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. 2022 Sep 20:13:985572.
doi: 10.3389/fpls.2022.985572. eCollection 2022.

Integrated metabolomics and transcriptomics insights on flavonoid biosynthesis of a medicinal functional forage, Agriophyllum squarrosum (L.), based on a common garden trial covering six ecotypes

Tingzhou Fang  1 , Shanshan Zhou  2 , Chaoju Qian  1 , Xia Yan  3   4 , Xiaoyue Yin  1 , Xingke Fan  1 , Pengshu Zhao  1 , Yuqiu Liao  1 , Liang Shi  1 , Yuxiao Chang  5 , Xiao-Fei Ma  1   4
Affiliations

Integrated metabolomics and transcriptomics insights on flavonoid biosynthesis of a medicinal functional forage, Agriophyllum squarrosum (L.), based on a common garden trial covering six ecotypes

Tingzhou Fang et al. Front Plant Sci. .

Erratum in

Abstract

Agriophyllum squarrosum (L.) Moq., well known as sandrice, is an important wild forage in sandy areas and a promising edible and medicinal resource plant with great domestication potential. Previous studies showed flavonoids are one of the most abundant medicinal ingredients in sandrice, whereby isorhamnetin and isorhamnetin-3-glycoside were the top two flavonols with multiple health benefits. However, the molecular regulatory mechanisms of flavonoids in sandrice remain largely unclear. Based on a common garden trial, in this study, an integrated transcriptomic and flavonoids-targeted metabolomic analysis was performed on the vegetative and reproductive periods of six sandrice ecotypes, whose original habitats covered a variety of environmental factor gradients. Multiple linear stepwise regression analysis unveiled that flavonoid accumulation in sandrice was positively correlated with temperature and UVB and negatively affected by precipitation and sunshine duration, respectively. Weighted co-expression network analysis (WGCNA) indicated the bHLH and MYB transcription factor (TF) families might play key roles in sandrice flavonoid biosynthesis regulation. A total of 22,778 differentially expressed genes (DEGs) were identified between ecotype DL and ecotype AEX, the two extremes in most environmental factors, whereby 85 DEGs could be related to known flavonoid biosynthesis pathway. A sandrice flavonoid biosynthesis network embracing the detected 23 flavonoids in this research was constructed. Gene families Plant flavonoid O-methyltransferase (AsPFOMT) and UDP-glucuronosyltransferase (AsUGT78D2) were identified and characterized on the transcriptional level and believed to be synthases of isorhamnetin and isorhamnetin-3-glycoside in sandrice, respectively. A trade-off between biosynthesis of rutin and isorhamnetin was found in the DL ecotype, which might be due to the metabolic flux redirection when facing environmental changes. This research provides valuable information for understanding flavonoid biosynthesis in sandrice at the molecular level and laid the foundation for precise development and utilization of this functional resource forage.

Keywords: Agriophyllum squarrosum; common garden; flavonoids; isorhamnetin; isorhamnetin-3-glycoside; metabolome; sandrice; transcriptome.

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Conflict of interest statement

Authors XY and X-FM were employed by the Marsgreen Biotech Jiangsu Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Map of samples collected and the contents of flavonoids in the 12 samples. (A) Original habitats of the six ecotypes and the location of the common garden trials are marked as stars and dots, respectively. The base map showed the gradient of annual precipitation, from high (blue) to low (red). (B) The number in each cell is the average content in μg/g. Modified from Zhou et al. (2021a). Stars are locations of original habitats of each sample.
FIGURE 2
FIGURE 2
Pairwise comparison of DEG (A) and DAF (B). Up- and downregulated genes or flavonoids are illustrated in orange and turquoise, respectively.
FIGURE 3
FIGURE 3
Module–trait relationships, partial. This figure shows relationships between 50 module eigengenes and the top six flavonoids. The complete module-trait relationships involving all the 26 detected flavonoids was provided in Supplementary Figure 6. The correlation and p-value of corresponding flavonoids and gene modules are shown as numbers up and down in each cell, respectively. The color scale on the right side represents the module–trait correlation from −1 (blue) to 1 (red).
FIGURE 4
FIGURE 4
Hub genes identified in flavonoids-correlated modules. Hub genes in (A) MEblue, (B) MEskyblue, and (C) MEfloralwhite, respectively. Flavonoid biosynthesis genes, transcription factors or regulators, and other high connectivity genes are shown as orange dots, blue triangle, and yellow diamonds, respectively. Shape size indicated connectivity, while line width indicated the weight of edges.
FIGURE 5
FIGURE 5
Phylogenetic relationship of 33 candidate AsOMT and 45 known OMTs in other plants. CCoAOMT subfamily, PFOMT subclade, and COMT subfamily are colored in orange, yellow, and blue, respectively. Sequences marked with triangles are the reported isorhamnetin biosynthesis genes.
FIGURE 6
FIGURE 6
AsOMT gene family identification. From left to right, the three columns showed the phylogenetic tree, heatmap of expression profiles (transcripts per million, TPM) in each of the 12 samples, and conserved domain visualization of the 33 candidates AsOMT, respectively. In the phylogenetic tree, the AsPFOMT subclade, AsCCoAOMT subfamily, and AsCOMT subfamily are shaded in yellow, orange, and blue, respectively. Numbers in each cell of the expression profile heatmap are genes’ original TPM values. Key conserved domains are shown as different colored bars, according to the actual length of peptides.
FIGURE 7
FIGURE 7
Flavonoid biosynthesis pathway in sandrice. Detected flavonoids and DEGDLRvsAEXR in this research are shown in bold fonts, black and red, respectively. The structural formula of the corresponding compound is also presented. DAF between AEXR and DLR are shown as dots, whereby red and gray dots indicate up- and downregulated flavonoids in DLR than in AEXR, respectively. The red cross and green curved arrow indicate the possible mechanism of trade-off between rutin and isorhamnetin biosynthesis. 4CL, 4-coumarate–CoA ligase; BGLB, Beta-glucosidase; C3′H, 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase; C4H, Trans-cinnamate 4-monooxygenase; CAD, Cinnamyl alcohol dehydrogenase; CCoAOMT, Caffeoyl-CoA O-methyltransferase; CHI, Chalcone-flavanone isomerase; CHS, Chalcone synthase; COMT, Caffeic acid 3-O-methyltransferase; CYP75A, Flavonoid 3′,5′-hydroxylase; CYP75B1, Flavonoid 3′-monooxygenase; CYP93B2, Flavone synthase II; CYP93C, 2-hydroxyisoflavanone synthase; CYP98A2, Cytochrome P450 98A2; F3H, Flavanone 3-dioxygenase; F3′H, Flavonoid 3′hydroxylase; F5H, Cytochrome P450 84A1; F6H, Feruloyl-CoA 6-hydroxylase; FG2, Flavonol-3-O-glucoside L-rhamnosyltransferase; FLS, Flavanol synthase; FN3′OMT, Flavone 3′-O-methyltransferase; FNSI, Flavone synthase I; GHF, Glycosyl hydrolase family protein; GTF, Glycosyltransferase; HCT, Shikimate O-hydroxycinnamoyltransferase; HI4OMT, 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase/isoflavone 4′-O-methyltransferase; HIDH, 2-hydroxyisoflavanone dehydratase; I3′OMT, Isoorientin 3′-O-methyltransferase; INMT, Amine N-methyltransferase OMT, SAM-dependent O-methyltransferase; PAL, Phenylalanine ammonia-lyase; POD, Peroxidase; ROMT, Tricin synthase; TaOMT, Tricetin 3′,4′,5′-O-trimethyltransferase; UGT73C6, Flavonol-3-O-L-rhamnoside-7-O-glucosyltransferase; UGT78D1, Flavonol-3-O -rhamnosyltransferase; UGT78D2, Flavonol-3-O-glycosyltransferase; EC:3.2.1.168, Hesperidin 6-O-alpha-L-rhamnosyl-beta-D-glucosidase.
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