Metabolite measurements
The same pooled plant materials used for RNA extraction were used to
determine ion contents, membrane lipids, and primary metabolites. For
cation/anion analysis, 20 mg frozen, powdered shoot or root material was
transferred into a reaction tube, suspended in 500 µl of Milli-Q water
by vortexing, and centrifuged for 1 min at 13,000 g. The supernatant was
transferred to a new reaction tube and the pellet re-extracted using the
same procedure. First and second supernatants were combined before
filtering through a 0.45 µm membrane. Ion chromatographic separation was
achieved on a Thermo Scientific ICS-5000 IC system (Thermo Fisher
Scientific, USA) using a Dionex CS12A , Ion Pac (2 × 250 mm) analytical
column with a AG12A guard column (2 x 50 mm). Quantification was
achieved using Chromeleon 7.2 version SR4 software. Standard curves were
prepared using dilution of the following standards, for anions Thermo
Scientific Dionex Seven Anion Standard II, and for cations Thermo
Scientific Dionex Six Cation II Standard. Lipid extraction followed the
procedure described by Vu et al. (2014) and non-polar metabolites for
gas chromatography-mass spectrometry (GC-MS) analysis were extracted
from powdered, freeze-dried materials using the protocol described by
Broeckling et al. (2005).