Integrated physiological and transcriptomic analyzes reveal the duality of TiO₂ nanoparticles on alfalfa (Medicago...

Alfalfa (Medicago sativa L.) is a feed crop due to its rich nutrition and high productivity. The utilization of titanium oxide nanoparticles (TiO₂ NPs) brings benefits to agricultural production but also has potential hazards. To investigate the duality and related mechanism of TiO₂ NPs on alfalfa, its different doses including 0, 50, 100, 200, 500, and 1000 mg L^{- 1} (CK, Ti-50, Ti-100, Ti-200, Ti-500, and Ti-1000) were sprayed on leaves. The results showed that greater doses of TiO₂ NPs (500 and 1000 mg L^-1) negatively affected the physiological parameters, including morphology, biomass, leaf ultrastructure, stomata, photosynthesis, pigments, and antioxidant ability. However, 100 mg L^-1 TiO₂ NPs revealed an optimal positive effect; compared with the CK, it dramatically increased plant height, fresh weight, and dry weight by 22%, 21%, and 41%, respectively. Additionally, TiO₂ NPs at low doses significantly protected leaf tissue, promoted stomatal opening, and enhanced the antioxidant system; while higher doses had phytotoxicity. Hence, TiO₂ NPs are dose-dependent on alfalfa. The transcriptomic analysis identified 4625 and 2121 differentially expressed genes (DEGs) in the comparison of CK vs. Ti-100 and CK vs. Ti-500, respectively. They were mainly enriched in photosynthesis, chlorophyll metabolism, and energy metabolism. Notably, TiO₂ NPs-induced phytotoxicity on photosynthetic parameters happened concurrently with the alterations of the genes involved in the porphyrin and chlorophyll metabolism and carbon fixation in photosynthetic organisms in the KEGG analysis. Similarly, it affected the efficiency of alfalfa energy transformation processes, including pyruvate metabolism and chlorophyll synthesis. Several key related genes in these pathways were validated. Therefore, TiO₂ NPs have positive and toxic effects by regulating morphology, leaf ultrastructure, stomata, photosynthesis, redox homeostasis, and genes related to key pathways. It is significant to understand the duality of TiO₂ NPs and cultivate varieties resistant to nanomaterial pollution.