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.