Abstract
BackgroundThe cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots.ResultsAtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents.ConclusionAtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported antisense phenotypes of decreased gravitropic and cell enlargement responses, suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division and growth.
Highlights
The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells
The AtCNGC10-specific antiserum binds to the hydrophilic N-terminus just upstream from the first transmembrane domain, while the AtCNGC5 antiserum binds to an exposed outer loop between the fifth transmembrane domain and the pore (Figure 1A)
AtCNGC5 is expressed in roots and leaves, while AtCNGC10 is expressed preferentially in roots
Summary
The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. The precise subcellular locations and trafficking of these membrane proteins are poorly understood. To regulate intracellular cation homeostasis, plants have evolved several distinct classes of transporters to facilitate the movement of monovalent and divalent cations across cellular membranes. One such class of cation transporters is the cyclic nucleotide-gated ion channels (CNGCs) [3]. The phenotypic characterization of loss-of-function mutants has implicated CNGCs to be involved in a wide range of plant processes [14] including tolerance to heavy metals [7,19], sensitivity to various cation stresses [17,20], plant development and germination [17,20], programmed cell death, and disease resistance [11,21]
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