Introduction
Aquaporins are membrane bound channel proteins that facilitate the
passive bidirectional movement of water and other small molecules across
biological membranes. Substrates currently known to be transported by
aquaporins include gases (O2; Zwiazek et al.,2017, CO2; Rodrigues et al., 2017; Uehleinet al., 2003), metalloids (silicon; Ma et al., 2006,
boron; Takano et al., 2006, arsenic; Li et al., 2009),
reactive oxygen species (H2O2; Bienertet al., 2007; Dynowski et al., 2008; Hooijmaijers et
al., 2012), monovalent cations (Na+; Byrt et
al., 2017; Kourghi et al., 2017; Weaver et al., 1994) and
other neutral substrates (urea; Dynowski et al., 2008b, glycerol;
Gerbeau et al., 1999, ammonia; Loqué et al., 2005). As
facilitators of transmembrane water transport, members of the Plasma
membrane Intrinsic Protein (PIP) sub-family have roles in mediating
water uptake at the root-soil interface, in transcellular water flow,
and in regulating hydraulic conductivity in response to abiotic stresses
(for recent reviews see: Chaumont & Tyerman, 2014; Maurel et
al., 2015; Gambetta et al., 2017). Similar to some mammalian
aquaporin isoforms, a subset of plant PIPs (Arabidopsis PIP2;1 and
PIP2;2) were recently found to facilitate the transport of monovalent
cations such as Na+, more evident at low external
calcium and high pH (Byrt et al., 2017; Kourghi et al.,2017). The ability of some plant aquaporins to facilitate
Na+ transport has implications in relation to plant
salinity stress responses and tolerance to osmotic stress (McGaugheyet al ., 2018).
PIPs are implicated in mediating water uptake from soil to roots and
changes to root hydraulic conductivity in response to stress
(Tournaire-Roux et al., 2002). In Arabidopsis, root hydraulic
conductance correlated positively with both protein abundance of PIP2
aquaporins and the abundance of phosphorylated PIP2 proteins (di Pietroet al., 2013). Exogenous treatment of barley plants with the
kinase inhibitor staurosporine significantly reduced root hydraulic
conductance (Horie et al., 2011). The phosphorylation state of
several conserved serine residues in the cytoplasmic regions of PIPs,
including those in the CTD, have been implicated in a mechanism where
phospho-regulation can directly influence water permeation through the
pore (Table 1; Johansson et al., 1998; Törnroth-Horsefieldet al., 2006; Nyblom et al., 2009; Yaneff et al.,2016) and have been demonstrated to change in response to salt or
osmotic stress, influencing PIP trafficking and localisation (Boursiacet al., 2005; Boursiac et al., 2008; Li et al.,2011; Prak et al., 2008). The phosphorylation state of two serine
residues on the CTD of AtPIP2;1, S280 and S283, has been reported to
change in plant roots exposed to salt treatments and the phosphorylation
of S283 has been associated with the salt-induced internalisation of
AtPIP2;1 in Arabidopsis roots (Prak et al., 2008).
Extensive studies on aquaporin regulation in animals has also identified
phosphorylation as a key regulator of animal aquaporin channel function
(both water and ion), protein cycling, trafficking, and membrane
localisation (Table 1). The water and ion channel function of soybean
(Glycine max ) NOD-26 (GmNOD-26), the first plant aquaporin to be
identified as permeable to both water and ions, was shown to be
regulated by the phosphorylation of a CTD residue S262 (Guentheret al., 2003; Lee et al., 1995; Weaver et al.,1994). Phosphorylation of S262 altered the voltage sensitivity of
GmNOD-26 ion channel activity (Lee et al., 1995) and increased
its osmotic water permeability (Guenther et al., 2003). GmNOD-26
phosphorylation was also reported to increase in plants exposed to
osmotic stress (Guenther et al., 2003). It is therefore
conceivable that phosphorylation could augment the ability of PIPs to
facilitate water and Na+ transport in isoforms capable
of such activity. In which case, changes in phosphorylation states would
not only regulate PIP protein trafficking and localisation in response
to salt and osmotic stresses but also water and Na+transport capacity (Byrt et al., 2017; McGaughey et al.,2018).
In this study the phosphorylation state of the two conserved CTD serine
residues (S280 and S283) in AtPIP2;1 was investigated through series of
single and double S280 and S283 phospho-mimic and -deficient AtPIP2;1
mutants in the context of the concurrent regulation of ion
(Na+ and K+) and water transport. It
is important to determine the relationships between PIP protein
regulation by phosphorylation and water and ion transport capacity
because these features influence plant tolerance to drought and NaCl
stresses (McGaughey et al ., 2018). Our results indicate that
phosphorylation has a key role to play in AtPIP2;1 regulation of
transport selectivity and capacity. Given existing information about the
regulation of animal aquaporins, and how precisely channel activity,
trafficking and localisation are co-ordinately controlled (Table 1), it
is expected that there is similar complexity in the regulation of plant
aquaporin function that are yet to be fully explored.