**Background:** Essential metals (Cu, Zn, Fe) are cofactors in enzymes vital for plant stress responses, including catalases, peroxidases, and superoxide dismutases. Metal bioavailability in the rhizosphere is enhanced through chelation by root exudate metabolites such as low molecular weight organic acids (LMWOAs), amino acids, and siderophores. Drought stress alters root exudate composition, which can shift rhizosphere microbial communities and potentially recruit beneficial microbes. This study investigated how drought and colonization by the probiotic bacterium Pseudomonas chlororaphis O6 (PcO6) affect metal-chelating metabolites in wheat shoots and rhizosphere solutions, and used geochemical modeling to predict impacts on Fe, Cu, and Zn speciation.
**Methods:** Wheat seeds (cv. Juniper, bred for drought tolerance on calcareous soils) were surface-sterilized, germinated on LB agar, and transferred to Magenta boxes containing sterile silica sand wetted with 3.34 mM Ca nitrate. The experiment used a 2 × 2 factorial design: with/without PcO6 inoculum (2.5 × 10^5 CFU/g sand) and with/without drought stress. Seedlings were grown for 10 days post-planting under LED lights (380 µmol/m²/s, 16 h light/8 h dark). Drought was imposed by opening boxes for 2 days to allow evapotranspiration (reducing water content to 19% field capacity), then boxes were closed for 3 more days. At harvest (14 days post-germination), shoot length, dry mass, and water content were measured. Rhizosphere solutions were extracted by vacuum filtration of sand. Metabolites (amino acids, organic acids, phenolic acids, and the wheat siderophore 2′-deoxymugineic acid [DMA]) were quantified by ion chromatography and LC-QqQ-MS. Geochemical modeling (Visual MINTEQ Ver. 3.1) was performed using measured metabolite concentrations, major ions, and metal oxide solid phases (CuO, ZnO, FeO(OH)) at pH 7.25 with 400 ppm CO₂.
**Key Results:** Drought reduced shoot length, dry mass, and water content, and shortened root length. PcO6 colonization increased shoot length and dry mass but did not affect shoot water content. Culturable PcO6 cells were recovered from inoculated roots at similar densities (~10^5–10^6 CFU/cm root) regardless of watering regime. In shoots, drought dramatically increased proline from 180 µg/g to ~10,000 µg/g, and increased asparagine, aspartate, leucine, phenylalanine, tyrosine, methionine, and DMA, while decreasing glutamate, serine, formate, and gluconate. PcO6 colonization caused fewer shoot metabolite changes, but increased butyrate (detected only in inoculated plants). In rhizosphere solutions, drought effects were subtle: increases in lysine, phenylalanine, and coumarate, and decreases in oxalate, propionate, salicylic acid, tryptophan, and arginine. PcO6 colonization caused large reductions in most amino acids and organic acids (e.g., asparagine, proline, serine, alanine, tryptophan, arginine, phenylalanine, gluconate, 2-oxoglutarate, lactate, propionate, coumaric acid decreased significantly). Gluconate remained the dominant organic acid in all treatments. The rhizosphere pH decreased from 7.2 to 6.5 with drought. Geochemical modeling predicted that Fe was almost entirely complexed as Ca–Fe–gluconates (>98%), with minimal DMA complexation (<2%). Zn existed predominantly as free Zn²⁺ ions (>90%) across all conditions. Cu showed complex speciation: free ions (2–7%), carbonate complexes (1–2%), and chelation with DMA, organic acids (citrate, malate, gluconate), and amino acids (glutamate, serine, phenylalanine, valine). Drought decreased Cu–gluconate complexation but increased Cu–DMA and Cu–amino acid complexes. PcO6 colonization reduced predicted soluble Cu and Fe compared to non-inoculated watered plants.
**Clinical Implications:** This study demonstrates that drought and root microbiome colonization differentially alter metal-chelating metabolites in wheat shoots versus rhizosphere, with potential consequences for plant metal nutrition and pathogen suppression. The dramatic increase in shoot proline under drought confirms its role as an osmoprotectant. The reduction of rhizosphere metabolites by PcO6 colonization may contribute to biocontrol by limiting substrate and bioavailable Fe for pathogens. The dominance of Ca–Fe–gluconate complexes (rather than the expected Fe–DMA) suggests gluconate plays an unexpected role in Fe bioavailability. The persistence of Zn as free ions indicates normal root uptake channels remain functional. These findings have implications for developing drought-tolerant crops and managing plant–microbe interactions to improve crop resilience, particularly in calcareous soils where metal availability is limiting.