Biology subjects

Munakata, R.

Publications and source records attributed to Munakata, R..

7 recordsLinked to original sources

bioRxiv2026

Biosynthesis of prenylated flavonoids by two membrane-bound prenyltransferases in glandular trichomes of Macaranga tanarius.

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

biochemistry↗

bioRxiv2026

A membrane-bound aromatic O-prenyltransferase catalyzes the last reaction step in citrus auraptene biosynthesis

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.

biochemistry↗

bioRxiv2026

First O-demethylation activity in Arabidopsis specialized metabolism resolves the missing step in esculetin biosynthesis

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.

plant biology↗

bioRxiv2026

An aromatic substrate prenyltransferase involved in the chemical diversification of flavonoids in Glycyrrhiza glabra

Prenylated isoflavonoids are widely distributed specialized metabolites within the Fabaceae and contribute to various characteristic biological activities for both plants and humans. Several aromatic prenyltransferases (PTs) have been identified in Glycyrrhiza species, which are the most widely consumed crude drugs in traditional Chinese medicine. However, these enzymes do not sufficiently explain the structural diversity of prenylated flavonoids produced in the Glycyrrhiza genus. To identify additional novel PTs, we used elicited cultured Glycyrrhiza glabra roots as source material, in which elicitor treatment of cultured roots increased the accumulation of multiple prenylated flavonoids. To identify the responsible enzyme, PT candidates were screened using G. uralensis transcriptomes, currently the sole publicly available transcriptomic resource within the genus, and a homolog designated GgBSPT1 (BSPT; a broad-substrate prenyltransferase) was subsequently isolated from elicited cultured G. glabra roots. GgBSPT1 differed from previously identified Glycyrrhiza PTs in both amino acid sequence and enzymatic properties. GgBSPT1 catalyzed 3'-prenylation of isoliquiritigenin and 6-prenylation of five flavonoids, i.e., this PT displayed broad substrate acceptance across 20 distinct flavonoid structures. Overall, elicited cultured G. glabra roots enabled the identification of a previously unrecognized PT that is functionally distinct from earlier reported Glycyrrhiza PTs. This study provides a new insight into the metabolic plasticity of Glycyrrhiza species and expands the enzymatic toolkit for future metabolic engineering of prenylated phytochemicals by the unusually broad substrate specificity of GgBSPT1. Main conclusionUsing cultured Glycyrrhiza glabra roots, we identified a new prenyltransferase involved in the formation of a variety of flavonoids, thereby revealing novel prenylated isoflavonoid pathways in licorice.

molecular biology↗

bioRxiv2025

Molecular evolution of terpene synthase underlying the diversification of isoprene emission in Fagaceae

Plants emit a wide range of volatile organic compounds, among which isoprene is the most abundant and atmospherically influential. Although oak species are major contributors to isoprene emission, there is considerable variation in isoprene emission capacity within the Fagaceae family. To unravel the evolutionary origins of isoprene emission, we investigated the molecular evolution of terpene synthase (TPS) genes across eight species within the Fagaceae. We identified a Fagaceae-specific TPS-b subclade in which potential isoprene synthase (IspS) activity evolved independently in two gene lineages within subgenus Quercus. Ancestral sequence reconstruction revealed that the acquisition of a diagnostic amino acid residue for IspS function arose convergently in these lineages and was subject to positive selection, suggesting adaptive evolution. Ancestral-enzyme assays targeting the gene lineage with high gene expression revealed that the early protein primarily produced monoterpenes from geranyl diphosphate (GPP), whereas their descendants shifted substrate preference to dimethylallyl diphosphate (DMAPP), evolving into dedicated isoprene synthases. Our results indicate that IspS activity was not ancestral in Fagaceae, but evolved approximately 56 million years ago within the subgenus Quercus, and has been retained ever since. These findings emphasize the roles of enzyme structural innovation and regulatory shifts in the diversification of volatile terpenoid biosynthesis.

evolutionary biology↗

bioRxiv2025

CRISPR/Cas9-mediated genome editing reveals the involvement of a polyphenol oxidase in the shikonin-specific biosynthesis in Lithospermum erythrorhizon

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon, only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3''-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

biochemistry↗

bioRxiv2025

Molecular basis behind the isoprene emission diversity in Fagaceae

Plants emit a large amount of volatile organic compounds (VOCs) into the atmosphere, reaching approximately 109 tons of carbon per year. These biogenic VOCs exhibit significant chemical diversity, with terpenoids being the dominant group, and isoprene accounting for nearly half of the total biogenic VOCs. Due to its high chemical reactivity, isoprene has a strong impact on atmospheric quality and climate. Quercus species (Fagaceae) are known to be the main isoprene emitters in the Northern Hemisphere. However, isoprene synthase is unknown in the entire Fagaceae family. Notably, even within a single genus such as Quercus, both isoprene-emitting and non-emitting species are present, yet the molecular basis of this dichotomy remains unclear. Here, we report the identification of the IspS gene from the isoprene-emitting species Quercus serrata (QsIspS1) through seasonal transcriptome analysis and its detailed biochemical characterization. We also identified two genes with high sequence similarity to QsIspS1 in the genomes of non-emitting species: Q. glauca (QgIspS1-like) and Lithocarpus edulis (LeIspS1-like). We discovered mutations in these sequences that likely impair their function. Biochemical analysis revealed that QgIspS1-like is a monoterpene synthase, whereas LeIspS1-like is a pseudogene incapable of isoprene synthesis, explaining these plants inability to emit isoprene. Furthermore, site-directed mutagenesis revealed an amino acid that plays a pivotal role in the substrate and product specificities of isoprene synthase. Our findings provide new insight into the molecular mechanisms of isoprene emission diversity in Fagaceae.

biochemistry↗