Fernando Gandía-Herrero

Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis: discovery of bifunctional O- and C-glucosyltransferases

Plants, as predominantly sessile organisms, have evolved complex detoxification pathways to deal with a diverse range of toxic chemicals. The elasticity of this stress response system additionally enables them to tackle relatively recently produced, novel, synthetic pollutants. One such compound is the explosive 2,4,6-trinitrotoluene (TNT). Large areas of soil and groundwater are contaminated with TNT, which is both highly toxic and recalcitrant to degradation, and persists in the environment for decades. Although TNT is phytotoxic, plants are able to tolerate low levels of the compound. To identify the genes involved in this detoxification process, we used microarray analysis and then subsequently characterized seven uridine diphosphate (UDP) glycosyltransferases (UGTs) from Arabidopsis thaliana (Arabidopsis). Six of the recombinantly expressed UGTs conjugated the TNT-transformation products 2- and 4-hydroxylaminodinitrotoulene, exhibiting individual bias for either the 2- or the 4-isomer. For both 2- and 4-hydroxylaminodinitrotoulene substrates, two monoglucose conjugate products, confirmed by HPLC-MS-MS, were observed. Further analysis indicated that these were conjugated by either an O- or C-glucosidic bond. The other major compounds in TNT metabolism, aminodinitrotoluenes, were also conjugated by the UGTs, but to a lesser extent. These conjugates were also identified in extracts and media from Arabidopsis plants grown in liquid culture containing TNT. Overexpression of two of these UGTs, 743B4 and 73C1, in Arabidopsis resulted in increases in conjugate production, and enhanced root growth in 74B4 overexpression seedlings. Our results show that UGTs play an integral role in the biochemical mechanism of TNT detoxification by plants.

Stabilization of the bioactive pigment of Opuntia fruits through maltodextrin encapsulation.

Betalains are water-soluble, nitrogen-containing pigments of growing interest in the food industry. They are present in most plants belonging to the order Caryophyllales, where they fulfill the role of anthocyanins, and are divided into two groups: violet betacyanins and yellow betaxanthins. They are bioactive molecules that account for health-promoting properties, recently described for cactus pears (Opuntia). In this work, the characteristic betalain of cactus pears, indicaxanthin, is obtained purely, and its stability is highly promoted by its encapsulation in a maltodextrin matrix. A suitable spray-drying procedure for encapsulation is described, and a bright yellow powder is obtained. The stability is analyzed under different conditions. In the absence of light, pure encapsulated pigment can be stored at 20 °C for months without appreciable loss of the bioactive substance and color variation. Furthermore, free radical scavenging and antioxidant properties of the pigment are studied under the ABTS(•+) radical and ferric reducing antioxidant power assays, in the presence and in the absence of maltodextrins. The stabilization of pure betalain pigments may boost the use of these bioactive and natural coloring molecules.

Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (Km = 84.3 μm). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin.

The role of phenolic hydroxy groups in the free radical scavenging activity of betalains.

Free radical scavenging compounds play important roles as health-protecting factors. Betalains are natural water-soluble pigments present in most plant families belonging to the order Caryophyllales. They are the subject of increasing attention following the discovery of their antiradical capacity, but a systematic analysis of the structural features involved in the activity is necessary. In this paper, both natural and previously unconsidered betaxanthins were obtained in order to study the role of phenolic hydroxy groups in the high free radical scavenging activity of betalains. Pigments were characterized spectrophotometrically, chromatographically, and by ESI-MS, and their antiradical and antioxidant properties were studied under the ABTS(*+) radical and FRAP assays. A high intrinsic activity is described that is not linked to the presence of hydroxy groups or aromaticity in the pigment structure. In addition, the presence of phenolic hydroxy groups implies an enhancement of the antiradical activity, reaching a TEAC value in the ABTS(*+) assay of 5.8 +/- 0.2 for the pure compound with two hydroxy groups.

Engineering a Catabolic Pathway in Plants for the Degradation of 1,2-Dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum ‘Xanthi’) plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.

Purification and antiradical properties of the structural unit of betalains.

Betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid] is a naturally occurring compound that is normally found condensed with amino acids, amines, cyclo-DOPA, and cyclo-DOPA derivatives to form the betalains. Betalains are the pigments responsible for the yellow to violet color of the fruits and flowers of plants belonging to the order Caryophyllales. Betalamic acid is the structural feature common to all of these pigments and contains the electron resonance system responsible for the spectroscopic properties. Betalamic acid was purified by chromatography and identified by UV-vis spectrophotometry and ESI mass spectrometry. The antioxidant and free radical scavenging capacities of betalamic acid were assessed using the FRAP and ABTS(·+) radical assays. A pK(a) of 6.8 was found for the deprotonation equilibrium involved in the nucleophilic activity of betalamic acid; this pK(a) explains the observed pH effect on the free radical scavenging capacity of these pigments.

Fluorescence detection of tyrosinase activity on dopamine-betaxanthin purified from Portulaca oleracea (common purslane) flowers.

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is one of the key enzymes for the biosynthesis of natural pigment betalains. These are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this work, dopamine-betaxanthin (also known as miraxanthin V) is reported as the pigment responsible for the bright coloration in yellow flowers of Portulaca oleracea (common purslane). The natural pigment is purified, and used as a substrate for the catecholase (diphenolase) activity of the enzyme tyrosinase. A new, continuous method to follow the activity is developed based on the fluorescent properties of the betaxanthin. Fluorescence of the enzyme activity derived products is reported for the first time. Relevance of the fluorescent phenomenon is discussed based on fluorescence images and the description of a physiological inner filter effect present in flowers of P. oleracea. The first description of the betalain content in flower pistils is also provided.

Production of Dihydroxylated Betalains and Dopamine in Cell Suspension Cultures of Celosia argentea var. plumosa

Betalains are plant pigments of hydrophilic nature with demonstrated chemopreventive potential in cancer cell lines and animal models. Among the betalains, those containing an aromatic moiety with two free hydroxyl groups possess the strongest antioxidant and free radical scavenging activities. The betaxanthins dopaxanthin and miraxanthin V and the betacyanins betanidin and decarboxy-betanidin are the only natural betalains with catecholic substructures. These four pigments have been produced in cell cultures established from hypocotyls of the plant Celosia argentea. Two stable and differentially colored cell lines, yellow and red, were maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (6.66 μM) and 2,4-dichlorophenoxyacetic acid (6.79 μM). Derived suspension cultures showed increased production of dihydroxylated betalains in the cells and secreted to the medium with a maximum reached after 8 days of culture. In addition, precursor molecules betalamic acid and dopamine, with content up to 42.08 mg/g dry weight, were also obtained. The joint presence of the bioactive betalains together with the production of dopamine and betalamic acid show the ability of cell cultures of C. argentea to become a stable source of valuable phytochemicals.

One-Step Synthesis of Betalains Using a Novel Betalamic Acid Derivatized Support

Betalains are plant pigments with high antioxidant and cancer chemopreventive properties used by the food industry as safe colorants. Betalains are restricted to species of the order Caryophyllales, and difficulty in obtaining individual molecules has limited their structural identification and application. This study was designed to develop a betalamic acid derivatized support generated from a primary amine polymer. The novel material presents color properties of a pseudobetaxanthin, and it is stable for at least 6 months. The bond formed can be displaced at mild conditions by the addition of amines in aqueous solutions over a broad pH range and at 25 °C. This releases the betalamic acid while forming the corresponding pigment. This one-step procedure significantly simplifies the process of obtaining semisynthetic betalains, and it is optimized here for the formation of betaxanthins and betacyanins derived from tyramine, dopamine, pyrrolidine, and indoline. The new method makes access to single betalains available to the entire scientific community and could stimulate research and applications in the field.

Development of Betalain Producing Callus Lines from Colored Quinoa Varieties (Chenopodium quinoa Willd)

Betalains are water-soluble plant pigments of hydrophilic nature with promising bioactive potential. Among the scarce edible sources of betalains is the grain crop quinoa (Chenopodium quinoa Willd), with violet, red, and yellow grains being colored by these pigments. In this work, callus cultures have been developed from differently colored plant varieties. Stable callus lines exhibited color and pigment production when maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (8.88 μM) and 2,4-dichlorophenoxyacetic acid (6.79 μM) with a reduction of the nitrogen source to 5.91 mM. Pigment analysis by HPLC-DAD and ESI-MS/MS fully describes the content of individual pigments in the cell lines and allows the first report on the pigments present in quinoa seedlings. Phyllocactin and vulgaxanthin I are described as novel pigments in the species and show the potential of C. quinoa culture lines in the production of compounds of nutritional value.