Rhamnosyltransferases in Chrysanthemum: Just a spoonful of sugar helps the flavonoid-based medicines abound

Members of the cosmopolitan genus Chrysanthemum (Asteraceae) are readily recognizable and highly valued for their showy composite inflorescences. With only around 40 species, the Chrysanthemum boasts numerous naturally grown and cultivated varieties. Thanks to thousands of years of ornamental, medicinal, culinary, and cultural uses of Chrysanthemum by humans, their extensively studied morphology and ploidy diversity continue to attract the attention of plant breeders and scientists alike (Su et al., 2019). However, much less is known about how Chrysanthemum plants synthesize their bioactive natural products, including the pharmaceutically important flavone glycosides. In this issue of The Plant Physiology, Wu et al. (2022) report the discovery of a group of closely related flavone rhamnosyltransferases in Chrysanthemum with implications for understanding flavonoid metabolism in plants and accessing nature’s chemical diversity for human health.

Flavones make up one structural subclass of flavonoids, a group of more than 8,000 compounds that serve as pigments and in UV filtration among other functions in plants. These ubiquitous small molecules are also associated with low risks of several illnesses, including cancer and cardiovascular diseases in humans. Flavones and other flavonoids are usually glycosylated, and the attached sugar moieties can alter the activities and bioavailability of the compounds. Thus, understanding glycosyltransferases is critical for unlocking and engineering flavonoid metabolism (Slámová et al., 2018).

The goal of Wu et al. (2022) was to shed light on the biosynthesis of linarin, isorhoifolin, and diosmin, three flavone rutinosides in Chrysanthemum with purported activities against Alzheimer’s disease, diabetes, and venous diseases (Mottaghipisheh et al., 2021; Gerges et al., 2022). The sugar moiety rutinoside is composed of a rhamnose and a glucose connected by an α(1→6) glycosidic bond. While glucosyltransferases that add glucose to the core flavonoid rings are common and extensively studied, the authors were after the more elusive rhamnosyltransferases that add rhamnose to the flavonoid-bonded glucose.

Focusing first on the tetraploid Chrysanthemum indicum because of its available transcriptomic data, Wu et al. (2022) searched for 1,6-rhamnosyltransferase (1,6RhaT) candidate genes using known 1,6RhaTs of different flavonoid classes in other plant families. The group found a full-length gene with a high number of RNA-seq raw reads and a translated amino acid sequence sharing 51% or higher identity with the known 1,6RhaTs. When expressed in Escherichia coli, the putative 1,6RhaT indeed yielded linarin, isorhoifolin, and diosmin from their corresponding monoglucoside precursors (Figure 1). Elevated levels of the three flavone rutinosides in C. indicum hairy roots were also observed when the identified gene was transformed and expressed in planta. Furthermore, using homology searches, Wu et al. (2022) identified 1,6RhaTs in five other C. indicum accessions (diploid and tetraploid) and two Chrysanthemum nankingense accessions (diploid). Of these, five displayed the same function as that of the originally identified C. indicum 1,6RhaT.

Figure 1.

Chrysanthemum 1,6RhaT in flavonoid diversification. The simplified C6–C3–C6 structure with two aromatic rings and an oxygen-containing heterocycle represents the core flavonoid scaffold. The types, numbers, and positions of sugar moieties (shown in red and blue colors) attached to the core structure affect the bioactivities and provide gateways to further diversification of flavonoids. Highlighted arrows and structures represent reactions catalyzed by the substrate-promiscuous Chrysanthemum 1,6RhaTs, which yield products native (linarin, isorhoifolin, and diosmin) and non-native to Chrysanthemum. The picture of the Chrysanthemum inflorescence is adapted from Wu et al. (2022).

The fact that Chrysanthemum 1,6RhaTs accept different flavone glucosides begged the question about the degree of their substrate promiscuity. To probe the answer, Wu et al. (2022) tested the identified 1,6RhaTs against flavonoid glucoside substrates that are not flavones. The authors found that the Chrysanthemum 1,6RhaTs also accept flavanone and flavonol glucosides despite differences in the flavonoid core scaffold and substitution positions of the glucose moiety (Figure 1). When assessing the previously reported 1,6RhaTs from sweet orange (Citrus sinensis) and pomelo (Citrus maxima) (Frydman et al., 2013; Ohashi et al., 2016), Wu et al. (2022) found that these enzymes are substrate promiscuous as well. A closer examination of the conversion rates showed that while all these enzymes have a wide substrate spectrum, Citrus 1,6RhaTs prefer flavanone over flavone substrates, and the reverse is true for the Chrysanthemum enzymes. These data were somewhat expected as flavanones are the major flavonoids in Citrus while flavones predominate in Chrysanthemum. Nevertheless, the results suggest that the common ancestral enzyme of Citrus and Chrysanthemum 1,6RhaTs likely tolerated a substantial range of substrates, and different evolutionary paths have led to different substrate preferences in descendants.

Wu et al. (2022) have successfully elucidated a critical step in the generally obscure biosynthesis of linarin, isorhoifolin, and diosmin in Chrysanthemum. The authors also advanced our understanding in the realm of natural product glycosylation with a set of branch-forming glycosyltransferases and their substrate tolerance. “Just a spoonful of sugar” moieties in various ways and numbers can enormously expand the chemical space of flavonoids and natural products, yet most glycosyltransferases have not been characterized (Lombard et al., 2014). Identifying additional glycosyltransferases such as Chrysanthemum 1,6RhaTs is ever more necessary for structural and functional alterations of glycosylated natural products, including flavonoids.

Although it is unclear whether the 1,6RhaTs from this study can accept flavonoids beyond flavones, flavanones, and flavonols, the demonstrated substrate tolerance might already be sufficient to provide an evolutionary advantage. Promiscuous enzymes can be recruited and optimized for emerging pathways as reported in other classes of metabolites in the family Asteraceae and elsewhere (Khersonsky and Tawfik, 2010; Nguyen et al., 2019). Given the diversity of flavonoid profiles and ploidy levels of the Chrysanthemum accessions as observed by Wu et al., (2022), there might be other variants of 1,6RhaTs in these species. This possibility is bolstered by the finding that there are accessions that produce flavone rutinosides but in which the 1,6RhaT homologs found by the authors are nonfunctional. Further genomic and functional analysis will also reveal any divergent evolution of 1,6RhaTs, particularly in accessions with more than two sets of chromosomes. Comparative structure–function studies of 1,6RhaT variants will inform our future glycosyltransferase engineering efforts toward desirable products. Such information also provides insights into the chemical diversification of Chrysanthemum and the Asteraceae, the second largest flowering plant family.

Conflict of interest statement. None declared.