Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content

In animals and yeast, voltage-dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plas ma membrane excitability, and control of pH and membrane potential in intra cellular compartments. To assess the function of CLCs in plants, we searche d for CLC insertion mutants in a library of Arabidopsis lines transformed b y Agrobacterium tumefaciens transferred DNA (T-DNA). Using a polymerase cha in reaction-based screening procedure, an Arabidopsis line that carries a T -DNA insertion within the C-terminus of the AtCLC-a coding sequence was ide ntified. Progeny from this plant line, clca-1, showed dramatically altered transcription of the AtCLC-a gene. Plants homozygous for the clca-1 mutatio n exhibited normal development and a morphology indistinguishable from the wild-type. However, their capacity to accumulate nitrate under conditions o f nitrate excess was reduced in roots and shoots, by approximately 50%, whi le chloride, sulphate and phosphate levels were similar to the wild-type. I n addition, the herbicide chlorate, an analogue of nitrate, induced a faste r and more pronounced chlorosis in mutant plants. Hypersensitivity to chlor ate as well as decreased nitrate levels co-segregated with the T-DNA insert ion. They were found at various time points of the clca-1 life cycle, suppo rting the idea that AtCLC-a has a general role in the control of the nitrat e status in Arabidopsis. Concordant with such a function, AtCLC-a mRNA was found in roots and shoots, and its levels rapidly increased in both tissues upon addition of nitrate but not ammonium to the culture medium. The speci ficity of AtCLC-a function with respect to nitrate is further supported by a similar free amino acid content in wild-type and clca-1 plants. Although the cellular localization of AtCLC-a remains unclear, our results suggest t hat AtCLC-a plays a role in controlling the intracellular nitrate status.