Proanthocyanidin synthesis in the forage legume Onobrychis viciifolia.: A study of chalcone synthase, dihydroflavonol 4-reductase and leucoanthocyanidin 4-reductase in developing leaves

The timing of proanthocyanidin (PA) biosynthesis in leaves of Onobrychis viciifolia (sainfoin) was examined using nucleotide probes, enzyme assays and immunochemical detection. To achieve this, a chalcone synthase (CHS) cDNA clone was isolated and a PCR-amplified DNA fragment corresponding to part of a dihydroflavonol reductase (DFR) gene was obtained from sainfoin. The high sequence homology of the CHS clone, both at the nucleotide and amino acid levels, with CHS sequences from other species, confirmed the identity of the clone. The identity of the PCR fragment was confirmed by sequence homology to known DFR sequences. The level of DFR mRNA, DFR and leucoanthocyanidin reductase (LAR) enzyme activities, CHS mRNA and CHS protein were at a maximum in young, unexpanded leaves of sainfoin. The levels of DFR mRNA, and DFR and LAR enzyme activity declined rapidly and were absent in older tissues. PA content of leaves declined slightly on a fresh and dry weight basis in the more mature phases of leaf development; however, PA content on a per leaf basis continued to increase throughout development. The apparent anomaly between the activity of critical enzymes (LAR and DFR) and the accumulation of PA indicates that the process of proanthocyanidin biosynthesis is still poorly understood.