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Most leaf surfaces are hydrophobic or only moderately hydrophilic as a result of leaf surface waxes, although some rare exceptions do exist (Holloway, 1969; Neinhuis & Barthlott, 1997; Koch et al., 2009). Forty years ago, in their paper entitled, ‘Penetration of stomata by liquids – dependence on surface tension, wettability, and stomatal morphology’, Schönherr & Bukovac (1972) concluded that the combination of cuticular hydrophobicity, water surface tension and stomatal geometry should prevent water droplets from infiltrating the stomata, unless pressure is applied or the equilibrium surface tension undergoes an extreme reduction, to < 30 mN m−1. Such a reduction is achievable only by a few surfactants, typically the organosilicones (Stevens et al., 1991). This concept became dominant (cf. Niederl et al., 1998; Fernandez & Eichert, 2009; Taiz & Zeiger, 2010) and its validity was increasingly assumed for all types of aqueous solutions and environments, turning it into a paradigm. The a priori exclusion of stomatal liquid water transfer restricted the uptake and loss of liquid water and aqueous solutions to the cuticular part of the leaf surface. However, there has also been a steadily increasing number of experimental reports from different backgrounds that have pointed to the stomatal uptake of water (Burgess & Dawson, 2004; Breshears et al., 2008; Limm et al., 2009; Simonin et al., 2009), nutrients (Fernandez et al., 2006; Will et al., 2012), ionic fluorescent dyes (Eichert et al., 1998; Eichert & Burkhardt, 2001) and abscisic acid (Wilkinson & Davies, 2008) without the use of surfactants. The main argument against such conflicting observations was the possibility of spatial, temporal or spatiotemporal cuticular heterogeneities (e.g. higher cuticular permeability in regions near the stomata, exclusively during periods of open stomata (Schönherr & Bukovac, 1978)), which has been challenging to disprove. Only the visualization of the stomatal uptake of fluorescent nanoparticles (specifically water-suspended hydrophilic polystyrene particles of 43 nm in diameter) via confocal laser scanning microscopy (Eichert et al., 2008) finally provided an unequivocal demonstration that the stomatal uptake of water, solutes and dispersed substances is indeed possible.
Stomata may be rendered susceptible to this type of transport by environmental factors that reduce the innate hydrophobicity of leaf surfaces. In a process that affects individual stomata, the ‘hydraulic activation of stomata’ (HAS) may be established once a threshold is exceeded (Burkhardt, 2010). HAS signifies the formation of continuous, thin (presumably < 100 nm) liquid water films on the stomatal walls that connect the apoplast and the leaf surface and enable the bidirectional transport of water, dissolved or dispersed substances, and hydraulic signals (Burkhardt, 2010). Depending on the history and age of the plant and the environmental conditions, HAS might be a common phenomenon. It provides a link between air pollution and plant drought stress, and could play a role in nocturnal transpiration or stomatal reaction to humidity (Burkhardt, 2010).
Suggestions addressing the development of HAS comprise fungal hyphae (Burgess & Dawson, 2004), bacteria (Eichert et al., 2008), stomatal mucilage (Zimmermann et al., 2007; Westhoff et al., 2009) and hygroscopic leaf surface particles (Burkhardt, 2010). Leaf surface particles may originate from the atmospheric deposition of aerosols or trace gases (e.g. SO2, which is oxidized within the water covering the leaf; Burkhardt & Drechsel, 1997), residues of evaporated rain, fog or agricultural sprays, plant exudations or phyllospheric organisms. The amount of leaf surface particles may reach several μg cm−2, a similar weight range as the cuticular leaf waxes (Burkhardt, 2010 and references therein, Sæbø et al., 2012). The deposition of submicrometre aerosols is almost independent of gravity, that is, deposition should happen equally on the abaxial and adaxial sides of the leaves as long as their microroughness is equal (Peters & Eiden, 1992; Burkhardt et al., 1995). Even sustained rainfall does not effectively remove deposited aerosols from deciduous or coniferous trees (Freer-Smith et al., 2005). Decreasing contact angles with increasing age are most probably caused by the accumulation of noncuticular material and its chemical constituents (Cape, 1983; Neinhuis & Barthlott, 1998; Bringe et al., 2006), although cuticular abrasion may also play a role (Pitcairn et al., 1986; Hoad et al., 1992). Leaf surface particles may influence plant–water relationships. The stomatal diffusive resistance of deciduous trees is reduced by particle pollution (Flückiger et al., 1977) and the transpiration of elder trees (Sambucus nigra) at the same degree of stomatal aperture increases on treatment with NaNO3 particles (Burkhardt et al., 2001). Approximately 20–40% lower stomatal conductance and transpiration were observed for bean (Vicia faba) and sunflower (Helianthus annuus) plants grown under the exclusion of aerosols compared with control plants grown in normal air (Pariyar et al., 2012).
Schönherr & Bukovac (1972) concentrated on static droplets of pure water on a flat surface, with droplet dimensions exceeding the stomatal aperture. Real leaf surfaces are more complex: epidermal roughness, trichomes or epicuticular waxes often create structural heterogeneities that influence leaf wetting (Quere, 2008; Fernandez et al., 2011). Phyllospheric organisms actively bind water on the surface through biofilm formation (Schreiber et al., 2005; Beattie, 2011). Water is also attracted by deposited aerosols, many of which consist partially or entirely of hygroscopic salts. Salts suddenly become deliquescent (liquid) at a salt-specific relative humidity (RH), the deliquescence relative humidity (DRH; c. 75% for NaCl or NaClO3, 40% for NH4HSO4 and 80% for (NH4)2SO4). The salts absorb exponentially more water with further increasing humidity (Pilinis et al., 1989; Zhao et al., 2008; Mauer & Taylor, 2010). This mechanism is similar to the activation of cloud condensation nuclei, although DRH is slightly different for deposited particles (Gao et al., 2007), and plant transpiration provides an additional water vapour source (Burkhardt & Eiden, 1994). Repeated deliquescence and efflorescence of single particles on the leaf surface can lead to the development of ‘thin water films’ or ‘filaments’, even on hydrophobic leaf surfaces and even during hot summer days (Burkhardt & Eiden, 1994; Burkhardt et al., 1999). The RH at the salt surface (water vapour phase) and the water activity of the hygroscopic particles (liquid water phase) are a physical identity (Pilinis et al., 1989). Once a saturated solution is formed at the deliquescence point, the corresponding water activities (e.g. 0.75 for NaCl or NaClO3, 0.4 for NH4HSO4 and 0.8 for (NH4)2SO4) increase, whereas the concentrations (6.1 M for NaCl, 8.6 M for NaClO3, 9.0 M for NH4HSO4 and 5.7 M for (NH4)2SO4 at 20°C, respectively; IFA, 2012) decrease with increasing RH.
The chemical, physical and physicochemical properties of such highly concentrated solutions are considerably different from those of dilute solutions. Ion activities, rather than ion concentrations, determine the chemical reactions (Pitzer, 1981). Physical effects include capillary condensation, capillary transport of substances, Marangoni flow (cyclic inward or outward movement within the droplet), the accumulation of dispersed substances at the edges (coffee-rings), the reduction of contact angles by preferential evaporation from droplet edges and ‘line-pinning’ of droplets during evaporation (Eiden et al., 1994; Deegan et al., 1997; Herminghaus et al., 2008; Xu et al., 2010; Hunsche & Noga, 2012).
The most important physicochemical effect of ions in the present context is related to water surface tension. This parameter reflects the dispersive forces across the phase boundary, as well as the specific forces within one phase, such as hydrogen bonding (Dutcher et al., 2010). Water surface tension was central to the reasoning of Schönherr & Bukovac (1972), but they referred to pure water. Ions change the water surface tension in a concentration-dependent and ion-specific manner. This is related to the respective distribution of the ions between the surface and the bulk of a water droplet and to the order of the Hofmeister (or lyotropic) series (Collins & Washabaugh, 1985; Bostrom et al., 2001; Pegram & Record, 2007; Liao et al., 2009; Dutcher et al., 2010; dos Santos et al., 2010; Zhang & Cremer, 2010). The Hofmeister series originally classified ions in electrolytic solutions according to their ability to dissolve proteins (Hofmeister, 1888; Kunz et al., 2004a,b). For anions, the Hofmeister series is
(Zhang & Cremer, 2006; Miller & Hoseney, 2008). Ions that are considered to be kosmotropic are at the left of the series, and chaotropic ions are at the right, and anions have a much greater effect than cations. Thus, the sulfate anion, which is on the kosmotropic side of the series, decreases the solubility of nonpolar molecules, increases the hydrophobic interaction (‘salting out’) and increases surface tension. The chlorate ion belongs to the group of chaotropic ions, which increase the solubility of nonpolar molecules, weaken the hydrophobic interaction (‘salting in’) and decrease surface tension. The chloride ion is sometimes considered to be kosmotropic (dos Santos et al., 2010) and sometimes considered to represent the dividing line between kosmotropic ‘structure makers’ and chaotropic ‘structure breakers’.
NaCl is the most important aerosol in coastal regions and leaf surface accumulation of up to 300 μg cm−2 NaCl has been found (Cheplick & Demetri, 1999). Coastal dune plant adaptations to salt spray may be manifested at both the genetic and acclimation level. Tolerance decreases with high wind, low rainfall and the presence of surfactants (Boyce, 1954; Sykes & Wilson, 1988; Griffiths & Orians, 2003; Griffiths, 2006; Lowry et al., 2009; de Vos et al., 2010). In several coastal areas around the world, the release of surfactants by landfills has been found to be responsible for forest decline symptoms as a result of enhanced stomatal salt uptake (Dowden et al., 1978; Grieve & Pitman, 1978; Truman & Lambert, 1978; Bussotti & Ferretti, 1998; Paoletti et al., 2005). By contrast, NaClO3 is well known as a defoliant (‘defol’), and is one of several salts and acids that have been used as ‘chemical desiccants’ in practical agriculture. This technique was primarily used by farmers several decades ago to kill potato vines before harvest, but has also been used in plant breeding applications to rapidly evaluate drought-tolerant cultivars, given that spraying with chemical desiccants creates drought symptoms under irrigated conditions. Other successfully used chemicals are potassium iodide, magnesium chloride, sulfuric acid and sodium hydroxide; by contrast, sodium chloride and ammonium sulfate (AMS) are not suitable for this purpose (Wilson et al., 1947; Murphy, 1968; Blum et al., 1983a,b; Nicolas & Turner, 1993; Regan et al., 1993; Royo & Blanco, 1998; Sawhney & Singh, 2002; Bond & Bollich, 2007). AMS has repeatedly been reported to increase the bio-efficacy of glyphosate (N-(phosphonomethyl)glycine; Gly) (Hall et al., 2000; Pratt et al., 2003; Liu, 2004b; Nurse et al., 2008). Gly, the world's most widely used herbicide (Alister et al., 2005), is a highly water-soluble agrochemical with pronounced translocation characteristics, but variable foliar uptake (Field & Bishop, 1988). Improved Gly uptake by AMS may come from water conditioning, with AMS preventing the inactivation of Gly through a chemical reaction with calcium (Thelen et al., 1995), but positive effects of AMS have also been observed for Gly in distilled water (Young et al., 2003; Nurse et al., 2008).
Hygroscopicity and ion-specific effects enable leaf surface particles to change both the cuticular hydrophobicity and water surface tension, the major pillars of Schönherr & Bukovac's (1972) argument. We therefore re-assessed the question of stomatal uptake of aqueous solutions using anions from different sides of the Hofmeister series, and evaluating their impact on physiology. We visualized key processes using an environmental scanning electron microscope (ESEM).
We hypothesized that:
- the stomatous abaxial side of apple leaves should take up more NaCl, NaClO3, AMS, Gly and combinations thereof than the astomatous adaxial side;
- the chaotropic NaClO3 should be taken up by the leaf more readily than the more kosmotropic NaCl and AMS;
- both NaClO3 and AMS may act as adjuvants for Gly uptake;
- salts on hydrophobic cuticles should attract liquid water considerably below saturation (100% RH) and be able to overcome cuticular hydrophobicity by spatial expansion.