Vacuola chloride transport in the extreme halophyte Messembryanthemum crystallinum
The halophyte Mesembryanthemum crystallinum L. accumulates high concentrations of NaCl (up to 1 M) in its leaf cells as a response to soil salinity. While there is evidence that vacuolar sodium transport is mediated by a tonoplast Na+/H+ antiporter (Barkla et al., 1995), little is known about the transport of Cl- transport into the vacuole. So far, it has been uncertain whether secondary active transport (e.g. H+/C1- antiporter) is involved or whether a passive mechanism (Cl- channel) is sufficient to mediate Cl- accumulation in the vacuole of M.crystallinum. This thesis describes the use of tonoplast vesicles from leaf mesophyll cells of M. crystallinum to study vacuolar Cl- transport. Cl- uptake into the vesicles was measured using the Cl--sensitive fluorescent dye lucigenin (N/N'-dimethyl-9,9'-bisacridinium dinitrate). This work was complemented by a patch-clamp study of ionic currents of leaf-mesophyll vacuoles from M.crystallinum. Cl- transport into tonoplast vesicles showed saturation-type kinetics with an apparent Km between 10 and 36 mM and a maximum initial change of the intravesicular Cl- concentration of 4.8 mM min-, corresponding to an estimated Cl- flux of 31 nmol m2 s-1. Vacuolar chloride transport was not affected by sulphate, malate, or nitrate, indicating a high specificity of this transport process for chloride over other anions. By imposing insidepositive membrane potentials using a K+/valinomycin clamp revealed a sigmoidal voltagedependent relationship with the steepest increase in vacuolar Cl- uptake around +30 mV. Only under severe salt treatment with 500 mM NaCl for 3 weeks did 9-week-old M. crystallinum plants show a significant increase (63%) of vacuolar C- uptake, along with an increased V-type H+-ATPase hydrolytic activity (up to 65%). The apparent Km of vacuolar Cl- uptake was also increased from 27 mM to 44 mM under these conditions. An inside-acid pH gradient, generated by a K+/nigericin clamp, reduced the initial rate of chloride transport into tonoplast vesicles of M.crystallinum. External Cl-, in contrast to external Na+ , did not dissipate an inside-acidic ΔpH generated by various techniques. This is strong evidence against a proton-driven antiport mechanism. The patch-clamp study of ionic currents of whole vacuoles and excised vacuolar membrane patches from M.crystallinum. leaf-mesophyll cells revealed a number of cation channels. At cytosolic free Ca 2+ concentrations of 1μM and above, ubiquitous slow-vacuolar type cation currents could be observed. In excised patches, eleven different single channel types, with conductances ranging from 2 up to 200 pS, could be described. However, no clear Cl-conductance could be identified. The lack of observed vacuolar Cl- channel activities is discussed in the light of possible lack of Cl--channel activation due to the loss of a cytosolic factor. The results obtained from the biochemical work on tonoplast vesicles support the hypothesis that a passive transport mechanism (i.e. channel) is sufficient to mediate vacuolar chloride transport in M.crystallinum. The observed upregulation of vacuolar Cl- transport under severe salt stress shows that it plays an important role in the salt adaptation of this halophytic plant.