A Low-Cost Imaging Method for the Temporal and Spatial Colorimetric Detection of Free Amines on Maize Root Surfaces

Truc H. Doan, Tu A. Doan, Michael J. Kangas, AdreAnna E. Ernest, Danny Tran, Christina L. Wilson, Andrea E. Holmes, Erin L. Doyle, Tessa L. Durham Brooks

Plant root exudates are important mediators in the interactions that occur between plants and microorganisms in the soil, yet much remains to be learned about spatial and temporal variation in their production. This work outlines a method utilizing a novel colorimetric paper to detect spatial and temporal changes in the production of nitrogen-containing compounds on the root surface. While existing methods have made it possible to conduct detailed analysis of root exudate composition, relatively less is known about where in the root system exudates are produced and how this localization changes as the root grows. Furthermore, there is much to learn about how exudate localization and composition varies in response to stress. Root exudates are chemically diverse secretions composed of organic acids, amino acids, proteins, sugars, and other metabolites. The sensor utilized for the method, ninhydrin, is a colorless substance in solution that reacts with free amino groups to form a purple dye. A detection paper was developed by formulating ninhydrin into a print solution that was uniformly deposited onto paper with a commercial ink jet printer. This "ninhydrin paper" was used to analyze the chemical makeup of root surfaces from maize seedlings grown vertically on germination paper. Through contact between the ninhydrin paper and seedling root surfaces, combined with images of both the seedlings and dried ninhydrin papers captured using a standard flatbed scanner, nitrogen-containing substances on the root surface can be localized and concentration of signal estimated for over 2 weeks of development. The method was found to be non-inhibiting to plant growth over the analysis period although damage to root hairs was observed. The method is sensitive in the detection of free amines at concentrations as little as 140 μM. Furthermore, ninhydrin paper is stable, showing consistent color changes up to 2 weeks after printing. This relatively simple, low-cost method could contribute to a better understanding of root exudates and mechanisms used by plants to interact with the complex soil environment during growth and development.