Phosphorylation status of C-terminal S280 and S283 sites regulate AtPIP2;1 facilitated water and ion transport
The influence of mutating AtPIP2;1 S280 and S283 sites to phospho-mimic and phospho-deficient versions on channel water and ion transport function was tested in oocytes (Figures 2-3, Figure S4). Oocytes expressing the single phospho-mimic versions of AtPIP2;1, S280D and S283D or the phospho-mimic double mutants A/D, D/A and D/D had greater ionic conductance in solutions containing Na+ and K+ and increased internal Na+content compared to oocytes expressing either AtPIP2;1 WT or the single phospho-deficient variants S280A and S283A or the double phospho-deficient mutant A/A (Figure 2). Oocytes expressing the single mutant S280A and double mutant A/A had greater Pos than the other versions (Figure S4). When the phosphorylation status of both CTD sites were controlled but only one site mimicked a phosphorylated state the effect of the phospho-mimicked residue presided somewhat over the effect of the phosphor-deficient site; for example, both the D/A and A/D phospho-mutants had water and ion transport more similar to that of the D/D mutants than the A/A mutants (Figure 2 and 3). Previously the Rattus AQP2 was reported to be phosphorylated at two serines on its CTD in renal epithelial cells in response to vasopressin, and the effect of phosphorylation of one residue presided over the channel function and trafficking (Table 1) (Lu et al., 2008). The phosphorylation of several CTD residues in Rattus AQP2 also exhibit a hierarchy where the phosphorylation of particular residues does not occur unless the phosphorylation of another site has preceded it (Hoffert et al.,2008).
AtPIP2;1 facilitated the transport of K+ and the single phospho-mimic mutants conferred greater K+associated conductance than the other versions, similar to the trend observed for phospho-mimic versions for Na+conductance (Figure 2). AtPIP2;1 and AtPIP2;2 have been proposed as molecular candidates for the elusive non-selective cation channel that have been observed in planta (Byrt et al., 2017; McGaugheyet al., 2018; Munns et al., 2019; Demidchik and Tester, 2002; Essah et al., 2003; Roberts and Tester, 1997). The observation that AtPIP2;1 can facilitate K+ transport in vivo adds support to this hypothesis. The NSCCs observed by Demidchik and Tester, (2002), had greater K+conductance relative to Na+ conductance (with a selectivity ratio of 1.49:1.00), which is similar to the trend in K+ relative to Na+ conductance for AtPIP2;1 when expressed in X. laevis oocytes (Figure 2c). The regulation of AtPIP2;1 ion transport by cGMP treatments (Figure 1) is also relevant to previous NSCC observations, because exogenous application of cGMP was previously shown to inhibit NSCCs in planta (Essah et al., 2003; Maathuis and Sanders, 2001), and intracellular cGMP concentrations have been reported to increase in response to salinity and osmotic stress treatments (Donaldson et al., 2004; Rubio et al., 2003). Interestingly, a recent review hypothesised that Na+ influx via AtPIP2;1 may be inhibited by cGMP under salt stress, which is an idea worthy of follow up investigation in planta (Isayenkov and Maathuis, 2019). The observation that AtPIP2;1 facilitates transport of the physiologically important element K+, and the potential for AtPIP2;1 transport of other monovalent ions such as NH4+, indicates that a potential role for PIPs in nutrient acquisition under normal conditions is also worthy of testing in planta .
A negative correlation between AtPIP2;1 facilitated water and ion transport was linked to the CTD phosphorylation state (Figure 3a). AtPIP2;1 mutants including S280D, S283D, D/A and D/D, had a greater tendency to facilitate the transport of ions over water compared to that of the phosphorylation deficient mutant A/A (Figure 3b, d). The variance seen for the ionic conductance and Pos of the D/D mutant indicates that there are likely to be other additional regulatory sites that were not controlled for in these experiments. Further research is needed to test how many other AtPIP2;1 regulatory sites influence water and ion transport functions and explore whether these sites have any sort of dependence on the status of the CTD sites.
Several General Regulatory Factors (GRFs; also known as 14-3-3 proteins) were recently reported to interact preferentially with AtPIP2;1 when the S280 and S283 sites were phosphorylated, and co-expression of AtPIP2;1 D/D mutant with GRFs 3,4 and 10 in oocytes increased their Pos compared to AtPIP2;1 A/A (Prado et al.,2019). In the current study it cannot be excluded that AtPIP2;1 could have interacted with an endogenous oocyte GRF protein, or an endogenous aquaporin interacting ion channel. However, the trends observed for AtPIP2;1 CTD status and associated ionic conductance do not appear common to all aquaporins. There are commonalities for CTD phosphorylation trends among some Arabidopsis PIPs, but not all PIPs with these commonalities confer ionic conductance in oocytes. For example, in Arabidopsis AtPIP2;1, AtPIP2;2, AtPIP2;3, AtPIP2;4 and AtPIP2;7 were found to be unphosphorylated, singly phosphorylated at S280 or diphosphorylated at S280 and S283 (Prak et al., 2008), but AtPIP2;7 did not facilitate ion transport when expressed in oocytes (Kourghi et al., 2017) as confirmed here also from a lack of Na+ uptake into yeast expressing AtPIP2;7. There is also a precedent for plant aquaporins having ion channel functions in the absence of any potential interacting partners, and associations with the CTD status. The soybean (Glycine max ) Gm-NOD26, produced ion channel activity when reconstituted in lipid bilayers (Weaver et al., 1994). The water and ion channel function of Gm-NOD26 was also found to be regulated by the phosphorylation of a CTD residue S262 (Guenther et al., 2003; Lee et al., 1995).
The exact physiological role of dual water and ion transporting aquaporins in plants remains unknown and may differ in different tissues (McGaughey et al., 2018). When Arabidopsis roots were exposed to a NaCl treatment the phosphorylation states of AtPIP2;1 S280 and S283 residues was observed to change (Prak et al., 2008). Specifically, when plants were treated with 100 mM NaCl the abundance of the S280/S283 disphosphorylated form decreased. Since phosphorylation of S280 and S283 increase AtPIP2;1 ion channel function, this reduction in S280/S283 diphosphorylated AtPIP2;1 may be a mechanism to reduce Na+ influx (and possibly K+ efflux) under salt stress. Salt treatment has also been reported to increase AtPIP2;1 location-cycling (Li et al., 2011; Luu et al.,2012), and induce AtPIP2;1 internalisation from the plasma-membrane into intra-cellular vesicles in root cells (Boursiac et al., 2005; Prak et al., 2008; Ueda et al., 2016) where internalisation was reported to be dependent on S283 phosphorylation state (Prak et al., 2008). By manipulating the phosphorylation state of the AtPIP2;1 CTD serine residues, we were also able to alter trafficking and abundance of the AtPIP2;1 protein between the PM and ER in yeast (Figure 5). In the yeast system we found that the phospho-mimic S280D mutation resulted in a more consistent localisation of AtPIP2;1 to the ER rather than trafficking to the PM. This feature specifically required the presence of a serine residue at position 283 and could not be replicated by mimicking a phospho-deficient state using alanine. We also observed that consistency in PM targeting was not only dependent on S283 phosphorylation, but also required dual phosphorylation of both S280 and S283, with the presence of a phospho-mimic residue at position 283 potentially influencing the phosphorylation state of S280. Interestingly the localisation of the double phospho-deficient mutant A/A was similar to other phospho-mimic mutants, such as D/A and A/D (Figure 5), where these other mutant versions exhibited much greater ionic conductance than A/A when expressed in X. laevis oocytes (Figure 2). This data, alongside the increased Pos of A/A relative to the other mutants (Figure 3), indicates that a mis-localisation of the A/A mutant in oocytes is not likely to be the cause of its lower ionic conductance. Furthermore, the fact we could make these observations in yeast using an aquaporin from the distant taxa of plants, indicates the potential for there to have been a shared evolutionary origin for the process of CTD phosphorylation influencing aquaporin trafficking.
The sophisticated relationship between AtPIP2;1 phosphorylation state and AtPIP2;1 trafficking, localisation, and water and ion transport function may be part of a mechanism for rapidly, reversibly and co-ordinately adjusting water and Na+ or K+ flux into or out of the cell under salt and osmotic stress.