Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid

Kinases from three different families can activate an anion channel that helps plants tolerate drought stress. Three-in-One Drought Response One way that plants survive drought conditions is by closing gas exchange pores called stomata. Drought stimulates production of the hormone abscisic acid, which stimulates the opening of anion channels to reduce the volume of guard cells so that they come together and the stomata close. Maierhofer et al. expressed various kinases and their interacting partners in Xenopus oocytes along with the anion channels SLAC1 or SLAH3 and determined that in addition to the known two types of kinases that activated these channels, a third type of kinase also stimulated channel opening. The kinases belonged to both calcium-independent and calcium-dependent families and phosphorylated different residues on SLAC1. Future studies will address how this complex regulation enables plants to coordinate responses and adapt to stressful conditions. Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B–like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA.

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