Rapid Phytotransformation of Benzotriazole Generates Synthetic Tryptophan and Auxin Analogs in Arabidopsis

Abstract

Benzotriazoles (BTs) are xenobiotic contaminants widely distributed in aquatic environments and of emerging concern due to their polarity, recalcitrance, and common use. During some water reclamation activities, such as stormwater bioretention or crop irrigation with recycled water, BTs come in contact with vegetation, presenting a potential exposure route to consumers. We discovered that BT in hydroponic systems was rapidly (approximately 1-log per day) assimilated by Arabidopsis plants and metabolized to novel BT metabolites structurally resembling tryptophan and auxin plant hormones; <1% remained as parent compound. Using LC-QTOF-MS untargeted metabolomics, we identified two major types of BT transformation products: glycosylation and incorporation into the tryptophan biosynthetic pathway. BT amino acid metabolites are structurally analogous to tryptophan and the storage forms of auxin plant hormones. Critical intermediates were synthesized (authenticated by (1)H/(13)C NMR) for product verification. In a multiple-exposure temporal mass balance, three major metabolites accounted for >60% of BT. Glycosylated BT was excreted by the plants into the hydroponic medium, a phenomenon not observed previously. The observed amino acid metabolites are likely formed when tryptophan biosynthetic enzymes substitute synthetic BT for native indolic molecules, generating potential phytohormone mimics. These results suggest that BT metabolism by plants could mask the presence of BT contamination in the environment. Furthermore, BT-derived metabolites are structurally related to plant auxin hormones and should be evaluated for undesirable biological effects.

Metabolism and Photolysis of 2,4-Dinitroanisole in Arabidopsis

“…21 For example, Arabidopsis quickly assimilates and metabolizes benzotriazole and 2-mercaptobenzothiazole in hydroponic media, while carrots and horseradish conjugate the antibacterial agent triclosan. 20, 35, 36, 39 Arabidopsis also takes up and transforms the structurally similar TNT. 9, 22 While the detected phase I and phase II metabolites were not unexpected based on the green liver model and plant metabolism of TNT, the low mass balance of DNAN and phase I metabolites from LC-QQQ analysis indicated that further investigation was required to identify unknown DNAN metabolites.…”

“…20, 43–45 Conjugation proceeds without prior phase I functionalization (e.g., refs 20, 35, 39) or after a phase I reaction. 22 Conjugation with sugars decreases toxicity of the compound and these phase II metabolites are likely intermediates to permanently bound residues.…”

“…The tissue was extracted without the freeze-thaw step two additional times with 500 μL of water:methanol (1:1) and the supernatants were combined. 35 Samples for metabolite identification were extracted only once, with 1 mL of water:methanol (1:1) mixture. Reported concentrations of DNAN and metabolites were corrected for a 95.7 ± 0.6 % extraction efficiency (determined from spike recoveries on unexposed plant tissues after three extractions).…”

Resilience to multiple stressors in an aquatic plant and its microbiome

“…It has been suggested that duckweeds may remove BZT from water (Gatidou et al., ), and other plants are already known to metabolize BZT (e.g., LeFevre et al., , ). We saw stronger decreases in BZT when salt was absent, which could be due to enhanced plant growth and therefore greater biotransformation, or the reverse: greater biotransformation at low salt facilitated enhanced duckweed growth and survival (Figs.…”

“…We thus hypothesized (1) that NaCl would magnify negative effects of BZT on duckweeds and microbes. Some microbes and plants cause biotransformation of BZT (Herzog et al, 2013;Kowalska and Felis, 2015;LeFevre et al, 2015), raising the possibility that duckweeds and microbes could potentially be used for bioremediation of this emerging contaminant. Further, microbes can reduce plant salinity stress in several systems (Mayak et al, 2004;Bal et al, 2013;Rho et al, 2018).…”