatives, such as sulfated 12-HSO4-JA. Despite causing JA-Ile overaccumulation due to impaired oxidation, CYP94 deficiency had negligible impacts on resistance, associated with enhanced JAZ repressor transcript levels. In contrast, plants overexpressing (OE) CYP94B3 or CYP94C1 were enriched in 12-OH-JA-Ile or 12-COOH-JA-Ile respectively. This shift towards oxidized JA-Ile derivatives was concomitant with strongly impaired defence gene induction and reduced disease resistance. CYP94B3-OE, but unexpectedly not CYP94C1-OE, plants displayed reduced JA-Ile levels compared with the wild type, suggesting that increased susceptibility in CYP94C1-OE plants may result from changes in the hormone oxidation ratio rather than absolute changes in JA-Ile levels. Consistently, while feeding JA-Ile to seedlings triggered strong induction of JA pathway genes, induction was largely reduced or abolished after feeding with the CYP94 products 12-OH-JA-Ile and 12-COOH-JA-Ile, respectively. This trend paralleled in vitro pull-down assays where 12-COOH-JA-Ile was unable to promote COI1-JAZ9 co-receptor assembly. Our results highlight the dual function of CYP94B3/C1 in antimicrobial defence: by controlling hormone oxidation status for signal attenuation, these enzymes also define JA-Ile as a metabolic hub directing jasmonate profile complexity.

The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine hormone turnover and generate 12-hydroxyjasmonic acid upon wounding in Arabidopsis leaves.

Jasmonates (JAs) are a class of signaling compounds that mediate complex developmental and adaptative responses in plants. JAs derive from jasmonic acid (JA) through various enzymatic modifications, including conjugation to amino acids or oxidation, yielding an array of derivatives. The main hormonal signal, jasmonoyl-L-isoleucine (JA-Ile), has been found recently to undergo catabolic inactivation by cytochrome P450-mediated oxidation. We characterize here two amidohydrolases, IAR3 and ILL6, that define a second pathway for JA-Ile turnover during the wound response in Arabidopsis leaves. Biochemical and genetic evidence indicates that these two enzymes cleave the JA-Ile signal, but act also on the 12OH-JA-Ile conjugate. We also show that unexpectedly, the abundant accumulation of tuberonic acid (12OH-JA) after wounding originates partly through a sequential pathway involving (i) conjugation of JA to Ile, (ii) oxidation of the JA-Ile conjugate, and (iii) cleavage under the action of the amidohydrolases. The coordinated actions of oxidative and hydrolytic branches in the jasmonate pathway highlight novel mechanisms of JA-Ile hormone turnover and redefine the dynamic metabolic grid of jasmonate conversion in the wound response.

Rice CYP703A3, a cytochrome P450 hydroxylase, is essential for development of anther cuticle and pollen exine.

Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum-expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3-2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3-2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in-chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7-hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in-chain hydroxylation of lauric acid required for the development of male organ in higher plants.

CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in entomopathogenic fungus Beauveria bassiana.

Infection of insects by the entomopathogenic fungus Beauveria bassiana proceeds via attachment and penetration of the host cuticle. The outermost epicuticular layer or waxy layer of the insect represents a structure rich in lipids including abundant amounts of hydrocarbons and fatty acids. A member of a novel cytochrome P450 subfamily, CYP52X1, implicated in fatty acid assimilation by B. bassiana was characterized. B. bassiana targeted gene knockouts lacking Bbcyp52x1 displayed reduced virulence when topically applied to Galleria mellonella, but no reduction in virulence was noted when the insect cuticle was bypassed using an intrahemoceol injection assay. No significant growth defects were noted in the mutant as compared with the wild-type parent on any lipids substrates tested including alkanes and fatty acids. Insect epicuticle germination assays, however, showed reduced germination of ΔBbcyp52x1 conidia on grasshopper wings as compared with the wild-type parent. Complementation of the gene-knock with the full-length gene restored virulence and insect epicuticle germination to wild-type levels. Heterologous expression of CYP52X1 in yeast was used to characterize the substrate specificity of the enzyme. CYP52X1 displayed the highest activity against midrange fatty acids (C12:0 and C14:0) and epoxy stearic acid, 4-8-fold lower activity against C16:0, C18:1, and C18:2, and little to no activity against C9:0 and C18:0. Analyses of the products of the C12:0 and C18:1 reactions confirmed NADPH-dependent regioselective addition of a terminal hydroxyl to the substrates (ω-hydroxylase). These data implicate CYP52X1 as contributing to the penetration of the host cuticle via facilitating the assimilation of insect epicuticle lipids.

Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone Jasmonoyl-isoleucine for catabolic turnover.

The jasmonate hormonal pathway regulates important defensive and developmental processes in plants. Jasmonoyl-isoleucine (JA-Ile) has been identified as a specific ligand binding the COI1-JAZ co-receptor to relieve repression of jasmonate responses. Two JA-Ile derivatives, 12OH-JA-Ile and 12COOH-JA-Ile, accumulate in wounded Arabidopsis leaves in a COI1- and JAR1-dependent manner and reflect catabolic turnover of the hormone. Here we report the biochemical and genetic characterization of two wound-inducible cytochromes P450, CYP94C1 and CYP94B3, that are involved in JA-Ile oxidation. Both enzymes expressed in yeast catalyze two successive oxidation steps of JA-Ile with distinct characteristics. CYP94B3 performed efficiently the initial hydroxylation of JA-Ile to 12OH-JA-Ile, with little conversion to 12COOH-JA-Ile, whereas CYP94C1 catalyzed preferentially carboxy-derivative formation. Metabolic analysis of loss- and gain-of-function plant lines were consistent with in vitro enzymatic properties. cyp94b3 mutants were largely impaired in 12OH-JA-Ile levels upon wounding and to a lesser extent in 12COOH-JA-Ile levels. In contrast, cyp94c1 plants showed wild-type 12OH-JA-Ile accumulation but lost about 60% 12COOH-JA-Ile. cyp94b3cyp94c1 double mutants hyperaccumulated JA-Ile with near abolition of 12COOH-JA-Ile. Distinct JA-Ile oxidation patterns in different plant genotypes were correlated with specific JA-responsive transcript profiles, indicating that JA-Ile oxidation status affects signaling. Interestingly, exaggerated JA-Ile levels were associated with JAZ repressor hyperinduction but did not enhance durably defense gene induction, revealing a novel negative feedback signaling loop. Finally, interfering with CYP94 gene expression affected root growth sensitivity to exogenous jasmonic acid. These results identify CYP94B3/C1-mediated oxidation as a major catabolic route for turning over the JA-Ile hormone.

The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning.

Fleshy tomato fruit typically lacks stomata; therefore, a proper cuticle is particularly vital for fruit development and interaction with the surroundings. Here, we characterized the tomato SlSHINE3 (SlSHN3) transcription factor to extend our limited knowledge regarding the regulation of cuticle formation in fleshy fruits. We created SlSHN3 overexpressing and silenced plants, and used them for detailed analysis of cuticular lipid compositions, phenotypic characterization, and the study on the mode of SlSHN3 action. Heterologous expression of SlSHN3 in Arabidopsis phenocopied overexpression of the Arabidopsis SHNs. Silencing of SlSHN3 results in profound morphological alterations of the fruit epidermis and significant reduction in cuticular lipids. We demonstrated that SlSHN3 activity is mediated by control of genes associated with cutin metabolism and epidermal cell patterning. As with SlSHN3 RNAi lines, mutation in the SlSHN3 target gene, SlCYP86A69, resulted in severe cutin deficiency and altered fruit surface architecture. In vitro activity assays demonstrated that SlCYP86A69 possesses NADPH-dependent ω-hydroxylation activity, particularly of C18:1 fatty acid to the 18-hydroxyoleic acid cutin monomer. This study provided insights into transcriptional mechanisms mediating fleshy fruit cuticle formation and highlighted the link between cutin metabolism and the process of fruit epidermal cell patterning.

Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice.

The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of omega -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the omega -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development.

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester.

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid omega-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology.

CYP77B1 a fatty acid epoxygenase specific to flowering plants.

Contrary to animals, little is known in plants about enzymes able to produce fatty acid epoxides. In our attempt to find and characterize a new fatty acid epoxygenase in Arabidopsis thaliana, data mining brought our attention on CYP77B1. Modification of the N-terminus was necessary to get enzymatic activity after heterologous expression in yeast. The common plant fatty acid C18:2 was converted into the diol 12,13-dihydroxy-octadec-cis-9-enoic acid when incubated with microsomes of yeast expressing modified CYP77B1 and AtEH1, a soluble epoxide hydrolase. This diol originated from the hydrolysis by AtEH1 of the epoxide 12,13-epoxy-octadec-cis-9-enoic acid produced by CYP77B1. A spatio-temporal study of CYP77B1 expression performed with RT-qPCR revealed the highest level of transcripts in flower bud while, in open flower, the enzyme was mainly present in pistil. CYP77B1 promoter-driven GUS expression confirmed reporter activities in pistil and also in stamens and petals. In silico co-regulation data led us to hypothesize that CYP77B1 could be involved in cutin synthesis but when flower cutin of loss-of-function mutants cyp77b1 was analyzed, no difference was found compared to cutin of wild type plants. Phylogenetic analysis showed that CYP77B1 is strictly conserved in flowering plants, suggesting a specific function in this lineage.

The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid omega-hydroxylase involved in suberin monomer biosynthesis.

The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of omega-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid omega-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in omega-hydroxyacids with a chain length

Characterization of a methyl jasmonate and wounding-responsive cytochrome P450 of Arabidopsis thaliana catalyzing dicarboxylic fatty acid formation in vitro.

A fatty-acid-metabolizing enzyme from Arabidopsis thaliana, CYP94C1, belonging to the cytochrome P450 family was cloned and characterized. CYP94C1 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for P450 expression. When recombinant yeast microsomes were incubated with lauric acid (C12:0) for 15 min, one major metabolite was formed. The product was purified and identified by GC/MS as 12-hydroxylauric acid. Longer incubation (40 min) led to the formation of an additional metabolite identified by GC/MS as dodecadioic acid. This diacid was also produced by incubation with 12-hydroxylauric acid. These compounds were not produced by incubating microsomes from yeast transformed with a void plasmid, demonstrating the involvement of CYP94C1. This new enzyme also metabolized fatty acids of varying aliphatic chain lengths (C12 to C18) and in-chain modifications, for example, degree of unsaturation or the presence of an epoxide as an additional polar functional group. Transcription of the gene encoding CYP94C1 is enhanced by stress, treatment with the hormone methyl jasmonate and wounding. Treatment with methyl jasmonate also induced lauric acid metabolism in microsomes prepared from Arabidopsis. The induction of hydroxylase activity was dose dependent and increased with exposure time, reaching 16x higher in microsomes from 24-h treated Arabidopsis compared with control plants. Analysis of the metabolites showed a mixture of 12-, 11- and 10-hydroxylauric acids, revealing for the first time the presence of fatty acid in-chain hydroxylase in Arabidopsis.

Visible light-induced oxidation of unsaturated components of cutins: a significant process during the senescence of higher plants.

9-Hydroperoxy-18-hydroxyoctadec-10(trans)-enoic and 10-hydroperoxy-18-hydroxyoctadec-8(trans)-enoic acids deriving from type II (i.e. involving 1O2) photooxidation of 18-hydroxyoleic acid were detected after visible light-induced senescence experiments carried out with Petroselinum sativum and subsequent cutin depolymerisation. These results showed that in senescent plants, where the 1O2 formation rate exceeds the quenching capacity of the photoprotective system, 1O2 can migrate outside the chloroplasts and affect the unsaturated components of cutins. Significant amounts of 9,18-dihydroxyoctadec-10(trans)-enoic and 10,18-dihydroxyoctadec-8(trans)-enoic acids resulting from the reduction of these photoproducts of 18-hydroxyoleic acid were also detected in different natural samples. These results well support the significance of the photooxidation of the unsaturated components of higher plant cutins in the natural environment.

Sequential oxidation of Jasmonoyl-Phenylalanine and Jasmonoyl-Isoleucine by multiple cytochrome P450 of the CYP94 family through newly identified aldehyde intermediates.

The role and fate of Jasmonoyl-Phenylalanine (JA-Phe), an understudied conjugate in the jasmonate pathway remain to be unraveled. We addressed here the possibility of JA-Phe oxidative turnover by cytochrome P450s of the CYP94 family. Leaf wounding or fungal infection in Arabidopsis resulted in accumulation of JA-Phe, 12-hydroxyl (12OH-JA-Phe) and 12-carboxyl (12COOH-JA-Phe) derivatives, with patterns differing from those previously described for Jasmonoyl-Isoleucine. In vitro, yeast-expressed cytochromes P450 CYP94B1, CYP94B3 and CYP94C1 differentially oxidized JA-Phe to 12-hydroxyl, 12-aldehyde and 12-carboxyl derivatives. Furthermore, a new aldehyde jasmonate, 12CHO-JA-Ile was detected in wounded plants. Metabolic analysis of CYP94B3 and CYP94C1 loss- and gain-of-function plant lines showed that 12OH-JA-Phe was drastically reduced in cyp94b3 but not affected in cyp94c1, while single or double mutants lacking CYP94C1 accumulated less 12COOH-JA-Phe than WT plants. This, along with overexpressing lines, demonstrates that hydroxylation by CYP94B3 and carboxylation by CYP94C1 accounts for JA-Phe turnover in planta. Evolutionary study of the CYP94 family in the plant kingdom suggests conserved roles of its members in JA conjugate homeostasis and possibly in adaptative functions. Our work extends the range and complexity of JA-amino acid oxidation by multifunctional CYP94 enzymes in response to environmental cues.

Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

Cytochrome P450 metabolizing fatty acids in plants: characterization and physiological roles.

In plants, fatty acids (FA) are subjected to various types of oxygenation reactions. Products include hydroxyacids, as well as hydroperoxides, epoxides, aldehydes, ketones and α,ω-diacids. Many of these reactions are catalysed by cytochrome P450s (P450s), which represent one of the largest superfamilies of proteins in plants. The existence of P450-type metabolizing FA enzymes in plants was established approximately four decades ago in studies on the biosynthesis of lipid polyesters. Biochemical investigations have highlighted two major characteristics of P450s acting on FAs: (a) they can be inhibited by FA analogues carrying an acetylenic function, and (b) they can be enhanced by biotic and abiotic stress at the transcriptional level. Based on these properties, P450s capable of producing oxidized FA have been identified and characterized from various plant species. Until recently, the vast majority of characterized P450s acting on FAs belonged to the CYP86 and CYP94 families. In the past five years, rapid progress in the characterization of mutants in the model plant Arabidopsis thaliana has allowed the identification of such enzymes in many other P450 families (i.e. CYP703, CYP704, CYP709, CYP77, CYP74). The presence in a single species of distinct enzymes characterized by their own regulation and catalytic properties raised the question of their physiological meaning. Functional studies in A. thaliana have demonstrated the involvement of FA hydroxylases in the synthesis of the protective biopolymers cutin, suberin and sporopollenin. In addition, several lines of evidence discussed in this minireview are consistent with P450s metabolizing FAs in many aspects of plant biology, such as defence against pathogens and herbivores, development, catabolism or reproduction.