Anthocyanin biosynthesis is induced by a series of endogenous developmental and environmental signals, such as sugar, light, and plant hormones [6-11]. The anthocyanin biosynthetic genes are divided into two groups, the early anthocyanin biosynthetic genes such aschalcone synthase (CHS) and chalcone isomerase (CHI), and the late anthocyanin biosynthetic genes inculding dihydroflavonol reductase (DFR), leucoanthocyandin dioxygenase (LDOX), and UDP-glucose: flavo-noid 3-O-glucosyltransferase (UF3GT) [12-14]. The WD-repeat/Myb/ bHLH transcription complex including transcription factors PRODUCTION of ANTHOCYANIN PIGMENT1 (PAP1), PAP2, GLABRA3 (GL3), ENHANCER of GLABRA3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) mainly regulates the expression of the late anthocyanin biosynthetic genes [15].
Tyr is the precursor of melanin that exits in both animals and plants [16-18]. However, until now it has been unclear whether Tyr induces anthocyanin biosynthesis. In this study the seedlings of Arabidopsis thaliana were treated with exogenous Tyr for analysis of anthocyanin accumulation and the expression of the anthocyanin biosynthesis genes and the WD-repeat/Myb/bHLH transcription factor genes to determine whether Tyr induces anthocyanin biosynthesis.
2. Materials and Methods
Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study.
Seeds were surface-sterilized with 20% chlorine bleach containing 0.1% Triton X-100 for 10 min, washed five times with sterile water, plated on Murashige and Skoog medium supplemented with 1% sucrose (MS, pH 5.8), chilled at 4˚C for 3 d, and then transferred to a growth chamber and cultured under a 16 h light/8h dark photoperiod at 22˚C.
For measurement of anthocyanin content, 7-d-old seedlings grown on MS were transferred onto MS containing 3% sucrose supplemented without or with different concentrations of Tyr for an additional 7 d growth. For analysis of gene expressions, 7-d-old seedlings grown on MS were transferred onto MS without or with 2 mM Tyr for an additional 7 d growth.
The measurement of anthocyanin was performed as described by Deikman and Hammer [19]. The preweighed seedlings were placed into 1 mL extraction buffer (18% 1-propanol, 1% HCl, and 81% water), boiled for 3 min and then incubated in darkness overnight at room temperature. The absorbance of the supernatant was measured at 535 nm and 650 nm. The anthocyanin content was determined by the formula (A535-A650) g−1 fresh weight (FW). There were six samples per each treatment.
The gene expressions were analyzed by quantitative real-time PCR (qPCR). The total RNA was isolated using TRIzol Reagent (Invitrogen, USA). After incubated with DNase (Thermo Fisher Scientific, USA) for 30 min at 37˚C and then for 10 min at 65˚C to remove genomic DNA, the RNA sample was quantified by a spectrophotometer. The reverse transcription was synthesized using ReverTra Ace qPCR RT Kit (Toyobo, Japan). The qPCR was carried out using SYBR qPCR Mix (Toyobo, Japan) with the ABI 7300 Sequence Detection System (Applied Biosystems, USA) following the manufacturer’s instructions. The primers of genes tested in qPCR were as follows: CHS, 5’-CGCATCACCAACAGTGAACAC-3’ and 5’-TCCTCCGTCAGATGCATGTG-3’; CHI, 5’-CCGGTTCATCGATCCTCTTC-3’ and 5’-ATCCCGGTTTCAGGGATACTATC-3’; DFR, 5’-CCTTATCACCGCGCTCTCT-3’ and 5’-TGTCCTTGTCTTATGATCGAGTAATGC-3’; LDOX, 5’-TCAATTTGGCCTAAGACACCAAGT-3’ and 5’-TCGCTAGCAAACGAAGACACTT-3’; UF3GT, 5’-CAACTGGTTTTCCGTTTCTGGTT-3’ and 5’-GCTTCCTCGACGGTTGATACAC-3’; PAP1, 5’-GACATTACGCCCATTCCTACAAC-3’ and 5’-TCGAGGTCGAGGCTTATAAACATT-3’; PAP2, 5’-GAGGAAAGGTGCATGGACTG-3’ and 5’-ATCGCATCAGCTTCTTGGT-3’; GL3, 5’-TCGGTTCGTTTGGTAATGAGG-3’ and 5’-GCTTGCAATTGACGGTTAAGC-3’; EGL3, 5’-GGAAGACGATTCAAGCAGCA- 3’ and 5’-GGATTCAGCGAGGGAGAGAG-3’; TTG1, 5’-GTCATGAACCTCTTTATCAT-3’ and 5’-ATGGATAATTCAGCTCCAGA-3’. ACTIN2 was used as an internal control and was amplified with primers 5’-AGCACTTGCACCAAGCAGCATG-3’ and 5’-ACGATTCCTGGACCTGCCTCATC-3’. Gene expression for each sample was calculated on three analytical replicates, and the relative expression was quantified using the 2−ΔΔCt method.
All the experiments were repeated at least three times. Data in the figures are expressed as means ± SE of three biological replicates for qPCR and six biological replicates for anthocyanin measurement.
3. Results and Discussion
In order to investigate whether Tyr induces anthocyanin biosynthesis in plants, the Arabidopsis thaliana seedlings were treated with exogenous Tyr. Upon Tyr treatment, pigmentation appeared in back of leaves and it was more obvious when treated with high concentrations of Tyr (Figure 1(a)). The content of anthocyanin was increased in a dose-dependent manner of Tyr (Figure 1(b)). These results indicated that Tyr is able to induce anthocyanin accumulation.
To investigate the molecular mechanism for Tyr-induced anthocyanin biosynthesis, the expression of anthocyanin biosynthetic genes including CHI, CHS, DFR, LDOX, and UF3GT was analyzed by qPCR. Upon Tyr treatment, the expression of CHI and CHS was comparable to control without Tyr treatment, however, the expression of DFR, LDOX, and UF3GT was significantly increased (Figure 2). Since DFR, LDOX, and UF3GT were classified as the late anthocyanin biosynthetic genes [13,15], we concluded that Tyr induces anthocyanin accumulation mainly by up-regulating the late anthocyanin biosynthetic genes.
Since WD-repeat/Myb/bHLH transcription complexes including transcription factors PAP1, PAP2, GL3, EGL3, and TTG1 mediates the expression of late anthocyanin biosynthetic genes [15], we further analyzed the expression of these transcription factors genes. As shown in Figure 3, the expression of PAP1, PAP2, and EGL3 was increased in the presence of Tyr, however, the expression of GL3 and TTG1 was not obviously induced by Tyr. These results suggested that the expression of late anthocyanin biosynthetic genes induced by Tyr might be mediated by transcription factors PAP1, PAP2, and EGL3.
Accumulation of anthocyanins in plants is stimulated by diverse developmental signals, sugar, plant hormone, and environmental stresses [6-11]. Jasmonate (JA), a kind of plant hormones, induces anthocyanin accumula-
Figure 1. The phenotype of Arabidopsis thaliana seedlings (Col-0) treated with different concentrations of Tyr for 7 days (a) and the corresponding anthocyanin content (b). The number 1, 2, 3, 4, 5, 6, and 7 in (a) represent treatment with 0, 0.5, 1, 2, 3, 4, 5 mM Tyr, respectively. Error bars in (b) represent SE. Tyr, tyrosine.
Figure 2. Relative expression level of anthocyanin biosynthesis genes including CHI, CHS, DFR, LDOX, and UF3GT in Arabidopsis thaliana seedlings (Col-0) treated with 2 mM Tyr to control without Tyr treatment. Error bars represent SE. Tyr, tyrosine.
tion and up-regulates the expression of late anthocyanin biosynthetic genes DFR, LDOX, and UF3GT, and transcription factors PAP1, PAP2, and GL3 [20]. In this study, we also found that Tyr induces the expression of
Figure 3. Relative expression level of the WD-repeat/MYB/ bHLH transcription factors PAP1, PAP2, EGL3, GL3, and TTG1 in Arabidopsis thaliana seedlings (Col-0) treated with 2 mM Tyr to control without Tyr treatment. Error bars represent SE. Tyr, tyrosine.
late anthocyanin biosynthetic genes DFR, LDOX, and UF3GT, and transcription factors PAP1 and PAP2 (Figures 2 and 3). However, the expression of transcription factor GL3 was not induced by Tyr (Figure 3). Instead, Tyr induces the expression of transcription factor EGL3 (Figure 3). Therefore, Tyr and JA modulate the expression of late anthocyanin biosynthetic genes by mediating the same Myb transcription factors PAP1 and PAP2, but the different bHLH transcription factors EGL3 and GL3.
4. Conclusion
This study demonstrated that the exogenous Tyr could induce the anthocyanin accumulation and the expression of late anthocyanin biosynthetic genes including DFR, LDOX, and UF3GT, and the transcription factor genes PAP1, PAP2, and EGL3 in Arabidopsis thaliana seedlings, and suggested that Tyr up-regulates transcription factors PAP1, PAP2, and EGL3, which mediates the expression of the late anthocyanin biosynthetic genes and then induces anthocyanin biosynthesis.
Acknowledgements
This work was supported by grants from the National Science Foundation of China (30671121), the Hunan Provincial Natural Science Foundation of China (12JJ2021), the Hunan Provincial Graduate Student’s Innovative Research (CX2013B301), and the Program for Innovative Research Team in University (IRT1239).
NOTES
#Corresponding author.