Biochar and ZnO-Se nanocomposites enhanced biochemical and antioxidant profiling of soybean: insight into GC-MS metabolomics, and KEGG pathway analysis.

Soybean (Glycine max L.) is a significant oilseed crop that is frequently limited by soil fertility, micronutrient deficiencies and oxidative stress. Although biochar and nano enable micronutrients have individually been reported to improve crop performance. There synergistic effect on the biochemical traits, antioxidants defense, and metabolic reactions of soybean have not yet been explored. This lack of knowledge gap affects the ability to develop integrated and sustainable nutrient management practices for producing soybean. Hence, we hypothesized that the synergistic effect of sugarcane bagasse-based biochar and green-synthesized zinc oxide-selenium nanocomposites (ZnO-Se NCs) would enhance soybean biochemical performance, antioxidant capacity and metabolite accumulation. For testing this hypothesis, a field experiment was carried out by using the biochar produced by the pyrolysis process at 450 °C and the foliar-applied ZnO-Se NCs synthesized from garlic extract. Biochemical properties, antioxidant activities, metabolite profile using GC-MS and KEGG pathway enrichment analyses were assessed. The biochar blended with 40 ppm of ZnO-Se NCs had highest soluble protein content (3.73 ± 0.22 µg g⁻¹ FW), total flavonoids (187.3 ± 1.82 µg mL⁻¹ QE), total phenolics (70.64 ± 0.57 µg mL⁻¹ GAE), DPPH radical scavenging activity (48.46 ± 0.94%)), and total antioxidant activity (57.18 ± 1.45 mg AAE g⁻¹ FW) in comparison with the control. Biochar + 50 ppm ZnO-Se NC showed the most abundant total soluble sugar content (215.5 ± 2.9 µg mL⁻¹). GC-MS characterization revealed the presence of important bioactive metabolites, such as linoleic acid, oleic acid, palmitic acid, and dl-α-tocopherol, and KEGG enrichment showed that amino acid metabolism and secondary metabolite biosynthesis pathways were the most significantly enriched pathways. This study supports novel evidence for soil improvement using biochar, with ZnO-Se nano-enabled micronutrient delivery that can affect both the biochemical and antioxidant responses of soybean plants, and alter the soybean metabolites. The results indicate that biochar and optimized ZnO-Se nanocomposite are promising potential strategies for boosting soybean physiological performance and metabolic resistance in field environments.