Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth β-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K+-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the α-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general. © 2008 The Authors

ePlant and the 3D Data Display Initiative: Integrative Systems Biology on the World Wide Web

Visualization tools for biological data are often limited in their ability to interactively integrate data at multiple scales. These computational tools are also typically limited by two-dimensional displays and programmatic implementations that require separate configurations for each of the user's computing devices and recompilation for functional expansion. Towards overcoming these limitations we have developed “ePlant” (http://bar.utoronto.ca/eplant) – a suite of open-source world wide web-based tools for the visualization of large-scale data sets from the model organism Arabidopsis thaliana. These tools display data spanning multiple biological scales on interactive three-dimensional models. Currently, ePlant consists of the following modules: a sequence conservation explorer that includes homology relationships and single nucleotide polymorphism data, a protein structure model explorer, a molecular interaction network explorer, a gene product subcellular localization explorer, and a gene expression pattern explorer. The ePlant's protein structure explorer module represents experimentally determined and theoretical structures covering >70% of the Arabidopsis proteome. The ePlant framework is accessed entirely through a web browser, and is therefore platform-independent. It can be applied to any model organism. To facilitate the development of three-dimensional displays of biological data on the world wide web we have established the “3D Data Display Initiative” (http://3ddi.org)

Evolutionary Diversification of Plant Shikimate Kinase Gene Duplicates

Shikimate kinase (SK; EC 2.7.1.71) catalyzes the fifth reaction of the shikimate pathway, which directs carbon from the central metabolism pool to a broad range of secondary metabolites involved in plant development, growth, and stress responses. In this study, we demonstrate the role of plant SK gene duplicate evolution in the diversification of metabolic regulation and the acquisition of novel and physiologically essential function. Phylogenetic analysis of plant SK homologs resolves an orthologous cluster of plant SKs and two functionally distinct orthologous clusters. These previously undescribed genes, shikimate kinase-like 1 (SKL1) and -2 (SKL2), do not encode SK activity, are present in all major plant lineages, and apparently evolved under positive selection following SK gene duplication over 400 MYA. This is supported by functional assays using recombinant SK, SKL1, and SKL2 from Arabidopsis thaliana (At) and evolutionary analyses of the diversification of SK-catalytic and -substrate binding sites based on theoretical structure models. AtSKL1 mutants yield albino and novel variegated phenotypes, which indicate SKL1 is required for chloroplast biogenesis. Extant SKL2 sequences show a strong genetic signature of positive selection, which is enriched in a protein–protein interaction module not found in other SK homologs. We also report the first kinetic characterization of plant SKs and show that gene expression diversification among the AtSK inparalogs is correlated with developmental processes and stress responses. This study examines the functional diversification of ancient and recent plant SK gene duplicates and highlights the utility of SKs as scaffolds for functional innovation