Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene-metabolite correlations in Arabidopsis

Abstract

To complete the metabolic map for an entire class of compounds, it is essential to identify gene-metabolite correlations of a metabolic pathway. We used liquid chromatography-mass spectrometry (LC-MS) to identify the flavonoids produced by Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-O-arabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase. Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDP-rhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the identification of a gene function and a more complete understanding of a plant metabolic network.

Figure 1.

The Arabidopsis Flavonoid Biosynthetic Pathway. 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; OMT1, O-methyltransferase; UGT, UDP-dependent glycosyltransferase; DFR, dihydroflavonol 4-reductase; LDOX/ANS, leucoanthocyanidin dioxygenase/anthocyanidin synthase; AAT, anthocyanin acyltransferase.

Figure 2.

UPLC-PDA-MS Analyses of the Extracts from Leaves, Flowers, Roots, and Stems of Wild-Type (Col-0) and tt4 Plants. (A) UPLC-PDA and mass chromatograms of aqueous methanol extracts of the Arabidopsis wild type and the tt4 knockout mutant. Absorbance at 320 nm was used for the detection of flavonols. Labels correspond to compounds shown in Figure 3. (B) Principal component analyses of the data obtained by LC-MS. Proportions of the first and second components are in parentheses. Wild-type results are in red, and those from the tt4 mutant are in blue.

Figure 3.

Flavonol Glycosides in Arabidopsis. Asterisks indicate that the compounds were identified based on a comparison of retention times and UV/mass spectra of the standards used in this study. R₁=H, kaempferol; R₁=OH, quercetin; R₁=OMe, isorhamnetin. The presence of a compound in the indicated tissue is denoted by a gray box.

Figure 4.

UPLC-PDA-MS Analyses of the Extracts from Flowers of Wild-Type (Col-0) and Flavonoid-Defective Mutants. Absorbance at 320 nm was used for detection. A5GlcT, anthocyanin 5-O-glucosyltransferase; F3RhaT, flavonol 3-O-rhamnosyltransferase; F7RhaT, flavonol 7-O-rhamnosyltransferase; Fd3GlcT, flavonoid 3-O-glucosyltransferase. Labels correspond to compounds shown in Figure 3. The labels in parentheses indicate compounds at <5% in the peak.

Figure 5.

A T-DNA Insertion Mutant of UGT78D3. (A) Schematic representation of UGT78D3 with a T-DNA insertion mutant used in this work. The thick black line indicates coding sequence. Numbers indicate the position of the T-DNA insertion. The gray box adjacent to T-DNA indicates the region containing the UGT78D3 promoter from −77 bp to +4 bp. LB, left border; RB, right border. (B) Real-time PCR analysis of UGT78D3 transcripts in organs of the Arabidopsis wild type (Col-0). Error bars represent sd of three technical replicates per sample. (C) Real-time PCR analysis of UGT78D3 transcripts in wild-type (Col-0) and ugt78d3 mutant flowers. Error bars represent sd of three technical replicates per sample. (D) Extracted fragment mass chromatograms of aqueous methanol extracts from flowers of the wild type (Col-0), the ugt78d3 mutant, and ugt78d3 complemented with p35S:UGT78D3. The extracted fragment mass ions m/z 565, m/z 581, and m/z 595 correspond to compounds f19, f25, and f29 in Figure 3, respectively.

Figure 6.

UPLC Analyses of the Reaction Products of UGT78D3 Recombinant Protein. Elution profiles of reaction products of GST protein (control) or GST-fused UGT78D3 protein (UGT78D3) and the standards (quercetin 3-O-arabinoside) are shown.

Figure 7.

UPLC-MS Analyses of the Reaction Products of UGT78D3 or UGT89C1 Recombinant Protein. Extracted fragment mass chromatograms of aqueous methanol extracts from flowers of wild type (Col-0) ([A] and [F]), the reaction products of UGT89C1 with kaempferol 3-O-arabinoside (B), (A) coeluted with (B) (C), the reaction products of UGT78D3 with kaempferol 7-O-rhamnoside (D), (A) coeluted with (D) (E), the reaction products of UGT89C1 with quercetin 3-O-arabinoside (G), and (F) coeluted with (G) (H). The extracted fragment mass ions m/z 565 ([A] to [E]) and m/z 581 ([F] to [H]) correspond to compounds f19 and f25 in Figure 3, respectively. Kae 3-Ara, kaempferol 3-O-arabinoside; Kae 7-Rha, kaempferol 7-O-rhamnoside; Que 3-Ara, quercetin 3-O-arabinoside.

Figure 8.

T-DNA Insertion Mutants of RHM1 and RHM2, and UPLC-MS Analyses of the rhm Mutant Lines. (A) Real-time PCR analysis of RHMs transcripts in Arabidopsis organs. Error bars represent sd of three technical replicates per sample. (B) Schematic representation of RHM1 and RHM2 with the mutations used in this work. rol1-1, a nonsense mutation at position 318; rol1-2, a missense mutation changing an Arg at position 283 to a Lys; rhm2-1 and rhm2-3, T-DNA insertion mutants. LB/RB, left/right borders. Numbers indicate the position of T-DNA insertion. (C) UPLC-PDA-MS analyses of the extracts from leaves and flowers in the wild type (Col-0) and the rhm mutants. Absorbance at 320 nm was used for detection. Labels correspond to compounds shown in Figure 3. The labels in parentheses indicate compounds at <5% in the peak. (D) The ratio of flavonol derivatives to total flavonol in leaves of the wild type (Col-0) and the mutants. Error bars represent sd for five independent experiments. (E) The ratio of flavonol derivatives to total flavonol in flowers of the wild type (Col-0) and the mutants. Error bars represent sd for five independent experiments. N.D., not determined.

Figure 9.

Proposed Comprehensive Flavonol Pathway in Arabidopsis. Each number in blue corresponds to the compounds shown in Figure 3. The compounds detected in Arabidopsis are shown in a blue background. Dotted lines indicate proposed but unidentified pathways. F3AraT, flavonol 3-O-arabinosyltransferase; Fd3GlcT, flavonoid 3-O-glucosyltransferase; F3RhaT, flavonol 3-O-rhamnosyltransferase; F7GlcT, flavonol 7-O-glucosyltransferase; F7RhaT, flavonol 7-O-rhamnosyltransferase; Ara, arabinose; DeoxyHex, deoxyhexose; Glc, glucose; Hex, hexose; Rha, rhamnose.