Ana Ortuño

UV irradiation alters the levels of flavonoids involved in the defence mechanism of Citrus aurantium fruits against Penicillium digitatum

The effect of UV irradiation on the levels of the flavanone, naringin, and the polymethoxyflavone, tangeretin, in the peel of Citrus aurantium fruits is described, as changes in the synthesis and/or accumulation of these compounds after infection with Penicillium digitatum. The growth of P. digitatum on previously irradiated fruit was reduced by up to 45%. Changes in flavonoid levels were detected, associated with inhibition of fungus growth, the naringin content falling by 69% and tangeretin levels increasing by 70%. The possible participation of naringin and tangeretin in the defence mechanism of this Citrus species is discussed.

Citrus cultivars with high flavonoid contents in the fruits

Abstract The flavanone contents of some lesser known citrus cultivars grown in Cuba have been determined in research searching for citrus species/cultivars with high flavanone contents. The cultivar ‘Galleta’ of the mandarin (Citrus reticulata Blanco) has a higher hesperidin content than the hybrid mandarin ‘Nova’, which is currently being used for the commercial obtainment of this flavanone. The grapefruit cultivar ‘Isaac’ had higher naringin levels than reported in the literature for any citrus species or cultivar. These two species seem potential candidates for the industrial obtainment of flavanones. With regard to neohesperidin, the ‘Afin’ and ‘Bouquet de Fleur’ varieties of Citrus aurantium L. from Spain are the best choices. A preliminary study indicated that the high flavanone contents resulted from the genetic characteristics of the cultivars, rather than from climatic effects.

Tylose formation and changes in phenolic compounds of grape roots infected with Phaeomoniella chlamydospora and Phaeoacremonium species.

The role of phenolic compounds in strengthening resistance of grapevine to young grapevine decline wasnanalysed. The formation of tyloses has also been associated with this disease. A microscopic study showed thatntyloses originated in parenchyma cells associated with the xylem and from there invaded the xylem lumen. As tylosesnformed, there were changes in the cell wall, associated with the accumulation of crystalline structures. The cellsnsurrounding the blocked xylem contained more polyphenolic compounds than the cells of intact xylem. Changes innthe level and composition of polyphenolic compounds occurred in relation to the severity of infection. Tannin concentrationsnalso increased with increasing numbers of xylem vessels containing tyloses. Root extracts added to fungalnculture media inhibited mycelial growth of Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Pm.ninflatipes. Inhibition was greater with extracts from roots with tyloses, and was also correlated with polyphenolncontent. Extracts of the leaves, stems and berries of vines treated with Brotomax, which increases the biosynthesis ofnphenolic compounds, inhibited mycelial growth of the fungi compared to untreated control plants.

Evolution of polymethoxy flavones during development of tangelo Nova fruits

Abstract The production of nobiletin, sinensetin, tangeretin, quercetogetin and heptamethoxyflavone by tangelo Nova fruits is described. The levels of these compounds were examined by high performance liquid chromatography–mass spectrometry. The results suggest that the highest levels of the nobiletin, sinensetin and tangeretin are associated with young developing states in fruits, while quercetogetin and heptamethoxyflavone come into full production towards the end of the lineal phase of growth. A study of the distributions of these secondary metabolites revealed that they were only located in the peel. ©

In vivo metabolism of labelled indole-3-acetic acid during polar transport in etiolated hypocotyls of Lupinus albus: Relationship with growth

The in vivo metabolism of indole-3-acetic acid (IAA) in etiolated hypocotyls of lupin (Lupinus albus L., from Bari, Italy) was investigated by appliying IAA labelled with two radioisotopes ([1-14C]-IAA+[5-3H]-IAA) to the apical end of decapitated seedlings, followed by extraction of the radioactivity in the different regions along the hypocotyl. This method allowed detection of IAA decarboxylation in zones distant from the cut surface and, therefore, containing intact cells. When IAA was added directly in solution to the cut surface, decarboxylation was high especially in those hypocotyl regions where transient accumulations characteristic of the polar transport of IAA occurred. In 10-day-old seedlings such accumulations were observed both in the elongation zone (2nd, 3rd, and 4th cm) and in the non elongating basal zone (8th, 9th and 10th cm). When the IAA, instead, was applied with an agar block deposited on the cut surface, IAA metabolism (decarboxylation as well as conjugation) was increased but almost exclusively in tissues within 10 mm of the cut surface. In both kinds of experiment, the increase in IAA decarboxylation seemed to coincide with a decrease in the transport of IAA, since in the assay without agar the transient accumulations of radioactivity were probably due to a decrease in the transport velocity, while in the assay with agar the transport intensity was much lower than in the assay without agar. These results point to a competitive relationship between IAA metabolism and transport. Consequently, it is suggested that hypocotyl regions that probably use auxin for development processes (e.g., cell elongation and differentiation) may have a more intense IAA metabolism in parallel with their higher IAA concentrations.

Influence of 2,3,5-triiodobenzoic acid on the transport and metabolism of IAA in lupin hypocotyls

The influence of 2,3,5-triiodobenzoic acid (TIBA) on the transport and metabolism of indolyl-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L) hypocotyls. Double isotope-labeled IAA [(5-3H)-IAA plus (1-14C)-IAA] was applied to the cut surface of decapitated seedlings. This confirmed that the species mobilized was unaltered IAA and permitted us to measure the in vivo decarboxylation of applied IAA. A pretreatment with TIBA applied to the cut surface produced a partial or drastic inhibition in the basipetal IAA movement at 0.5 or 100 μM, respectively. Since TIBA inhibits auxin polar transport by interfering with the efflux carrier, the above results suggest that 100 μM TIBA is sufficient to saturate the binding sites in the transporting cells. Compared to the control plants, in vivo decarboxylation of IAA was enhanced in 0.5 μM TIBA-treated plants, while no decarboxylation was detected after treatment with 100 μM TIBA. The in vitro decarboxylation of (1-14C)-IAA catalyzed by purified peroxidase was moderately activated by 100 μM and unaffected by 0.5 μM TIBA. The paradoxical effect of TIBA in vivo vs in vitro assays suggests that the in vivo effect of TIBA on IAA oxidation might be the consequence of the action of TIBA on the auxin transport system. Thus, transport reduction by 0.5 μM TIBA caused a temporary accumulation of IAA in that apical region of the hypocotyl which has the highest capacity to decarboxylate IAA. In the presence of 100 μM TIBA, a concentration which presumably saturates the efflux carriers, most of the added IAA can be expected to be located in the transporting cells where, according to the present data, IAA decarboxylation cannot take place.

Involvement of an extracellular fungus laccase in the flavonoid metabolism in Citrus fruits inoculated with Alternaria alternata

Fungi of the genus Alternaria are responsible for substantial pre-harvest losses in Citrus. In this study a degradative metabolism of flavonoids (flavanones, flavones and polymethoxyflavones) was observed when Fortune mandarin, Citrus limon and Citrus paradisi, fruits were inoculated with Alternaria alternata, a pre-harvest pathogenic fungus. Associated to this flavonic metabolism the de novo synthesis of the phytoalexin scoparone was detected. This metabolism of flavonoids is caused by an extracellular fungus laccase. The kinetic characterisation of this enzyme revealed that the activity was induced by Citrus flavonoids and was dependent on flavonoid concentrations. The enzyme exhibited a Km of 1.9 mM using ABTS as substrate with an optimum pH of 3.5 in citrate buffer 100 mM. The enzyme is active between 15 and 45 °C, the optimum temperature being around 35 °C, although 50% of the initial activity is lost after 45 min at 35 °C. The A. alternata laccase was inhibited by 0.5 mM l-cysteine and by caffeic acid. Study of the substrate specificity of this enzyme revealed that Citrus flavonoids are substrates of A. alternata laccase. These results suggest that the laccase enzyme could be involved in the pathogenesis of A. alternata in Citrus.

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0xa0μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0xa0μM) were tested for calli induction, and with 5xa0μM BAxa0+xa00.5xa0μM NAA or IAA for shoot development. The average number of shoots (≥5xa0mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5xa0μM IAAxa0+xa010xa0μM BA for calli induction and on 0.5xa0μM IAAxa0+xa05xa0μM BA for shoot development, and by culturing petiole explants on 0.5xa0μM NAAxa0+xa010xa0μM BA followed by a second culture on 0.5xa0μM NAAxa0+xa05xa0μM BA. The highest frequency of shoot rooting was achieved with 10.0xa0μM NAA and 1.0xa0μM gibberellic acid (GA3). Rooted plants were acclimatised in a culture chamber, reaching 96xa0% survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100xa0% survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4xa0months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824xa0μgxa0g−1 DW total FC) and lowest in organogenic calli (up to 50xa0μgxa0g−1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.

Furanocoumarins: Biomolecules of Therapeutic Interest

Abstract Furanocoumarins are a subgroup of phenolic compounds included in the coumarins group. They can be subdivided into two types: linear, generically known as psoralens, among which are included psoralen, xanthotoxin, and bergapten, and angular, generically known as angelicins, including angelicin, sphondin, and pimpinellin. Linear furanocoumarins have been identified in a great variety of plant families, the highest concentrations being found in Apiaceae, Rutaceae, Leguminosae, and Moraceae. Angular furanocoumarins are less widely distributed and are principally confined to the Apiaceae and Leguminosae. Psoralen-containing plants have been used for centuries in a popular medicine to treat vitiligo, a skin disease characterized by lack of pigmentation. Different methods have been used for the extraction, separation, and analysis of furanocoumarins: supercritical fluid extraction, solid-phase extraction, thin-layer chromatography, high-performance liquid chromatography, high-performance liquid chromatography–mass spectrometry, high-speed countercurrent chromatography, gas chromatography, gas chromatography–mass spectrometry, capillary electrophoresis, and pressurized capillary electrochromatography. Furanocoumarins are typical phototoxic compounds that produce photodermatitis in combination with UV light exposure and cytotoxic and mutagenic disorders. At molecular level, furocoumarins bind to cellular constituents such as proteins and lipids, damaging lysosomes, leading to the formation of reactive oxygen species and contributing to the formation of novel antigens by covalent modification of proteins. Furocoumarins are well known for interfering with drug metabolism, in particular with cytochrome P450 (CYP). On the other hand, furanocoumarins possess mutagenic and carcinogenic properties due to their ability to intercalate into dsDNA and create covalent cross-links primarily with thymidine residues. Since the discovery of furanocoumarins and other structurally related biomolecules, extensive research on different aspects of therapeutical interest has been realized due to the exclusive behavior of these compounds when irradiated with UVA light. These properties have made possible the use of these molecules in PUVA (combination therapy of psoralen and UVA radiation) or extracorporeal photopheresis for the treatment not only of many skin diseases such as vitiligo or psoriasis but also of several autoimmune diseases (systemic lupus erythematosus, Crohns disease, type 1 diabetes mellitus, or multiple sclerosis) and for the treatment of cutaneous T-cell lymphoma, solid organ transplant rejection, and graft versus host disease. Importantly, these compounds alone or in combination with other drugs represent promising candidates to develop new therapies or improve the existing ones. This chapter looks at the structures of furanocoumarins, their presence in plants, analytic methods, their pharmacological properties, and their therapeutic uses.