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VIGS was performed to verify the function of VwTYDC. As shown in Fig. 1, there was no change in flower phenotype after injection of the blank control pTRV2 (Fig. 1a), but the blotches disappeared or became smaller when plants were injected with the positive controls pTRV2-VwANS and pTRV2-VwCHS (Fig. 1b, c). In the pTRV2-VwTYDC treatment, the flower color changed to pink (Fig. 1d), indicating pigment accumulation in petals when VwTYDC was silenced.
Phenotypic changes and anthocyanin accumulation of pansy petals treated with exogenous tyrosine
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To determine how tyrosine effects anthocyanin accumulation in pansy petals, non-blotched petal areas were treated with exogenous tyrosine. Petal phenotypes were observed after 24 h of treatment with water or tyrosine: there were no obvious changes in the non-blotched areas following water treatment (Fig. 2a), but cyanic stripes appeared in the non-blotched areas of pansy petals following treatment with tyrosine (Fig. 2b).
Figure 2.
Effect of tyrosine treatment on non-blotched areas of Viola × wittrockiana petals. (a) ddH2O treatment; (b) tyrosine treatment. The treated areas are marked by a red box.
ESI-HPLC-MS/MS was used to scan for 108 anthocyanidins, flavonoids, and procyanidins in the petal areas that received tyrosine or water treatment, and 24 compounds were detected in at least one of the treatments (Supplemental Table S3). The LC-MS/MS data have been uploaded to MetaboLights under number MTBLS3419. The contents of all 24 compounds in different samples were analyzed by hierarchical clustering analysis (Figs 3 & 4). The contents of two metabolites, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, differed based on the threshold Fold Change ≥ 2 or Fold Change ≤ 0.5 (Fig. 3). However, if compounds with an expression level of N/A in the CK treatment were included, there were nine differentially abundant anthocyanidins, including cyanidins, delphinidins, petunidins, peonidins and pelargonidins. Among these, cyanidin-3-O-rutinoside showed the highest content of 5.098 ng/g. Delphinidin-3-O-rutinoside, delphinidin-3-O-rhamnoside, cyanidin-3,5-O-diglucoside, petunidin-3-O-rutinoside, peonidin-3,5-O-diglucoside, peonidin-3-O-galactoside, and pelargonidin-3-O-(coumaryl)-glucoside were detected in the tyrosine treatments but not in the water treatments (Fig. 4).
Figure 3.
Violin plot of anthocyanins in petals of Viola × wittrockiana treated with tyrosine or ddH2O. The box in the middle represents the interquartile range. The black horizontal line in the middle is the median, and the outer shape represents the distribution of the data.
Figure 4.
Heatmap of the contents of the 24 compounds detected in different Viola × wittrockiana samples analyzed by hierarchical clustering analysis. The horizontal axis shows the sample information, and the vertical axis shows the metabolite information. The tree on the left of the figure shows the metabolite clustering, and the scale is the metabolite content after standardization. A deeper red color indicates a higher content, and gray indicates that the compound was not detected. Groups indicate the different treatments. CK: ddH2O treatment; Tyr: tyrosine treatment.
RNA sequencing, gene functional annotation, and classification
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Three cDNA libraries were constructed from petals treated with tyrosine, and 57,665,984, 41,950,452 and 49,379,404 high-quality reads were obtained. Three more cDNA libraries were constructed from the control samples, and 52,086,074, 46,811,096, and 48,288,596 high-quality reads were obtained. The final transcriptome assembly contained 90,732 genes with an average length of 735 nt and an N50 of 1,172 nt. The sequencing raw data have been deposited into the NCBI Sequence Read Archive (SRA) under accession number PRJNA754504.
To assign putative functions to the assembled genes, their sequences were searched against public databases; 50,829 genes were annotated by the NR database, and 36,481 genes were annotated by the KOG database. GO annotations (16,976 genes) and KEGG pathway annotations (15,175 genes) were also obtained to gain more insight into the putative gene functions. In the GO analysis, the terms metabolic process, catalytic activity, cellular process, binding, and single-organism process were the top five annotations with the largest number of genes (Supplemental Fig. S1). In the KEGG pathway analysis, the pathways with the greatest unigene enrichment were metabolic pathways (5790, 38.15%), followed by biosynthesis of secondary metabolites (3089, 20.86%), and biosynthesis of antibiotics (1622, 10.69%).
Identification of DEGs related to anthocyanin biosynthesis
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A total of 19,438 DEGs were detected between water- and tyrosine-treated areas of pansy petals: 6401 downregulated and 13,037 upregulated (Supplemental Fig. S2). Among these, 4525 were mapped to 131 KEGG pathways. There were nine DEGs related to anthocyanin biosynthesis in the tyrosine-treated areas, and all but unigene0045619 were upregulated (Table 1). VwHCT (unigene0045619, unigene0015507), VwC3′H (unigene0083763, unigene0029000), and VwCHS (unigene0058680, unigene0058682, unigene0011199) were involved in the flavonoid biosynthesis pathway (ko00941), whereas VwUGT75C1 (unigene0060888, unigene0055085) was involved in the anthocyanin biosynthesis pathway (ko00942).
Table 1. Putative anthocyanin structural genes that were differentially expressed in response to tyrosine treatment in Viola × wittrockiana petals.
Gene ID Annotation RPKM
(Tyr treatment)RPKM
(H2O treatment)log2 (T/CK) FDR Up/Down unigene
0045619VwHCT 0.54 1.48 −1.45 0.05 Down unigene
0015507VwHCT 2.31 0.56 2.26 5.35e-12 Up unigene
0083763VwC3’H 3.60 0.34 3.42 1.18e-28 Up unigene
0029000VwC3’H 1.80 0.02 6.86 8.38e-15 Up unigene
0058680VwCHS 213.61 100.30 1.09 4.65e-35 Up unigene
0058682VwCHS 87.86 42.41 1.05 1.18e-41 Up unigene
0011199VwCHS 10.51 3.40 1.63 6.24e-33 Up unigene
0060888VwUGT75C1 13.30 5.71 1.22 2.95e-21 Up unigene
0055085VwUGT75C1 177.86 66.99 1.41 3.19e-105 Up VwHCT, shikimate O-hydroxycinnamoyltransferase; VwC3'H, coumaroylquinate 3'-monooxygenase; VwCHS, chalcone synthase; VwUGT75C1, anthocyanidin 3-O-glucoside 5-O-glucosyltransferase. Analysis of differentially expressed MYB transcription factors
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There were 13 differentially expressed MYB TFs: eight upregulated and five downregulated (Supplemental Table S4). We constructed a phylogenetic tree that included the MYB TFs of Arabidopsis thaliana to provide insight into the potential functions of these differentially expressed MYB TFs (Supplemental Fig. S3). We found that unigene0005403 was highly homologous to the Arabidopsis TFs AT5G49330.1, AT2G47460.1, and AT3G62610.1, which encode AtMYB111, AtMYB12, and AtMYB11. A blastx search of the transcriptome data indicated that this unigene was also highly similar to an MYB12-like TF gene from Cicer arietinum (E-value < 10−5), and we therefore speculated that it was likely to be an MYB12-like gene in pansy.
qPCR of key anthocyanin biosynthesis and transcription factor genes
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The relative expression levels of 10 DEGs (nine biosynthesis genes and one TF gene) related to the anthocyanin biosynthesis pathway were verified in tyrosine- and ddH2O-treated petals by qPCR. The expression levels of these DEGs were again promoted by tyrosine, with the exception of unigene0045619 (HCT-1-like) (Fig. 5), consistent with the transcriptome sequencing results (Table 1).
Figure 5.
The relative expression levels of anthocyanin biosynthesis and transcription factor genes in Viola × wittrockiana petals that received tyrosine (Tyr) or ddH2O (CK) treatment. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Tyrosine upregulates ABA biosynthesis-related genes and promotes ABA production
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The transcriptome data revealed that expression levels of some key genes in the ABA biosynthesis pathway were significantly higher in the tyrosine-treated areas than in the H2O-treated areas based on FDR < 0.05 and |log2FC| > 1 (Table 2). This result suggested that the ABA content might be higher in the tyrosine-treated areas. We therefore analyzed ABA content and confirmed that it was significantly higher in the tyrosine-treated areas than in the H2O-treated areas (Fig. 6).
Table 2. Differentially expressed genes related to ABA biosynthesis in Viola × wittrockiana.
Gene ID Annotation RPKM
(Tyr treatment)RPKM
(H2O treatment)log2
(T/CK)FDR Up/Down unigene
0004917VwNCED 2.20 1.03 1.10 0.00 Up unigene
0068071VwNCED 2.41 0.56 2.10 7.18e-6 Up unigene
0068072VwNCED 1.16 0.35 1.72 0.00 Up unigene
0082413VwABA2 0.33 0.01 4.51 0.00 Up unigene
0073280VwAAO3 8.81 4.28 1.04 2.46e-31 Up unigene
0017204VwCYP707A 18.30 0.81 4.50 7.13e-212 Up unigene
0087548VwHY5 0.63 0 9.29 0.01 Up VwNCED, 9-cis-epoxycarotenoid dioxygenase; VwABA2, xanthoxin dehydrogenase 2; VwAAO3, abscisic-aldehyde oxidase; VwCYP707A, (+)-abscisic acid 8'-hydroxylase. Figure 6.
ABA contents in Viola × wittrockiana from different treatment groups. ** p ≤ 0.01. CK, ddH2O treatment. Tyr, tyrosine treatment.
We also analyzed differentially expressed bZIP TFs of pansy by building a phylogenetic tree with their sequences and those of all bZIP TFs from Arabidopsis (Supplemental Fig. S4). The upregulated unigene0087548 was highly homologous to the Arabidopsis TFs AT3G17609.2 and AT3G17609.3, which encode homologs of AtHY5, an important bZIP TF that binds to the promoter of the bZIP TF gene ABA insensitive 5 (ABI5) to activate ABA biosynthesis[27].
Exogenous ABA upregulated the expression of anthocyanin biosynthesis genes
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To determine whether ABA could activate anthocyanin-related genes in Viola × wittrockiana, we treated non-blotched areas of pansy petals with ABA and used ddH2O as a control treatment. The treated petal areas were then collected for qRT-PCR analysis. As shown in Fig. 7, ABA treatment upregulated the expression levels of multiple anthocyanin biosynthesis genes, including VwCHS, VwANS, and VwUGT, as well as the TF genes VwMYB12 and VwHY5.
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Tyrosine has recently been shown to promote anthocyanin biosynthesis, and the molecular mechanism by which it induces anthocyanin biosynthesis in Arabidopsis has been investigated. Exogenous tyrosine is now known to induce anthocyanin biosynthesis and accumulation by upregulating anthocyanin biosynthesis-related genes, including DFR, LODX, and UGT[18]. Tyrosine has also been found to promote the biosynthesis of flavonoids, the substrates of anthocyanin biosynthesis[28]. These results suggest that tyrosine may serve as an efficient regulatory metabolite for anthocyanin biosynthesis.
In the present research, VIGS silencing of VwCHS and VwANS produced obvious fading of petal blotches, thus confirming that the VIGS protocol worked well in pansy. By contrast, VIGS silencing of VwTYDC induced pigment accumulation in pansy petals (Fig. 1), demonstrating that an increase in tyrosine promoted pigment biosynthesis in pansy. Moreover, the non-blotched parts of the petals showed some cyanic spots or stripes after tyrosine treatment (Fig. 2). Flowers were also significantly smaller after VIGS treatment. This phenomenon may reflect the ability of the VIGS technique to silence relevant developmental genes, thereby affecting organ development; alternatively, wounding caused by the silencing treatment may have affected flower development (Fig. 2)[29,30]. Anthocyanin metabolome analysis demonstrated that contents of cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were significantly enhanced by tyrosine treatment. Furthermore, delphinidin-3-O-rutinoside, delphinidin-3-O-rhamnoside, cyanidin-3,5-O-diglucoside, petunidin-3-O-rutinoside, peonidin-3,5-O-diglucoside, peonidin-3-O-galactoside, and pelargonidin-3-O-(coumaryl)-glucoside were detected in the tyrosine treatment but not the water control (Figs 3 & 4). Consistent with these findings, the transcriptome results indicated that VwHCT, VwC3′H, VwCHS, and VwUGT were upregulated in the tyrosine-treated areas. All these results suggest that tyrosine may promote anthocyanin accumulation by upregulating a number of anthocyanin biosynthesis-related genes.
Twenty-four different anthocyanins and related compounds were detected in the different samples (Fig. 4), among which almost all of the anthocyanins had been reported previously in Viola[31]. Although the Tyr-1 data differed somewhat from those of Tyr-2 and Tyr-3, probably owing to difficulties in accurately sampling specific petal areas, cyanidin-3-O-rutinoside was the major enriched anthocyanin with the highest average content (5.098 ng/g) in tyrosine-treated areas, similar to the main anthocyanidins previously reported in the blotched areas of pansy petals[1].
Tyrosine promotes anthocyanin biosynthesis via ABA synthesis in pansy
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Previous studies have reported that endogenous or exogenous factors can induce anthocyanin accumulation through ABA. In Lycium, for example, developmental cues transcriptionally activated LbNCED1 and thereby enhanced accumulation of ABA. ABA then stimulated transcription of the MYB-bHLH-WD40 TF complex, which in turn upregulated the expression of structural genes in the flavonoid biosynthetic pathway, ultimately promoting anthocyanin production and fruit coloration[32]. In bilberry (Vaccinium myrtillus), red light induced high expression of specific genes of anthocyanin biosynthesis and ABA signal perception and metabolism, including 9-cis-epoxycarotenoid dioxygenase (NCED), the ABA receptor pyrabactin resistance-like (PYL), and an abscisic acid 8'-hydroxylase gene (CYP707A) that functions in ABA catabolism[33]. ABA has been reported to promote anthocyanin accumulation by upregulating the expression of CHS, ANS, or UGT[34]. In our research, exogenous tyrosine application significantly upregulated the expression of several ABA biosynthesis genes, including VwNCED, VwABA2, VwAAO3, and VwCYP707A (Table 2). At the same time, the concentration of ABA was significantly higher in the tyrosine-treated areas of pansy petals (Fig. 6). Moreover, ABA treatment also significantly upregulated the expression of anthocyanin biosynthesis genes, including VwCHS, VwANS, and VwUGT, similar to the effect of tyrosine treatment on gene expression (Table 1). These results support a model in which tyrosine promotes anthocyanin biosynthesis via ABA accumulation in pansy petals.
Transcription factors may be important mediators of tyrosine-promoted anthocyanin biosynthesis
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Anthocyanin biosynthesis genes are transcriptionally regulated by the MYB-bHLH-WD40 complex[35]. In Arabidopsis, exogenous tyrosine upregulates the expression of MBW complex genes including PAP1, PAP2, GL3, EGL3, and TTG1[18]. In this study, we found eight significantly upregulated MYB-like unigenes, among which unigene0005403 had high homology to MYB12 from Cicer arietinum. Phylogenetic analysis with Arabidopsis MYBs also supported the hypothesis that unigene0005403 may have similar functions to AtMYB12 (Supplemental Fig. S3), which upregulates early anthocyanin biosynthesis genes such as CHS, CHI, and F3H[36]. These results suggest that unigene0005403 may also promote anthocyanin accumulation by upregulating anthocyanin biosynthesis genes. According to the transcriptome and qPCR data for VwCHS, VwCHI, and VwF3H, only VwCHS was significantly upregulated in response to tyrosine, and we thus speculate that unigene0005403 may act mainly on VwCHS in pansy.
bZIP TFs may also regulate anthocyanin biosynthesis by interacting synergistically with the MYBs[37]. In Arabidopsis, AtHY5 is a bZIP gene that can activate the expression of MYB12/PFG1 and MYB75/PAP1[38]. HY5 is also known to activate the expression of MYB12 and MYB111[39], which are involved in the regulation of flavonol synthase[40]. Moreover, HY5 can respond to ABA by specifically binding to ABI5 chromatin[41]. In this research, unigene0087548, annotated as an HY5-like gene, and unigene0005403, annotated as an MYB12 homolog, were both significantly upregulated in ABA-treated pansy petals (Fig. 7), suggesting that HY5 may have responded to ABA treatment and interacted synergistically with VwMYB12 to promote anthocyanin biosynthesis in pansy.
A proposed model of the promotion of anthocyanin biosynthesis by tyrosine in pansy
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On the basis of our experimental results, we propose a working model of the promotion of anthocyanin biosynthesis by tyrosine treatment in pansy. Tyrosine treatment activates ABA production by upregulating VwNCED, VwABA2, VwAAO3, and VwCYP707A. Then the bZIP TF VwHY5 responds to ABA accumulation and activates VwMYB12 to upregulate VwCHS expression and then induce anthocyanin production in non-blotched areas of pansy petals (Fig. 8).
Figure 8.
Possible pathways by which tyrosine treatment may promote anthocyanin biosynthesis in Viola × wittrockiana. The red color represents upregulation of genes and metabolites. The dashed arrow represents the possible pathway. PAL/TAL indicates that PAL may function like TAL in pansy, although this remains to be verified. PAL: phenylalanine ammonia-lyase; TAL: tyrosine ammonia-lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumarate-CoA ligase; HCT: shikimate O-hydroxycinnamoyltransferase; C3′H: 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase; CHS: chalcone synthase; F3H: naringenin 3-dioxygenase; F3′H: flavonoid 3'-hydroxylase; F3′5′H: flavonoid 3',5'-hydroxylase; DFR: flavanone 4-reductase; ANS: anthocyanidin synthase; UGT: UDP glucuronosyltransferase, falconoid 3-O-glycosyltransferase; CCoAOMT: caffeoyl-CoA O-methyltransferase; VwNCED: 9-cis-epoxycarotenoid dioxygenase; VwABA2: xanthoxin dehydrogenase; VwAAO3: abscisic-aldehyde oxidase; VwCYP707A: (+)-abscisic acid 8'-hydroxylase.
Another possible pathway is that exogenous tyrosine promotes the production of p-coumaric acid, which then leads to increased anthocyanin content. Tyrosine is known to give rise to p-coumaric acid through the catalysis of tyrosine ammonia-lyase (TAL); p-coumaric acid is then acted upon by 4-coumarate-CoA ligase (4CL) to form p-coumaroyl CoA, the main precursor of the anthocyanin biosynthesis pathway. When the content of tyrosine increases, p-coumaric acid production may be upregulated, thus promoting anthocyanin biosynthesis. However, no unigenes in the Viola × wittrockiana transcriptome were annotated as TAL in the present study. We speculate that phenylalanine ammonia-lyase of pansy (VwPAL) may have the same function as TAL, as PAL has been shown to function like TAL in some other plants[42]. However, more experiments are needed to verify this hypothesis.
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About this article
Cite this article
Cui Z, Gu J, Li J, Zhao A, Fu Y, et al. 2022. Tyrosine promotes anthocyanin biosynthesis in pansy (Viola × wittrockiana) by inducing ABA synthesis. Tropical Plants 1:9 doi: 10.48130/TP-2022-0009
Tyrosine promotes anthocyanin biosynthesis in pansy (Viola × wittrockiana) by inducing ABA synthesis
- Received: 07 September 2022
- Accepted: 02 November 2022
- Published online: 29 November 2022
Abstract: Viola × wittrockiana (pansy) is an important ornamental plant, particularly during winter and spring. In previous studies, we found that the tyrosine decarboxylase gene of pansy (VwTYDC) was expressed differently in blotched and non-blotched areas of pansy petals, suggesting that tyrosine may have a role in anthocyanin biosynthesis. In this study, we found that virus-induced gene silencing of VwTYDC caused an accumulation of pink pigmentation in pansy petals. Likewise, exogenous tyrosine treatment (TYRT) induced the formation of black stripes in non-blotched petal areas. Metabolome analysis indicated that the contents of two anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, increased significantly in the TYRT areas. RT-qPCR results revealed that the anthocyanin-related genes VwHCT, VwC3′H, VwCHS, and VwUGT were upregulated in the same areas. Transcriptome analysis revealed that four genes involved in the abscisic acid (ABA) biosynthesis pathway (VwNCED, VwABA2, VwAAO3, and VwCYP707A) were significantly upregulated in the same TYRT areas. ABA content was measured by ESI-HPLC-MS/MS, and ABA content was significantly higher in TYRT areas than in control areas. In addition, when exogenous ABA was spread onto non-blotched petal areas, anthocyanin biosynthesis genes were upregulated just as with tyrosine. Thus, transcriptome and metabolite analyses revealed a possible novel regulatory network for anthocyanin biosynthesis in which tyrosine induces ABA synthesis and ABA then promotes anthocyanin biosynthesis in pansy petals.
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Key words:
- Tyrosine /
- Transcriptome /
- Anthocaynins metabolome /
- Abscisic acid /
- Viola × wittrockiana