A histochemical assay for polyphenolic profiling in cereal grains

In different cell layers, cereal grains may accumulate various economically important polyphenols such as colored anthocyanins and melanins and colorless proanthocyanidins. To effectively create new cultivars with different combinations of these compounds, a simple, fast, and precise screening method is required. Here, a histochemical assay that includes a combination of hot ethanolic, acidic, alkaline, and ammoniacal silver treatments of grain cryosections followed by microscopy was successfully applied to distinguish these substances in cereal grains. Barley lines previously characterized chemically for the presence of anthocyanins, proanthocyanidins, and melanins in grains were used as a model. In black barley grains, this approach allowed to visually distinguish insoluble melanins that do not react to a pH change from anthocyanins, which can be insoluble or soluble but always react to changing pH. For the first time, ammoniacal silver staining commonly used for melanin identification in human and animal tissues was adapted for melanin identification in plant tissues. Along with melanins, this reagent stains other polyphenols thereby helping to detect colorless polyphenols including proanthocyanidins in the testa of barley grains as confirmed by p-dimethylaminocinnamaldehyde (DMACA) staining. The applicability of this assay to polyphenol profiling was demonstrated not only in the barley grain but also in wheat and common vetch grains. The proposed histochemical assay allows rapid polyphenol screening using a single grain, making it a practical and efficient alternative to time-consuming chromatographic methods for preliminary selection from large sample sets prior to detailed quantitative and qualitative chemical analysis.