Integrated transcriptomic and metabolomic profiling reveals the flower color formation mechanism of alfalfa different purple flowers.

Background: Flower color plays a crucial role in plant identification, evaluation, and evolution, and it is directly or indirectly associated with the agronomic characteristics of plants. Alfalfa ( Methods: We integrated metabolomics and transcriptomics analyses to investigate five alfalfa flower types: white (BH) and four shades of purple (ZH1-ZH4, from light to dark). Anthocyanin and carotenoid contents were quantified by LC-ESI-MS/MS. Differentially accumulated pigments were subjected to KEGG enrichment and statistical analysis. RNA-seq reads were mapped to the Medicago reference genome, and differentially expressed genes (DEGs) were identified using DESeq2. Common significant DEGs (CSDEGs) among the four purple-versus-white comparisons were clustered to identify expression trends. Quantitative Real-time PCR (qRT-PCR) was performed to validate the transcriptome data, and an O2PLS model was used to integrate metabolomic and transcriptomic datasets. Results: Metabolomics analysis revealed that delphinidin-3, 5-O-diglucoside and malvidin-3, 5-O-diglucoside were the two most abundant anthocyanins, and their contents increased steadily from ZH1 to ZH4. The ratio of blue to red pigments decreased along the same gradient. Transcriptomics identified 580 CSDEGs, of which 108 showed a regular trend across the five flower types, and 24 were transcription factors, with MYB and bHLH being the most represented. CSDEGs are involved in multiple processes such as stimulation of the internal and external cellular environment, pigment synthesis and regulation, pigment transport and deposition. Notably, 29 genes in CSDEGs were definitively involved in the anthocyanin biosynthesis chain, and some important candidate genes regulating the formation of purple color in alfalfa flowers were identified. Joint analysis of the two datasets revealed that the relative expression levels of two novel genes (MSTRG.59017, MSTRG.14861) were positively correlated with the contents changes of delphinidin-3, 5-O-diglucoside or malvidin-3, 5-O-diglucoside in BH, ZH1, ZH2, ZH3, and ZH4. Conclusion: Anthocyanins are the dominant pigments responsible for the purple coloration of alfalfa flowers. Delphinidin and malvidin derivatives, especially delphinidin-3, 5-O-diglucoside and malvidin-3, 5-O-diglucoside, are the key molecules driving the purple gradient, as well as the proportional relationship between blue and red pigments. Genes responding to the intracellular and extracellular environments influencing the purple formation of alfalfa flowers, involved in transcriptional, translational and post-translational regulation of anthocyanin biosynthesis, and involved in synthesis, transport and deposition of anthocyanin were screened out. We confirmed that there should be key genes specific to the synthesis of each anthocyanin glycoside, which will be the focus of future research. This study has established a theoretical foundation for explaining the different purple color of alfalfa flowers. This study enriches the theory of plant flower color formation and provides a theoretical basis for alfalfa flower color improvement and molecular breeding.