Quantification and enzyme targets of fatty acid amides from duckweed root exudates involved in the stimulation of denitrification

Fatty acid amides from plant root exudates, such as oleamide and erucamide, have the ability to participate in strong plant-microbe interactions, stimulating nitrogen metabolism in rhizospheric bacteria. However, mechanisms of secretion of such fatty acid amides, and the nature of their stimulatory activities on microbial metabolism, have not been examined. In the present study, collection, pre-treatment, and determination methods of oleamide and erucamide in duckweed root exudates are compared. The detection limits of oleamide and erucamide by gas chromatography (GC) (10.3 ng mL−1 and 16.1 ng mL−1, respectively) are shown to be much lower than those by liquid chromatography (LC) (1.7 and 5.0 μg mL−1, respectively). Quantitative GC analysis yielded five times larger amounts of oleamide and erucamide in root exudates of Spirodela polyrrhiza when using a continuous collection method (50.20 ± 4.32 and 76.79 ± 13.92 μg kg−1 FW day−1), compared to static collection (10.88 ± 0.66 and 15.27 ± 0.58 μg kg−1 FW day−1). Furthermore, fatty acid amide secretion was significantly enhanced under elevated nitrogen conditions (>300 mg L−1), and was negatively correlated with the relative growth rate of duckweed. Mechanistic assays were conducted to show that erucamide stimulates nitrogen removal by enhancing denitrification, targeting two key denitrifying enzymes, nitrate and nitrite reductases, in bacteria. Our findings significantly contribute to our understanding of the regulation of nitrogen dynamics by plant root exudates in natural ecosystems.