Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action
Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action
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DOI：
10.1073/pnas.1116590109
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发表时间：
2012-06-26
影响因子：
11.1
通讯作者：
Schroeder, Julian I.
中科院分区：
文献类型：
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作者：
Brandt, Benjamin;Brodsky, Dennis E.;Schroeder, Julian I.
植物激素脱落酸（ABA）是在应对非生物胁迫时产生的，并通过最近发现的ABA受体（pyrabactin resistance/ ABA receptor的调控成分；PYR/RCAR）介导干旱时的气孔关闭。SLAC1编码中央保护细胞s型阴离子通道，介导aba诱导的气孔关闭。钙依赖性蛋白激酶21 （CPK21）、CPK23或开放气孔1激酶（OST1）的共表达可激活SLAC1阴离子电流。然而，ABA活化植物离子通道的重构尚未实现。已知的核心ABA信号成分是否足以激活SLAC1阴离子通道，或者是否需要其他成分仍然未知。已知Ca2+依赖性蛋白激酶CPK6在体内aba诱导的气孔关闭中起作用。在这里，我们发现CPK6强有力地激活SLAC1介导的电流并磷酸化SLAC1 N端。在SLAC1中发现了一个磷酸化位点（S59），对CPK6的激活至关重要。A组PP2Cs ABI1、ABI2和PP2CA下调cpk6介导的卵母细胞SLAC1活性。出乎意料的是，ABI1直接使SLAC1的N端去磷酸化，表明ABI1直接靶向SLAC1的交替分支早期ABA信号核心（下调）。此外，我们利用ABA受体pyrabactin resistant 1 （PYR1）和PP2C磷酸酶，通过两个替代信号核心CPK6或OST1，成功重建了ABA诱导的卵母细胞SLAC1通道激活。ABI1的点突变破坏PYR1-ABI1相互作用，破坏ABA信号转导。此外，通过添加CPK6，在没有SnRK2激酶的情况下，从ABA受体到离子通道激活的功能ABA信号转导核心被重建。
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.