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).