Relationship between phosphorylation, water permeability and ionic conductance of AtPIP2;1
Mutant versions of AtPIP2;1 where different phosphorylation states for CTD sites S280 and S283 were mimicked differed in their osmotic water permeability (Pos) and ionic conductance when expressed in oocytes (Figure 2, Figure S2, Figure 3, Figure S3 and Figure S4). For each individual oocyte included in the experiment measurements of both Pos and ionic conductance were captured so that the relationship between Pos and ionic conductance could be investigated.
The Pos of oocytes expressing AtPIP2;1 WT, and AtPIP2;1 S280 and S283 single and double phospho-mimic and -deficient mutants was determined via the photometric swelling assay (Figure S4). The single and double phospho-deficient mutants A/A had greater mean Pos relative to AtPIP2;1 WT (Figure S4). Comparatively, the single and double phospho-mimic mutants S280D, S283D, D/A, A/D and D/D all had lower mean Pos compared to AtPIP2;1 WT (Figure S4). The lower Pos for the D/A and A/D mutants indicates that when either of the S280 or S283 sites are phosphorylated this is likely having a dominant functional effect over the dephosphorylated state of the other site. It was also evident that the variation between individual oocytes in both Pos and ion conductance was dependent on the mutation (Figure 2 c, d & Figure S4)
To test for a relationship between Pos, ionic conductance and CTD phosphorylation state, TEVC was first performed followed by swelling assays on the same oocytes after a 2 h recovery incubation. Data was collected from multiple independent oocyte batches. Individual conductance was plotted against the corresponding Pos for each oocyte (Figure S3). For WT and D/D the variation in both ionic conductance and Pos showed a clear and significant inverse correlation (Figure S3). A significant inverse linear regression was also observed when all genotypes were combined (Figure S3). To better illustrate the relationship, all data points were binned on the basis of ionic conductance (10 µS bins) regardless of genotype (Figure 3a. The negative correlation between Pos and ionic conductance was best fit to a single exponential decay (p < 0.005) (Figure 3a) such that a high ionic conductance corresponded to a lower Pos similar in level to that of water injected controls (dashed horizontal blue line in Figure 3b). This indicates that phosphorylation at AtPIP2;1 CTD affects the ion/water permeability in a reciprocal but variable manner, whereby at the maximum ionic conductance the Pos of PIP2;1 is effectively zero, and when Pos was maximal the ionic conductance of PIP2;1 expressing oocytes effectively reduced to zero (i.e. similar to water injected control oocytes; dashed vertical red line Figure 3a and c).
To illustrate the trend with the different CTD mimics the frequency distributions are shown for decreasing Pos (Figure 3b) and increasing ion conductance (Figure 3c) The red (vertical) and blue (horizontal) dashed lines indicate the means of ionic conductance and Pos respectively for H2O injected oocytes (Figure 3a, b, c). AtPIP2;1 A/A, S283A, A/D and S280D mutants follow the same relative order for the change in mean Pos and ionic conductance (Figure 3d).
The AtPIP2;1 single and double phosphorylation mutants with at least one phospho-mimic residue (S280D, S283D, A/D, D/A, D/D) had greater mean ionic conductance and reduced mean Pos relative to AtPIP2;1 WT (Figure 3b, c). The S280D, S283D and D/D mutants exhibited increased frequency of a clustered population with significantly down-regulated Pos, in contrast to AtPIP2;1 WT and other mutants that showed a wide distribution of Pos (Figure 3b). The different distribution patterns observed in ionic conductance and Pos for the S280 and S283 phosphorylation mimics suggests that other factors or phosphorylation states may be altered in oocytes to cause variation.