Jamshaid HUSSAIN, Fen Yong YAO, Guo Ning QI

Investigation of the regulation mechanism of Arabidopsis thaliana anion channel SLAH2

In recent years a slow-type anion channel (SLAC) family composing five members, including slow anion channel associated 1 (SLAC1) and four SLAC1 homologs (SLAHs), has been identified in Arabidopsis thaliana. The anion channels are activated by calciumdependent protein kinases (CPKs) and play crucial roles in mediating anion fluxes across plasma membranes. SLAH2 is specifically expressed in root stele cells and has been characterized as a nitrate-selective anion channel activated by CPK21 and CBL-interacting protein kinase 23 (CIPK23). However, the interaction between CPKs and SLAH2 and how it is regulated remains to be elucidated. In this study we found that SLAH2 could be activated by a few CPKs from three subgroups of the CPK family. Out of those CPKs, the constitutively active form of CPK2 from subgroup 1 activates SLAH2, whereas the native as well as constitutively active form of CPK3 from subgroup 2 and CPK7, CPK10, and CPK32 from subgroup 3 activate SLAH2. We also explored the role of SLAH2 in root-toshoot nitrate transport by measuring the nitrate contents in xylem sap, but no significant differences were observed between the slah2-1 T-DNA insertion mutant and SLAH2 overexpression lines. Together, these data demonstrate that SLAH2 is regulated by multiple CPKs with variable Ca2+ sensitivities. Further research is required to understand the role of SLAH2 in nitrate transport.

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Boudsocq M, Droillard MJ, Regad L, Lauriere C (2012). Characterization of Arabidopsis calcium-dependent protein kinases: activated or not by calcium? Biochem J 447: 291-299.
Brandt B, Brodsky DE, Xue S, Negi J, Iba K, Kangasjarvi J, Ghassemian M, Stephan AB, Hu H, Schroeder JI (2012). Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. P Natl Acad Sci USA 109: 10593-10598.
Cheng SH, Willmann MR, Chen HC, Sheen J (2002). Calcium signaling through protein kinases. The Arabidopsis calciumdependent protein kinase gene family. Plant Physiol 129: 469- 485.
Chiu CC, Lin CS, Hsia AP, Su RC, Lin HL, Tsay YF (2004). Mutation of a nitrate transporter, AtNRT1:4, results in a reduced petiole nitrate content and altered leaf development. Plant Cell Physiol 45: 1139-1148.
Clough SJ, Bent AF (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743.
Cubero-Font P, Maierhofer T, Jaslan J, Rosales MA, Espartero J, DiazRueda P, Muller HM, Hurter AL, Al-Rasheid KA, Marten I et al. (2016). Silent S-type anion channel subunit SLAH1 gates SLAH3 open for chloride root-to-shoot translocation. Curr Biol 26: 2213-2220.
Geiger D, Maierhofer T, Al-Rasheid KA, Scherzer S, Mumm P, Liese A, Ache P, Wellmann C, Marten I, Grill E et al. (2011). Stomatal closure by fast abscisic acid signaling is mediated by the guard cell anion channel SLAH3 and the receptor RCAR1. Science Signaling 4: ra32.
Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann C, Al-Rasheid KA, Grill E et al. (2010). Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities. P Natl Acad Sci USA 107: 8023-8028.
Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese A, Al-Rasheid KA et al. (2009). Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. P Natl Acad Sci USA 106: 21425-21430.
Gutermuth T, Lassig R, Portes MT, Maierhofer T, Romeis T, Borst JW, Hedrich R, Feijo JA, Konrad KR (2013). Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20. Plant Cell 25: 4525-4543.
Klimecka M, Muszynska G (2007). Structure and functions of plant calcium-dependent protein kinases. Acta Biochim Pol 54: 219- 233.
Kohler B, Raschke K (2000). The delivery of salts to the xylem. Three types of anion conductance in the plasmalemma of the xylem parenchyma of roots of barley. Plant Physiol 122: 243-254.
Latz A, Mehlmer N, Zapf S, Mueller TD, Wurzinger B, Pfister B, Csaszar E, Hedrich R, Teige M, Becker D (2013). Salt stress triggers phosphorylation of the Arabidopsis vacuolar $K^+$ channel TPK1 by calcium-dependent protein kinases (CDPKs). Mol Plant 6: 1274-1289.
Lee SC, Lan W, Buchanan BB, Luan S (2009). A protein kinasephosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. P Natl Acad Sci USA 106: 21419-21424.
Lefoulon C, Gutla PV, Honsbein A, Wang Y, Grefen C, Riedelsberger J, Karnik R, Gonzalez W, Blatt MR (2014). Voltage-sensor transitions of the inward-rectifying $K^+$ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor. Plant Physiol 166: 960-975.
Li JY, Fu YL, Pike SM, Bao J, Tian W, Zhang Y, Chen CZ, Zhang Y, Li HM, Huang J et al. (2010). The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell 22: 1633-1646.
Lin SH, Kuo HF, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HL, Wang YY, Tsai CB et al. (2008). Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective rootto-shoot nitrate transport. Plant Cell 20: 2514-2528.
Maierhofer T, Lind C, Huttl S, Scherzer S, Papenfuss M, Simon J, AlRasheid KA, Ache P, Rennenberg H, Hedrich R et al. (2014). A single-pore residue renders the Arabidopsis root anion channel SLAH2 highly nitrate selective. Plant Cell 26: 2554-2567.
Mehlmer N, Wurzinger B, Stael S, Hofmann-Rodrigues D, Csaszar E, Pfister B, Bayer R, Teige M (2010). The $Ca^{2+} -$dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis. Plant J 63: 484-498.
Meng SA, Peng JS, He YN, Zhang GB, Yi HY, Fu YL, Gong JM (2016). Arabidopsis NRT1.5 Mediates the suppression of nitrate starvation-induced leaf senescence by modulating foliar potassium level. Mol Plant 9: 461-470.
Mori IC, Murata Y, Yang Y, Munemasa S, Wang YF, Andreoli S, Tiriac H, Alonso JM, Harper JF, Ecker JR et al. (2006). CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and $Ca^{2+}-$permeable channels and stomatal closure. PLoS Biol 4: e327.
Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M, Uchimiya H, Hashimoto M, Iba K (2008). CO2 regulator
SLAC1 and its homologues are essential for anion homeostasis in plant cells. Nature 452: 483-486.
Qiu J, Henderson SW, Tester M, Roy SJ, Gilliham M (2016). SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl- accumulation and salt tolerance in Arabidopsis thaliana. J Exp Bot 67: 4495-4505.
Scherzer S, Maierhofer T, Al-Rasheid KA, Geiger D, Hedrich R (2012). Multiple calcium-dependent kinases modulate ABAactivated guard cell anion channels. Mol Plant 5: 1409-1412.
Sun SJ, Qi GN, Gao QF, Wang HQ, Yao FY, Hussain J, Wang YF (2016). Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLAC1, which is nitrateselective in rice. Planta 243: 489-500.
Sunarpi, Horie T, Motoda J, Kubo M, Yang H, Yoda K, Horie R, Chan WY, Leung HY, Hattori K et al. (2005). Enhanced salt tolerance mediated by AtHKT1 transporter-induced $Na^+$ unloading from xylem vessels to xylem parenchyma cells. Plant J 44: 928-938.
Vahisalu T, Kollist H, Wang YF, Nishimura N, Chan WY, Valerio G, Lamminmaki A, Brosche M, Moldau H, Desikan R et al. (2008). SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452: 487-491.
Wang YY, Hsu PK, Tsay YF (2012). Uptake, allocation and signaling of nitrate. Trends Plant Sci 17: 458-467.
Wang YY, Tsay YF (2011). Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport. Plant Cell 23: 1945- 1957.
Zhang A, Ren HM, Tan YQ, Qi GN, Yao FY, Wu GL, Yang LW, Hussain J, Sun SJ, Wang YF (2016). S-type anion channels SLAC1 and SLAH3 function as essential negative regulators of inward $K^+$ channels and stomatal opening in Arabidopsis. Plant Cell 28: 949-965.
Zhang B, Karnik R, Wang Y, Wallmeroth N, Blatt MR, Grefen C (2015). The Arabidopsis R-SNARE VAMP721 interacts with KAT1 and KC1 $K++$ channels to moderate $K^+$ current at the plasma membrane. Plant Cell 27: 1697-1717.
Zheng XJ, He K, Kleist T, Chen F, Luan S (2015). Anion channel SLAH3 functions in nitrate-dependent alleviation of ammonium toxicity in Arabidopsis. Plant Cell Environ 38: 474- 486.
Zou JJ, Wei FJ, Wang C, Wu JJ, Ratnasekera D, Liu WX, Wu WH (2010). Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and $Ca^{2+}-$mediated stomatal regulation in response to drought stress. Plant Physiol 154: 1232-1243.