Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor
Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor
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DOI：
10.1105/tpc.15.00637
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发表时间：
2015-11
期刊：
影响因子：
11.6
通讯作者：
Cun Wang;Honghong Hu;Xue Qin;Brian S. Zeise;Danyun Xu;W. Rappel;W. Boron;J. Schroeder
中科院分区：
文献类型：
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作者：
Cun Wang;Honghong Hu;Xue Qin;Brian S. Zeise;Danyun Xu;W. Rappel;W. Boron;J. Schroeder
在X.laevis卵母细胞中，细胞外CO2信号对离子通道调节的功能重建表现为SLAC1、蛋白激酶、Carbon AnHYDRase 4的表达以及与CO2通透性PIP2；1水通道蛋白的相互作用。暗呼吸引起叶片CO2浓度(Ci)的增加，大气CO2浓度的持续增加进一步增加了Ci。叶片二氧化碳浓度升高会导致气孔孔关闭。在这里，我们证明了高细胞内CO_2/HCO_3−在非洲爪哇卵母细胞中共表达拟南芥蛋白激酶OST_1、CPK_6或CP_(23)时，增强了由拟南芥保卫细胞S型阴离子通道SlaC_1介导的电流。裂解泛素筛选确定PIP2；1水通道蛋白是βCA4型碳酸酐酶的相互作用因子，裂解荧光素酶、双分子荧光互补和免疫共沉淀实验证实了这一点。PIP2；1表现出CO2渗透性。仅植物中PIP2；1的突变不足以破坏CO2和脱落酸诱导的气孔关闭，这可能是由于冗余。有趣的是，在卵母细胞中，βCA4和PIP2；1与OST1-SLAC1或CPK6/23-SLAC1共表达可以增强细胞外二氧化碳对SLAC1阴离子通道的激活。一个失活的PIP2；1点突变被鉴定为破坏了水和二氧化碳的通透性以及细胞外二氧化碳对SLAC1活性的调节。这些发现表明，二氧化碳渗透性的PIP2；1是βCA4的关键相互作用因子，并证明了在PIP2；1、βCA4、SLAC1和蛋白激酶共表达的情况下，细胞外CO2信号对离子通道调节的功能重构。这些数据进一步表明，Slac1是一种碳酸氢盐响应蛋白，参与了S类型阴离子通道的二氧化碳调节。
Functional reconstitution of extracellular CO2 signaling to ion channel regulation is shown by expression of SLAC1, protein kinases, CARBONIC ANHYDRASE4, and interacting CO2-permeable PIP2;1 aquaporin in X. laevis oocytes. 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.