MYB transcription factors of plants play important roles in flavonoid synthesis, aroma regulation, floral organ morphogenesis, and responses to biotic and abiotic stresses. Cymbidium ensifolium is a perennial herbaceous plant belonging to Orchidaceae, with special flower colors and high ornamental value. In this study, a total of 136 CeMYB transcription factors were identified from the genome of C. ensifolium, including 27 1R-MYBs, 102 R2R3-MYBs, 2 3R-MYBs, 2 4R-MYBs, and 3 atypical MYBs. Through phylogenetic analysis in combination with MYB in Arabidopsis thaliana, 20 clusters were obtained, indicating that these CeMYBs may have a variety of biological functions. The 136 CeMYBs were distributed on 18 chromosomes, and the conserved domain analysis showed that they harbored typical amino acid sequence repeats. The motif prediction revealed that multiple conserved elements were mostly located in the N-terminal of CeMYBs, suggesting their functions to be relatively conserved. CeMYBs harbored introns ranging from 0 to 13 and contained a large number of stress- and hormone-responsive cis-acting elements in the promoter regions. The subcellular localization prediction demonstrated that most of CeMYBs were positioned in the nucleus. The analysis of the CeMYBs expression based on transcriptome data showed that CeMYB52, and CeMYB104 of the S6 subfamily may be the key genes leading to flower color variation. The results lay a foundation for the study of MYB transcription factors of C. ensifolium and provide valuable information for further investigations of the potential function of MYB genes in the process of anthocyanin biosynthesis.

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation.

The basic leucine zipper (bZIP) transcription factors constitute the most widely distributed and conserved eukaryotic family. They play crucial roles in plant growth, development, and responses to both biotic and abiotic stresses, exerting strong regulatory control over the expression of downstream genes. In this study, a genome-wide characterization of the CebZIP transcription factor family was conducted using bioinformatic analysis. Various aspects, including physicochemical properties, phylogenetics, conserved structural domains, gene structures, chromosomal distribution, gene covariance relationships, promoter cis-acting elements, and gene expression patterns, were thoroughly analyzed. A total of 70 CebZIP genes were identified from the C. ensifolium genome, and they were randomly distributed across 18 chromosomes. The phylogenetic tree clustered them into 11 subfamilies, each exhibiting complex gene structures and conserved motifs arranged in a specific order. Nineteen pairs of duplicated genes were identified among the 70 CebZIP genes, with sixteen pairs affected by purifying selection. Cis-acting elements analysis revealed a plethora of regulatory elements associated with stress response, plant hormones, and plant growth and development. Transcriptome and qRT-PCR results demonstrated that the expression of CebZIP genes was universally up-regulated under low temperature conditions. However, the expression patterns varied among different members. This study provides theoretical references for identifying key bZIP genes in C. ensifolium that confer resistance to low-temperature stress, and lays the groundwork for further research into their broader biological functions.

Cymbidium floribundum is an ornamental plant with showy and colorful flowers. The color of its lip changes significantly after pollination. However, the mechanism of lip coloration remains unclear. In this study, the mechanism underlying lip color change in C. floribundum was investigated before and after pollination. Metabolome analysis detected 61 flavonoids in the lip, including 24 flavonoids, 13 flavonols, nine flavonoid carbonosides, eight anthocyanins, three flavanols, two isoflavones, one chalcone, and one dihydroflavone. Accumulation of peonidin 3-O-glucoside chloride, cyanin chloride, and cyanidin 3-O-malonylhexoside after pollination may be the key factors contributing to the change in lip color. Furthermore, transcriptome analysis identified 43 genes related to the anthocyanin biosynthesis pathway (ABP). Phylogenetic and co-expression analysis indicated that CfMYB1, CfMYB3, and CfMYB4 may be involved in the regulation of anthocyanin biosynthesis in the lips. Subcellular localization results showed that CfMYB1 was located in the nucleus, while CfMYB3 and CfMYB4 were located in the nucleus and cytoplasm. Further functional analysis verified that CfMYB1 could activate ABP genes and promote the synthesis and accumulation of anthocyanin, which may be the main transcription factors leading to the change of lip color in C. floribundum after pollination. These findings provide insight into the anthocyanin accumulation and coloration mechanisms during C. floribundum flower development. The results provide genetic resources and a theoretical basis for the improvement and breeding of flower color in C. floribundum.