A high-resolution method for the localization of proanthocyanidins in plant tissues

Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.Proanthocyanidins, or condensed tannins, are polymers of flavan-3-ol subunits, which are produced by the flavonoid secondary pathway in many plants. Proanthocyanidins are best known for their protein-binding ability and are commercially significant because of their antioxidant properties and their potential health benefits when included at a low level in the diets of humans and livestock [1-5]. Proanthocyanidins are produced naturally in the leaves, flowers, fruit, seeds, bark and roots of many plant species [6-11]. A number of quantitative methods have been developed to analyze the level and subunit composition of proanthocyanidins in bulk tissue samples [5,7,12-14]. These methods can provide information about the degree of polymerisation and the hydroxylation pattern and stereochemistry of flavan-3-ol subunits.Vanillin and 4-dimethylaminocinnama