Vanillin Assay of Arabidopsis Seeds for Proanthocyanidins

Abstract

[Abstract] Proanthocyanidins (PAs) are colorless flavonoid polymers and deposit in Arabidopsis seed coat specifically. Oxidation of PAs gives rise to brown color of mature seeds. PA accumulation can be affected by a number of growth conditions, such as temperature and sun light. PAs, which are converted from anthocyanidins, can protect seeds from outer environment and have a positive effect in seed longevity (Debeaujon, 2003). Vanillin turns red upon binding to leucoanthoanthocyanidins, catechins and monomers and terminal subunits of PAs (Butler et al., 1982; Deshpande et al., 1986). Based on this principle, PA deposition in Arabidopsis seed coat can be visualized. Materials and Reagents 1. Arabidopsis seeds (Col 0 and tt mutants) 2. 6 M HCl 3. Sterile distilled water 4. Dye solution (for coloration and reaction with PAs) containing 1% w/v vanillin (Sangon Biotech, catalog number: VT0974-100g) and 6 M HCl stored in brown bottle Note: The dye solution should be used after preparation as soon as possible (not longer than half an hour). 5. Vanillin reagent (see Recipes) Equipment 1. Brown bottle (or glass bottle covered with aluminum foil) 2. 1.5 ml microcentrifuge tubes 3. Glass slides (25.4 mm x 76.2 mm) and coverslips (20 mm x 20 mm) 4. Dissecting needle and tweezer 5. An SZ61-zoom stereomicroscope (OLYMPUS, model: SZ61) 6. A compound light microscope (OLYMPUS, model: BX61)