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

Linear response subordination to intermittent energy release in off-equilibrium aging dynamics

The interpretation of experimental and numerical data describing off-equilibrium aging dynamics crucially depends on the connection between spontaneous and induced fluctuations. The hypothesis that linear response fluctuations are statistically subordinated to irreversible outbursts of energy, so-called quakes, leads to predictions for averages and fluctuations spectra of physical observables in reasonable agreement with experimental results [see e.g. Sibani et al., Phys. Rev. B74:224407, 2006]. Using simulational data from a simple but representative Ising model with plaquette interactions, direct statistical evidence supporting the hypothesis is presented and discussed in this work. A strict temporal correlation between quakes and intermittent magnetization fluctuations is demonstrated. The external magnetic field is shown to bias the pre-existent intermittent tails of the magnetic fluctuation distribution, with little or no effect on the Gaussian part of the latter. Its impact on energy fluctuations is shown to be negligible. Linear response is thus controlled by the quakes and inherits their temporal statistics. These findings provide a theoretical basis for analyzing intermittent linear response data from aging system in the same way as thermal energy fluctuations, which are far more difficult to measure.Comment: 9 pages, 10 figures. Text improve

The relative influences of disorder and of frustration on the glassy dynamics in magnetic systems

The magnetisation relaxations of three different types of geometrically frustrated magnetic systems have been studied with the same experimental procedures as previously used in spin glasses. The materials investigated are Y$_2$Mo$_2$O$_7$ (pyrochlore system), SrCr$_{8.6}$Ga$_{3.4}$O$_{19}$ (piled pairs of Kagom\'e layers) and (H$_3$O)Fe$_3$(SO$_4$)$_2$(OH)$_6$ (jarosite compound). Despite a very small amount of disorder, all the samples exhibit many characteristic features of spin glass dynamics below a freezing temperature $T_g$, much smaller than their Curie-Weiss temperature $\theta$. The ageing properties of their thermoremanent magnetization can be well accounted for by the same scaling law as in spin glasses, and the values of the scaling exponents are very close. The effects of temperature variations during ageing have been specifically investigated. In the pyrochlore and the bi-Kagom\'e compounds, a decrease of temperature after some waiting period at a certain temperature $T_p$ re-initializes ageing and the evolution at the new temperature is the same as if the system were just quenched from above $T_g$. However, as the temperature is raised back to $T_p$, the sample recovers the state it had previously reached at that temperature. These features are known in spin glasses as rejuvenation and memory effects. They are clear signatures of the spin glass dynamics. In the Kagom\'e compound, there is also some rejuvenation and memory, but much larger temperature changes are needed to observe the effects. In that sense, the behaviour of this compound is quantitatively different from that of spin glasses.Comment: latex VersionCorrigee4.tex, 4 files, 3 figures, 5 pages (Proceedings of the International Conference on Highly Frustrated Magnetism (HFM2003), August 26-30, 2003, Institut Laue Langevin (ILL), Grenoble, France

Extraction of the Spin Glass Correlation Length

The peak of the spin glass relaxation rate, S(t)=d{-M_{TRM}(t,t_w)}/H/{d ln t}, is directly related to the typical value of the free energy barrier which can be explored over experimental time scales. A change in magnetic field H generates an energy E_z={N_s}{X_fc}{H^2} by which the barrier heights are reduced, where X_{fc} is the field cooled susceptibility per spin, and N_s is the number of correlated spins. The shift of the peak of S(t) gives E_z, generating the correlation length, Ksi(t,T), for Cu:Mn 6at.% and CdCr_{1.7}In_{0.3}S_4. Fits to power law dynamics, Ksi(t,T)\propto {t}^{\alpha(T)} and activated dynamics Ksi(t,T) \propto {ln t}^{1/psi} compare well with simulation fits, but possess too small a prefactor for activated dynamics.Comment: 4 pages, 4 figures. Department of Physics, University of California, Riverside, California, and Service de Physique de l'Etat Condense, CEA Saclay, Gif sur Yvette, France. To appear in Phys. Rev. Lett. January 4, 199

Aging in an infinite-range Hamiltonian system of coupled rotators

We analyze numerically the out-of-equilibrium relaxation dynamics of a long-range Hamiltonian system of $N$ fully coupled rotators. For a particular family of initial conditions, this system is known to enter a particular regime in which the dynamic behavior does not agree with thermodynamic predictions. Moreover, there is evidence that in the thermodynamic limit, when $N\to \infty$ is taken prior to $t\to \infty$, the system will never attain true equilibrium. By analyzing the scaling properties of the two-time autocorrelation function we find that, in that regime, a very complex dynamics unfolds in which {\em aging} phenomena appear. The scaling law strongly suggests that the system behaves in a complex way, relaxing towards equilibrium through intricate trajectories. The present results are obtained for conservative dynamics, where there is no thermal bath in contact with the system. This is the first time that aging is observed in such Hamiltonian systems.Comment: Figs. 2-4 modified, minor changes in text. To appear in Phys. Rev.

Production and application of stable isotope-labeled internal standards for RNA modification analysis

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue

Aging, rejuvenation and memory effects in Ising and Heisenberg spin glasses

We have compared aging phenomena in the Fe_{0.5}Mn_{0.5}TiO_3 Ising spin glass and in the CdCr_{1.7}In_{0.3}S_4 Heisenberg-like spin glass by means of low-frequency ac susceptibility measurements. At constant temperature, aging obeys the same `$\omega t$ scaling' in both samples as in other systems. Investigating the effect of temperature variations, we find that the Ising sample exhibits rejuvenation and memory effects which are qualitatively similar to those found in other spin glasses, indicating that the existence of these phenomena does not depend on the dimensionality of the spins. However, systematic temperature cycling experiments on both samples show important quantitative differences. In the Ising sample, the contribution of aging at low temperature to aging at a slightly higher temperature is much larger than expected from thermal slowing down. This is at variance with the behaviour observed until now in other spin glasses, which show the opposite trend of a free-energy barrier growth as the temperature is decreased. We discuss these results in terms of a strongly renormalized microscopic attempt time for thermal activation, and estimate the corresponding values of the barrier exponent $\psi$ introduced in the scaling theories.Comment: 8 pages, including 6 figure

Aging, rejuvenation and memory phenomena in spin glasses

In this paper, we review several important features of the out-of-equilibrium dynamics of spin glasses. Starting with the simplest experiments, we discuss the scaling laws used to describe the isothermal aging observed in spin glasses after a quench down to the low temperature phase. We report in particular new results on the sub-aging behaviour of spin glasses. We then discuss the rejuvenation and memory effects observed when a spin glass is submitted to temperature variations during aging, from the point of view of both energy landscape pictures and of real space pictures. We highlight the fact that both approaches point out the necessity of hierarchical processes involved in aging. Finally, we report an investigation of the effect of small temperature variations on aging in spin glass samples with various anisotropies which indicates that this hierarchy depends on the spin anisotropy.Comment: submitted for the Proceedings of Stat Phys 22, Bangalore (India

Spin Glasses: Model systems for non-equilibrium dynamics

Spin glasses are frustrated magnetic systems due to a random distribution of ferro- and antiferromagnetic interactions. An experimental three dimensional (3d) spin glass exhibits a second order phase transition to a low temperature spin glass phase regardless of the spin dimensionality. In addition, the low temperature phase of Ising and Heisenberg spin glasses exhibits similar non-equilibrium dynamics and an infinitely slow approach towards a thermodynamic equilibrium state. There are however significant differences in the detailed character of the dynamics as to memory and rejuvenation phenomena and the influence of critical dynamics on the behaviour. In this article, some aspects of the non-equilibrium dynamics of an Ising and a Heisenberg spin glass are briefly reviewed and some comparisons are made to other glassy systems that exhibit magnetic non-equilibrium dynamics.Comment: To appear in J. Phys.: Condens. Matter, Proceedings from HFM2003, Grenobl

The dietary impact of the Norman Conquest: A multiproxy archaeological investigation of Oxford, UK

Archaeology has yet to capitalise on the opportunities offered by bioarchaeological approaches to examine the impact of the 11th-century AD Norman Conquest of England. This study utilises an integrated multiproxy analytical approach to identify and explain changes and continuities in diet and foodways between the 10th and 13th centuries in the city of Oxford, UK. The integration of organic residue analysis of ceramics, carbon (δ13C) and nitrogen (δ15N) isotope analysis of human and animal bones, incremental analysis of δ13C and δ15N from human tooth dentine and palaeopathological analysis of human skeletal remains has revealed a broad pattern of increasing intensification and marketisation across various areas of economic practice, with a much lesser and more short-term impact of the Conquest on everyday lifestyles than is suggested by documentary sources. Nonetheless, isotope data indicate short-term periods of instability, particularly food insecurity, did impact individuals. Evidence of preferences for certain foodstuffs and cooking techniques documented among the elite classes were also observed among lower-status townspeople, suggesting that Anglo-Norman fashions could be adopted across the social spectrum. This study demonstrates the potential for future archaeological research to generate more nuanced understanding of the cultural impact of the Norman Conquest of England, while showcasing a method which can be used to elucidate the undocumented, everyday implications of other large-scale political events on non-elites