Color components determination and full-length comparative transcriptomic analyses reveal the potential mechanism of carotenoid synthesis during Paphiopedilum armeniacum flowering

Abstract

Background: Paphiopedilum armeniacum (P. armeniacum), an ornamental plant native to China, is known for its distinctive yellow blossoms. However, the mechanisms underlying P. armeniacum flower coloration remain unclear.

Methods: We selected P. armeniacum samples from different flowering stages and conducted rigorous physicochemical analyses. The specimens were differentiated based on their chemical properties, specifically their solubilities in polar solvents. This key step enabled us to identify the main metabolite of flower color development of P. armeniacum, and to complete the identification by High-performance liquid chromatography (HPLC) based on the results. Additionally, we employed a combined approach, integrating both third-generation full-length transcriptome sequencing and second-generation high-throughput transcriptome sequencing, to comprehensively explore the molecular components involved.

Results: We combined physical and chemical analysis with transcriptome sequencing to reveal that carotenoid is the main pigment of P. armeniacum flower color. Extraction colorimetric method and HPLC were used to explore the characteristics of carotenoid accumulation during flowering. We identified 28 differentially expressed carotenoid biosynthesis genes throughout the flowering process, validated their expression through fluorescence quantification, and discovered 19 potential positive regulators involved in carotenoid synthesis. Among these candidates, three RCP2 genes showed a strong potential for governing the PDS and ZDS gene families. In summary, our study elucidates the fundamental mechanisms governing carotenoid synthesis during P. armeniacum flowering, enhancing our understanding of this process and providing a foundation for future research on the molecular mechanisms driving P. armeniacum flowering.

Keywords: Carotenoid; Flowering process; Full-length transcriptome; Paphiopedilum armeniacum; Transcriptome.

Figure 1. Color results of P. armeniacum flower organs and reagent delamination experiments at different stages.

Color results of P. armeniacum flower organs and reagent delamination experiments at different stages. (A–C) Flower bud stage, initial flowering stage, and full flowering stage, respectively. (D–E) Delaminated color results of reagents in the aforementioned stages.

Figure 2. Results of full-length transcriptome sequencing.

(A) Distribution map of transcript length; (B) functional annotation of transcripts in different databases; (C) Venn diagram of annotated results in different databases; (D) pathway enrichment map of transcripts in the KEGG database; and (E) histogram of the top 20 transcription factors with the highest transcript counts.

Figure 3. GO and KEGG enrichment diagrams of differentially expressed genes among three groups of samples.

(A) Bubble diagram depicting GO enrichment of differential genes; and (B) histogram displaying KEGG enrichment of differential genes, with the number at the end of the column representing the specific count of genes associated with the respective pathway.

Figure 4. Differentially expressed genes involved in carotenoid synthesis and their qPCR results.

(A) Heat map of differentially expressed genes in the carotenoid biosynthesis pathway. Green box represents intermediate compounds, and base is denoted by black and bold font. (B) Histogram presenting the GO enrichment results of subcellular localization for the differentially ex-pressed genes. (C) Histogram displaying the KEGG pathway enrichment results for the afore-mentioned differentially expressed genes. (D-H) qPCR results of genes related to carotenoid synthesis. These genes are i1c11866 (D), i2c21133 (E), i0c928 (F), i0c7956 (G) and i1c23264 (H). The TPM values of all genes and the relative expression values obtained by qPCR were divided by the mean of the data set to remove the dimensions between the data.

Figure 5. Enrichment results of expression trends for differentially expressed transcription factors and the correlation network diagram of key transcription factors and carotenoid synthesis genes.

(A) Enrichment results of differentially expressed transcription factors for expression trends. Colored trends represent significant enrichment. The number in the upper left corner of each box represents the trend number and is unrelated to internal enrichment genes. (B) Distribution of transcription factors within trend 5. (C) Distribution of transcription factors within trend 2. (D) Network diagram of correlation between MYB305, RCP1, RCP2 transcription factors and carotenoid biosynthesis genes.