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).