A <i>HORT1</i> Retrotransposon Insertion in the <i>PeMYB11</i> Promoter Causes Harlequin/Black Flowers in <i>Phalaenopsis</i> Orchids

Abstract

The harlequin/black flowers in <i>Phalaenopsis</i> orchids contain dark purple spots and various pigmentation patterns, which appeared as a new color in 1996. We analyzed this phenotype by microscopy, HPLC, gene functional characterization, genome structure analysis, and transient overexpression system to obtain a better understanding of the black color formation in <i>Phalaenopsis</i> orchids. Most mesophyll cells of harlequin flowers showed extremely high accumulation of anthocyanins as well as a high expression of <i>Phalaenopsis equestris</i> MYB11 (<i>PeMYB11</i>) as the major regulatory R2R3-MYB transcription factor for regulating the production of the black color. In addition, we analyzed the expression of basic helix-loop-helix factors, WD40 repeat proteins, and <i>MYB27</i>- and <i>MYBx</i>-like repressors for their association with the spot pattern formation. To understand the high expression of <i>PeMYB11</i> in harlequin flowers, we isolated the promoter sequences of <i>PeMYB11</i> from red and harlequin flowers. A retrotransposon, named <i>Harlequin Orchid RetroTransposon 1</i> (<i>HORT1</i>), was identified and inserted in the upstream regulatory region of <i>PeMYB11</i> The insertion resulted in strong expression of <i>PeMYB11</i> and thus extremely high accumulation of anthocyanins in the harlequin flowers of the <i>Phalaenopsis</i> Yushan Little Pearl variety. A dual luciferase assay showed that the insertion of <i>HORT1</i> enhanced <i>PeMYB11</i> expression by at least 2-fold compared with plants not carrying the insertion. Furthermore, the presence of <i>HORT1</i> explains the high mutation rates resulting in many variations of pigmentation patterning in harlequin flowers of <i>Phalaenopsis</i> orchids.