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. 2021 Aug 15;22(16):8751.
doi: 10.3390/ijms22168751.

Integrated Metabolomic and Transcriptomic Analysis Reveals the Flavonoid Regulatory Network by Eutrema EsMYB90

Yuting Qi  1 , Chuanshun Li  1 , Chonghao Duan  1 , Caihong Gu  1 , Quan Zhang  1
Affiliations

Integrated Metabolomic and Transcriptomic Analysis Reveals the Flavonoid Regulatory Network by Eutrema EsMYB90

Yuting Qi et al. Int J Mol Sci. .

Abstract

Flavonoids are representative secondary metabolites with different metabolic functions in plants. Previous study found that ectopic expression of EsMYB90 from Eutremasalsugineum could strongly increase anthocyanin content in transgenic tobacco via regulating the expression of anthocyanin biosynthesis genes. In the present research, metabolome analysis showed that there existed 130 significantly differential metabolites, of which 23 metabolites enhanced more than 1000 times in EsMYB90 transgenic tobacco leaves relative to the control, and the top 10 of the increased metabolites included caffeic acid, cyanidin O-syringic acid, myricetin and naringin. A total of 50 markedly differential flavonoids including flavones (14), flavonols (13), flavone C-glycosides (9), flavanones (7), catechin derivatives (5), anthocyanins (1) and isoflavone (1) were identified, of which 46 metabolites were at a significantly enhanced level. Integrated analysis of metabolome and transcriptome revealed that ectopic expression of EsMYB90 in transgenic tobacco leaves is highly associated with the prominent up-regulation of 16 flavonoid metabolites and the corresponding 42 flavonoid biosynthesis structure genes in phenylpropanoid/flavonoid pathways. Dual luciferase assay documented that EsMYB90 strongly activated the transcription of NtANS and NtDFR genes via improving their promoter activity in transiently expressed tobacco leaves, suggesting that EsMYB90 functions as a key regulator on anthocyanin and flavonoid biosynthesis. Taken together, the crucial regulatory role of EsMYB90 on enhancing many flavonoid metabolite levels is clearly demonstrated via modulating flavonoid biosynthesis gene expression in the leaves of transgenic tobacco, which extends our understanding of the regulating mechanism of MYB transcription factor in the phenylpropanoid/flavonoid pathways and provides a new clue and tool for further investigation and genetic engineering of flavonoid metabolism in plants.

Keywords: EsMYB90; Eutrema salsugineum; flavonoid pathway; integrated analysis; metabolites; metabolome; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Content analysis of pigments and total flavonoids in EsMYB90 transgenic tobacco and wild type. (A) Level of anthocyanin; (B) total flavonoid content in young leaves (YL), mature leaves (ML), roots and stems; (C) chlorophyll content; and (D) proanthocyanin content in mature leaves (ML) of transgenic line 2 (L2), line 4 (L4) and wild type (WT). The values are means ± SD of three independent biological replicates. Statistical significance: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05. Bar = 1 cm.
Figure 2
Figure 2
Volcano plot and classification of differential metabolites in leaves of EsMYB90 transgenic tobacco relative to wild type. (A) The volcano plot exhibits metabolites detected in metabolome. The abscissa represents the logarithm of the quantitative difference multiples of metabolites (log2FC); the ordinate represents the variable importance in project (VIP) value. Green dots represent the down-regulated differential metabolites; red dots represent the up-regulated differential metabolites; black dots represent the metabolites that were detected with no significant difference. (B) Classification of the differential metabolites. The ordinate represents the name of the metabolites and the abscissa represents the number of metabolites. The red represents up-regulated metabolites and the green represents down-regulated metabolites.
Figure 3
Figure 3
Correlation analysis of significantly differential metabolites and DEGs in flavonoid biosynthesis pathways in leaves of EsMYB90 transgenic tobacco. (A) The map of integrative analysis in flavonoid metabolites and their biosynthesis-related key enzymes. Red box: significantly up-regulated DEGs encoding corresponding enzyme; green box: significantly down-regulated DEGs encoding corresponding enzyme; red/green box: significantly up/down-regulated DEGs encoding corresponding enzyme; black box: the enzyme encoding genes detected but with no significant difference. Red dot: significantly up-regulated differential metabolites; black circle: no significantly differential metabolites. Blue arrow lines: ko00940; black arrow lines: ko00941; purple arrow lines: ko00942; light blue arrow lines: ko00943; yellow arrow lines: ko00944. (B) Heatmap of DEGs enriched in flavonoid biosynthesis pathways. The color bar represents the logarithm of gene expression foldchange (log2FC), where the red indicates up-regulated genes and the blue indicates down-regulated genes. (C) Heatmap of differential metabolites annotated in flavonoid biosynthesis pathways. The color bar represents the level of metabolites, where the red indicates the metabolites with a higher level, and the blue indicates metabolites with a lower level.
Figure 4
Figure 4
EsMYB90 activates transcription of flavonoid biosynthesis genes to modulate flavonoid metabolites in tobacco (Nicotiana benthamiana) leaves. (A) MYB-binding motifs analysis in NtANS promoter region. (B) MYB-binding motifs analysis in NtDFR promoter region. (A,B): Triangle indicates the predicted MYB recognized cis-elements. (C) Relative luciferase activity (LUC/REN) of NtANS promoter (pNtANS) by EsMYB90. (D) Relative luciferase activity (LUC/REN) of NtDFR promoter (pNtDFR) by EsMYB90. (E) Correlation network between EsMYB90 gene and the differential metabolites. (F) Correlation network analysis of differential flavonoid metabolites and differential expression genes in phenylpropanoid/flavonoid biosynthesis pathways. (E,F): Red oval: significantly differential metabolites; blue oval: differential expression genes (DEGs); grey straight line indicates the correlations between the differential metabolites and DEGs. The values are means ± SD of three biological replicates. Asterisks indicate significant difference: **** p ≤ 0.0001, *** p ≤ 0.001.
Figure 5
Figure 5
Schematic model and heatmap analysis of metabolites in flavonoid biosynthesis pathways. (A) Schematic model of the pathways leading to biosynthesis of anthocyanin, flavonol and PA. (B) Heatmap analysis of differential metabolites in flavonoid biosynthesis pathways. The color bar represents the logarithm of metabolite content foldchange (log2FC), where the red indicates up-regulated metabolites and the blue indicates down-regulated metabolites.
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