Natural antisense transcripts (NATs) represent an important regulatory layer that can modulate gene expression in plants. However, their functional relevance at specific stress-responsive loci and evolutionary emergence remain poorly characterized. The DOXC21 clade of 2-oxoglutarate–dependent dioxygenases (2OGDs) is associated with abiotic stress adaptation, and the NAT overlapping DOXC21-A (AT3G19000), annotated as AT3G19002, represents a candidate locus-specific regulatory element. Here, we combined in silico analyses and quantitative gene expression assays to investigate the evolutionary origin and potential regulatory role of this NAT at the DOXC21 locus in Arabidopsis thaliana. Phylogenetic reconstruction of 590 plant DOXC homologs revealed that DOXC21-A and its neighboring homologous gene, DOXC21-B, form a Brassicales-specific clade of stress-responsive dioxygenases distinct from canonical hormone biosynthesis enzymes. In silico analysis of AT3G19002 identified a conserved 29-nucleotide motif with partial similarity to plant microRNAs, thermodynamically stable stem-loop secondary structures, and predicted candidate targets associated with stress response, metabolism, and transcriptional regulation. Reanalysis of publicly available small RNA sequencing datasets confirmed persistent, low-abundance small RNA reads mapping to the AT3G19002 locus, supporting the presence of small RNA-associated signals. Quantitative PCR analysis in wild-type plants, two independent NAT-disrupted mutants (nat1, nat2), and a doxc21-a knockout line showed that osmotic stress significantly upregulated DOXC21-A expression in both nat mutants, whereas transcript levels remained stable in wild-type plants. NAT expression was highest in the doxc21-a knockout under control conditions and markedly decreased under osmotic stress, while remaining low in the nat mutants under all conditions. These reciprocal expression patterns support a regulatory relationship between DOXC21-A and its antisense transcript. Sequence alignment and motif analysis also identified conserved features potentially associated with small RNA biogenesis and predicted candidate targets, including genes involved in signaling, secondary metabolism, and transcriptional regulation. Our combined computational and experimental analyses suggest that AT3G19002 contributes to the regulation of DOXC21-A under stress, potentially through transcriptional interference or small RNA-mediated mechanisms. Together with the reciprocal expression of the sense and antisense transcripts, these results identify the DOXC21-A locus as a candidate stress-responsive regulatory module and provide a foundation for future functional studies on NAT-mediated regulation in plant stress responses.

Abstract Plants produce a variety of O -prenylated aromatics that exhibit biological activities beneficial to human health, and the presence of the O -prenyl moiety is often crucial to their functions. However, most aromatic O -prenylation genes remain unknown in plants. In this study, we report the molecular identification of an aromatic O -prenyltransferase (PT) involved in the biosynthesis of auraptene (7-geranyloxycoumarin), a citrus metabolite known for its preservative effect on human cognitive function. Based on in silico screening focusing on the membrane-bound PT family, CpPT4 was isolated as a candidate from grapefruit ( Citrus x paradisi ), an auraptene-rich species. Enzymatic characterization demonstrated that recombinant CpPT4 specifically catalyzes umbelliferone 7- O -geranyltransferase activity to form auraptene, which differs from the enzymatic functions of known O -PTs. This enzyme also catalyzed aromatic N -prenylation to produce a new-to-nature auraptene analog. Regarding organ- and organellar-specific localization, it is strongly suggested that CpPT4 functions in the outer pericarp plastids, where auraptene is expected be formed. Furthermore, we found that CpPT4 orthologs are widely distributed in citrus genomes. Intriguingly, mandarins and their descendant species possess dysfunctional orthologs, which is consistent with the low accumulation of auraptene and its downstream metabolites in these species. This study provides an example of the contribution of the UbiA superfamily to O -prenylated aromatic biosynthesis. Moreover, CpPT4 can be useful as a tool in the synthetic biology-based production of auraptene and its analogs, as well as a molecular marker in the breeding of auraptene-rich citrus varieties.

ABSTRACT Coumarins are phenylpropanoid-derived specialized metabolites that contribute to plant defence, shape plant–microbe interactions in the rhizosphere, and promote iron acquisition. In Arabidopsis thaliana , a model plant for iron-responsive coumarin metabolism, the enzymatic origin of the catecholic coumarin esculetin has long remained unresolved. Here we identify the first O -demethylation reaction in Arabidopsis specialized metabolism and show that 2-oxoglutarate- and Fe(II)-dependent dioxygenases catalyze scopoletin 6- O -demethylation to form esculetin. We designate these enzymes scopoletin 6- O -demethylases (S6ODs) and validate their activity through biochemical characterization, together with metabolomic profiling and independent loss-of-function mutant lines providing genetic evidence in planta . Disruption of S6OD activity remodels coumarin profiles and alters plant performance under limited iron availability, indicating that esculetin biosynthesis contributes to plant responses under these conditions. Our findings resolve the long-sought missing step in esculetin biosynthesis. It establishes O -demethylation as a previously unrecognized reaction in Arabidopsis specialized metabolism and suggest that 2OGD-mediated O -demethylation is recurrently recruited during evolution of plant metabolism, with implications for metabolic engineering and improvement of iron acquisition traits in crops.

Specialized metabolites are molecules involved in plants' interaction with their environment. Elucidating their biosynthetic pathways is a challenging but rewarding task, leading to societal applications and ecological insights. Furanocoumarins emerged multiple times in Angiosperms, raising the question of how different enzymes evolved into catalyzing identical reactions. To identify enzymes producing lineage-specific metabolites, an evolutionary-based approach was developed and applied to furanocoumarin biosynthesis in Ficus carica (Moraceae). This led to the characterization of CYP71B129-131a, three P450 enzymes whose evolution of the function was investigated using phylogenetics, structural comparisons and site-directed mutagenesis. CYP71B129 and CYP71B130,131a were found to hydroxylate umbelliferone (coumarin) and xanthotoxin (furanocoumarin), respectively. Results suggest that CYP71Bs xanthotoxin hydroxylase activity results from duplications and functional divergence of umbelliferone hydroxylase genes. Structural comparisons highlighted an amino acid affecting CYP71Bs substrate specificity, which may play a key role in allowing xanthotoxin hydroxylation in several P450 subfamilies. CYP71B130-131a characterization validates the proposed enzyme-discovery approach, which can be applied to different pathways and help to avoid the classic bottlenecks of specialized metabolism elucidation. The CYP71Bs also exemplify how furanocoumarin-biosynthetic enzymes can stem from coumarin-biosynthetic ones and provides insights into the molecular mechanisms underlying the multiple emergences of xanthotoxin hydroxylation in distant P450 subfamilies.

BACKGROUND:Scopoletin and umbelliferone belong to coumarins, which are plant specialized metabolites with potent and wide biological activities, the accumulation of which is induced by various environmental stresses. Coumarins have been detected in various plant species, including medicinal plants and the model organism Arabidopsis thaliana. In recent years, key role of coumarins in maintaining iron (Fe) homeostasis in plants has been demonstrated, as well as their significant impact on the rhizosphere microbiome through exudates secreted into the soil environment. Several mechanisms underlying these processes require clarification. Previously, we demonstrated that Arabidopsis is an excellent model for studying genetic variation and molecular basis of coumarin accumulation in plants. RESULTS:Here, through targeted metabolic profiling and gene expression analysis, the gene-metabolite network of scopoletin and umbelliferone accumulation was examined in more detail in selected Arabidopsis accessions (Col-0, Est-1, Tsu-1) undergoing different culture conditions and characterized by variation in coumarin content. The highest accumulation of coumarins was detected in roots grown in vitro liquid culture. The expression of 10 phenylpropanoid genes (4CL1, 4CL2, 4CL3, CCoAOMT1, C3'H, HCT, F6'H1, F6'H2,CCR1 and CCR2) was assessed by qPCR in three genetic backgrounds, cultured in vitro and in soil, and in two types of tissues (leaves and roots). We not only detected the expected variability in gene expression and coumarin accumulation among Arabidopsis accessions, but also found interesting polymorphisms in the coding sequences of the selected genes through in silico analysis and resequencing. CONCLUSIONS:To the best of our knowledge, this is the first study comparing accumulation of simple coumarins and expression of phenylpropanoid-related genes in Arabidopsis accessions grown in soil and in liquid cultures. The large variations we detected in the content of coumarins and gene expression are genetically determined, but also tissue and culture dependent. It is particularly important considering that growing plants in liquid media is a widely used technology that provides a large amount of root tissue suitable for metabolomics. Research on differential accumulation of coumarins and related gene expression will be useful in future studies aimed at better understanding the physiological role of coumarins in roots and the surrounding environments.

Specialized metabolites are molecules involved in plants interaction with their environment. Elucidating their biosynthetic pathways is a challenging but rewarding task, leading to societal applications and ecological insights. Furanocoumarins emerged multiple times in Angiosperms, raising the question of how different enzymes evolved into catalyzing identical reactions.To identify enzymes producing lineage-specific metabolites, an evolutionary-based approach was developed and applied to furanocoumarin biosynthesis in Ficus carica (Moraceae). This led to the characterization of CYP71B129-131a, three P450 enzymes whose evolution of the function was investigated using phylogenetics, structural comparisons and site-directed mutagenesis. CYP71B129 and CYP71B130,131a were found to hydroxylate umbelliferone (coumarin) and xanthotoxin (furanocoumarin), respectively. Results suggest that CYP71Bs xanthotoxin hydroxylase activity results from duplications and functional divergence of umbelliferone hydroxylase genes. Structural comparisons highlighted an amino acid affecting CYP71Bs substrate specificity, which may play a key role in allowing xanthotoxin hydroxylation in several P450 subfamilies. CYP71B130-131a characterization validates the proposed enzyme-discovery approach, which can be applied to different pathways and help to avoid the classic bottlenecks of specialized metabolism elucidation. The CYP71Bs also exemplify how furanocoumarin-biosynthetic enzymes can stem from coumarin-biosynthetic ones and provides insights into the molecular mechanisms underlying the multiple emergences of xanthotoxin hydroxylation in distant P450 subfamilies. ### Competing Interest Statement The authors have declared no competing interest.

Significance Plants produce approximately 300 O -prenylated aromatics, with their O -prenyl moieties often being crucial to their bioactivities. This study identified a gene from grapefruit encoding an aromatic O -prenyltransferase ( O- PT) belonging to the UbiA superfamily. The O -PT was shown responsible for the biosynthesis of pharmaceutically active O -prenylated coumarins that cause grapefruit–drug interactions, an adverse effect disturbing the pharmacokinetics of more than 85 medications. Another UbiA O -PT for coumarins was isolated from Angelica keiskei , an apiaceous medicinal plant. Phylogenetic analysis of the rutaceous and apiaceous O -PTs suggested that aromatic O -prenylation activity emerged in parallel in these distant plant taxa. The molecular evolution of aromatic O -PTs from plant UbiA proteins may aid citrus breeding and a synthetic biology approach to bioactive O -prenylated coumarins.

Coumarins are phytochemicals occurring in the plant kingdom, which biosynthesis is induced under various stress factors. They belong to the wide class of specialized metabolites well known for their beneficial properties. Due to their high and wide biological activities, coumarins are important not only for the survival of plants in changing environmental conditions, but are of great importance in the pharmaceutical industry and are an active source for drug development. The identification of coumarins from natural sources has been reported for different plant species including a model plant Arabidopsis thaliana. In our previous work, we demonstrated a presence of naturally occurring intraspecies variation in the concentrations of scopoletin and its glycoside, scopolin, the major coumarins accumulating in Arabidopsis roots. Here, we expanded this work by examining a larger group of 28 Arabidopsis natural populations (called accessions) and by extracting and analysing coumarins from two different types of tissues–roots and leaves. In the current work, by quantifying the coumarin content in plant extracts with ultra-high-performance liquid chromatography coupled with a mass spectrometry analysis (UHPLC-MS), we detected a significant natural variation in the content of simple coumarins like scopoletin, umbelliferone and esculetin together with their glycosides: scopolin, skimmin and esculin, respectively. Increasing our knowledge of coumarin accumulation in Arabidopsis natural populations, might be beneficial for the future discovery of physiological mechanisms of action of various alleles involved in their biosynthesis. A better understanding of biosynthetic pathways of biologically active compounds is the prerequisite step in undertaking a metabolic engineering research.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01488-x

Within specialized metabolites, coumarins and furanocoumarins represent a wide group of structurally diverse compounds and are specially produced in plants belonging to the Rutaceae family. Here we performed the furanocoumarin and coumarin-targeted chemical characterization of three Ruteae species collected from Algeria. Detection and quantification of 27 coumarins and furanocoumarins extracted from stems and leaves was carried out by UHPLC-MS. We highlighted significant chemical differences between these plants. Ruta chalepensis L. is the highest producer with 24.83 mmol/g dry material in stems and 15.70 mmol/g dry materials in leaves while Haplophyllum tuberculatum (Forsk.) is the lowest producer. We also showed a surprising chemical diversity between R. chalepensis L and R. angustifolia Pers. This chemical diversity might, therefore, be a helpful tool for phylogenetic identification of plants.

Furanocoumarins are defense molecules mainly described in four plant families that are phylogenetically distant. Molecular characterization of the biosynthetic pathway has been started for many years in Apiaceae and Rutaceae. The results obtained thus far in Apiaceae indicated a major role of cytochromes P450 (P450s) in the CYP71 family. In the present work, we describe the importance of another subfamily of P450s, CYP82D, identified by using a deep analysis of the citrus (Rutaceae) genome and microarray database. CYP82D64 is able to hydroxylate xanthotoxin to generate 5-OH-xanthotoxin. Minor and limited amino acid changes in the CYP82D64 coding sequence between Citrus paradisi and Citrus hystrix provide the enzyme in the latter with the ability to hydroxylate herniarin, but with low efficiency. The kinetic constants of the enzyme are consistent with those of other enzymes of this type in plants and indicate that it may be the physiological substrate. The activity of the enzyme is identical to that of CYP71AZ6 identified in parsnip, showing possible evolutionary convergence between these two families of plants. It is highly possible that these molecules are derived from the synthesis of ubiquitous coumarins throughout the plant kingdom.

The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.

Highlight A strongly iron-responsive gene of previously unknown function, At3g12900, encodes a scopoletin 8-hydroxylase involved in coumarin biosynthesis and plays an important role in the iron uptake strategy in Arabidopsis. Abstract Iron (Fe) deficiency represents a serious agricultural problem, particularly in alkaline soils. Secretion of coumarins by Arabidopsis thaliana roots is induced under Fe-deficiency. An essential enzyme for the biosynthesis of major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6’-Hydroxylase1 (F6′H1) that belongs to a large enzyme family of the 2-oxoglutarate and Fe(II)-dependent dioxygenases. Another member of this family that is a close homologue of F6’H1 and is encoded by a strongly Fe-responsive gene, At3g12900, is functionally characterized in the presented work. We purified the At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin via hydroxylation at the C8-position. Consequently, it was named scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves followed by the metabolite profiling and the biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various regimes of Fe availability. Our results indicate that S8H is involved in coumarin biosynthesis as part of the Fe acquisition machinery.

In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.

SummaryFuranocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co‐localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3‐CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) α‐ketoglutarate‐dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p‐coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.

BACKGROUND:Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects.RESULTS:A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.CONCLUSION:Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.