Structural responses to salt accumulation in foliage
As indicated by significant correlation with both Na
(P <0.001, r=0.79) and Cl (P =0.0007, r=0.67), the
leaves of lime trees from Riga’s street greeneries showed a higher
percentage area in the form of necrotic leaf rims (Fig. 2) with
increasing salt accumulation. However, the LMEM models were not
significant (Table S1B). In parts of leaves still asymptomatic,
structural changes clearly distinct from those by biotic injury –
noteworthy aphids (Bouraoui et al., 2019) - were observed in the leaf
mesophyll (Fig. 6) and epidermis (Fig. S2). The most striking symptom of
salt accumulation was an increase in the percentage area of vacuome
(Fig. 6D-F), with one large vacuole filling most of cell volume in
samples with highest contamination (Fig. 6I, J). Given the ontological
progress in epidermis by the time of sampling and random sectioning of
spongy parenchyma cells, this marker was primarily visible in palisade
parenchyma. Other palisade cell structures were increasingly condensed
and degenerated (Fig. 6G-J, L, M). At subcellular level, the vacuoles
showed intense autophagic activity and contained many inclusions as well
as multivesicular bodies (Fig. 6I, J, L, M). Large plastoglobules,
protruding towards and being extruded into the vacuole (Fig. 6L, M), had
developed inside of chloroplasts. In the epidermis, mostly degenerative
changes including the a) higher condensation of nuclear material (Fig.
S2B, E), b) increase in size and frequency of plastoglobules within
leucoplasts (Fig. S2F, G) or c) larger autophagic activity and frequency
of multivesicular bodies were observed.
The LMEM models in combination with salt microlocalisation data
confirmed the direct or indirect implication of subcellular
accumulations of Na/Cl, with respect to the observed structural changes.
The increase in the vacuole size was linearly related to Na/Cl
concentration at leaf level (Fig. 7B, E; Table S1C) and the vacuolar
allocation of contaminants indicated a direct salt accumulation effect
(Fig. 4I, J). Given missing salt contaminants in cytoplasm (Fìg. 4I, J),
the observed cell size increase with higher foliar salt concentrations
(Fig. 7A, D; Table S1C) should be driven by that of vacuome, which was
also confirmed by 1) the correlation between vacuome and cell size
(P =0.03, r=0.57) and 2) concomitant thinning and degeneration of
cytoplasmic strands (Fig. 6F, L, M), as a consequence of autophagic
activity (Fig. 6I, J, L, M). As indicated by non-significant models for
the cell circularity (Table S1C), the cell size increased both
periclinally and anticlinally. Missing cytoplasmic accumulation of salt
contaminants, the observed increase in the size of chloroplasts (Cl
only) and plastoglobules (Na only) implied an indirect driving effect of
salt contaminants (Fig. 7C, F; Table S1C). Only the models calculated
using leaf rim data were significant; regarding those calculated on the
basis of leaf center measurements, the cross-sectional area of palisade
cells formed the best responsive estimate, increasing marginally
(P =0.054; Cl) or as a tendency (P =0.088; Na) with rising
salt accumulation in foliage.
Considering the cell responses in the palisade parenchyma from leaf rim
samples globally, the salt contamination matrix (Na and Cl foliar
concentrations) in the RDA model explained 78.12 % of observed
variation in the structural markers from descriptor matrix (P=0.001;
Fig. 8). However, only the first RDA axis (77.49 %) was significant
(P =0.001). The two explanatory variables showed balanced
contributions. The vacuole size (VS in Fig. 8) was the structural
parameter most strongly contributing to RDA axis 1. The site centroids
showed a distribution along first axis reflecting the gradient of
average salt contamination in foliage. The passively projected
macroscopic leaf injury parameter (Injury), which was significantly
correlated with both Na and Cl (P < 0.001 and 0.002),
showed a positive correlation tendency with respect to RDA first axis
(P =0.073).