CAPTIONS
Fig. 1. Location of and level of salt contamination at the
study sites.
Fig. 2. Visible injury by salt stress within foliage and at a
street site from Riga’s street greenery (Meierovica 1; September 16,
2014). For microscopy assessments, leaf center (light green) and rim
(teal blue) disks were excised apart the main center vein and next to
salt-triggered necrosis, within still green foliar tissues.
Fig. 3. Principal changes in the concentration of macro- and
micronutrients, as a function of Na and Cl accumulation within foliage
of trees at the study sites. The data points represent site averages
(+ SE). Statistics refer to linear mixed effects models (LMEM).
Solid lines and shaded areas indicate significant (P≤0.05) linear
regressions and 95% confidence intervals.
Fig. 4. Tissue-(A -C ) and cell-level
(H -K ) allocation of salt contaminants (Na, Cl) and
some macro-nutrients (K, Ca) within leaf rim samples of Tilia x
vulgaris leaves from moderately (B Meierovica 1) and strongly
(C, H-K Aspazijas) contaminated versus control site
(A NBG) in the street greenery of Riga. D -GElement spectra obtained for the element maps (D forA , E for B , F for C ,G for H -K ). The salt contaminants were found
within all leaf blade tissues. At cell level, they primarily accumulated
within vacuoles (v) and were missed within cell walls (cw). Other
structures: uE – upper epidermis, pP – palisade parenchyma, sP –
spongy parenchyma, lE – lower epidermis. The whitish globular
structures represent ice contamination. Average foliar concentrations of
Na/Cl: 86±8/760±121 mg kg-1 (NBG, A ,D ), 4020±739/7333±1746 mg kg-1 (Meierovica 1,B , E ), 9733±1964/13917±2022 mg kg-1(Aspazijas, C , F -H ). Technical
specifications: High pressure frozen leaf samples planed by
cryo-ultramicrotomy and examined using a cryo-FEG-SEM in high-vacuum
cryo-mode (– 160 °C) at acceleration voltage of 20 kV, magnification of
1350x (A-F) and 4500x (G-K), 9 mm working distance, HDBSD detectors for
imaging and X-ray Energy Spectrometer for micro-analysis; element
spectra and maps obtained by scanning during 1500 s (200 µs dwell time);
measurements cumulated over 30 frames and mapped at 512 x 384 ppi
resolution (each point in elementary map representing the total number
of counts for the element during mapping); element maps overlaid on
HDBSD images; scale of spectra: 71517 cps.
Fig. 5. Nutrient content (mass percentages) of vacuolar medium
within leaf rim samples of Tilia x vulgaris leaves from salt
contaminated sites (average foliar concentrations of Na/Cl at Meierovica
1, Gertrudes, Aspazijas: 4020±739/7333±1746, 7067±677/7083±682,
9733±1964/13917±2022 mg kg-1. The values represent
averages per tissue (+ SE) of measurements performed along two
transect through the leaf blade, in one leaf sample per site (N = 3).
The insert graph shows the vacuolar nutrient content at the asymptomatic
NBG site (average foliar concentrations of Na/Cl: 86±8/760±121 mg
kg-1). Abbreviations: uE – upper epidermis, pP –
palisade parenchyma, sP – spongy parenchyma, lE – lower epidermis.Technical specifications: High-pressure-frozen leaf samples
planed by cryo-ultramicrotomy and examined using a cryo-FEG-SEM in
cryo-mode at acceleration voltage of 10 kV and magnification of 2200x
(260 pA of current, 9.5 mm working distance). Collection of EDS spectra
by each measurement point during 30 s. Half-quantitative nutrient mass
percent composition (weight %) of vacuolar sap estimated on the basis
of deconvoluted spectrum of each element (XPP deconvolution).
Fig. 6. Structural effects of salt contamination in symptomatic
leaves of Tilia x vulgaris from moderately (B, E, H, L )
and strongly (C, F, I, J, M ) contaminated sites in the street
greenery of Riga. Asymptomatic leaves from NBG (A, D, G, K ).
Samples from the leaf center (A-F ) and leaf rim region
(G-M ). In pP cells of symptomatic leaves, salt contamination
caused an increase in the cell size driven by that of vacuome, with one
large vacuole containing many vesicular inclusions (* in H-J )
filling most of cell volume finally (v; E, F, H-J ). The
cytoplasm showed degenerative changes (# in M ) and an increase
of autophagic vesicles (av). The chloroplasts (ch; L, M ) were
also degenerated, with larger plastoglobules (pl; E, F,L, M ) protruding and being expelled (&) into the vacuole.
Other structures: lE: lower epidermis, m: mitochondria, mvb:
multivesicular body, n: nucleus, pP: palisade parenchyma, sP: spongy
parenchyma, st: starch, ta: tannin body, uE: upper epidermis. Average
foliar concentrations of Na/Cl: 86±8/760±121 mg kg-1(A, D, G, K ), 4020±739/7333±1746 mg kg-1(B, E, H, L ), 9733±1964/13917±2022 mg kg-1(C, F, I, J, M ). Technical specifications: A-F :
1.5 µm semi-thin cuttings stained with toluidine blue and acid fuchsine
and observed in diascopic light microscopy; G-M: 75 nm
ultra-thin sections stained with uranyl acetate and lead citrate, and
observed in TEM.
Fig. 7. Structural responses within palisade cells of leaf rim
samples, as a function of Na and Cl accumulation within foliage of trees
at the study sites. The data points represent site averages (+SE). Statistics refer to linear mixed effects models (LMEM). Solid lines
and shaded areas indicate significant (P≤0.05) linear regressions and
95% confidence intervals. Abbreviations: pP: palisade parenchyma, Pl
size: percentage area of plastoglobules within chloroplasts. Vacuole
size: percentage area of largest vacuole within pP cells.
Fig. 8. Multivariate structural responses to salt contamination
in palisade parenchyma of leaf rim samples - RDA models. Correlation
biplot based on a redundancy analysis of the structural data measured in
mesophyll, showing the relationship between markers of salt injury
(response variables, black arrows) and salt concentration in foliage
(Na, Cl explanatory variables; blue arrows). The leaf injury
(i.e. percentage area of leaf showing necrosis, light blue arrow)
was passively projected, as a supplementary variable. Altogether, the
first and second canonical axis explained 78.12% of total variance in
the mesophyll structure dataset, whilst only the first axis was
significant (P < 0.001). The color of each tree score
shows the average Na-contamination within lime tree foliage at the five
sampling sites (■ 86 + 7 mg kg-1, ■ 895+ 477 mg kg-1, ■ 4507 + 534 mg
kg-1, ■ 8000 + 793 mg kg-1; N
= 3 trees per site), with each site centroid indicated by a cross and
label. Abbreviations for the descriptor variables: ChS chloroplast size,
CS cell size, circ Cell circularity, PlD plastoglobule density, PlS
plastoglobule size, VS vacuome size.
Fig. S1. Element mapping by means of compact FIB-Tof-SIMS
performed during the microlocalisation trials, with a focus on Na and Cl
ions within cross-sections of leaf rim samples collected in the street
greenery of Riga. The Na and Cl concentration of leaf sample is
indicated directly in the Fig. A, B Microlocalisation trial
applying a freeze-substitution protocol. The Na and Cl contaminants have
been dislodged from the vacuole to cytosol and cell wall compartments
during the freeze-substitution. C, D Microlocalisation
trial using the cryo-fixed sample block, after trimming by
cryo-ultramicrotomy and controlled freeze-drying (as a consequence of of
intercellular spaces, large vacuoles and lignified structures, the
sections fell apart and could not be retrieved). Dislodging of salt
contaminants was reduced but the tissue and cell structure was too much
distorted for enabling the observation of salt distribution among the
storage compartments. Sampling sites: Barona (A , B) ;
Meierovica 1 (C , D ).
Technical specifications: Sample preparation: A, Bfreeze substitution of samples in LN2 using an EM AFS2 (Leica
Microsystems) at ZMB (1% OsO4 in 100% acetone -90oC 8 hours, graded temperature increase to 0oC in 16 hours, rinsing in 100% acetone, infiltration
by a series of graded acetone/Epon-Araldite mixture at +4oC and embedding in Epon-Araldite). Thin cuttings (100
nm) sectioned using a Reichert UltraCut S ultramicrotome, mounted on
custom-made Si-wafers and coated with 4-6 nm Au. C, D stepwise
freeze-drying of 400 µm-deep trimmed block sample (6.2 h in total, using
a modified Bal-Tec BAF 060), prior to mounting on Al-stub. Element
mapping: Visualization and mapping of 23Na+ and
35Cl- ions by means of Focused Ion Beam-Secondary Ion
Mass Spectrometer microscope (compact FIB-SIMS) at the Swiss Research
Institute for Applied Materials Sciences and Technology (EMPA) in Thun,
Switzerland (Ga ion beam, 20 keV, 180 pA, 100x100 µm view field). 20keV
180-200 pA, 100x100 μm.
Fig. S2. Structural effects of salt contamination in the upper
epidermis cells of Tilia x vulgaris foliage from moderately
(B, E ) and strongly (C, F, G ) contaminated sites in
the street greenery of Riga (leaf rim samples). A, BAsymptomatic material from the National Botanical Garden. Within
epidermis cells, salt accumulation further hastened the ontological
senescence by exacerbating the ongoing degenerative changes, such as the
increase in size of plastoglobules (pl) within leucoplasts (l;B, E, F, G ), condensation of chromatin within nucleus (*n;B, E ), or higher frequency of autophagic vesicles (av) and
multivesicular bodies (mvb; B, E ). Other structures: #,
fibrillous mucilage material, m: mitochondria v: vacuole. Average foliar
concentrations of Na/Cl: 86±8/760±121 mg kg-1(A, D ), 4020±739/7333±1746 mg kg-1(B, E ), 9733±1964/13917±2022 mg kg-1(C, F, G ). Technical specifications: 75 nm ultra-thin
sections stained with uranyl acetate and lead citrate, and observed with
TEM.