Resumen de Uptake and metabolism of the antiepileptic drug carbamazepine in plants and role of endophytic bacteria /
Occurrence of pharmaceuticals in our environment is an issue threatening one of our most valuable natural resources as is fresh water. Some of this compounds have very low removal efficiency in wastewater treatment plants. The antiepileptic drug carbamazepine (CBZ) is one of the most recalcitrant compound. Therefore, it is often found in wastewater effluents, surface water and in some cases drinking water. With a growing urban population, forecasts are such that the use of treated wastewater will be a necessity in the future to alleviate the stress put on the water cycle.
Phytoremediation is the biological treatment of wastewater with plants in constructed wetlands and represent a cheap and environmental friendly alternative to remove these contaminants from treated wastewater before its use for human activities. Efforts have been put on the design of constructed wetlands to improve removal efficiencies of hazardous compounds, including choice of plant species or water flow regime. Other studies have considered the role of microbial communities found in water or sediments, but little attention has been put into plant-associated and endophytic communities. However, the importance of the microbiome in plant fitness and resistance to biotic and abiotic stresses has been demonstrated recently.
In this work, we study uptake and metabolism of CBZ in plants using a holobiontic conceptual approach in which plant and endophytic bacteria interact for mutual benefit. Common reed plants (Phragmites australis) were grown in liquid Hoagland solution under control conditions. After treatment with CBZ (5 mg/L) for nine days, up to 90% of the compound was removed. Endophytic bacteria were extracted from roots and rhizomes of these exposed plants, identified by 16S rRNA sequencing, and further characterized for their plant growth promoting traits and CBZ removal.
Rhizobium radiobacter and Diaphorobacter nitroreducens were selected among the isolates for a comprehensive study of CBZ uptake and metabolism in interaction with plant roots. An axenic horseradish (Armoracia rusticana) hairy root (HR) culture was used as model to unravel which metabolic pathways are used for CBZ transformation by plants in the absence and presence of their endophytic partners. Inoculation with D. nitroreducens and R. radiobacter led to a 2-fold and 4-fold increase in the removal capacity oh HRs alone, respectively.
In total, thirteen transformation products were identified in the liquid media by LC-UHR-QTOF-MS/MS. These metabolites were classified in four distinct metabolic pathways. For the first time, a CBZ-glutathione conjugate was found in plants. Glutathione and 10,11-diOH pathways were preferred by horseradish HRs while inoculation with R. radiobacter and D. nitroreducens favour the 2,3-diOH and the acridine pathway respectively.
The activity of the reactive oxygen species (ROS) scavenging enzymes glutathione reductase (GR), peroxidases (POX) and ascorbate peroxidase (APOX) and the detoxification enzymes glutathione-S-transferases (GSTs) were determined in cytosolic extracts of P. australis plants exposed to 100 µM CBZ to characterize oxidative stress and defense mechanisms induced by the pharmaceutical. These enzymes play a major role in the detoxification and degradation of xenobiotics in plants. The slight increase of ROS scavenging enzymes observed only in leaves tissues suggests that active metabolites formed in the root system by endophytic strains and plant cells are rapidly transported into the aerial part, inducing an antioxidant response in leaves followed by further metabolism. GSTs were induced in rhizomes as well, bringing indicating that 10,11- diOH and subsequent GSH pathways are the main metabolic pathways for CBZ degradation in P. australis.
Antioxidant responses in HRs were induced by CBZ treatment but also after inoculation with endophytic bacteria. Similar observations were made on GST activities. The hypothesized protective role of endophytic bacteria when plants are confronted to abiotic stress by enhancing their antioxidant responses and detoxification mechanisms was evidenced by proteomics analyses. Superoxide dismutase, GR, monodehydroascorbate reductase, ascorbate peroxidase, all enzymes from the Halliwell-Asada cycle were indeed identified in the growth media of inoculated roots. Additionally, proteomics results revealed a shift on HRs metabolism from primary metabolism to structural and chemical defense processes after CBZ treatment.
It can be concluded that P. australis is a well-suited species for removal of CBZ at relevant environmental concentrations and that removal and degradation of the compound can be improved by enhancing the presence and functionality of selected beneficial strains among the endophytic bacterial community.
