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

Flavonoids are representative secondary metabolites with different metabolic functions in plants. Previous study found that ectopic expression of EsMYB90 from Eutrema salsugineum 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.


Introduction
Flavonoids, as the most ancient and vital secondary metabolites in plants, are determinants of crop quality and commercial value because they influence many characteristics of plant tissue including color, nutritive value, aroma and antioxidant properties [1]. More than 6000 different flavonoids widely distributed in plants have been identified [2,3]. Accumulation of flavonoids is favorable for plants because they not only provide plants with color for most flowers, fruits and seeds to attract pollinators, but also light-shielding, metal-binding and antioxidant capacities and functions in osmotic regulation [3][4][5]. A common factor with abiotic stress is the accumulation of ROS [6]. Flavonoids, acting as scavengers of ROS, can be induced by many environmental stimuli to protect plants against diverse abiotic and biotic stresses [6,7]. In general, flavonoids are classified into several subgroups including anthocyanin, proanthocyanidin (PA or condensed tannin), Flavonols, having a wide range of potent physiological activities, are probably the most important flavonoids participating in stress responses [3,26]. The first committed step for biosynthesis of flavonol branching is catalyzed by flavonol synthase (FLS) [27]. Flavonol, PA and anthocyanin share several biosynthetic steps in the flavonoid pathway [15]. Dihydroflavonols as the competing substrates for DFR, FLS, F3 H and F3 5 H are the precursors of both anthocyanin and flavonol [28]. In Arabidopsis, leaves, stems, flowers and seedlings mainly accumulate kaempferol glycosides, whereas in mature seeds, quercetin-3-O-rhamnoside (quercitrin) is the major flavonol [7]. Additionally, flavone and isoflavone are synthesized at a branch point of the anthocyanin/PA pathway using flavanone as the direct precursor [1].
The level of metabolites as the end product of gene expression can be regarded as the ultimate response of biological systems to genetic or environmental changes, and metabolomics describes the metabolite changes that occur in organisms [35,36]. Flavonoids, a representative group of secondary metabolites, have roles in many facets of plant physiology, including the well-known antioxidant and health-promoting effects of these compounds [6]. Salt cress (Eutrema salsugineum), a Brassicaceae species closely related to Arabidopsis, is an important model halophyte. Tobacco, as a model plant for genetic transformation, is very convenient for transformation of genes and molecular biology research. In our previous research, we have successfully obtained Eutrema EsMYB90 transgenic tobacco plants [35]. Here, the function of EsMYB90, an R2R3-MYB transcription factor on regulating biosynthesis of flavonoid metabolites, was investigated by the integrated analysis of metabolite profiling and RNA-seq as well as the dual luciferase assay in the leaves of EsMYB90 transgenic tobacco in comparison with wild type plants. The result manifested that the EsMYB90 gene is responsible for the accumulation of flavonoids and other antioxidant metabolites, and EsMYB90 plays a pivotal regulating role in flavonoid metabolite biosynthesis via modulating the expression of flavonoid biosynthesis genes such as NtANS and NtDFR. Therefore, the study exhibited a growing insight for us to understand and genetically manipulate the biosynthesis of anthocyanin and flavonoids to generate a novelty of colors and enhance antioxidant activity in plants.

Increased Flavonoid Compounds in EsMYB90 Transgenic Tobacco Leaves
In our previous study, we demonstrated that ectopic expression of Eutrema EsMYB90 (p35S:EsMYB90) in tobacco led to purplish red at varying degrees in leaves, stem, flowers and fruit pods and was accompanied with a strongly elevated anthocyanin level [37]. To further understand and define the regulating effect of the EsMYB90 gene on different pigments and flavonoid compounds, the contents of anthocyanin, chlorophyll, proanthocyanin (PA) and total flavonoid were checked in EsMYB90 transgenic tobacco and wild type plants. We found that the contents of total flavonoid and anthocyanin in young leaves (YL), mature leaves (ML), stems and roots in EsMYB90 transgenic tobacco L2 and L4 lines were extremely higher than that of wild type. Moreover, the content of anthocyanin, total flavonoid and proanthocyanidin in the tissues detected from L4 EsMYB90 transgenic tobacco line were all significantly higher than that of the L2 line, except for total flavonoid content in young leaves (YL). The contents of anthocyanin and total flavonoid in the ML of transgenic L4 line were respectively elevated more than 979-and 591-fold ( Figure 1A,B, Supplementary Table S1). Additionally, in the mature leaves of transgenic lines L2 and L4, PA levels were significantly up-regulated, whereas chlorophyll content almost remained unaffected ( Figure 1C,D and Supplementary Table S1).
Collectively, the results demonstrate that ectopic expression of the EsMYB90 gene not only led to the markedly higher accumulation of anthocyanin but also the contents of total flavonoid and PA in transgenic tobacco. ments and flavonoid compounds, the contents of anthocyanin, chlorophyll, proanthocyanin (PA) and total flavonoid were checked in EsMYB90 transgenic tobacco and wild type plants. We found that the contents of total flavonoid and anthocyanin in young leaves (YL), mature leaves (ML), stems and roots in EsMYB90 transgenic tobacco L2 and L4 lines were extremely higher than that of wild type. Moreover, the content of anthocyanin, total flavonoid and proanthocyanidin in the tissues detected from L4 EsMYB90 transgenic tobacco line were all significantly higher than that of the L2 line, except for total flavonoid content in young leaves (YL). The contents of anthocyanin and total flavonoid in the ML of transgenic L4 line were respectively elevated more than 979-and 591-fold ( Figure 1A,B, Supplementary Table S1). Additionally, in the mature leaves of transgenic lines L2 and L4, PA levels were significantly up-regulated, whereas chlorophyll content almost remained unaffected ( Figure 1C,D and Supplementary Table S1). Collectively, the results demonstrate that ectopic expression of the EsMYB90 gene not only led to the markedly higher accumulation of anthocyanin but also the contents of total flavonoid and PA in transgenic tobacco.

Metabolite Analysis Based on OPLS-DA Model
In order to preliminarily understand the overall metabolic differences among samples and the degree of variability between samples within groups, PCA analysis was performed for all samples in wild type (WT) and EsMYB90 transgenic tobacco (T). From the analysis result, it can be observed that the WT and T of inter-groups were clearly separated, and the repeated samples in intra-groups were gathered together in the PCA score plot, thus indicating that the experiment was reproducible and reliable (Supplementary Figure S1). . The values are means ± SD of three independent biological replicates. Statistical significance: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05. Bar = 1 cm.

Metabolite Analysis Based on OPLS-DA Model
In order to preliminarily understand the overall metabolic differences among samples and the degree of variability between samples within groups, PCA analysis was performed for all samples in wild type (WT) and EsMYB90 transgenic tobacco (T). From the analysis result, it can be observed that the WT and T of inter-groups were clearly separated, and the repeated samples in intra-groups were gathered together in the PCA score plot, thus indicating that the experiment was reproducible and reliable (Supplementary Figure S1). Q 2 is an important parameter for evaluating the models of OPLS-DA, and the value of Q 2 ≥ 0.9 indicates OPLS-DA as an excellent model. The OPLS-DA model in our study showed that Q 2 values of WT and T of inter-groups were equal to 0.994, R 2 X = 0.863 and R 2 Y = 1, which demonstrated that the model was reliable and meaningful (Supplementary Figure S2). The VIP value indicates the importance of a variable for the entire model, and a variable with a VIP greater than 1 is regarded as responsible for separation [38]. In this study, 130 discriminating metabolites were screened and distinguished by the combining fold change ≥ 2 (or ≤0.5) with VIP ≥ 1 in the OPLS-DA model.

Metabolic Profiling and Significantly Differential Metabolite Analysis
To explore the functions of EsMYB90 genes in modulating flavonoid biosynthesis, we investigate the differential metabolites in leaves of EsMYB90 transgenic tobacco relative to wild type. A total of 550 metabolites obtained from UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry) were analyzed in leaves using a widely targeted metabolomics method (Figure 2A and Supplementary Table S2). Combining FC (fold change) ≥2 (or ≤0.5) and VIP ≥ 1 in OPLS-DA model, 130 significantly differential metabolites including 50 flavonoids, 9 hydroxycinnamoyl derivatives, 13 quinate derivatives and 3 coumarins were screened and identified ( Figure 2 and Supplementary Table S3). Among these, 50 significantly differential flavonoid metabolites were divided into 7 categories including anthocyanin (1), flavone (14), flavonol (13), flavone C-glycosides (9), flavanone (7), isoflavone (1) and catechin derivatives (5) ( Figure 2B, Supplementary Table S3). In the 50 significantly differential flavonoid metabolites, contents of 46 metabolites were increased more than twofold in transgenic tobacco leaves, of which 15 metabolites with the negligible expression levels were observed in leaves of wild type plants (Supplementary Table S3). Above all, the most prominently differential metabolites were cyanidin O-syringic acid in anthocyanin, followed by myricetin in flavonol and chrysoeriol 5-Ohexoside in flavone (Figure 2A and Supplementary Table S3).  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.

Integrated Analysis of Metabolite Profiling and RNA-seq in Phenylpropanoid/Flavonoid Biosynthesis Pathways
The increased anthocyanin content in leaves, stems, flowers and fruit pods of EsMYB90 transgenic tobacco plants was caused by significantly up-regulated expression of anthocyanin biosynthesis genes in the previous study [37]. In order to clarify in depth 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 (log 2 FC); 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. Except for the above flavonoid metabolites, the contents of caffeic acid in hydroxycinnamoyl derivatives, 3-O-p-coumaroyl shikimic acid O-hexoside as well as chlorogenic acid methyl ester in quinate derivatives, esculetin (6,7-dihydroxycoumarin) and esculetin O-quinacyl esculetin O-quinic acid in coumarin also strongly increased (Figure 2A and Supplementary Table S3). The hydroxycinnamoyl derivative, quinate derivative and coumarin possess a wide range of antibacterial and antiviral activities and the capacity of absorbing ultraviolet rays; thus, these metabolites also possibly play an important role in improving the antioxidative activity of plants.
In summary, the level of flavonoid metabolites such as cyanidin O-syringic acid, myricetin and chrysoeriol 5-O-hexoside and other metabolites including caffeic acid, chlorogenic acid methyl ester and esculetin were all strikingly up-regulated, which suggested that the Eutrema EsMYB90 transcription factor plays a critical regulating role on the enhanced level of antioxidant metabolites in transgenic plants.

Integrated Analysis of Metabolite Profiling and RNA-seq in Phenylpropanoid/Flavonoid Biosynthesis Pathways
The increased anthocyanin content in leaves, stems, flowers and fruit pods of Es-MYB90 transgenic tobacco plants was caused by significantly up-regulated expression of anthocyanin biosynthesis genes in the previous study [37]. In order to clarify in depth the specific roles of EsMYB90 in flavonoid pathways and unravel the regulatory mechanisms in place, we have performed the combined analysis of significantly differential metabolites and markedly differential expression genes (DEGs). This result showed that 16 markedly up-regulated metabolites and 15 key enzymes encoded by 53 significantly DEGs were mapped onto the phenylpropanoid/flavonoid metabolism pathways (ko00940, ko00941, ko00942, ko00943 and ko00944), as shown in Figure 3 Tables S4 and S5).
In the phenylpropanoid pathway (ko00940), the DEGs encoding PAL (107802063, 107761482 and 107820497, 107769293) were up-regulated more than threefold, and the level of corresponding metabolites of cinnamic acid and caffeic acid markedly increased ( Figure 3, Supplementary Tables S4 and S5). In the flavonoid biosynthesis pathway (ko00941), the 42 DEGs and 12 differential metabolites were shown as black arrow lines in a map of the integrated metabolic pathways. In detail, 12 differential metabolites were up-regulated more than 10 times, including caffeoyl quinic acid in the quinate derivatives; naringin, hesperetin, eriodictyol, homoeriodictyol and prunin in the flavanones;  Table S4). 2 -hydroxydaidzein in isoflavonoid biosynthesis pathways (ko00943) and isotrifoliin in flavone and flavonol pathways (ko00944) were enhanced more than twofold, while the expression of genes encoding F3 H (2) and 3GT (2) that correspond to these metabolites' biosynthesis are also significantly up-regulated. It is noteworthy that F3 H (2) and 3GT (2) also exist in the flavonoid biosynthesis pathway (ko00941) and anthocyanin biosynthesis pathway (ko00942), respectively ( Figure 3 and Supplementary  Table S4). Table S4). 2′-hydroxydaidzein in isoflavonoid biosynthesis pathways (ko00943) and isotrifoliin in flavone and flavonol pathways (ko00944) were enhanced more than twofold, while the expression of genes encoding F3′H (2) and 3GT (2) that correspond to these metabolites' biosynthesis are also significantly up-regulated. It is noteworthy that F3′H (2) and 3GT (2) also exist in the flavonoid biosynthesis pathway (ko00941) and anthocyanin biosynthesis pathway (ko00942), respectively (Figure 3 and  Supplementary Table S4).  In addition, cyanidin O-syringic acid, the only differential metabolite detected in anthocyanin but presently not annotated in the KEGG pathway, had a dramatically higher level in the leaves of EsMYB90 transgenic tobacco when compared to the wild type ( Figure 3, Supplementary Table S5). The specific enzymes for synthesizing anthocyanin are DFR, ANS, 3GT/BZ1 and UFGT [39]. In our study, all genes encoding DFR(2), ANS/LDOX (5), ANS/FLS (2), 3GT/BZ1(2) and UFGT (4) were markedly up-regulated (Figure 3, Supplementary Table S4); thus, these up-regulated DEGs are possibly responsible for the strongly enhanced cyanidin O-syringic acid content in the leaves of EsMYB90 transgenic tobacco.

3, Supplementary
Taken together, these markedly increased flavonoid metabolites were tightly associated with the up-regulated expression of corresponding genes controlling their biosynthesis in flavonoid/phenylpropanoid pathways.

EsMYB90 Enhanced Flavonoid Metabolite Level via Activating Transcription of Flavonoid Biosynthesis Genes
Anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) encoding genes have been widely characterized as the key anthocyanin biosynthetic genes [28]. In order to expound the regulatory mechanism of EsMYB90 genes on biosynthesis of anthocyanin and flavonoids, firstly, the MYB-binding motif analysis in promoters of NtANS and NtDFR genes were analyzed using PlantCARE and visualized by Tbtools software. The result showed that five MYB transcript factor binding elements were found in the promoter of NtANS, while six MYB-binding elements existed in the promoter of NtDFR ( Figure 4A,B, Supplementary Table S6). Further, the Agrobacterium containing pCAMBIA3301H-p35S:EsMYB90 as an effector as well as the Agrobacterium containing pGreenII0800-pNtANS:LUC or pGreenII0800-pNtDFR:LUC as the reporter were co-transfected into tobacco leaves, and the dual luciferase assay was performed. The results demonstrated that the relative activity ratio of the pNtANS:LUC firefly luciferase to renilla luciferase (p35S:REN) was up-regulated about four times ( Figure 4C, Supplementary  Table S7), while the ratio of pNtDFR:LUC firefly luciferase to renilla luciferase (p35S:REN) was elevated 23 times ( Figure 4D, Supplementary Table S7). This study documented that EsMYB90 TF could operate as a positive transcriptional regulator of NtANS and NtDFR genes by directly binding to the MYB-binding elements of their promoters.
To investigate the different kinds of metabolites regulated by EsMYB90 in place, the significant correlations were analyzed between the EsMYB90 gene and the differential metabolites. On account of the screening criteria required for Pearson correlation coefficient |PCC| ≥ 0. To further identify the DEGs and differential metabolites affected by EsMYB90 in transgenic tobacco leaves in phenylpropanoid/flavonoid pathways, we constructed a regulating network between the 15 significantly differential metabolites (14 up-regulated and one down-regulated) and the DEGs related to these metabolites in the ko00940, ko00941, ko00943 and ko00944 pathways. The results displayed that 424 significant correlations were established between 36 DEGs and 15 markedly differential metabolites on the basis of the criteria of |PCC| ≥ 0.8 and PCCP ≤ 0.05 ( Figure 4F, Supplementary Table S9). Moreover, except for L-epicatechin and medicarpin which only correlated with HCT (107830064) and

(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.
In summary, EsMYB90 could strikingly increase the level of flavonoid metabolites via modulating the flavonoid biosynthesis genes in transgenic tobacco leaves, indicating that EsMYB90 plays crucial roles in improving antioxidant capacity by strongly elevating the level of flavonoid metabolites in transgenic plants.

Key Role of EsMYB90 in Enhancing Antioxidative Metabolite Level
Anthocyanins are best known for their function in the color of fruits, flowers and autumn leaves [5,40,41]. In the present study, the content of anthocyanin and total flavonoid in young leaves (YL), mature leaves (ML), stems and roots in EsMYB90 transgenic tobacco were extremely higher than that of wild type ( Figure 1A,B, Supplementary Table S1). Further, the metabolome analysis showed that a dramatically higher level of cyanidin O-syringic acid, as the sole significantly differential metabolite in anthocyanin, was found in leaves of EsMYB90 transgenic tobacco, while a negligible level was found in wild type plants (Figure 2 and Supplementary Table S2) Table S2). Thus, it is proposed that cyanidin O-syringic acid is a crucial pigment compound for the purplish red phenotype in the leaves of EsMYB90 transgenic tobacco. Flavonol was reported to participate in stress responses and is involved in complex interplay between flavonols and ROS [7]. Dihydroflavonols including DHK (dihydrokaempferol), DHQ (dihydroquercetin) and DHM (dihydromyricetin) are precursors of anthocyanins and flavonols and could further form unstable anthocyanins via catalysis of DFR and ANS [28]. There were 13 flavonol metabolites which increased more than six times in the leaves of transgenic tobacco compared with wild type plants, including Table S10). PAs contribute to the protection of plants against abiotic stresses and microbial attacks [42]. In our study, epigallocatechin (EGC), L-epicatechin and protocatechuic acid in PAs all increased more than 50 times (Figure 5a and Supplementary  Table S10). In addition, 13 of 14 flavones, 7 of 9 flavone C-glycosides, 7 flavanones and one isoflavone were all significantly up-regulated in the leaves of EsMYB90 transgenic tobacco relative to wild type ( Figure 5 and Supplementary Table S10). Therefore, it is inferred that these flavonols, PAs, flavones, flavone C-glycosides, flavanones and isoflavone up-regulated by EsMYB90 were the important flavonoid metabolites for improving the antioxidant capacity of plants.
Flavonoids play a prominent role in many stress responses owing to their functions in protecting the photosynthetic machinery of plants exposed to an excess of light (especially UV-B), scavenging reactive compounds including ROS occurring under various stress conditions, binding various heavy metals and sequestering the toxic elements [43], exhibiting the osmotic adjustment to remain turgid in plant cells under low water availability and enhancing the efficiency of nutrient retrieval during senescence [3,5,[44][45][46][47]. In summary, a diverse array of flavonoids in higher plants have evolved according to their physiological and ecological functions to adapt to various environmental pressures [8]. Moreover, flavonoids are also beneficial to the agronomic value of plant products and the health value of food [2,20]. A series of flavonoid subgroups such as PAs, anthocyanins, flavonols, flavones, flavanones and isoflavones have been recognized in over 500 food items by the US Department of Agriculture as dietary flavonoids with consistent evidence of beneficial effects in humans [6]. In this study, the 92 metabolites including 46 flavonoids, 6 hydroxycinnamoyl derivatives, 12 quinate derivatives and 3 coumarins were markedly up-regulated in 130 significantly different metabolites, and most of them have strongly antioxidant capacities (Figure 2 and Supplementary Table S3).  Table S10). PAs contribute to the protection of plants against abiotic stresses and microbial attacks [42]. In our study, epigallocatechin (EGC), L-epicatechin and protocatechuic acid in PAs all increased more than 50 times (Figure 5a and Supplementary Table S10). In addition, 13 of 14 flavones, 7 of 9 flavone C-glycosides, 7 flavanones and one isoflavone were all significantly up-regulated in the leaves of EsMYB90 transgenic tobacco relative to wild type ( Figure 5 and Supplementary Table  S10). Therefore, it is inferred that these flavonols, PAs, flavones, flavone C-glycosides, flavanones and isoflavone up-regulated by EsMYB90 were the important flavonoid metabolites for improving the antioxidant capacity of plants. Flavonoids play a prominent role in many stress responses owing to their functions in protecting the photosynthetic machinery of plants exposed to an excess of light (especially UV-B), scavenging reactive compounds including ROS occurring under various stress conditions, binding various heavy metals and sequestering the toxic elements [43], exhibiting the osmotic adjustment to remain turgid in plant cells under low water availability and enhancing the efficiency of nutrient retrieval during senescence [3,5,[44][45][46][47]. In summary, a diverse array of flavonoids in higher plants have evolved according to their physiological and ecological functions to adapt to various environmental pressures [8]. Moreover, flavonoids are also beneficial to the agronomic value of plant products and the health value of food [2,20]. A series of flavonoid subgroups such as PAs, anthocyanins, flavonols, flavones, flavanones and isoflavones have been recognized in over 500 food items by the US Department of Agriculture as dietary flavonoids with consistent evidence of beneficial effects in humans [6]. In this study, the 92 metabolites including 46 flavonoids, 6 hydroxycinnamoyl derivatives, 12 quinate derivatives and 3 coumarins were markedly up-regulated in 130 significantly different metabolites, and most of them have strongly antioxidant capacities (Figure 2 and Supplementary Table S3).
Our transcriptomic analysis herein revealed that many of the genes involved in phenylpropanoid/flavonoid metabolism, from PAL to UFGT genes, can be directly or indi- Our transcriptomic analysis herein revealed that many of the genes involved in phenylpropanoid/flavonoid metabolism, from PAL to UFGT genes, can be directly or indirectly up-regulated by the EsMYB90 transcription factor. Moreover, the transcript abundance of flavonoid biosynthesis genes coincided with the corresponding metabolite levels detected (Figure 3). Significantly, DFR, ANS and UFGT are the crucial structural proteins that facilitate anthocyanin pigmentation in plants. In this study, EsMYB90 strongly activated the flux of flavonoid intermediates toward the production of cyanidin O-syringic acid in anthocyanin by elevating the transcription of DFR, ANS and UFGT genes, with a resulting purple-red leaf phenotype (Figure 1, Supplementary Tables S4 and S5). Meanwhile, the level of epicatechin gallate (ECG) and L-epicatechin in PAs were markedly enhanced, which is in accordance with the significantly up-regulated expression of DFR and ANS genes ( Thus, it is reasonable to suggest that the tobacco plants overexpressing the EsMYB90 gene alone showed similar changes in levels of both metabolites and transcripts, which supports that EsMYB90 is fulfilling a key central role by regulating the most biosynthetic genes related to the phenylpropanoid/flavonoid pathways in transgenic tobacco leaves.

Regulating Mechanism of EsMYB90 in Phenylpropanoid/Flavonoid Pathways and Its Novelty
Several R2R3 MYB transcription factors are known to regulate the branches of the phenylpropanoid/flavonoid metabolic networks, and various end-products of the associated pathways accumulate in specific cells, tissues and organs in higher plants [30]. Arabidopsis, AtMYB75, AtMYB90, AtMYB113, AtMYB114 and AtMYBL2 act as crucial modulators of flavonoid biosynthesis [32,[48][49][50]. Furthermore, AtMYB75/PAP1 is a master regulator of the flavonoid/anthocyanin biosynthesis pathway, and AtMYB75/PAP1 and MYB90/PAP2 can work in a combinatorial way with TTG1, a WD40 protein, and different bHLH partners such as TT8, GL3 or EGL3 [7,14,51]. On the other hand, AtMYB60 is the first MYB protein identified that functions as a transcriptional repressor in anthocyanin biosynthesis [30]. Thus, it can be seen that the MYB transcription factor, acting as activator or repressor, plays a critical role in the modulation of flavonoid biosynthesis [30].
In Arabidopsis, AtMYB75, AtMYB90, AtMYB113 and AtMYB114 mainly regulate the expression of late biosynthesis genes (LBGs) [5,32]. However, Eutrema EsMYB90 is not only involved in regulating the expression of LBGs (DFR, ANS/LDOX and UFGT) but also in promoting the expression of anthocyanin early biosynthesis genes (EBGs) such as PAL, CHS, CHI and F3H in 35S:EsMYB90 tobacco transgenic plants [37]. Additionally, Arabidopsis AtMYB75, AtMYB90, AtMYB113 and AtMYB114 function redundantly in participating in the MYB-bHLH-WD40 (MBW) complex to regulate anthocyanin and proanthocyanidin (PA) biosynthesis, and at least four MBW complexes assembled with various MYBs were involved in PA accumulation of the innermost cell layer in an Arabidopsis seed coat [2,52]. EsMYB90 protein has the closest homologous relationship with EsMYB5, EsMYB15, EsMYB106, EsMYB108 and EsMYB-related protein 340 in Eutrema; however, the ANDV and KPRPR [S/T] F motifs characterizing anthocyanin-promoting MYBs only exist in EsMYB90 protein, but not in the other five EsMYBs, while the conserved [D/E]LX 2 [R/K]X 3 LX 6 LX 3 R motif for interacting with an R/B-like bHLH protein only exists in EsMYB90 and EsMYB5 proteins [37]. Thus, it was suggested that EsMYB90 is a major player in flavonoid biosynthesis in Eutrema, which is different from that in Arabidopsis [37].
In view of the targets of MYB TFs suggested to be different in different plant species, the choice of gene source is important for an efficient change of the metabolites in the flavonoid pathways [3,53]. Moreover, understanding how phenylpropanoid/flavonoid metabolism changes can be regulated by the MYB transcription factor would be useful for improving crop yield and quality via enhancing the antioxidative capacity of plants. Therefore, this study was designed to evaluate the influence of halophyte Eutrema EsMYB90 on the phenylpropanoid/flavonoid pathways and potential regulatory mechanisms in transgenic plants. In this study, the combined analysis of transcriptome and metabolome as well as a dual luciferase assay were employed to clarify the regulatory mechanism of EsMYB90 on flavonoid biosynthesis and verify the function of EsMYB90 on metabolite biosynthesis. The result found that ectopic expression of the EsMYB90 gene in transgenic tobacco increased 46 flavonoid levels as well as the content of 6 hydroxycinnamoyl derivatives, 12 quinate derivatives and 3 coumarins with the antibacterial and antiviral activities (Figure 2, Supplementary Table S3). Moreover, the evidence from our experiments demonstrated that the flavonoid biosynthetic pathways in transgenic tobacco can be switched on by EsMYB90, which will help to deepen our understanding of plant flavonoid metabolite biosynthesis. Thus, it is inferred that the EsMYB90 gene, as a novel activator of flavonoid biosynthesis, possibly acts effectively in most plant species to promote flavonoid levels.

EsMYB90-A Potential Important Gene for Genetic Breeding to Improve the Flavonoid Level of Crops
In the last several decades, the majority of flavonoid molecules and the genes involved in biosynthesis of these diverse compounds in Arabidopsis have been intensively identified [54]. Moreover, a huge number of genetic engineering attempts have been described to produce novel flower colors in several plant species, such as petunia, gerbera, rose and carnation, by modifying the anthocyanin biosynthesis pathway [55][56][57]. However, the diversified functionality of various flavonoids and the transcription regulation of the enormous diversification of flavonoids should be addressed by further investigation.
In higher plants, flavonoid and anthocyanin biosynthesis are regulated by a conserved MYB-bHLH-WD40 (MBW) complex, and the different sets of MBW complexes exerted in different plants [2]. Of these, R2R3-MYB plays pivotal role in transcriptional regulation of flavonoid biosynthesis. EsMYB90 protein, an R2R3-MYB from Eutrema, has a conserved DNA-binding domain (R2 and R3 repeats) in the N-terminal, and a conserved [D/E]LX 2 [R/K]X 3 LX 6 LX 3 R motif required for interaction with R/B-like bHLH proteins [37,57]. The phylogenetic tree of EsMYB90 with 29 MYB proteins involved in flavonoid synthesis in 16 plants demonstrated that EsMYB90 has a closer relationship with Arabidopsis AtMYB75, AtMYB90, AtMYB113, AtMYB114, Brassica oleracea BoMYB1 and Brassica rapa BrMYB114 [37]. Further, the ANDV motif, a characteristic identifier for anthocyanin-promoting MYBs in dicots, exists in EsMYB90, AtMYB90, AtMYB75, At-MYB113 and AtMYB114 [37,58]. However, EsMYB90 protein exists only at 80.5%, 78.9%, 78.4%, 74.4%, 65.9%, and 50% identities respectively in BoMYB1, AtMYB90, BrMYB114, AtMYB75, AtMYB113 and AtMYB114 [37]. In our current findings, the direct activation of NtANS and NtDFR exerted by EsMYB90 protein resulted in a phenotypic effect of anthocyanin accumulation in transgenic tobacco plants. Furthermore, we demonstrated that the critical regulation function of the Eutrema EsMYB90 gene on the flavonoid regulatory network allowed the significant accumulation of 46 flavonoid compounds. These findings could be the basis for further engineering of flavonoids and optimization of the metabolic pathway. Biotechnological applications for the promotion of human health by engineering of flavonoids is a promising area [59,60]. Considering various flavonoids not only provide the abundant colors of plants, but also possess extensive antioxidative effects, antiviral activities and neuroprotective properties, their accumulation is a key objective for the genetic improvement of crops [3,61,62]. All in all, the EsMYB90 transcription factor on the regulatory network involved in several already annotated flavonoid biosynthesis genes and flavonoid metabolites, as well as its conservation of structure, revealed that the EsMYB90 gene is a potential excellent gene for genetic breeding to enhance the flavonoid level of crops, and the study of modifying flavonoid content by Eutrema EsMYB90 in plant tissues also opens up a new avenue to improve the agricultural and economic performance of crops.
This study describes the importance of flavonoid, and the critical role of EsMYB90 gene in controlling the flavonoid biosynthesis in transgenic tobacco. However, the functional conservation of EsMYB90 gene needs to be explored and studied furtherly to evaluate its application prospect in some important ecnomic crops.

Plant Materials and Growth Conditions
In our previous research, eighteen 35S:EsMYB90 transgenic tobacco lines were obtained, and almost all transgenic tobacco lines exhibited purple-red leaves and corollas, purple-black sepals and fruit pods, whereas the leaves, sepals and fruit pods of wild type were green, with pink corollas. Specifically, L2 and L4 transgenic lines displayed more obvious pigmentation [37]. In the research, L2 and L4 transgenic lines were used to determine the content of anthocyanin, total flavonoid, proanthocyanidin and chlorophyll as well as L4 lines for subsequent analysis of metabolome and transcriptome.
Tobacco (Nicotiana tabacum) were grown in a mixture of vermiculite, perlite and peat moss (1:1:1) at a greenhouse with a temperature of 25 • C and a photoperiod of 16 h light/8 h dark. After 6 weeks' growth, the samples from the sixth leaves of wild type and transgenic tobacco plants at the period of 8-9 leaves, respectively, were collected for sequencing of transcriptome and metabolome. All samples were frozen immediately in liquid nitrogen and stored at −80 • C. The leaves from six individual plants were sampled as one biological replicate, and three biological replicates were used in this study.

Measurement of Anthocyanin, Total Flavonoid, PA and Chlorophyll
The young leaves (YL), mature leaves (ML), stems and roots of tobacco from six different plants that grew for about 8 weeks were respectively collected to quantify the anthocyanin, total flavonoid, PA and chlorophyll.
Anthocyanin and total flavonoid were extracted and quantified using the method described by Neff and Chory with minor modifications [63,64]. The absorbance of the samples was determined at 530 nm, 657 nm and 535 nm with a spectrophotometer (UV-1800, Shimadzu), and methanol with 1% HCl was used as a blank control. The anthocyanin content (mg/g FW) was calculated based on the formula of (A 530 − 0.25 ×A 657 )/fresh weight (g), while the total flavonoid content was measured with (mg/g FW) = (1/958 × A 535 × 10000 × V) /fresh weight (g), where the V indicates the total volume of the extract (mL).
For the determination of chlorophyll content, 80% acetone was used as the blank control, the absorbance of samples was detected at 663 nm and 645 nm and the chlorophyll content (mg/g FW) was calculated by the eq. (20.21× A 645 + 8.02 × A 663 ) / fresh weight (g) [65]. For the determination of proanthocyanidin content, 60% ethanol was used as the blank control, the absorbance of catechin standard samples were detected at 500 nm to draw the standard curve, and the PA content of each sample was calculated on the basis of the above standard curve using the improved vanillin colorimetric method [66]. At least three biological replicates were performed for each sample.

RNA-Seq and Bioinformatic Analysis
Total RNAs from the sixth leaves of wild type and EsMYB90 transgenic tobacco at the 7-8 leaf stage were isolated using a Quick RNA isolation kit (Bioteke Corporation, Beijing, China). The RNA library construction and sequencing were performed in the BGI Corporation (Shenzhen, China) using the BGISEQ-500 platform. Three independent biological replicates were carried out for the leaves of wild type and EsMYB90 transgenic tobacco, respectively. The methods of getting the clean reads, gene functional annotation, identification of differential expressed genes, and GO and KEGG pathway functional enrichment analysis has been reported and shown in our previous publication [37]. The DEGs with log 2 Fold Change ≥1 (or ≤−1) and Padj ≤ 0.05 were designated as significantly differentially expressed genes.

Analysis of Metabolite Profiling
The qualitative and quantitative analysis of metabolites from tobacco leaf samples were performed using ultra performance liquid chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A, http://www.shimadzu.com.cn/) (Accessed on June 2018) and tandem mass spectrometry (MS/MS) (Applied Biosystems 6500 QTRAP, http://www. appliedbiosystems.com.cn/) (Accessed on June 2018) in MetWare (Wuhan, China). The mass spectrometry data was processed using the analyst 1.6.1 software. The metabolites were identified and analyzed based on the metabolite information public database and the local MetWare database MWDB (Metware Biotechnology Co., Ltd. Wuhan, China).

Differential Metabolite Analysis and Metabolic Pathways Construction
Principal component analysis (PCA), a multivariate technique for extracting the important information in inter-and intra-groups, was tested for the leaves of wild type (WT) and transgenic tobacco (T) to get the overall metabolic differences among samples and the de-gree of variability in samples within groups. Orthogonal partial least squares-discriminant analysis (OPLS-DA) is an effective method for screening differential metabolites to maximize the difference between groups. Q 2 as an important parameter is used to evaluate the reliability of OPLS-DA model, and the value of Q 2 ≥ 0.9 representing OPLS-DA is an outstanding model. In the study, the Q 2 value of the OPLS-DA model is applied to distinguish the discriminated metabolites between samples of T and WT groups. Variable importance in projection (VIP), the weighted sum of squares of the OPLS-DA analysis, indicates the importance of a variable to the entire model [38]. In the study, the differential metabolites were screened by fold change ≥ 2 (or ≤0.5) and VIP ≥ 1.
The metabolomics data analysis was performed by MetaboAnalyst, and the metabolic pathways were constructed according to KEGG metabolic database.

Integrated Analysis of Metabolome and Transcriptome
For the combined analysis of metabolome and transcriptome, COR program from R was used to calculate the value of the Pearson correlation coefficient (PCC) in this study. In order to adequately define the networks of the EsMYB90 gene regulating different kinds of metabolites via activating the transcription of flavonoid biosynthesis genes, we performed the analysis of significant correlation between: (1) the EsMYB90 gene and the differential metabolites, (2) the differential expression genes (DEGs) and the differential metabolites in the phenylpropanoid/flavonoid pathways, based on the screening criteria required for the absolute value of the Pearson correlation coefficient (|PCC|) ≥ 0.8 and the p-value of correlation (PCCP) ≤ 0.05. The corresponding correlation network analysis was visualized with the Cytoscape software (version 3.3.0).
Dual luciferase assay has been widely applied to measure the TF (transcription factor)promoter interactions to validate the transcriptional regulatory roles of TFs on target promoters. Here, the promoters (−2000 to 0) of NtANS (107819370) and NtDFR (107803097) genes were respectively cloned and recombined into the MCS (multiple cloning sites) of pGreenII0800-LUC as a reporter vector. The ORF of EsMYB90 was inserted into the BamHI and EcoRI sites of pCAMBIA3301H vector to get pCAMBIA3301H-p35S:EsMYB90 as the effector vector, and the empty pCAMBIA3301H vector was used as a negative control. Primers for these constructs are listed in Supplementary Table S11. Then, all these constructs were individually were transferred into Agrobacterium tumefaciens GV3101. The GV3101 containing the pGreenII0800-pNtANS (or pNtDFR):LUC reporter gene vector was co-transfected with GV3101 containing the pCAMBIA3301H-p35S:EsMYB90 effector vector into the leaves of tobacco (Nicotiana benthamiana). After 2-3 days of the tobacco leaves being transfected, we used the dual-luciferase reporter assay system (Meilunbio) to detect the enzyme activities of firefly luciferase (pNtANS:LUC or pNtDFR:LUC) and renilla luciferase (p35S:REN), respectively, in a highly sensitive plate chemiluminescence instrument (Centro LB960, Berthold Technologies, wildbad, Jerman). The relative luciferase activity (LUC/REN) was calculated by the ratio of LUC to REN. Each assay was carried out with three independent experiments, and each independent experiment was conducted with three biological replicates. Error bars are the S.E. of three biological replicates.

Conclusions
In this paper, the conjoint analysis of metabolites and transcriptional profiling of EsMYB90 transgenic tobacco leaves confirms that the EsMYB90 transcription factor is a pivotal regulator of phenylpropanoid/flavonoid metabolism, and the ectopic expression of the Eutrema EsMYB90 gene in tobacco plants led to a large amount of flavonoid production, which plays an important role in enhancing the antioxidative activity of plants. Moreover, consistent with the higher gene expression that operates at the early and late steps in the flavonoid biosynthesis pathway, a higher level of corresponding flavonoid metabolites were produced in the leaves of transgenic plants. This study shed new light on the transcriptional regulation of flavonoid biosynthesis, and Eutrema EsMYB90 has great potential as a candidate gene to improve plant antioxidative capacity.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary files. The raw data of RNA library were available at NCBI Short Read Archive (PRJNA609528).

Conflicts of Interest:
The authors declare no conflict of interest.