Phospho-mimic and deficient AtPIP2;1 mutants had altered ionic conductance and Na+ accumulation in X. laevis oocytes
The phosphorylation state of AtPIP2;1 CTD residues S280 and S283 is altered in planta by salt treatments (Prak et al., 2008). To explore the potential regulatory roles of S280 and S283 phosphorylation on AtPIP2;1-facilitated ion transport, single and double S280 and S283 phospho-mimics mutated to aspartic acid (D), or phospho-deficient mutated to alanine (A) versions of AtPIP2;1 were generated. The ionic conductance of oocytes expressing AtPIP2;1 wild-type (WT) or AtPIP2;1 phospho-mimic (S280D, S283D) and phospho-deficient (S280A, S283A) single and double mutants in the presences of Na+ and K+ were measured by TEVC (Figure 2, Figure S2).
In the ‘Na100’ solution the single and double phospho-deficient-mimicking mutants S280A, S283A and A/A induced currents and had ionic conductance of similar magnitude to that of AtPIP2;1 WT (Figure 2a-d). Whereas, the expression of the single phosphorylation-mimicking mutants S280D and S283D and the double phosphorylation-mimicking mutants D/A, A/D and D/D induced greater currents and ionic conductance than WT or the phospho-deficient mutants (Figure 2a-d).
AtPIP2;1 WT was also able to elicit somewhat larger currents and conductances (20-30% larger) with K+ as the major univalent cation (Figure 2a,c). The phosphorylation mutants had similar effects on conductance in a ‘K100’ solution to that observed in the ‘Na100’ solution. The phospho-mimics S280D and S283D had greater ionic conductance than either AtPIP2;1 WT or the phospho-deficient mutants (Figure 2c).
The total Na+ content of AtPIP2;1 WT and phospho-mutant expressing oocytes after 24 h incubation in ‘Na100’ solution was determined by analysis with an atomic absorption spectrophotometer (Figure 2e). Consistent with the trends observed for ionic conductance in the same solution (‘Na100’; Figure 2c,d), the phospho-mimic single (S280D and S283D) and double mutants (A/D, D/A, D/D) accumulated greater Na+ per oocyte than WT (Figure 2e). The phospho-deficient mutants (S280A, A/A) accumulated similar Na+ to AtPIP2;1 WT oocytes with the exception of S283A, which had significantly higher Na+accumulation and an opposite trend to that observed for ionic conductance (Figure 2a,c,e).