Alfonso Ros Barceló

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.

Peroxidase: a multifunctional enzyme in grapevines

Peroxidases are heme-containing enzymes that catalyse the one-electron oxidation of several substrates at the expense of H2O2. They are probably encoded by a large multigene family in grapevines, and therefore show a high degree of polymorphism. Grapevine peroxidases are glycoproteins of high thermal stability, whose molecular weight usually ranges from 35 to 45 kDa. Their visible spectrum shows absorption bands characteristic of high-spin class III peroxidases. Grapevine peroxidases are capable of accepting a wide range of natural compounds as substrates, such as the cell wall protein extensin, plant growth regulators such as IAA, and phenolics such as benzoic acids, stilbenes, flavonols, cinnamyl alcohols and anthocyanins. They are located in cell walls and vacuoles. These locations are in accordance with their key role in determining the final cell wall architecture, especially regarding lignin deposition and extensin insolubilization, and the turnover of vacuolar phenolic metabolites, a task that also forms part of the molecular program of disease resistance. Although peroxidase is a constitutive enzyme in grapevines, its levels are strongly modulated during plant cell development and in response to both biotic and abiotic environmental factors. To gain an insight into the metabolic regulation of peroxidase, several authors have studied how grapevine peroxidase and H2O2 levels change in response to a changing environment. Nevertheless, the results obtained are not always easy to interpret. Despite such difficulties, the response of the peroxidase-H2O2 system to both UV-C radiation and Trichoderma viride elicitors is worthy of study. Both UV-C and T. viride elicitors induce specific changes in peroxidase isoenzyme / H2O2 levels, which result in specific changes in grapevine physiology and metabolism. In the case of T. viride-elicited grapevine cells, they show a particular mechanism for H2O2 production, in which NADPH oxidase-like activities are apparently not involved. However, they offer a unique system whereby the metabolic regulation of peroxidase by H2O2, with all its cross-talks and downstream signals, may be elegantly dissected.

The presence of sinapyl lignin in Ginkgo biloba cell cultures changes our views of the evolution of lignin biosynthesis

Suspension cell cultures (SCCs) from one of the oldest seed plants, Ginkgo biloba, show unpredictable alterations in the nature of the lignins, such as is the recruitment of sinapyl alcohol for lignin biosynthesis, compared with the woody tissues of the same species, which lack syringyl (S) lignins. These results show that, in this gymnosperm, the genes involved in sinapyl alcohol biosynthesis are latent and that their regulatory regions respond, by initiating gene expression, to the developmental signals and the environmental clues, which condition its in vitro culture. G. biloba SCCs not only synthesize S lignins but also their extracellular proteome contains both class III peroxidases capable of oxidizing sinapyl alcohol and enzymes involved in H2O2 production, observation which suggests that the peroxidase branch for the oxidative coupling of sinapyl alcohol units into lignins is operative. The incomplete knowledge of the G. biloba peroxidase-encoding genes led us to purify, characterize and partially sequence the peroxidase responsible for monolignol oxidation. When the major peroxidase from G. biloba SCCs (GbPrx) was purified to homogeneity, it showed absorption maxima in the visible region at 414 (Soret band), and at 543 and 570 nm, which calls to mind those shown by low-spin ferric peroxidases. However, the results also showed that the paraperoxidase-like character of GbPrx is not an obstacle for oxidizing the three monolignols compared with high-spin ferric peroxidases. Taken together, these results mean that the time at which the evolutionary gain of the segment of the route that leads to the biosynthesis of S lignins took place in seed plants needs to be revised.

Induction of sesquiterpenes, phytoesterols and extracellular pathogenesis-related proteins in elicited cell cultures of Capsicum annuum

Capsicum annuum suspension cell cultures were used to evaluate the effect of cyclodextrins and methyl jasmonate as elicitors of defence responses. The induced defence responses included the accumulation of sesquiterpenes and phytosterols and the activation of pathogenesis-related proteins, leading to reinforcement and modification of the cell wall architecture during elicitation and protection cells against biotic stress. The results showed that the addition of both cyclodextrins and methyl jasmonate induced the biosynthesis of two sesquiterpenes, aromadendrene and solavetivone. This response was clearly synergistic since the increase in the levels of these compounds was much greater in the presence of both elicitors than when they were used separately. The biosynthesis of phytosterols was also induced in the combined treatment, as the result of an additive effect. Likewise, the exogenous application of methyl jasmonate induced the accumulation of pathogenesis-related proteins. The analysis of the extracellular proteome showed the presence of amino acid sequences homologous to PR1 and 4, NtPRp27-like proteins and class I chitinases, peroxidases and the hydrolytic enzymes LEXYL1 and 2, arabinosidases, pectinases, nectarin IV and leucin-rich repeat protein, which suggests that methyl jasmonate plays a role in mediating defence-related gene product expression in C. annuum. Apart from these methyl jamonate-induced proteins, other PR proteins were found in both the control and elicited cell cultures of C. annuum. These included class IV chitinases, beta-1,3-glucanases, thaumatin-like proteins and peroxidases, suggesting that their expression is mainly constitutive since they are involved in growth, development and defence processes.

The Vinca Alkaloids: From Biosynthesis and Accumulation in Plant Cells, to Uptake, Activity and Metabolism in Animal Cells

ABSTRACT: 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.

Subcellular location of basic and acidic soluble isoperoxidases in lupinus

Abstract The subcellular location of non-particulate (soluble) peroxidases (EC 1.11.1.7) was studied in lignifying lupin ( Lupinus albus L.) hypocotyls. Supernatants of cellular homogenates contain two acidic (A 1 and A 2 ) and two basic (B 1 and B 2 ) isoperoxidases. Studies of centrifugation on isopycnic sucrose gradients of cellular homogenates of plasmolyzed tissues, vacuum infiltration of hypocotyl sections, and binding probes to membranes and cell walls, suggest that while basic soluble isoperoxidases possess a cytoplasmic location, acidic isoperoxidases possess an extracellular location, as freely soluble forms in the intercellular spaces. These results are discussed on the basis of the subcellular location of the auxin catabolism, mediated mainly by the two basic isoperoxidases, which in lupin cells at least, seems to be restricted to the cytosolic compartment.

Reversed-phase and size-exclusion chromatography as useful tools in the resolution of peroxidase-mediated (+)-catechin oxidation products

The peroxidase-catalysed oxidation of plant phenolics involves one-electron oxidation reactions, and yields unstable mono-radical species, which couple to generate heterogeneous product mixtures of different degrees of polymerisation. One such phenolic susceptible to oxidation by peroxidase is (+)-catechin. Low-pressure chromatography on Sephadex LH-20, using methanol as mobile phase, resolves the main peroxidase-mediated (+)-catechin oxidation products into a dimeric compound (dehydrodicatechin A) and an oligomeric fraction with a polymerisation degree equal or greater than 5. These pure fractions were used to develop rapid high-performance liquid chromatographic methods, both reversed-phase and size-exclusion chromatography for the direct analysis of the peroxidase-mediated (+)-catechin oxidation products. The joint use of both chromatographic systems permitted the qualitative and quantitative identification of the peroxidase-mediated (+)-catechin oxidation products, and can thus be considered as a useful tool for analysing the complex mixtures of natural bioactive plant products synthesized in reactions catalyzed by plant peroxidases.

Digestibility of silages in relation to their hydroxycinnamic acid content and lignin composition

BACKGROUND The effectiveness of the analysis of cell wall-bound hydroxycinnamic acids and the composition of lignin to evaluate the in vivo digestibility of a silage collection with unknown botanical composition was evaluated. RESULTS Syringyl units content and total etherified phenols showed the highest correlation coefficients with in vivo dry matter digestibility (IVDMD) (r = - 0.792 and r = - 0.703, respectively), while guaiacyl units and total phenols showed the highest correlation coefficients with in vivo organic matter digestibility (IVOMD) (r = - 0.871 and r = - 0.817, respectively). Using the above-mentioned chemical parameters, 10 equations were also developed to predict in vivo digestibility. The prediction of IVDMD produced a high adjusted R(2) value (0.710) using syringyl, total lignin, etherified total phenols, esterified ferulic acid and total phenol content as predictors. The prediction of IVOMD produced a higher adjusted R(2) value (0.821) using guaiacyl, total phenols, total ferulic acid and etherified p-coumaric acid content as predictors. CONCLUSION Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, syringyl and guaiacyl content and etherified phenols emerge as good predictors of digestibility.

Reactive Oxygen Species in Plant Cell Walls

Plant cell walls are dynamic structures composed of polysaccharides, phenolics, and proteins. The plant cell wall is important not only for maintaining cell shape, but it also responds to endogenous and environmental clues through the release of signaling molecules, such as H2O2, which may act following autocrine and paracrine pathways. However, the primary function of reactive oxygen species (ROS) production in the plant cell wall is to modify cell wall components by processes of cell wall stiffening/softening, which, in the last intance, control plant growth and morphogenesis. Four possible enzymatic machineries could explain how this H2O2 is produced: NADPH oxidases (NOX), peroxidases, poly(di)amine oxidases, and oxalate oxidases, but most of the molecular evidence, particularly in epidermal, vascular, and suberizing tissues, supports the exclusive participation of NOX in this process. Given the limited efficacy of the ROS-scavenging systems in plant cell walls, it may be concluded that ROS accumulation in the cell walls of these tissues is only the static image of their high redox state and large oxidative metabolism.