Characterization of CIC transporter proteins : functional analysis of clc mutants in Arabidopsis thaliana

In higher plants anion channels, present in various tissues and cell types, play importants roles in signaling pathways leading to the adaptation of plant cells to abiotic and biotic stresses. One of the an protein families with anion channel function, is the Chloride Channel (ClC) family. Seven ClC genes are identified in the Arabidopsis genome. The main aim of this research was to physiologically characterize Arabidopis thaliana ClC proteins and to acquire knowledge on the position of ClC proteins in the complex network of membrane transport and solute fluxes. We used a reverse genetics approach with T-DNA knock-out mutants of three of the 7 ClC-genes that are present in the genome of Arabidopsis thaliana: AtClCa, AtClCb and AtClCd. In chapter 1 we predicted, concerning function and localization of plant ClC transporters, are made.In chapter 2. The seven mutants are preliminary characterized by testing the effect of the mutation on seed germination, vegetative growth and ion fluxes in root and leaf tissue as measured by the Micro-Electrode Ion Flux Estimation (MIFE). In chapter 3 the MIFE technique was expanded with a flow-through system that. the results of these experiments indicate that AtClCd functions as an anion/H+ antiporter and might be involved in the accumulation of both Cl- and NO3 in the vacuole. In chapter 4 a root growth phenotype is described for mutant plants in which either AtClCa or AtClCd. A model for the interaction between endomembrane anion/H+ antiporters, plasma membrane proton fluxes and cell expansion is discussed. In chapter 5 we study the effect of heavy metal treatments on the 7 mutant plants. Based on these results a role of AtClCa and AtClCd in cadmium detoxification is suggested and a mechanism is proposed. In chapter 6 all our result are placed in the context of the earlier published functions and localization of ClC transporter proteins. It is suggested that the main role of the ClC proteins is to function as electrical short circuits, which plays an important role in energizing the membranes. A role in NO3- and Cl- homeostasis seems of minor importance. Another important issue is the intracellular localization of these proteins.

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