GmSOD from Glycine max fine-tunes H2O2 homeostasis to coordinate antioxidant defense and citrate exudation under aluminum stress in Nicotiana benthamiana

Aluminum (Al) toxicity in acidic soils severely inhibits root growth. While enhanced antioxidant defense and organic acid exudation are both known Al tolerance mechanisms, how these processes are coordinated remains unclear. This study investigates whether and how the soybean ( Glycine max ) superoxide dismutase gene GmSOD (GenBank accession no. M64267.1) orchestrates these responses via modulation of hydrogen peroxide (H 2 O 2 ) signaling. Wild-type (WT) and GmSOD -overexpressing transgenic tobacco ( Nicotiana benthamiana ) lines ST7 were exposed to Al stress (0–400 μM). A combination of physiological assays, biochemical activity measurements, gene expression analysis (quantitative RT-PCR), protein-protein interaction studies (co-immunoprecipitation), and multivariate factor analysis was employed to dissect the response pathways. GmSOD overexpression conferred superior Al tolerance, as evidenced by better-maintained root growth in ST7 under moderate stress (≤200 μM). The transgenic line ST7 exhibited a stronger antioxidant system, resulting in lower H 2 O 2 and malondialdehyde (MDA) accumulation. Crucially, under moderate Al stress, ST7 maintained root tips H 2 O 2 within a narrow signaling range (0.35–0.6 μmol.g -1 FW). This specific H 2 O 2 level promoted the phosphorylation of plasma membrane (PM) H + -ATPase (Gene: NtA HA2, GenBank Accession no: M80490) and enhanced its interaction with 14-3-3 proteins, leading to increased H + -pump activity and a significant boost in citrate exudation. In contrast, under severe stress (400 μM), H 2 O 2 accumulated to toxic levels (>1.0 μmol.g -1 FW), inhibiting this pathway. Factor analysis distilled ten stress-response variables into two principal components—"Antioxidant Capacity" and "Al Toxicity Index"—quantitatively demonstrating that ST7 maintains a favorable balance at higher Al concentrations than WT. We propose a novel model in which GmSOD -mediated fine-tuning of H 2 O 2 homeostasis plays a dual role: it directly alleviates oxidative damage and, at a specific low concentration, acts as a signal to activate the PM H + -ATPase-dependent citrate exudation pathway. This coupling of internal redox control with external detoxification constitutes a key integrative mechanism for Al tolerance, offering strategic insights for crop improvement.