Aquaporin contribution to facilitated membrane diffusion of hydrogen peroxide and cations in plant cells

Abstract

Aquaporins (AQPs) are channels facilitating the diffusion of water and small solutes across the cellular membranes. Recent evidence suggests that plant AQPs also facilitate the rapid permeation of hydrogen peroxide (H2O2) and even cations, which are essential for plant physiology. In our project, we aim at establishing molecular and cellular tools to determine whether plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) facilitate the diffusion of H2O2 and cations through the plasma membrane in plant cells and characterize this transport. We prepared genetic constructs to stably co-express in Nicotiana tabacum BY2 suspension cells the fluorescent H2O2 sensor HyPer, with maize PIP2;5, an AQP that, when expressed in yeast, facilitates the membrane diffusion of H2O2. PIP2;5 expression was controlled by a heat shock inducible promoter. After induction of PIP2;5 expression, a HyPer signal was recorded when the cells were incubated with H2O2, which suggests that PIP2;5 facilitates H2O2 transmembrane diffusion. Using this new system, we tested several maize and Arabidopsis PIP2s and showed that they facilitate the H2O2 transmembrane diffusion, but it was not the case for the PIP2;5W85A mutant. We also showed that treatment by abscisic acid and the elicitor flagellin-derived flg22 peptide induced the intracellular H2O2 accumulation in plant cells when PIP2;5 was expressed. These results indicate that plant suspension cells and the HyPer sensor co-expressed with PIPs are a powerful toolkit for evaluating the transport specificity of PIPs in living cells, determining their molecular determinants as well as real-time monitoring H2O2 dynamics in plant single cells. The cation transport ability of PIP2;5 was first tested in a yeast expression system. PIP2;5 increased the Na+ sensitivity of the cells compared with the control yeast. We confirmed this result in BY2 cells using a novel fluorescent photo-switchable Li+ sensor. After induction of PIP2;5 expression and incubation with the sensor and Li+, a higher fluorescent signal was observed compared with the signal obtained in non-induced BY2 cells, suggesting that PIP2;5 facilitates the membrane diffusion of cations. Furthermore, this new feature was tested in intact maize plants deregulated in PIP2;5 expression. The results obtained from PIP2;5 deregulated plants support our conclusion that PIP2;5 is a novel cation channel or a cation transport facilitator.(SC - Sciences) -- UCL, 202