ANALYSIS OF THE DEGRADATION OF THE AQUAPORIN ATPIP2;1 IN ARABIDOPSIS THALIANA

Aquaporins are highly conserved membrane proteins that facilitate the water transport across the biological membranes. In this work we study the degradation process of Plasma membrane Intrinsic Proteins (PIPs), in particular, the isoform AtPIP2;1, in Arabidopsis thaliana roots. We expressed AtPIP2;1-GFP and AtPIP2;1-photoactivable GFP (paGFP) under the control of a 35S CaMV or a PIP2;1 native promoter in a k.o. pip2;1 line. The intensities of the fluorescence and the subcellular localization were monitored by means of epifluorescence and confocal microscopies. First, roots expressing the GFP constructs were treated with cycloheximide (CHX), a protein synthesis inhibitor. After 24 h, no significant difference compared to a control treatment was observed; experiments with the paGFP constructs indicate the same tendency. To make sure that the CHX treatments efficiently inhibited the protein synthesis, we monitored the signal of Aleurin-GFP fusion expressed in Arabidopsis plantlets. CHX treatment provoked a tremendous decrease of fluorescence signal, indicating the efficiency of the CHX. Next, we analyzed the effect of H2O2 treatments, and observed a slight but not significant decrease of the signal. Confocal microscopy, allowed us to observe that AtPIP2;1 constructs labeled intracellular dots, similar to those previously described in our laboratory. Lastly, treatments with 1 μM NAA, a synthetic analog of auxin, did not provoke any decrease of the intensity of the signal; we obtain a similar result with 1 μM IAA. Also, confocal microscopy observations showed a strong intracellular labeling. Altogether, these results allowed to conclude that AtPIP2;1-GFP construct is intrinsically very stable in the plasma membrane, in resting conditions. Treatment with H2O2 did not destabilize AtPIP2;1-GFP construct in the plasma membrane. In the contrary, auxin maintain the basal level of AtPIP2;1-GFP construct and provoke a strong relocalisation in intracellular compartments