Allocation of salt contaminants to tissues and cell storage
compartments in foliage
The artefact-free microlocalization of NaCl contaminants within their
vacuolar sinks, 1) similar to findings in studies also using
cryo-microscopy (Dérue et al., 2006; James et al., 2006), 2) consistent
with microscopic injury and 3) in agreement with prevailing opinion
(Apse & Blumwald, 2007; Jabeen et al., 2014; Munns et al., 2016), was
conditioned to sample preparation and observation in full
cryo-conditions. The high salt accumulation levels, superior in
mesophyll than epidermis, suggested NaCl importation alongside
apoplastic (epidermis) as well as symplastic (mesophyll) routes
(Meidner, 1975; Leigh & Tomos, 1993; Buckley, 2015). Superior mesophyllversus epidermis allocation has also been found in Aster
tripolium (Perera et al., 1997) but contrasted with findings in
cultivars of durum wheat and barley (Leigh & Tomos, 1993; James et al.,
2006). A reverse accumulation pattern was even observed in barley or
halophytic Atriplex spongiosa (Fricke et al., 1996; Storey et
al., in McCully et al., 2010). Massive NaCl allocation to
stress-sensitive mesophyll suggested weak salt management at tissue
level, partially explaining the reported salt stress sensitivity of lime
trees. The distribution of contaminants and rate of vacuolar filling, as
observed within leaf rim samples next to necrosis, exemplified
situations at the far end of salt accumulation gradients within leaves.
The occurrence of latter gradients was confirmed by the 1) less
significant models, relating salt concentrations to microscopic changes
in the leaf center versus rim samples, or 2) thickness of leaf
rim necrosis varying as a function of salt concentration, similar to
boron accumulation and injury patterns (Rees et al., 2011).
The Na+ and Cl- contaminants showed
a distribution at tissue level and microlocalization within cells
similar to several mobile nutrients but a reverse dynamic. Whilst K is
the main inorganic osmoticum in plant vacuoles, it may increasingly
share this role with NaCl in saline conditions or in the case of a low
K+ supply (Kronzucker & Britto, 2011; Ahmad &
Maathuis, 2014; Ivanova et al., 2016), probably because of the similar
Na and K physico-chemistry (Benito et al., 2014). Though the
Na+, K+ cell transporters are mostly
ion-specific (Apse & Blumwald, 2007; Ahmad & Maathuis, 2014; Benito et
al., 2014), concomitant increase of Na+ and decrease
of K+ concentrations, as in the case of Riga’s lime
tree foliage, is usually observed in saline conditions (Munns et al.,
2016). Steady K+ concentrations and higher
K+:Na+ ratios in tolerant halophytes
(Perera et al., 1997; McCully et al., 2010) let thus appear the patent
mechanistic link between K+ and NaCl dynamic within
mesophyll vacuoles as another characteristic trait of salt sensitivity
in lime tree (Dobson, 1991; Cekstere et al., 2008). In epidermis,
especially Ca2+ but also Mg2+ formed
the main cationic osmotica whilst K+ played a minor
role, in likely relation to ontological aging (Fricke et al., 1996).
Similar to mesophyll, NaCl accumulation caused a drop in the
concentration of inorganic osmotica, similar to observations in barley
(Fricke et al., 1996) and, in this case, in line with chemical
measurements at leaf level.
Besides a marked decrease of all detected nutrient mass fractions within
vacuoles and replacement by Na+ and
Cl- ions, salt accumulation within mesophyll and
epidermis tissues could also change the osmotic homeostasis. Higher
vacuolar osmolarity in foliage of Riga’s polluted versus NBG
sites was suggested by the 1) higher NaCl concentrations at leaf level,
2) higher cumulated mass percentages of vacuolar ions, 3) salt-driven
increase in the vacuole and cell size and 4) increased autophagic
reactions. Leaves show osmotic adjustments as a function of NaCl
exposure and osmotic pressure variation in the nutrient solution (Ottow
et al., 2005). More vacuolar osmolytes can reduce osmotic stress as a
consequence of increased salt concentration in the soil solution (Munns
et al., 2016; Polle & Chen, 2015) and thus alleviate physiological
drought stress (Dobson, 1991).