Two Indel Variations in the <i>Regulated Carotenoid Pigment</i> Gene Hold the Key to the Interspecies Flower Colour Differentiation in <i>Liriodendron</i> Plants

Flower colour variation is pivotal for plant evolution, ecological adaptation, and pollinator attraction. In the Liriodendron genus, L. tulipifera features a distinct orange petal stripe which is absent in L. chinense, driven by differential carotenoid accumulation, yet the underlying genetic mechanism remains unclear. To address this, we assembled a high-quality 1.57 Gb genome of L. tulipifera 'MSL' and analysed comparative transcriptome data to identify carotenoid biosynthesis-related regulatory factors. Transcriptomic analysis revealed specific activation of the carotenoid biosynthesis pathway in the orange stripe region of L. tulipifera, with specific expression of the lycopene ε-cyclase gene (LCYE). A subgroup 2 R2R3-MYB transcription factor, designated RCP (Regulated Carotenoid Pigment), was found to bind the LCYE promoter and promote carotenoid biosynthesis. Notably, two indel variants (24 and 15 bp) in the third exon of L. chinense LcRCP altered its function, reducing LCYE activation and carotenoid content, and these variants are widespread in natural L. chinense populations. Transient expression assays in N. benthamiana and Liriodendron leaves showed L. tulipifera LtRCP promoted carotenoid accumulation, while LcRCP inhibited it. Our findings uncover a natural regulatory variation governing interspecific flower colour divergence in Liriodendron, providing new insights into carotenoid pathway regulation and its role in floral trait evolution.