Calcium Live Imaging at Multi-Scales from Cellular to Organ Level in Arabidopsis thaliana.

Plants must adapt to environmental constraints. For this, they are able to perceive several types of stress in isolation or in combination manner. At the cellular level, after the perception of stress, cell signaling is set up to allow the establishment of the specific response. The calcium ion is known to be one of the ubiquitous second messengers which is involved in most of the stresses perceived by the plant. Changes of free cytosolic calcium but also in other cellular compartments are able to activate or inactivate several mechanisms involved in the cell to cope with the changes of environmental conditions. Several calcium reporters have been intensively used to visualize calcium signals in different conditions. In this chapter, we will present only genetically encoded fluorescent reporters for calcium imaging in living plant tissues to measure variations in calcium at several scales. The FRET (fluorescence resonance energy transfer) YC3.60 and the intensiometric GCamP3 sensors will be used in this method chapter. The image analyses will be also detailed for fluorescence quantification of calcium variation.

Plant Viruses Can Alter Aphid-Triggered Calcium Elevations in Infected Leaves.

Alighting aphids probe a new host plant by intracellular test punctures for suitability. These induce immediate calcium signals that emanate from the punctured sites and might be the first step in plant recognition of aphid feeding and the subsequent elicitation of plant defence responses. Calcium is also involved in the transmission of non-persistent plant viruses that are acquired by aphids during test punctures. Therefore, we wanted to determine whether viral infection alters calcium signalling. For this, calcium signals triggered by aphids were imaged on transgenic Arabidopsis plants expressing the cytosolic FRET-based calcium reporter YC3.6-NES and infected with the non-persistent viruses cauliflower mosaic (CaMV) and turnip mosaic (TuMV), or the persistent virus, turnip yellows (TuYV). Aphids were placed on infected leaves and calcium elevations were recorded by time-lapse fluorescence microscopy. Calcium signal velocities were significantly slower in plants infected with CaMV or TuMV and signal areas were smaller in CaMV-infected plants. Transmission tests using CaMV-infected Arabidopsis mutants impaired in pathogen perception or in the generation of calcium signals revealed no differences in transmission efficiency. A transcriptomic meta-analysis indicated significant changes in expression of receptor-like kinases in the BAK1 pathway as well as of calcium channels in CaMV- and TuMV-infected plants. Taken together, infection with CaMV and TuMV, but not with TuYV, impacts aphid-induced calcium signalling. This suggests that viruses can modify plant responses to aphids from the very first vector/host contact.

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

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

Arabidopsis pollen tube germination and growth depend on the mitochondrial calcium uniporter complex.

The mitochondrial calcium uniporter complex (MCUc) was recently characterized in details in metazoans and consists of pore-forming units (MCUs) and regulatory factors that channel calcium (Ca2+ ) ion into the mitochondria. MCUs participate in many stress and developmentally related processes involving Ca2+ . Although multiple homologues of MCUs and one regulatory subunit are usually present in plants, the first functional characterization and contribution to Ca2+ related processes of these proteins have been reported recently. Here, we focused on two predicted Arabidopsis MCUs and studied their role in the germination and the growth of pollen tube, a tip-growing cell type highly dependent on Ca2+ homeostasis. Heterologous expression of MCU1 or MCU2 in yeast is sufficient to generate a mitochondrial Ca2+ influx. MCU1 and MCU2 fluorescent reporters are co-expressed in the vegetative cell mitochondria of the pollen grain but are undetectable in the embryo sac. We demonstrate that MCU1 and MCU2 can form a heterotypic complex. Phenotypic analyses revealed an impaired pollen tube germination and growth in vitro only for the mcu2 mutants suggesting a predominant role of MCU2. Our results show that mitochondrial Ca2+ controlled by MCUs is an additional player in Arabidopsis pollen tube germination and growth.

The Arabidopsis guard cell outward potassium channel GORK is regulated by CPK33.

A complex signaling network involving voltage-gated potassium channels from the Shaker family contributes to the regulation of stomatal aperture. Several kinases and phosphatases have been shown to be crucial for ABA-dependent regulation of the ion transporters. To date, the Ca2+ -dependent regulation of Shaker channels by Ca2+ -dependent protein kinases (CPKs) is still elusive. A functional screen in Xenopus oocytes was launched to identify such CPKs able to regulate the three main guard cell Shaker channels KAT1, KAT2, and GORK. Seven guard cell CPKs were tested and multiple CPK/Shaker couples were identified. Further work on CPK33 indicates that GORK activity is enhanced by CPK33 and unaffected by a nonfunctional CPK33 (CPK33-K102M). Furthermore, Ca2+ -induced stomatal closure is impaired in two cpk33 mutant plants.

Spatio-Temporal Imaging of Promoter Activity in Intact Plant Tissues.

Localization and quantification of expression levels of genes help to determine their function. Localization of gene expression is often achieved through the study of their promoter activity. Three main reporter genes ß-glucuronidase (GUS), green fluorescent protein (GFP), and luciferase (LUC) have been intensively used to characterize promoter activities, each having its own specificities and advantages. Among them, the LUC reporter gene is best suitable for the analysis of the promoter activity of genes in intact living plants. Here, we describe a LUC-based method that allows to precisely localize and quantify promoter activity at the whole plant level, and to study the mechanisms that are involved in long-distance regulation of gene expression in Arabidopsis thaliana. Imaging LUC signals with a low-light CCD camera allows monitoring promoter activity in time and space in the transgenic plant harboring the promoter fused with the LUC gene. In addition, it allows quantifying change of promoter activities in plant during several hours.

CPK13, a noncanonical Ca2+-dependent protein kinase, specifically inhibits KAT2 and KAT1 shaker K+ channels and reduces stomatal opening.

Ca(2) (+)-dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca(2+), CPKs can be sorted into three types: strictly Ca(2+)-dependent CPKs, Ca(2+)-stimulated CPKs (with a significant basal activity in the absence of Ca(2+)), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K(+) Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cell-expressed KAT2 and KAT1 channels.

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

Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) 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 Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells.

Studies performed in animals have highlighted the major role of sphingolipids in regulating the balance between cell proliferation and cell death. Sphingolipids have also been shown to induce cell death in plants via calcium-based signalling pathways but the contribution of free cytosolic and/or nuclear calcium in the overall process has never been evaluated. Here, we show that increase in tobacco BY-2 cells of the endogenous content of Long Chain Bases (LCBs) caused by external application of d-erythro-sphinganine (DHS) is followed by immediate dose-dependent elevations of cellular free calcium concentration within the first minute in the cytosol and 10min later in the nucleus. Cells challenged with DHS enter a death process through apoptotic-like mechanisms. Lanthanum chloride, a general blocker of calcium entry, suppresses the cellular calcium variations and the PCD induced by DHS. Interestingly, dl-2-amino-5-phosphopentanoic acid (AP5) and [(+)-dizocilpine] (MK801), two inhibitors of animal and plant ionotropic glutamate receptors, suppress DHS-induced cell death symptoms by selectively inhibiting the variations of nuclear calcium concentration. The selective action of these compounds demonstrates the crucial role of nuclear calcium signature in controlling DHS-induced cell death in tobacco cells.

An inducible, modular system for spatio-temporal control of gene expression in stomatal guard cells.

Stomata, flanked by pairs of guard cells, are small pores on the leaf surfaces of plants and they function to control gas exchange between plants and the atmosphere. Stomata will open when water is available to allow for the uptake of carbon dioxide for photosynthesis. During periods of drought, stomata will close to reduce desiccation stress. As such, optimal functioning of stomata will impact on water use efficiency by plants. The development of an inducible, modular system for robust and targeted gene expression in stomatal guard cells is reported here. It is shown that application of ethanol vapour to activate the gene expression system did not affect the ability of stomata to respond to ABA in bioassays to determine the promotion of stomatal closure and the inhibition of stomatal opening. The system that has been developed allows for robust spatio-temporal control of gene expression in all cells of the stomatal lineage, thereby enabling molecular engineering of stomatal function as well as studies on stomatal development.

Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice.

SUMMARY: Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Deltaypc1Deltaydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca(2+), Mg(2+), Mn(2+) and Zn(2+), but inhibited in the presence of Fe(2+). OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants.

Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco.

Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Delta4-unsaturated LCB D-erythro-sphingosine and the saturated LCB D-ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-,N-Dimethyl-D-erythro-sphingosine, a structural analogue of D-erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans.

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.

Calcium signaling in plant cell organelles delimited by a double membrane.

Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.

Autonomous regulation of free Ca2+ concentrations in isolated plant cell nuclei: a mathematical analysis.

Experiments performed on nuclei isolated from animal or plant cells have provided evidence that the nucleus generates directly specific nucleoplasmic calcium transients in response to external stimuli. Recent data suggest that isolated plant nuclei might be considered as a closed system where the nuclear concentration of free calcium would be regulated by reversible movements between the nucleoplasm and nuclear stores. We have addressed the relevance of this hypothesis by developing a mathematical approach to simulate nucleoplasmic calcium dynamics generated under various pH and temperature conditions. Here, we show that the experimental results could be explained provided that calcium channels as well as systems transporting calcium are present on the inner nuclear membrane. The putative channels would allow the entry of calcium into the nucleoplasm whereas the elusive transporting system(s) would contribute to replenish the nuclear stores. The simple proposed model is versatile enough to explain and predict autonomous changes in free calcium in the nucleoplasm of isolated plant nuclei.

Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling.

Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus.

[Luminous plant and animals or the expression of aequorin and "chameleon" probes: a new light in calcium signaling]. / Les plantes et les animaux lumineux ou l'expression de l'aequorine et des sondes "caméléon": Un nouvel éclairage sur la signalisation calcique.

Calcium ion is a universal second messenger in numerous cell physiological processes. The paper describes the structure and the activation mechanisms of the bioluminescent (aequorin) and fluorescent based GFP calcium sensitive probes (Cameleon) and the data obtained with such probes in genetically transformed animal and vegetal organisms. The importance of these in vivo Ca2+ imaging molecules in the understanding of calcium signalling is discussed.