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.