Smrutisanjita Behera

Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

The mitochondrial Ca2+ uptake protein At-MICU shapes mitochondrial Ca2+ dynamics, providing molecular in vivo evidence for the existence and function of a mitochondrial uniporter complex in plants. Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca2+ signaling may play a central role in this process. Free Ca2+ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca2+ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca2+ uniporter machinery in mammals. MICU binds Ca2+ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca2+ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca2+ in the matrix. Furthermore, Ca2+ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca2+ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca2+ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca2+ uptake by moderating influx, thereby shaping Ca2+ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca2+ signaling in plants.

Analyses of Ca2+ Accumulation and Dynamics in the Endoplasmic Reticulum of Arabidopsis Root Cells Using a Genetically Encoded Cameleon Sensor

Generation of an ER-targeted Cameleon reporter protein enables the analysis of Ca2+ accumulation and dynamics in the lumen of the ER in plant cells. In planta, very limited information is available about how the endoplasmic reticulum (ER) contributes to cellular Ca2+ dynamics and homeostasis. Here, we report the generation of an ER-targeted Cameleon reporter protein suitable for analysis of Ca2+ accumulation and dynamics in the lumen of the ER in plant cells. Using stably transformed Arabidopsis (Arabidopsis thaliana) plants expressing this reporter protein, we observed a transiently enhanced accumulation of Ca2+ in the ER in response to stimuli inducing cytosolic Ca2+ rises in root tip cells. In all experimental conditions, ER Ca2+ dynamics were substantially different from those monitored in the cytosol. A pharmacological approach enabled us to evaluate the contribution of the different ER-resident Ca2+-ATPase classes in the regulation of the ER Ca2+ homeostasis. Taken together, our results do not provide evidence for a role of the ER as a major source that releases Ca2+ for stimulus-induced increases in cytosolic Ca2+ concentration. Instead, our results show that the luminal ER Ca2+ elevations typically follow cytosolic ones, but with distinct dynamics. These findings suggest fundamental differences for the function of the ER in cellular Ca2+ homeostasis in plants and animals.

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

A unique mitochondrial ion channel affects organelle physiology and its lack is associated with senescence in the model plant Arabidopsis. Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.

Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma

Plants expressing a chloroplast-localized Cameleon Ca2+ probe allow single-organelle analysis of chloroplast Ca2+ dynamics. In eukaryotes, subcellular compartments such as mitochondria, the endoplasmic reticulum, lysosomes, and vacuoles have the capacity for Ca2+ transport across their membranes to modulate the activity of compartmentalized enzymes or to convey specific cellular signaling events. In plants, it has been suggested that chloroplasts also display Ca2+ regulation. So far, monitoring of stromal Ca2+ dynamics in vivo has exclusively relied on using the luminescent Ca2+ probe aequorin. However, this technique is limited in resolution and can only provide a readout averaged over chloroplast populations from different cells and tissues. Here, we present a toolkit of Arabidopsis (Arabidopsis thaliana) Ca2+ sensor lines expressing plastid-targeted FRET-based Yellow Cameleon (YC) sensors. We demonstrate that the probes reliably report in vivo Ca2+ dynamics in the stroma of root plastids in response to extracellular ATP and of leaf mesophyll and guard cell chloroplasts during light-to-low-intensity blue light illumination transition. Applying YC sensing of stromal Ca2+ dynamics to single chloroplasts, we confirm findings of gradual, sustained stromal Ca2+ increases at the tissue level after light-to-low-intensity blue light illumination transitions, but monitor transient Ca2+ spiking as a distinct and previously unknown component of stromal Ca2+ signatures. Spiking was dependent on the availability of cytosolic Ca2+ but not synchronized between the chloroplasts of a cell. In contrast, the gradual sustained Ca2+ increase occurred independent of cytosolic Ca2+, suggesting intraorganellar Ca2+ release. We demonstrate the capacity of the YC sensor toolkit to identify novel, fundamental facets of chloroplast Ca2+ dynamics and to refine the understanding of plastidial Ca2+ regulation.

Physiological Characterization of a Plant Mitochondrial Calcium Uniporter in Vitro and in Vivo

An Arabidopsis homolog of the mammalian mitochondrial calcium uniporter MCU acts as Ca2+ channel, and its absence impacts mitochondrial function and morphology. Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca2+ regulation network in plants.

Ca2+ Imaging in Plants Using Genetically Encoded Yellow Cameleon Ca2+ Indicators

Temporally and spatially defined changes in cellular calcium (Ca(2+)) concentration represent stimulus-specific signals and regulate a myriad of biological processes. The development of ratiometric Ca(2+) reporter proteins like Yellow Cameleons (YCs) has greatly advanced our ability to analyze Ca(2+) dynamics in vivo with unprecedented spatial and temporal resolution. In plants, the application of these Ca(2+) reporter proteins has been pioneered for the investigation of Ca(2+) dynamics in guard cells, and recently their use has been extended to other single-cell models like growing pollen tubes and root hairs. However, in plants, the use of YC reporter proteins has largely remained restricted to the investigation of cytoplasmic alterations of Ca(2+) concentrations. Here, we provide an introduction to current methods for imaging Ca(2+) dynamics with increasing sophistication.

High-Resolution Imaging of Cytoplasmic Ca2+ Dynamics in Arabidopsis Roots

This protocol describes a method for imaging cytoplasmic Ca(2+) dynamics in roots with high resolution using confocal laser scanning microscopy (CLSM). The Förster (fluorescence) resonance energy transfer (FRET)-based genetically modified Ca(2+) indicator Yellow Cameleon YC3.6, stably expressed in plants under the control of the ubiquitin promoter UBQ10, is used for Ca(2+) measurements. This protocol enables imaging of 5- to 7-d-old seedlings with high-magnification objectives (25×, 40×, and 63×).

Live Cell Imaging of Cytoplasmic Ca2+ Dynamics in Arabidopsis Guard Cells

This protocol describes a classical method for measuring cytoplasmic Ca(2+) dynamics in Arabidopsis thaliana guard cells. The Förster (fluorescence) resonance energy transfer (FRET)-based genetically modified Ca(2+) indicator Yellow Cameleon YC3.6, under the control of the guard cell-specific promoter GC1, is used for Ca(2+) measurements.

In Vivo Analysis of Calcium Levels and Glutathione Redox Status in Arabidopsis Epidermal Leaf Cells Infected with the Hypersensitive Response-Inducing Bacteria Pseudomonas syringae pv. tomato AvrB ( PstAvrB )

Plants react to the attack of pathogen microorganisms by mounting appropriate and efficient downstream defense responses often involving a form of localized cell death called hypersensitive response (HR).Here we describe an innovative and noninvasive protocol based on in vivo bioimaging technique coupled with utilization of genetically encoded fluorescent sensors that allows to monitor and analyze intracellular calcium (Ca2+) dynamics and changes of the glutathione redox status taking place in plant organs during plant interaction with the HR-inducing bacteria Pseudomonas syringae (PstAvrB).