Abstract
Background: The millet, an important coarse grain crop, exhibits drought resistance, water conservation efficiency, and adaptability to poor soil conditions. Although the purple leaf sheath is a prevalent phenotype, the underlying mechanisms of color variation remain unclear. Previous studies suggested that leaf sheath coloration might result from differential accumulation of plant pigments, yet the key substances and molecular mechanisms governing color formation in millet have not been systematically elucidated. Results: We investigated the metabolic pathway underlying millet leaf sheath coloration using multi-omics analysis. Physiological data revealed that the purple phenotype was associated with up-regulated anthocyanin biosynthesis and down-regulated chlorophyll accumulation. However, carotenoid content was extremely low, showing no significant changes, and it was significantly lower than the contents of total anthocyanins and chlorophyll. It is speculated that there is no significant association between carotenoids and the color differences in leaf sheaths. Transcriptome data suggest that the high expression levels of anthocyanin biosynthesis genes (PAL, 4CL, CHS, CHI, F3H, CYP73A, CYP75B1, DFR, ANS, and UFGT) and the low expression of chlorophyll synthesis genes (chlH, chlI, and chlM) likely contributed to the observed color phenotype. Furthermore, transcription factors from the WRKY, MYB, and bHLH families were predicted to interact with these differentially expressed genes, collectively modulating color formation. Proteomic analysis revealed that these candidate genes also exhibited significant differential expression at the protein level, demonstrating strong concordance between transcriptional and translational regulation. However, the expression levels of the few detected mRNAs and proteins do not align exactly between the transcriptome and proteome. It was speculated that these proteins may have undergone post-transcriptional alterations and activations, releasing active proteins immediately without the need for transcription of related genes. Conclusions: This study had laid a foundation for the preliminary exploration of the differential expression and biosynthetic mechanism of the color of millet leaf sheaths. Furthermore, these findings identify valuable candidate genes that may facilitate future breeding efforts for colored millet varieties.