Identification and functional analysis of three new anthocyanin R2R3‐<scp>MYB</scp> genes in <i>Petunia</i>

Abstract

We identified three novel members of the R2R3-MYB clade of anthocyanin regulators in the genome of the purple flowering <i>Petunia inflata S6</i> wild accession, and we called them <i>ANTHOCYANIN SYNTHESIS REGULATOR</i> (<i>ASR</i>). Two of these genes, <i>ASR1</i> and <i>ASR2,</i> are inactivated by two different single base mutations in their coding sequence. All three of these genes are absent in the white flowering species <i>P. axillaris N</i> and <i>P. parodii</i>, in the red flowering <i>P. exserta</i>, and in several <i>Petunia hybrida</i> lines, including R27 and W115. <i>P. violacea</i> and other <i>P</i>. <i>hybrida</i> lines (M1, V30, and W59) instead harbor functional copies of the <i>ASR</i> genes. Comparative, functional and phylogenic analysis of anthocyanin R2R3-MYB genes strongly suggest that the <i>ASR</i> genes cluster is a duplication of the genomic fragment containing the other three R2R3-MYB genes with roles in pigmentation that were previously defined, the <i>ANTHOCYANIN4-DEEP PURPLE</i>-<i>PURPLE HAZE</i> (<i>AN4</i>-<i>DPL</i>-<i>PHZ</i>) cluster. An investigation of the genomic fragments containing anthocyanin MYBs in different <i>Petuni</i>a accessions reveals that massive rearrangements have taken place, resulting in large differences in the regions surrounding these genes, even in closely related species. Yeast two-hybrid assays showed that the ASR proteins can participate in the WMBW (WRKY, MYB, B-HLH, and WDR) anthocyanin regulatory complex by interacting with the transcription factors AN1 and AN11. All three ASRs can induce anthocyanin synthesis when ectopically expressed in <i>P. hybrida</i> lines, but ASR1 appeared to be the most effective. The expression patterns of <i>ASR1</i> and <i>ASR2</i> cover several different petunia tissues with higher expression at early stages of bud development. In contrast, <i>ASR3</i> is only weakly expressed in the stigma, ovary, and anther filaments. The characterization of these novel <i>ASR</i> MYB genes completes the picture of the MYB members of the petunia anthocyanin regulatory MBW complex and suggests possible mechanisms of the diversification of pigmentation patterns during plant evolution.