Identification of genes that may regulate the expression of the transcription factor production of anthocyanin pigment 1 (PAP1)/MYB75 involved in Arabidopsis anthocyanin biosynthesis

A putative RNA-binding protein with a single RNA Recognition Motif (At3G63450) is involved in anthocyanin biosynthesis via its ability to modulate the transcript level of a major positive regulator PAP1 in Arabidopsis. The R2R3 MYB-activator production of anthocyanin pigment 1 (PAP1)/MYB75 plays a major role in anthocyanin biosynthesis in Arabidopsis in combination with one of three bHLH activators including transparent test 8 (TT8), enhancer of glabra3 (EGL3), glabra3 (GL3), and the WD-repeat transcription factor transparent testa 1 (TTG1), forming ternary MYB-basic HLH-WD40 complexes. Transcriptional activation of PAP1 expression is largely triggered by changes in light color and intensity, temperature fluctuations, nutrient status, and sugar and hormone treatments. However, the immediate upstream and downstream regulatory factors for PAP1 transcription are largely unknown. In the present study, using a T-DNA insertional mutagenesis approach, we transformed pap1-Dominant (pap1D) plants to modulate the levels of endogenous PAP1 transcripts. We employed Restriction Site Extension (RSE)-PCR analysis of 247 homogenous T3 genetic mutant lines exhibiting variations in anthocyanin accumulation compared to pap1D and identified 92 lines with T-DNA integrated in either intra- or inter-genic locations. This analysis revealed 80 novel candidate proteins, including a putative RNA-binding protein with a single RNA Recognition Motif (At3G63450), which may directly or indirectly regulate PAP1 expression at the transcriptional level.