Xylem sap extraction and collection
Plants were collected and roots rinsed with distilled water, then excised at the caulinar (floral) stem above the rosette leaves, and the xylem sap was collected directly from the cut with a micropipette. To improve xylem sap exudation in Hg-stressed plants, roots were subjected to external pressure in a portable SKPM 1400 Schölander pressure chamber (SKYE Instruments Ltd., Powys, UK) by applying a 3 MPa constant pressure with compressed N2 gas for less than 20 min. The first 10 µL were discarded to avoid cellular contamination, and the xylem sap collected (45 µL) was transferred to Eppendorf tubes containing 5 µL of acid mixture (10% metaphosphoric acid, 1% formic acid and 10 mM EDTA), for preservation of biothiol and biothiol-Hg complexes. Samples were subsequently frozen at -80°C and stored until analysis. Cross-contamination with cellular and phloem exudates was checked routinely by measuring L-malate dehydrogenase (c-MDH) activity (López-Millán, Morales, Abadı́a, & Abadı́a, 2000) (see Extended Materials and Methods details in Supplementary online material).