Structural responses to salt accumulation in foliage
Notwithstanding concomitant Mn and K deficiency and likely physiological drought stress, the visible and microscopic injuries in lime tree foliage from salt-polluted street lines of Riga could be attributed to the sole effects of salt stress. Visible injury was similar to that reported in lime trees or other broadleaved species from Warsaw (Dmuchowski et al., 2013), Opole (Czerniawska-Kusza et al., 2004), London (Gibbs & Palmer, 1994) or coastal ecosystems under salt stress (Vollenweider & Günthardt-Goerg, 2005). Symptoms appeared partly similar to those resulting from drought stress or K deficiency but clearly distinct from Mn deficiency injury (Vollenweider & Günthardt-Goerg, 2005; Hartmann et al., 2007; Papadakis et al., 2007). However, several microscopic traits (i.e. cell hypertrophy / normal chloroplastic grana stacks / ubiquitous starch grains) clearly excluded any significant contribution to injuries by environmental constraints other than salt stress (Fink, 1999; Vollenweider et al., 2016). Moreover, changes in the foliar concentration of Na/Cl explained 79%/65% of variation in the necrosis percentage area within leaves.
The salt-driven increase in the vacuole/cell size and microscopic injury in cytoplasm were indicative of sink adjustments and degenerative responses. Larger vacuome and cells form structural hallmarks of so-called leaf succulence trait within salt-exposed foliage (Ottow et al., 2005; Benzarti et al., 2014; Polle & Chen, 2015). By contrast with healthy succulent plants however (e.g. Kondo et al., 1998) and as indicated by LMEM and RDA models, larges vacuoles in lime tree mesophyll did not appear for ontological reasons but resulted from phenotypic adjustments to increasing salt accumulation. Moreover, they were associated to a syndrome of typical but unspecific degenerative traits in adjacent cytoplasm (i.e. cytoplasm and organelle condensation / autophagic activity / plastoglobule size increase and extrusion into vacuoles). Indeed, most of these injuries are being observed not only in the case of salt (Hernandez et al., 1995; Naidoo et al., 2011; Yamane et al., 2012; Ivanova et al., 2016) but also ozone (Vollenweider et al., 2019) or drought stress (Fink, 1999; Vollenweider et al., 2016) or with ontological senescence (Mikkelsen & Jorgensen, 1996; Inada et al., 1998). Similar to latter studies, they indicated an acceleration of cell senescence (ACS; Günthardt-Goerg & Vollenweider, 2007). The lack of more specific e.g. swelling of chloroplastic thylakoids (Fink, 1999), as commonly observed during experimental salt exposure (Hernandez et al., 1995; Guan et al., 2013; Bejaoui et al., 2016), may relate to the primarily vacuolar allocation of salt contaminants. Acclimated halophytes from saline environments can also miss this trait (Naidoo et al., 2011).
The drawback of higher vacuolar osmolarity, caused by steady NaCl accumulation, may comprise impeded leaf conductance, leading in turn to observed ACS in synergy with disturbed hormonal balance. Mesophyll succulence-like, as a consequence of concomitant NaCl accumulation and autophagy, may decrease leaf conductance by reducing the circadian variation of cell volume and water exchanges (Canny & Huang, 2006) driven by evapotranspiration and causing diel changes in the leaf thickness (Westhoff et al., 2009; Ehrenberger et al., 2012). Within epidermis and together with K+, NO3- and malate2-, Cl- is one of the main osmolytes which fluctuations actuate guard cell/vacuome size variation and stomatal opening (Daloso et al., 2017). Interestingly in some adapted halophytes, better salt-tolerance appear to be conferred by the lower NaCl and steady K+ concentration to be found in guard versusepidermal neighbor cells (Perera et al., 1997; McCully et al., 2010). Furthermore, Cl- > Na+ are abscisic acid inducers, the main hormone regulator of stomatal closure (Geilfus et al., 2018) and lower stomatal conductance forms an ecophysiological hallmark of salt stress (Munns & Tester, 2008). Lowered stomatal conductance is consistent with enhanced senescence, as indicated by the aforementioned ACS responses (Bond, 2000, Munns & Tester, 2008; Daszkowska-Golec & Szarejko, 2013). Farther, it can enhance photoinhibition and photo-oxidative stress, especially in high light environment as in the outer tree canopy or at many saline habitats (Foyer et al., 1994; Suzuki et al., 2012). Enhanced oxidative stress is another consequence and hallmark of salt accumulation in foliage (Hernandez et al., 1995; Benzarti et al., 2014; Polle & Chen, 2015), and its structural markers (Günthardt-Goerg & Vollenweider, 2007; Moura et al., 2018) show many similarities with those observed in lime tree foliage.