Specificity of the plasma membrane Aquaporins PIP1;1

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.