UC Santa Barbara

As the agricultural use of ENMs becomes more prevalent, the exposure of plants to these nanomaterials has emerged as a significant abiotic stress. Researchers have previously explored plant responses to ENMs through non-targeted proteomics studies, revealing qualitative insights into protein-level responses to abiotic stress. However, there remains a knowledge gap in understanding the molecular mechanisms underlying these responses. This study aims to bridge that gap by employing targeted proteomics, which involves the quantitative measurement of a specific set of ENM-responsive proteins. Unlike non-targeted approaches, targeted proteomics allows for high-quality quantification of pre-selected signature peptides associated with targeted proteins. This approach is valuable for hypothesis-driven experiments and provides detailed insights into the perturbations in biological pathways triggered by ENMs.A key focus of the study was the optimization of targeted plant proteomics methods to ensure high reproducibility of results. By refining signature peptide selection, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical methods, and sample preparation, the study establishes a robust workflow for the specific quantification of ENMs-responsive proteins. The investigation then applied the optimized targeted proteomics approach to explore the responses of crop plants, specifically Triticum aestivum (wheat), to copper (Cu) based nano-pesticide (Cu(OH)2-NP) and molybdenum (Mo) based nano-fertilizer (MoO3-NP). The study measured protein and metabolite levels in different plant tissues exposed to these ENMs through root or leaf routes. Joint pathway analysis was employed to comprehensively understand the changes in both protein and metabolite levels, providing a holistic view of the molecular responses. The study optimized targeted proteomics methods, revealing the phenol extraction method with fresh plant tissue and trypsin digestion as the best for sample preparation. Applying this approach to wheat exposed to ENMs, significant upregulation of 16 proteins associated with 11 metabolic pathways was observed for Mo exposure through root. Notably, a dose-dependent response of this treatment highlighted the delicate balance between nutrient stimulation and toxicity, as the high Mo dose led to robust protein upregulation (especially amino acid metabolism related proteins) but depressed physiological measurements (include biomass, length and color of plant tissue), while low doses showed no physiological depression but downregulation of proteins. Integration of targeted proteomics and metabolomics identified responsive metabolites and proteins for ENM treatments, with Cu effects prominent through leaf exposure and Mo effects through root exposure. A joint pathway analysis was conducted using MetaboAnalyst 6.0 to integrate information from various omics platforms to comprehensively understand biological pathways. It revealed 23 perturbed pathways, emphasizing the interconnectedness of metabolic and proteomic responses. Coordinated responses in protein and metabolite concentrations, particularly in amino acids, demonstrated a dynamic proteomic-to-metabolic-to-proteomic relationship. Contrasting expression patterns in glutamate dehydrogenase highlighted dose-dependent regulatory trends influencing both proteins and metabolites following specific Mo exposure through roots. Overall, this study contributes to advancing our understanding of plant responses to ENMs at the molecular level. By quantifying specific proteins and employing joint pathway analysis to integrate proteomics with metabolomics, the research sheds light on the intricate biological pathways affected by exposure to ENMs. The optimized targeted proteomics approach ensures the reliability and reproducibility of results, paving the way for further research in the field of nanomaterial impacts on plant biology and sustainable agriculture. The significance of our research lies in the potential for guiding agricultural practices and environmental safety protocols by providing a comprehensive understanding of how plants respond to exposure to ENMs. By taking into account ENM design, dose optimization, and exposure routes, this project aims to contribute to the advancement of sustainable agricultural practices, and facilitate the utilization of nanotechnology's benefits while mitigating potential risks to plants, ecosystems, and human health.