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.

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.

A phenol-enriched cuticle is ancestral to lignin evolution in land plants.

Lignin, one of the most abundant biopolymers on Earth, derives from the plant phenolic metabolism. It appeared upon terrestrialization and is thought critical for plant colonization of land. Early diverging land plants do not form lignin, but already have elements of its biosynthetic machinery. Here we delete in a moss the P450 oxygenase that defines the entry point in angiosperm lignin metabolism, and find that its pre-lignin pathway is essential for development. This pathway does not involve biochemical regulation via shikimate coupling, but instead is coupled with ascorbate catabolism, and controls the synthesis of the moss cuticle, which prevents desiccation and organ fusion. These cuticles share common features with lignin, cutin and suberin, and may represent the extant representative of a common ancestor. Our results demonstrate a critical role for the ancestral phenolic metabolism in moss erect growth and cuticle permeability, consistent with importance in plant adaptation to terrestrial conditions.

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.

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.

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.

CYP98A22, a phenolic ester 3'-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens.

BACKGROUND: Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis. RESULTS: In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn't affect the synthesis of scopoletin. CONCLUSIONS: The overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to phytophageous insects.

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.

Phenolamides: bridging polyamines to the phenolic metabolism.

Phenolamides constitute a diverse and quantitatively major group of secondary metabolites resulting from the conjugation of a phenolic moiety with polyamines or with deaminated aromatic aminoacids. This review summarizes their bioactivities and their reported roles in plant development, adaptation and defence compared to those of their polyamine precursors. The most conclusive recent developments point to their contribution to cell-wall reinforcement and to direct toxicity for predators and pathogens, either as built-in or inducible defence. Phenolamides were often considered as accumulated end-chain products. Recent data bring a light on their biosynthesis and suggests their possible contribution in the branching of the phenylpropanoid metabolism.

Evolution of a novel phenolic pathway for pollen development.

Metabolic plasticity, which largely relies on the creation of new genes, is an essential feature of plant adaptation and speciation and has led to the evolution of large gene families. A typical example is provided by the diversification of the cytochrome P450 enzymes in plants. We describe here a retroposition, neofunctionalization, and duplication sequence that, via selective and local amino acid replacement, led to the evolution of a novel phenolic pathway in Brassicaceae. This pathway involves a cascade of six successive hydroxylations by two partially redundant cytochromes P450, leading to the formation of N1,N5-di(hydroxyferuloyl)-N10-sinapoylspermidine, a major pollen constituent and so-far-overlooked player in phenylpropanoid metabolism. This example shows how positive Darwinian selection can favor structured clusters of nonsynonymous substitutions that are needed for the transition of enzymes to new functions.

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis.

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3'-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3',4'-dihydroxyphenyllactate and the 3'-hydroxylation of caffeoyl-4'-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K (m)-values for 4-coumaroyl-3',4'-dihydroxyphenyllactate and caffeoyl-4'-hydroxyphenyllactate were determined to be at 5 microM and 40 microM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.

Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis.

Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat.

A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.

A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth.

Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.

Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases.

Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid.

Maturation of cytosolic iron-sulfur proteins requires glutathione.

Glutathione is the major protective agent against oxidative stress in Saccharomyces cerevisiae. Deletion of the GSH1 gene (strain Deltagsh1) encoding the enzyme that catalyzes the first step of glutathione biosynthesis leads to growth arrest, which can be relieved by either glutathione or reducing agents such as dithiothreitol. Because defects in the biosynthesis of cellular iron-sulfur (Fe/S) proteins are associated with increases in glutathione levels, we examined the consequences of glutathione depletion on this essential process. No significant defects were detected in the amounts, activities, and maturation of mitochondrial Fe/S proteins in glutathione-depleted Deltagsh1 cells. On the contrary, the maturation of extra-mitochondrial Fe/S proteins was decreased substantially. The defect was rectified neither by addition of dithiothreitol nor under anaerobic conditions excluding oxidative damage of Fe/S clusters. A double mutant in GSH1 and ATM1 encoding a mitochondrial ATP binding cassette (ABC) transporter involved in cytosolic Fe/S protein maturation is nonviable even in the presence of dithiothreitol. Similar to atm1 and other mutants defective in cytosolic Fe/S protein maturation, mitochondria from glutathione-depleted Deltagsh1 cells accumulated high amounts of iron. Together, our data demonstrate that glutathione, in addition to its protective role against oxidative damage, performs a novel and specific function in the maturation of cytosolic Fe/S proteins.