Root exudates sampling
After 7 days of exposure to treatments, root exudates were collected by a soil-hydroponic hybrid approach (Oburger & Jones 2018) with modifications (Figure 1). The maize plants were sampled and the whole root system was carefully washed after removing the soil. Afterwards, the single primary and seminal roots attached to the shoot were placed on two different compartmented chambers (2×10×2 cm) with five cells, each 1.8 cm deep and 1.8 cm wide (Figure 1). Each cell was connected to the next one by a shallow bridge so that a root could be laid out across the block traversing all the cells. The intact primary and seminal root axes were inserted in separate chambers. The area starting from the root tip to the first visible lateral root (apical root zone) was isolated from the remaining root portion (subapical root zone) by silicon grease (Dow Corning, Midland, MI, USA) (Figure 1). Hence, the root exudates of the following root types and zones have been collected: seminal-apical (Sa), seminal-subapical (Ssa), primary-apical (Pa), and primary-subapical (Psa).
Before beginning the exudate collection, we submerged the apical root zone with the trap solution (0.5 mM CaCl2) to detect possible leaks between the cells hosting the apical and sub-apical root zones. During the exudates collection, the roots were continuously aerated. To collect root exudates, cells were filled with 6 mL of the trap solution, and every 30 minutes the trap solution was collected for up to 4 hours, yielding 48 mL for each root zone. This solution was then centrifuged (15 min at 13000 rpm at 4 °C) and the supernatant was freeze-dried. The exudate solution was then resuspended in 5 mL 1:1 H2O/CH3OH and filtered with 0.45 µm syringe filters (Phenex-RC 0.45 µm - Phenomenex) and used for the targeted and untargeted metabolomics analyses.