Regulation shoot anion loading via the AtSLAH1–AtSLAH3 complex

Qiu J and Gilliham M

ARC CoE PEB, University of Adelaide, South Australia, Australia.

AtSLAH1 and AtSLAH3, two Arabidopsis slow type anion channel-associated 1 (SLAC1) homologues form a protein complex to regulate root-to-shoot delivery of chloride (Cl-) and nitrate (NO3-). Both proteins are expressed in Arabidopsis root pericycle and their expression is down-regulated by NaCl and ABA (Qiu et al., 2016). AtSLAH3 carries predominantly NO3- when expressed by itself but has increased Cl- transport in a complex with SLAH1 (Cubero-Font et al., 2016). Here, we explore the regulation of this complex using heterologous expression in Xenopus laevis oocytes and two-electrode voltage clamp. We observed two ABA signaling associated protein kinases, SnRK2.2 and SnRK2.3, negatively regulate anion currents through AtSLAH1-AtSLAH3. Conversely, a positive guard cell anion channel regulator from the same kinase subgroup, SnRK2.6 (OST1), appears not to regulate the complex. To confirm whether AtSLAH1-AtSLAH3 can be targeted by SnRK2s, the only SnRK2s-specific substrate motif RxxT/S in AtSLAH1 was site-mutagenized (R176K) to disrupt the recognition by SnRk2s. When SLAH1R176 was co-injected with SLAH3 and SnRK2.2 the conductance inhibition was less pronounced compared to SLAH1-SLAH3-SnRK2.2. This result implies SnRk2.2 has an important role in mediating root-to-shoot Cl- transport by regulating AtSLAH1-AtSLAH3 activity. Previous studies have reported different protein kinases from the same subgroup might distinguish their roles with different tissue localization. As SnRK2.2 and SnRk2.3 are abundantly expressed within the plant including the root, while SnRK2.6 is highly expressed in the shoot but less detectable in the root; this is consistent with why SnRk2.6 does not affect the ionic conductance of AtSLAH1-AtSLAH3 complex. In addition, the AtSLAH1-AtSLAH3-SnRK2.2 was also found to be regulated by calcium-dependent protein kinase 21 (CPK21) by restoring the anion transport, suggesting a potential cross talk between kinases with/without Ca2+ dependency. As AtSLAH1-AtSLAH3 is involved in mediating root-to-shoot anion movement, multiple regulating mechanisms of the complex may help the plant to acclimate to stressful growth conditions by adjusted anion transfer to the shoot. Experiments to explore whether this regulation occurs in planta is underway.