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).