Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus

Abstract

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

Keywords: O-methyltransferase; citrus; neofunctionalization; polymethoxyflavone; tandemly duplicated gene cluster.

Fig. 1.

Differences in PMF content among citrus species. (A) Citrinae phylogeny and the total PMF content in different citrus accessions. The phylogenetic tree was constructed using genome-wide SNP data from 13 citrus accessions. Bootstrap support values greater than 90 are indicated. Mandarins are highlighted in shaded orange with a rounded rectangle. The log₂-normalized mean total PMF content in the fruit flavedo at 60 DPA is indicated with color in rounded squares to the right of each tree branch. (B) Levels of four main PMFs and total PMF content in the fruit flavedo of wild mandarins and early-admixture mandarins. The chemical structures of NOB, TAN, SIN, 5-DNOB and a fully O-methylated flavone (representing the total PMF content) are shown (Top). Cre, mandarins (C. reticulata). Wild Cre, wild mandarins. Early Cre, early-admixture mandarins. Modern Cre, modern cultivated mandarins. DW indicates dry weight. Each box represents the median and interquartile range. Whiskers extend to 1.5 times the interquartile range.

Fig. 2.

Mapping of the major loci controlling PMF biosynthesis in citrus. (A) Accumulation of PMFs in nine out of 94 F₁ hybrids derived from an RT × TO cross. RP represents F1 individual hybrids. Typical HPLC elution profiles of PMFs from the flavedo of two hybrids (RP77 and RP63) are shown to illustrate the contrast in PMF content among different individuals. (B) BSA of PMF content. The x-axis represents nine scaffolds from C. reticulata Clementine. The y-axis indicates the transformed Δ(SNP index), which represents differences in SNP frequencies between the two extreme PMF phenotype pools. The red horizontal line indicates a significance threshold of 0.25 for the Δ(SNP index). The red and blue arrows indicate that the candidate loci contain or do not contain genes that encode OMTs, respectively. (C) Schematic representation of the candidate genes on scaffold 4. OMT6 is highly orthologous and closely linked to the OMT cluster. (D) Integrated metabolome and transcriptome analysis of OMT genes associated with PMF content. A heatmap illustrates the expression levels of 18 genes in the candidate region of scaffold 4 and the related genes OMT6 and OMT9. The FPKM values of genes were Log₁₀-transformed.

Fig. 3.

Crucial roles of CreOMT3, CreOMT4, and CreOMT5 in PMF biosynthesis. (A–C) Methylation of flavones by recombinant CreOMT3, CreOMT4, and CreOMT5 demonstrated using HPLC. O-methylation reactions were programmed with LYS (A), DMF1 (B), and CIR (C). Products were identified using comparisons to chromatograms (gray) produced with standards. Substrates incubated with recombinant enzymes are indicated in bold above each panel. Reactions catalyzed by either CreOMT3, CreOMT4, or CreOMT5 are indicated with purple, red, or orange, respectively. Empty vector (EV, black chromatograms) was used as a control. (D) Transient overexpression of CreOMT3, CreOMT4, or CreOMT5 in the flavedo of Guoqing No.1 satsuma mandarin. Expression was quantified after infiltration with strains harboring pB121-based constructs that overexpress either CreOMT3, CreOMT4, or CreOMT5 and compared to an empty vector control (EV). (E) PMF profiles from the Guoqing No.1 satsuma mandarin flavedo with overexpression of CreOMT genes. (F) RNAi suppression of CreOMT3, CreOMT4, and CreOMT5 expression in the flavedo of Ota Ponkan mandarin. Expression was quantified after infiltration of the flavedo from Ota Ponkan mandarin with strains harboring pK7GWIWG2D (II)-based constructs that RNAi suppress CreOMT3, CreOMT4 or CreOMT5. (G) PMF profiles of RNAi-suppressed flavedo from Ota Ponkan mandarin fruits. Data are presented as mean values ± SD (n = 3 or 4). *P < 0.05, **P < 0.01. Fresh weight (FW). Abbreviations and MS/MS data for all substrates and products are listed in Dataset S2.

Fig. 4.

Evolutionary history of the OMT cluster and the potential capacity for PMF biosynthesis. (A) Gene family analysis for 12 species. Protein sequences encoded by single-copy orthologous genes were retrieved from 12 species to conduct alignments and reconstruction of a phylogenetic tree. (B) Collinear relationships for OMT genes that contribute to PMF biosynthesis in citrus groups I to IV. The syntenic blocks are connected by light brown lines. The purple, red, orange, and blue blocks with arrows represent OMT3, OMT4, OMT5, and OMT6 (or OMT6-like), respectively. (C) Global view of the candidate domesticated regions in wild and cultivated mandarins. Regions with top 1% XPCLR values were defined as containing domestication signals. The sources of all samples are provided in Dataset S4. Mangshan mandarin is a primitive type. Two distinct domestication events seem to have occurred, leading to two separate groups of cultivated mandarins (MD1 and MD2).

Fig. 5.

Influence of expression profiles and promoter variations in CreOMT3–CreOMT6 on PMF content in different types of citrus germplasm. (A) Total PMF content (Left) and expression levels of OMT3-OMT4-OMT5-OMT6 (Right) in various citrus species. The accession IDs of citrus genotypes listed along the y-axis are defined in Dataset S6. (B) Schematic representation of the promoters and coding sequences of OMT3, OMT4, OMT5, and OMT6 in the four citrus groups (I to IV). M1 to M11 are individual MITEs identified in the promoters of OMT3, OMT4, OMT5, and OMT6 from Group IV. Their precise insertion sites are shown in Dataset S7. The two types of OMT4 promoters found in Ponkan mandarin are indicated with a short red line to the left of the diagram of CrpOMT4. Ab, A. buxfoliata; Pt, P. trifoliata; Ci, C. ichangensis; Cm, C. medica; Ch, C. hongheensis; Cg, C. grandis; Cms, C. mangshanensis; Crm, C. reticulata Mangshan; Crp, C. reticulata Ponkan; Cre, C. reticulata Clementine; Cs, C. sinensis. (C) Amino acid residues that are crucial for O-methylation activity in CreOMT4 and CreOMT6. Site-directed mutagenesis was used to introduce the indicated amino acid substitutions in CreOMT4 and CreOMT6. O-methylation assays were programmed with CreOMT4 and CreOMT6 harboring the indicated amino acid substitutions. Either GAD or 3HF was used as a substrate. Data are presented as mean values ±SD (n = 3). (D) Elevated promoter activity in OMT4 genes containing a 1,041-bp fragment. The OMT4 promoter was amplified from Clementine mandarin (738 bp) and Mangshan mandarin (1,779 bp). Promoter activities were quantified using a dual-luciferase assay. Data are presented as mean values ± SD (n = 3).

Fig. 6.

Evolutionary history of the OMT cluster and OMT6 in citrus. (A) Origin of OMT6 in a proposed primitive citrus-related genus. The gray inverted triangle indicates negligible levels of gene expression. (B) Distribution of OMT6 in early-diverging citrus. The OMT6 gene indicated with a gray box encodes a nonfunctional protein. (C) Distribution of OMT6 in pummelo-citron. (D) Emergence of the OMT cluster and PMF biosynthesis in wild and early-admixture mandarins. (E) Reduction in the OMT cluster expression and PMF content in modern cultivated mandarins and mandarin-derived hybrids. The red box and the white box surrounded by a dashed line indicate the presence and absence, respectively, of a 1,041-bp fragment in the OMT4 promoter. The red gradients in the inverted triangles in (D) and (E) indicate a decline in the expression of the OMT cluster.