Allocation of salt contaminants to leaf tissues
The high mobility of salt ions within plant tissues (Mengel & Kirkby,
2001; White & Broadley, 2001; Li et al., 2017) and lack of fresh
material (Rokebul Anower et al., 2017) formed challenging constraints
with regard to salt microlocalisation free of dislodging artefacts.
Using a freeze substitution procedure (Wu & Becker, 2012) and with
visualization and mapping of 23Na+ and
35Cl- ions using a Focused Ion Beam-Secondary Ion Mass
Spectrometer (FIB-SIMS) microscope, dislodging of Na and Cl contaminants
from the vacuole to cytosol and cell wall compartments was observed
(Fig. S1A, B versus 4I, J). As a consequence, we sectioned the
deeply frozen sample-carrier sandwiches directly, focusing on the leaf
rim samples and using a Leica EM FC6 ultra-cryo-microtome at
ScopeM, -150 °C cooling and LN2
flushing. Given the unrecoverable sections and distortion artefacts of
stepwise freeze-dried block sample (Fig. S1C, D versus 6A-C),
full cryo-conditions were also required with a view to visualizing and
mapping the salt contaminants and leaf nutrients. Therefore and prior to
observation, the planed samples transferred in LN2 to the UMR
Silva-Silvatech Microscopy platform in Champenoux, France, were coated
with 1.5 nm Pt (-120 °C, 2.5E-2 mbar Ar plasma) and the ice
contamination freeze-etched (30 min, -85 °C, 2.7E-6 mbar), using an EM
VCT100 vacuum cryo shutter (LEICA, UK) and an EM ACE600 double sputter
coater. The samples were then transferred to a cryo- Focused Electron
Gun Scanning Electron Microscope (cryo-FEGSEM; SIGMA HD VP, ZEISS,
Germany) equipped with a High Definition Back Scattered Electron
Detector (HDBSD) and an Energy Dispersive Spectrometer
(X-MaxN EDS – SDD 80mm²; Oxford-Instruments, UK)
interfaced to the SEM by the INCA software (Oxford–Instruments, UK).
Element maps and line scans of salt contaminants and nutrients were
obtained at – 160 °C, using magnifications of 1.35 or 4.5 kX and 20 keV
acceleration voltages (30-35° take off angle, 9 mm work distance, 200 µs
dwell time, 30 frames and 1500 s measurement time). The smooth and
homogeneous material structure of samples allowed us the quantification
of contaminants and main nutrients within the vacuolar compartment. At
two randomly selected leaf blade transects per sample, point
measurements were performed within the vacuole of one cell per tissue
(10 kV HT, 30 s measurement time). The range of electron penetration in
such conditions can be estimated to 2-3 µm (Huang & van Steveninck,
1989). The spectrometer was calibrated with reference to calcium
standardization before micro-analysis. The half quantitative nutrient
mass percent composition (weight %) of vacuolar sap was estimated on
the basis of deconvoluted spectra for each element (INCA-XPP matrix
deconvolution). The estimates per tissue type in the two investigated
leaf blade transects were averaged.