Low nitrate concentration may avoid protein aggregation
Phloem sap contains very high content of sucrose (about 400 mM in castor bean and palms, for example), amino acids (about 60 mM in most species, up to 140 mM in some trees) and K+ (about 60 mM, up to 500 mM in Salix , (Peel & Weatherley, 1959)). Mg2+ and Na+ are present in lower abundance and Ca2+ is at low concentration (1 mM or less) (Ziegler, 1975). Anions that counterbalance K+high content are mostly Cl-, phosphate, sulphate and organic acids (malate). Nitrate is thus a minor participant in phloem electroneutrality. Also, phloem sap contains significant amounts of proteins, at about 1 g L-1, and an important proportion is made of the so-called P-proteins (SEOR proteins inArabidopsis ) that are believed to play a role in phloem occlusion by Ca2+-dependent aggregation (Anstead, Froelich, Knoblauch, & Thompson, 2012; Jekat et al., 2013; Knoblauch, Froelich, Pickard, & Peters, 2014; van Bel et al., 2014). Of course, phloem sap contains many other proteins, and recent proteomics analyses have shown that this includes not only enzymes, but also translation initiation or elongation factors, proteins involved in redox homeostasis, chaperones, etc. (Rodríguez-Celma, Ceballos-Laita, Grusak, Abadía, & López-Millán, 2016). It is worth mentioning that with such high salt concentration (in particular K+), there is a risk of uncontrolled protein aggregation. In fact, both experiments and theory have provided evidence that ion species have different propensity to trigger protein aggregation (chaotropism), via denaturation and/or instability (Kunz, 2010). Ca2+ is the most chaotropic cation while tertiary amines are the least chaotropic, K+ being intermediate. This provides a physical justification for the role of Ca2+ in phloem occlusion. Similarly for anions, phosphate, sulphate and organic acids are the least chaotropic, but nitrate is much more chaotropic, just behind perchlorate and iodide (Kunz, 2010). Accordingly, experiments with lysozyme have demonstrated that the displacement of the solubilization-aggregation equilibrium towards aggregation is larger with nitrate than chloride forms of sodium salts (Kastelic, Kalyuzhnyi, Hribar-Lee, Dill, & Vlachy, 2015). As such, having high concentrations of nitrate is not desirable for phloem protein stability when K+ (and/or Mg2+) is present at high concentration. Conversely,in vitro , in sucrose concentrated solutions (at concentrations similar to that found in phloem sap), ethylamine nitrate has been found to be beneficial to protein renaturation and decreases viscosity due to the rescuing property of ethylamine as a non-chaotropic cation (Byrne, Wang, Belieres, & Angell, 2007).