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Abstract Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacteriumPseudomonas chlororaphisO6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H₂O₂-released from roots during plant stress responses. Atomic force microscopy revealed 40–300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles ofPcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids thanPcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H₂O₂-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents. 

Strategies to reduce crop losses due to drought are needed as climate variability affects agricultural productivity. Wheat (Triticum aestivum var. Juniper) growth in a nutrient-sufficient, solid growth matrix containing varied doses of CuO, ZnO, and SiO2 nanoparticles (NPs) was used to evaluate NP mitigation of drought stress. NP amendments were at fertilizer levels, with maxima of 30 Cu, 20 Zn, and 200 Si (mg metal/kg matrix). Seeds of this drought-tolerant cultivar were inoculated with Pseudomonas chlororaphis O6 (PcO6) to provide a protective root microbiome. An 8 day drought imposed on 14 day-old wheat seedlings decreased shoot and root mass, shoot water content, and the quantum yield of photosystem II when compared to watered plants. PcO6 root colonization was not impaired by drought or NPs. A dose-dependent increase in the Cu, Zn, and Si from the NPs was observed from analysis of the rhizosphere solution, and this process was not affected by drought. Consequently, fertilizer concentrations of the NPs did not further improve drought tolerance in wheat seedlings under the growth conditions of adequate mineral nutrition and the presence of a beneficial microbiome. These findings suggest that potential NP benefits in promoting plant drought tolerance occur only under certain environmental conditions.