Identification and functional characterization of cation-chloride cotransporters in plants

Chloride (Cl-) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl- cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K+:Cl- cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K+, Na+ and Cl-, and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na+:K+:Cl- cotransporters, indicating that At CCC encodes for a bona fide Na+:K+:Cl- cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl- homeostasis under high salinity, as they accumulated higher and lower Cl- amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl- transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl- homeostasis.