Overexpression of a directed mutant of 14-3-3ω in Arabidopsis leaves affects phosphorylation and protein content of nitrate reductaseThis paper is one of a selection published in a Special Issue comprising papers presented at the 50th Annual Meeting of the Canadian Society of Plant Physiologists (CSPP) held at the University of Ottawa, Ontario, in June 2008.

Abstract

The 14-3-3 family of proteins are highly conserved signaling proteins in eukaryotes that bind to their client proteins, usually through specific phosphorylated target sequences. While the 14-3-3 proteins are thought to interact with a wide array of cellular proteins, there have been few studies addressing the in-vivo role of 14-3-3. As one approach to study this in-vivo role, we generated transgenic Arabidopsis plants constitutively overexpressing a directed mutant of 14-3-3 isoform ω that inhibits phosphorylated nitrate reductase (pNR) in a largely divalent-cation-independent manner in vitro. The transgenic plants had increased relative phosphorylation of NR at the regulatory Ser-534 site and decreased NR activity measured in the presence of 5 mmol·L–1 MgCl2 relative to nontransgenic plants. In addition, total NR protein was increased and the protein half-life was increased about two-fold. Two-dimensional difference gel electrophoresis analysis of proteins extracted from leaves of plants expressing the mutant 14-3-3 identified numerous cellular proteins that were altered in abundance. In particular, several β-glucosidase and glutathione S-transferase isoforms were decreased in abundance relative to wild type plants suggesting a possible alteration in stress or defense responses.