Transcriptome and metabolome combine to analyze the mechanism of leaf coloration formation in Aeonium arboretum ‘Pink Sybil’

The decorative quality of succulents largely stems from their leaf color. Aeonium arboreum's pink Sybil leaves feature an eye-catching stripe pattern and are particularly favored by customers, although the underlying mechanisms of its distinctive coloration are unknown. This study analyzed Aeonium arboreum ‘Pink Sybil’ leaves at the cellular and molecular levels. UHPLC-HRMS identified 11 flavonoid-related metabolites, showing elevated levels in RS samples. Cyanidin 3-galactoside emerged as the predominant compound, representing 93.4% of the total flavonoid content in RS samples(the red part of leaf margin), which was substantially greater than in the GM samples༈the green part of leaf center༉. Freehand slices revealed that anthocyanins, which contribute to the red coloring, were predominantly accumulated in the epidermal cells of the red tissue, in contrast to their presence in the green leaf tissue. Furthermore, cyanidin 3,5-diglucoside was not identified in GM but only in RS. The comparison of two transcripts identified 1,817 DEGs, with 1,123 up-regulated and 694 down-regulated genes. KEGG enrichment analysis revealed that the 20 most significantly enriched DEGs were involved in metabolic pathways, notably the phenylpropanoid and flavonoid biosynthesis pathways, which were closely related to the metabolism of anthocyanins. The majority of the structural genes and transcription factors involved in flavonoid metabolism were shown to be up-regulated using qRT-PCR. Phylogenetic analysis of transcription factors and co-expression network analysis of various metabolites and genes identified one MYB transcription factor, Aa PHL7, and three NAC transcription factors, Aa NAC102, Aa NAC045, and Aa NAC017, which may be involved in the regulation of anthocyanin synthesis in the leaves of the Aeonium arboreum ‘Pink Sybil’. The expression of these structural genes was highly and positively linked with the levels of anthocyanidins, such as Cyanidin 3,5-diglucoside and Cyanidin 3-galactoside. These compounds synergistically increase the expression of CHS1, CHS2, UFGT1, UFGT2, and 4CL during anthocyanin production. The study's findings identified the primary differential metabolites in the red tissue RS and green tissue GM of Aeonium arboretum ‘Pink Sybil’ leaves. This insight lays the groundwork for the initial identification of structural genes and transcription factors that show a strong and positive link with these metabolites. Our findings pave the way for a deeper understanding of the biochemical processes behind leaf discoloration in Aeonium arboreum ‘Pink Sybil’.