Publications by authors named "Henrique Noronha"

16 Publications

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Grapevine aquaporins: Diversity, cellular functions, and ecophysiological perspectives.

Biochimie 2021 Jun 15. Epub 2021 Jun 15.

Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017, Lisbon, Portugal; Departamento de Recursos Biologicos, Ambiente e Territorio (DRAT), Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017, Lisbon, Portugal.

High-scored premium wines are typically produced under moderate drought stress, suggesting that the water status of grapevine is crucial for wine quality. Aquaporins greatly influence the plant water status by facilitating water diffusion across the plasma membrane in a tightly regulated manner. They adjust the hydraulic conductance of the plasma membrane rapidly and reversibly, which is essential in specific physiological events, including adaptation to soil water scarcity. The comprehension of the sophisticated plant-water relations at the molecular level are thus important to optimize agricultural practices or to assist plant breeding programs. This review explores the recent progresses in understanding the water transport in grapevine at the cellular level through aquaporins and its regulation. Important aspects, including aquaporin structure, diversity, cellular localization, transport properties, and regulation at the cellular and whole plant level are addressed. An ecophysiological perspective about the roles of grapevine aquaporins in plant response to drought stress is also provided.
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http://dx.doi.org/10.1016/j.biochi.2021.06.004DOI Listing
June 2021

Flavescence Dorée-Derived Leaf Yellowing in Grapevine ( L.) Is Associated to a General Repression of Isoprenoid Biosynthetic Pathways.

Front Plant Sci 2020 17;11:896. Epub 2020 Jun 17.

Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Braga, Portugal.

Flavescence dorée (FD), caused by the phytoplasma Phytoplasma vitis, is a major threat to vineyard survival in different European grape-growing areas. It has been recorded in French vineyards since the mid-1950s, and rapidly spread to other countries. In Portugal, the phytoplasma was first detected in the DOC region of 'Vinhos Verdes' in 2006, and reached the central region of the country in 2009. The infection causes strong accumulation of carbohydrates and phenolics in the mesophyll cells and a simultaneous decrease of chlorophylls, events accompanied by a down regulation of genes and proteins involved in the dark and light-dependent reactions and stabilization of the photosystem II (PSII). In the present study, to better elucidate the basis of the leaf chlorosis in infected grapevine cv. Loureiro, we studied the isoprenoid transcript-metabolite correlation in leaves from healthy and FD-infected vines. Specifically, targeted metabolome revealed that twenty-one compounds (out of thirty-two), including chlorophylls, carotenoids, quinones and tocopherols, were reduced in response to FD-infection. Thereafter, and consistently with the biochemical data, qPCR analysis highlighted a severe FD-mediated repression in key genes involved in isoprenoid biosynthetic pathways. A more diverse set of changes, on the contrary, was observed in the case of ABA metabolism. Principal component analysis (PCA) of all identified metabolites clearly separated healthy from FD-infected vines, therefore confirming that the infection strongly alters the biosynthesis of grapevine isoprenoids; additionally, forty-four genes and metabolites were identified as the components mostly explaining the variance between healthy and infected samples. Finally, transcript-metabolite network correlation analyses were exploited to display the main hubs of the infection process, which highlighted a strong role of , and genes and the chlorophylls intermediates aminolevulunic acid and porphobilinogen in response to FD infection. Overall, results indicated that the FD infection impairs the synthesis of isoprenoids, through the repression of key genes involved in the biosynthesis of chlorophylls, carotenoids, quinones and tocopherols.
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http://dx.doi.org/10.3389/fpls.2020.00896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311760PMC
June 2020

The grapevine NIP2;1 aquaporin is a silicon channel.

J Exp Bot 2020 12;71(21):6789-6798

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.
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http://dx.doi.org/10.1093/jxb/eraa294DOI Listing
December 2020

CXCR3 chemokine receptor contributes to specific CD8+ T cell activation by pDC during infection with intracellular pathogens.

PLoS Negl Trop Dis 2020 06 23;14(6):e0008414. Epub 2020 Jun 23.

Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control.
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http://dx.doi.org/10.1371/journal.pntd.0008414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337401PMC
June 2020

Sweet Cherry ( L.) PaPIP1;4 Is a Functional Aquaporin Upregulated by Pre-Harvest Calcium Treatments that Prevent Cracking.

Int J Mol Sci 2020 Apr 24;21(8). Epub 2020 Apr 24.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, Universidade do Minho, 4710-057 Braga, Portugal.

The involvement of aquaporins in rain-induced sweet cherry ( L.) fruit cracking is an important research topic with potential agricultural applications. In the present study, we performed the functional characterization of PaPIP1;4, the most expressed aquaporin in sweet cherry fruit. Field experiments focused on the pre-harvest exogenous application to sweet cherry trees, cultivar Skeena, with a solution of 0.5% CaCl, which is the most common treatment to prevent cracking. Results show that PaPIP1;4 was mostly expressed in the fruit peduncle, but its steady-state transcript levels were higher in fruits from CaCl-treated plants than in controls. The transient expression of PaPIP1;4-GFP in tobacco epidermal cells and the overexpression of PaPIP1;4 in YSH1172 yeast mutation showed that PaPIP1;4 is a plasma membrane protein able to transport water and hydrogen peroxide. In this study, we characterized for the first time a plasma membrane sweet cherry aquaporin able to transport water and HO that is upregulated by the pre-harvest exogenous application of CaCl supplements.
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http://dx.doi.org/10.3390/ijms21083017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215675PMC
April 2020

Correction to: A molecular perspective on starch metabolism in woody tissues.

Planta 2018 09;248(3):569

Centre of Molecular and Environmental Biology (CBMA), University of Minho, Braga, Portugal.

The original article was corrected.
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http://dx.doi.org/10.1007/s00425-018-2963-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096776PMC
September 2018

A molecular perspective on starch metabolism in woody tissues.

Planta 2018 Sep 18;248(3):559-568. Epub 2018 Jul 18.

Centre of Molecular and Environmental Biology (CBMA), University of Minho, Braga, Portugal.

Main Conclusion: The elucidation of the molecular mechanisms of starch synthesis and mobilization in perennial woody tissues is of the utmost scientific and agricultural importance. Starch is the main carbohydrate reserve in plants and is fundamental in human nutrition and several industrial processes. In leaves, starch accumulated during the day is degraded throughout the night and the resulting sugars, glucose and maltose, are exported to the cytosol by the specialized transmembrane translocators pGT and MEX, respectively. Nevertheless, the degradation of the starch granule is a complex process not completely elucidated. While the mechanisms of starch mobilization during germination in the dead endosperm of cereal seeds are well described, the molecular and biochemical mechanisms involved in starch storage in the heterotrophic tissues of woody plants and its utilization in spring and winter are still puzzling. It is known that some biochemical steps of starch synthesis are conserved in heterotrophic tissues and in the leaves, but some aspects are particular to sink organs. From an agronomic standpoint, the knowledge on starch storage and mobilization in woody tissues is pivotal to understand (and to optimize) some common practices in the field that modify source-sink relationships, such as pruning and defoliation. Soluble sugars resulting from starch are also pivotal to cold adaptation, and in several fruits, such as banana and kiwifruit, starch may provide soluble sugars during ripening. In this review, we explore the recent advances on the molecular mechanisms and regulations involved in starch synthesis and mobilization, with a focus on perennial woody tissues.
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http://dx.doi.org/10.1007/s00425-018-2954-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096779PMC
September 2018

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

Methods Mol Biol 2018 ;1789:81-99

CIBIO/InBIO-Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H-ATPase and the V-H-pyrophosphatase, also producing further information on vacuole purity.
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http://dx.doi.org/10.1007/978-1-4939-7856-4_7DOI Listing
February 2019

Low source-sink ratio reduces reserve starch in grapevine woody canes and modulates sugar transport and metabolism at transcriptional and enzyme activity levels.

Planta 2017 Sep 19;246(3):525-535. Epub 2017 May 19.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas (CITAB), Vila Real, Portugal.

Main Conclusion: Severe leaf removal decreases storage starch and sucrose in grapevine cv. Cabernet Sauvignon fruiting cuttings and modulates the activity of key enzymes and the expression of sugar transporter genes. Leaf removal is an agricultural practice that has been shown to modify vineyard efficiency and grape and wine composition. In this study, we took advantage of the ability to precisely control the number of leaves to fruits in Cabernet Sauvignon fruiting cuttings to study the effect of source-sink ratios (2 (2L), 6 (6L) and 12 (12) leaves per cluster) on starch metabolism and accumulation. Starch concentration was significantly higher in canes from 6L (42.13 ± 1.44 mg g DW) and 12L (43.50 ± 2.85 mg g DW) than in 2L (22.72 ± 3.10 mg g DW) plants. Moreover, carbon limitation promoted a transcriptional adjustment of genes involved in starch metabolism in grapevine woody tissues, including a decrease in the expression of the plastidic glucose-6-phosphate translocator, VvGPT1. Contrarily, the transcript levels of the gene coding the catalytic subunit VvAGPB1 of the VvAGPase complex were higher in canes from 2L plants than in 6L and 12L, which positively correlated with the biochemical activity of this enzyme. Sucrose concentration increased in canes from 2L to 6L and 12L plants, and the amount of total phenolics followed the same trend. Expression studies showed that VvSusy transcripts decreased in canes from 2L to 6L and 12L plants, which correlated with the biochemical activity of insoluble invertase, while the expression of the sugar transporters VvSUC11 and VvSUC12, together with VvSPS1, which codes an enzyme involved in sucrose synthesis, increased. Thus, sucrose seems to control starch accumulation through the adjustment of the cane sink strength.
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http://dx.doi.org/10.1007/s00425-017-2708-6DOI Listing
September 2017

The Grapevine Uncharacterized Intrinsic Protein 1 (VvXIP1) Is Regulated by Drought Stress and Transports Glycerol, Hydrogen Peroxide, Heavy Metals but Not Water.

PLoS One 2016 9;11(8):e0160976. Epub 2016 Aug 9.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas CITAB, Vila Real, Portugal.

A MIP (Major Intrinsic Protein) subfamily called Uncharacterized Intrinsic Proteins (XIP) was recently described in several fungi and eudicot plants. In this work, we cloned a XIP from grapevine, VvXIP1, and agrobacterium-mediated transformation studies in Nicotiana benthamiana revealed that the encoded aquaporin shows a preferential localization at the endoplasmic reticulum membrane. Stopped-flow spectrometry in vesicles from the aqy-null yeast strain YSH1172 overexpressing VvXIP1 showed that VvXIP1 is unable to transport water but is permeable to glycerol. Functional studies with the ROS sensitive probe CM-H2DCFDA in intact transformed yeasts showed that VvXIP1 is also able to permeate hydrogen peroxide (H2O2). Drop test growth assays showed that besides glycerol and H2O2, VvXIP1 also transports boric acid, copper, arsenic and nickel. Furthermore, we found that VvXIP1 transcripts were abundant in grapevine leaves from field grown plants and strongly repressed after the imposition of severe water-deficit conditions in potted vines. The observed downregulation of VvXIP1 expression in cultured grape cells in response to ABA and salt, together with the increased sensitivity to osmotic stress displayed by the aqy-null yeast overexpressing VvXIP1, corroborates the role of VvXIP1 in osmotic regulation besides its involvement in H2O2 transport and metal homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978503PMC
August 2017

Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants.

Methods Mol Biol 2016 ;1405:121-35

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.
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http://dx.doi.org/10.1007/978-1-4939-3393-8_12DOI Listing
November 2016

Identification and functional characterization of grapevine transporters that mediate glucose-6-phosphate uptake into plastids.

Planta 2015 Oct 26;242(4):909-20. Epub 2015 May 26.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas CITAB, Vila Real, Portugal.

Main Conclusion: Two grapevine glucose-6-Pi plastidial transporters differently expressed in plant organs and in response to environmental and hormonal signals are characterized. They are involved in starch accumulation in berries and canes. In grapevine, starch accumulation in the trunk is important for winter storage of carbon and in the flower for reproductive development. Berries also accumulate starch in their plastids, which are also involved in the synthesis of aroma compounds important for fruit quality. The present work characterizes two glucose-phosphate translocators (VvGPT1, VvGPT2) that control the accumulation of starch in grape amyloplasts. Three different splicing variants identified for VvGPT2 (VvGPT2α, VvGPT2β and VvGPT2Ω) were more expressed in the leaves than in other organs. In contrast, VvGPT1 transcripts were more abundant in mature berries, canes and flowers than in the leaves. Expression of 35S-VvGPT1-GFP and 35S-VvGPT2Ω-GFP in tobacco leaf epidermal cells showed that the fusion proteins localized at the plastidial envelope. Complementation of the Arabidopsis pgi1-1 mutant impaired in leaf starch synthesis restored its ability to synthesize starch, demonstrating that VvGPT1 and VvGPT2Ω mediate the transport of glucose-6-Pi across the plastidial envelope. In grape cell suspensions, ABA, light and galactinol, together with sucrose and fructose, significantly increased the transcript abundance of VvGPT1, whereas VvGPT2Ω expression was affected only by sucrose. In addition, elicitation with methyl jasmonate strongly upregulated VvGPT1, VvGPT2Ω and VvPAL1, suggesting a role for GPTs in the production of secondary compounds in grapevine. Moreover, in grapevines cultivated in field conditions, VvGPT1 expression was higher in berries more exposed to the sun and subjected to higher temperatures. Although both VvGPT1 and VvGPT2 mediate the same function at the molecular level, they exhibit different expression levels and regulation in plant organs and in response to environmental and hormonal signals.
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http://dx.doi.org/10.1007/s00425-015-2329-xDOI Listing
October 2015

The first insight into the metabolite profiling of grapes from three Vitis vinifera L. cultivars of two controlled appellation (DOC) regions.

Int J Mol Sci 2014 Mar 10;15(3):4237-54. Epub 2014 Mar 10.

Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Quinta de Prados, Vila Real 5001-801, Portugal.

The characterization of the metabolites accumulated in the grapes of specific cultivars grown in different climates is of particular importance for viticulturists and enologists. In the present study, the metabolite profiling of grapes from the cultivars, Alvarinho, Arinto and Padeiro de Basto, of two Portuguese Controlled Denomination of Origin (DOC) regions (Vinho Verde and Lisboa) was investigated by gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) and an amino acid analyzer. Primary metabolites, including sugars, organic acids and amino acids, and some secondary metabolites were identified. Tartaric and malic acids and free amino acids accumulated more in grapes from vines of the DOC region of Vinho Verde than DOC Lisboa, but a principal component analysis (PCA) plot showed that besides the DOC region, the grape cultivar also accounted for the variance in the relative abundance of metabolites. Grapes from the cultivar, Alvarinho, were particularly rich in malic acid and tartaric acids in both DOC regions, but sucrose accumulated more in the DOC region of Vinho Verde.
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http://dx.doi.org/10.3390/ijms15034237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975394PMC
March 2014

The grape aquaporin VvSIP1 transports water across the ER membrane.

J Exp Bot 2014 Mar 27;65(4):981-93. Epub 2013 Dec 27.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas CITAB, Portugal.

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.
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http://dx.doi.org/10.1093/jxb/ert448DOI Listing
March 2014

Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

Plant Physiol 2013 Jul 17;162(3):1486-96. Epub 2013 May 17.

Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.

Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.
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http://dx.doi.org/10.1104/pp.113.220558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707533PMC
July 2013

Flow cytometry as a novel tool for structural and functional characterization of isolated yeast vacuoles.

Microbiology (Reading) 2013 May 28;159(Pt 5):848-856. Epub 2013 Feb 28.

Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

The yeast vacuole is functionally analogous to the mammalian lysosome. Both play important roles in fundamental cellular processes such as protein degradation, detoxification, osmoregulation, autophagy and apoptosis which, when deregulated in humans, can lead to several diseases. Some of these vacuolar roles are difficult to study in a cellular context, and therefore the use of a cell-free system is an important approach to gain further insight into the different molecular mechanisms required for vacuolar function. In the present study, the potentialities of flow cytometry for the structural and functional characterization of isolated yeast vacuoles were explored. The isolation protocol resulted in a yeast vacuolar fraction with a degree of purity suitable for cytometric analysis. Moreover, isolated vacuoles were structurally and functionally intact and able to generate and maintain electrochemical gradients of ions across the vacuolar membrane, as assessed by flow cytometry. Proton and calcium gradients were dissipated by NH4Cl and calcimycin, respectively. These results established flow cytometry as a powerful technique for the characterization of isolated vacuoles. The protocols developed in this study can also be used to enhance our understanding of several molecular mechanisms underlying the development of lysosome-related diseases, as well as provide tools to screen for new drugs that can modulate these processes, which have promising clinical relevance.
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http://dx.doi.org/10.1099/mic.0.062570-0DOI Listing
May 2013