Minisymposium 17: Water

Abs # 32001: Regulation of plasma membrane aquaporins from maize

Presenter:	Van Wilder, Valerie , vanwilder@fysa.ucl.ac.be
Authors	Van Wilder, Valerie  (A)   Degand, Herve  (A)   Fetter, Karolina  (A)   Zelazny, Enric  (A)   Chaumont, Francois  (A)
Affiliations:	(A): Universite catholique de Louvain

The osmotic water permeability of the cell membranes is dependent on the presence of active aquaporins. The water channel activity of aquaporins is mostly derived from experiments with Xenopus oocytes. Cells expressing an aquaporin swell more rapidly in hypo-osmotic medium than control cells. The plant plasma membrane aquaporins (PIP) are divided in two subgroups according to their sequence and their water channel activity: all PIP2 proteins examined have been shown to have high water channel activity in Xenopus oocytes, whereas PIP1 proteins are often inactive or have low activity. We are studying the mechanisms regulating the activity of maize PIPs. We showed that co-expression of the non-functional ZmPIP1;2 and functional ZmPIP2;1, ZmPIP2;4, or ZmPIP2;5 resulted in an increase in Pf which was dependent on the amount of injected ZmPIP1;2 cRNA. Confocal analysis of oocytes expressing ZmPIP1;2-GFP alone or ZmPIP1;2-GFP plus ZmPIP2;5 showed that the amount of ZmPIP1;2-GFP present in the plasma membrane was significantly higher in co-expressing cells. Physical interaction of ZmPIP1;2-GFP and ZmPIP2;1 fused to a His-tag was demonstrated by affinity chromatography (Fetter et al.(2004) Plant Cell 16, 215-228). These data revealed a new mechanism of aquaporin regulation and we are currently investigating whether PIP heteromerization is also observed in plant cells. We also showed that plasma membrane PIPs from etiolated maize shoots were in vivo and in vitro phosphorylated. This phosphorylation occurred on a serine residue and was due to a Ca-dependent kinase associated with the plasma membrane. Three PIP2 isoforms (ZmPIP2;1, ZmPIP2;3 and ZmPIP2;4) were detected in the phosphorylated band. We are currently identifying the phosphorylation sites of the PIP2 proteins by mass spectrometry. The water channel activity of these isoforms was partially inhibited by H7, a PKC inhibitor, suggesting an important effect of phosphorylation on channel function. The two predicted phosphorylation sites of ZmPIP2;1 have been mutagenized (S126A, S285A, S126E and S285E) and fused to a His-tag, and the mutants tested in oocytes. A decrease of the water channel activity of ZmPIP2;1S126A was observed. We are checking the phosphorylation status of these different mutated proteins in oocytes after purification on Ni-NTA column. Finally, we are also investigating the role of the phosphorylation in the regulation of the osmotic water permeability of protoplast plasma membrane.