Triacontanol Boosts Soybean Nodulation via GmHSP26-Mediated Antioxidant Enhancement

Soybean (Glycine max (L.) Merr.) is a globally crucial food crop and a model plant for studying symbiotic nitrogen fixation in legumes. Triacontanol (TRIA) is a natural plant growth regulator that enhances photosynthetic efficiency, stress tolerance, antioxidant enzyme activities and yield in crops. However, its regulatory role in nodulation and nitrogen fixation in legumes remains unclear. In this study, soybean seedlings inoculated with Bradyrhizobium japonicum strain USDA110 were treated with different concentrations of TRIA (0, 0.33, 0.5, 1 and 2 μg/mL). Then, oxidative stress indicators and comparative transcriptomic analysis were performed to check the oxidative status and screen the candidate genes under TRIA treatment. Our results showed that the 0.5 μg/mL TRIA treatment produced the greatest nodule number. TRIA treatment significantly induced antioxidant responses in soybean roots. Comparative transcriptome identified 867 differentially expressed genes (DEGs), Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of DEGs revealed that a large number of genes were enriched in pathways related to oxidative activity. Combined with the expression pattern, we identified a Glutathione S-Transferase family gene, GmHSP26 (Glyma.07G139700), whose expression was induced by both TRIA and rhizobial infection, with its promoter activity was activated throughout the entire process of nodule development. Further function study using overexpression and gene editing proved that GmHSP26 was a positive regulator of soybean nodulation. Collectively, this study identifies the optimal TRIA concentration for promoting soybean nodulation, reveals the function and mechanism of GmHSP26 in response to TRIA-regulated nodulation, and provides a theoretical basis and genetic resource for enhancing nodulation and nitrogen fixation in leguminous crops through exogenous growth regulators.