Rhamnosyltransferases involved in the biosynthesis of flavone rutinosides in Chrysanthemum species

Abstract

Linarin (acacetin-7-O-rutinoside), isorhoifolin (apigenin-7-O-rutinoside), and diosmin (diosmetin-7-O-rutinoside) are chemically and structurally similar flavone rutinoside (FR) compounds found in Chrysanthemum L. (Anthemideae, Asteraceae) plants. However, their biosynthetic pathways remain largely unknown. In this study, we cloned and compared FRs and genes encoding rhamnosyltransferases (RhaTs) among eight accessions of Chrysanthemum polyploids. We also biochemically characterized RhaTs of Chrysanthemum plants and Citrus (Citrus sinensis and Citrus maxima). RhaTs from these two genera are substrate-promiscuous enzymes catalyzing the rhamnosylation of flavones, flavanones, and flavonols. Substrate specificity analysis revealed that Chrysanthemum 1,6RhaTs preferred flavone glucosides (e.g. acacetin-7-O-glucoside), whereas Cs1,6RhaT preferred flavanone glucosides. The nonsynonymous substitutions of RhaTs found in some cytotypes of diploids resulted in the loss of catalytic function. Phylogenetic analysis and specialized pathways responsible for the biosynthesis of major flavonoids in Chrysanthemum and Citrus revealed that rhamnosylation activity might share a common evolutionary origin. Overexpression of RhaT in hairy roots resulted in 13-, 2-, and 5-fold increases in linarin, isorhoifolin, and diosmin contents, respectively, indicating that RhaT is mainly involved in the biosynthesis of linarin. Our findings not only suggest that the substrate promiscuity of RhaTs contributes to the diversity of FRs in Chrysanthemum species but also shed light on the evolution of flavone and flavanone rutinosides in distant taxa.

Figure 1

Geographical distribution of and morphological variations among diploid C. indicum, tetraploid C. indicum, and diploid C. nankingense. A, Chrysanthemum indicum population from Huangshi, Hubei Province; Anqing, Anhui Province; Jinzhong, Shanxi Province; Guangzhou, Guangdong Province; Yichun, Jiangxi Province; and Huanggang, Hubei Province; C. nankingense population from Huanggang, Hubei Province and Nanjing, Jiangsu Province. B, Geographical distribution of the eight accessions of Chrysanthemum in China. C, Somatic chromosomes at the mitotic metaphase of the representative diploid and tetraploid C. indicum. The chromosome number of C. indicum (GD) was 2n = 36 (top panel) and that of C. indicum (HB) was 2n = 18 (bottom panel). Bar: 5 µm.

Figure 2

Major flavone glycosides accumulated in the flowers of different accessions of Chrysanthemum plants. A, Extracted-ion chromatogram of flavone glucosides and rutinosides standards. B, Heatmap visualization indicated the metabolic abundance of targeted FGs and FRs in different accessions of eight Chrysanthemum plants. The contents of major FG and FR compounds ranged from 0 to 6000 μg/g dry weight (DW). Linarin was highly accumulated in the C. indicum (HB_2x). Each datum is averaged from triplicates. N.D. indicates “not detected.”

Figure 3

Molecular phylogenetic tree of Chrysanthemum RhaTs and other known branch-forming glycosyltransferases. A, Multiple sequences were aligned using Clustal W and used for tree construction with the maximum-likelihood method using MEGA7 and modified by iTOL. Bootstrap values (based on 1,000 replications) are indicated at each node. Chrysanthemum RhaTs are labeled with red subclades. The UGT79 family members are labeled with blue dots. Functional clade I of 1,2/1,6 branch-forming flavonoid UGTs is shaded yellow and detailed information is provided in Supplemental Table S3. Abbreviations and Genbank accession numbers of clades II–IV are as follows: A. thaliana 3RhaT (AEE31240), Petunia hybrida 3GlcT (AAD55985), Citrus paradise 3GlcT (GQ141630), G. max 3GlcT (P16166), A. thaliana 5Glc (AEE83370), Perilla frutescens 5GlcT (BAA36421), Glandularia hybrida 5GlcT (BAA36423), Nicotiana tabacum 7GlcT (AAB36653), and A. thaliana 7GlcT (AAL90934). GlcT, glucosyltransferase; XylT, xylosyltransferase; and GAT, galacturonic acid transferase. B, Schematic representation of the possible convergent evolution of RhaT proteins into 1,6-RhaTs in Chrysanthemum and Citrus plants. Enzymes labeled in the red star represent Cs1,6RhaT, flavanone-7-O-glucoside-1,6-RhaT from oranges (C. sinensis), Cm1,6RhaT, and flavanone-7-O-glucoside-1,6-RhaT from pummelo (C. maxima), whereas the putative RhaTs of Chrysanthemum are shown in the red subclade of (A). GT, glycosyltransferases; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; and CHI, chalcone isomerase.

Figure 4

In vitro enzyme assays indicated that some RhaTs can catalyze FGs to the corresponding FRs (i.e. linarin, isorhoifolin, and diosmin). The reaction catalyzed by Chrysanthemum RhaTs involves the transfer of rhamnose from a sugar donor, UDP-Rha, to acceptor substrates. The MS/MS spectrum of the standard and products in the reaction of RhaTs. The selected acceptor substrates include acacetin-7-O-glucoside (A), apigenin-7-O-glucoside (B), and diosmetin-7-O-glucoside (C).

Figure 5

Sugar acceptor specificity of RhaTs of Chrysanthemum and Citrus plants. A, The relative percent conversions of rhamnosylated products catalyzed by RhaTs. The rhamnosylating activity toward acacetin-7-O-glucoside is considered to be 100%. Error bars represent the standard deviation from three replicates, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with acacetin-7-O-glucoside (1) by Dunnett’s multiple comparison test. N.D. indicates “not detected.” B, Chemical structures of substrates used as rhamnosyl acceptors.

Figure 6

Kinetic analysis of Chrysanthemum RhaTs by using acacetin-7-O-glucoside (1), apigenin-7-O-glucoside (2), and diosmetin-7-O-glucoside (3) as acceptors. Data are means ± sd of three independent experiments.

Figure 7

In vivo functions of CiRhaT-GD_4x enzymes in C. indicum. A–C, Overexpression of CiRhaT-GD_4x in hairy root cultures by A. rhizogenes. A, Hairy root cultures of C. indicum. Scale bar: 5 cm. B, Expression level of CiRhaT in transgenic hairy roots. C, Effect of the overexpression of CiRhaT on the biosynthesis of FRs. Data are presented as the mean ± sd (n = 3 biologically independent samples). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the WT group by using Student’s t test. D–F, Overexpression of CiRhaT-GD_4x in Chrysanthemum plant by Agrobacterium tumefaciens. D, The phenotype of the transgenic line. E, Comparative expression analysis of RhaT in transgenic and WT plants through RT-qPCR. F, Flavonoid rutinoside contents in the transgenic lines. For (E) and (F), experiment was performed in triplicate and error bars represent standard deviation, asterisks indicate a significant difference from WT control line (*P < 0.05, ***P < 0.001) analyzed through one-way ANOVA with Dunnett’s multiple comparison test.

Figure 8

Biosynthetic pathway of proposed FRs in Chrysanthemum and specialized flavonone rutinosides in Citrus. Genes encoding OMT, 7GlcT, and F3ʹH were presumably involved in pharmaceutically active linarin, isorhoifolin, and diosmin biosynthesis. Dashed arrows indicate proposed steps. Enzymes highlighted in red represents 1,6RhaT that has been biochemically characterized in this study. FNS II, flavonoid synthase II; F3ʹH, flavonoid-3ʹ-hydroxylase; 1,6RhaT, flavonoid-7-O-glucoside-1,6-RhaT; Cm1,6RhaT, flavanone-7-O-glucoside-1,6-RhaT from pummelo (Citrus maxima); Cs1,6RhaT, flavanone-7-O-glucoside-1,6-RhaT from oranges (Citrus sinensis).