Calcium 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.

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.

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.

Background: During the interaction between rhizobia and leguminous plants the two partners engage in a molecular conversation that leads to reciprocal recognition and ensures the beginning of a successful symbiotic integration. In host plants, intracellular Ca²⁺ changes are an integral part of the signalling mechanism. In rhizobia it is not yet known whether Ca²⁺ can act as a transducer of symbiotic signals. Results: A plasmid encoding the bioluminescent Ca²⁺ probe aequorin was introduced into Mesorhizobium loti USDA 3147^T strain to investigate whether a Ca²⁺ response is activated in rhizobia upon perception of plant root exudates. We find that M. loti cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca²⁺ concentration. Only root exudates from the homologous host Lotus japonicus induce Ca²⁺ signalling and downstream activation of nodulation genes. The extracellular Ca²⁺ chelator EGTA inhibits both transient intracellular Ca²⁺ increase and inducible nod gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible. Conclusion: These findings indicate a newly described early event in the molecular dialogue between plants and rhizobia and highlight the use of aequorin-expressing bacterial strains as a promising novel approach for research in legume symbiosis.

Photosynthetic cell suspension cultures are a useful experimental system to analyse a variety of physiological processes, bypassing the structural complexity of the plant organism in toto. Nevertheless, cell cultures containing functional chloroplasts are quite difficult to obtain, and this process is usually laborious and time-consuming. In this work a novel and rapid method to set up photosynthetic cell suspension cultures from the model plant Arabidopsis thaliana was developed. The direct germination of Arabidopsis seeds on a sucrose-containing agarized culture medium supplemented with 0.25 µg/ml 6-benzylaminopurine and 0.5 µg/ml 2,4-dichlorophenoxyacetic acid caused the straightforward formation of green calli at the level of seedling hypocotyls. The subsequent transfer of these calli in liquid culture medium containing the same concentrations of phytohormones and gradually decreasing sucrose levels allowed for the establishment of chloroplast-containing cell suspension cultures, containing functional chloroplasts, in a much faster way than previously described procedures. Pulse amplitude modulation analyses, measurements of oxygen evolution and electron transport rate, together with confocal and electron microscopy observations, confirmed the photosynthetic efficiency of these cell suspension cultures. The described procedure lends itself as a simple and effective way to obtain a convenient tool for a wide array of structural and functional studies on chloroplasts.

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 Ca2+ concentration, which is a fundamental prerequisite for any Ca2+-based signalling system, is accomplished by complex mechanisms including Ca2+ binding proteins acting as Ca2+ buffers. In this work we investigated the occurrence of Ca2+ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus. A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca2+-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca2+-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 Ca2+ binding ability of the M. loti protein was demonstrated by 45Ca2+-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 Ca2+ adducts. The present data indicate that ferredoxin II is a major Ca2+ binding protein in M. loti that may participate in Ca2+ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca2+ regulatory systems.