Agronomical valorization of eluates from the industrial production of microorganisms: Chemical, microbiological, and ecotoxicological assessment of a novel putative biostimulant.

Plant Biostimulants (BSs) are a valid supplement to be considered for the integration of conventional fertilization practices. Research in the BS field keeps providing alternative products of various origin, which can be employed in organic and conventional agriculture. In this study, we investigated the biostimulant activity of the eluate obtained as a by-product from the industrial production of lactic acid bacteria on bare agricultural soil. Eluates utilization is in line with the circular economy principle, creating economical value for an industrial waste product. The research focused on the study of physical, chemical, biochemical, and microbiological changes occurring in agricultural soil treated with the biowaste eluate, applied at three different dosages. The final aim was to demonstrate if, and to what extent, the application of the eluate improved soil quality parameters and enhanced the presence of beneficial soil-borne microbial communities. Results indicate that a single application at the two lower dosages does not have a pronounced effect on the soil chemical parameters tested, and neither on the biochemical proprieties. Only the higher dosage applied reported an improvement in the enzymatic activities of ß-glucosidase and urease and in the chemical composition, showing a higher content of total, nitric and ammonia N, total K, and higher humification rate. On the other hand, microbial communities were strongly influenced at all dosages, showing a decrease in the bacterial biodiversity and an increase in the fungal biodiversity. Bioinformatic analysis revealed that some Operative Taxonomic Units (OTUs) promoted by the eluate application, belong to known plant growth promoting microbes. Some other OTUs, negatively influenced were attributed to known plant pathogens, mainly Fusarium spp. Finally, the ecotoxicological parameters were also determined and allowed to establish that no toxic effect occurred upon eluate applications onto soil.

Comparison of Biostimulant Treatments in Acmella oleracea Cultivation for Alkylamides Production.

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.

Plant biodiversity and regulation of photosynthesis in the natural environment.

MAIN CONCLUSION: Investigation of photosynthesis regulation in different plant groups exposed to variable conditions showed that all species have similar photosynthetic electron transport modulation while excess energy dissipation is species specific. Photosynthesis is regulated in response to dynamic environmental conditions to satisfy plant metabolic demands while also avoiding possible over-excitation of the electron transport chain and the generation of harmful reactive oxygen species. Photosynthetic organisms evolved several mechanisms to modulate light harvesting and electron transport efficiency to respond to conditions changing at different timescales, going from fast sun flecks to slow seasonal variations. These regulatory mechanisms changed during evolution of photosynthetic organisms, also adapting to various ecological niches, making the investigation of plant biodiversity highly valuable to uncover conserved traits and plasticity of photosynthetic regulation and complement studies on model species. In this work, a set of plants belonging to different genera of angiosperms, gymnosperms, ferns and lycophytes were investigated by monitoring their photosynthetic parameters in different seasons looking for common trends and differences. In all plants, analysed photosynthetic electron transport rate was found to be modulated by growth light intensity, ensuring a balance between available energy and photochemical capacity. Growth light also influenced the threshold where heat dissipation of excitation energy, a mechanism called non-photochemical quenching (NPQ), was activated. On the contrary, NPQ amplitude did not correlate with light intensity experienced by the plants but was a species-specific feature. The zeaxanthin-dependent component of NPQ, qZ, was found to be the most variable in different plants and its modulation influenced the intensity and the kinetic properties of the response.

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.

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.

A Rapid and Efficient Method to Obtain Photosynthetic Cell Suspension Cultures of Arabidopsis thaliana.

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.

Spatial and temporal specificity of Ca2+ signalling in Chlamydomonas reinhardtii in response to osmotic stress.

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.

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.

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.