The major objective in @entity57472 research is the identification of the biological process, in which an individual enzyme is integrated. @entity57472 -mediated signalling is thereby often addressed by single knock-out mutation- or co-suppression-based reverse genetics approaches. If a @entity57472 of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining @entity855 -analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant @entity460 -dependent @entity57472 (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with @entity855 as substrate, but were able to use @entity855 analogues as @entity1391 donors or as kinase inhibitors. For functional characterization in planta, we have substituted an @entity958 mutant line of @entity44640 with the respective as-variant under the native @entity44640 promoter. Seedlings of @entity958 wild type and @entity44640 as-lines were treated with the @entity855 analogue inhibitor @entity68678 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and @entity44640 as-plants before and after inhibitor treatment revealed differential @entity44640 -dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.

A chemical-genetic approach to elucidate XXXX function in planta.

@entity44640 :: ('830366(Tax:3702)', 'Gene') :: ['CPK1', 'AtCPK1'] @entity958 :: ('3702', 'Species') :: ['Arabidopsis thaliana', 'Arabidopsis'] @entity1391 :: ('CHEBI:26020', 'Chemical') :: ['phosphate'] @entity57472 :: ('838980(Tax:3702)', 'Gene') :: ['Protein kinase', 'protein kinase'] @entity460 :: ('MESH:D002118', 'Chemical') :: ['calcium'] @entity855 :: ('MESH:D000255', 'Chemical') :: ['ATP'] @entity68678 :: ('CHEBI:52310', 'Chemical') :: ['1-NA-PP1']
@entity57472 :: (838980(Tax:3702),Gene) :: ['protein kinase']
