Introduction

During the winter season, different chemical and abrasive materials are being spread for de-icing road and sidewalk pavement, with cheap sodium chloride (NaCl) most commonly used (Dobson, 1991, Cunningham et al., 2008, Fayun et al., 2015, Ordóñez-Barona et al., 2018, Dmuchowski et al., 2019). Alone, moistened or mixed with sand, the amounts of NaCl reach 19.6 tons per lane km in New York State (1.1 million tons year-1, Cunningham et al., 2008), more than 200’000 tons in Poland (Marosz, 2011), 16’000-31’500 tons in Edmonton (Equiza et al., 2017), 2 kg m-2 in Denmark (Pedersen et al., 2000) and 4.06 kg m-2 in Riga (Cekstere et al., 2008) annually. Such high amounts cause substantial environmental contamination by leaching and uptake in the surrounding soil and vegetation during the subsequent vegetation seasons (Pedersen et al., 2000, Bryson & Barker 2002; Ordóñez-Barona et al., 2018, Nikolaeva et al., 2019). The contaminants absorbed by roots and translocated to foliage cause leaf necrosis, crown defoliation, twig dieback and street tree decay (Dobson, 1991, Bryson & Barker, 2002, Paludan-Müller et al., 2002, Cekstere et al., 2008, Dmuchowski et al., 2014, Milewska-Hendel et al., 2017). In turn, the important ecosystem services provided by street trees, such as cooling of urban climate, mitigation of air pollution, reduction of street runoff or promotion of biodiversity can be seriously affected (Moser et al., 2015, Nowak et al., 2017; Bouraoui et al., 2019).
In street greeneries, NaCl contamination affects the whole urban ecosystem. In the soil compartment, Na accumulation destroys the soil aggregates, thus reducing the soil porosity and promoting puddling in the case of fine-textured soils (Marschner & Marschner, 2012; Bryson & Barker, 2002). The effects on soil chemistry include the degradation of soil organic matter, increase in pH, dislodging of cations within absorption complexes or nitrate leaching as a consequence of enhanced nitrification rates (Cunningham et al., 2008; Dmuchowski et al. 2014; Eimers et al., 2015). Directly or indirectly, the soil salinization can depress the biological processes and alter the microbial communities in the rhizosphere (Ke et al., 2013). Tree lines planted on salt-contaminated soils can suffer under osmotic and ionic stress (Munns & Tester, 2008), similar to ‘physiological drought stress’ (Dobson, 1991), which can reduce the leaf gas exchanges and growth rates (James et al., 2006, Munns & Tester, 2008, Cekstere et al., 2015). Within leaf tissues, the accumulation of salt ions can impair several enzymatic activities, inhibit membrane functions, promote nutrient imbalance, decrease chlorophyll concentration, and significantly affect essential physiological activities (Muszynska et al., 2014; Negrão et al., 2017). Molecular responses and tolerance mechanisms have been extensively investigated and reviewed (Munns & Tester, 2008; Polle & Chen, 2015; Flowers et al., 2015; Munns et al., 2016; Wu et al., 2018). Structurally, various studies, so far not yet comprehensively reviewed, have reported about cellular responses such as enhanced oxidative stress (Hernandez et al., 1995; Benzarti et al., 2012) and numerous alterations in cytoplasm and e.g.chloroplast or mitochondria (Naidoo et al., 2011; Yamane et al., 2012; Ivanova et al., 2016;). However, quantitative evidence relating the levels of NaCl contamination to structural injury is still largely missing and the mechanisms of NaCl toxicity in foliage thus remain partly elusive (Munns & Tester, 2008). Moreover, most reports illustrating microscopic injury miss microlocalisation evidence, complicating the distinction between direct and indirect effects by salt contaminants.
Adaptations to elevated salinity show similarities with those in the case of infertile soils (Chapin, 1980). They may have evolved in the framework of exclusion or inclusion strategies, the latter conferring lower tolerance to chronic salt stress (Chen et al., 2018). Tolerance mechanisms in includers may encompass vacuolar allocation of contaminants whilst keeping a high K+/Na+ ratio, allocation to non-photosynthetic tissues, leaf succulence or translocation to older foliage organs (Ottow et al., 2005; Flowers et al., 2015; Polle & Chen, 2015; Wu et al., 2018). Safe allocation of salt contaminants is thus crucially important with a view to salt tolerance in salt includers (Polle & Chen, 2015; Munns et al., 2016). Given their high mobility however, the microlocalisation of Na+ and Cl- ions within tissues and cells is demanding methodologically (Frey & Zierold, 2003; Wu & Becker, 2012) and still limited evidence is so far available. The latter has been obtained applying various microscopical techniques, but control evidence for excluding dislodging artefacts is generally missing.
After characterizing the extent of soil pollution by salt contaminants (Cekstere & Osvalde, 2013) or the toxic impact of high salt concentration in foliage (Cekstere et al., 2008), the present study has focused on mechanistic aspects of chronic salt stress in foliage of urban lime trees (Tilia spp. ) in the street greenery of Riga, Latvia. Its main objectives included to 1) relate the allocation of salt contaminants within leaf tissues and cell compartments to injury at cell, tissue and whole leaf level, 2) identify the most characteristic symptoms and cellular responses to salt stress and 3) distinguish direct and indirect salt stress effects from those by other environmental factors. We had the following working hypotheses: 1) by changing the nutrient balance in the soil, the de-icing salt contaminants cause nutrient imbalance within foliage of street trees; 2) the salt contaminants are primarily allocated to safe storage compartments, including the epidermis at leaf (2a) and vacuole at cell (2b) level; 3) salt accumulation in leaves causes specific structural changes relating to storage (3a) and toxicity (3b). Using 8 street sites forming a gradient of foliage contamination representative of the salt pollution in Riga’s street greenery (Cekstere et al., 2008), we 1) characterized the soil and foliage chemistry, 2) analyzed the distribution of salt contaminants and main nutrients within foliar tissues and cells and 3) quantified structural and ultrastructural markers of salt stress within assimilating tissues.