Function of the anion transporter AtCLC‐d in the trans‐Golgi network

Summary Anion transporting proteins of the CLC type are involved in anion homeostasis in a variety of organisms. CLCs from Arabidopsis have been shown to participate in nitrate accumulation and storage. In this study, the physiological role of the functional chloride transporter AtCLC‐d from Arabidopsis was investigated. AtCLC‐d is weakly expressed in various tissues, including the root. When transiently expressed as a GFP fusion in protoplasts, it co‐localized with the VHA‐a1 subunit of the proton‐transporting V‐type ATPase in the trans‐Golgi network (TGN). Stable expression in plants showed that it co‐localized with the endocytic tracer dye FM4‐64 in a brefeldin A‐sensitive compartment. Immunogold electron microscopy confirmed the localization of AtCLC‐d to the TGN. Disruption of the AtCLC‐d gene by a T‐DNA insertion did not affect the nitrate and chloride contents. The overall morphology of these clcd‐1 plants was similar to that of the wild‐type, but root growth on synthetic medium was impaired. Moreover, the sensitivity of hypocotyl elongation to treatment with concanamycin A, a blocker of the V‐ATPase, was stronger in the clcd‐1 mutant. These phenotypes could be complemented by overexpression of AtCLC‐d in the mutant background. The results suggest that the luminal pH in the trans‐Golgi network is adjusted by AtCLC‐d‐mediated transport of a counter anion such as Cl− or NO3−.