Subcellular distribution and biotransformation of phenanthrene in pakchoi after inoculation with endophytic Pseudomonas sp. as probed using HRMS coupled with isotope-labeling.

Contamination of food-crops with polycyclic aromatic hydrocarbons (PAHs) poses a grave concern to food safety, especially when PAHs are internalized. We have demonstrated in our previous study that inoculation of crop with a phenanthrene-degrading endophytic Pseudomonas sp. Ph6-gfp could overcome this problem. Here, the subcellular distribution and biotransformation mechanism of phenanthrene in pakchoi (Brassica chinensis L.) seedlings with inoculation of Ph6-gfp were further investigated both in vitro and in vivo. The possible biotransformation products of phenanthrene were identified by high-resolution mass spectrometry (HRMS) coupled with 13C2-phenanthrene labeling. Results indicated that Ph6-gfp colonized pakchoi interior and reduced the content of phenanthrene in different cell compartments. Notably, the inoculation hindered the subcellular distribution of phenanthrene from intercellular space to subcellular fractions (i.e., cell wall, cell membrane, cell solution, and cell organelles), likely resulting from the interception and biodegradation of phenanthrene by the bacterium between the cell wall and intercellular space. Additionally, the conjugation reactions of phenanthrene-metabolites and endogenous plant compounds were enhanced as a result of the inoculation. We propose that endophytic degradation, plant metabolism, and conjugation reaction are the three possible biotransformation mechanisms that could account for the changes in phenanthrene inside the plant cell compartments. This is the first observation of endophytic bacteria (EB)-enhanced biotransformation and conjugation of phenanthrene in pakchoi at the subcellular level, which drive novel insights in regulating food-crop contamination with endophytes in PAH-contaminated matrices.