Low nitrate concentration may avoid protein aggregation
Phloem sap contains very high content of sucrose (about 400 mM in castor
bean), amino acids (about 60 mM) and K+ (about 60 mM).
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 et al. ,
2012; Jekat et al. , 2013; Knoblauch et al. , 2014; van Belet 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.
(RodrÃguez-Celma et al. , 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 et
al. , 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
sucrose concentrated solutions (like phloem sap), ethylamine nitrate is
beneficial to protein renaturation and decreases viscosity due to the
rescuing property of ethylamine as a non-chaotropic cation (Byrneet al. , 2007).