Publications by authors named "Michela Zottini"

34 Publications

In Vivo NADH/NAD Biosensing Reveals the Dynamics of Cytosolic Redox Metabolism in Plants.

Plant Cell 2020 10 13;32(10):3324-3345. Epub 2020 Aug 13.

Institute of Plant Biology and Biotechnology (IBBP), Westfälische Wilhelms-Universität Münster, D-48143 Münster, Germany

NADH and NAD are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis () and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism.
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http://dx.doi.org/10.1105/tpc.20.00241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534465PMC
October 2020

WHIRLY2 plays a key role in mitochondria morphology, dynamics, and functionality in .

Plant Direct 2020 May 30;4(5):e00229. Epub 2020 May 30.

Department of Biology University of Padova Padova Italy.

WHIRLY2 is a single-stranded DNA binding protein associated with mitochondrial nucleoids. In the mutant of , a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild-type and mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the mutant. In line with that, the expression level of in seeds of wild-type plants is higher than that of , whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the mutant, although not in seeds, the expression level of is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of and predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant.
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http://dx.doi.org/10.1002/pld3.229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261051PMC
May 2020

Genome communication in plants mediated by organelle-n-ucleus-located proteins.

Philos Trans R Soc Lond B Biol Sci 2020 06 4;375(1801):20190397. Epub 2020 May 4.

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

An increasing number of eukaryotic proteins have been shown to have a dual localization in the DNA-containing organelles, mitochondria and plastids, and/or the nucleus. Regulation of dual targeting and relocation of proteins from organelles to the nucleus offer the most direct means for communication between organelles as well as organelles and nucleus. Most of the mitochondrial proteins of animals have functions in DNA repair and gene expression by modelling of nucleoid architecture and/or chromatin. In plants, such proteins can affect replication and early development. Most plastid proteins with a confirmed or predicted second location in the nucleus are associated with the prokaryotic core RNA polymerase and are required for chloroplast development and light responses. Few plastid-nucleus-located proteins are involved in pathogen defence and cell cycle control. For three proteins, it has been clearly shown that they are first targeted to the organelle and then relocated to the nucleus, i.e. the nucleoid-associated proteins HEMERA and Whirly1 and the stroma-located defence protein NRIP1. Relocation to the nucleus can be experimentally demonstrated by plastid transformation leading to the synthesis of proteins with a tag that enables their detection in the nucleus or by fusions with fluoroproteins in different experimental set-ups. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.
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http://dx.doi.org/10.1098/rstb.2019.0397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7209962PMC
June 2020

Exploring the potential of vineyards for biodiversity conservation and delivery of biodiversity-mediated ecosystem services: A global-scale systematic review.

Sci Total Environ 2020 Mar 5;706:135839. Epub 2019 Dec 5.

University of Bologna, Dept. of Biological, Geological and Environmental Sciences, Via Irnerio 42, I-40126 Bologna, Italy.

Vineyards are experiencing strong expansion and management intensification worldwide, especially in areas with a Mediterranean climate, which are often characterized by a high conservation value. This is posing concerns about their environmental impact and it is fostering research on biodiversity patterns and ecosystem services in this agroecosystem. With this systematic review, we aim at providing a global and comprehensive overview of the current research on biodiversity and biodiversity-mediated ecosystem services in vineyards, considering the effects of landscape features and management practices. We carried out a systematic literature search on the Web of Science Core Collection database. Literature was filtered according to several criteria, resulting in a final collection of 218 papers published between 1995 and 2018 and referring to different organism groups (from microbes to vertebrates) and two spatial scales (local and landscape). The results of the studies are often contrasting and taxon- and scale-dependent, thus hindering conclusions at the global scale. However, at least three main points of practical relevance can be fixed: (i) organic viticulture weakly enhances biodiversity at the landscape scale, whereas contrasting effects have been found at the local scale; (ii) ground vegetation management by cover cropping and the conservation of native ground cover strongly promotes biodiversity; (iii) habitat heterogeneity at the landscape and local scales is a key element for biodiversity. Several studies support the view that promoting biodiversity in vineyard-dominated landscapes could also positively impact on several ecosystem services. Our study further revealed knowledge gaps that should be filled by future research. In particular, important geographical areas for wine production, as well as several organism groups, have been completely neglected. Studies at the landscape level are still scarce (specifically those addressing landscape configuration), and also the research about supporting, provisioning, and cultural biodiversity-mediated ecosystem services is still in its infancy.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135839DOI Listing
March 2020

The Role of the Endophytic Microbiome in the Grapevine Response to Environmental Triggers.

Front Plant Sci 2019 9;10:1256. Epub 2019 Oct 9.

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

Endophytism within represents a topic of critical relevance due to the multiple standpoints from which it can be approached and considered. From the biological and botanical perspectives, the interaction between microorganisms and perennial woody plants falls within the category of stable relationships from which the plants can benefit in multiple ways. The life cycle of the host ensures persistence in all seasons, repeated chances of contact, and consequent microbiota accumulation over time, leading to potentially high diversity compared with that of herbaceous short-lived plants. Furthermore, grapevines are agriculturally exploited, highly selected germplasms where a profound man-driven footprint has indirectly and unconsciously shaped the inner microbiota through centuries of cultivation and breeding. Moreover, since endophyte metabolism can contribute to that of the plant host and its fruits' biochemical composition, the nature of grapevine endophytic taxa identities, ecological attitudes, potential toxicity, and clinical relevance are aspects worthy of a thorough investigation. Can endophytic taxa efficiently defend grapevines by acting against pests or confer enough fitness to the plants to endure attacks? What are the underlying mechanisms that translate into this or other advantages in the hosting plant? Can endophytes partially redirect plant metabolism, and to what extent do they act by releasing active products? Is the inner microbial colonization necessary priming for a cascade of actions? Are there defined environmental conditions that can trigger the unleashing of key microbial phenotypes? What is the environmental role in providing the ground biodiversity by which the plant can recruit microsymbionts? How much and by what practices and strategies can these symbioses be managed, applied, and directed to achieve the goal of a better sustainable viticulture? By thoroughly reviewing the available literature in the field and critically examining the data and perspectives, the above issues are discussed.
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http://dx.doi.org/10.3389/fpls.2019.01256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794716PMC
October 2019

Overexpression of 14-3-3 proteins enhances cold tolerance and increases levels of stress-responsive proteins of Arabidopsis plants.

Plant Sci 2019 Dec 28;289:110215. Epub 2019 Sep 28.

Department of Biology, University of Rome Tor Vergata, 00133, Rome, Italy.

14-3-3 proteins are a family of conserved proteins present in eukaryotes as several isoforms, playing a regulatory role in many cellular and physiological processes. In plants, 14-3-3 proteins have been reported to be involved in the response to stress conditions, such as drought, salt and cold. In the present study, 14-3-3ε and 14-3-3ω isoforms, which were representative of ε and non-ε phylogenetic groups, were overexpressed in Arabidopsis thaliana plants; the effect of their overexpression was investigated on H-ATPase activation and plant response to cold stress. Results demonstrated that H-ATPase activity was increased in 14-3-3ω-overexpressing plants, whereas overexpression of both 14-3-3 isoforms brought about cold stress tolerance, which was evaluated through ion leakage, lipid peroxidation, osmolyte synthesis, and ROS production assays. A dedicated tandem mass tag (TMT)-based proteomic analysis demonstrated that different proteins involved in the plant response to cold or oxidative stress were over-represented in 14-3-3ε-overexpressing plants.
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http://dx.doi.org/10.1016/j.plantsci.2019.110215DOI Listing
December 2019

Phosphorylation of p23-1 cochaperone by protein kinase CK2 affects root development in Arabidopsis.

Sci Rep 2019 07 8;9(1):9846. Epub 2019 Jul 8.

Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, I-35131, Padova, Italy.

Root growth is a fundamental process in plants and assures nutrient and water uptake required for efficient photosynthesis and metabolism. Postembryonic development of roots is controlled by the functionality of the meristem. Several hormones and signaling molecules regulate the size of the meristem, and among them, auxins play a major role. Protein kinase CK2, along with the chaperone protein HSP90, has been found to be involved in the regulation of auxin transport. Here, we show that p23-1, a cochaperone of HSP90, is phosphorylated by CK2 in Arabidopsis. We identified Ser201 as the major CK2 target site in p23-1 and demonstrated that phosphorylation of this site is necessary for normal root development. Moreover, we shed light on the nature of CK2 in Arabidopsis, showing that the three catalytic isoforms, CK2 αA, αB and αC, are proteins of approximately 40 kDa. Our results increase knowledge of the connection among HSP90, p23-1 and CK2 in Arabidopsis, suggesting the existence of a possible common root development mechanism controlled by these signaling molecules.
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http://dx.doi.org/10.1038/s41598-019-46327-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614504PMC
July 2019

HO Signature and Innate Antioxidative Profile Make the Difference Between Sensitivity and Tolerance to Salt in Rice Cells.

Front Plant Sci 2018 23;9:1549. Epub 2018 Oct 23.

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

Salt tolerance is a complex trait that varies between and within species. HO profiles as well as antioxidative systems have been investigated in the cultured cells of rice obtained from Italian rice varieties with different salt tolerance. Salt stress highlighted differences in extracellular and intracellular HO profiles in the two cell cultures. The tolerant variety had innate reactive oxygen species (ROS) scavenging systems that enabled ROS, in particular HO, to act as a signal molecule rather than a damaging one. Different intracellular HO profiles were also observed: in tolerant cells, an early and narrow peak was detected at 5 min; while in sensitive cells, a large peak was associated with cell death. Likewise, the transcription factor salt-responsive ethylene responsive factor 1 (TF SERF1), which is known for being regulated by HO, showed a different expression profile in the two cell lines. Notably, similar HO profiles and cell fates were also obtained when exogenous HO was produced by glucose/glucose oxidase (GOX) treatment. Under salt stress, the tolerant variety also exhibited rapid upregulation of K transporter genes in order to deal with K/Na impairment. This upregulation was not detected in the presence of oxidative stress alone. The importance of the innate antioxidative profile was confirmed by the protective effect of experimentally increased glutathione in salt-treated sensitive cells. Overall, these results underline the importance of specific HO signatures and innate antioxidative systems in modulating ionic and redox homeostasis for salt stress tolerance.
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http://dx.doi.org/10.3389/fpls.2018.01549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206305PMC
October 2018

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

Systemic Calcium Wave Propagation in Physcomitrella patens.

Plant Cell Physiol 2018 Jul;59(7):1377-1384

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

The adaptation to dehydration and rehydration cycles represents a key step in the evolution of photosynthetic organisms and requires the development of mechanisms by which to sense external stimuli and translate them into signaling components. In this study, we used genetically encoded fluorescent sensors to detect specific transient increases in the Ca2+ concentration in the moss Physcomitrella patens upon dehydration and rehydration treatment. Observation of the entire plant in a single time-series acquisition revealed that various cell types exhibited different sensitivities to osmotic stress and that Ca2+ waves originated from the basal part of the gametophore and were directionally propagated towards the top of the plant. Under similar conditions, the vascular plant Arabidopsis thaliana exhibited Ca2+ waves that propagated at a higher speed than those of P. patens. Our results suggest that systemic Ca2+ propagation occurs in plants even in the absence of vascular tissue, even though the rates can be different.
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http://dx.doi.org/10.1093/pcp/pcy104DOI Listing
July 2018

Transcriptome and Cell Physiological Analyses in Different Rice Cultivars Provide New Insights Into Adaptive and Salinity Stress Responses.

Front Plant Sci 2018 5;9:204. Epub 2018 Mar 5.

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

Salinity tolerance has been extensively investigated in recent years due to its agricultural importance. Several features, such as the regulation of ionic transporters and metabolic adjustments, have been identified as salt tolerance hallmarks. Nevertheless, due to the complexity of the trait, the results achieved to date have met with limited success in improving the salt tolerance of rice plants when tested in the field, thus suggesting that a better understanding of the tolerance mechanisms is still required. In this work, differences between two varieties of rice with contrasting salt sensitivities were revealed by the imaging of photosynthetic parameters, ion content analysis and a transcriptomic approach. The transcriptomic analysis conducted on tolerant plants supported the setting up of an adaptive program consisting of sodium distribution preferentially limited to the roots and older leaves, and in the activation of regulatory mechanisms of photosynthesis in the new leaves. As a result, plants resumed grow even under prolonged saline stress. In contrast, in the sensitive variety, RNA-seq analysis revealed a misleading response, ending in senescence and cell death. The physiological response at the cellular level was investigated by measuring the intracellular profile of HO in the roots, using a fluorescent probe. In the roots of tolerant plants, a quick response was observed with an increase in HO production within 5 min after salt treatment. The expression analysis of some of the genes involved in perception, signal transduction and salt stress response confirmed their early induction in the roots of tolerant plants compared to sensitive ones. By inhibiting the synthesis of apoplastic HO, a reduction in the expression of these genes was detected. Our results indicate that quick HO signaling in the roots is part of a coordinated response that leads to adaptation instead of senescence in salt-treated rice plants.
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http://dx.doi.org/10.3389/fpls.2018.00204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844958PMC
March 2018

Molecular analysis of the early interaction between the grapevine flower and Botrytis cinerea reveals that prompt activation of specific host pathways leads to fungus quiescence.

Plant Cell Environ 2017 Aug 12;40(8):1409-1428. Epub 2017 Apr 12.

Genomics and Biology of Fruit Crops Department, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, San Michele all'Adige, 38010, Trentino, Italy.

Grape quality and yield can be impaired by bunch rot, caused by the necrotrophic fungus Botrytis cinerea. Infection often occurs at flowering, and the pathogen stays quiescent until fruit maturity. Here, we report a molecular analysis of the early interaction between B. cinerea and Vitis vinifera flowers, using a controlled infection system, confocal microscopy and integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cultivar Pinot Noir were infected with green fluorescent protein (GFP)-labelled B. cinerea and studied at 24 and 96 hours post-inoculation (h.p.i.). We observed that penetration of the epidermis by B. cinerea coincided with increased expression of genes encoding cell-wall-degrading enzymes, phytotoxins and proteases. Grapevine responded with a rapid defence reaction involving 1193 genes associated with the accumulation of antimicrobial proteins, polyphenols, reactive oxygen species and cell wall reinforcement. At 96 h.p.i., the reaction appears largely diminished both in the host and in the pathogen. Our data indicate that the defence responses of the grapevine flower collectively are able to restrict invasive fungal growth into the underlying tissues, thereby forcing the fungus to enter quiescence until the conditions become more favourable to resume pathogenic development.
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http://dx.doi.org/10.1111/pce.12937DOI Listing
August 2017

Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma.

Plant Physiol 2016 08 1;171(4):2317-30. Epub 2016 Jun 1.

Department of Biosciences, University of Milan, 20133 Milan, Italy (G.L., F.G.D., S.B., A.C.);Department of Biology, University of Padua, Italy, 35131 Padua, Italy (G.L., M.Z.);Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, 48149 Münster, Germany (S.We., J.K.);Plant Energy Biology Lab, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 53113 Bonn, Germany (S.Wa., M.S.); andInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy (A.C.).

In eukaryotes, subcellular compartments such as mitochondria, the endoplasmic reticulum, lysosomes, and vacuoles have the capacity for Ca(2+) transport across their membranes to modulate the activity of compartmentalized enzymes or to convey specific cellular signaling events. In plants, it has been suggested that chloroplasts also display Ca(2+) regulation. So far, monitoring of stromal Ca(2+) dynamics in vivo has exclusively relied on using the luminescent Ca(2+) probe aequorin. However, this technique is limited in resolution and can only provide a readout averaged over chloroplast populations from different cells and tissues. Here, we present a toolkit of Arabidopsis (Arabidopsis thaliana) Ca(2+) sensor lines expressing plastid-targeted FRET-based Yellow Cameleon (YC) sensors. We demonstrate that the probes reliably report in vivo Ca(2+) dynamics in the stroma of root plastids in response to extracellular ATP and of leaf mesophyll and guard cell chloroplasts during light-to-low-intensity blue light illumination transition. Applying YC sensing of stromal Ca(2+) dynamics to single chloroplasts, we confirm findings of gradual, sustained stromal Ca(2+) increases at the tissue level after light-to-low-intensity blue light illumination transitions, but monitor transient Ca(2+) spiking as a distinct and previously unknown component of stromal Ca(2+) signatures. Spiking was dependent on the availability of cytosolic Ca(2+) but not synchronized between the chloroplasts of a cell. In contrast, the gradual sustained Ca(2+) increase occurred independent of cytosolic Ca(2+), suggesting intraorganellar Ca(2+) release. We demonstrate the capacity of the YC sensor toolkit to identify novel, fundamental facets of chloroplast Ca(2+) dynamics and to refine the understanding of plastidial Ca(2+) regulation.
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http://dx.doi.org/10.1104/pp.16.00652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972287PMC
August 2016

Do vineyards in contrasting landscapes contribute to conserve plant species of dry calcareous grasslands?

Sci Total Environ 2016 Mar 31;545-546:244-9. Epub 2015 Dec 31.

Department of Agronomy, Food, Natural Resources, Animals and the Environment (DAFNAE), University of Padova, viale dell'Università 16, 35020 Legnaro, Padova, Italy.

The increasing development of vineyards in Mediterranean areas worldwide is considered a major driver of conversion of several habitats of conservation concern, including calcareous dry grasslands that are targeted for biodiversity conservation by the European Union, according to Natura 2000 policies. Here, we aim at evaluating the potential of extensive vineyards located in contrasting landscapes (semi-natural vs crop-dominated) for providing suitable habitat conditions to plant species associated with dry grasslands. This study was carried out in one of the economically most important winemaking districts of Italy, characterized by a hilly landscape with steep slope vineyards. We compared plant communities of vineyards in contrasting landscapes with those of the remnants of dry grasslands. Our study demonstrates that landscape composition strongly affects local plant communities in vineyards, with a positive effect of semi-natural habitats bordering the cultivated areas. Our findings thus supply an additional tool for improving the effectiveness of viticultural landscapes for nature conservation. In particular, our results indicate that vineyards on steep slopes could provide moderate chance for the conservation of plant specialists inhabiting calcareous dry grasslands, depending on the landscape composition: vineyards embedded in semi-natural landscapes have more potential for conservation than those in crop-dominated landscapes. Our study also indicates that conservation efforts should aim at (a) decreasing the current management intensity that likely hampers the beneficial effects of semi-natural habitats in the surrounding landscape on local plant assemblages, and (b) strictly conserving the remnants of dry grasslands that are irreplaceable refugia for habitat specialists and species of conservation concern.
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http://dx.doi.org/10.1016/j.scitotenv.2015.12.051DOI Listing
March 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

The co-chaperone p23 controls root development through the modulation of auxin distribution in the Arabidopsis root meristem.

J Exp Bot 2015 Aug 10;66(16):5113-22. Epub 2015 Jul 10.

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

Homologues of the p23 co-chaperone of HSP90 are present in all eukaryotes, suggesting conserved functions for this protein throughout evolution. Although p23 has been extensively studied in animal systems, little is known about its function in plants. In the present study, the functional characterization of the two isoforms of p23 in Arabidopsis thaliana is reported, suggesting a key role of p23 in the regulation of root development. Arabidopsis p23 mutants, for either form, show a short root length phenotype with a reduced meristem length. In the root meristem a low auxin level associated with a smaller auxin gradient was observed. A decrease in the expression levels of PIN FORMED PROTEIN (PIN)1, PIN3, and PIN7, contextually to an inefficient polar localization of PIN1, was detected. Collectively these results suggest that both Arabidopsis p23 isoforms are required for root growth, in particular in the maintenance of the root meristem, where the proteins are located.
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http://dx.doi.org/10.1093/jxb/erv330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513928PMC
August 2015

Mitochondria change dynamics and morphology during grapevine leaf senescence.

PLoS One 2014 10;9(7):e102012. Epub 2014 Jul 10.

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

Leaf senescence is the last stage of development of an organ and is aimed to its ordered disassembly and nutrient reallocation. Whereas chlorophyll gradually degrades during senescence in leaves, mitochondria need to maintain active to sustain the energy demands of senescing cells. Here we analysed the motility and morphology of mitochondria in different stages of senescence in leaves of grapevine (Vitis vinifera), by stably expressing a GFP (green fluorescent protein) reporter targeted to these organelles. Results show that mitochondria were less dynamic and markedly changed morphology during senescence, passing from the elongated, branched structures found in mature leaves to enlarged and sparse organelles in senescent leaves. Progression of senescence in leaves was not synchronous, since changes in mitochondria from stomata were delayed. Mitochondrial morphology was also analysed in grapevine cell cultures. Mitochondria from cells at the end of their growth curve resembled those from senescing leaves, suggesting that cell cultures might represent a useful model system for senescence. Additionally, senescence-associated mitochondrial changes were observed in plants treated with high concentrations of cytokinins. Overall, morphology and dynamics of mitochondria might represent a reliable senescence marker for plant cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102012PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092070PMC
November 2015

The onset of grapevine berry ripening is characterized by ROS accumulation and lipoxygenase-mediated membrane peroxidation in the skin.

BMC Plant Biol 2014 Apr 2;14:87. Epub 2014 Apr 2.

Research and Innovation Centre, Fondazione Edmund Mach, via E, Mach 1, 38010 San Michele a/Adige, TN, Italy.

Background: The ripening of fleshy fruits is a complex developmental program characterized by extensive transcriptomic and metabolic remodeling in the pericarp tissues (pulp and skin) making unripe green fruits soft, tasteful and colored. The onset of ripening is regulated by a plethora of endogenous signals tuned to external stimuli. In grapevine and tomato, which are classified as non-climacteric and climacteric species respectively, the accumulation of hydrogen peroxide (H2O2) and extensive modulation of reactive oxygen species (ROS) scavenging enzymes at the onset of ripening has been reported, suggesting that ROS could participate to the regulatory network of fruit development. In order to investigate this hypothesis, a comprehensive biochemical study of the oxidative events occurring at the beginning of ripening in Vitis vinifera cv. Pinot Noir has been undertaken.

Results: ROS-specific staining allowed to visualize not only H2O2 but also singlet oxygen (1O2) in berry skin cells just before color change in distinct subcellular locations, i.e. cytosol and plastids. H2O2 peak in sample skins at véraison was confirmed by in vitro quantification and was supported by the concomitant increase of catalase activity. Membrane peroxidation was also observed by HPLC-MS on galactolipid species at véraison. Mono- and digalactosyl diacylglycerols were found peroxidized on one or both α-linolenic fatty acid chains, with a 13(S) absolute configuration implying the action of a specific enzyme. A lipoxygenase (PnLOXA), expressed at véraison and localizing inside the chloroplasts, was indeed able to catalyze membrane galactolipid peroxidation when overexpressed in tobacco leaves.

Conclusions: The present work demonstrates the controlled, harmless accumulation of specific ROS in distinct cellular compartments, i.e. cytosol and chloroplasts, at a definite developmental stage, the onset of grape berry ripening. These features strongly candidate ROS as cellular signals in fruit ripening and encourage further studies to identify downstream elements of this cascade. This paper also reports the transient galactolipid peroxidation carried out by a véraison-specific chloroplastic lipoxygenase. The function of peroxidized membranes, likely distinct from that of free fatty acids due to their structural role and tight interaction with photosynthesis protein complexes, has to be ascertained.
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http://dx.doi.org/10.1186/1471-2229-14-87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021102PMC
April 2014

FISSION1A, an Arabidopsis tail-anchored protein, is localized to three subcellular compartments.

Mol Plant 2014 Aug 22;7(8):1393-1396. Epub 2014 Mar 22.

Department of Biology, University of Padua, Via U. Bassi 58/B, I-35131 Padua, Italy. Electronic address:

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http://dx.doi.org/10.1093/mp/ssu027DOI Listing
August 2014

Management intensity and topography determined plant diversity in vineyards.

PLoS One 2013 1;8(10):e76167. Epub 2013 Oct 1.

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

Vineyards are amongst the most intensive forms of agriculture often resulting in simplified landscapes where semi-natural vegetation is restricted to small scattered patches. However, a tendency toward a more sustainable management is stimulating research on biodiversity in these poorly investigated agro-ecosystems. The main aim of this study was to test the effect on plant diversity of management intensity and topography in vineyards located in a homogenous intensive hilly landscape. Specifically, this study evaluated the role of slope, mowing and herbicide treatments frequency, and nitrogen supply in shaping plant diversity and composition of life-history traits. The study was carried out in 25 vineyards located in the area of the Conegliano-Valdobbiadene DOCG (Veneto, NE Italy). In each vineyard, 10 plots were placed and the abundance of all vascular plants was recorded in each plot. Linear multiple regression was used to test the effect of management and topography on plant diversity. Management intensity and topography were both relevant drivers of plant species diversity patterns in our vineyards. The two most important factors were slope and mowing frequency that respectively yielded positive and negative effects on plant diversity. A significant interaction between these two factors was also demonstrated, warning against the detrimental effects of increasing mowing intensity on steep slope where plant communities are more diverse. The response of plant communities to mowing frequency is mediated by a process of selection of resistant growth forms, such in the case of rosulate and reptant species. The other two management-related factors tested in this study, number of herbicide treatments and N fertilization, were less influential. In general, our study corroborates the idea that some simple changes in farming activities, which are compatible with grape production, should be encouraged for improving the natural and cultural value of the landscape by maintaining and improving wild plant diversity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076167PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788025PMC
July 2014

Limits in the use of cPTIO as nitric oxide scavenger and EPR probe in plant cells and seedlings.

Front Plant Sci 2013 29;4:340. Epub 2013 Aug 29.

Department of Biology, University of Padova Padova, Italy.

Over the last decade the importance of nitric oxide (NO) in plant signaling has emerged. Despite its recognized biological role, the sensitivity and effectiveness of the methods used for measuring NO concentration in plants are still under discussion. Among these, electron paramagnetic resonance (EPR) is a well-accepted technique to detect NO. In the present work we report the constraints of using 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) in biological samples as spin trap for quantitative measurement of NO. EPR analyses on Arabidopsis cell cultures and seedlings show that cPTIO(NNO) is degraded in a matter of few minutes while the (INO) compound, produced by cPTIO and NO reaction, has not been detected. Limitations of using this spin trap in plant systems for quantitative measurements of NO are discussed. As NO scavenger, cPTIO is widely used in combination with 4-amino-5-methylamino-2('),7(')-difluorofluorescein (DAF-FM) fluorescent dye in plant research. However, the dependence of DAF-FM fluorescence on cPTIO and NO concentrations is not clearly defined so that the range of concentrations should be tightly selected. In this context, a systematic study on cPTIO NO scavenging properties has been performed, as it was still lacking for plant system applications. The results of this systematic analysis are discussed in terms of reliability of the use of cPTIO in the quantitative determination and scavenging of NO in plants and plant cultured cells.
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http://dx.doi.org/10.3389/fpls.2013.00340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756283PMC
September 2013

Peroxisome Ca(2+) homeostasis in animal and plant cells.

Subcell Biochem 2013 ;69:111-33

Department of Biosciences, University of Milan, Milan, Italy.

Ca(2+) homeostasis in peroxisomes has been an unsolved problem for many years. Recently novel probes to monitor Ca(2+) levels in the lumen of peroxisomes in living cells of both animal and plant cells have been developed. Here we discuss the contrasting results obtained in mammalian cells with chemiluminecsent (aequorin) and fluorescent (cameleon) probes targeted to peroxisomes. We briefly discuss the different characteristics of these probes and the possible pitfalls of the two approaches. We conclude that the contrasting results obtained with the two probes may reflect a heterogeneity among peroxisomes in mammalian cells. We also discuss the results obtained in plant peroxisomes. In particular we demonstrate that Ca(2+) increases in the cytoplasm are mirrored by similar rises of Ca(2+) concentration the lumen of peroxisomes. The increases in peroxisome Ca(2+) level results in the activation of a catalase isoform, CAT3. Other functional roles of peroxisomal Ca(2+) changes in plant physiology are briefly discussed.
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http://dx.doi.org/10.1007/978-94-007-6889-5_7DOI Listing
August 2014

Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²⁺ handling relationship in plant cells.

Plant J 2012 Jul 30;71(1):1-13. Epub 2012 Apr 30.

Dipartimento di Biologia, Università degli Studi di Padova, Via U. Bassi 58/B, 35131 Padova, Italy.

Here we describe use of a mitochondrial targeted Cameleon to produce stably transformed Arabidopsis plants that enable analyses of mitochondrial Ca²⁺ dynamics in planta and allow monitoring of the intra-mitochondrial Ca²⁺ concentration in response to physiological or environmental stimuli. Transgenic plants co-expressing nuclear and mitochondrial targeted Cameleons were also generated and analyzed. Here we show that mitochondrial Ca²⁺ accumulation is strictly related to the intensity of the cytoplasmic Ca²⁺ increase, demonstrating a tight association between mitochondrial and cytoplasmic Ca²⁺ dynamics. However, under all experimental conditions, mitochondrial Ca²⁺ dynamics were substantially different from those monitored in the cytoplasm, demonstrating that mitochondria do not passively sense cytosolic Ca²⁺, but actively modulate the intra-mitochondrial level of the cation. In particular, our analyses show that the kinetics of Ca²⁺ release from mitochondria are much slower than in the cytoplasm and nucleus. The mechanisms and functional implications of these differences are discussed.
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http://dx.doi.org/10.1111/j.1365-313X.2012.04968.xDOI Listing
July 2012

The p23 co-chaperone protein is a novel substrate of CK2 in Arabidopsis.

Mol Cell Biochem 2011 Oct 7;356(1-2):245-54. Epub 2011 Jul 7.

Department of Biological Chemistry and Venetian Institute of Molecular Medicine, University of Padova, Viale G. Colombo, 3, 35131 Padova, Italy.

The ubiquitous Ser/Thr protein kinase CK2, which phosphorylates hundreds of substrates and is essential for cell life, plays important roles also in plants; however, only few plant substrates have been identified so far. During a study aimed at identifying proteins targeted by CK2 in plant response to salicylic acid (SA), we found that the Arabidopsis co-chaperone protein p23 is a CK2 target, readily phosphorylated in vitro by human and maize CK2, being also a substrate for an endogenous casein kinase activity present in Arabidopsis extracts, which displays distinctive characteristics of protein kinase CK2. We also demonstrated that p23 and the catalytic subunit of CK2 interact in vitro and possibly in Arabidopsis mesophyll protoplasts, where they colocalize in the cytosol and in the nucleus. Although its exact function is presently unknown, p23 is considered a co-chaperone because of its ability to associate to the chaperone protein Hsp90; therefore, an involvement of p23 in plant signal transduction pathways, such as SA signaling, is highly conceivable, and its phosphorylation may represent a fine mechanism for the regulation of cellular responses.
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http://dx.doi.org/10.1007/s11010-011-0969-0DOI Listing
October 2011

H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+)-dependent scavenging system.

Plant J 2010 Jun 2;62(5):760-72. Epub 2010 Mar 2.

Dipartimento di Biologia, Università degli Studi di Padova, Via U. Bassi 58/B, 35131 Padova, Italy.

Oxidative stress is a major challenge for all cells living in an oxygen-based world. Among reactive oxygen species, H2O2, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H2O2 in plant cells, it is necessary to have a reliable, specific and non-invasive methodology for its in vivo detection. Hence, the genetically encoded H2O2 sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)-based Cameleon Ca2+ indicator, D3cpv-KVK-SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca2+ increase is followed by Ca2+ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre- and post-bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca2+ rise in vivo stimulates catalase activity, increasing peroxisomal H2O2 scavenging efficiency.
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http://dx.doi.org/10.1111/j.1365-313X.2010.04190.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884081PMC
June 2010

Salicylic acid differentially affects suspension cell cultures of Lotus japonicus and one of its non-symbiotic mutants.

Plant Mol Biol 2010 Mar 10;72(4-5):469-83. Epub 2009 Dec 10.

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

Salicylic acid (SA) is known to play an important role in the interaction between plant and micro-organisms, both symbiotic and pathogen. In particular, high levels of SA block nodule formation and mycorrhizal colonization in plants. A mutant of Lotus japonicus, named Ljsym4-2, was characterized as unable to establish positive interactions with Rhizobium and fungi (NOD(-), MYC(-)); in particular, it does not recognize signal molecules released by symbiotic micro-organisms so that eventually, epidermal cells undergo PCD at the contact area. We performed a detailed characterization of wild-type and Ljsym4-2 cultured cells by taking into account several parameters characterizing cell responses to SA, a molecule strongly involved in defense signaling pathways. In the presence of 0.5 mM SA, Ljsym4-2 suspension-cultured cells reduce their growth and eventually die, whereas in order to induce the same effects in wt suspension cells, SA concentration must be raised to 1.5 mM. An early and short production of nitric oxide (NO) and reactive oxygen species (ROS) was detected in wt-treated cells. In contrast, a continuous production of NO and a double-peak ROS response, similar to that reported after a pathogenic attack, was observed in the mutant Ljsym4-2 cells. At the molecular level, a constitutive higher level of a SA-inducible pathogenesis related gene was observed. The analysis in planta revealed a strong induction of the LjPR1 gene in the Ljsym4-2 mutant inoculated with Mesorhizobium loti.
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http://dx.doi.org/10.1007/s11103-009-9585-8DOI Listing
March 2010

Nitric oxide is involved in cadmium-induced programmed cell death in Arabidopsis suspension cultures.

Plant Physiol 2009 May 4;150(1):217-28. Epub 2009 Mar 4.

Dipartimento di Biologia, Università degli Studi di Padova, I-35131 Padova, Italy.

Exposure to cadmium (Cd(2+)) can result in cell death, but the molecular mechanisms of Cd(2+) cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 microm CdCl(2) and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl(2) treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N(G)-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd(2+)-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd(2+) cytotoxicity in plants.
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http://dx.doi.org/10.1104/pp.108.133397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675725PMC
May 2009

Transcriptome analysis of Medicago truncatula leaf senescence: similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling.

New Phytol 2009 17;181(3):563-75. Epub 2008 Nov 17.

Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, I-35131 Padova, Italy.

Here, for the first time, a comprehensive transcriptomics study is presented of leaf senescence in the legume model Medicago truncatula, providing a broad overview of differentially expressed transcripts involved in this process. The cDNA-amplification fragment length polymorphism (AFLP) technique was used to identify > 500 genes, which were cloned and sorted into functional categories according to their gene ontology annotation. Comparison between the datasets of Arabidopsis and M. truncatula leaf senescence reveals common physiological events but differences in the nitrogen metabolism and in transcriptional regulation. In addition, it was observed that a minority of the genes regulated during leaf senescence were equally involved in other processes leading to programmed cell death, such as nodule senescence and nitric oxide signalling. This study provides a wide transcriptional profile for the comprehension of key events of leaf senescence in M. truncatula and highlights a possible regulative role for MADS box transcription factors in the terminal phases of the process.
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http://dx.doi.org/10.1111/j.1469-8137.2008.02684.xDOI Listing
March 2009

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells.

Plant Cell Rep 2008 May 7;27(5):845-53. Epub 2008 Feb 7.

Dipartimento di Biologia, Università Degli Studi di Padova, Via U. Bassi 58/B, 35131 Padua, Italy,

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (beta::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line.
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http://dx.doi.org/10.1007/s00299-008-0510-4DOI Listing
May 2008

Salicylic acid activates nitric oxide synthesis in Arabidopsis.

J Exp Bot 2007 21;58(6):1397-405. Epub 2007 Feb 21.

Dipartimento di Biologia, Università Degli Studi di Padova, Via U. Bassi 58/B, I-35131 Padova, Italy.

The relationship between nitric oxide (NO) and salicylic acid (SA) was investigated in Arabidopsis thaliana. Here it is shown that SA is able to induce NO synthesis in a dose-dependent manner in Arabidopsis. NO production was detected by confocal microscopic analysis and spectrofluorometric assay in plant roots and cultured cells. To identify the metabolic pathways involved in SA-induced NO synthesis, genetic and pharmacological approaches were adopted. The analysis of the nia1,nia2 mutant showed that nitrate reductase activity was not required for SA-induced NO production. Experiments performed in the presence of a nitric oxide synthase (NOS) inhibitor suggested the involvement of NOS-like enzyme activity in this metabolic pathway. Moreover, the production of NO by SA treatment of Atnos1 mutant plants was strongly reduced compared with wild-type plants. Components of the SA signalling pathway giving rise to NO production were identified, and both calcium and casein kinase 2 (CK2) were demonstrated to be involved. Taken together, these results suggest that SA induces NO production at least in part through the activity of a NOS-like enzyme and that calcium and CK2 activity are essential components of the signalling cascade.
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http://dx.doi.org/10.1093/jxb/erm001DOI Listing
October 2007