Anatomical and Digital Image Analysis of Flavonoid-Mediated Grain Coloration in Rye (Secale cereale L.)

Rye exhibits high diversity in grain coloration among small cereals, which is mainly linked to the presence of colored flavonoids synthesized in the outer layers of the kernel. This variability is not yet sufficiently described from colorimetric, cytological, and biochemical points of view. In this study, the localization of flavonoid pigments, anthocyanins and proanthocyanidins (PAs), was analyzed across different grain tissues in 26 rye lines with identified anthocyanin grain color genes. Grain coloration was objectively characterized using the CIELAB color coordinates and the GrainScan software for image analysis of individual grains. The distribution of anthocyanins and PAs was investigated through light microscopy and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) on longitudinal and cross sections of the grains. The results revealed that violet-grained lines accumulate anthocyanins in the pericarp, while green-grained lines contain anthocyanins in the aleurone layer. MALDI-IMS confirmed the presence of specific anthocyanins: cyanidins in the pericarp of violet-grained lines and delphinidins in the aleurone layer of green-grained lines. All studied lines, except for the anthocyanin-less vi3 mutants, exhibited PAs in the brown-colored testa. Four main color groups of the rye grains (yellow, green, brown, and violet) could be clearly differentiated using the CIE color coordinate h°.