TrIFS strengthens isoflavonoid accumulation and improves aluminum tolerance involved with GmALDH22A2-GmRBOHL pathway in soybean

Isoflavonoids, a major class of secondary metabolites predominantly found in legumes, serve as vital defenders against both biotic and abiotic stresses, though their molecular mechanisms in mitigating aluminum (Al) toxicity remain incompletely understood. In this study, we introduced the isoflavone synthase gene isolated from a white clover ( TrIFS ) into soybean. The resulting transgenic soybeans exhibited a noticeable increase in isoflavone accumulation in various organs and stronger tolerance to aluminum toxicity. The dry weight of roots of two transgenic lines, TL124 and TL129, were heavier than that of the wild type (WT) after 200 µM AlCl 3 treatment, by 46.1% and 38.6% respectively. Higher seed yield per plant was also obtained in these two transgenic lines by 18.9% and 18.2% than in the WT under Al stress, respectively. Additionally, TrIFS transgenic soybeans showed significantly reduced levels of aluminum-induced malondialdehyde (MDA) and reactive oxygen species (ROS). RNA transcriptome analysis revealed that the transcriptional level of GmALDH22A2 ( Glyma.09g036000 , Wm82.a2.v1) was sharply decreased in the TrIFS transgenic lines under Al treatment. The GmALDH22A2 protein interaction with a respiratory burst oxidase homologue (GmRBOHL, Glyma.10g152200 , Wm82.a2.v1) was comfirmed by yeast two hybrid, fluorescence complementation and co-immunoprecipitation assays. Furthermore, this ALDH-RBOH interaction could cause increase in ROS production in tobacco, which suggested that the decreased transcription of GmALDH22A2 in the TrIFS transgenic soybeans would attenuate ROS generation. In summary, we provided new insights in improving Al tolerance through isoflavone enhancement. • Overexpression of TrIFS in soybean strengthens isoflavone accumulation across various organs. • Elevated isoflavone levels obviously alleviate Al phytotoxicity. • Higher levels of soybean isoflavones would induce to suppress the expression of GmALDH22A2 . • GmALDH22A2-GmRBOHL protein interaction enhances ROS generation.