A role for ascorbate conjugates of (+)-catechin in proanthocyanidin polymerization

Proanthocyanidins (PAs) are natural polymers of flavan-3-ols, commonly (+)-catechin and (-)-epicatechin. However, exactly how PA oligomerization proceeds is poorly understood. Here we show, both biochemically and genetically, that ascorbate (AsA) is an alternative "starter unit" to flavan-3-ol monomers for leucocyanidin-derived (+)-catechin subunit extension in the Arabidopsis thaliana anthocyanidin synthase (ans) mutant. These (catechin)_n:ascorbate conjugates (AsA-[C]_n) also accumulate throughout the phase of active PA biosynthesis in wild-type grape flowers, berry skins and seeds. In the presence of (-)-epicatechin, AsA-[C]_n can further provide monomeric or oligomeric PA extension units for non-enzymatic polymerization in vitro, and their role in vivo is inferred from analysis of relative metabolite levels in both Arabidopsis and grape. Our findings advance the knowledge of (+)-catechin-type PA extension and indicate that PA oligomerization does not necessarily proceed by sequential addition of a single extension unit. AsA-[C]_n defines a new type of PA intermediate which we term "sub-PAs".