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Catharanthus roseusleaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell-type localizations and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialised idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts ofC. roseusleaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterised. This involved the assembly of an improvedC. roseustranscriptome from short- and long-read data, IDIO+. It was observed thatC. roseusmesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast with surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the keys to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.HighlightCatharanthus mesophyll idioblasts are a hotspot of anticancer alkaloid accumulation. The idioblast transcriptome reveals commitment with stress responses and provides a roadmap towards the increase of anticancer alkaloid levels.

Transcriptional regulation is a central piece of the highly valuable monoterpenoid indole alkaloid pathway of C. roseus , and the ultimate tool for its understanding and manipulation. Here, we describe the adaptation of the TARGET methodology to identify specific and genome-wide leaf targets of C. roseus candidate transcription factors (TFs).

Plant organs are built of different cell types, characterized by specific transcription programs and metabolic profiles. The possibility of isolation of such cell types to perform differential transcriptomic, proteomic and metabolomic analyses is highly important to understand many aspects of plant physiology, namely, the structure and regulation of economically valuable specialized metabolic pathways. Here, we describe the isolation of idioblast leaf protoplasts of the medicinal plant Catharanthus roseus by fluorescence-activated cell sorting, taking advantage of the differential autofluorescence properties of those specialized cells.

Description, Hybridization footprint suggests introgression with local wild relatives in Western European grape varieties., Grapevine (Vitis vinifera L.) diversity richness results from a complex domestication history over multiple historical periods. Here, we used whole-genome resequencing to elucidate different aspects of its recent evolutionary history. Our results support a model in which a central domestication event in grapevine was followed by postdomestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties showing large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by gene flow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

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.

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.jplph.2018.08.011., 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 10mM CaCl2 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., This work was supported by European investment funds by FEDER/COMPETE/POCI-Operacional Competitiveness and Internationalization Programme, under the Projects INTERACT-NORTE-01-0145-FEDER-000017-Linha VitalityWine-ON 0013 and POCI-01-0145-FEDER-006958, and by National Funds by FCT-Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013, and CherryCrackLess (PTDC/AGR-PRO/7028/2014). VM was supported by a FCT postdoctoral Grant (SFRH/BPD/107905/2015)., info:eu-repo/semantics/publishedVersion

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

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., This work was supported by (1) Fundo Europeu de Desenvolvimento Regional funds through the Operational Competitiveness Programme COMPETE and by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the projects FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013) and FCOMP-01-0124-FEDER-019664 (PTDC/BIA-BCM/119718/2010); (2) the FCT scholarships cosupported by FCT and POPH-QREN (European Social Fund), SFRH/BD/41907/2007 (I.C.) and SFRH/BD/74257/2010 (H.N.); (3) a Scientific Mecenate Grant from Grupo Jerónimo Martins.

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 Ca2+ 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 100200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ 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), Ca2+, 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., The authors thank Professor Kyle W. Cunningham (Department of Biology, Johns Hopkins University, USA) for providing the yeast strain K667. The authors also acknowledge Professor Manuela Côrte-Real and Dr. Nuria Genicio Bouza (Department of Biology, University of Minho, Portugal) for technical support in flow cytometry and critical analysis of the results. This work is supported by European investment funds by FEDER/COMPETE/POCI-Operacional Competitiveness and Internationalization Programme, under the Projects INTERACT-NORTE-01-0145FEDER-000017-Linha VitalityWine-ON 0013 and POCI-01-0145FEDER-006958, and by National Funds by FCT–Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013, and CherryCrackLess (PTDC/AGR-PRO/7028/2014). VM was supported by a FCT postdoctoral Grant (SFRH/BPD/107905/ 2015)., info:eu-repo/semantics/publishedVersion

The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca

“Flavescence doree” (FD) is a quarantine disease associated with the presence of a phytoplasma transmitted by the insect vector Scaphoideus titanus. This disease affects grapevines and is of a great concern to the stability and sustainability of the wine industry, due to the harvest losses and death of infected plants. In Portugal, FD has seriously affected the “Vinhos Verdes” region, but so far the research on this topic is very limited. The current study confirmed that the FD phytoplasma strain involved in the outbreak in Vitis vinifera cv. Loureiro belongs to the 16SrV-D subgroup and shows very low RFLP variability in the tuf and secY genes. Transmission electron microscopy analysis of leaf midribs from infected grapevine plants revealed that the shape and structure of phloem cells were altered, presenting collapsed cells, callose accumulation in sieve plates and lipid accumulation in chloroplasts of phloem parenchyma cells. Moreover, data from two subsequent years showed that FD presence was associated with an average delay of 10 to 15 days on the time to visible inflorescences and to veraison, compared to healthy plants. At veraison, FD also lead to a significant decrease in the budburst percentage (7% to 12%), fertility index (35% in 2015), leaf area (56% to 63%), and chlorophyll content (18% to 35% lower SPAD values). Hence, infected plants showed a drastic reduction in the yield, corresponding to a decrease between 51% and 92% compared to healthy plants, which mostly resulted from a lower number of bunches (63% to 92% less), but also a decreased bunch weight (35% lower in 2015). Concerning berry quality, there were no significant differences in terms of total soluble solids and titrable acidity in both years. Here, this study concluded that the FD infection delays the grapevine development and leads to drastic production losses, which may be partly linked to the ultrastructural modifications observed in the phloem cells of infected plants.

Summary 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.

In the present work, we study the relationship between the production of indole alkaloids and the transcript profiles of several genes involved in their biosynthesis in Catharanthus roseus cell cultures elicited with 50 mM cyclodextrins (CDs) and/or 100 µM methyl jasmonate (MJ) for 8 days. Gene expression and indole alkaloid production were significantly enhanced by CDs treatment while MJ induced alkaloid synthesis to a lesser extent and had almost no effect on gene expression. The combined treatment with both the elicitors pointed to synergistic effect on gene transcript accumulation, which resulted in a 6,071.42 and 8,100 % enhancement of ajmalicine and catharanthine productivity, respectively, compared with non-treated cells at day 8. This increase in alkaloid production under the combined influence of CDs and MJ was 329.66 and 595.07 % for ajmalicine and 202.5 and 291.36 % more for catharanthine in compared with elicitation with CDs or MJ alone. Interestingly the over-synthesized alkaloids readily secreted into the culture medium, thereby avoiding the possibility of retro-inhibition and cytotoxicity due to their accumulation in cells.

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.

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.

6 páginas, 4 figuras, 2 tablas.-- El PDF es la versión de autor., Catharanthus roseus is among the most important medicinal plants, mainly due to its anticancer alkaloids. Phenolics and organic acids were studied in this plant’s roots, using HPLC–MS and HPLC–UV, respectively. Neither phenolic acids nor flavonoids could be identified, but an organic acid profile composed of oxalic, cis-aconitic, citric, pyruvic, malic and fumaric acids was found. Roots proved to be much richer in these compounds than were other plant parts, previously described, reaching 187 g/kg. Antioxidant activity of the roots was evaluated using representative radicals: DPPH, superoxide radical and nitric oxide radical. The results indicate a strong anti-radical activity, surpassing that of leaves, petals, stems and seeds previously reported. Synergistic studies revealed the contribution of organic acids to the displayed effect, although other compounds, not determined herein, might be responsible for part of the activity. The results indicate that C. roseus roots could be exploited as a source of health-promoting antioxidants for use by both food and pharmaceutical industries., The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT) for financial support (PTDC/AGR-AAM/64150/2006). L. Gaspar is indebted to FCT for the Grant (BII).

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.

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.

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.

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.

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.

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.

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.

The leaves of Catharanthus roseus (L.) G. Don produce the first natural drugs used in cancer therapy – the dimeric terpenoid indole alkaloids vinblastine and vincristine. The study of C. roseus further revealed two other terpenoid indole alkaloids with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative. The biosynthetic pathway of the medicinal alkaloids has been investigated in much detail and a number of steps are now well characterized at the enzyme and gene level and, recently, several regulatory genes have also been isolated and characterized. Since early studies of the biosynthesis of vinblastine, during the 1970s and 1980s, the dimerization reaction has attracted much attention due to its possible regulatory importance and potential application for the semi synthetic production of the dimeric alkaloids. After initial, inconclusive work suggesting the involvement of peroxidase-like enzymes, the search for the dimerization enzyme in leaf tissue detected a single dimerization activity credited to the single class III plant peroxidase present in the leaves of the plant – the basic isoenzyme CRPRX1. The enzyme was purified to homogeneity, the respective cDNA and genomic sequences were characterized, and a channeling mechanism was proposed for the peroxidase-mediated-vacuolar synthesis of the first dimeric alkaloid intermediate, α-3′,4′-anhydrovinblastine. On the other hand, the oxidation of ajmalicine into serpentine has been attributed to basic peroxidase isoenzymes localized in the vacuole of C. roseus cells. An overview of the work implying class III plant peroxidases in the biosynthesis of terpenoid indole alkaloids in C. roseus is presented here. Abbreviations: CRPRX1 –Catharanthus roseus peroxidase 1; DAB – diaminobenzidine; IEF – isoelectric focusing; UV – ultraviolet.

We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.

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.

An H2O2-dependent enzyme capable of coupling catharanthine and vindoline into α-3′,4′-anhydrovinblastine (AVLB) was purified to apparent homogeneity from Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS-PAGE). In addition to AVLB synthase activity, the purified enzyme shows peroxidase activity, and the VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin ferric heme protein, belonging to the plant peroxidase superfamily. Kinetic studies revealed that both catharanthine and vindoline were substrates of the enzyme, AVLB being the major coupling product.

The fate of catharanthine and vindoline during the peroxidase-mediated enzymatic synthesis of α-3′,4′-anhydrovinblastine (AVLB) has been studied. The results showed that catharanthine and vindoline are suitable electron donors for the oxidizing intermediates of horseradish peroxidase (compound I and compound II). The reduction of peroxidase compound II is one of the limiting steps in the coupling reaction. In fact, k3 (compound II reduction constant) values were 2.53 × 103 m −1 s−1 and 3.57 × 103 m −1 s−1 for catharanthine and vindoline, respectively. These values are extremely low if we compare them with those found for other substrates of peroxidase-catalyzed oxidations. From these results and the analysis of the reaction products in conditions in which coupling occurred, we propose a mechanism where vindoline-free radicals resulting from reaction of vindoline with the oxidized states of peroxidase are deactivated by reaction with catharanthine. Catharanthine radicals actually set off the coupling reaction.

Partially purified protein extracts of Catharanthus roseus leaves were able to couple catharanthine and vindoline to produce α-3′,4′-anhydrovinblastine (AVLB) in a reaction strictly dependent on H2O2. This result, and the co-purification of peroxidase with AVLB synthetase activity, strongly suggest a peroxidase-like nature for the coupling enzyme. Only one peroxidase isoenzyme was detected in C. roseus leaves, and it was shown that this isoenzyme consists of a molecularly-heterogeneous basic peroxidase (EC 1-11-1-7) mainly located in the vacuole. These results suggest that a basic peroxidase located in the vacuole may be the main enzyme responsible for AVLB synthesis in C. roseus leaves. This isoenzyme was also found in cell walls where a peroxidase inhibitor was detected.

The genome size and organization of the important medicinal plant Catharanthus roseus is shown to correspond to 1C = 0.76 pg (~738 Mbps) and 2n = 16 chromosomes. The data provide a sound basis for future studies including cytogenetic mapping, genomics and breeding., 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.

Catharanthus roseus (L.) G. Don. accumulates in the leaves the dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which were the first natural anticancer products to be clinically used, and are still among the most valuable agents used in cancer chemotherapy. The great pharmacological importance of the TIAs, associated with the low abundance of the anticancer alkaloids in the plant, stimulated intense research on the TIA pathway, and C. roseus has now become one of the most extensively studied medicinal plants. About 130 TIAs have already been isolated from C. roseus, and it has been shown that the biosynthesis of vinblastine is highly complex, involving at least 30 steps from the amino-acid tryptophan and the monoterpenoid geraniol (Loyola-Vargas et al., 2007; van der Heijden et al., 2004). All the TIAs of C. roseus derive from the common precursor strictosidine, after which the TIA pathway splits into several branches including a short one leading to ajmalicine and serpentine (used as an antihypertensive and as a sedative respectively), and two long branches leading to vindoline and catharanthine the leaf abundant monomeric precursors of vinblastine and vincristine (Loyola-Vargas et al., 2007; van der Heijden et al., 2004). In our lab, we have performed the characterization of a key biosynthetic step of the anticancer TIAs the biosynthesis of the first dimeric TIA, α-3’,4’-anhydrovinblastine (AVLB), from vindoline and catharanthine. We identified a leaf class III peroxidase (Prx) with AVLB synthase activity and we purified and characterized this enzyme, which was named Catharanthus roseus peroxidase 1, CroPrx1 (Bakalovic et al., 2006; Peroxibase, http://peroxibase.toulouse.inra.fr index.php). We have further shown the localization of CroPrx1 in the same subcellular compartment where alkaloids accumulate, the vacuole, through biochemical and molecular methodologies

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.

7 páginas, 4 figuras, 1 tabla.-- El PDF es la versión de autor., 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 (IC50 = 0.775 μM vs physostigmine IC50 = 6.45 μM) 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 pA2 of 5.2, whereas the precursor ajmalicine up to 80 μM was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 μM concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine (IC50 = 59.6 μM) and ajmalicine (IC50 = 72.3 μM), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine (IC50 = 0.35 μM) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up to 100 μM 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., The authors are grateful to Fundaçäo para a Ciência e a Tecnologia (FCT) for financial support (PTDC/AGR-AAM/64150/ 2006). J. Faria (BII) is grateful to FCT for the grant.

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.

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.

5 páginas, 2 figuras, 2 tablas.-- El PDF es la copia de autor., 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., The authors are grateful to Fundaçäo para a Ciência e a Tecnologia (PTDC/AGR-AAM/64150/2006) for financial support. J. Faria (BII) is grateful to FCT for the grant.

6 páginas, 4 figuras, 3 tablas.-- El PDF es la copia de autor., 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 (IC50 at 422, 442 and 2683 μg/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., The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT) for financial support of this work (PTDC/AGR-AAM/ 64150/2006). D.M. Pereira is indebted to FCT for the grant.

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.

8 pages, 4 figures, 1 table., 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., D.M.P. is grateful to Fundaçäo para a Ciência e Tecnologia (PTDC/AGR-AAM/64150/2006) for the grant.

The leaves of Catharanthus roseus (L.) G. Don (formerly Vinca rosea L.) were used in traditional medicine as an oral hypoglycemic agent and investigation of this activity ultimately led to the serendipitous discovery of the cytostatic terpenoid indole alkaloids vinblastine and vincristine. These compounds were the first natural anticancer agents to be clinically used and, together with a number of semisynthetic derivatives, are universally known as the Vinca alkaloids. Due to its important pharmaceutical alkaloids, C. roseus has now become one of the most extensively studied medicinal plants and much has been discovered about the biosynthetic pathway of terpenoid indole alkaloids, the regulation and compartmentation of the pathway, and the mechanisms of accumulation of those compounds inside the plant cell. The biosynthesis of vinblastine involves more than twenty enzymatic steps, nine of which are now well characterized at the enzyme and gene level and, recently, regulatory genes of the initial part of the pathway (ORCAs) have been cloned, in what consists a highly promising strategy for the manipulation of the pathway. On the other hand, the activity of vinblastine and vincristine in human cells has been thoroughly studied. The cytostatic activity has been shown to result from interference with tubulin, but the precise mechanism of action is still not perfectly understood. Uptake and extrusion in human cells has been characterized, specially the extrusion mechanism responsible for resistance to the drugs, and their metabolism in the human body has also been studied. Together, the above mentioned studies enable to establish some interesting evolutionary links between the enzymes involved in plant biosynthesis of the anticancer alkaloids and the enzymes involved in animal metabolism of the drugs, and also, possibly, between their vacuolar transport in plant cells and multidrug resistance in human cancer cells.

Publisher Summary Programmed cell death (PCD) is a critical part of normal development at all stages of the plant life cycle. This chapter discusses protocols for PCD assays. DNA degradation is an important diagnostic factor in programmed cell death, and cleavage of DNA in internucleosomal fragments of 180 bp is considered a hallmark of apoptosis. The ability to isolate undegraded high molecular weight DNA from viable tissue is a requirement for studying PCD-induced DNA degradation because it ensures that any DNA degradation observed is not an artifact of the isolation procedure. The barley aleurone layer is a model system to study hormonally induced PCD in plants. In this tissue, gibberellic acid (GA) induces PCD, whereas abscisic acid (ABA) prevents this program. The chapter describes a method for DNA isolation from barley aleurone cells that minimizes DNA degradation during the purification process. It has been shown to work well for high molecular weight DNA analysis in a variety of other tissue and cell preparations, for example, Arabidopsis thaliana leaves.