Identification and characterization of the structure genes involved in anthocyanin biosynthesis in flowers of Cymbidium species

The Orchidaceae family is renowned for its remarkable floral diversity, showing a wide range of colors primarily influenced by pigments especially anthocyanins. <i>Cymbidium haematodes</i> is a terrestrial orchid characterized by vibrant flower colors, making it an ideal resource for studying anthocyanin biosynthesis in orchids. In this study, we systematically identified and characterized the structural genes involved in anthocyanin biosynthesis in <i>C. haematodes</i> and compared their roles between <i>C. haematodes</i> and its close relative <i>C. sinense</i>. Pigment analysis revealed that anthocyanins were the predominant pigments in the red-colored tepals of <i>C. haematodes</i>, exhibiting significantly higher levels in dark red (DR) and red (R) accessions compared to lighter color accessions. Transcriptome sequencing identified 20 structural genes, including <i>CHS</i>, <i>F3H</i>, <i>F3'H</i>, <i>DFR</i>, <i>ANS</i>, and <i>UFGT</i>. Phylogenetic analysis indicated close evolutionary relationships with other <i>Cymbidium</i> species. Notably, <i>CsF3'H2</i> and <i>CsF3'H3</i> exhibited high expression levels in red tepals, while <i>CsUFGT1-3</i> and <i>CsDFR1-2</i> were up-regulated in <i>C. sinense</i>. This suggests species-specific regulatory mechanisms governing pigment production. Subcellular localization assays confirmed cytoplasmic distribution for <i>CsANS</i>, chloroplast localization for <i>CsF3'H2</i>, and cell membrane association for <i>CsF3'H3</i>, implicating diverse functional roles related to anthocyanin transportation and biosynthesis. These findings highlight the functional divergence of structural genes in anthocyanin biosynthesis between <i>C. haematodes</i> and <i>C. sinense</i>, and have specifically identified <i>CsF3'Hs</i> as key genes of red coloration in <i>C. haematodes</i> flowers. This study provides foundational insights into the molecular mechanisms underlying flower color variation among orchids, offering potential targets for future genetic manipulation aimed at enhancing ornamental breeding practices.