Metabolic reprogramming in finger millet following ethyl methanesulfonate mutagenesis

Finger millet is a climate-resilient cereal crop with strong potential for improving food security in marginal environments; however, its improvement has been constrained by low natural genetic variability. This limitation has prompted the use of mutation breeding to expand genetic diversity in underutilized crops. Chemical mutagenesis, particularly using ethyl methanesulfonate (EMS) has been widely applied for this purpose, yet its concentration-dependent metabolic effects during plant development remain insufficiently understood. This study aimed to characterize metabolic changes in seedlings of four finger millet genotypes subjected to 0% (control), 0.10%, 0.40%, and 0.70% EMS using untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. Seeds were treated for one hour and grown in a randomized complete design with three replicates per treatment. Aboveground tissues were harvested six weeks after planting, and metabolic profiles were compared across treatments.

Principal component analysis revealed clear concentration-dependent separation, with PC1 and PC2 explaining 63.5% of the variance. Control samples formed a compact cluster, while 0.10% EMS-treated seedlings showed slight but distinct separation along PC2. Supervised models (PLS-DA, sPLS-DA, and OPLS-DA) confirmed strong discrimination among treatments and identified key metabolites driving separation. A total of 2 262 metabolic features were detected, of which 400 were putatively annotated at MSI level 2 based on accurate mass and MS/MS spectral matching. Differential analysis identified 81, 31, and 50 significantly altered metabolites at 0.10%, 0.40% and 0.70% EMS concentrations, respectively, spanning organic acids, phenolics, flavonoids, lipids, alkaloids, amino acids and terpenoids. The lowest concentration (0.10%) induced the broadest response, marked by increased phenolic acids and flavonoids, alongside mixed regulation of organic acids, including elevated malic and succinic acids and reduced oxaloacetic acid. However, 0.40% of EMS resulted in widespread metabolic repression, with mesaconic acid among the few consistently upregulated metabolites. The highest concentration (0.70%) triggered extensive metabolic remodeling, including perturbation of tricarboxylic acid cycle intermediates and activation of secondary metabolites including chlorogenic acid. Pairwise comparisons revealed structured metabolic transitions, including suppression of phenylpropanoid pathways and shifts in central carbon metabolism with increasing EMS levels. Heatmap analysis highlighted genotype and replicate-specific responses, indicating heterogeneity in metabolic adaptation. Certain metabolites, including 2-caffeoyl-L-tartaric acid, showed strong accumulation under moderate and high EMS treatments, suggesting localized metabolic plasticity.

These findings demonstrate that EMS induces concentration-dependent and genotype-specific metabolic reprogramming in finger millet, providing insight into early biochemical responses to mutagenesis and supporting the application of metabolomics in optimizing mutation breeding strategies.