Glutathione promotes somatic embryogenesis by regulating <i>ThGPX4</i> in <i>Taxodium</i> hybrid ‘zhongshanshan’

Somatic embryogenesis (SE) in conifers is significantly genotype-dependent. In this study, multiple genotypes of Taxodium hybrid 'zhongshanshan' callus were treated with GSH (glutathione), BSO (Buthionine-sulfoximine, GSH synthesis inhibitor) and OTC (L-2-oxothiazolidine-4-carboxylate, increase the intracellular accumulation of GSH) to clarify that GSH significantly promoted SE efficiency and improved callus status. Determination of physiological indices revealed that GSH facilitated SE by enhancing antioxidant capacity, with the most significant differences observed at the dominant embryo stage. Transcriptome analysis indicated that the differentially co-expressed genes (DEGs) in the GSH-treated and BSO-treated groups were mainly enriched in the Glutathione metabolism, Protein processing in endoplasmic reticulum, Ascorbate and aldarate metabolism, and Pentose and glucuronate interconversions, and most of these DEGs were associated with antioxidant activity. Furthermore, we found that the glutathione peroxidase gene (ThGPX4) was significantly up-regulated after GSH treatment. Functional analysis showed that ThGPX4 had the highest expression at the dominant embryo stage and was located in both the nucleus and cytoplasm. Overexpression of ThGPX4 significantly enhanced SE in Arabidopsis thaliana. Using transient genetic transformation technology to overexpress ThGPX4 in Taxodium hybrid 'zhongshanshan' embryogenic calli, we observed that ThGPX4 could regulate the GSH/GSSG (reduced glutathione/oxidized glutathione) ratio, reduce H₂O₂ (hydrogen peroxide) content, and inhibit the activities of SOD (superoxide dismutase) and POD (peroxidase). These results indicated that GSH treatment can activate the expression of genes like ThGPX4 and consequently increases the antioxidant capacity of calli and enhances SE efficiency. The present study provides a valuable basis on the solution of the genotype recalcitrance in conifer SE.