Asr1 mediates glucose-hormone crosstalk by affecting sugar trafficking in tobacco plants

Asr (for ABA, stress, ripening) genes are exclusively found in the genomes of higher plants and the encoded proteins have been found localized both to the nucleus and cytoplasm. However, before the mechanisms underlying the activity of ASR proteins can be determined, the role of these proteins in planta should be deciphered. Results from the present study suggest that ASR is positioned within the signaling cascade of interactions among glucose, abscisic acid and gibberellins. Nicotiana tabacum transgenic lines with reduced levels of ASR protein showed impaired glucose metabolism and altered abscisic acid and gibberellin levels. These changes were associated with dwarfism, reduced CO2 assimilation and accelerated leaf senescence as a consequence of a fine regulation exerted by ASR to the glucose metabolism. This regulation resulted in an impact on glucose signaling mediated by hexokinase1 and SnRk1 (for Snf1-related kinase) which would subsequently have been responsible for photosynthesis, leaf senescence and hormone level alterations. It thus can be postulated that ASR is not only involved in the control of hexose uptake in heterotrophic organs, as we have previously reported, but also in the control of carbon fixation by the leaves mediated by a similar mechanism.Fil: Dominguez, Pia Guadalupe. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Frankel, Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución; ArgentinaFil: Mazuch, Jeannine. Max Planck Institute for Molecular Plant Physiology; AlemaniaFil: Balbo, Ilse. Max Planck Institute for Molecular Plant Physiology; AlemaniaFil: Iusem, Norberto Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernie, Alisdair R.. Max Planck Institute for Molecular Plant Physiology; AlemaniaFil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Changes of Dietary Fat and Carbohydrate Content Alter Central and Peripheral Clock in Humans.

CONTEXT: The circadian clock coordinates numerous metabolic processes with light-dark and feeding regimens. However, in humans it is unknown whether dietary patterns influence circadian rhythms. OBJECTIVE: We examined the effects of switching from a high-carbohydrate, low-fat diet to a low-carbohydrate, high fat (LC/HFD) isocaloric diet on the central and peripheral circadian clocks in humans. DESIGN: Diurnal patterns of salivary cortisol and gene expression were analyzed in blood monocytes of 29 nonobese healthy subjects before and 1 and 6 weeks after the dietary switch. For this, we established a method of rhythm prediction by 3-time point data. RESULTS: The centrally driven cortisol rhythm showed a phase delay 1 and 6 weeks after the dietary switch to a LC/HFD as well as an amplitude increase. The dietary switch altered diurnal oscillations of core clock genes (PER1, PER2, PER3, and TEF) and inflammatory genes (CD14, CD180, NFKBIA, and IL1B). The LC/HFD also affected the expression of nonoscillating genes contributing to energy metabolism (SIRT1) and fat metabolism (ACOX3 and IDH3A). Expression of clock genes but not of salivary cortisol in monocytes tightly correlated with levels of blood lipids and with expression of metabolic and inflammatory genes. CONCLUSIONS: Our results suggest that the modulation of the dietary fat and carbohydrate content alters the function of the central and peripheral circadian clocks in humans

Circadian properties of the mathematical model reproduce the RAS-mediated effect on the clock.

<p>(A) Simplified network representation of the mathematical model. The model consists of seven core-clock components (grey boxes) and nine cell cycle components (orange boxes). Activating (green lines) and inhibiting (red lines) interactions known from literature and a potential transcriptional activation (reported in MotifMap) are represented. The dashed red line from MYC to CLOCK/BMAL represents the overall interference with BMAL-mediated transcription by competitive E-box binding. Module 1 consists of the INK4a/RB1/E2F1 pathway (green) and module 2 of the ARF/MDM2/p53 pathway (orange). (B) In silico peak phases of clock genes from the model are consistent with experimental data. The highest mRNA expression intervals (experimental published data) from the core-clock genes <i>Ror</i> (blue), <i>Rev-Erb</i> (red), <i>Bmal</i> (green), <i>Per</i> (purple), <i>Cry</i> (turquoise) and the cytoplasmic and nuclear PER/CRY protein complexes (orange) are depicted in darker colours within the circles. Yellow dots represent the peak of expression as simulated by the model. (C) Shown are in silico expression profiles for the five core-clock genes <i>Bmal</i>, <i>Per</i>, <i>Cry</i>, <i>Ror</i>, and <i>Rev-Erb</i> and for the PER/CRY protein complex. The period was set to 23.65 h and the phase of <i>Bmal</i> to CT21. (D) The model reproduces experimental clock phenotypes qualitatively. In silico expression data show that upon simulation of RAS overexpression, the Ink4a/Arf^+/+ system acquires a longer period and Ink4a/Arf^-/- system a shorter period compared to the corresponding simulated WT system. RAS overexpression was simulated by decreasing the parameter <i>ktt</i> to 0.4 (<i>ktt</i> = 1 WT, <i>ktt</i> < 1 increase of RAS). (E) RAS overexpression increases <i>Ink4a</i> expression levels in silico. WT, wild-type.</p

Predictive classification of cell cycle-fate phenotypes for Ink4a/Arf^+/+ and Ink4a/Arf^-/- MEFs based on the expression of senescence-associated genes can be validated by experimental results.

<p>(A) A set of 32 senescence-related genes derived from the literature clusters according to the knockout of <i>Ink4a/Arf</i>. (B) Two-dimensional representation of the SVM classification based on the expression of the senescence-related genes <i>Rb1</i> and <i>Suv39h1</i> for the three training conditions (represented as squares) and the remaining five predicted conditions (represented as dots). (C) FACS analysis to determine the percentage of cells in each cell cycle phase for the three training conditions (Ink4a/Arf^+/+, Ink4a/Arf^+/++RAS, and Ink4a/Arf^-/-) and two of the predicted conditions (Ink4a/Arf^-/-+RAS and Ink4a/Arf^-/- <i>shBmal1</i>+RAS) (<i>n</i> = 3; mean and SEM). The cell cycle phases were determined by fitting a univariate cell cycle model using the Watson pragmatic algorithm. Shown are representative examples for each condition. Numerical values are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002940#pbio.2002940.s013" target="_blank">S1 Data</a>. FACS, fluorescence-activated cell sorting; MEF, mouse embryonic fibroblasts; PI, propidium iodide.</p

Ink4a/Arf^+/+ and Ink4a/Arf^-/- MEFs show different circadian phenotypes upon RAS overexpression.

<p>(A) Schematic representation of the experimental setup to investigate the influence of RAS on the circadian phenotype via <i>Ink4a/Arf</i>. MEFs from <i>Ink4a/Arf</i> WT (Ink4a/Arf^+/+) and their <i>Ink4a/Arf</i> knock-out littermates (Ink4a/Arf^-/-) were lentivirally transduced with a <i>Bmal1</i>-promoter driven luciferase construct (<i>Bmal1</i>:<i>Luc</i>). To investigate the effect of the core-clock in this cell model system, <i>Bmal1</i> was downregulated by shRNA. The effect of the oncogene RAS was examined by overexpression of RAS. Bioluminescence was measured over five days. Shown are representative data for eight different conditions, as indicated. (B) RAS overexpression leads to an increase of the period in Ink4a/Arf^+/+ MEFs (26.9 h, dark blue) compared to the corresponding control (24.1 h, light blue). (C) RAS overexpression shortens the period of Ink4a/Arf^-/- MEFs (24.0 h, dark red) compared to the corresponding control (21.4 h, light red). RAS overexpression disrupts the circadian clock in <i>Bmal1</i> knock-down conditions in (D) Ink4a/Arf^+/+ MEFs and (E) Ink4a/Arf^-/- MEFs. (F) Summary of circadian period phenotype measurements for Ink4a/Arf^+/+ MEFs and Ink4a/Arf^-/- MEFs with and without RAS overexpression (<i>n</i> = 5; mean and SEM). (G) The cell number of Ink4a/Arf^+/+ MEFs and Ink4a/Arf^-/- MEFs with and without shBmal1 was monitored over five days (<i>n</i> = 3; mean and SEM). Ink4a/Arf^-/- MEFs proliferate faster than their corresponding Ink4a/Arf^+/+ littermates, independent from <i>Bmal1</i> knockdown. (H) The cell cycle arrest phenotypes were estimated by SA-ß-Gal staining in Ink4a/Arf^+/+ MEFs and their Ink4a/Arf^-/- littermates with or without RAS overexpression and <i>Bmal1</i> downregulation. (I) Percentage of SA-ß-Gal staining positive cells (<i>n</i> = 3; mean and SEM). RAS overexpression significantly increased the number of senescent cells in the Ink4a/Arf^+/+ MEFs compared to the WT. There is no effect of <i>Bmal1</i> downregulation in the Ink4a/Arf^-/- cell population. Statistical significance was determined by <i>t</i> test with <i>p</i>-values corrected for multiple testing with the Benjamini and Hochberg method. ***<i>p</i> < 0.001. Numerical values are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002940#pbio.2002940.s013" target="_blank">S1 Data</a>. MEF, mouse embryonic fibroblasts; ND, not defined; n.s., not significant; RAS, rat sarcoma viral oncogene; shRNA, short hairpin RNA; T, period; WT, wild-type.</p

The RAS-mediated effect on the circadian period is dependent on <i>Ink4a/Arf</i>.

<p>The RAS-mediated effect on the circadian period is dependent on <i>Ink4a/Arf</i>.</p

Schematic model of the Ink4a/Arf-RAS interplay and its connections to circadian clock and cell cycle phenotypes.

<p>(A) Overexpression of the oncogene RAS results in a lengthening of the circadian period in WT MEFs and leads to a senescent cell phenotype. (B) Knockout of the tumour suppressor element <i>Ink4a/Arf</i> that connects components of both cellular oscillators, leads to a change in the RAS-induced effect on the clock resulting in a shorter circadian period and proliferation of cells. MEF, mouse embryonic fibroblasts; RAS, rat sarcoma viral oncogene; WT, wild-type.</p

The genome-wide effect of RAS overexpression and Bmal1 downregulation on Ink4a/Arf ^+/+ and Ink4a/Arf ^-/- MEFs can be mirrored by genes from the mathematical model.

<p>(A) The first three principal components as determined by genome-wide expression analysis based on microarray data of Ink4a/Arf^+/+ and Ink4a/Arf^-/- MEFs with different perturbations highlighting the differences between the eight experimental conditions. The arrays cluster in four groups depending on the presence of <i>Ink4a/Arf</i> and RAS overexpression. (B) Based on the four groups determined in the PCA, the top 50 differentially expressed genes across the different experimental conditions were determined. (C) A network connecting 36 out of the topmost 50 differentially expressed genes (orange) to the previously published NCRG (blue) by at most one connecting element (grey) was generated using information from the IntAct database. The genes that are part of the mathematical model are highlighted. An enrichment analysis was performed to determine the most highly represented pathways for the whole network (D) and the set of connecting elements (F). (E) The clustering based on the expression levels of genes from the mathematical model mimics the genome-wide clustering. (G) The expression changes of selected core-clock and cell cycle-related genes upon perturbations by RAS and <i>shBmal1</i> in Ink4a/Arf^+/+ and Ink4a/Arf^-/- MEFs were validated by RT-qPCR and visualised as the log₂ fold change compared to the WT MEFs. Numerical values are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002940#pbio.2002940.s013" target="_blank">S1 Data</a>. MEF, mouse embryonic fibroblasts; NCRG, network of circadian regulated genes.</p