Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes.

The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene, which plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bona fide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar to tracheophyte HCT orthologs, uses shikimate as a native acyl acceptor to produce a p-coumaroyl-5-O-shikimate intermediate. Phenotypic and metabolic analyses of loss-of-function mutants show that PpHCT is necessary for the production of caffeate derivatives, including previously reported caffeoyl-threonate esters, and for the formation of an intact cuticle. Deep conservation of HCT function in embryophytes is further suggested by the ability of HCT genes from P. patens and the liverwort Marchantia polymorpha to complement an Arabidopsis thaliana CRISPR/Cas9 hct mutant, and by the presence of phenolic esters of shikimate in representative species of the three bryophyte lineages.

A Promiscuous CYP706A3 Reduces Terpene Volatile Emission from Arabidopsis Flowers, Affecting Florivores and the Floral Microbiome.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.plantcell;31/12/2947/FX1F1fx1.

Innate promiscuity of the CYP706 family of P450 enzymes provides a suitable context for the evolution of dinitroaniline resistance in weed.

Increased metabolism is one of the main causes for evolution of herbicide resistance in weeds, a major challenge for sustainable food production. The molecular drivers of this evolution are poorly understood. We tested here the hypothesis that a suitable context for the emergence of herbicide resistance could be provided by plant enzymes with high innate promiscuity with regard to their natural substrates. A selection of yeast-expressed plant cytochrome P450 enzymes with well documented narrow to broad promiscuity when metabolizing natural substrates was tested for herbicide metabolism competence. The positive candidate was assayed for capacity to confer herbicide tolerance in Arabidopsis thaliana. Our data demonstrate that Arabidopsis thaliana CYP706A3, with the most promiscuous activity on monoterpenes and sesquiterpenes for flower defence, can also oxidize plant microtubule assembly inhibitors, dinitroanilines. Ectopic overexpression of CYP706A3 confers dinitroaniline resistance. We show, in addition, that the capacity to metabolize dinitroanilines is shared by other members of the CYP706 family from plants as diverse as eucalyptus and cedar. Supported by three-dimensional (3D) modelling of CYP706A3, the properties of enzyme active site and substrate access channel are discussed together with the shared physicochemical properties of the natural and exogenous substrates to explain herbicide metabolism.

Toward a Systematic Nomenclature for (Neo)Lignanamides.

Phenylalkenoic acid amides, often referred to as phenol amides or hydroxycinnamic acid amides, are bioactive phytochemicals, whose bioactivity can be enhanced by coupling to form dimers or oligomers. Phenylalkenoic acid amides consist of a (hydroxy)cinnamic acid derivative (i.e., the phenylalkenoic acid subunit) linked to an amine-containing compound (i.e., the amine subunit) via an amide bond. The phenylalkenoic acid moiety can undergo oxidative coupling, either catalyzed by oxidative enzymes or due to autoxidation, which leads to the formation of (neo)lignanamides. Dimers described in the literature are often named after the species in which the compound was first discovered; however, the naming of these compounds lacks a systematic approach. We propose a new nomenclature, inspired by the existing system used for hydroxycinnamic acid dimers and lignin. In the proposed systematic nomenclature for (neo)lignanamides, compound names will be composed of three-letter codes and prefixes denoting the subunits, and numbers that indicate the carbon atoms involved in the linkage between the monomeric precursors. The proposed nomenclature is consistent, future-proof, and systematic.

Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense.

Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long-distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate-limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4-coumaroyl-shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4-coumaroyl-esters and -amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4-coumaroyl-shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4-coumaroyl-shikimate poorly in vitro, but were able to use alternative substrates, in particular 4-coumaroyl-anthranilate. Thus, caffeoyl-shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non-seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4-coumaroyl-anthranilate, while 4-coumaroyl-shikimate was converted to lower extents. Despite having in vitro activity with 4-coumaroyl-shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss-of-function phenotype, suggesting distinct properties also in vivo.

The MYB transcription factor Emission of Methyl Anthranilate 1 stimulates emission of methyl anthranilate from Medicago truncatula hairy roots.

Plants respond to herbivore or pathogen attacks by activating specific defense programs that include the production of bioactive specialized metabolites to eliminate or deter the attackers. Volatiles play an important role in the interaction of a plant with its environment. Through transcript profiling of jasmonate-elicited Medicago truncatula cells, we identified Emission of Methyl Anthranilate (EMA) 1, a MYB transcription factor that is involved in the emission of the volatile compound methyl anthranilate when expressed in M. truncatula hairy roots, giving them a fruity scent. RNA sequencing (RNA-Seq) analysis of the fragrant roots revealed the upregulation of a methyltransferase that was subsequently characterized to catalyze the O-methylation of anthranilic acid and was hence named M. truncatula anthranilic acid methyl transferase (MtAAMT) 1. Given that direct activation of the MtAAMT1 promoter by EMA1 could not be unambiguously demonstrated, we further probed the RNA-Seq data and identified the repressor protein M. truncatula plant AT-rich sequence and zinc-binding (MtPLATZ) 1. Emission of Methyl Anthranilate 1 binds a tandem repeat of the ACCTAAC motif in the MtPLATZ1 promoter to transactivate gene expression. Overexpression of MtPLATZ1 in transgenic M. truncatula hairy roots led to transcriptional silencing of EMA1, indicating that MtPLATZ1 may be part of a negative feedback loop to control the expression of EMA1. Finally, application of exogenous methyl anthranilate boosted EMA1 and MtAAMT1 expression dramatically, thus also revealing a positive amplification loop. Such positive and negative feedback loops seem to be the norm rather than the exception in the regulation of plant specialized metabolism.

CYP76B74 Catalyzes the 3''-Hydroxylation of Geranylhydroquinone in Shikonin Biosynthesis.

Shikonin and its derivatives are the most abundant naphthoquinone pigments formed in species of the medicinally and economically valuable Boraginaceae. A key step in the shikonin biosynthetic pathway, namely the C-3'' hydroxylation of the prenylated phenolic intermediate geranylhydroquinone, is expected to be catalyzed by a cytochrome P450. To identify cytochrome P450 candidates with transcription profiles similar to those of genes known to be involved in shikonin biosynthesis, we carried out coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines and examined the spatial expression of candidate genes in different organs of Arnebia euchroma In biochemical assays using geranylhydroquinone as the substrate, CYP76B74 exhibited geranylhydroquinone 3''-hydroxylase activity and produced 3''-hydroxy-geranylhydroquinone. In CYP76B74 RNA interference A. euchroma hairy roots, shikonin derivative accumulation decreased dramatically, which demonstrated that CYP76B74 is required for shikonin biosynthesis in the plant. Phylogenetic analysis confirmed that CYP76B74 belonged to the CYP76B subfamily and was most likely derived from an ancestral geraniol 10-hydroxylase. In a subcellular localization analysis, a GFP-CYP76B74 fusion localized to endoplasmic reticulum membranes. Our results demonstrate that CYP76B74 catalyzes the key hydroxylation step in shikonin biosynthesis with high efficiency. The characterization of the CYP76B74 described here paves the way for further exploration of the ring closure reactions generating the naphthoquinone skeleton as well as for the alternative metabolism of geranylhydroquinone 3''-hydroxylase to dihydroechinofuran.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.

CYP76C1 (Cytochrome P450)-Mediated Linalool Metabolism and the Formation of Volatile and Soluble Linalool Oxides in Arabidopsis Flowers: A Strategy for Defense against Floral Antagonists.

The acyclic monoterpene alcohol linalool is one of the most frequently encountered volatile compounds in floral scents. Various linalool oxides are usually emitted along with linalool, some of which are cyclic, such as the furanoid lilac compounds. Recent work has revealed the coexistence of two flower-expressed linalool synthases that produce the (S)- or (R)-linalool enantiomers and the involvement of two P450 enzymes in the linalool oxidation in the flowers of Arabidopsis thaliana. Partially redundant enzymes may also contribute to floral linalool metabolism. Here, we provide evidence that CYP76C1 is a multifunctional enzyme that catalyzes a cascade of oxidation reactions and is the major linalool metabolizing oxygenase in Arabidopsis flowers. Based on the activity of the recombinant enzyme and mutant analyses, we demonstrate its prominent role in the formation of most of the linalool oxides identified in vivo, both as volatiles and soluble conjugated compounds, including 8-hydroxy, 8-oxo, and 8-COOH-linalool, as well as lilac aldehydes and alcohols. Analysis of insect behavior on CYP76C1 mutants and in response to linalool and its oxygenated derivatives demonstrates that CYP76C1-dependent modulation of linalool emission and production of linalool oxides contribute to reduced floral attraction and favor protection against visitors and pests.

A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine.

Monoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound. Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool. Our analysis of (E)-8-carboxylinalool in Riesling × Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries. Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production.

ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis.

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.

Production of 3-geranyl-4-hydroxybenzoate acid in yeast, an important intermediate of shikonin biosynthesis pathway.

Shikonin and its derivatives are the main active components in the medicinal plant Arnebia euchroma and possess extensive pharmaceutical properties. In this study, we developed an optimized yeast system to obtain high-level production of 3-geranyl-4-hydroxybenzoate acid (GBA), an important intermediate involved in shikonin biosynthesis pathway. For host selection, recombinant expression of p-hydroxybenzoate:geranyltransferase (PGT) derived from A. euchroma was performed in Saccharomyces cerevisiae WAT11U strain and high yield monoterpene strain. In shake flask culture with 1 mM p-hydroxybenzoate acid (PHBA), they could yield GBA at 0.1567 and 20.8624 mg L-1, respectively. Additionally, AePGT6 showed higher enzymatic activity than its homologs. Moreover, by combining improvement in the homologous mevalonate pathway with reconstruction in the heterologous shikimic pathway, a de novo GBA synthesis pathway was constructed in StHP6tHC with co-overexpressed SctHMG1, optimized EcUbiC and AePGT6. A high titer of 179.29 mg L-1 GBA was achieved in StHP6tHC under shake flask fermentation with 1 mM PHBA. These results suggest that yeast could be engineered systematically to enable an efficient monoterpene-quinone or naphthoquinone production.

Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

Dual function of the cytochrome P450 CYP76 family from Arabidopsis thaliana in the metabolism of monoterpenols and phenylurea herbicides.

Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance.

Protein-protein and protein-membrane associations in the lignin pathway.

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein-protein, and protein-membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.

A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

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.

Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae.

Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into ß-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of ß-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L⁻¹. The second is the accumulation of ß-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from ß-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process.

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.

Innate immune responses activated in Arabidopsis roots by microbe-associated molecular patterns.

Despite the fact that roots are the organs most subject to microbial interactions, very little is known about the response of roots to microbe-associated molecular patterns (MAMPs). By monitoring transcriptional activation of beta-glucuronidase reporters and MAMP-elicited callose deposition, we show that three MAMPs, the flagellar peptide Flg22, peptidoglycan, and chitin, trigger a strong tissue-specific response in Arabidopsis thaliana roots, either at the elongation zone for Flg22 and peptidoglycan or in the mature parts of the roots for chitin. Ethylene signaling, the 4-methoxy-indole-3-ylmethylglucosinolate biosynthetic pathway, and the PEN2 myrosinase, but not salicylic acid or jasmonic acid signaling, play major roles in this MAMP response. We also show that Flg22 induces the cytochrome P450 CYP71A12-dependent exudation of the phytoalexin camalexin by Arabidopsis roots. The phytotoxin coronatine, an Ile-jasmonic acid mimic produced by Pseudomonas syringae pathovars, suppresses MAMP-activated responses in the roots. This suppression requires the E3 ubiquitin ligase COI1 as well as the transcription factor JIN1/MYC2 but does not rely on salicylic acid-jasmonic acid antagonism. These experiments demonstrate the presence of highly orchestrated and tissue-specific MAMP responses in roots and potential pathogen-encoded mechanisms to block these MAMP-elicited signaling pathways.