title: Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor.
abstract: 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.
authors.affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116.
authors.forename: Cun
authors.identifier: http://orcid.org/0000-0001-6975-0959
authors.initials: C
authors.lastname: Wang
authors.affiliation: 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 huhh@mail.hzau.edu.cn jischroeder@ucsd.edu.
authors.forename: Honghong
authors.identifier: http://orcid.org/0000-0003-0538-6646
authors.initials: H
authors.lastname: Hu
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Ohio 44106.
authors.forename: Xue
authors.identifier: http://orcid.org/0000-0001-8049-6285
authors.initials: X
authors.lastname: Qin
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Ohio 44106.
authors.forename: Brian
authors.initials: B
authors.lastname: Zeise
authors.affiliation: College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Danyun
authors.initials: D
authors.lastname: Xu
authors.affiliation: Physics Department, University of California San Diego, La Jolla, California 92093.
authors.forename: Wouter-Jan
authors.initials: WJ
authors.lastname: Rappel
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Ohio 44106.
authors.forename: Walter F
authors.initials: WF
authors.lastname: Boron
authors.affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 huhh@mail.hzau.edu.cn jischroeder@ucsd.edu.
authors.forename: Julian I
authors.initials: JI
authors.lastname: Schroeder
mesh_terms: D000040:Abscisic Acid / Q000378:metabolism; D000838:Anions / Q000378:metabolism; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010937:Plant Growth Regulators / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D054046:Plant Stomata / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000838:Anions; D020346:Aquaporins; D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D010937:Plant Growth Regulators; C528438:SLAC1 protein, Arabidopsis; C491680:plasma membrane intrinsic protein 2 Arabidopsis; D002245:Carbon Dioxide; D000040:Abscisic Acid; D011494:Protein Kinases; C472139:OST1 protein, Arabidopsis; D002256:Carbonic Anhydrases