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 Ca 2+ -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 (downregulation). 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.Arabidopsis | chloride channel T he perception of the phytohormone abscisic acid (ABA) is achieved by the recently discovered 14-member START protein family of ABA receptors named pyrabactin resistance (PYR), or regulatory component of ABA receptor (RCAR) (1, 2). PYR/RCARs have been shown to bind to clade A PP2Cs and inhibit the activity of these PP2Cs in the presence of ABA (1-5). Structural studies show that PYR1, PYL1, and PYL2 function as ABA receptors, with ABA binding in a protein cavity that locks down the ABA molecule (6-10).ABA reduces transpirational water loss of plants by inducing stomatal closure (11). ABA can cause an increase in guard cell intracellular Ca 2+ concentration (12-17), which leads to the down-regulation of inward-rectifying K + channels and activation of both slow-sustained (S-type) and rapid-transient (R-type) anion channels (18)(19)(20). Previous findings have led to the model that S-type anion channels play a key role in controlling stomatal closure (18,21,22). slac1 mutant plants have greatly reduced S-type anion channel activity (23) and display impaired stomatal closure in response to ABA, elevated CO 2 , ozone, reactive oxygen species, calcium, and reduced humidity, underlining that SLAC1 repres...

Na(+) and K(+) homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na(+) transport or Na(+) and K(+) transport when expressed in Xenopus laevis oocytes and yeast. However, the Na(+)/K(+) selectivities of the K(+)-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K(+) permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na(+) uptake, but little Rb(+) uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na(+)-K(+) cotransport in plant cells such that extracellular K(+) stimulated OsHKT2;2-mediated Na(+) influx and vice versa. Furthermore, at millimolar Na(+) concentrations, OsHKT2;2 mediated Na(+) influx into plant cells without adding extracellular K(+). This study shows that the Na(+)/K(+) selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K(+) and Ca(2+) down-regulated OsHKT2;1-mediated Na(+) influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na + -K + cotransport and at high Na + concentrations preferred Na + -selective transport, depending on the ionic conditions. But the physiological functions of this K

Intracellular malate-starch interconversion plays an important role in stomatal movements. We investigated whether malate or oxaloacetate from the cytosolic membrane side regulate anion channels in the plasma membrane of Arabidopsis thaliana guard cells. Physiological concentrations of cytosolic malate have been reported in the range of 0.4-3 mM in leaf cells. Guard cell patch clamp and two-electrode oocyte voltage-clamp experiments were pursued. We show that a concentration of 1 mM cytosolic malate greatly activates S-type anion channels in Arabidopsis thaliana guard cells. Interestingly, 1 mM cytosolic oxaloacetate also activates S-type anion channels. Malate activation was abrogated at 10 mM malate and in SLAC1 anion channel mutant alleles. Interestingly, malate activation of S-type anion currents was disrupted at below resting cytosolic-free calcium concentrations ([Ca ] ), suggesting a key role for basal [Ca ] signaling. Cytosolic malate was not able to directly activate or enhance SLAC1-mediated anion currents in Xenopus oocytes, whereas in positive controls, cytosolic NaHCO enhanced SLAC1 activity, suggesting that malate may not directly modulate SLAC1. Cytosolic malate activation of S-type anion currents was impaired in ost1 and in cpk5/6/11/23 quadruple mutant guard cells. Together these findings show that these cytosolic organic anions function in guard cell 'plasma membrane' ion channel regulation.

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