Arabidopsis DORN1 extracellular ATP receptor; activation of plasma membrane K+‐and Ca2+‐permeable conductances

The first reports in the 1970s of extracellular ATP (eATP) effects on algal cytoplasmic streaming and Venus fly trap closure received little attention (Jaffe, 1973; Williamson, 1975). By the time interest in plant eATP revived, work on animals had revealed the existence of plasma membrane (PM) receptors for eATP that function in such processes as pain perception and vasodilation (Burnstock, 2016). Plant research is now catching up. eATP effects on roots (gravitropism, growth and development), hypocotyl (elongation), pollen (germination and tube growth), stomatal aperture, and cell viability are now documented (reviewed by Clark et al., 2014; Cho et al., 2017). Furthermore, eATP is implicated in immunity, abiotic stress responses and nodulation (reviewed by Clark et al., 2014; Cho et al., 2017). eATP increases in response to wounding, mechanical stimulation, abiotic stress, abscisic acid, glutamate and chitin (Dark et al., 2011; Cho et al., 2017). eATP can increase plant free Ca2+ (cytosolic, nuclear, mitochondrial), phosphatidic acid, nitric oxide, and reactive oxygen species (ROS) as potential second messengers in signalling (Demidchik et al., 2003, 2009; Loro et al., 2012; Cho et al., 2017). Until recently, perception of eATP was very much a ‘black box’ mechanism as no equivalents of animal receptors were apparent in higher plant genomes. A breakthrough came with the discovery of Arabidopsis thaliana DORN1 (Does not Respond to Nucleotides1) as a PM eATP receptor (Choi et al., 2014). Analysis of dorn1 mutants has shown that this receptor governs eATP-induced [Ca2+]cyt elevation in young seedlings and also a specific transcriptional response enriched in wound-responsive genes (Choi et al., 2014). Delineation of the first higher plant eATP receptor will now spur on attempts to identify the immediate targets of its serine/threonine kinase activity and downstream components of the signalling pathway it commands. eATP affects plasma membrane Ca2+, K+ and Na+ fluxes (Demidchik et al., 2011; Zhao et al., 2016) but none have yet been shown to rely on DORN1. eATP-activated Ca2+ fluxes are mediated by channel proteins in Arabidopsis root epidermal and guard cell PM but their genetic identities are unknown (Demidchik et al., 2009; Wang et al., 2014; Zhu et al., 2017). eATP- and eADP-induced K+ fluxes have been detected at the Arabidopsis root epidermis using extracellular K+-selective microelectrodes (Demidchik et al., 2011). eADP was found (by using patch clamp electrophysiology) to activate channel-mediated K+ efflux and Ca2+ influx conductances (Demidchik et al., 2011). Patch clamp electrophysiology has been used here to test whether DORN1 is required for activation of Arabidopsis root epidermal PM channel conductances by exogenous ATP. Roots were chosen as they have greater eATP-induced [Ca2+]cyt elevations than leaves (Tanaka et al., 2010) and robust DORN1 expression (Cho et al., 2017). DORN1 expression is high at the root apex and is maintained there as the root ages; expression declines with age in the distal epidermis (Cho et al., 2017). Therefore, epidermal protoplasts were isolated from excised root apices (< 3 mm) of 9–16-d-old Col-0 (parental wild type) and two dorn1 mutants. The dorn1-1 mutant has a point mutation in its cytosolic serine-threonine kinase domain while the dorn1-3 mutant has a T-DNA insertion in the extracellular legume-type lectin domain; both lesions result in failure in eATP-induced [Ca2+]cyt elevation (Choi et al., 2014). Both mutants were confirmed as homozygous. Mean (± SE) protoplast diameter was 16 ± 0.5 μm (n = 40), smaller than those in a previous study from the mature epidermis (20 μm) isolated using the same method (Demidchik et al., 2011). The ‘whole cell’ mode of patch clamping was applied, permitting measurement of net current as ions are conducted through PM channels at different trans-membrane voltages, and reported as the overall current/voltage (I/V) relationship. Under control conditions, one type of overall nonlinear conductance was most commonly observed in Col-0 (Fig. 1a, left panel and Supporting Information Fig. S1a) and both dorn1 mutants (Fig. S1b,c). The time constant of half-activation of Col-0 outward currents at 23 mV was 88.4 ± SE 8.5 ms (n = 12). This is reminiscent of the depolarization-activated nonselective cation channels (NSCCs) in different plant species (Wegner & de Boer, 1997; Zhang et al., 2002; Shabala et al., 2006). In all genotypes, both inward currents (evoked by hyperpolarized voltages) and outward currents (evoked by depolarized voltages) were greatly inhibited by the cation channel blocker, Gd3+ (Fig. S1a–c). The Gd3+-insensitive conductance was not investigated further. Tail-current analysis of the Col-0 outward conductance revealed a reversal potential (Erev) of −90.2 ± 8.9 mV (n = 8). Thus, permeability ratios PK : PCl and PCa : PK of 13.7 ± 5.4 and 0.04 ± 0.01, respectively, were estimated using the Goldman equation. A high selectivity for K+ was also found in the NSCC studies and also for PM K+ channels in root cells of Arabidopsis (Maathuis & Sanders, 1995). Voltage ramping minimized activation of the Col-0 outward conductance to delineate the inward conductance (Fig. S2) and yielded an Erev of −8.2 ± 4.7 mV (n = 6) and PCa : PK of 1.9 ± 0.7. This indicates Ca2+ influx contributing to the inward conductance. Single channel studies are now required to delineate channel contribution to the Gd3+-sensitive K+- and Ca2+-permeable conductances. Before addition of ATP, at least two recordings were performed to confirm the stability of currents (Fig. 1b, −3 and 0 min). ATP was added as its disodium salt and in controls with an equivalent Na+ concentration applied as sodium chloride (NaCl), there was no current activation (Fig. S3), confirming that any responses would be caused by ATP. Addition of 300 μM ATP increased the outward currents and initiated small ‘spiky’ inward currents at hyperpolarized voltage after 3 min (Fig. 1a,b). The kinetics of the inward conductance resembled the hyperpolarization-activated Ca2+ conductance (HACC) found in previous studies on epidermal PM (e.g. Demidchik et al., 2009). Current activation was transient (Fig. 1c), as was previously observed with eATP- and eADP-activated PM conductances in protoplasts from mature epidermis (Demidchik et al., 2009, 2011). Eight minutes after ATP application, maximum current values (at +43 and −217 mV) were significantly greater than those before treatment (P < 0.01, Student's t-test; Fig. 1c,d) and inward rectification of the hyperpolarization-activated currents became apparent. Analysis of the eATP-activated currents (control I/V subtracted from eATP I/V at 8 min; Fig. 1d, insert) revealed a positive shift of Erev to −64.5 ± 16.8 mV, indicating a greater Ca2+ permeability in eATP-induced inward currents (ECa at +161 mV). It is therefore likely that eATP transiently increased Ca2+ conductivity to deliver Ca2+ to the cytosol, although increased Cl˗ permeation is also possible. However, neither outward nor inward currents in Col-0 were stimulated by the same concentration of eADP (300 μM; Fig. S4). Under control conditions, there was no significant difference between the overall Col-0 conductance and that of dorn1-1 (Fig. 2a,b: tail current analysis of outward current, Erev −88.8 ± 9.3 mV; PK : PCl 9.4 ± 3.0; n = 5). Consistent with Col-0, the epidermal PM currents recorded from both dorn1 mutants were blocked by Gd3+ (Fig. S1b,c). In contrast to Col-0, dorn1-1 did not respond to 300 μM ATP (Fig. 2a–c), neither did dorn1-3 (Fig. 2d,e). As DORN1 binds ATP with a Kd of 45.7 nM (Choi et al., 2014), the lack of response to this high concentration of ATP supports the currents lying downstream of this receptor in Col-0. DORN1 governs eATP's transient activation of a Ca2+-permeable inward conductance at hyperpolarized voltages (Figs 1c,d, 2). With resting PM voltage in epidermal cells from Arabidopsis roots varying between −153 and −129 mV (Maathuis & Sanders, 1993), eATP-induced HACC-like currents could initiate [Ca2+]cyt elevation for wound signalling (Choi et al., 2014) and promote the eATP-induced depolarization of Arabidopsis PM (Lew & Dearnaley, 2000). DORN1 also governs eATP's transient activation of a K+-permeable outward conductance (probably NSCCs) at depolarized voltages (Figs 1c,d, 2). In whole Arabidopsis roots, the same concentration of eATP (300 μM) was shown by Demidchik et al. (2011) to induce K+ efflux at the root epidermis. The K+ efflux conductance found in the present study would be a strong candidate for the root eATP-induced K+ efflux pathway. This may have significance for eATP and DORN1 function in pathogen- or stress-induced K+ loss in cell death (Demidchik, 2014). DORN1 commands [Ca2+]cyt elevation in response to both eATP and eADP (Choi et al., 2014). The eADP concentration used here (300 μM) was found previously to activate PM K+ efflux and Ca2+ influx conductances in Col-0 protoplasts from mature root epidermis (Demidchik et al., 2011) but in the present study it had no effect on those from the root apex (Fig. S4). A possible explanation is a cell-specific secondary effect of ADP that negates DORN1's activation of channels. Nevertheless, the relationship between DORN1 and PM conductances found here for eATP may well prove valuable in the search for the molecular identities of the contributory channels. Root PM conductances activated by eATP independently of DORN1 may also yet be discovered. Work was funded by the Brookes and Broodbank Funds, University of Cambridge. Prof. Gary Stacey (University of Missouri, Columbia, MO, USA) donated the dorn1 mutants. Dr Adeeba Dark and Mr Greg Habrych provided technical support. The study was conceived by L.W. and J.M.D.; L.W. and K.A.W. generated and analysed the data; L.W. and J.M.D. wrote the paper. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Fig. S1 Gd3+ inhibits whole-cell currents from epidermal plasma membrane of Col-0 and dorn1 mutants. Fig. S2 Voltage ramping to determine Ca2+ permeability of the Col-0 inward conductance. Fig. S3 Exogenous NaCl has no effect on Col-0 currents. Fig. S4 Exogenous ADP does not induce currents in Col-0 apical root epidermal plasma membrane. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.