Assessing the Impacts of Cu and Mo Engineered Nanomaterials on Crop Plant Growth Using a Targeted Proteomics Approach

Abstract

In this study, we investigated the effects of molybdenum (Mo)-based nanofertilizer and copper (Cu)-based nanopesticide exposure on wheat through a multifaceted approach, including physiological measurements, metal uptake and translocation analysis, and targeted proteomics analysis. Wheat plants were grown under a 16 h photoperiod (light intensity 150 μmol·m^-2·s^-1) for 4 weeks at 22 °C and 60% humidity with 6 different treatments, including control, Mo, and Cu exposure through root and leaf. The exposure dose was 6.25 mg of element per plant through either root or leaf. An additional low-dose (0.6 mg Mo/plant) treatment of Mo through root was added after phytotoxicity was observed. Using targeted proteomics approach, 24 proteins involved in 12 metabolomic pathways were quantitated to understand the regulation at the protein level. Mo exposure, particularly through root uptake, induced significant upregulation of 16 proteins associated with 11 metabolic pathways, with the fold change (FC) ranging from 1.28 to 2.81. Notably, a dose-dependent response of Mo exposure through the roots highlighted the delicate balance between nutrient stimulation and toxicity as a high Mo dose led to robust protein upregulation but also resulted in depressed physiological measurements, while a low Mo dose resulted in no depression of physiological measurements but downregulations of proteins, especially in the first leaf (0.23 < FC < 0.68) and stem (0.13 < FC < 0.68) tissues. Conversely, Cu exposure exhibited tissue-specific effects, with pronounced downregulation (18 proteins involved in 11 metabolic pathways) particularly in the first leaf tissues (root exposure: 0.35 < FC < 0.74; leaf exposure: 0.49 < FC < 0.72), which indicated the quick response of plants to Cu-induced stress in the early stage of exposure. By revealing the complexities of plants' response to engineered nanomaterials at both physiological and molecular levels, this study provides insights for optimizing nutrient management practices in crop production and advancing toward sustainable agriculture.

Figure 1

Wheat plant growth and exposure. (A) Images of plant growth with two different exposure techniques (root exposure and leaf exposure) and 3 treatment groups [control group, Mo exposure group (6.25 mg Mo/plant), and Cu exposure group (6.25 mg Cu/plant)]; (B) Leaf exposure steps: (1) prepare ENM suspensions in a 50 mL centrifuge tube; (2) insert all the leaves into the tube, swirling the leaves gently and soaking the leaves in solution for 10 s; (3) remove the leaves and let them dry for 10 s; (4) bring plant upright and let it dry for 15 min and then repeat steps 2–4 for another 2 times for a total of 3 daily exposures.

Figure 2

Wheat plant harvest. (A) Plants after harvest and wash [from left to right: root exposure control, Cu exposure through root (6.25 mg of Cu/plant), Mo exposure through root (6.25 mg of Mo/plant), leaf exposure control, Cu exposure through leaf (6.25 mg of Cu/plant), and Mo exposure through leaf (6.25 mg of Mo/plant)]; (B) biomass distribution of 6 groups; (C) leaves’ color distribution of 6 groups.

Figure 3

Box-and-whisker plot of (a) shoot length, (b) root length, (c) shoot biomass, and (d) root biomass of 6 treatment groups. t-test results indicated as *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Figure 4

Heatmap of metal concentrations in plant tissues. (A) Cu and Mo concentration in plant tissues; (B) nutrient element concentration in plant tissues. (C) Correlation analysis between Cu and Mo and other nutrient elements. RC: root exposure control; RCu: Cu exposure through root; RMo: Mo exposure through root; LC: leaf exposure control; LCu: Cu exposure through leaf; LMo: Mo exposure through leaf. Element concentration data are listed in Table S3.

Figure 5

Heatmap of protein concentrations in different plant tissues with different treatments. Refer to Table 1 for the Pathway and Protein IDs.

Figure 6

PLS-DA of protein concentrations in each plant tissue with different treatments.

Figure 7

Volcano plots to visualize the relationship between significance (p-values <0.05) and FCs in each tissue. Gray points: not significant; blue color points: significant but 0.75 < FC < 1.25; yellow color points: significant and FC ≥ 1.25 or ≤0.75; red color points: FC ≥ 1.5 or ≤0.5.

Figure 8

FC bar plots of proteins with FC ≥ 1.25 or ≤0.75 significant changes in different plant tissues.

Figure 9

Protein expression in the whole plant. (A) FC bar plot of proteins with FC ≥ 1.25 or ≤0.75 significant changes in the whole plant; (B) Venn diagram of proteins with FC ≥ 1.25 or ≤0.75 significant changes in the whole plant.