Expression and sub-cellular localization of cyclic nucleotide-gated ion channels in Arabidopsis thaliana

Abstract

Cyclic nucleotide gated ion channels (CNGCs) are a 20-member gene family in the model plant, Arabidopsis thaliana, and playa role in the transport of both monovalent (ie. Na+, K+ and divalent (ie. Ca2+, Mg2+) cations across cellular membranes. CNGCs play vital roles in numerous processes required by living organisms, such as sight and smell in animals, and cell division, growth, and development in plants. Structurally, CNGCs contain six transmembrane domains, a pore region, and two regulatory domains, a calmodulin binding domain, and a cyclic nucletotide-binding domain (CNBD). Previous work on Arabidopsis CNGCs has shown that mutations in several of the genes can cause aberrant regulation of cell death, loss of pathogen defense mechanisms, and altered sensitivity to cation concentration in growth media. Very little research has been done to determine the location and function of these channels at the cellular level. Two previously unstudied CNGCs, CNGC5 and CNGC11, were chosen for sub-cellular localization using immunolocalization and CNGC-GFP fusion protein expression. Both CNGCS and 11 were found to immunolocalize to the plasma membrane of isolated plant cells. Focusing on CNGCS, T-DNA insertional mutant plants were also identified and found to have a knockdown in CNGCS expression resulting in a slight growth deficiency under normal conditions. Sub-cellular location, along with T-DNA mutant phenotypes, will provide critical insight into determining the role of these CNGCs at the cellular level.