Publications by authors named "Giuseppe Dalessandro"

31 Publications

Progress towards Sustainable Control of subsp. in Olive Groves of Salento (Apulia, Italy).

Pathogens 2021 May 29;10(6). Epub 2021 May 29.

Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Monteroni-Lecce, Italy.

subsp. is the causal agent of "olive quick decline syndrome" in Salento (Apulia, Italy). On April 2015, we started interdisciplinary studies to provide a sustainable control strategy for this pathogen that threatens the multi-millennial olive agroecosystem of Salento. Confocal laser scanning microscopy and fluorescence quantification showed that a zinc-copper-citric acid biocomplex-Dentamet-reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. The biocomplex showed in vitro bactericidal activity towards all subspecies. A mid-term evaluation of the control strategy performed in some olive groves of Salento indicated that this biocomplex significantly reduced both the symptoms and subsp. cell concentration within the leaves of the local cultivars Ogliarola salentina and Cellina di Nardò. The treated trees started again to yield. A H-NMR metabolomic approach revealed, upon the treatments, a consistent increase in malic acid and γ-aminobutyrate for Ogliarola salentina and Cellina di Nardò trees, respectively. A novel endotherapy technique allowed injection of Dentamet at low pressure directly into the vascular system of the tree and is currently under study for the promotion of resprouting in severely attacked trees. There are currently more than 700 ha of olive groves in Salento where this strategy is being applied to control . subsp. . These results collectively demonstrate an efficient, simple, low-cost, and environmentally sustainable strategy to control this pathogen in Salento.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pathogens10060668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228964PMC
May 2021

Recent exposure to smoking and COVID-19.

Crit Care Resusc 2020 09;22(3):253-256

Department of Anesthesia and Intensive Care, IRCCS San Raffaele Scientific Institute, Milan, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
September 2020

Recent exposure to smoking and COVID-19.

Crit Care Resusc 2020 Jul 10. Epub 2020 Jul 10.

Department of Anesthesia and Intensive Care, IRCCS San Raffaele Scientific Institute, Milan, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
July 2020

Tranexamic acid in open aortic aneurysm surgery: a randomised clinical trial.

Br J Anaesth 2020 01 10;124(1):35-43. Epub 2019 Oct 10.

Department of Anesthesia and Intensive Care, IRCCS San Raffaele Scientific Institute, Milan, Italy; Department of Anesthesia and Intensive Care, Vita-Salute San Raffaele University, Milan, Italy.

Background: Bleeding and transfusions affect mortality in aortic surgery. Although tranexamic acid significantly reduced bleeding in multiple settings, its role in major vascular surgery was never studied. The aim of this study was to determine if tranexamic acid reduces blood loss in open abdominal aortic aneurysm (AAA) surgery.

Methods: A total of 100 patients undergoing elective open AAA repair were randomised to receive tranexamic acid (a loading dose of 500 mg and a continuous infusion of 250 mg h) or placebo. The primary outcome was intraoperative blood loss, and the secondary outcomes were the number of patients receiving red blood cells, occurrence of thromboembolic events, and mortality. Data were analysed using the intention-to-treat principle.

Results: Fifty patients were randomised into each group. Median (inter-quartile range) intraoperative blood loss was 400 (300-1050) ml in the tranexamic acid group vs 500 (360-1000) ml in the placebo group (P=0.44). Transfusion rate was seven/50 (14%) in the tranexamic group vs 12/50 (24%) in the placebo group (P=0.20). No thrombosis was recorded. In a post hoc analysis, postoperative blood loss was reduced in the tranexamic group both at 4 h (60 [40-80] ml vs 100 [60-140] ml, P<0.001) and 24 h (180 [120-275] vs 275 [190-395] ml, P=0.003) after surgery. At 1 yr, three patients were dead, all in the placebo group (P=0.24) and all after 28 days.

Conclusions: Tranexamic acid did not reduce intraoperative blood loss or blood transfusions in open AAA repair, although it may reduce postoperative blood loss without increasing adverse effects.

Clinical Trial Registration: NCT02335359.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bja.2019.08.028DOI Listing
January 2020

Noninvasive Ventilation After Thoracoabdominal Aortic Surgery: A Pilot Randomized Controlled Trial.

J Cardiothorac Vasc Anesth 2019 Jun 2;33(6):1639-1645. Epub 2018 Nov 2.

Department of Anesthesia and Intensive Care, Humanitas Clinical and Research Center, Rozzano Milan, Italy.

Objective: To assess the beneficial effects of noninvasive ventilation in treating postoperative pulmonary complications in patients undergoing thoracoabdominal aortic aneurysm (TAAA) open repair surgery.

Design: Randomized controlled trial.

Setting: University tertiary-care hospital.

Participants: Forty patients who underwent elective TAAA open repair.

Interventions: Patients were randomized to the "noninvasive ventilation" group, receiving 2-hour cycles of noninvasive ventilation every 8 hours for at least 3 days in addition to the best available postoperative treatment currently in use at the authors' institution versus the "standard" group, not receiving noninvasive ventilation treatment MEASUREMENTS AND MAIN RESULTS: The primary outcome of clinical worsening, described as a composite outcome of need for therapeutic noninvasive ventilation, need for mechanical ventilation owing to respiratory causes, need for intensive care unit admission owing to respiratory causes, and in-hospital mortality, occurred in 2 (11%) patients in the noninvasive ventilation group versus 12 (57%) in the standard group (p = 0.002; relative risk 0.18; 95% confidence interval 0.047-0.72).

Conclusion: Noninvasive ventilation is a promising, affordable, and easy-to-use tool to prevent postoperative respiratory complications after TAAA open surgical repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.jvca.2018.10.041DOI Listing
June 2019

Drought and Heat Differentially Affect XTH Expression and XET Activity and Action in 3-Day-Old Seedlings of Durum Wheat Cultivars with Different Stress Susceptibility.

Front Plant Sci 2016 10;7:1686. Epub 2016 Nov 10.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento Lecce, Italy.

Heat and drought stress have emerged as major constraints for durum wheat production. In the Mediterranean area, their negative effect on crop productivity is expected to be exacerbated by the occurring climate change. Xyloglucan endotransglucosylase/hydrolases (XTHs) are chief enzymes in cell wall remodeling, whose relevance in cell expansion and morphogenesis suggests a central role in stress responses. In this work the potential role of XTHs in abiotic stress tolerance was investigated in durum wheat. The separate effects of dehydration and heat exposure on XTH expression and its endotransglucosylase (XET) activity and action have been monitored, up to 24 h, in the apical and sub-apical root regions and shoots excised from 3-day-old seedlings of durum wheat cultivars differing in stress susceptibility/tolerance. Dehydration and heat stress differentially influence the XTH expression profiles and the activity and action of XET in the wheat seedlings, depending on the degree of susceptibility/tolerance of the cultivars, the organ, the topological region of the root and, within the root, on the gradient of cell differentiation. The root apical region was the zone mainly affected by both treatments in all assayed cultivars, while no change in XET activity was observed at shoot level, irrespective of susceptibility/tolerance, confirming the pivotal role of the root in stress perception, signaling, and response. Conflicting effects were observed depending on stress type: dehydration evoked an overall increase, at least in the apical region of the root, of XET activity and action, while a significant inhibition was caused by heat treatment in most cultivars. The data suggest that differential changes in XET action in defined portions of the root of young durum wheat seedlings may have a role as a response to drought and heat stress, thus contributing to seedling survival and crop establishment. A thorough understanding of the mechanisms underlying these variations could represent the theoretical basis for implementing breeding strategies to develop new highly productive hybrids adapted to future climate scenarios.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.01686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102909PMC
November 2016

Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

Front Plant Sci 2015 26;6:660. Epub 2015 Aug 26.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento Lecce, Italy.

The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1-10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2015.00660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550104PMC
September 2015

Enzyme-aided extraction of lycopene from high-pigment tomato cultivars by supercritical carbon dioxide.

Food Chem 2015 Mar 24;170:193-202. Epub 2014 Aug 24.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università del Salento, via Prov.le Lecce-Monteroni, 73100 Lecce, Italy. Electronic address:

This work reports a novel enzyme-assisted process for lycopene concentration into a freeze-dried tomato matrix and describes the results of laboratory scale lycopene supercritical CO2 (SC-CO2) extractions carried out with untreated (control) and enzyme-digested matrices. The combined use of food-grade commercial plant cell-wall glycosidases (Celluclast/Novozyme plus Viscozyme) allows to increase lycopene (∼153%) and lipid (∼137%) concentration in the matrix and rises substrate load onto the extraction vessel (∼46%) compared to the control. The addition of an oleaginous co-matrix (hazelnut seeds) to the tomato matrix (1:1 by weight) increases CO2 diffusion through the highly dense enzyme-treated matrix bed and provides lipids that are co-extracted increasing lycopene yield. Under the same operative conditions (50 MPa, 86 °C, 4 mL min(-1) SC-CO2 flow) extraction yield from control and Celluclast/Novozyme+Viscozyme-treated tomato matrix/co-matrix mixtures was similar, exceeding 75% after 4.5h of extraction. However, the total extracted lycopene was ∼3 times higher in enzyme-treated matrix than control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.foodchem.2014.08.081DOI Listing
March 2015

Cellular localization and biochemical characterization of a chimeric fluorescent protein fusion of Arabidopsis cellulose synthase-like A2 inserted into Golgi membrane.

ScientificWorldJournal 2014 14;2014:792420. Epub 2014 Jan 14.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento (DiSTeBA), Provinciale Lecce-Monteroni, 73100 Lecce, Italy.

Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of β -mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a 14C-mannan starting from GDP-D-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2014/792420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3914377PMC
October 2014

Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit.

BMC Genomics 2013 Nov 12;14:781. Epub 2013 Nov 12.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di,S,Te,B,A,), Università del Salento, via Prov,le Lecce-Monteroni,73100 Lecce, Italy.

Background: Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement.

Results: Total carotenoids progressively increased during fruit ripening up to ~55 μg g(-1) fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening.

Conclusions: Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-14-781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840736PMC
November 2013

Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting.

Plant Physiol Biochem 2013 Dec 23;73:337-43. Epub 2013 Oct 23.

Laboratory of Cell and Molecular Biology, University of Neuchâtel, Rue Emile-Argand 11, CH-2000 Neuchâtel, Switzerland; CNR-IGV, Institute of Plant Genetics, Thematic Center for the Preservation of Mediterranean Plant Biodiversity, via Nazionale 44, 75025 Policoro, MT, Italy.

Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.plaphy.2013.10.010DOI Listing
December 2013

Possible use of the carbohydrates present in tomato pomace and in byproducts of the supercritical carbon dioxide lycopene extraction process as biomass for bioethanol production.

J Agric Food Chem 2013 Apr 9;61(15):3683-92. Epub 2013 Apr 9.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, Lecce, Italy.

This study provides information about the carbohydrate present in tomato pomace (skins, seeds, and vascular tissues) as well as in the byproducts of the lycopene supercritical carbon dioxide extraction (SC-CO₂) such as tomato serum and exhausted matrix and reports their conversion into bioethanol. The pomace, constituting approximately 4% of the tomato fruit fresh weight, and the SC-CO₂-exhausted matrix were enzyme saccharified with 0.1% Driselase leading to sugar yields of ~383 and ~301 mg/g dw, respectively. Aliquots of the hydrolysates and of the serum (80% tomato sauce fw) were fermented by Saccharomyces cerevisiae . The bioethanol produced from each waste was usually >50% of the calculated theoretical amount, with the exception of the exhausted matrix hydolysate, where a sugar concentration >52.8 g/L inhibited the fermentation process. Furthermore, no differences in the chemical solubility of cell wall polysaccharides were evidenced between the SC-CO₂-lycopene extracted and unextracted matrices. The deduced glycosyl linkage composition and the calculated amount of cell wall polysaccharides remained similar in both matrices, indicating that the SC-CO₂ extraction technology does not affect their structure. Therefore, tomato wastes may well be considered as potential alternatives and low-cost feedstock for bioethanol production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jf4005059DOI Listing
April 2013

AtSYP51/52 functions diverge in the post-Golgi traffic and differently affect vacuolar sorting.

Mol Plant 2013 May 19;6(3):916-30. Epub 2012 Oct 19.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Salento, Campus Ecotekne, 73100 Lecce, Italy.

Plant sensitive factor attachment protein receptors (SNAREs) encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the application of innovative experimental approaches highlighted that members of the same gene sub-family often have different functional specificities. In this work, two closely related Qc-SNAREs--the AtSYP51 and the AtSYP52--are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs (syntaxin of plants) act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs (i-SNAREs) when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/mp/sss117DOI Listing
May 2013

A bifasic response to cadmium stress in carrot: Early acclimatory mechanisms give way to root collapse further to prolonged metal exposure.

Plant Physiol Biochem 2012 Sep 10;58:269-79. Epub 2012 Jul 10.

Dipartimento di Biologia Evolutiva e Funzionale, Università di Parma, Viale delle Scienze 11/A, I-43124 Parma, Italy.

Very few studies have provided information about the effects of cadmium (Cd) at histoanatomical and ultrastructural levels, along with potential localization of the metal in planta. In particular, from this standpoint, almost nothing is known in Daucus carota L. (carrot), a particularly important species for in vitro and in vivo functional investigations. In this work we hypothesized that 36 μM Cd, supplied for 1, 2, 3, 4, 7 and 14 days to 30-day-old in vitro-cultured plants, might induce an early acclimation, but a final collapse of roots and leaves. In fact, as a general feature, a biphasic root response to Cd stress actually took place: in the first phase (1-4 days of Cd exposure), the cytological and functional events observed - by light microscopy, TEM, epifluorescence, as well as by the time-course of thiol-peptide compounds - can be interpreted as acclimatory responses aimed at diminishing the movement of Cd across the root. The second phase (from 4 to 14 days of Cd exposure) was instead characterized by cell hypertrophy, cell-to-cell separation events, increase in α-β-γ-tocopherol levels and, not least, endocytogenic processes, coupled with a dramatic drop in the amount of thiol-peptide compounds. These events led to a progressive root collapse, even if they did not ingenerate macro/microscopic injury symptoms in leaf blades and petioles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.plaphy.2012.07.002DOI Listing
September 2012

Novel durum wheat genes up-regulated in response to a combination of heat and drought stress.

Plant Physiol Biochem 2012 Jul 21;56:72-8. Epub 2012 Apr 21.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Prov. le Monteroni, 73100 Lecce, Italy.

We report the effect of heat, drought and combined stress on the expression of a group of genes that are up-regulated under these conditions in durum wheat (Triticum turgidum subsp. durum) plants. Modulation of gene expression was studied by cDNA-AFLP performed on RNAs extracted from flag leaves. By this approach, we identified several novel durum wheat genes whose expression is modulated under different stress conditions. We focused on a group of hitherto undescribed up-regulated genes in durum wheat, among these, 7 are up-regulated by heat, 8 by drought stress, 15 by combined heat and drought stress, 4 are up-regulated by both heat and combined stress, and 3 by both drought and combined stress. The functional characterization of these genes will provide new data that could help the developing of strategies aimed at improving durum wheat tolerance to field stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.plaphy.2012.04.006DOI Listing
July 2012

Isoprenoid, lipid, and protein contents in intact plastids isolated from mesocarp cells of traditional and high-pigment tomato cultivars at different ripening stages.

J Agric Food Chem 2012 Feb 9;60(7):1764-75. Epub 2012 Feb 9.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, Lecce, Italy.

This study reports quali-quantitative analyses on isoprenoids, phospholipids, neutral lipids, phytosterols, and proteins in purified plastids isolated from fresh fruits of traditional (Donald and Incas) and high-pigment (Kalvert and HLY-18) tomato cultivars at four ripening stages. In all of the investigated cultivars, lycopene, β-catotene, lutein, and total carotenoids varied significantly during ripening. Chromoplasts of red-ripe tomato fruits of high-pigment cultivars accumulated twice as much as lycopene (307.6 and 319.2 μg/mg of plastid proteins in Kalvert and HLY-18, respectively) than ordinary cultivars (178.6 and 151.7 μg/mg of plastid proteins in Donald and Incas, respectively); differences in chlorophyll and α-tocopherol contents were also evidenced. Phospholipids and phytosterols increased during ripening, whereas triglycerides showed a general decrease. Regardless of the stage of ripening, palmitic acid was the major fatty acid in all cultivars (ranging from 35 to 52% of the total fatty acids), followed by stearic, oleic, linoleic, linolenic, and myristic acids, but their relative percentage was affected by ripening. Most of the bands detected on the SDS-PAGEs of plastid proteins were constantly present during chloroplast-to-chromoplast conversion, some others disappeared, and only one, with a molecular weight of ~41.6 kDa, was found to increase in intensity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jf204189zDOI Listing
February 2012

Nicotiana tabacum protoplasts secretome can evidence relations among regulatory elements of exocytosis mechanisms.

Plant Signal Behav 2011 Aug 1;6(8):1140-5. Epub 2011 Aug 1.

DiSTeBA, Università del Salento, Lecce, Italy.

An alternative study involving proteome analysis of the 24 hour Nicotiana tabacum protoplast culture medium was performed with the aim to confirm relations among regulatory elements of exocytotic processes. Protoplasts present many convenient features to study cellular processes during transient over-expression or suppression of specific gene's products. We performed a proteomic analysis of the culture medium fraction of protoplasts transiently expressing transgenes for 24 hours to characterize the effect of various regulatory proteins dominant negative mutants. A total number of 49 spots were found reproducible in the medium. 24 of these spots were identified with nano RP-HPLC-ESI-MS/MS. Only three and six spots were respectively identified as canonical and non-canonical secreted cell wall proteins. The low number of spots present in the culture medium fraction allowed us the ambitious experiment to analyze the influence of various SNAREs (SYP121, SYP122, SNAP33) and Rab (Rab11) dominant negative mutants. Missing a reasonable number of identified proteins the analyses gave rise to a similarity matrix statistically analyzed considering variation within the presence of 24 spots reproducible in presence of transient over-expression of SNAREs (SYP121 and SYP122) and Rab11 native cDNAs. The similarity confirmed the closer relation between the function of SYP122 and Rab11 as evidenced by the secRGUS based analysis. This analysis included the effect of SNAP33 DN mutant and showed that this Qb-c-SNARE influence both SYP121 and SYP122 SNARE complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/psb.6.8.15750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260711PMC
August 2011

Dynamic protein trafficking to the cell wall.

Plant Signal Behav 2011 Jul;6(7):1012-5

DiSTeBA, Università del Salento, Lecce, Italy.

Recently we have studied the secretion pattern of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2) in tobacco protoplast using the protein fusions, secGFP-PMEI1 and PGIP2-GFP. Both chimeras reach the cell wall by passing through the endomembrane system but using distinct mechanisms and through a pathway distinguishable from the default sorting of a secreted GFP. After reaching the apoplast, sec-GFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP undergoes endocytic trafficking. Here we describe the final localization of PGIP2-GFP in the vacuole, evidenced by co-localization with the marker Aleu-RFP, and show a graphic elaboration of its sorting pattern. A working model taking into consideration the presence of a regulated apoplast-targeted secretion pathway is proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257782PMC
http://dx.doi.org/10.4161/psb.6.7.15550DOI Listing
July 2011

Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco.

Plant J 2011 Jan 1;65(2):295-308. Epub 2010 Dec 1.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, 73100 Lecce, Italy.

The secretory pathway in plants involves sustained traffic to the cell wall, as matrix components, polysaccharides and proteins reach the cell wall through the endomembrane system. We studied the secretion pattern of cell-wall proteins in tobacco protoplasts and leaf epidermal cells using fluorescent forms of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2). The two most representative protein fusions, secGFP-PMEI1 and PGIP2-GFP, reached the cell wall by passing through ER and Golgi stacks but using distinct mechanisms. secGFP-PMEI1 was linked to a glycosylphosphatidylinositol (GPI) anchor and stably accumulated in the cell wall, regulating the activity of the endogenous pectin methylesterases (PMEs) that are constitutively present in this compartment. A mannosamine-induced non-GPI-anchored form of PMEI1 as well as a form (PMEI1-GFP) that was unable to bind membranes failed to reach the cell wall, and accumulated in the Golgi stacks. In contrast, PGIP2-GFP moved as a soluble cargo protein along the secretory pathway, but was not stably retained in the cell wall, due to internalization to an endosomal compartment and eventually the vacuole. Stable localization of PGIP2 in the wall was observed only in the presence of a specific fungal endopolygalacturonase ligand in the cell wall. Both secGFP-PMEI1 and PGIP2-GFP sorting were distinguishable from that of a secreted GFP, suggesting that rigorous and more complex controls than the simple mechanism of bulk flow are the basis of cell-wall growth and differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-313X.2010.04421.xDOI Listing
January 2011

Optimisation of biological and physical parameters for lycopene supercritical CO2 extraction from ordinary and high-pigment tomato cultivars.

J Sci Food Agric 2010 Aug;90(10):1709-18

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Prov. le Lecce-Monteroni, Lecce, Italy.

Background: Lycopene is used for several industrial applications. Supercritical CO(2) (SC-CO(2)) extraction from red-ripe tomato fruits is an excellent technique to replace the use of harmful solvents. In this study, starting from red-ripe tomatoes of ordinary and high-lycopene cultivars, the effect of different agronomical and technical aspects on lycopene content, stability and yield was evaluated throughout the production process from fresh tomatoes to the final SC-CO(2)-extracted oleoresin containing lycopene.

Results: Red-ripe tomato cultivars differed in their lycopene content. Irrigation excess or deficit caused an increase in the amount of lycopene in the fruits. Fresh tomatoes were processed into a lyophilised matrix suitable for SC-CO(2) extraction, which could be stored for more than 6 months at -20 degrees C without lycopene loss. Under the optimal extraction conditions, efficiencies of up to 80% were achieved, but the recovery of lycopene in the extracted oleoresin was very low (approximately 24%). Co-extraction of the tomato matrix mixed with a lipid co-matrix allowed the recovery of approximately 90% of lycopene in the oleoresin. Using the high-lycopene cultivars, the yield of total extracted lycopene increased by approximately 60% with respect to the ordinary cultivars. Lipids and other biologically active molecules were present in the oleoresin.

Conclusion: A method for extracting, from a tomato matrix, a natural and solvent-free oleoresin containing lycopene dissolved in a highly unsaturated vegetable oil has been described. The oleoresin represents an excellent product for testing on cancer and cardiovascular disease prevention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jsfa.4006DOI Listing
August 2010

Expression of a glycosylated GFP as a bivalent reporter in exocytosis.

Plant Cell Rep 2010 Jan 2;29(1):79-86. Epub 2009 Dec 2.

INRA, Biochemistry and Plant Molecular Physiology, IBIP, Bât. 7, Montpellier, France.

The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an alpha-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a beta-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00299-009-0799-7DOI Listing
January 2010

In muro feruloylation and oxidative coupling in monocots: a possible role in plant defense against pathogen attacks.

Plant Signal Behav 2009 Mar;4(3):228-30

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, Lecce, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652537PMC
http://dx.doi.org/10.4161/psb.4.3.7883DOI Listing
March 2009

Evidence for intra- and extra-protoplasmic feruloylation and cross-linking in wheat seedling roots.

Planta 2009 Jan 31;229(2):343-55. Epub 2008 Oct 31.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, via prov.le Lecce-Monteroni, 73100, Lecce, Italy.

The sub-cellular feruloylation and oxidative coupling sites of cell wall polysaccharides were investigated in planta by monitoring the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall either in the presence or absence of brefeldin A (BFA). By using root apical segments excised from wheat seedlings (Triticum durum Desf.), incubated with trans-[U-(14)C]cinnamic acid, we demonstrated that [14C]ferulate, likely [14C]diferulate, as well as trimers and larger products of ferulate are incorporated into the protoplasmic polysaccharides very rapidly within 1-3 min of [14C]cinnamate feeding. This agrees with the assumption that (glucurono)arabinoxylans [(G)AX] feruloylation and oxidative coupling occur intracellularly, likely in the Golgi apparatus. Simultaneously, polymer bound radioactive hydroxycinnamic acids appeared to be incorporated into the cell wall of root apical segments as early as 2 min after trans-[U-(14)C]cinnamic acid feeding. On the contrary, starting from L-[1-(14)C]arabinose as tracer, the secretion of the pentose-containing polymers into the wall was between 5 to 10 min. These results indicated that (G)AX feruloylation and oxidative coupling occur both intra-protoplasmically and in muro. The occurrence of in muro feruloylation and oxidative coupling was confirmed by the use of BFA a well known inhibitor of secretion. The drug caused a strong inhibition of the synthesis and secretion into the wall of the 14C-pentosyl-labelled polymers as well as of 14C-feruloyl-polymers. In spite of this, the total amount of 14C-feruloyl-polymers incorporated into the wall was only slightly affected by BFA. This indicates the existence of a mechanism involved into secretion of the activated hydroxycinnamoyl precursors to the wall, alternative to that involved in polysaccharide secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00425-008-0834-xDOI Listing
January 2009

Tomato Rab11a characterization evidenced a difference between SYP121-dependent and SYP122-dependent exocytosis.

Plant Cell Physiol 2008 May 1;49(5):751-66. Epub 2008 Apr 1.

Di.S.Te.B.A., Università del Salento, via prov. Lecce-Monteroni, 73100 Lecce, Italy.

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat beta-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/pcp/pcn051DOI Listing
May 2008

Water stress and cell wall polysaccharides in the apical root zone of wheat cultivars varying in drought tolerance.

J Plant Physiol 2008 Jul 26;165(11):1168-80. Epub 2007 Dec 26.

Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Università del Salento, Lecce, Italy.

Glycosyl composition and linkage analysis of cell wall polysaccharides were examined in apical root zones excised from water-stressed and unstressed wheat seedlings (Triticum durum Desf.) cv. Capeiti ("drought-tolerant") and cv. Creso ("drought sensitive"). Wall polysaccharides were sequentially solubilized to obtain three fractions: CDTA+Na(2)CO(3) extract, KOH extract and the insoluble residue (alpha-cellulose). A comparison between the two genotypes showed only small variations in the percentages of matrix polysaccharides (CDTA+Na(2)CO(3) plus KOH extract) and of the insoluble residues (alpha-cellulose) in water-stressed and unstressed conditions. Xylosyl, glucosyl and arabinosyl residues represented more than 90 mol% of the matrix polysaccharides. The linkage analysis of matrix polysaccharides showed high levels of xyloglucans (23-39 mol%), and arabinoxylans (38-48 mol%) and a low amount of pectins and (1-->3), (1-->4)-beta-D-glucans. The high level of xyloglucans was supported by the release of the diagnostic disaccharide isoprimeverose after Driselase digestion of KOH-extracted polysaccharides. In the "drought-tolerant" cv. Capeiti the mol% of side chains of rhamnogalacturonan I and II significantly increased in response to water stress, whereas in cv. Creso, this increase did not occur. The results support a role of the pectic side chains during water stress response in a drought-tolerant wheat cultivar.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jplph.2007.09.006DOI Listing
July 2008

Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways.

Planta 2007 Mar;225(4):1001-17

Di.S.Te.B.A., Università di Lecce, via prov.le Lecce-Monteroni, 73100 Lecce, Italy.

The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (sec-GFP) and secreted rat preputial beta-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00425-006-0407-9DOI Listing
March 2007

Antioxidant composition in cherry and high-pigment tomato cultivars.

J Agric Food Chem 2006 Apr;54(7):2606-13

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università di Lecce, via prov.le Lecce-Monteroni, 73100 Lecce, Italy.

Fourteen cultivars of cherry tomatoes and four cultivars of high-pigment tomato hybrids were cultivated in southern Italy, and the red-ripe fruits were analyzed for their content in different classes of antioxidants and for their antioxidant activity. Among the different cultivars, significant differences were found between lycopene, beta-carotene, alpha-tocopherol, vitamin C (ascorbic acid and dehydroascorbic acid), and total phenolic and flavonoid contents. LS203 and Corbus appear to be the cultivars with the highest content of lipophilic and hydrophilic antioxidants among cherry tomatoes, respectively. All cultivars of high-pigment tomato hybrids showed an expected exceptionally high lycopene content. Among them, the highest content of lipophilic and hydrophilic antioxidants was found in cv. HLY 13. Hydrophilic and lipophilic antioxidant activities were both significantly influenced by genotype. Such results highlight an existing unexploited variability in tomato germplasm and stress the need to evaluate the biodiversity and to support conventional breeding programs to improve tomato nutritional value.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jf052920cDOI Listing
April 2006

Sorting of GFP Tagged NtSyr1, an ABA Related Syntaxin.

Plant Signal Behav 2006 Mar;1(2):77-85

Laboratorio di Botanica; Di.S.Te.B.A.; Università di Lecce; Lecce, Italy.

Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633883PMC
http://dx.doi.org/10.4161/psb.1.2.2621DOI Listing
March 2006

Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

Phytochemistry 2005 Nov 18;66(21):2581-94. Epub 2005 Oct 18.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università di Lecce, via prov.le Lecce-Monteroni, 73100 Lecce, Italy.

Two new reducing glycoconjugates [N-D-galacturonoyl-putrescinamide (GalA-Put) and N,N'-di-D-galacturonoyl-putrescinamide (GalA-Put-GalA)] and homogalacturonan-putrescine (GalAn-Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25 degrees C) but rapidly hydrolysed by these agents at 100 degrees C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalAn-Put, releasing fragments such as GalA3-Put-GalA3. To trace the possible formation of GalA-polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional approximately 73% of the 14C taken up (in Arabidopsis), or approximately 21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide-Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin-polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phytochem.2005.08.017DOI Listing
November 2005

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells.

J Plant Physiol 2004 Oct;161(10):1143-56

Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Università di Lecce, via prov. le Lecce-Monteroni, I-73100 Lecce, Italy.

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jplph.2004.01.012DOI Listing
October 2004
-->