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

Abstract Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 through the plasma membrane in living plant cells. We designed genetic constructs to co‐express the fluorescent H 2 O 2 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 H 2 O 2 , suggesting that ZmPIP2;5 facilitates H 2 O 2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H 2 O 2 channel. ZmPIP2;1, ZmPIP2;4 and AtPIP2;1 also facilitated H 2 O 2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H 2 O 2 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.