Romain Larbat

Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes

Coumarins (1,2-benzopyrones) are ubiquitously found in higher plants where they originate from the phenylpropanoid pathway. They contribute essentially to the persistence of plants being involved in processes such as defense against phytopathogens, response to abiotic stresses, regulation of oxidative stress, and probably hormonal regulation. Despite their importance, major details of their biosynthesis are still largely unknown and many P450-dependent enzymatic steps have remained unresolved. Ortho-hydroxylation of hydroxycinnamic acids is a pivotal step that has received insufficient attention in the literature. This hypothetical P450 reaction is critical for the course for the biosynthesis of simple coumarin, umbelliferone and other hydroxylated coumarins in plants. Multiple P450 enzymes are also involved in furanocoumarin synthesis, a major class of phytoalexins derived from umbelliferone. Several of them have been characterized at the biochemical level but no monooxygenase gene of the furanocoumarin pathway has been identified yet. This review highlights the major steps of the coumarin pathway with emphasis on the cytochrome P450 enzymes involved. Recent progress and the outcomes of novel strategies developed to uncover coumarin-committed CYPs are discussed.

Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis

Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (+)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9-10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (+)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (+)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (+)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (+)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L

BackgroundCynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.ResultsA gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content.ConclusionThe data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters.

Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent Km of 2.1 ± 0.4 μm for (+)-columbianetin and a kcat of 112 ± 14 min–1. Moreover, the use of 3′-deuterated (+)-columbianetin as substrate led to an almost complete “metabolic switch,” resulting in the synthesis of anti-3′-hydroxy-3′-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3′. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.

Is the C:N ratio a reliable indicator of C allocation to primary and defence-related metabolisms in tomato?

Plant growth and defence are both fuelled by compounds synthesized from a common pool of carbon and nitrogen, implying the existence of a competition for carbon and nitrogen allocation to both metabolisms. The ratio of carbon to nitrogen (C:N) of an organ is often regarded as a convenient indicator of growth and quality. The purpose of this work was to assess whether or not it is possible to extend its use to characterize the trade-off between growth and defence processes. Therefore, we calculated C:N ratios in the pool of resources and in the total plant, and correlated them to the concentrations of diverse compounds of the primary and secondary metabolisms in young tomatoes. Plants were grown hydroponically at N availabilities either limiting (0.1 mM) or not (7 mM) for growth in two glasshouses maintained either under ambient or enriched (700 vpm) air CO(2). These conditions yielded a large array of C:N in fully developed leaves, developing leaves, stem and roots, sampled 27, 35 and 47 days after sowing. Growth parameters and tissue concentrations of primary metabolites (carbohydrates, starch), defence-related compounds (polyphenols, glycoalkaloids), lignin, nitrate, ammonium, C and N were analyzed. Net CO(2) exchange rate was also measured at the last sampling date. Low N limited plant growth more than photosynthesis. The C:N in the resource pool was far higher than the total C:N. Starch was the most responsive compound, attaining high concentration under high C:N, whereas lignin remained stable. Chlorogenic acid, rutin, kaempferol-rutinoside and tomatine concentrations correlated positively to C:N. The same patterns were observed for most organs and molecules, except soluble carbohydrates in fully developed leaves whose concentration was not influenced. Among the organs, developing leaves showed the highest concentrations of secondary compounds and were the most responsive to C:N variations. Neither the biochemical nature of the compounds (C-based or N- containing metabolites) nor the calculation mode of C:N, influenced the patterns observed. Within the range of N availabilities considered (up to N limitation but not deficiency), the C:N can be considered as a good indicator of the secondary compounds concentrations in organs, especially for those involved in the chemical defence.

Oil composition and characterisation of phenolic compounds of Opuntia ficus-indica seeds.

The seed composition of four varieties of Opuntia ficus-indica growing in Algeria was investigated. Seeds ground into a fine powder were first, subjected to oil extraction and fatty acids analysis. The phenolic compounds were then extracted from the defatted powder of seeds in order to be quantified and characterised by liquid chromatography coupled to mass spectrometry (LC-MS(n)) and to nuclear magnetic resonance (LC-NMR) approaches. In addition, an evaluation of the antioxidant activity of the phenolic extracts was investigated. Gas chromatography analysis of the seed oil showed high percentages of linoleic acid in the four varieties ranging from 58% to 63%. The phenolic profile of the Opuntia ficus-indica seeds displayed a high complexity, with more than 20 compounds detected at 330 nm after the LC separation. Among them, three isomers of feruloyl-sucrose were firmly identified and another was strongly supposed to be a sinapoyl-diglycoside. High correlations were found between phenolic content in the defatted seed extracts and their antioxidant activity. The data indicate that the defatted cactus seed wastes still contain various components that constitute a source for natural foods.

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

Fast diversification and versatility of a subfamily of cytochrome P450 enzymes in Brassicaceae has been important in their metabolism of both monoterpenols and 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.

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

Fast diversification and versatility of a subfamily of cytochrome P450 enzymes in Brassicaceae has been important in their metabolism of both monoterpenols and 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.

Influence of repeated short-term nitrogen limitations on leaf phenolics metabolism in tomato.

High concentrations of phenolics have been shown to play a role in plant resistance to pathogens. One way to obtain increased phenolic concentrations in plant tissues is to limit mineral nitrogen (N) availability; however, over long periods, this treatment will have a negative effect on plant growth. The aim of our study was to determine the effect of repeated short-term N limitations on plant growth and phenolic metabolism in leaves. Tomato plants (Solanum lycopersicum, cv. Pixie) were subjected to two successive 10-day N-limitation periods (0.15 mM NO(3)(-), 0.01 mM NH(4)(+)), followed by periods of full nutrient supply (15 mM NO(3)(-), 1.2 mM NH(4)(+)). Additionally, other plants were subjected to either of these two limitation periods, and a set of control plants was given a full nutrient supply during the entire period. The phenolic metabolism was monitored by measuring the leaf concentrations of chlorogenic acid, three flavonol glycosides (quercetin and kaempferol derivatives) and two major anthocyanins, together with the expression of eight structural genes and three transcription factors of the phenylpropanoid pathway. The relative growth rate of the plants decreased during the N-limitation periods but was restored as soon as N was resupplied. Each N-limitation period resulted in an up-regulation of the phenolic biosynthetic pathway, as demonstrated by an increase in the leaf phenolic concentration and an up-regulation of the related genes. The genes in the phenolic pathway were down-regulated immediately when N was resupplied; however, the leaf concentrations of several phenolics, particularly flavonol glycosides, were maintained at significantly higher levels than in the control plants for up to 17 days after the end of the first limitation. The amplitude of the increase in leaf phenolic concentration did not depend on the number of N-limitation periods to which the plant was subjected, which indicates that the plants did not acclimate to nitrogen limitation. Successive N-limitation periods resulted in additive increases in flavonol glycoside concentrations.

Organ-specific responses of tomato growth and phenolic metabolism to nitrate limitation

Phenolic compounds are secondary metabolites involved in plant innate chemical defence against pests and diseases. Their concentration varies depending on plant tissue and also on genetic and environmental factors, e.g. availability of nutrient resources. This study examines specific effects of low (LN) and high (HN) nitrogen supply on organ (root, stem and leaf) growth and accumulation of major phenolics [chlorogenic acid (CGA); rutin; kaempferol rutinoside (KR)] in nine hydroponically grown tomato cultivars. LN limited shoot growth but did not affect root growth, and increased concentrations of each individual phenolic in all organs. The strength of the response was organ-dependent, roots being more responsive than leaves and stems. Significant differences were observed between genotypes. Nitrogen limitation did not change the phenolic content in shoots, whereas it stimulated accumulation in roots. The results show that this trade-off between growth and defence in a LN environment can be discussed within the framework of the growth-differentiation balance hypothesis (i.e. GDBH), but highlight the need to integrate all plant organs in future modelling approaches regarding the impact of nitrogen limitation on primary and secondary metabolism.