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

Abstract

SummaryIn animals and yeast, voltage-dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plasma membrane excitability, and control of pH and membrane potential in intracellular compartments. To assess the function of CLCs in plants, we searched for CLC insertion mutants in a library of Arabidopsis lines transformed by Agrobacterium tumefaciens transferred DNA (T-DNA). Using a polymerase chain reaction-based screening procedure, an Arabidopsis line that carries a T-DNA insertion within the C-terminus of the AtCLC-a coding sequence was identi®ed. Progeny from this plant line, clca-1, showed dramatically altered transcription of the AtCLC-a gene. Plants homozygous for the clca-1 mutation exhibited normal development and a morphology indistinguishable from the wild-type. However, their capacity to accumulate nitrate under conditions of nitrate excess was reduced in roots and shoots, by approximately 50%, while chloride, sulphate and phosphate levels were similar to the wild-type. In addition, the herbicide chlorate, an analogue of nitrate, induced a faster and more pronounced chlorosis in mutant plants. Hypersensitivity to chlorate as well as decreased nitrate levels co-segregated with the T-DNA insertion. They were found at various time points of the clca-1 life cycle, supporting the idea that AtCLC-a has a general role in the control of the nitrate 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®city 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 that AtCLC-a plays a role in controlling the intracellular nitrate status.

Dissection of the Control of Anion Homeostasis by Associative Transcriptomics in Brassica napus

“…Fourteen mutants were obtained for genes potentially controlling nitrate levels, and nine and 15 mutants were recovered from the phosphate and sulfate sets, respectively (Supplemental Table S4). For the CLC-A gene, a previously described clca-1 mutant in the Wassilewskija (Ws) background (Geelen et al, 2000) was provided by Sébastien Thomine at the Centre National de la Recherche Scientifique. These mutants were grown in controlled-environment conditions, and anion levels in leaves were determined (Fig.…”

Roles of ion channels and transporters in guard cell signal transduction

“…One candidate gene family is the CLC family, because CLC homologs in prokaryotes mediate Cl À flux. However to date, none of the seven CLC family members in Arabidopsis has been localized to the PM or shown to function as an anion channel in planta [84,85]. Another candidate gene family is the ATP-binding cassette (ABC) superfamily [86,87].…”

CLC-mediated anion transport in plant cells

“…OsClC1 and OsClC2 proteins, both closely related with AtClCc, were localized at the vacuolar membrane using both immunochemical detection methods and transient expression of GFP fusions (Nakamura et al 2006). Whereas loss-of-function mutants for OsClC1 and 2 genes exhibit reduced growth at all life stages (Nakamura et al 2006) (Geelen et al 2000). In agreement with the chloroplast localization of AtClCe, the clce mutants showed altered photosynthetic activity (Marmagne et al 2007), but their nitrate content is also reduced as observed in clca mutants ( D. Monachello & G. Ephritikhine 2007, personal communication).…”