The <i>Arabidopsis TDS4</i> gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development

<jats:title>Summary</jats:title><jats:p>The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The <jats:italic>Arabidopsis</jats:italic> mutant <jats:italic>tannin deficient seed 4</jats:italic> (<jats:italic>tds4‐1</jats:italic>) has a reduced PA level and altered pattern PA accumulation. We identified the <jats:italic>TDS4</jats:italic> gene as <jats:italic>LDOX</jats:italic> by complementation of the <jats:italic>tds4‐1</jats:italic> mutation either with a cosmid encoding LDOX or a <jats:italic>35S:LDOX</jats:italic> construct. Independent <jats:italic>Arabidopsis</jats:italic> lines with a T‐DNA insertion in the <jats:italic>LDOX</jats:italic> gene had a similar phenotype, and one was allelic to <jats:italic>tds4‐1.</jats:italic> The seed phenotype of <jats:italic>ban tds4</jats:italic> double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other <jats:italic>TDS</jats:italic> genes. Analysis of the PA intermediates in <jats:italic>tds4‐1</jats:italic> revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of <jats:italic>Arabidopsis</jats:italic> PA and its precursors indicates that <jats:italic>Arabidopsis</jats:italic>, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of <jats:italic>tds4</jats:italic> were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that <jats:italic>tds4</jats:italic> endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.</jats:p>