Wild soybean cotyledons at the emergence stage tolerate alkali stress by maintaining carbon and nitrogen metabolism, and accumulating organic acids.

Soil alkalization is a global ecological problem that constrains food security and sustainable socio-economic development. As a wild relative of soybean, wild soybean (Glycine soja) exhibits strong salt and alkali stress resistance and its cotyledons play a key role during the emergence (VE) stage. This study aimed to compare variations in growth parameters, cotyledon ultrastructure, photosynthetic physiology, mineral ion and metabolite contents, and gene expression in two ecotypes of wild soybean to elucidate the regulatory mechanisms underlying alkali stress resistance in salt-tolerant wild soybean cotyledons during the VE stage. The results showed that salt-tolerant wild soybean cotyledons exhibited relatively stable growth parameters, dense and orderly chloroplast structure, high photosynthetic rates, as well as high K and Ca contents under alkali stress. Metabolomics, transcriptomics, and weighted gene co-expression network analyses revealed that salt-tolerant wild soybean cotyledons adapted to alkali stress during the VE stage by enhancing photosynthetic carbon assimilation pathways, increasing methionine and proline biosynthesis, and enhancing gamma-aminobutyric acid biosynthesis, thereby maintaining a stable carbon and nitrogen balance. In addition, upregulation of the expression of ICL, MS, and ACO led to the accumulation of various organic acids, such as pyruvic, aconitic, succinic, oxalic, malic, and fumaric acids, thereby promoting the synthesis of organic acid metabolism modules. This study provides novel insights into the key metabolic modules by which wild soybeans resist alkali stress.