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.