A Unique Voltage Sensor Sensitizes the Potassium Channel AKT2 to Phosphoregulation

Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (Kweak channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of +100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique Kweak gating. Instead, a lysine residue in S4, highly conserved among all Kweak channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the “open-lock” characteristic and converted AKT2 into an inward-rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the Kin channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is ∼6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it

Identification of molecular integrators shows that nitrogen activelycontrolsthephosphatestarvationresponseinplants

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other’s transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.This work was supported in the Honude group (Biochemistry & Plant Molecular Physiology) by Agence Nationale de la Recherche (IMANA ANR-14-CE19-0008 with a doctoral fellowship to A.S.), by the Centre National de la Recherche Scientifique (CNRS LIA-CoopNet to G.K.), and by the National Science Foundation (NSF IOS 1339362-NutriNet). Research in V.R.’s laboratory was funded by the Ministry of Economy and Competitiveness and AEI/FEDER/European (grants BIO2013-46539-R and BIO2016-80551-R)

Identification of precursor transcripts for 6 novel miRNAs expands the diversity on the genomic organisation and expression of miRNA genes in rice

<p>Abstract</p> <p>Background</p> <p>The plant miRNAs represent an important class of endogenous small RNAs that guide cleavage of an mRNA target or repress its translation to control development and adaptation to stresses. MiRNAs are nuclear-encoded genes transcribed by RNA polymerase II, producing a primary precursor that is subsequently processed by DCL1 an RNase III Dicer-like protein.</p> <p>In rice hundreds of miRNAs have been described or predicted, but little is known on their genes and precursors which are important criteria to distinguish them from siRNAs. Here we develop a combination of experimental approaches to detect novel miRNAs in rice, identify their precursor transcripts and genes and predict or validate their mRNA targets.</p> <p>Results</p> <p>We produced four cDNA libraries from small RNA fractions extracted from distinct rice tissues. By <it>in silico </it>analysis we selected 6 potential novel miRNAs, and confirmed that their expression requires OsDCL1. We predicted their targets and used 5'RACE to validate cleavage for three of them, targeting a PPR, an SPX domain protein and a GT-like transcription factor respectively.</p> <p>In addition, we identified precursor transcripts for the 6 miRNAs expressed in rice, showing that these precursors can be efficiently processed using a transient expression assay in transfected <it>Nicotiana benthamiana </it>leaves. Most interestingly, we describe two precursors producing tandem miRNAs, but in distinct arrays. We focus on one of them encoding osa-miR159a.2, a novel miRNA produced from the same stem-loop structure encoding the conserved osa-miR159a.1. We show that this dual osa-miR159a.2-osa-miR159a.1 structure is conserved in distant rice species and maize. Finally we show that the predicted mRNA target of osa-miR159a.2 encoding a GT-like transcription factor is cleaved <it>in vivo </it>at the expected site.</p> <p>Conclusion</p> <p>The combination of approaches developed here identified six novel miRNAs expressed in rice which can be clearly distinguished from siRNAs. Importantly, we show that two miRNAs can be produced from a single precursor, either from tandem stem-loops or tandemly arrayed in a single stem-loop. This suggests that processing of these precursors could be an important regulatory step to produce one or more functional miRNAs in plants and perhaps coordinate cleavage of distinct targets in the same plant tissue.</p

Actes du 12e Colloque annuel des étudiant-e-s de cycles supérieurs du CRISES

La 12e édition du Colloque annuel des étudiant-e-s de cycles supérieurs du CRISES s’est tenue les 25 et 26 mars 2010 à l’Université Laval. Trente et une (31) communications y ont été présentées sur des thèmes aussi diversifiés que le Vieillissement au travail, les Grappes industrielles, l’Immigration, les Défis de la gouvernance, la Gestion des ressources et les solidarités locales, incluant un atelier spécial sur les Avancées et les limites de l’innovation sociale. Les textes qui nous sont parvenus avant la date limite du 18 mars afin d’être soumis à un processus d’évaluation, ainsi que respectivement le texte et/ou les diapositives PowerPoint des conférenciers d’ouverture et de clôture sont contenus dans ces Actes. Nous tenons ici à remercier chaleureusement nos collègues du comité organisateur : Kamel Béji, Manon Boulianne et Frédéric Hanin et notre assistante : Carole-Anne Gauthier."VIEILLISSEMENT AU TRAVAIL" "APPROCHES DE LA GOUVERNANCE" "GRAPPES INDUSTRIELLES ET DÉVELOPPEMENT LOCAL" "GESTION DES RESSOURCES" "SOLIDARITÉS LOCALES" "IMMIGRATION ET IDENTITÉ" "L'INNOVATION SOCIALE: AVANCÉES ET LIMITES

Discordance between testosterone measurement methods in castrated prostate cancer patients

Failure to suppress testosterone below 0.7 nM in castrated prostate cancer patients is associated with poor clinical outcomes. Testosterone levels in castrated patients are therefore routinely measured. Although mass spectrometry is the gold standard used to measure testosterone, most hospitals use an immunoassay method. In this study, we sought to evaluate the accuracy of an immunoassay method to measure castrate testosterone levels, with mass spectrometry as the reference standard. We retrospectively evaluated a cohort of 435 serum samples retrieved from castrated prostate cancer patients from April to September 2017. No follow-up of clinical outcomes was performed. Serum testosterone levels were measured in the same sample using liquid chromatography coupled with tandem mass spectrometry and electrochemiluminescent immunoassay methods. The mean testosterone levels were significantly higher with immunoassay than with mass spectrometry (0.672 ± 0.359 vs 0.461 ± 0.541 nM; P 0.7 nM was significantly higher with immunoassay (22.1%) than with mass spectrometry (13.1%; P 0.7 nM by immunoassay can result in an inaccurately identified castration status. Suboptimal testosterone levels in castrated patients should be confirmed by either mass spectrometry or an immunoassay method validated at low testosterone levels and interpreted with caution before any changes are made to treatment management