Wang Cun, Hu Honghong, Qin Xue, Zeise Brian, Xu Danyun, Rappel Wouter-Jan, Boron Walter F, Schroeder Julian I
Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116.
Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Plant Cell. 2016 Feb;28(2):568-82. doi: 10.1105/tpc.15.00637. Epub 2016 Jan 13.
Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3 (-) enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels.
暗呼吸导致叶片二氧化碳浓度（Ci）升高，而大气中[CO2]的持续增加进一步提高了Ci。叶片二氧化碳浓度升高会导致气孔关闭。在此，我们证明，在非洲爪蟾卵母细胞中共表达拟南芥蛋白激酶OST1、CPK6或CPK23中的任何一种时，高细胞内CO2/HCO3 (-) 会增强拟南芥保卫细胞S型阴离子通道SLAC1介导的电流。分裂泛素筛选确定PIP2;1水通道蛋白是βCA4碳酸酐酶的相互作用蛋白，这在分裂荧光素酶、双分子荧光互补和免疫共沉淀实验中得到了证实。PIP2;1表现出CO2通透性。单独在植物中突变PIP2;1不足以损害CO2和脱落酸诱导的气孔关闭，这可能是由于冗余性。有趣的是，在卵母细胞中βCA4和PIP2;1与OST1-SLAC1或CPK6/23-SLAC1共表达可使细胞外CO2增强SLAC1阴离子通道活性。鉴定出一种无活性的PIP2;1点突变，该突变消除了水和CO2通透性以及细胞外CO2对SLAC1活性的调节。这些发现确定了具有CO2通透性的PIP2;1是βCA4的关键相互作用蛋白，并证明了在PIP2;1、βCA4、SLAC1和蛋白激酶共表达时细胞外CO2信号传导对离子通道调节的功能重建。这些数据进一步表明SLAC1是一种对碳酸氢盐有反应的蛋白，有助于CO2对S型阴离子通道的调节。
