572 research outputs found
Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1
BACKGROUND: Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many – if not all – molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase. RESULTS: Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates. CONCLUSION: The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-016-0900-7) contains supplementary material, which is available to authorized users
SWITCH 1/DYAD is a WINGS APART-LIKE antagonist that maintains sister chromatid cohesion in meiosis
During cell division, Soronin antagonises WAPL to prevent premature loss of sister chromatid cohesion. Here the authors show that, despite a lack of sequence similarity, the Arabidopsis SWI1 protein functions as a novel Soronin-like WAPL antagonist, suggesting convergent evolution with animals
Predicting selective drug targets in cancer through metabolic networks
The authors develop a genome-scale model of cancer metabolism and use it to predict genes that are essential for cancer cell growth. An array of target combinations are then identified that could potentially provide novel selective treatments for specific cancers
FLOWERING REPRESSOR AAA(+) ATPase 1 is a novel regulator of perennial flowering in Arabis alpina
Arabis alpina is a polycarpic perennial, in which PERPETUAL FLOWERING1 (PEP1) regulates flowering and perennial traits in a vernalization-dependent manner. Mutagenesis screens of the pep1 mutant established the role of other flowering time regulators in PEP1-parallel pathways. Here we characterized three allelic enhancers of pep1 (eop002, 085 and 091) which flower early. We mapped the causal mutations and complemented mutants with the identified gene. Using quantitative reverse transcriptase PCR and reporter lines, we determined the protein spatiotemporal expression patterns and localization within the cell. We also characterized its role in Arabidopsis thaliana using CRISPR and in A. alpina by introgressing mutant alleles into a wild-type background. These mutants carried lesions in an AAA(+) ATPase of unknown function, FLOWERING REPRESSOR AAA(+) ATPase 1 (AaFRAT1). AaFRAT1 was detected in the vasculature of young leaf primordia and the rib zone of flowering shoot apical meristems. At the subcellular level, AaFRAT1 was localized at the interphase between the endoplasmic reticulum and peroxisomes. Introgression lines carrying Aafrat1 alleles required less vernalization to flower and reduced number of vegetative axillary branches. By contrast, A. thaliana CRISPR lines showed weak flowering phenotypes. AaFRAT1 contributes to flowering time regulation and the perennial growth habit of A. alpina
Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum
Key message In plants, phosphorylated MAPKs display
constitutive nuclear localization; however, not all
studied plant species show co-localization of activated
MAPKs to mitotic microtubules.
Abstract The mitogen-activated protein kinase (MAPK)
signaling pathway is involved not only in the cellular
response to biotic and abiotic stress but also in the regulation
of cell cycle and plant development. The role of
MAPKs in the formation of a mitotic spindle has been
widely studied and the MAPK signaling pathway was
found to be indispensable for the unperturbed course of cell
division. Here we show cellular localization of activated
MAPKs (dually phosphorylated at their TXY motifs) in
both interphase and mitotic root meristem cells of Lupinus
luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization
of activated MAPKs has been found in all species. Colocalization
of these kinases to mitotic microtubules was
most evident in L. esculentum, while only about 50 % of
mitotic cells in the root meristems of P. sativum and V.
faba displayed activated MAPKs localized to microtubules
during mitosis. Unexpectedly, no evident immunofluorescence
signals at spindle microtubules and phragmoplast
were noted in L. luteus. Considering immunocytochemical
analyses and studies on the impact of FR180204 (an
inhibitor of animal ERK1/2) on mitotic cells, we hypothesize
that MAPKs may not play prominent role in the
regulation of microtubule dynamics in all plant species
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