CLC-b-Mediated NO3-/H+ Exchange Across the Tonoplast of Arabidopsis Vacuoles

Nitrate is frequently the major nitrogen source for plants and is generally assimilated during the day. In the absence of light, nitrate can transiently accumulate in the vacuolar lumen via tonoplast transporters. CLC-a, a member of the CLC family of anion transporters, is critically involved in this nitrate storage in the vacuole, while other CLC family members apparently have different roles in diverse cell organelles. Here, CLC-b, a close relative of CLC-a, was functionally expressed in oocytes and analyzed. CLC-b conducted strongly outwardly rectifying anionic currents that were largest in the presence of nitrate. Fluorescence ratio changes of oocytes loaded with a pH-dependent fluorescent dye suggested that NO3- transport is associated with H+ exchange. CLC-b was localized at the tonoplast, as was CLC-c, when tagged with the green fluorescent protein. CLC-b expression was strongest in young roots, hypocotyl and cotyledons. The physiological role of CLC-b was analyzed using two independent knock-out alleles. Both lines grew as the wild type in various conditions. The total chloride and nitrate content was identical in clcb lines and the wild type, potentially suggesting that mutants were able to compensate the loss of CLC-b.