Tyrosine Induces Anthocyanin Biosynthesis in Arabidopsis thaliana

Anthocyanins are widely found in plants and are responsible for the purple coloration of plants. Anthocyanin biosynthesis is induced by environmental stresses, plant hormones, sugar, and so on. Tyrosine (Tyr) is the precursor of melanin that exits in both animals and plants. However, until now it has been unknown whether Tyr induces anthocyanin biosynthesis. In this study, the seedlings of Arabidopsis thaliana were treated with exogenous Tyr and then the anthocyanin accumulation was determined. The results showed that Tyr induced anthocyanin accumulation in Arabidopsis thaliana in a dose-dependent manner. Furthermore, the expression of the late anthocyanin biosynthetic genes including DFR, LDOX, and UF3GT, and the transcription factor genes PAP1, PAP2, and EGL3 was induced by Tyr. Taken together, these results demonstrated that Tyr is able to induce anthocyanin accumulation 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.


Introduction
Anthocyanins are widely found in plants and are responsible for the purple coloration of plants.Anthocyanins are not only used as natural colorants [1] but also play an important role in plant defense against both biotic and abiotic stresses including protecting plants from damage by UV irradiation and acting as antimicrobial agents and feeding deterrents against pathogens and herbivores [1][2][3].In addition, Anthocyanins can help humans prevent cardiovascular diseases, anti-platelet aggregation and enhance immune modulating activity [4,5].
Tyr is the precursor of melanin that exits in both animals and plants [16][17][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 bio-synthesis genes and the WD-repeat/Myb/bHLH transcription factor genes to determine whether Tyr induces anthocyanin biosynthesis.

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.
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.

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.

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.

Figure 2 .
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.

Figure 3 .
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 and3).However, the expression of transcription factor GL3 was not induced by Tyr (Figure3).Instead, Tyr induces the expression of transcription factor EGL3 (Figure3).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.