Identification and knockout of rhamnose synthase CiRHM1 enhances accumulation of flavone aglycones in chrysanthemum flower

Flavonoids play critical roles in plant adaptation to environmental changes and are valuable medicinal resources (Chagas et al., 2022). Flavonoids are predominantly found in glycosylated forms, which exhibit increased structural complexity, solubility and stability. However, the aglycone forms of flavonoids exhibit greater antioxidant capacity and bioavailability (Xie et al., 2022). Enhancing the content of flavonoid aglycones in crops can improve their nutritional value and health benefits for humans.

In plants, UDP-rhamnose serves as a key sugar donor in flavonoid glycosylation, synthesized from UDP-glucose via the enzyme rhamnose synthase (RHM). In Arabidopsis (Arabidopsis thaliana), mutations in the RHM1 lead to significant reduction in rhamnosylated flavonols (Saffer and Irish, 2018). However, as Arabidopsis lacks flavone synthase and flavones, the impacts of UDP-rhamnose on flavone aglycone or glycoside biosynthesis are unknown.

Chrysanthemum indicum, a notable medicinal plant, has been used in traditional Chinese medicine for over 2000 years (He et al., 2016). The dried flowers of C. indicum, known as ‘Yejuhua’ in the Pharmacopoeia of the People's Republic of China (2020 edition), are recognized for their anti-inflammatory, antioxidant, antimicrobial, anticancer and immunomodulatory properties (Xie et al., 2012). These pharmaceutical effects are largely attributed to the high flavone content, particularly compounds like apigenin, luteolin and their derivatives (Shao et al., 2020). Previous studies have shown significant variations in morphology and metabolic composition among different eco-geographic populations (ecotypes) of C. indicum in China, influencing their medicinal and nutritional value (Fang et al., 2012).

To explore the natural variation of flavones in C. indicum, we collected ecotypes from various regions across China, and quantified the major bioactive flavone, apigenin, in the flowers using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS). A metabolite-based genome-wide association study (mGWAS) was performed on 72 ecotypes exhibiting distinct apigenin content (Figure 1a; Dataset S1). The association analysis showed that the natural variation in apigenin content across these ecotypes was governed by two loci located on chromosomes 2 and 9 (Figure 1b–c). Within the locus on chromosome 2, we identified a TREHALOSE-6-PHOSPHATE SYNTHASE1 gene (CiTPS1, Cse_sc000461.1_g010.1) and CiRHM1 (Cse_sc000461.1_g020.1) (Figure 1b; Table S1). Collinearity analysis indicated that the TPS1-RHM1 gene cluster is conserved among dicotyledonous plants (Figure S1). RNA-seq of 18 ecotypes showed that the expression of CiRHM1 was negatively correlated with apigenin content (Figure 1d, Table S2), while CiTPS1 expression displayed no correlation (Figure S2a, Table S2). Furthermore, we identified one paralog of CiRHM1 located on chromosome 6 of C. indicum genome, named CiRHM1-like (Cse_sc005633.1_g060.1, Figure S3), whose expression showed a lower correlation with apigenin content compared to CiRHM1 (Figure S2b). Therefore, CiRHM1 was selected as a candidate gene for further functional analysis. Subsequent investigation identified the SNP (LG02: 36801176) located within the intron of CiRHM1 (Table S1). Ecotypes with the homozygous T/T genotype at this SNP exhibited significantly higher apigenin content compared to those with the G/G genotype (Figure 1e).

Figure 1

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CiRHM1 influences flavone aglycone content in C. indicum. (a) Frequency distribution of apigenin content across 72 C. indicum ecotypes. Classes of apigenin content are shown on the x-axis, and the corresponding counts of ecotypes are shown on the y-axis. (b) Manhattan plots displaying GWAS signals for SNPs associated with apigenin content. (c) Q–Q plot illustrating the GWAS results for apigenin. (d) Correlation analysis between the expression levels of CiRHM1 and apigenin content. (e) Boxplot demonstrating significant associations between apigenin content and the SNP LG02:36801176. (f) Genotyping of Cirhm1 mutants. (g) Phenotypes of WT and Cirhm1 mutants. (h) Plant height measurements of WT and Cirhm1 mutants. (i) Rhamnose and UDP-glucose contents in the flowers of WT and Cirhm1 mutants. (j) Flavone aglycone content in the flowers of WT and Cirhm1 mutants. Data are presented as mean ± SD from three biological replicates. Statistical significance between WT and Cirhm1 mutants was determined using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

We knocked out CiRHM1 in C. indicum using our CRISPR/Cas9 gene editing platform (Liu et al., 2023). Two single-guide RNAs (sgRNAs) were designed to specifically target the first exon of the CiRHM1 gene, and two Cirhm1 mutants were generated (Figure 1f). RT-qPCR results showed that knocking out CiRHM1 significantly reduced its expression in Cirhm1 mutants, while the expression levels of other RHM family genes remained unchanged (Figure S4). Although no significant differences in flowering were observed between the wild-type (WT) and mutants, the mutant plants exhibited reduced height compared to the WT (Figure 1g,h).

We analysed the sugar profiles of flowers from both WT and Cirhm1 mutants (Dataset S2). The results showed a significant decrease in L-rhamnose and UDP-rhamnose levels in Cirhm1 mutants compared to WT (Figure 1i), confirming the role of CiRHM1 as a rhamnose synthase. Consistently, the levels of trehalose, glucose and sucrose were significantly higher in the flowers of Cirhm1 mutants than in WT (Figure S5).

Next, we measured the flavonoid content in the flowers of WT and Cirhm1 mutants (Dataset S3). Flavone rhamnoside levels were significantly reduced in both Cirhm1 mutants (Figure S6). In contrast, the content of flavone aglycones, including apigenin, luteolin, norwogonin, diosmetin and hispidulin, was significantly higher in Cirhm1 mutants compared to WT (Figure 1j). Additionally, levels of flavone glucosides, such as apigenin 7-glucoside, luteolin 7-glucoside, genistein 7-glucoside and wogonin 7-O-glucuronide, were also elevated in Cirhm1 mutants compared to WT (Figure S7).

In summary, our study demonstrates that CiRHM1 influences flavone aglycone content in C. indicum. Furthermore, we developed novel germplasm of C. indicum with enhanced flavone aglycone content through genome editing.