Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

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Functional Characterization of the Arabidopsis Abscisic Acid Transporters NPF4.5 and NPF4.6 in Xenopus Oocytes

Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidlyrespond to environmental perturbation. However, the underlying transient interactionsbetween transcription factors (TFs) and genome-wide targets typically elude biochemicaldetection. Here, we capture both stable and transient TF-target interactions genome-widewithin minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation(ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq).The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKEPROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. Thesetransient NLP7 targets captured in root cells using temporal TF perturbation account for 50%of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiatedtranscriptional cascade. Our approaches to capture transient TF-target interactions genomewidecan be applied to validate dynamic GRN models for any pathway or organism of interest

ABA transport and transporters.

International audienceAbscisic acid (ABA) metabolism, perception, and transport form a triptych allowing higher plants to use ABA as a signaling molecule. The molecular bases of ABA metabolism are now well described and, over the past few years, several ABA receptors have been discovered. Although ABA transport has long been demonstrated in planta, the first breakthroughs in identifying plasma membrane-localized ABA transporters came in 2010, with the identification of two ATP-binding cassette (ABC) proteins. More recently, two ABA transporters in the nitrate transporter 1/peptide transporter (NRT1/PTR) family have been identified. In this review, we discuss the role of these different ABA transporters and examine the scientific impact of their identification. Given that the NRT1/PTR family is involved in the transport of nitrogen (N) compounds, further work should determine whether an interaction between ABA and N signaling or nutrition occurs

Regulation by Calcium-dependent protein kinases of Skaker channels expressed in guard cells

Stomatal movements result from osmotically-driven fluxes of water, which follow massive exchanges of solutes, including K+ ions, between guard cells and surrounding tissues. These exchanges involve a set of dedicated transport systems, controlled through signalling pathways, including Ca2+-dependent ones. For example, Ca2+ signals have been reported to lead to stomatal closure through the inhibition of inward and activation of outward K+ channels. This could be mediated in particular by Ca2+-dependent kinases targeting these channels.Here, we addressed the post-translational regulation of three K+-selective voltage-gated channels expressed in Arabidopsis guard cells (inwardly-rectifying KAT1 and KAT2, and outwardly-rectifying GORK) by Ca2+-dependent protein kinases (CPKs). Of the 34 CPK genes inArabidopsis thaliana, some CPK are thought to be expressed in guard cells. Seven of these CPKs were co-expressed in Xenopus oocytes with the three studied Shaker channels, and their influence on K+ transport was examined with a classical voltage-clamp technique. Pinpointed interactions were checked by on-chip phosphorylation assays on peptide arrays designed from the channel primary sequence. This enabled us to list a number of candidate phosphorylation sites and, subsequently, to design mutant channels expected to display CPK-insensitive of CPKphosphorylated phenotype. The functional characterisation of these mutant channels and the phenotype of plants overexpressing CPKs will be presented

Regulation by Calcium-dependent protein kinases of Skaker channels expressed in guard cells

Stomatal movements result from osmotically-driven fluxes of water, which follow massive exchanges of solutes, including K+ ions, between guard cells and surrounding tissues. These exchanges involve a set of dedicated transport systems, controlled through signalling pathways, including Ca2+-dependent ones. For example, Ca2+ signals have been reported to lead to stomatal closure through the inhibition of inward and activation of outward K+ channels. This could be mediated in particular by Ca2+-dependent kinases targeting these channels.Here, we addressed the post-translational regulation of three K+-selective voltage-gated channels expressed in Arabidopsis guard cells (inwardly-rectifying KAT1 and KAT2, and outwardly-rectifying GORK) by Ca2+-dependent protein kinases (CPKs). Of the 34 CPK genes inArabidopsis thaliana, some CPK are thought to be expressed in guard cells. Seven of these CPKs were co-expressed in Xenopus oocytes with the three studied Shaker channels, and their influence on K+ transport was examined with a classical voltage-clamp technique. Pinpointed interactions were checked by on-chip phosphorylation assays on peptide arrays designed from the channel primary sequence. This enabled us to list a number of candidate phosphorylation sites and, subsequently, to design mutant channels expected to display CPK-insensitive of CPKphosphorylated phenotype. The functional characterisation of these mutant channels and the phenotype of plants overexpressing CPKs will be presented

Two Ca2+-dependent protein kinases (cpks) oppositely regulate a ubiquitous k+ channel in arabidopsis thaliana

In plant cells, as regards both the electrical polarization of the plasma-membrane and the osmotic homeostasis, voltage-gated K + channels participate in transduction chains, often downstream Ca 2+ signals. Thus, worth considering is, among the range of post-translational modifications, that potentially regulate their activity, the phosphorylation of these so-called Shaker channels by Ca 2+ -dependent protein kinases (CPKs). We investigated the interactions between these two families of proteins by a combination of techniques: heterologous expression in Xenopus oocytes followed by electrophysiology recordings, in planta interaction by FRET-FLIM imaging, in vitro phosphorylation study using channel-derived peptide arrays, looking for overlap of expression patterns and characterization of gain- or loss-of-function mutants. Here we have identified several CPKs targeting and regulating the Shaker channel activities. Interestingly, we found that CPK13, a Ca 2+ -insensitive CPK, and CPK6, a Ca 2+ -strictly dependent CPK, respectively inhibits and activates the same Shaker channel, KAT2. In the context of (i) the large expression pattern of KAT2 (e.g. in leaf vasculature and in guard cells) and (ii) the documented ability of KAT2 to form heteromeric channels with the Shaker sub‐units AKT2 and KAT1, we shall discuss the potential implication of KAT2 regulation by CPKs in phloem and stomata physiology

Two Ca2+-dependent protein kinases (cpks) oppositely regulate a ubiquitous k+ channel in arabidopsis thaliana

In plant cells, as regards both the electrical polarization of the plasma-membrane and the osmotic homeostasis, voltage-gated K + channels participate in transduction chains, often downstream Ca 2+ signals. Thus, worth considering is, among the range of post-translational modifications, that potentially regulate their activity, the phosphorylation of these so-called Shaker channels by Ca 2+ -dependent protein kinases (CPKs). We investigated the interactions between these two families of proteins by a combination of techniques: heterologous expression in Xenopus oocytes followed by electrophysiology recordings, in planta interaction by FRET-FLIM imaging, in vitro phosphorylation study using channel-derived peptide arrays, looking for overlap of expression patterns and characterization of gain- or loss-of-function mutants. Here we have identified several CPKs targeting and regulating the Shaker channel activities. Interestingly, we found that CPK13, a Ca 2+ -insensitive CPK, and CPK6, a Ca 2+ -strictly dependent CPK, respectively inhibits and activates the same Shaker channel, KAT2. In the context of (i) the large expression pattern of KAT2 (e.g. in leaf vasculature and in guard cells) and (ii) the documented ability of KAT2 to form heteromeric channels with the Shaker sub‐units AKT2 and KAT1, we shall discuss the potential implication of KAT2 regulation by CPKs in phloem and stomata physiology

Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter

Calcium (Ca2+) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and G FP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins

AtNPF5.5, a nitrate transporter affecting nitrogen accumulation in Arabidopsis embryo

International audienceDipeptide (Leu-Leu) and nitrate transport activities of 26 Arabidopsis NPF (NRT1/PTR Family) proteins were screened in Saccharomyces cerevisiae and Xenopus laevis oocytes, respectively. Dipeptide transport activity has been confirmed for 2 already known dipeptide transporters (AtNPF8.1 and AtNPF8.3) but none of the other tested NPFs displays dipeptide transport. The nitrate transport screen resulted in the identification of two new nitrate transporters, AtNPF5.5 and AtNPF5.10. The localization of the mRNA coding for NPF5.5 demonstrates that it is the first NPF transporter reported to be expressed in Arabidopsis embryo. Two independent homozygous npf5.5 KO lines display reduced total nitrogen content in the embryo as compared to WT plants, demonstrating an effect of NPF5.5 function on the embryo nitrogen content. Finally, NPF5.5 gene produces two different transcripts (AtNPF5.5a and AtNPF5.5b) encoding proteins with different N-terminal ends. Both proteins are able to transport nitrate in xenopus oocytes

Potasium nutrition in rice under salt stress: role of arbuscular mycorrhizal symbiosis?

International audienceArbuscular mycorrhizal fungi (AMF) establish a symbiotic association with the roots of 80% of terrestrial plants and form complex tree-shaped feeding structures called arbuscules in colonized root cells. This association with AMF not only provides more efficient uptake of nutrients for the plant, but also confers protection against pathogens and increased tolerance to environmental stress such as salt stress [1]. Rice (Oryza sativa), the most salt-sensitive crop species amongst cereals, has a productivity strongly reduced around the world due to soil salinity/salinization and increased sea level (in deltas). High Na+ concentrations (salt stress conditions) impairs K+ uptake and inhibits many K+-activated enzymes. Rice exhibits molecular mechanisms to alleviate salt stress such as maintaining a high cellular K+/Na+ ratio, e.g. by a more efficient K+ uptake, which was recently shown to occur upon root/AMF interaction [2]. Knowledge on the role of root/AMF interaction on K+/Na+ transport upon salt stress is sparse [2, 3]. The aim of the research is to understand the mechanisms by which AMF improve plant K+ nutrition upon salt stress, taking Rhizophagus irregularis (model AMF)-rice interaction as a model. We will investigate the mechanisms by which AMF mediate K+ uptake and translocation towards root cells. Rice loss-of-function mutants for each of the three major K+ uptake systems of the plant, OsAKT1, OsHAK5 and OsHAK1, were inoculated with R. irregularis and submitted to salt stress, and their K+ contents and K+/Na+ ratios were monitored to identify key player(s) for AMF-mediated improved rice K+ nutrition and salt tolerance