Nonbinary fungal signals and calcium-mediated transduction in plant immunity and symbiosis.

Chitin oligomers (COs) are among the most common and active fungal elicitors of plant responses. Short-chain COs from symbiotic arbuscular mycorrhizal fungi activate accommodation responses in the host root, while long-chain COs from pathogenic fungi are acknowledged to trigger defence responses. The modulation of intracellular calcium concentration - a common second messenger in a wide variety of plant signal transduction processes - plays a central role in both signalling pathways with distinct signature features. Nevertheless, mounting evidence suggests that plant immunity and symbiosis signalling partially overlap at multiple levels. Here, we elaborate on recent findings on this topic, highlighting the nonbinary nature of chitin-based fungal signals, their perception and their interpretation through Ca2+ -mediated intracellular signals. Based on this, we propose that plant perception of symbiotic and pathogenic fungi is less clear-cut than previously described and involves a more complex scenario in which partially overlapping and blurred signalling mechanisms act upstream of the unambiguous regulation of gene expression driving accommodation or defence responses.

Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity.

Plants activate an immune or symbiotic response depending on the detection of distinct signals from root-interacting microbes. Both signalling cascades involve Ca2+ as a central mediator of early signal transduction. In this study, we combined aequorin- and cameleon-based methods to dissect the changes in cytosolic and nuclear Ca2+ concentration caused by different chitin-derived fungal elicitors in Lotus japonicus roots. Our quantitative analyses highlighted the dual character of the evoked Ca2+ responses taking advantage of the comparison between different genetic backgrounds: an initial Ca2+ influx, dependent on the LysM receptor CERK6 and independent of the common symbiotic signalling pathway (CSSP), is followed by a second CSSP-dependent and CERK6-independent phase, that corresponds to the well-known perinuclear/nuclear Ca2+ spiking. We show that the expression of immunity marker genes correlates with the amplitude of the first Ca2+ change, depends on elicitor concentration, and is controlled by Ca2+ storage in the vacuole. Our findings provide an insight into the Ca2+-mediated signalling mechanisms discriminating plant immunity- and symbiosis-related pathways in the context of their simultaneous activation by single fungal elicitors.

Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca2+ -affinity targeted aequorin reporter.

Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca2+ -affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells - consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling - was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+ ]ER ) were recorded in response to different environmental stresses, displaying stimulus-specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+ ]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system.

Quantitative Analysis of Plant Cytosolic Calcium Signals in Response to Water Activated by Low-Power Non-Thermal Plasma.

Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca2+ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca2+ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca2+ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.

Chloroplast Ca2+ Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes.

Chloroplasts require a fine-tuned control of their internal Ca2+ concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca2+ signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca2+ homeostasis and in the modulation of chloroplast Ca2+ signals in vivo, we targeted the bioluminescent Ca2+ reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (Arabidopsis thaliana). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca2+ levels in the thylakoid lumen were maintained at about 0.5 µm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca2+ dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca2+ signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca2+ changes in the thylakoid lumen. Localized Ca2+ increases also were observed on the thylakoid membrane surface, mirroring transient Ca2+ changes observed for the bulk stroma, but with specific Ca2+ dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca2+ changes, suggesting a new scenario for light-to-dark-induced Ca2+ fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca2+ storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca2+ signaling networks within chloroplasts and plant cells.

The contribution of organelles to plant intracellular Calcium signalling.

Calcium (Ca2+) is among the most important intracellular messengers in living organisms. Understanding of the players and dynamics of Ca2+ signalling pathways in plants may help to unravel the molecular basis of their exceptional flexibility to respond and to adapt to different stimuli. In the present review we focus on new tools that have recently revolutionized our view of organellar Ca2+ signalling as well as on the current knowledge regarding the pathways mediating Ca2+ fluxes across intracellular membranes. The contribution of organelles and cellular subcompartments to the orchestrated response via Ca2+ signalling within a cell is also discussed, underlining the fact that one of the greatest challenges in the field is the elucidation of how influx and efflux Ca2+ transporters/channels are regulated in a concerted manner to translate specific information into a Ca2+ signature.

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

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca(2+) signaling may play a central role in this process. Free Ca(2+) dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca(2+) 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 Ca(2+) uniporter machinery in mammals. MICU binds Ca(2+) and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca(2+) sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca(2+) in the matrix. Furthermore, Ca(2+) elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca(2+) signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca(2+) uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca(2+) uptake by moderating influx, thereby shaping Ca(2+) signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca(2+) signaling in plants.

The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Trichoderma longibrachiatum Triggers a NAADP-Mediated Calcium Signalling Pathway in Lotus japonicus.

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca2+-dependence of these gene expression was demonstrated by using the extracellular Ca2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca2+-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca2+ release in a signalling pathway triggered by a biotic stimulus.

Dissecting stimulus-specific Ca2+ signals in amyloplasts and chloroplasts of Arabidopsis thaliana cell suspension cultures.

Calcium is used by plants as an intracellular messenger in the detection of and response to a plethora of environmental stimuli and contributes to a fine-tuned internal regulation. Interest in the role of different subcellular compartments in Ca(2+) homeostasis and signalling has been growing in recent years. This work has evaluated the potential participation of non-green plastids and chloroplasts in the plant Ca(2+) signalling network using heterotrophic and autotrophic cell suspension cultures from Arabidopsis thaliana plant lines stably expressing the bioluminescent Ca(2+) reporter aequorin targeted to the plastid stroma. Our results indicate that both amyloplasts and chloroplasts are involved in transient Ca(2+) increases in the plastid stroma induced by several environmental stimuli, suggesting that these two functional types of plastids are endowed with similar mechanisms for handling Ca(2+) A comparison of the Ca(2+) trace kinetics recorded in parallel in the plastid stroma, the surface of the outer membrane of the plastid envelope, and the cytosol indicated that plastids play an essential role in switching off different cytosolic Ca(2+) signals. Interestingly, a transient stromal Ca(2+) signal in response to the light-to-dark transition was observed in chloroplasts, but not amyloplasts. Moreover, significant differences in the amplitude of specific plastidial Ca(2+) changes emerged when the photosynthetic metabolism of chloroplasts was reactivated by light. In summary, our work highlights differences between non-green plastids and chloroplasts in terms of Ca(2+) dynamics in response to environmental stimuli.

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

BACKGROUND: Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca(2+) concentration, which is a fundamental prerequisite for any Ca(2+)-based signalling system, is accomplished by complex mechanisms including Ca(2+) binding proteins acting as Ca(2+) buffers. In this work we investigated the occurrence of Ca(2+) binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus. RESULTS: A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca(2+)-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca(2+)-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca(2+) binding ability of the M. loti protein was demonstrated by (45)Ca(2+)-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca(2+) adducts. CONCLUSIONS: The present data indicate that ferredoxin II is a major Ca(2+) binding protein in M. loti that may participate in Ca(2+) homeostasis and suggest an evolutionarily ancient origin for protein-based Ca(2+) regulatory systems.

The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.

Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi.

Quantitative analysis of the naringenin-inducible proteome in Rhizobium leguminosarum by isobaric tagging and mass spectrometry.

The rhizobium-legume interaction is a critical cornerstone of crop productivity and environmental sustainability. Its potential improvement relies on elucidation of the complex molecular dialogue between its two partners. In the present study, the proteomic patterns of gnotobiotic cultures of Rhizobium leguminosarum bv. viciae 3841 grown for 6 h in presence or absence of the nod gene-inducing plant flavonoid naringenin (10 µM) were analyzed using the iTRAQ approach. A total of 1334 proteins were identified corresponding to 18.67% of the protein-coding genes annotated in the sequenced genome of bv. viciae 3841. The abundance levels of 47 proteins were increased upon naringenin treatment showing fold change ratios ranging from 1.5 to 25 in two biological replicates. Besides the nod units, naringenin enhanced the expression of a number of other genes, many of which organized in operons, including ß(1-2) glucan production and secretion, succinoglycan export, the RopA outer membrane protein with homology to an oligogalacturonide-specific porin motif, other enzymes for carbohydrate and amino acid metabolism, and proteins involved in the translation machinery. Data were validated at the transcriptional and phenotypic levels by RT-PCR and an assay of secreted sugars in culture supernatants, respectively. The current approach provides not only a high-resolution analysis of the prokaryotic proteome but also unravels the rhizobium molecular dialogue with legumes by detecting the enhanced expression of several symbiosis-associated proteins, whose flavonoid-dependency had not yet been reported.

Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis.

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca(2+) involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca(2+) chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca(2+) influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed.

Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.

Introduction: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. Methods: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. Results: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. Conclusions: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

Oligogalacturonides: novel signaling molecules in Rhizobium-legume communications.

Oligogalacturonides are pectic fragments of the plant cell wall, whose signaling role has been described thus far during plant development and plant-pathogen interactions. In the present work, we evaluated the potential involvement of oligogalacturonides in the molecular communications between legumes and rhizobia during the establishment of nitrogen-fixing symbiosis. Oligogalacturonides with a degree of polymerization of 10 to 15 were found to trigger a rapid intracellular production of reactive oxygen species in Rhizobium leguminosarum bv. viciae 3841. Accumulation of H(2)O(2), detected by both 2',7'-dichlorodihydrofluorescein diacetate-based fluorescence and electron-dense deposits of cerium perhydroxides, was transient and did not affect bacterial cell viability, due to the prompt activation of the katG gene encoding a catalase. Calcium measurements carried out in R. leguminosarum transformed with the bioluminescent Ca(2+) reporter aequorin demonstrated the induction of a rapid and remarkable intracellular Ca(2+) increase in response to oligogalacturonides. When applied jointly with naringenin, oligogalacturonides effectively inhibited flavonoid-induced nod gene expression, indicating an antagonistic interplay between oligogalacturonides and inducing flavonoids in the early stages of plant root colonization. The above data suggest a novel role for oligogalacturonides as signaling molecules released in the rhizosphere in the initial rhizobium-legume interaction.

Chemical Perturbation of Chloroplast Ca2+ Dynamics in Arabidopsis thaliana Suspension Cell Cultures and Seedlings.

Ca2+ signaling is part of universal signal transduction pathways to respond to external and internal stimuli or stress and in plants plays a central role in chloroplasts, such as in the regulation of photosynthetic enzymes or the transition from light to dark. Only recently, the underlying molecular machinery, e.g., transporters and channels that enable chloroplast Ca2+ fluxes, has started to be elucidated. However, chemical tools to specifically perturb these chloroplast Ca2+ fluxes are largely lacking. Here, we describe an efficient aequorin-based system in Arabidopsis thaliana suspension cell cultures to screen for chemicals that alter light-to-dark-induced chloroplast stroma Ca2+ signals. Subsequently, the effect of the hits on chloroplast Ca2+ signals is validated in Arabidopsis seedlings. The research lays a foundation for the identification of novel proteins involved in Ca2+ transport in chloroplast stroma under light-to-dark transition and for investigating the interaction of chloroplast Ca2+ signaling with photosynthesis in general.

The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity.

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.

TAT-mediated aequorin transduction: an alternative approach for effective calcium measurements in plant cells.

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

Arabidopsis Photosynthetic and Heterotrophic Cell Suspension Cultures.

Cell suspension cultures represent a widely used experimental tool suitable to perform a variety of structural and physiological studies in a more simplified system compared to the organism in toto. In this chapter we describe the methods routinely used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures, containing either chloroplasts or amyloplasts, respectively. The use of these in vitro systems may allow to obtain insights into the unique features of chloroplasts versus non-green plastids, as well as their integration in the structural and metabolic compartmentalization of the plant cell.