Regulation Of Anthocyanin Biosynthesis In Arabidopsis By GLABRA2

Anthocyanins are edible pigments with different colours including red, purple and blue. Anthocyanins are hydrosoluble secondary metabolites produced by plants. Anthocyanins have multiple roles in plants. For example, the presence of anthocyanins make plant colourful which may help to attract insects for pollination, and anthocyanins may enhance plants’ resistance to environmental stresses such as UV radiation, strong light and cold. Anthocyanins have also been wildly used in food and medicinal industries. For example, anthocyanins can be added into health care products for the perpose of enhancing oxidation resistance, anthocyanins can also be used to prevent cardiovascular, cerebrovascular diseases and cancer.Anthocyanins are produce via the flavonoid pathway. Anthocyanin biosynthetic process in Arabidopsis thaliana can be divided into three stages. First, The production of 4-coumarate CoA from phenylalanine by sequential catalyzation of phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL); Second, the forming of dihydrokaempferols from 4-Coumaroyl CoA and 3-Malonyl CoA catalyzed by chalcone synthsae (CHS), chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H). Third, dihydrokaempferols are catalyzed in sequential by dihydroflavonol reductase (DFR), Anthocyanidin synthase (ANS) and UDP-flavonoid glucosyl transferase (UFGT) to form anthocyanins. CHS, CHI and F3H are early biosynethesis genes (EBGs); DFR, ANS and UFGT are late biosynethesis genes (LBGs) in the anthocyanin biosynthesis pathway. EBGs and LBGs are structural genes, their expression is controlled by regulatory genes which encoding several different types of transcription factors.In Arabidopsis, the expression of EBGs is controlled by R2R3 MYB transcription factors, including MYB DOMAIN PROTEIN 11 (MYB 11), MYB 12 and MYB111. The expression of LBGs is regulated by the MYB-bHLH-WD40 (MBW) transcription activator complex formed by an R2R3 MYB transcription factor, ANTHOCYANIN PIGMENT 1 (PAP 1), PAP2, MYB DOMAIN PROTEIN 113 (MYB 113) or MYB114, a bHLH transcription fector, GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) or TRANSPARENT TESTA8 (TT8), and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1), A similar MBW complex regulates epidermal cell fate in Arabidopsis by activating the transcription of GLABRA2 (GL2). A role of GL2 in regulating anthocyanin biosynthesis has not been reported.From an activation-tagged mutagenized population of Arabidopsis plants, we isolated a dominant, gain-of-function mutant with reduced anthocyanins. We found that the T-DNA was inserted in between GOLGIN CANDIDATE 5 (GC5) and GL2 in the chromosome 1, in a position that is 1886 bp upstream of the start codon of GL2 gene, with the four outward-facing 35S enhancers facing GL2. RT-PCR results revealed that this phenotype is caused by an elevated expression of the GL2, thus the mutant was named gl2-1D.35SE-GL2p:GL2 transgenic plants accumulate less anthocyanin, confirmed that the phenotype observed in the gl2-1D mutant is caused by elevated expression of GL2. Consistent with the view that GL2 acts as a negative regulator of anthocyanin biosynthesis, gl2-1D seedlings are hyposensitive whereas gl2-3 seedlings are hypersensitive to sucrose-induced anthocyanin biosynthesis. RT-PCR analysis indicated that expression of late, but not early, biosynthesis genes in the flavonoid pathway reduced in gl2-1D but elevated in gl2-3 mutants. Further analysis showed that expression of MBW component genes PAP1, PAP2, MYB113, MYB114 and TT8 rather than other transcription factor genes involved in the flavonoid pathway was reduced in gl2-1D but elevated in gl2-3 mutants. Chromatin Immuno-Precipitation (ChIP) assay showed that GL2 binds to MYB113, PAP2 and TT8 promoters. Protoplast transfection assays showed that GL2 functions as a transcriptional repressor. Taken together, our results indicate that GL2 is a transcriptional repressor that negatively regulates anthocyanin biosynthesis in Arabidopsis by directly repressing the expression of some of the MBW component genes.