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

Sampling Distributions of Random Electromagnetic Fields in Mesoscopic or Dynamical Systems

We derive the sampling probability density function (pdf) of an ideal localized random electromagnetic field, its amplitude and intensity in an electromagnetic environment that is quasi-statically time-varying statistically homogeneous or static statistically inhomogeneous. The results allow for the estimation of field statistics and confidence intervals when a single spatial or temporal stochastic process produces randomization of the field. Results for both coherent and incoherent detection techniques are derived, for Cartesian, planar and full-vectorial fields. We show that the functional form of the sampling pdf depends on whether the random variable is dimensioned (e.g., the sampled electric field proper) or is expressed in dimensionless standardized or normalized form (e.g., the sampled electric field divided by its sampled standard deviation). For dimensioned quantities, the electric field, its amplitude and intensity exhibit different types of Bessel $K$ sampling pdfs, which differ significantly from the asymptotic Gauss normal and $\chi^{(2)}_{2p}$ ensemble pdfs when $\nu$ is relatively small. By contrast, for the corresponding standardized quantities, Student $t$, Fisher-Snedecor $F$ and root-$F$ sampling pdfs are obtained that exhibit heavier tails than comparable Bessel $K$ pdfs. Statistical uncertainties obtained from classical small-sample theory for dimensionless quantities are shown to be overestimated compared to dimensioned quantities. Differences in the sampling pdfs arising from de-normalization versus de-standardization are obtained.Comment: 12 pages, 15 figures, accepted for publication in Phys. Rev. E, minor typos correcte

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

TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth

Plant photoperiodic growth is coordinated by interactions between circadian clock and light signaling networks. How post‐translational modifications of clock proteins affect these interactions to mediate rhythmic growth remains unclear. Here, we identify five phosphorylation sites in the Arabidopsis core clock protein TIMING OF CAB EXPRESSION 1 (TOC1) which when mutated to alanine eliminate detectable phosphorylation. The TOC1 phospho‐mutant fails to fully rescue the clock, growth, and flowering phenotypes of the toc1 mutant. Further, the TOC1 phospho‐mutant shows advanced phase, a faster degradation rate, reduced interactions with PHYTOCHROME‐INTERACTING FACTOR 3 (PIF3) and HISTONE DEACETYLASE 15 (HDA15), and poor binding at pre‐dawn hypocotyl growth‐related genes (PHGs), leading to a net de‐repression of hypocotyl growth. NUCLEAR FACTOR Y subunits B and C (NF‐YB/C) stabilize TOC1 at target promoters, and this novel trimeric complex (NF‐TOC1) acts as a transcriptional co‐repressor with HDA15 to inhibit PIF‐mediated hypocotyl elongation. Collectively, we identify a molecular mechanism suggesting how phosphorylation of TOC1 alters its phase, stability, and physical interactions with co‐regulators to precisely phase PHG expression to control photoperiodic hypocotyl growth

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

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