Specificity of the plasma membrane Aquaporins PIP1;1

Details

Supervisors

Chaumont, François

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Plasma membrane aquaporins (PIPs), because of their essential role in membrane diffusion of water and other small solutes, are widely studied and already well characterized. However, the maize PIP1;1 isoform differs from the others. Indeed, its function and substrate specificity remain unknown. Interestingly, PIP1;1 is the most highly and constitutively expressed isoform in all maize tissues (Hachez et al., 2006, 2008; Heinen et al., 2014). This level of expression suggests that this AQP plays a key role in the regulation and/or transport of molecules necessary for plant growth. In the present work, we aimed at characterizing further PIP1;1 function and regulation using different homologous and heterologous expression systems. To study the effect of amino acid substitution on the water channel activity of PIP1;1, we used the X. laevis oocyte system. We tested if the QH/RD mutation, located in the loop E, is sufficient to activate the PIP1;1 water channel activity as well as the effect of the W94 mutation on the PIP1;1QH/RD activity. Oocytes swelling assays indicated that PIP1;1QH/RD seems to be an active water channel by itself and the W to A mutation in the PIP1;1QH/RD mutant inhibits the protein activity. By performing swelling assays with protoplasts prepared from Nicotiana tabacum BY-2 suspension cells overexpressing (OE) PIP1;1 and PIP1,1W94A, we tested whether the expression of those proteins led to a change in the cell membrane water permeability coefficient (Pf). Even if the results were not statistically significant, we observed that PIP1;1 tends to increase the Pf as observed with the positive control, AtPIP2;1, which facilitates water diffusion. Interestingly, expression of PIP1;1W94A systematically led to a lower Pf than the negative control cells, suggesting that the W94A mutation inactivates the channel activity of PIP1;1 and/or endogenous PIP2s. Finally, using the H2O2 biosensor HyPer technology, we carried out H2O2 transport assays in BY-2 cells expressing PIP1;1 and PIP1;1W94A. The latter proteins were under the control of a heat shock-inducible promoter and the assays compared H2O2 accumulation in induced and non-induced cells. While PIP1;1 appeared not to facilitate H2O2 diffusion, expression induction of PIP1;1W94A decreased H2O2 transport into the cells. Altogether, these data suggest that the overexpression of PIP1;1W94A has a negative impact on the activity of endogenous PIP aquaporins, reducing H20 and H2O2 membrane diffusion.