Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

Benjamin Brandt, Dennis E. Brodsky, Shaowu Xue, Juntaro Negi, Koh Iba, Jaakko Kangasjärvi, Majid Ghassemian, Aaron B. Stephan, Honghong Hu, Julian I. Schroeder

Research output: Contribution to journalArticle

212 Citations (Scopus)

Abstract

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca2+-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

Original languageEnglish
Pages (from-to)10593-10598
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number26
DOIs
Publication statusPublished - Jun 26 2012

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Abscisic Acid
Phosphoric Monoester Hydrolases
Anions
Phosphotransferases
Ion Channels
Oocytes
Signal Transduction
Plant Growth Regulators
Droughts
Point Mutation
Protein Kinases

All Science Journal Classification (ASJC) codes

  • General

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Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. / Brandt, Benjamin; Brodsky, Dennis E.; Xue, Shaowu; Negi, Juntaro; Iba, Koh; Kangasjärvi, Jaakko; Ghassemian, Majid; Stephan, Aaron B.; Hu, Honghong; Schroeder, Julian I.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 26, 26.06.2012, p. 10593-10598.

Research output: Contribution to journalArticle

Brandt, Benjamin ; Brodsky, Dennis E. ; Xue, Shaowu ; Negi, Juntaro ; Iba, Koh ; Kangasjärvi, Jaakko ; Ghassemian, Majid ; Stephan, Aaron B. ; Hu, Honghong ; Schroeder, Julian I. / Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. In: Proceedings of the National Academy of Sciences of the United States of America. 2012 ; Vol. 109, No. 26. pp. 10593-10598.
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abstract = "The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca2+-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.",
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AU - Brandt, Benjamin

AU - Brodsky, Dennis E.

AU - Xue, Shaowu

AU - Negi, Juntaro

AU - Iba, Koh

AU - Kangasjärvi, Jaakko

AU - Ghassemian, Majid

AU - Stephan, Aaron B.

AU - Hu, Honghong

AU - Schroeder, Julian I.

PY - 2012/6/26

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