The HD‐ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat

SUMMARY Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB–bHLH–WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain‐leucine zipper (HD‐ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production. Significance Statement This study identifies MtGL2, an HD‐ZIP IV transcription factor in Medicago truncatula, that plays a crucial role in facilitating the metabolic flow from anthocyanidin to proanthocyanidin (PA) biosynthesis. Contrary to its Arabidopsis homolog, GL2, the loss of MtGL2 function does not lead to discernible phenotypic changes in trichomes, root hairs, or mucilage. MtGL2 demonstrates synergistic cooperation with the MYB–bHLH–WD40 (MBW) complexes in promoting PA biosynthesis, which provides insights for genetic engineering for PA production.