A new method to measure aquaporin-facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells

Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2O2) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2O2 through the plasma membrane in living plant cells. We designed genetic constructs to co-express the fluorescent H2O2 sensor HyPer and AQPs, with expression controlled by a heat shock-inducible promoter in Nicotiana tabacum BY-2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2O2, suggesting that ZmPIP2;5 facilitates H2O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2O2 channel. ZmPIP2;1, ZmPIP2;4 and AtPIP2;1 also facilitated H2O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2O2 accumulation in BY-2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo-switchable Li+ sensor in BY-2 cells. BY-2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells.