Structural changes at tissue and cell level in foliage
Structural changes within foliar tissues and cells were investigated by
means of widefield light (LM) and transmitted electron microscopy (TEM)
at WSL and ZMB in Switzerland. For LM, the leaf material was dehydrated
with 2-methoxyethanol (3 changes), ethanol, n -propanol,n -butanol (Feder and O’Brien 1968) and embedded in Technovit 7100
(Kulzer HistoTechnik). Semi thin sections (1.5 µm thick) were cut using
a Reichert UltraCut S ultramicrotome, stained in 1% acid fuchsine and
0.05% toluidine blue in acetate buffer pH 4.4 (Feder and O’Brien, 1968)
and mounted in DPX. The sections were observed using the 5x-100x
objectives of a Leica microscope Leitz DMRB and photographed using the
INFINITY 2-1R camera and Lumenera Infinity Analyze (release 6.4)
software (Lumenera Corp., Ottawa, Canada). Based on the differential
diagnosis of salt versus other types of biotic and abiotic injury
(Fink 1999; Günthardt-Goerg et al., 2007), salt accumulation markers
(cell size and form) were measured in palisade parenchyma.
For TEM, the leaf samples were post-fixed in buffered 2%
OsO4, dehydrated by a series of graded ethanol,
infiltrated by a series of graded propylene oxide/Epon 812 mixture (with
DDSA, NMA and DMP hardener) and embedded in Epon. Ultra-thin sections
(70 nm) were cut using the aforementioned ultramicrotome, mounted on
copper grids and stained using saturated uranyl acetate in 50 % ethanol
and lead citrate (Reynolds procedure). Sections were observed using a
Philips CM12 transmission electron microscope (TEM) and micrographs
taken using the Gatan Microscopy Suite Software (Gatan Inc., Pleasanton,
USA). Selecting a subsample of 5 sites spanning over the whole
contamination range, changes in the frequency, size and shape of
cellular structures responsive to salt accumulation were quantified by
stereology (Toth, 1982; Kubínová, 1994), using medially-sectioned
palisade cells, a 0.5 µm grid size and the ImageJ software
(https://imagej.nih.gov/ij/).