1. Mechanisms of Salinity Tolerance in Barley Zhonghua Chen 1, Ian Newman 1, Igor Pottosin 2, Sergey Shabala 1 1 University of Tasmania and 2 Universidad de Colima. Ian.Newman@utas.edu.au ASPB Mexico June 2008 Poster # P10 041 www.mife.com Root Epidermal cell Processes 1a. Na + influx occurs via Non Selective Cation Channels (NSCC). This leads to rising Na i. 1b. The extra + charge causes membrane depolarisation: E m rises (less negative). 2a. Depolarisation stimulates the H + ATPase causing H + extrusion and lower H i. 2b. Depolarisation activates K + outward channels (KORC) leading to K + loss. 3. K + loss and H + extrusion both lead to E m recovery. The recovered E m then limits both KORC opening and H + extrusion. 4a. The lower H i (from 2a) and the E m recovery (from 3) both increase the Δμ H (= µ H o – µ H i ). 4b. This larger Δμ H provides driving potential for “SOS1” Na + extrusion. 5. Vacuolar sequestration plays a minor role in roots. Evidence Background Crop plant salinity tolerance is a polygenic trait, generally attained through maintaining a sufficient ratio of K + to Na + in the cell cytoplasm. Three barley varieties tolerant to salinity and three sensitive were selected from a range of 70 cultivars, whose tolerance level was determined from a range of agronomic measurements. We considered processes at the root, regardless of any foliar sequestration. The model here identifies the key ionic mechanisms, and key transporters, underlying salinity tolerance in barley. Conclusions (See Chen et al. Plant Phys.145, 1714) For salinity tolerance, to maintain the K + /Na + ratio, the membrane potential E m links many processes and to maintain its negativity is crucial. Maintaining E m diminishes K + Loss from the cell and contributes to the Δμ H which drives “SOS1”– like Na + /H + exchange to remove Na +. Higher intrinsic H + - extruding ATPase activity also assists to maintain Δμ H. Methods Details are given by Chen et al., Plant Physiol. 145, 1714-1725. Most experiments used 3-d seedlings grown in 0.5 mM KCl, 0.1 mM CaCl 2. Net H + and K + fluxes from the mature region of intact roots were measured by the MIFE system. Membrane potentials of root epidermal cells were measured by standard microelectrode impalement. Relevant standard techniques were used for 22 Na tracer influx (into the entire root) and for ATP content and activity of root tissue. Segments of mature root were protoplasted for whole cell patch clamping, selecting those of 20 μm diameter which indicates epidermal origin. MIFE Ion Flux Measurement The movement of an ion in solution can be described in terms of its electrochemical potential  (chemical and electrical driving forces), and other parameters of the ion and solution. It can be shown (see Newman, 2001, Plant, Cell & Environment 24(1), 1-14) that the net flux J of an ion may be found from a measurement of the change in voltage of an ion selective microelectrode that is moved through a small known distance dx in the solution. The MIFE system, used in this study for H + and K +, allows non- invasive measurement of net ion fluxes with resolution of 10 seconds in time and 20  m in position. A leaflet describing the commercial MIFE system is available here, with other information at www.mife.com. Membrane potential E m -+ E m links all the processes that contribute to Salinity tolerance NSCC Na o Na i “SOS1” H o µ H o Δ µ H i H i ATPase KORC Cytoplasm Vacuole 2b 3 2a 4a 3 4b 1a 1b 5 µ K o Δ µ K i KiKi K + is lost faster by sensitive than by tolerant cultivars in 0.1 mM CaCl 2. The K + losses, and the sensitive/tolerant difference, are much less in 1.0 mM CaCl 2. KORC currents show the same voltage dependence for both sensitive and tolerant cultivars. Their different K + losses are adequately explained solely by their different E m depolarisations, which cause different conductances and different electrochemical driving forces: Δμ K. Time, min after 1 h in 80 mM NaCl + 0.1 mM CaCl 2 Net K + flux, nmol m -2 s -1 (inwards positive) Mean K + flux values in 0.1 mM Ca 2+ 1.0 mM T-100T-60 S-110 S-400 This ATPase activity for the 6 cultivars is correlated with the NaCl-induced E m depolarisation for them (r 2 = ~ 0.8). PM H + ATPase specific activity, µM ADP min -1 mg -1 ATPase is more active in tolerant than in sensitive cultivars. This results in sensitive having greater H + extrusion and a larger Δμ H to drive the “SOS1” Na + /H + exchanger than sensitive. 22 Na + influx, µ mol g -1 root FW After 24 h in 80 mM NaCl Time, min after 22 Na addition 80 mM NaCl at t=0 22 Na influx is the same for both tolerant and sensitive cultivars. Net uptake during 24 h in 80 mM NaCl (with 0.5 mM KCl + 0.1 CaCl 2 ) is less for tolerant than sensitive. µmol g -1 FW Numar(T): 130 ZUG293(T): 125 Gairdner(S): 170 ZUG403(S): 190 Hence tolerant have more effective Na + extrusion to the soil; sensitive have more foliar Na + accumulation. Time from impalement, min Membrane potential E m, mV (T) (S) 80 mM NaCl Depolarisation caused by Na + entry is consistently larger in sensitive cultivars than in tolerant ones. Colima Logo needed here