ORIGINAL PAPER Identification of genes that may regulate the expression of the transcription factor production of anthocyanin pigment 1 (PAP1)/MYB75 involved in Arabidopsis anthocyanin biosynthesis Dong Ho Shin • Misuk Cho • Myoung Goo Choi • Prasanta Kumar Das • Seul-Ki Lee • Sang-Bong Choi • Youn-Il Park Received: 17 November 2014 / Revised: 19 December 2014 / Accepted: 6 January 2015 Ó Springer-Verlag Berlin Heidelberg 2015 Abstract Key message 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. Abstract The R2R3 MYB-activator production of antho- cyanin 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 com- plexes. 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 pro- teins, 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. Keywords Anthocyanin  PAP1 regulator  T-DNA insertion mutagenesis  Arabidopsis Introduction Anthocyanins serve various functions including coloring plant tissues, attracting pollinators and animals for polli- nation and seed dispersal, and protecting plants against different biotic and abiotic stresses such wounding, UV, insects, herbivores, and fungal infection (Winkel-Shirley 2002; Allan et al. 2008; Rowan et al. 2009). The biosyn- thesis of anthocyanins is mediated by multiple enzymes in the phenylpropanoid pathway (Koes et al. 2005); the genes encoding these enzymes are grouped into two classes D. H. Shin and M. Cho contributed equally to this work. Communicated by J. S. Shin. Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1743-7) contains supplementary material, which is available to authorized users. D. H. Shin  M. Cho  M. G. Choi  S.-K. Lee  Y.-I. Park (&) Department of Biological Sciences, Chungnam National University, 99 Daehagro, Youseong, Daejeon, 305-764, Korea e-mail: yipark@cnu.ac.kr Present Address: M. Cho Department of Eco-Biological Science, Woosuk University, Jincheon-eup, Jincheon-gun, Chungcheongbuk-do 365-800, South Korea P. K. Das DBT-AAU Centre for Agricultural Biotechnology, Assam Agricultural University, Jorhat, India S.-B. Choi Division of Biosciences and Bioinformatics, Myongji University, Yongin 449-728, Korea 123 Plant Cell Rep DOI 10.1007/s00299-015-1743-7