Plastidial thioredoxin-like proteins are essential for normal embryogenesis and seed development in Arabidopsis thaliana.

Thiol/disulfide-based redox regulation is a key mechanism for modulating protein functions in response to changes in cellular redox status. Two thioredoxin (Trx)-like proteins [atypical Cys His-rich Trx (ACHT) and Trx-like2 (TrxL2)] have been identified as crucial for oxidizing and deactivating several chloroplast enzymes during light-to-dark transitions; however, their roles remain to be fully understood. In this study, we investigated the functions of Trx-like proteins in seed development. Using the CRISPR/Cas9 system, we generated an Arabidopsis quadruple mutant defective in ACHT1, ACHT2, TrxL2.1, and TrxL2.2 (acht/trxl2). This mutant showed increased seed lethality prior to maturation, with embryogenesis impaired primarily during the heart and torpedo stages, which are critical phases for plastid differentiation into chloroplasts. Using transgenic plants expressing EGFP-fused proteins, we confirmed that ACHT and TrxL2 are localized in plastids during embryogenesis. Additionally, seed development in the acht/trxl2 mutant was further impaired under extended darkness and could not be recovered through complementation with variants of ACHT or TrxL2 lacking the redox-active Cys residue (replaced by Ser). These findings indicate that the protein-oxidation functions of ACHT and TrxL2 are important for plastid differentiation into chloroplasts, embryogenesis, and seed development.