Comparative Metabolomics Reveals Differences in Germination and Phytochemical Profiles Between Contrasting Pearl Millet (Penninsetum glaucum) Varieties

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a crucial cereal in arid regions, values for its bioactive compounds. To understand the impact of nutrient enhancement, we conducted a comparative analysis between an iron- and zinc-enriched pearl millet variety, HHB-299, and a non-enriched variety, ICMV-221. Our investigation evaluated seed germination rate, callus induction behavior, and their associated endogenous chemical compositions. Notably, HHB-299 exhibited significantly higher seed germination and callus induction rates, accompanied by elevated levels of macro- and microelements, phenolic compounds (including syringic acid and myricetin), and amino acids. Comprehensive GC-MS-based volatile metabolomic profiling revealed a greater diversity of compounds in HHB-299 seeds and early seedlings. FTIR analysis corroborated the presence of diverse bioactive functional groups. Furthermore, non-targeted LC-MS metabolomics identified dynamic metabolite changes across germination stages in both varieties. Principal component analysis clearly distinguished between the genotypes and germination stages, explaining over 94% of the variance in both GC-MS and LC-MS data. HHB-299 consistently displayed a higher number of detected metabolites across the initial germination time points. Classification of metabolites into lipids, flavonoids, alkaloids, terpenoids, amino acid derivatives, and organic acids, coupled with Venn diagrams and heatmap analyses, highlighted both shared and unique metabolic features. Importantly, HHB-299 showed a greater upregulation of the majority of common metabolites. These findings underscore the potential of nutrient enrichment to not only enhance nutritional content but also to improve developmental potential through the modulation of endogenous phytonutrients in pearl millet.