Highlight Arabidopsis suspension-cultured cells stably expressing aequorin in the plastid stroma were used as a suitable tool to dissect differential Ca2+ responses of non-green plastids versus chloroplasts to several environmental cues.

Chloroplasts require a fine-tuned control of their internal Ca 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 Ca signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca homeostasis and in the modulation of chloroplast Ca signals in vivo, we targeted the bioluminescent Ca reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (). 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 Ca 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 Ca dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca changes in the thylakoid lumen. Localized Ca increases also were observed on the thylakoid membrane surface, mirroring transient Ca changes observed for the bulk stroma, but with specific Ca dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca changes, suggesting a new scenario for light-to-dark-induced Ca fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca signaling networks within chloroplasts and plant cells.

Summary Ca2+‐dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants.We used single‐cell imaging of Ca2+‐responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress.We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo‐osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo‐osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol.Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

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.

Photosynthetic cell suspension cultures are a useful experimental system to analyze 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.

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SummaryArbuscular 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). Ca 2+ measurements in G. margarita mycelia pre-incubated with TATaequorin demonstrated the occurrence of changes in the intracellular free Ca 2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosisrelated 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 Ca 2+ homeostasis and signalling in AM fungi.

Acmella oleracea is a promising cosmetic, nutraceutical, and pharmaceutical ingredient, and plants with high levels of active compounds are needed in the market. Cultivation can be valuable if sufficient levels of alkylamides are present in plant material. In this regard the application of biostimulants can be an innovative approach to increase yield of cultivation or bioactive compound levels. A. oleracea plants were cultivated in Northern Italy in an experimental site using three different types of biostimulants, triacontanol-based mixture (Tria), an extract from plant tissues (LL017), and seaweed extract (Swe). Plants were grown in the field in two different growing seasons (2018 and 2019). After treatments inflorescences were harvested and the quali-quantitative analysis of alkylamides and polyphenols was performed. Treated and control plants were compared for yields, morphometric measurements, quali-quantitative composition in secondary metabolites. Overall results show that both triacontanol-based mixture and the LL017 positively influenced plant growth (Tria >+ 22%; LL017 >+ 25%) and flower production (Tria >+ 34%; LL017 >+ 56%). The amount of alkylamides and polyphenols in flowers were between 2.0–5.2% and 0.03–0.50%, respectively. Biostimulant treatments ensure higher cultivation yields and allow maintenance of the alkylamide and polyphenol levels based on % (w/w), thus offering an advantage in the final quantity of extractable chemicals. Furthermore, data revealed that samples harvested in late season show a decrease of polyphenols.