Lorella Navazio

A Diffusible Signal from Arbuscular Mycorrhizal Fungi Elicits a Transient Cytosolic Calcium Elevation in Host Plant Cells

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca2+ indicator aequorin to detect intracellular Ca2+ changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca2+ were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2+-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca2+ transient were constitutively released in the medium, and the induced Ca2+ signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca2+ response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.

Symbiosis with an endobacterium increases the fitness of a mycorrhizal fungus, raising its bioenergetic potential

Arbuscular mycorrhizal fungi (AMF) occur in the rhizosphere and in plant tissues as obligate symbionts, having key roles in plant evolution and nutrition. AMF possess endobacteria, and genome sequencing of the endobacterium Candidatus Glomeribacter gigasporarum revealed a reduced genome and a dependence on the fungal host. To understand the effect of bacteria on fungal fitness, we used next-generation sequencing to analyse the transcriptional profile of Gigaspora margarita in the presence and in the absence of its endobacterium. Genomic data on AMF are limited; therefore, we first generated a gene catalogue for G. margarita. Transcriptome analysis revealed that the endobacterium has a stronger effect on the pre-symbiotic phase of the fungus. Coupling transcriptomics with cell biology and physiological approaches, we demonstrate that the bacterium increases the fungal sporulation success, raises the fungal bioenergetic capacity, increasing ATP production, and eliciting mechanisms to detoxify reactive oxygen species. By using TAT peptide to translocate the bioluminescent calcium reporter aequorin, we demonstrated that the line with endobacteria had a lower basal intracellular calcium concentration than the cured line. Lastly, the bacteria seem to enhance the fungal responsiveness to strigolactones, the plant molecules that AMF perceive as branching factors. Although the endobacterium exacts a nutritional cost on the AMF, endobacterial symbiosis improves the fungal ecological fitness by priming mitochondrial metabolic pathways and giving the AMF more tools to face environmental stresses. Thus, we hypothesise that, as described for the human microbiota, endobacteria may increase AMF innate immunity.

Endoplasmic reticulum stress-induced programmed cell death in soybean cells

In animal cells, the endoplasmic reticulum may participate in programmed cell death by sensing and transducing apoptotic signals. In an attempt to analyze the role of the endoplasmic reticulum in plant programmed cell death we investigated the effect of cyclopiazonic acid, a specific blocker of plant endoplasmic reticulum-type IIA Ca2+-pumps, in soybean cells. Cyclopiazonic acid treatment elicited endoplasmic reticulum stress and a biphasic increase in cytosolic Ca2+ concentration, followed by the induction of a cell death program. Cyclopiazonic acid-induced programmed cell death occurred with accumulation of H2O2, cytochrome c release from mitochondria, caspase 9- and caspase 3-like protease activation, cytoplasmic shrinkage and chromatin condensation. Chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethil ester) failed to inhibit cyclopiazonic acid-induced cell death. Taken together, our results provide evidence for a role of the endoplasmic reticulum and mitochondria in regulating cyclopiazonic acid-induced programmed cell death in soybean cells, probably via a cross-talk between the two organelles.

An Endopolygalacturonase from Sclerotinia sclerotiorum Induces Calcium-Mediated Signaling and Programmed Cell Death in Soybean Cells

A basic endopolygalacturonase (PG) isoform, produced early by Sclerotinia sclerotiorum when infecting soybean seedlings, was used to examine the signaling role of the enzyme in aequorin-expressing soybean cells. A cytosolic Ca2+ elevation was induced, with a rapid increase (phase 1) and a very slow decrease (phase 2) of Ca2+ concentration, indicating the involvement of Ca2+ ions in PG signaling. Within 1 h of PG-cell contact a remarkable level of cell death was recorded, significantly higher than the control cell culture turnover. The observed morphological and biochemical changes were indicative of the activation of programmed cell death; in particular, cytochrome c release in the cytoplasm and activation of both caspase 9-like and caspase 3-like proteases were found. When a polygalacturonase-inhibiting protein (PGIP) and the PG were simultaneously applied to cells, both the Ca2+ increase and cell death were annulled. The possible roles of prolonged sustained cytosolic Ca2+ concentrations in inducing cell death and of the PG-PGIP interaction in preventing PG signaling are discussed.

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride

BackgroundCalcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. Trichoderma spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.ResultsHere, we investigated the effects on plant cells of metabolite complexes secreted by Trichoderma atroviride wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca2+ and activation of defense responses. Trichoderma culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen Botrytis cinerea, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (Glycine max L.) cell suspension cultures, Trichoderma and Botrytis metabolite mixtures were distinctively perceived and activated transient intracellular Ca2+ elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca2+ signature and cellular effects were modified by the culture medium from the knock-out mutant of Trichoderma, defective for the production of the secreted 42 kDa endochitinase.ConclusionNew insights are provided into the mechanism of interaction between Trichoderma and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca2+ changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.

Primary structure of the N-linked carbohydrate chains of Calreticulin from spinach leaves.

Calreticulin is a multifunctional Ca2+-binding protein of the endoplasmic reticulum of most eukaryotic cells. The 56 kDa Calreticulin glycoprotein isolated from spinach (Spinacia oleracea L.) leaves was N-deglycosylated by PNGase-F digestion. The carbohydrate moiety was isolated by gel permeation chromatography and purified by high-pH anion-exchange chromatography. The fractions were investigated by 500 MHz1H-NMR spectroscopy, in combination with monosaccharide analysis and fast-atom bombardment-mass spectrometry. The following carbohydrate structure could be established as the major component (Man8GlcNAc2): Heterogeneity was demonstrated by the presence of two minor components being Man7GlcNAc2 lacking a terminal residue (D1 or D3), compared to the major component. A cross-reactivity with an antibody against the endoplasmic reticulum retention signal HDEL was also found.

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.

Flavonoid‐induced calcium signalling in Rhizobium leguminosarum bv. viciae

• Legume-rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals - flavonoids - before infection. Host plants sense counterpart rhizobial signalling molecules - Nod factors - through transient changes in intracellular free-calcium. Here we investigate the potential involvement of Ca(2+) in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae. • By using aequorin-expressing rhizobial strains, we monitored intracellular Ca(2+) dynamics and the Ca(2+) dependence of nod gene transcriptional activation. • Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca(2+) that were essential for the induction of nod genes. Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca(2+) variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca(2+) signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca(2+) signalling event. • These findings demonstrate a key role played by Ca(2+) in sensing and transducing plant-specific flavonoid signals in rhizobia and open up a new perspective in the flavonoid-NodD paradigm of nod gene regulation.

Evidence that Spinach Leaves Express Calreticulin but Not Calsequestrin

The presence of either calreticulin (CR) or calsequestrin (CS)-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca2+-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn2+ (i.e. under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca2+. Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca2+-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.

Expression and localization of calreticulin in tobacco anthers and pollen tubes

The developmental expression pattern and localization of calreticulin were studied in Nicotiana tabacum L. anthers, pollen and pollen tubes. High transcript and protein levels were detected throughout anther development. Immunolocalization of calreticulin in the anthers showed particular dense label in tapetum and pollen at developmental stage 2, when the tapetum is highly active and the pollen tetrads are formed. Much lower transcript and protein levels were detected in dry and hydrated pollen and in pollen tubes. Immunofluorescence labeling of both chemically fixed and cryo-fixed and freeze-substituted pollen tubes showed the presence of calreticulin in Golgi apparatus and endoplasmic reticulum (ER). Calreticulin was seen throughout the stacks in the Golgi apparatus and in the areas with coated-Golgi vesicles but much less so in the ER. Calreticulin was not found in the secretory vesicles. A relatively intense label was occasionally seen adjacent to the wall of the tube. No significant label was observed in mitochondria, vacuoles, generative cells, cell wall or callose plugs. The present results are consistent with a role of calreticulin in Ca2+-dependent folding of secreted glycoproteins in tapetum, pollen and pollen tubes.