Publications by authors named "Mariana Sottomayor"

23 Publications

  • Page 1 of 1

Subfunctionalization of Paralog Transcription Factors Contributes to Regulation of Alkaloid Pathway Branch Choice in .

Front Plant Sci 2021 25;12:687406. Epub 2021 May 25.

Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.

produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.
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http://dx.doi.org/10.3389/fpls.2021.687406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8186833PMC
May 2021

Calcium- and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells.

J Plant Physiol 2018 Dec 1;231:57-67. Epub 2018 Sep 1.

Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Escola de Ciências, Universidade do Minho, 4710-057, Braga, Portugal; Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas CITAB-UM, Departamento de Biologia, Escola de Ciências, Universidade do Minho, 4710-057, Braga, Portugal; Centro de Engenharia Biológica (CEB), Universidade do Minho, 4710-057, Braga, Portugal.

The efficacy of calcium sprays for improving fleshy fruit resistance to abiotic/biotic stress and enhancement of fruit shelf life has increasingly been explored. However, because calcium is a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid (ABA) and jasmonates, it may interfere with the biosynthesis of specialized metabolites highly important for fruit and wine quality, such as phenolic compounds. In this study, a combination of biochemical and molecular biology approaches were applied to grape cell cultures and detached grape berries, in order to investigate the effect of calcium in the modulation of enzymes involved in the biosynthesis of phenolic compounds and in cell wall organization. Concentrations up to 10 mM CaCl did not affect cell growth, size or viability, but triggered modifications in total phenolics content, particularly in anthocyanin levels in grape cell suspensions. The effects of calcium applied alone or in combination with ABA or methyl jasmonate (MeJA) were visible in several branches of specialized metabolic pathways, confirming that the calcium-hormone interplay regulates the expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), dihydroflavonol reductase (DFR) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The activity of PAL and UFGT enzymes was also specifically modulated by calcium, ABA and MeJA. These results closely correlated to the modifications observed in the expression of VvAM1 and VvABCC1 encoding vacuolar anthocyanin transporters. Modulation of the expression and activity of pectin methyl esterases (PME) and polygalacturonases (PG) by calcium was also evident, confirming an important role of calcium in cell wall organization via the regulation of enzyme activity, besides its well-known role in the formation of cross links between pectin molecules. Overall, this study uncovers important biochemical mechanisms induced by calcium and stress hormones on grape berries, and highlights the need to consider the consequences of calcium treatments and stress for fruit quality.
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http://dx.doi.org/10.1016/j.jplph.2018.08.011DOI Listing
December 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

The grapevine VvCAX3 is a cation/H exchanger involved in vacuolar Ca homeostasis.

Planta 2017 Dec 11;246(6):1083-1096. Epub 2017 Aug 11.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas, CITAB-UMinho Pole, Departamento de Biologia, Escola de Ciências, Universidade do Minho, Braga, Portugal.

Main Conclusion: The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca , Na and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca, demonstrating a role in Ca transport. The truncated VvCAX3 was further shown to be involved in the transport of Na, Li, Mn and Cu in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca, and Na in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.
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http://dx.doi.org/10.1007/s00425-017-2754-0DOI Listing
December 2017

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant Physiol 2016 08 29;171(4):2371-8. Epub 2016 Jun 29.

CIBIO/InBIO-Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, 4485-661 Vairão, Portugal (S.B., T.M.-C., J.G.G., M.S.); Instituto de Investigação e Inovação em Saúde, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal (I.C., A.L.G., P.D.);Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal (I.C., M.S.);Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal (R.G., T.L., C.A., C.B., N.P.M.);REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal (P.A., P.V.); andREQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal (I.M.V., J.A.R.)

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.
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http://dx.doi.org/10.1104/pp.16.01028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972299PMC
August 2016

Protoplast Transformation as a Plant-Transferable Transient Expression System.

Methods Mol Biol 2016 ;1405:137-48

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

The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.
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http://dx.doi.org/10.1007/978-1-4939-3393-8_13DOI Listing
November 2016

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

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

New Phytol 2015 Jan 25;205(2):653-65. Epub 2014 Sep 25.

Department of Ornamental Horticulture, Agriculture Research Organization, The Volcani Center, PO Box 6, Beit Dagan, 50250, Israel.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.
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http://dx.doi.org/10.1111/nph.13038DOI Listing
January 2015

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

Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don.

AoB Plants 2012 2;2012:pls002. Epub 2012 Mar 2.

IBMC - Instituto de Biologia Molecular e Celular , Universidade do Porto , Rua do Campo Alegre, 823, 4150-180 Porto , Portugal.

Background And Aims: Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size.

Methodology: The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology.

Principal Results: The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp.

Conclusions: The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.
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http://dx.doi.org/10.1093/aobpla/pls002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292738PMC
August 2012

The promoter region of the Zinnia elegans basic peroxidase isoenzyme gene contains cis-elements responsive to nitric oxide and hydrogen peroxide.

Planta 2012 Aug 24;236(2):327-42. Epub 2012 Feb 24.

Department of Plant Biology, University of Murcia, 30100, Murcia, Spain.

NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.
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http://dx.doi.org/10.1007/s00425-012-1604-3DOI Listing
August 2012

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

J Exp Bot 2011 May 28;62(8):2841-54. Epub 2011 Feb 28.

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), PO Box 164, E-30100 Campus University Espinardo (Murcia), Spain.

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.
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http://dx.doi.org/10.1093/jxb/erq458DOI Listing
May 2011

Fusion with fluorescent proteins for subcellular localization of enzymes involved in plant alkaloid biosynthesis.

Methods Mol Biol 2010 ;643:275-90

IBMC - Institute of Cellular and Molecular Biology, Porto University, Porto, Portugal.

To establish the role in alkaloid metabolism of candidate genes identified in silico or by Omics approaches, it may be essential to determine the subcellular localization of the encoded proteins. The fusion with fluorescent proteins (FP) may now be used as a quite effective and reliable tool to investigate this question. The methodology involves the choice of the FP, the design and production of the appropriate FP fusions, and the use of a transient or stable transformation protocol applied to a homologous or heterologous plant system. This chapter describes the application of this methodology to an enzyme involved in indole alkaloid biosynthesis, with general considerations on the development of the approach.
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http://dx.doi.org/10.1007/978-1-60761-723-5_19DOI Listing
September 2010

Downregulation of the basic peroxidase isoenzyme from Zinnia elegans by gibberellic acid.

J Integr Plant Biol 2010 Feb;52(2):244-51

Department of Plant Biology, University of Murcia, E-30100, Murcia, Spain.

Hypocotyl formation during the epigeal germination of seedlings is under strict hormonal regulation. In a 3 d old Zinnia elegans seedling system, gibberellic acid (GA(3)) exerts an opposite effect to that exerted by light on hypocotyl photomorphogenesis because GA(3) promotes an etiolated-like growth with an inhibition of radial (secondary) growth. For this reason, the effect of GA(3) on the basic peroxidase isoenzyme from Z. elegans (ZePrx), an enzyme involved in hypocotyl lignin biosynthesis, was studied. The results showed that GA(3) reduces ZePrx activity, similarly to the way in which it reduces seedling secondary growth. This hormonal response is supported by the analysis of the ZePrx promoter, which contains four types of GA(3)-responsive cis-elements: the W Box/O2S; the Pyr Box; the GARE; and the Amy Box. Taken together, these results suggest that ZePrx is directly regulated by GA(3), with this effect matching the inhibitory effect of GA on the hypocotyl secondary growth.
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http://dx.doi.org/10.1111/j.1744-7909.2010.00888.xDOI Listing
February 2010

Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission.

Phytomedicine 2010 Jul 4;17(8-9):646-52. Epub 2009 Dec 4.

REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Aníbal Cunha, 164, 4050-047 Porto, Portugal.

The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibited acetylcholinesterase (AchE) in an in vitro microassay, an effect ascribable mainly to serpentine (IC(50) = 0.775 microM vs physostigmine IC(50) = 6.45 microM) as assessed with the pure compound. Pure alkaloids were tested for muscarinic and nicotinic antagonism using rat ex-vivo preparations, namely, ileum and diaphragm/phrenic-nerve, respectively. Serpentine competitively blocked muscarinic receptors with a pA(2) of 5.2, whereas the precursor ajmalicine up to 80 microM was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 microM concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine (IC(50) = 59.6 microM) and ajmalicine (IC(50) = 72.3 microM), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine (IC(50) = 0.35 microM) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up to 100 microM did not change diaphragm contractions suggesting reduced affinity for neuromuscular nicotinic receptors. Despite strong in vitro AchE inhibition, serpentine failed to restore diaphragm contractions upon submaximal tubocurarine blockade, suggesting that poor tissue penetration may prevent serpentine from inhibiting AchE in deep neuromuscular synapses in the ex-vivo preparation. To our knowledge, the present study is the first to assess the effect of C. roseus root extracts, as well as of serpentine, ajmalicine and catharanthine on AchE. The results described herein suggest that the currently overlooked C. roseus roots may constitute a promising source of compounds with pharmaceutical interest. Moreover, given serpentine's potent in vitro AchE inhibitory activity and low cholinergic receptor affinity, it is conceivable that minor structural modifications may yield a potent and selective AchE inhibitor, potentially useful for the pharmacological management of conditions such as Alzheimer's disease and/or myasthenia gravis.
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http://dx.doi.org/10.1016/j.phymed.2009.10.008DOI Listing
July 2010

Simple and reproducible HPLC-DAD-ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots.

J Pharm Biomed Anal 2010 Jan 12;51(1):65-9. Epub 2009 Aug 12.

Department of Food Science and Technology, CEBAS (CSIC), Murcia, Spain.

Catharanthus roseus is one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin (leurocristine) and vinblastin (vincaleucoblastine), whose anticancer activity represents powerful therapeutics to many diseases, such as Hodgkin lymphoma. Usually, the remaining plant parts go to waste. Here we describe a phytochemical study on this species roots. Alkaloids in aqueous extracts, the usual form of consumption of this matrix, were studied using HPLC-DAD-ESI-MS/MS, which allowed the identification of 19-S-vindolinine, vindolinine, ajmalicine and an ajmalicine isomer, tabersonine, catharanthine, serpentine and a serpentine isomer. Quantification of the identified compounds revealed that serpentine and its isomer were predominant (64.7%) over the other alkaloids, namely vindolinine and its isomer (23.9%), catharanthine (7.7%) and ajmalicine (3.8%). The used procedure revealed to be simple, sensitive and reproducible.
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http://dx.doi.org/10.1016/j.jpba.2009.08.005DOI Listing
January 2010

A vacuolar class III peroxidase and the metabolism of anticancer indole alkaloids in Catharanthus roseus: Can peroxidases, secondary metabolites and arabinogalactan proteins be partners in microcompartmentation of cellular reactions?

Plant Signal Behav 2008 Oct;3(10):899-901

IBMC-Instituto de Biologia Molecular e Celular; Universidade do Porto; Porto Portugal.

Plants possess a unique metabolic diversity commonly designated as secondary metabolism, of which the anticancer alkaloids from Catharanthus roseus are among the most studied. Recently, in a classical function-to-protein-to-gene approach, we have characterized the main class III peroxidase (Prx) expressed in C. roseus leaves, CrPrx1, implicated in a key biosynthetic step of the anticancer alkaloids. We have shown the vacuolar sorting determination of CrPrx1 using GFP fusions and we have obtained further evidence supporting the role of this enzyme in alkaloid biosynthesis, indicating the potential of CrPrx1 as a molecular tool for the manipulation of alkaloid metabolism. Here, we discuss how plant cells may regulate Prx reactions. In fact, Prxs form a large multigenic family whose members accept a broad range of substrates and, in their two subcellular localizations, the cell wall and the vacuole, Prxs co-locate with a large variety of secondary metabolites which can be accepted as substrates. How then, are Prx reactions regulated? Localization data obtained in our lab suggest that arabinogalactan proteins (AGPs) and Prxs may be associated in membrane microdomains, evocative of lipid rafts. Whether plasma membrane and/or tonoplast microcompartmentation involve AGPs and Prxs and whether this enables metabolic channeling determining Prx substrate selection are challenging questions ahead.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634410PMC
http://dx.doi.org/10.4161/psb.3.10.6576DOI Listing
October 2008

Hormonal regulation of the basic peroxidase isoenzyme from Zinnia elegans.

Planta 2009 Sep 22;230(4):767-78. Epub 2009 Jul 22.

Department of Plant Biology, University of La Coruña, 15071 La Coruña, Spain.

Xylem differentiation in plants is under strict hormonal regulation. Auxins and cytokinins, together with brassinosteroids (BRs), appear to be the main hormones controlling vascular differentiation. In this report, we study the effect of these hormones on the basic peroxidase isoenzyme from Zinnia elegans (ZePrx), an enzyme involved in lignin biosynthesis. Results showed that auxins and cytokinins induce ZePrx, similarly to the way in which they induce seedling secondary growth (in particular, metaxylem differentiation). Likewise, the exogenous application of BR reduces the levels of ZePrx, in a similar way to their capacity to inhibit seedling secondary growth. Consistent with this notion, the exogenous application of BR reverses the auxin/cytokinin-induced ZePrx expression, but has no effect on the auxin/cytokinin-induced secondary growth. This differential hormonal response is supported by the analysis of the ZePrx promoter, which contains (a) cis-elements directly responsive to these hormones and (b) cis-elements targets of the plethora of transcription factors, such as NAC, MYB, AP2, MADS and class III HD Zip, which are up-regulated during the auxin- and cytokinin-induced secondary growth. Taken together, these results suggest that ZePrx is directly and indirectly regulated by the plethora of hormones that control xylem differentiation, supporting the role of ZePrx in xylem lignification.
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http://dx.doi.org/10.1007/s00425-009-0982-7DOI Listing
September 2009

Targeted metabolite analysis of Catharanthus roseus and its biological potential.

Food Chem Toxicol 2009 Jun 17;47(6):1349-54. Epub 2009 Mar 17.

REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Aníbal Cunha, 164, 4050-047 Porto, Portugal.

Catharanthus roseus is nowadays one of the most studied medicinal plants. In this work, further knowledge on different parts of this species (leaves, stems, seeds and petals) was achieved, namely phenolics by HPLC-DAD and organic acids and amino acids by HPLC-UV. Also, the biological potential, expressed as acetylcholinesterase inhibitory activity was accessed and, in some parts, an acetylcholinesterase inhibitory capacity higher than 85% was found (IC(50) at 422, 442 and 2683 microg/mL in leaves, stems and petals, respectively). C. roseus aqueous extract revealed to be a rich source of phenolics, namely caffeoylquinic acids and flavonoids derivatives (up to 4127 mg/kg in stems, 4484 mg/kg in seeds, 8688 mg/kg in leaves and 41125 mg/kg in petals), organic acids (962, 6678, 25972 and 12463 mg/kg in seeds, petals, stems and leaves, respectively), such as citric acid (over 85% in some plant parts), and amino acids (31557, 39327, 50540 and 159697 mg/kg in stems, petals, seeds and leaves, respectively), of which arginine was a major compound.
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http://dx.doi.org/10.1016/j.fct.2009.03.012DOI Listing
June 2009

Volatile composition of Catharanthus roseus (L.) G. Don using solid-phase microextraction and gas chromatography/mass spectrometry.

J Pharm Biomed Anal 2009 Apr 31;49(3):674-85. Epub 2008 Dec 31.

REQUIMTE/Serviço de Farmacognosia, Faculdade de Farmácia da Universidade do Porto, R. Aníbal Cunha 164, 4050-047 Porto, Portugal.

A total of 88 volatile and semi-volatile components were formally or tentatively identified in flowers, leaves and stems of Catharanthus roseus (L.) G. Don (cv. Little Bright Eye), by headspace solid-phase microextraction (HS-SPME) and by dichloromethane extraction, combined with gas chromatography-mass spectrometry (GC-MS). These include some diterpenic compounds (manool and manoyl oxides), a sesquiterpen (alpha-bisabolol), and some pyridine, pyrazine, indol and carotenoid derivatives. Applying multivariate analysis (principal component analysis and agglomerative hierarchic cluster analysis) to the HS-SPME-GC-MS data, it was possible to characterize each part of the vegetal material using a relative small number of compounds. Hence, flowers were richer in terpenic molecules (including limonene), alpha-bisabolol, methyljasmonate, cis-jasmone, 2-phenylethanol, phenylacetaldehyde, trans-2-octenal, benzylic alcohol and 2-isobutyl-3-methoxypyrazine. Leaves can be characterized by the methyl and propyl esters of fatty acids, mono- and disaturated, trans-phytol, carotenoid derivative compounds, hydrofarnesylacetone, methylanthranilate, manool and epi-manool oxide, while stems have high levels of volatile aldehydes, such as hexanal, octanal, cis-2-nonenal, cis-2-decenal, cis, trans-2,6-nonadienal, trans, trans-2,4-decadienal and cis, trans-2,4-decadienal. Dichloromethane extraction allowed also the identification of some alkaloid-like compounds that were not detected by HS-SPME.
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http://dx.doi.org/10.1016/j.jpba.2008.12.032DOI Listing
April 2009

New phenolic compounds and antioxidant potential of Catharanthus roseus.

J Agric Food Chem 2008 Nov 14;56(21):9967-74. Epub 2008 Oct 14.

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University Espinardo, Murcia, Spain.

Screening of the phenolic compounds from seeds, stems, leaves and petals of Catharanthus roseus (L.) G. Don (cv. Little Bright Eye) was achieved by HPLC-DAD-ESI-MS/MS. This is the first detailed study of noncolored phenolics in C. roseus, which allowed the characterization of three caffeoylquinic acids and fifteen flavonol glycosides (di- and trisaccharides of kaempferol, quercetin and isorhamnetin). Fifteen compounds are reported for the first time in this species. The scavenging ability of the different plant matrices was assessed against DPPH(*) radical and against reactive oxygen (superoxide radical) and a reactive nitrogen (nitric oxide) species. A concentration-dependent protective effect was observed for seeds and tissues, with petals shown to be the most active.
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http://dx.doi.org/10.1021/jf8022723DOI Listing
November 2008

Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus.

Plant Physiol 2008 Feb 7;146(2):403-17. Epub 2007 Dec 7.

John Innes Centre, Norwich NR4 7UH, United Kingdom.

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.
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http://dx.doi.org/10.1104/pp.107.107060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245823PMC
February 2008

Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana.

Planta 2006 Jan 15;223(2):374-80. Epub 2005 Oct 15.

Instituto de Biologia Molecular e Celular, Universidade do Porto, R. Campo Alegre 823 Porto, Portugal.

The expression of "classical" arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana was characterized by RT-PCR. Transcripts of Agp6 and Agp11 were consistently found to be more abundant in pollen tubes and seem to be pollen-specific. Transcripts of other AGP genes were also detected in pollen but in lesser amounts and in a non-specific fashion. Two reference genes, ubiquitin-conjugating enzyme 9 and tubulin beta-4 chain, were evaluated and selected for gene expression studies in pollen. Expression characterization was complemented with immunolocalization studies using monoclonal antibodies specific to several glycosidic epitopes of AGPs. These studies were performed on in vitro germinated pollen tubes with the antibodies MAC207 and LM2. MAC207 produced labelling at the tip of the pollen tube, while LM2 produced a ring-like fluorescence around the emerging region of the tube, suggesting a role of AGPs during Arabidopsis pollen tube germination. To our knowledge, this is the first report establishing the presence of AGPs on Arabidopsis pollen tubes.
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http://dx.doi.org/10.1007/s00425-005-0137-4DOI Listing
January 2006