Transcriptomic Responses and Putative Mechanisms of Nicotinamide-Mediated Saline–Alkaline Tolerance in Soybean

Objective: Saline–alkali stress (composed of neutral and alkaline salts) severely constrains soybean productivity. Although nicotinamide can enhance plant salt tolerance, its most effective concentration and molecular basis in soybean remain unclear. This study aimed to systematically investigate the regulatory effect of exogenous nicotinamide on soybean saline–alkaline tolerance and to identify its candidate genes and putative mechanisms under salt-stressed conditions. Methods: Soybean cultivar ‘Hefeng 25’ seedlings were exposed to 200 mmol/L mixed salt stress and treated with nicotinamide at 0 (CK), 10, 50, and 200 mg/L. Growth parameters, ROS levels, ion contents, photosynthetic pigments, osmoregulatory substances, and antioxidant enzyme activities were measured. Transcriptome sequencing and RT-qPCR were performed to identify differentially expressed genes (DEGs), followed by enrichment analysis. Results: Exogenous nicotinamide significantly improved soybean saline–alkaline tolerance relative to the salt-stressed CK group, with 50 mg/L (SA2) showing the most pronounced physiological improvements among all tested concentrations. SA2 treatment enhanced plant height, biomass, K+ content, and antioxidant enzyme activities while reducing Na+ accumulation and ROS levels compared with CK. Transcriptome analysis identified PM1 as the shared gene across the five non-most effective comparisons. Enrichment analysis implicated DEGs primarily in ko03110 (chaperones and folding catalysts), ko00199 (cytochrome P450), and ko04141 (protein processing in the endoplasmic reticulum). RT-qPCR confirmed that PM1, P450 genes (CYP74A1, CYP83D1), HSPs (HSP23, HSP18), and ROS scavengers (NAC1, GSTU43) were upregulated under non-most effective doses (SA1/SA3) but downregulated to levels comparable to the stressed CK in SA2; conversely, SA2 specifically restored the expression levels of stress-repressed WRKY57 and LBD15. Transcriptomically, SA2 exhibited a profile most similar to the saline–alkali-stressed CK group, with the fewest number of differentially expressed genes among all treatments, whereas non-optimal nicotinamide concentrations were associated with extensive transcriptional changes in ER protein processing, detoxification-related, and oxidative stress-related pathways. The limited transcriptional changes in SA2 indicate greater transcriptional similarity to the stressed control, which paralleled its superior physiological performance. Conclusions: Nicotinamide effectively alleviates soybean saline–alkaline stress at 50 mg/L, with the most significant physiological benefits and the fewest transcriptional changes relative to the stressed control. The non-most effective nicotinamide concentrations show extensive stress-related transcriptional reprogramming in protein folding, detoxification, and stress-response pathways relative to the salt-stressed control, putatively implicating ER stress, detoxification, and oxidative stress-related responses at the transcript level, with PM1 acting as a potential candidate transcriptional marker of dose-dependent transcriptional regulation under saline–alkali stress.