Simple Induction and Detection of Anthocyanins in Arabidopsis thaliana: A Tool for Mutant Screening and Metabolic...

Anthocyanins are specialized flavonoid pigments that play critical roles in plant coloration, photoprotection, and responses to environmental stress. Arabidopsis thaliana serves as a valuable genetic model for dissecting anthocyanin biosynthesis and regulatory networks. Conventional methods for anthocyanin quantification, such as crude spectrophotometric assays, often compromise pigment integrity, yield inconsistent results, and provide limited information on compound composition. Here, we describe a simple, reproducible, and high-fidelity protocol for the induction, extraction, quantification, and chromatographic profiling of anthocyanins in Arabidopsis thaliana seedlings. The workflow employs well-defined anthocyanin-inductive conditions (AIC), methanol/formic acid extraction, lyophilization for dry-weight normalization, and dual quantification via spectrophotometry and High-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis. This protocol enables accurate comparison between wild-type and mutant genotypes, facilitating both mutant screening and metabolic pathway analysis. The approach minimizes pigment degradation, enhances reproducibility across replicates, and offers a robust tool for research in plant metabolism, stress physiology, and flavonoid biochemistry. Key features • This protocol establishes well-defined anthocyanin-inductive conditions (AIC) using sucrose and continuous light, enabling reproducible pigment accumulation in Arabidopsis thaliana seedlings. • This protocol employs methanol/formic acid extraction and lyophilization to maintain anthocyanin stability and minimize degradation during sample processing. • This protocol integrates spectrophotometric OD₅₃₂ normalization with HPLC-DAD profiling for quantification of total anthocyanins and characterization of individual anthocyanin species. • This protocol is suitable for mutant screening, metabolic pathway analysis, and stress-response studies in Arabidopsis thaliana.