Perfluoroalkyl compounds (PFAS) exposure alters xylem hydraulics and gas exchange in willow plants

Poly- and Per-Fluorinated Alkyl Substances (PFAS) are among the most common pollutants derived from industry causing worldwide environmental contamination. PFAS are a class of more than 4700 artificial aliphatic compounds partially or fully fluorinated, which may contain functional groups. This particular chemical structure makes PFAS extremely persistent and resistant to degradation, and provides both hydrophobic and lipophobic properties, as well as high chemical and thermal stability. PFAS can move across water and are known to translocate and accumulate in living organisms, according to their chain length and functional group. Toxic effects on human health have been widely reported. PFAS occurrence in the xylem of the stem was demonstrated by DESI-MS and TEMEDS technologies by Wang et al., (2020), but there is still limited knowledge about PFAS effects on the plant vascular system and physiology. The aim of this research was to investigate if accumulation of PFAS may occur in conductive elements of willow plants, thus altering their hydrophilicity and hydraulic properties. Willow cuttings (Salix triandra) were grown hydroponically in water under greenhouse conditions to induce rooting and shooting. 50 plants were selected for uniformity and transferred to single pots filled with Hoagland nutrient solution. After a few days of adaptation in agronomically suitable condition, they were equally divided in two groups and the nutrient solution was changed. In one of the groups the new nutrient solution was spiked with a PFAS mixture containing PFBA, PFBS, PFPeA, PFHxA, PFHpA, PFOA, PFOS, PFNA, PFDA, PFDoA, each one at 100 µg L-1, while nutrient solution without PFAS was added to the control group. The experiment lasted 8 days to avoid refilling with the nutrient solution. Chlorophyll fluorescence and gas exchange measurements were carried out on two fully expanded leaves of uniform size for all 50 plants before and after the PFAS exposure. The uptake of PFAS was determined in terms of depletion from the nutrient solution and accumulation in the plant tissues at the end of the experiment. PFAS were extracted from root and leaf samples with methanol using an accelerated solvent extraction system. The amount of PFAS in nutrient solution, roots and leaves samples was measured by LC-MS/MS. Hydraulic vulnerability curves were obtained from samples with a minimal length of 200 mm using the air injection method. The pressure in the chamber was gradually increased with steps of 0.5 MPa to reach a maximum of 4 MPa. PFAS were detected in all the nutrient solution samples at a lower concentration compared to the beginning of the experiment, indicating that they were partially absorbed by the plants but remained available throughout the experiment. All the PFAS were detected in both leaves and roots: short chain molecules were accumulated at higher concentration in the leaves, whereas long chain PFAS were more abundant in the roots, consistent with the previous literature. Phenotypical differences between control and treated plants were not observed. In PFAS-exposed plants, parameters related to both gas exchange and chlorophyll fluorescence were significantly altered with respect to the control plants. In particular, transpiration rate (E), net CO2 assimilation rate (A), stomatal conductance to water vapor (gsw), electron transport rate (ETR) and photosynthesis efficiency net of NPQ losses (Fv’/Fm’) were increased in treated plants, whereas vapor pressure deficit at leaf temperature (VPD) and non-photochemical quenching (NPQ) were decreased. An increased A and gsw could be interpreted as an increase in CO2 influx and a decreased water loss. However, there is no demonstrated evidence to consider the PFAS exposure as a stimulating effect on photosynthesis, since our experiments were carried out in hydroponics and water availability was not limiting. To evaluate possible effects on the xylem vulnerability to drought-induced embolism formation due to PFAS presence, we compared the values of the water potential at which 50% of hydraulic conductance is lost (P50) between control and treated plants. PFAS-exposed plants are characterized by a higher P50 value with respect to untreated plants and this reflects an increased susceptibility to xylem embolism under drought conditions. These results support the hypothesis that PFAS can adhere to inner walls of xylem conduits and to plant tissues, making them more hydrophobic. Although the driving force for water movement in plants is the low water potential established by stomatal opening, adhesion forces play a major role in preventing cavitation events. The reduction of adhesion forces provoked by PFAS increases the vulnerability to embolism and the stomatal conductance