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 (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.