Publications by authors named "Barbara Baldan"

39 Publications

Evolution of isoprene emission in Arecaceae (palms).

Evol Appl 2021 Apr 14;14(4):902-914. Epub 2020 Dec 14.

Department of Biodiversity and Molecular Ecology, Research and Innovation Centre Fondazione Edmund Mach San Michele all'Adige Italy.

Isoprene synthase (IspS) is the sole enzyme in plants responsible for the yearly emission in the atmosphere of thousands of tonnes of the natural hydrocarbon isoprene worldwide. Species of the monocotyledonous family Arecaceae (palms) are among the highest plant emitters, but to date no gene from this family has been identified. Here, we screened with PTR-ToF-MS 18 genera of the Arecaceae for isoprene emission and found that the majority of the sampled species emits isoprene. Putative genes from six different genera were sequenced and three of them were functionally characterized by heterologous overexpression in , demonstrating that they encode functional IspS genes. Site-directed mutagenesis and expression in Arabidopsis demonstrated the functional relevance of a novel IspS diagnostic tetrad from Arecaceae, whose most variable amino acids could not preserve catalytic function when substituted by a putatively dicotyledonous-specific tetrad. In particular, mutation of threonine 479 likely impairs the open-closed transition of the enzyme by altering the network of hydrogen bonds between helices H1α, H, and I. These results shed new light on the evolution of IspS in monocots, suggesting that isoprene emission is an ancestral trait within the Arecaceae family. The identification of IspS from Arecaceae provides promising novel enzymes for the production of isoprene in heterologous systems and allows the screening and selection of commercially relevant palm varieties with lower environmental impact.
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http://dx.doi.org/10.1111/eva.13169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061277PMC
April 2021

Taxonomic Re-Examination of Nine Types (Ascomycota, Xylariales) Stored in the Saccardo Mycological Collection.

Microorganisms 2021 Mar 23;9(3). Epub 2021 Mar 23.

Botanical Garden, University of Padova, Via Orto Botanico, 15, 35123 Padova, Italy.

In a recent monograph on the genus , type specimens worldwide were revised and re-classified using a morphological approach. Among them, some came from Pier Andrea Saccardo's fungarium stored in the Herbarium of the Padova Botanical Garden. In this work, we taxonomically re-examine via a morphological and molecular approach nine different Saccardo types. ITS1 and/or ITS2 sequences were successfully obtained applying Illumina MiSeq technology and phylogenetic analyses were carried out in order to elucidate their current taxonomic position. Only the ITS1 sequence was recovered for , while for , only the ITS2 sequence was recovered. We proposed here new combinations for , and , while for , , , and , we did not suggest taxonomic changes compared to the current ones. The name Sacc. is invalid, as it is a later homonym of P. Karst. & Starbäck. Therefore, we introduced as a nomen novum for Sacc. This is the first time that these types have been subjected to a molecular study. Our results demonstrate that old types are an important source of DNA sequence data for taxonomic re-examinations.
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http://dx.doi.org/10.3390/microorganisms9030666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005106PMC
March 2021

Arabidopsis Photosynthetic and Heterotrophic Cell Suspension Cultures.

Methods Mol Biol 2021 ;2200:167-185

Department of Biology, University of Padova, Padova, Italy.

Cell suspension cultures represent a widely used experimental tool suitable to perform a variety of structural and physiological studies in a more simplified system compared to the organism in toto. In this chapter we describe the methods routinely used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures, containing either chloroplasts or amyloplasts, respectively. The use of these in vitro systems may allow to obtain insights into the unique features of chloroplasts versus non-green plastids, as well as their integration in the structural and metabolic compartmentalization of the plant cell.
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http://dx.doi.org/10.1007/978-1-0716-0880-7_8DOI Listing
March 2021

FePO NPs Are an Efficient Nutritional Source for Plants: Combination of Nano-Material Properties and Metabolic Responses to Nutritional Deficiencies.

Front Plant Sci 2020 30;11:586470. Epub 2020 Sep 30.

Department of Biotechnology, University of Verona, Verona, Italy.

Phosphorous and iron are a macro- and micronutrient, respectively, whose low bioavailability can negatively affect crop productivity. There is ample evidence that the use of conventional P and Fe fertilizers has several environmental and economical disadvantages, but even though great expectations surround nanotechnology and its applications in the field of plant nutrition, little is known about the mechanisms underlying the uptake and use of these sub-micron particles (nanoparticles, NPs) by crop species. This work shows that cucumber and maize plants both use the nutrients borne by FePO NPs more efficiently than those supplied as bulk. However, morpho-physiological parameters and nutrient content analyses reveal that while cucumber plants (a species with regard to Fe acquisition) mainly use these NPs as a source of P, maize (a species) uses them preferentially for Fe. TEM analyses of cucumber root specimens revealed no cell internalization of the NPs. On the other hand, electron-dense nanometric structures were evident in proximity of the root epidermal cell walls of the NP-treated plants, which after ESEM/EDAX analyses can be reasonably identified as iron-oxyhydroxide. It appears that the nutritional interaction between roots and NPs is strongly influenced by species-specific metabolic responses.
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http://dx.doi.org/10.3389/fpls.2020.586470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554371PMC
September 2020

The Rare Sugar Tagatose Differentially Inhibits the Growth of and by Interfering With Mitochondrial Processes.

Front Microbiol 2020 6;11:128. Epub 2020 Feb 6.

Department of Sustainable Agro-Ecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy.

Rare sugars are monosaccharides with limited availability in nature and their biological functions are largely unknown. Among them, tagatose was developed as a low-calorie sweetener and showed beneficial effects on human health. Tagatose is metabolized by only certain microbial taxa and inhibits the growth of important crop pathogens (e.g., ), but its mode of action and the microbial responses are unknown. The aim of this study was to understand the tagatose mode of action against spp., with the final aim of developing new plant protection products. Tagatose inhibited growth and caused severe ultrastructural alterations, with the formation of circular and concentric mitochondrial cristae. Decreased ATP content and reduced oxygen consumption rate (OCR) were found in tagatose-incubated as compared to the control, with the consequent accumulation of reactive oxygen species (ROS) and induction of genes related to apoptosis and oxidative stress response. On the other hand, tagatose did not, or only slightly, affect the growth, cellular ultrastructure and mitochondrial processes in , indicating a species-specific response to this rare sugar. The mode of action of tagatose against was mainly based on the inhibition of mitochondrial processes and this rare sugar seems to be a promising active substance for the further development of eco-friendly fungicides, thanks to its anti-nutritional properties on some phytopathogens and low risk for human health.
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http://dx.doi.org/10.3389/fmicb.2020.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015900PMC
February 2020

FePO nanoparticles produced by an industrially scalable continuous-flow method are an available form of P and Fe for cucumber and maize plants.

Sci Rep 2019 08 2;9(1):11252. Epub 2019 Aug 2.

Department of Biotechnology, University of Verona, Verona, Italy.

Nanomaterials are widely used in medical and pharmaceutical fields, but their application in plant nutrition is at its infancy. Phosphorous (P) and iron (Fe) are essential mineral nutrients limiting in a wide range of conditions the yield of crops. Phosphate and Fe fertilizers to-date on the market display low efficiency (P fertilizers) and low persistence in soil (Fe fertilizers) and negatively affect the environment. In the tentative to overcome these problems, we developed a continuous industrially scalable method to produce FePO NPs based on the rapid mixing of salt solutions in a mixing chamber. The process, that included the addition of citrate as capping agent allowed to obtain a stable suspension of NPs over the time. The NPs were tested for their effectiveness as P and Fe sources on two hydroponically grown crop species (cucumber and maize) comparing their effects to those exerted by non-nanometric FePO (bulk FePO). The results showed that FePO NPs improved the availability of P and Fe, if compared to the non-nano counterpart, as demonstrated by leaf SPAD indexes, fresh biomasses and P and Fe contents in tissues. The results open a new avenue in the application of nanosized material in the field of plant nutrition and fertilization.
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http://dx.doi.org/10.1038/s41598-019-47492-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677738PMC
August 2019

Plant biodiversity and regulation of photosynthesis in the natural environment.

Planta 2019 Apr 3;249(4):1217-1228. Epub 2019 Jan 3.

Department of Biology, University of Padova, Via Ugo Bassi 58B, 35121, Padua, Italy.

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.
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http://dx.doi.org/10.1007/s00425-018-03077-zDOI Listing
April 2019

Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera.

BMC Microbiol 2018 10 16;18(1):133. Epub 2018 Oct 16.

Botanical Garden and Department of Biology, University of Padova, Padova, Italy.

Background: Bacillus licheniformis GL174 is a culturable endophytic strain isolated from Vitis vinifera cultivar Glera, the grapevine mainly cultivated for the Prosecco wine production. This strain was previously demonstrated to possess some specific plant growth promoting traits but its endophytic attitude and its role in biocontrol was only partially explored. In this study, the potential biocontrol action of the strain was investigated in vitro and in vivo and, by genome sequence analyses, putative functions involved in biocontrol and plant-bacteria interaction were assessed.

Results: Firstly, to confirm the endophytic behavior of the strain, its ability to colonize grapevine tissues was demonstrated and its biocontrol properties were analyzed. Antagonism test results showed that the strain could reduce and inhibit the mycelium growth of diverse plant pathogens in vitro and in vivo. The strain was demonstrated to produce different molecules of the lipopeptide class; moreover, its genome was sequenced, and analysis of the sequences revealed the presence of many protein-coding genes involved in the biocontrol process, such as transporters, plant-cell lytic enzymes, siderophores and other secondary metabolites.

Conclusions: This step-by-step analysis shows that Bacillus licheniformis GL174 may be a good biocontrol agent candidate, and describes some distinguished traits and possible key elements involved in this process. The use of this strain could potentially help grapevine plants to cope with pathogen attacks and reduce the amount of chemicals used in the vineyard.
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http://dx.doi.org/10.1186/s12866-018-1306-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192205PMC
October 2018

The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Triggers a NAADP-Mediated Calcium Signalling Pathway in .

Int J Mol Sci 2018 Sep 1;19(9). Epub 2018 Sep 1.

Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.

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 , which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca increase that dissipated within 30 min, followed by the activation of the defence-related genes , , and . The Ca-dependence of these gene expression was demonstrated by using the extracellular Ca 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 Ca 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 Ca-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 Ca release in a signalling pathway triggered by a biotic stimulus.
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http://dx.doi.org/10.3390/ijms19092596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164116PMC
September 2018

Chloroplast Ca Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes.

Plant Physiol 2018 05 20;177(1):38-51. Epub 2018 Mar 20.

Department of Biology, University of Padova, 35131 Padova, Italy

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.
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http://dx.doi.org/10.1104/pp.18.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933129PMC
May 2018

Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection.

Plant Cell Rep 2017 Dec 14;36(12):1917-1928. Epub 2017 Sep 14.

Department of Environmental Biology, University of Rome La Sapienza, Piazzale A. Moro 5, 00185, Rome, Italy.

Key Message: PLGA NPs' cell uptake involves different endocytic pathways. Clathrin-independent endocytosis is the main internalization route. The cell wall plays a more prominent role than the plasma membrane in NPs' size selection. In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500-600 nm.
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http://dx.doi.org/10.1007/s00299-017-2206-0DOI Listing
December 2017

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

Front Plant Sci 2017 18;8:1444. Epub 2017 Aug 18.

Department of Biology, University of PadovaPadova, Italy.

Photosynthetic cell suspension cultures are a useful experimental system to analyze a variety of physiological processes, bypassing the structural complexity of the plant organism . 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 was developed. The direct germination of 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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http://dx.doi.org/10.3389/fpls.2017.01444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563381PMC
August 2017

Impact of Phenylpropanoid Compounds on Heat Stress Tolerance in Carrot Cell Cultures.

Front Plant Sci 2016 22;7:1439. Epub 2016 Sep 22.

Department of Biotechnology, University of Verona Verona, Italy.

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated , focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on projection to latent structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton.
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http://dx.doi.org/10.3389/fpls.2016.01439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031593PMC
September 2016

Characterization of lipopeptides produced by Bacillus licheniformis using liquid chromatography with accurate tandem mass spectrometry.

Rapid Commun Mass Spectrom 2016 Oct;30(20):2237-52

Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy.

Rationale: The plant endophyte Bacillus licheniformis, isolated from leaves of Vitis vinifera, was studied to individuate and characterize the presence of bioactive lipopeptides having amino acidic structures.

Methods: Crude extracts of liquid cultures were analyzed by ultra-high-performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight (QTOF) mass analyzer. Chromatographic conditions were optimized in order to obtain an efficient separation of the different isobaric lipopeptides, avoiding merged fragmentations of co-eluted isomeric compounds and reducing possible cross-talk phenomena. Composition of the amino acids was outlined through the interpretation of the fragmentation behavior in tandem high-resolution mass spectrometry (HRMS/MS) mode, which showed both common-class and peculiar fragment ions. Both [M + H](+) and [M + Na](+) precursor ions were fragmented in order to differentiate some isobaric amino acids, i.e. Leu/Ile. Neutral losses characteristic of the iso acyl chain were also evidenced.

Results: More than 90 compounds belonging to the classes of surfactins and lichenysins, known as biosurfactant molecules, were detected. Sequential LC/HRMS/MS analysis was used to identify linear and cyclic lipopeptides, and to single out the presence of a large number of isomers not previously reported. Some critical issues related to the simultaneous selection of different compounds by the quadrupole filter were highlighted and partially solved, leading to tentative assignments of several structures. Linear lichenysins are described here for the first time.

Conclusions: The approach was proved to be useful for the characterization of non-target lipopeptides, and proposes a rationale MS experimental scheme aimed to investigate the difference in amino acid sequence and/or in the acyl chain of the various congeners, when standards are not available. Results expanded the knowledge about production of linear and cyclic bioactive compounds from Bacillus licheniformis, clarifying the structures of isomeric forms, and enabling the use of selected endophytes to produce fungicides for eco-friendly biocontrol. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/rcm.7705DOI Listing
October 2016

Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots.

PLoS One 2015 16;10(10):e0140252. Epub 2015 Oct 16.

Dipartimento di Biologia, Universita degli Studi di Padova, Padova, Italy.

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140252PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652591PMC
June 2016

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits.

J Exp Bot 2015 Dec 1;66(22):7255-70. Epub 2015 Oct 1.

Department of Agronomy, Food, Natural resources, Animals and Environment (DAFNAE), University of Padova, 35020 Legnaro, Italy

Apple (Malus×domestica Borkh) fruits are stored for long periods of time at low temperatures (1 °C) leading to the occurrence of physiological disorders. 'Superficial scald' of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H2O2 homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H2O2 homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress.
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http://dx.doi.org/10.1093/jxb/erv422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765793PMC
December 2015

Characterization of an AGAMOUS gene expressed throughout development of the fleshy fruit-like structure produced by Ginkgo biloba around its seeds.

BMC Evol Biol 2015 Jul 16;15:139. Epub 2015 Jul 16.

Department of Biology, University of Padua, Via G. Colombo, 3, 35131, Padua, Italy.

Background: The involvement of MADS-box genes of the AGAMOUS lineage in the formation of both flowers and fruits has been studied in detail in Angiosperms. AGAMOUS genes are expressed also in the reproductive structures of Gymnosperms, yet the demonstration of their role has been problematic because Gymnosperms are woody plants difficult to manipulate for physiological and genetic studies. Recently, it was shown that in the gymnosperm Ginkgo biloba an AGAMOUS gene was expressed throughout development and ripening of the fleshy fruit-like structures produced by this species around its seeds. Such fleshy structures are evolutionarily very important because they favor the dispersal of seeds through endozoochory. In this work a characterization of the Ginkgo gene was carried out by over-expressing it in tomato.

Results: In tomato plants ectopically expressing the Ginkgo AGAMOUS gene a macroscopic anomaly was observed only in the flower sepals. While the wild type sepals had a leaf-like appearance, the transgenic ones appeared connately adjoined at their proximal extremity and, concomitant with the development and ripening of the fruit, they became thicker and acquired a yellowish-orange color, thus indicating that they had undergone a homeotic transformation into carpel-like structures. Molecular analyses of several genes associated with either the control of ripening or the ripening syndrome in tomato fruits confirmed that the transgenic sepals behaved like ectopic fruits that could undergo some ripening, although the red color typical of the ripe tomato fruit was never achieved.

Conclusions: The ectopic expression of the Ginkgo AGAMOUS gene in tomato caused the homeotic transformation of the transgenic sepals into carpel-like structures, and this showed that the gymnosperm gene has a genuine C function. In parallel with the ripening of fruits the related transgenic sepals became fleshy fruit-like structures that also underwent some ripening and such a result indicates that this C function gene might be involved, together with other gens, also in the development of the Ginkgo fruit-like structures. It seems thus strengthened the hypothesis that AGAMOUS MADS-box genes were recruited already in Gymnosperms for the development of the fleshy fruit habit which is evolutionarily so important for the dispersal of seeds.
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http://dx.doi.org/10.1186/s12862-015-0418-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502469PMC
July 2015

AtSIA1 AND AtOSA1: two Abc1 proteins involved in oxidative stress responses and iron distribution within chloroplasts.

New Phytol 2014 Jan 4;201(2):452-465. Epub 2013 Oct 4.

Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada Le Grazie 15, Verona, Italy.

The Abc1 protein kinases are a large family of functionally diverse proteins with multiple roles in the regulation of respiration and oxidative stress tolerance. A functional characterization was carried out for AtSIA1, an Arabidopsis thaliana Abc1-like protein, focusing on its potential redundancy with its homolog AtOSA1. Both proteins are located within chloroplasts, even if a different subplastidial localization seems probable. The comparison of atsia1 and atosa1 mutants, atsia1/atosa1 double mutant and wild-type plants revealed a reduction in plastidial iron-containing proteins of the Cytb6 f complex in the mutants. Iron uptake from soil is not hampered in mutant lines, suggesting that AtSIA1 and AtOSA1 affect iron distribution within the chloroplast. Mutants accumulated more ferritin and superoxide, and showed reduced tolerance to reactive oxygen species (ROS), potentially indicating a basal role in oxidative stress. The mutants produced higher concentrations of plastochromanol and plastoquinones than wild-type plants, but only atsia1 plants developed larger plastoglobules and contained higher concentrations of α- and γ-tocopherol and VTE1. Taken together, these data suggest that AtSIA1 and AtOSA1 probably act in signaling pathways that influence responses to ROS production and oxidative stress.
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http://dx.doi.org/10.1111/nph.12533DOI Listing
January 2014

A unique midgut-associated bacterial community hosted by the cave beetle Cansiliella servadeii (Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors.

BMC Microbiol 2013 Jun 10;13:129. Epub 2013 Jun 10.

Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35131 Padova, Italy.

Background: Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches.

Results: Abundant microbial cell masses were observed on the external appendages. Cansiliella's midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals' digestive traits.

Conclusions: Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.
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http://dx.doi.org/10.1186/1471-2180-13-129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695770PMC
June 2013

Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules.

Arch Microbiol 2013 Jun 10;195(6):385-91. Epub 2013 Apr 10.

C.N.R., Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo ISPAAM, Traversa La Crucca 3, Località Baldinca, Li Punti, Sassari, Italy.

In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants.
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http://dx.doi.org/10.1007/s00203-013-0886-6DOI Listing
June 2013

Plant cell suspension cultures.

Methods Mol Biol 2013 ;953:77-93

Department of Biology, University of Padova, Padova, Italy.

Plant cell suspension cultures are widely used in plant biology as a convenient tool for the investigation of a wide range of phenomena, bypassing the structural complexity of the plant organism in toto. The homogeneity of an in vitro cell population, the large availability of material, the high rate of cell growth and the good reproducibility of conditions make suspension-cultured cells suitable for the analysis of complex physiological processes at the cellular and molecular levels. Moreover, plant cell cultures provide a valuable platform for the production of high-value secondary metabolites and other substances of commercial interest. Here we describe how to initiate and maintain plant cell cultures starting from explants obtained from in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and regeneration of plants via organogenesis and somatic embryogenesis are also presented.
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http://dx.doi.org/10.1007/978-1-62703-152-3_5DOI Listing
March 2013

Oligogalacturonides: novel signaling molecules in Rhizobium-legume communications.

Mol Plant Microbe Interact 2012 Nov;25(11):1387-95

Dipartimento di Biologia, Universita di Padova, Padova, Italy.

Oligogalacturonides are pectic fragments of the plant cell wall, whose signaling role has been described thus far during plant development and plant-pathogen interactions. In the present work, we evaluated the potential involvement of oligogalacturonides in the molecular communications between legumes and rhizobia during the establishment of nitrogen-fixing symbiosis. Oligogalacturonides with a degree of polymerization of 10 to 15 were found to trigger a rapid intracellular production of reactive oxygen species in Rhizobium leguminosarum bv. viciae 3841. Accumulation of H(2)O(2), detected by both 2',7'-dichlorodihydrofluorescein diacetate-based fluorescence and electron-dense deposits of cerium perhydroxides, was transient and did not affect bacterial cell viability, due to the prompt activation of the katG gene encoding a catalase. Calcium measurements carried out in R. leguminosarum transformed with the bioluminescent Ca(2+) reporter aequorin demonstrated the induction of a rapid and remarkable intracellular Ca(2+) increase in response to oligogalacturonides. When applied jointly with naringenin, oligogalacturonides effectively inhibited flavonoid-induced nod gene expression, indicating an antagonistic interplay between oligogalacturonides and inducing flavonoids in the early stages of plant root colonization. The above data suggest a novel role for oligogalacturonides as signaling molecules released in the rhizosphere in the initial rhizobium-legume interaction.
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http://dx.doi.org/10.1094/MPMI-03-12-0066-RDOI Listing
November 2012

Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall.

Fungal Biol 2012 Apr 28;116(4):543-9. Epub 2012 Feb 28.

Dipartimento di Biotecnologie Agrarie, Università di Padova, Legnaro, Padova, Italy.

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group.
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http://dx.doi.org/10.1016/j.funbio.2012.02.004DOI Listing
April 2012

Sesuvium portulacastrum maintains adequate gas exchange, pigment composition, and thylakoid proteins under moderate and high salinity.

J Plant Physiol 2010 Nov;167(16):1336-41

Laboratory Plant Adaptation to Abiotic Stresses (LAPSA), Biotechnology Centre of Borj Cedria, P.O. Box 901, 2050 Hammam-Lif, Tunisia.

Cuttings of Sesuvium portulacastrum L. (Aizoaceae) were taken from plants cultivated under severe saline conditions. The obtained seedlings were grown on sand and irrigated with nutrient solution over 5 weeks under no (0 mM NaCl), moderate (200 mM NaCl), or high (400 mM NaCl) salinity conditions. A follow-up of gas exchange was performed weekly and pigment levels and patterns of fully expanded leaves were determined after 3 and 5 weeks of treatment. At the end of the 5-week period, immunoblot analysis of the main polypeptides of photosystem I and II was performed with the aim to investigate salt-induced variations in photosystem composition. Net CO2 assimilation rate (Pn) increased under salinity up to 3 weeks of treatment then decreased to reach the value of 0mM-treated plants at the end of the experiment. For stomatal conductance (gs) and intercellular CO2 concentration (Ci), the opposite occurred. These results were concomitant with an increase in practically all pigment levels, mainly under high salinity, with the exception of zeaxanthin. The de-epoxidation index (DEPS index) was much lower under saline than non-saline conditions in the 3rd week, indicating light stress in 0mM-treated plants. At the end of the experiment, this index showed much lower values with no significant differences between treatments, which coincided with no significant differences in gas exchange as well. Protein amounts of D1, CP47, and CP43 did not show noticeable variations with salt treatment, whereas LHCII underwent a slight but significant decrease (-15%) at the highest NaCl concentration. LHCI polypeptides were unaffected by the salt treatments, where conversely, the highest concentration induced a significant decrease in PsaA/B amount (-18%).
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http://dx.doi.org/10.1016/j.jplph.2010.05.009DOI Listing
November 2010

Programmed cell death and adaptation: two different types of abiotic stress response in a unicellular chlorophyte.

Plant Cell Physiol 2010 Jun 10;51(6):884-95. Epub 2010 May 10.

Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35131 Padova, Italy.

Eukaryotic microalgae are highly suitable biological indicators of environmental changes because they are exposed to extreme seasonal fluctuations. The biochemical and molecular targets and regulators of key proteins involved in the stress response in microalgae have yet to be elucidated. This study presents morphological and biochemical evidence of programmed cell death (PCD) in a low temperature strain of Chlorella saccharophila induced by exposure to NaCl stress. Morphological characteristics of PCD, including cell shrinkage, detachment of the plasma membrane from the cell wall, nuclear condensation and DNA fragmentation, were observed. Additionally, a significant production of H(2)O(2) and increase in caspase 3-like activity were detected. We demonstrated that singly applied environmental stresses such as warming or salt stress trigger a pathway of PCD. Intriguingly, the prior application of salt stress seems to reduce heat shock-induced cell death significantly, suggesting a combined effect which activates a defense mechanism in algal cells. These results suggest that C. saccharophila can undergo PCD under stress conditions, and that this PCD shares several features with metazoan PCD. Moreover, the simultaneous exposure of this unicellular chlorophyte to different abiotic stresses results in a tolerance mechanism.
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http://dx.doi.org/10.1093/pcp/pcq069DOI Listing
June 2010

Identification of two fungal endophytes associated with the endangered orchid Orchis militaris L.

J Microbiol Biotechnol 2010 Mar;20(3):630-6

Dipartimento di Biotecnologie Agrarie, Università di Padova, viale dell'Università 16, 35020 Legnaro, Padova, Italy.

A survey of the endangered orchid Orchis militaris populations was carried out in north-eastern Italy. The occurrence of fungal root endophytes was investigated by light and electron microscopies and molecular techniques. Two main sites of presence were individuated in the Euganean Hills, differing as to the percentage of flowering individuals and of capsules completing maturity. Fluorescence microscopy revealed an intracellular cortical colonization by hyphal pelotons. Two ITS PCR products co-amplified. Sequencing revealed for the former an identity and a high similarity (99%) with a Tulasnellaceae (Basidiomycota) fungus found within tissues of the same host in independent studies in Hungary and Estonia, suggesting an interesting case of tight specificity throughout the Eurosiberian home range. The second amplicon had 99% similarity with Tetracladium species (Ascomycota) recently demonstrated as potential endophytes. TEM revealed two different hyphal structures. Double fungal colonization appears to occur in Orchis militaris and the possible requirement of a specific fungal partner throws light on the causes of this plant's rarity and threatened status.
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March 2010

Chlorella saccharophila cytochrome f and its involvement in the heat shock response.

J Exp Bot 2009 22;60(14):4189-200. Epub 2009 Sep 22.

Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35131 Padova, Italy.

Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes.
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http://dx.doi.org/10.1093/jxb/erp264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755035PMC
February 2010

Phosphate availability affects the tonoplast localization of PLDzeta2, an Arabidopsis thaliana phospholipase D.

FEBS Lett 2008 Mar 31;582(5):685-90. Epub 2008 Jan 31.

Laboratoire de Physiologie Cellulaire Végétale, CNRS, CEA, INRA, Université J. Fourier, CEA-Grenoble, iRTSV/LPCV, 17 Rue des Martyrs, Grenoble, France.

Under phosphate deprivation, higher plants change their lipid composition and recycle phosphate from phospholipids. A phospholipase D, PLDzeta2, is involved in this recycling and in other cellular functions related to plant development. We investigated the localization of Arabidopsis PLDzeta2 by cell fractionation and in vivo GFP confocal imaging. AtPLDzeta2 localizes to the tonoplast and the Nter regulatory domain is sufficient for its sorting. Under phosphate deprivation, AtPLDzeta2 remains located in the tonoplast but its distribution is uneven. We observed PLDzeta2-enriched tonoplast domains preferentially positioned close to mitochondria and beside chloroplasts. In absence of PLDzeta2, membrane developments were visualized inside vacuoles.
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http://dx.doi.org/10.1016/j.febslet.2008.01.039DOI Listing
March 2008

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes.

FEMS Microbiol Ecol 2008 Mar 9;63(3):383-400. Epub 2008 Jan 9.

Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università degli Studi di Sassari, Sassari, Italy.

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.
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http://dx.doi.org/10.1111/j.1574-6941.2007.00424.xDOI Listing
March 2008

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

BMC Plant Biol 2007 Jul 30;7:41. Epub 2007 Jul 30.

Dipartimento di Biologia, Università di Padova, Padova, Italy.

Background: 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.

Results: Here, 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.

Conclusion: New 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.
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http://dx.doi.org/10.1186/1471-2229-7-41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950503PMC
July 2007