Vacuola chloride transport in the extreme halophyte Messembryanthemum crystallinum

Description

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 K_m 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.

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Tags

580

Ice plant : Halophytes

Additional Fields

Identifer	oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:299437
Date	January 1999
Creators	Wissing, Frank
Publisher	University of Oxford
Source Sets	Ethos UK
Detected Language	English
Type	Electronic Thesis or Dissertation
Source	http://ora.ox.ac.uk/objects/uuid:b59195f3-63fd-40c5-9055-e470d2ef2ada