Patrícia Duarte

Molecular Cloning and Characterization of a Vacuolar Class III Peroxidase Involved in the Metabolism of Anticancer Alkaloids in Catharanthus roseus

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 α-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 α-3′,4′-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

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

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.

Vacuolar Transport of the Medicinal Alkaloids from Catharanthus roseus Is Mediated by a Proton-Driven Antiport

A specific H+ antiport system mediates the vacuolar uptake of terpenoid indole alkaloids in Catharanthus roseus. 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.

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

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.

The embryo sac of Cynara cardunculus: ultrastructure of the development and localisation of the aspartic proteinase cardosin B

Cynara cardunculus is a native plant with flowers that are used traditionally in the manufacture of ewe’s cheese in the Iberian Peninsula. Milk clotting ability of the plant is attributed to the high concentrations of aspartic proteinases (APs), named cardosins, found in the flowers. Although these enzymes are well characterised on a molecular and biochemical basis, the biological role of the majority of plant APs is yet unassigned. We suspected APs play an important role in ovule function, and we characterised the maturation of the ovules of C. cardunculus and its Polygonum-type embryo sacs. The internal layer of the integument differentiates into an endothelium as described for other Asteraceae, with differentiation of two nucellar layers, a podium and a hypostase coinciding with the onset of pollen receptivity. In flowering plants, programmed cell death (PCD) events are essential for the success of nucellar maturation and consequent differentiation of a fully functional embryo sac. In C. cardunculus, nucellar PCD is integral to the maturation of the embryo sac, which in turn is closely correlated with the accumulation of the AP cardosin B specifically in the hypostase. The onset of cardosin B expression temporally coincides with the degeneration of nucellar cells. In fully mature embryo sacs, cardosin B is localised in both the hypostase and epistase, two regions that differentiate through PCD. Thus, cardosin B localisations closely correlate with events of PCD in the nucellus of C. cardunculus suggesting involvement in ovule and embryo sac development and further suggest the biological significance of APs like cardosin B, in this particular process. This work contributes new data to the plant AP research field and indicates an involvement of cardosin B in the PCD-dependent degeneration of the nucellus.

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?

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. Addendum to: Costa MM, Hilliou F, Duarte P, Pereira LG, Almeida I, Leech M, Memelink J, Barceló AR, Sottomayor M. Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol 2008; 146:403-17.

On site noninvasive assessment of peri‐implant inflammation by optical spectroscopy

BACKGROUND AND OBJECTIVE Optical spectroscopy has been proposed to measure regional tissue hemodynamics in periodontal tissue. The objective of this study was to further evaluate the diagnostic potential of optical spectroscopy in peri-implant inflammation in vivo by assessing multiple inflammatory parameters (tissue oxygenation, total tissue hemoglobin, deoxyhemoglobin, oxygenated hemoglobin and tissue edema) simultaneously. MATERIAL AND METHODS A cross-sectional study was performed in a total of 64 individuals who presented with dental implants in different stages of inflammation. In brief, visible-near-infrared spectra were obtained, processed and evaluated from healthy (n = 151), mucositis (n = 70) and peri-implantitis sites (n = 75) using a portable spectrometer. A modified Beer-Lambert unmixing model that incorporates a nonparametric scattering loss function was employed to determine the relative contribution of each inflammatory component to the overall spectrum. RESULTS Tissue oxygenation at peri-implantitis sites was significantly decreased (p < 0.05) when compared with that at healthy sites, which was largely due to an increase in deoxyhemoglobin and a decrease in oxyhemoglobin at the peri-implantitis sites compared with the mucositis and healthy sites. In addition, the tissue hydration index derived from the optical spectra in mucositis was significantly higher than that in other groups (p < 0.05). CONCLUSION In summary, the results of this study revealed that hemodynamic alterations can be detected around diseased peri-implant sites by optical spectroscopy, and this method may be considered an alternative and feasible approach for the monitoring and diagnosis of peri-implant diseases.

Simple and Fast In Situ Hybridization

RNA in situ hybridization is a powerful technique but can be time consuming and potentially hazardous. We developed a procedure in which nonisotopic in situ hybridization is performed in Vibratome-sectioned tissues that are not dehydrated or embedded in paraffin or resin, thus avoiding additional pretreatment steps to allow the probes to penetrate the cell structure. Predigestion with proteases was not necessary. Hybridization of sections and immunologic detection were performed in microplates to avoid the use of slide-coating agents. Biotinylated cDNA probes were synthesized by PCR. Synthetic oligonucleotide probes chemically labeled with biotin or digoxigenin were also used. Stained sections were mounted on slides for microscopic observation, and various transcripts were successfully detected using different visualization methods. This method is fast and can be easily implemented in other laboratories because it requires minimal expertise in processing biological samples for microscopy.

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

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.

Fusion with Fluorescent Proteins for Subcellular Localization of Enzymes Involved in Plant Alkaloid Biosynthesis

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.