SummaryThe vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K + channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patchclamp studies identified TPK1 as a voltage-independent and Ca 2+ -activated K + channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca 2+ -and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

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SUMMARYControl of ion loading into the xylem has been repeatedly named as a crucial factor determining plant salt tolerance. In this study we further investigate this issue by applying a range of biophysical [the microelectrode ion flux measurement (MIFE) technique for non-invasive ion flux measurements, the patch clamp technique, membrane potential measurements] and physiological (xylem sap and tissue nutrient analysis, photosynthetic characteristics, stomatal conductance) techniques to barley varieties contrasting in their salt tolerance. We report that restricting Na + loading into the xylem is not essential for conferring salinity tolerance in barley, with tolerant varieties showing xylem Na + concentrations at least as high as those of sensitive ones. At the same time, tolerant genotypes are capable of maintaining higher xylem K + /Na + ratios and efficiently sequester the accumulated Na + in leaves. The former is achieved by more efficient loading of K + into the xylem. We argue that the observed increases in xylem K + and Na + concentrations in tolerant genotypes are required for efficient osmotic adjustment, needed to support leaf expansion growth. We also provide evidence that K + -permeable voltage-sensitive channels are involved in xylem loading and operate in a feedback manner to maintain a constant K + /Na + ratio in the xylem sap.

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K 1 channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca 21 -and voltage-dependent outwardly rectifying plasma membrane K 1 channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca 21 and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca 21 , requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K 1 over Na 1 , and its activity turned out to be independent of the membrane voltage over the range of 680 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca 21 -activated, K 1 -selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.

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