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.

Increasing evidence indicates that water activated by plasma discharge, termed as plasma-activated water (PAW), can promote plant growth and enhance plant defence responses. Nevertheless, the signalling pathways activated in plants in response to PAW are still largely unknown. In this work, we analysed the potential involvement of calcium as an intracellular messenger in the transduction of PAW by plants. To this aim, Arabidopsis thaliana (Arabidopsis) seedlings stably expressing the bioluminescent Ca²⁺ reporter aequorin in the cytosol were challenged with PAW generated by a plasma torch. Ca²⁺ measurement assays demonstrated the induction by PAW of rapid and sustained cytosolic Ca²⁺ elevations in Arabidopsis seedlings. The dynamics of the recorded Ca²⁺ signals were found to depend upon different parameters, such as the operational conditions of the torch, PAW storage, and dilution. The separate administration of nitrate, nitrite, and hydrogen peroxide at the same doses as those measured in the PAW did not trigger any detectable Ca²⁺ changes, suggesting that the unique mixture of different reactive chemical species contained in the PAW is responsible for the specific Ca²⁺ signatures. Unveiling the signalling mechanisms underlying plant perception of PAW may allow to finely tune its generation for applications in agriculture, with potential advantages in the perspective of a more sustainable agriculture.

Societal Impact Statement: An increasing world population facing limited natural resources poses a global challenge to food security. This challenge is increasing due to climate change, which in turn is strongly affected by the food system that accounts, according to the Food and Agriculture Organization (FAO), for one‐third of the global greenhouse gas emissions. To make food production sustainable, a new model of agriculture with responsible use of natural resources and minimum use of agrochemicals must be implemented. This approach fosters the development of new technologies, such as that based on plasma‐activated water, whose effects, perspectives, and quest for fundamentals are discussed herein.Summary: Applications based on non‐thermal atmospheric plasmas ‐ commonly referred to as cold plasmas ‐ to the food system are emerging as effective technologies that may contribute to reconciling the increasing demand for food with the need to minimize its environmental impact. In particular, the technology based on the so‐called plasma‐activated water (PAW) has proven effective in promoting plant germination, growth, and resistance to biotic and abiotic stresses. Several interpretations have been proposed for the various beneficial effects played by PAW on plants, and the quest for the fundamental processes underlying them is ongoing. In this contribution, we present and discuss the approach based on calcium signaling, a ubiquitous and versatile signal transduction mode that has recently been demonstrated to be involved in PAW sensing by plants. The plant Ca²⁺‐mediated response is related to the mixture of reactive chemical species, as confirmed by selective plasma irradiation. Directions for future research are discussed, with a special focus on the link between Ca²⁺ signaling and plant responses to PAW/cold plasma. The potential offered by an interdisciplinary approach, combining real‐time monitoring of intracellular Ca²⁺ changes with machine learning algorithms, is pinpointed.

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.