The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity

International audienceThe hydraulic conductivity of plant roots (Lpr ) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lpr of knockout Arabidopsis plants for four Ca(2+) -dependent protein kinases. cpk7 plants showed a 30% increase in Lpr because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lpr of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins

Microscopic Dynamics in the Strain Hardening Regime of Glassy Polymers

International audienceWe study by Dielectric Spectroscopy the molecular dynamics of relaxation processes during plastic ow of glassy polymers up to the strain hardening regime, for 3 dierent protocols of deformation. The measured dielectric spectra cover 4 decades in frequencies and allow us to measure the evolution as a function of the applied strain of the dominant relaxation time τα and of the width wτ of the distribution of relaxation times.The rst protocol is performed at constant strain rate λ. We conrm that for increasing stress both τα and wτ rst decrease reaching a minimum in the stress softening regime before increasing in the strain hardening regime. In the second protocol we stop the deformation at some point λw in the strain hardening regime and we let the sample age for a waiting time tw, during which the applied stress remains high. Upon resuming the deformation at constant λ, strain-stress displays a yield stress and a stress softening regime comparable in magnitude to that of the reference protocol before rejoining the reference curve. In contrast the dielectric spectrum measured during the second protocol recovers that measured during the reference curve much later than strain-stress. In the third protocol the stress is canceled during tw. In this case after recovering the constant λ the dielectric spectrum and the stress-strain curve rejoin almost immediately the reference curve. We interpret these dierent behaviors as the consequence of changes in the free energy barriers for α-relaxation induced by the stress applied to the sample. These changes are the sum of two contributions: a) the rst one, which allows for plastic ow, is due to the applied stress σ and, according to a recently published theory, scales as −σ 2 ; b) The second contribution κ(λ), which is a function of the chain orientation at the monomer level, is positive and is responsible for the stress hardening regime. The rst one evolves immediately upon varying the stress whereas the second relaxes very slowly upon cessation of the applied stress. Our interpretation for the results of the third protocol is that aging dynamics is frozen when the stress is removed, as it is known for polycarbonate at room temperature. Our experiments set precise conditions for a theory of strain hardening

Quantitative proteomics unravels novel post-translational regulatory mechanisms of plant aquaporins

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Accumulation of cyclophilin A isoforms in conditioned medium of irradiated breast cancer cells

International audienceSecreted proteins play a key role in cell signaling and communication. We recently showed that ionizing radiations induced a delayed cell death of breast cancer cells, mediated by the death receptor pathways through the expression of soluble forms of death ligands. Using the same cell model, the objective of our work was the identification of diffusible factors, secreted following cell irradiation, potentially involved in cell death signaling. Differential proteomic analysis of conditioned media using 2DE resulted in detection of numerous spots that were significantly modulated following cell irradiation. The corresponding proteins were identified using MALDI-TOF MS and LC-MS/MS approaches. Interestingly, five isoforms of cyclophilin A were observed as increased in conditioned medium of irradiated cells. These isoforms differed in isoelectric points and in accumulation levels. An increase of cyclophilin A secretion was confirmed by Western blotting of conditioned media of irradiated or radiosentive mammary cells. These isoforms displayed an interesting pattern of protein maturation and post-translational modifications, including an alternating removal of N-terminal methionine, associated with a combination of acetylations and methylations. The role of the protein is discussed in relation with its potential involvement in the mechanisms of intercells relationships and radiosensitivity

Quantitative proteomics unravels novel post-translational regulatory mechanisms of plant aquaporins

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Quantitative proteomics unravels novel post-translational regulatory mechanisms of plant aquaporins

absen

Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots

Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots. European Proteomics Association 2013 Scientific Meetin

Quantitative proteomics unravels novel post-translational regulatory mechanisms of plant aquaporins

absen

Seed comparative genomics in three coffee species identify desiccation tolerance mechanisms in intermediate seeds

International audienceIn contrast to desiccation-tolerant 'orthodox' seeds, so-called 'intermediate' seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slowdown of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses

The cell proliferation antigen Ki-67 organises heterochromatin

International audienceAntigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression