F. Sabater

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.

Lateral diffusion of polarly transported indoleacetic acid and its role in the growth of Lupinus albus L. hypocotyls.

The transport and metabolism of indole-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L, cv. Multolupa) hypocotyls, following application of dual-isotope-labelled indole-3-acetic acid, [5-3H]IAA plus [1-14C]IAA, to decapitated plants. To study the radial distribution of the transported and metabolized IAA, experiments were carried out with plants in which the stele was separated from the cortex by a glass capillary. After local application of labelled IAA to the cortex, radioactivity remained immobilized in the cortex, near the application point, showing that polar transport cannot occur in the outer tissues. However, following application of IAA to the stele, radioactivity appeared in the cortex in those hypocotyl sections below the first 1 cm (in which the capillary was inserted), and the basipetal IAA movement was similar to that observed after application of IAA to the complete cut surface. In both assays, longitudinal distribution of 14C and 3H in the stele outside the first 1 cm was positively correlated with that of cortex, indicating that there was a lateral migration of IAA from the transport pathway (in the stele) to the outer tissues and that this migration depended on the amount of IAA in the stele. Both tissues (stele and cortex) exhibited intensive IAA metabolism, decarboxylation being higher in the stele than in the cortex while IAA conjugation was the opposite. Decapitation of the seedlings caused a drastic reduction of hypocotyl growth in the 24 h following decapitation, unless the hypocotyls were treated apically with IAA. Thus, exogenous IAA, polarly transported, was able to substitute the endogenous source of auxin (cotyledons plus meristem) to permit hypocotyl growth. It is proposed that IAA escapes from the transporting cells (in the stele) to the outer tissues in order to reach the growth-responsive cells. The IAA metabolism in the outer tissues could generate the IAA gradient necessary for the maintenance of its lateral flow, and consequently the auxin-induced cell elongation.

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus.

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and 1H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.

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.

Oxygen consumption and enzyme inactivation in the indolyl-3-acetic acid oxidation catalyzed by peroxidase

Abstract Oxidation of indolyl-3-acetic acid (IAA) catalyzed by peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) can be brought about alternatively by two different pathways that correspond to the oxidase or peroxidase activities of the enzyme. The relative participation of the enzyme in the two pathways depends on the enzyme/substrate ratio and/or the oxygen concentration. Low levels of oxygen favour the oxidase pathway due to the high affinity for oxygen of the ferroperoxidase, the enzymatic form that initiates this pathway. The inactivation of the enzyme estimated either by appearance of P-670 or by measurements of the IAA degraded when the reaction has finished ( P ∞ ) is a good indication that the peroxidase pathway operates. Therefore, the conditions that reduce the enzyme inactivation (for example, high IAA concentrations), also favour the oxidase pathway.

Auxin carriers in membranes of lupin hypocotyls.

The pH-driven accumulation of [3H]indolyl-3-acetic acid (IAA) has been found to occur in membrane vesicles of lupin (Lupinus albus L.) hypocotyls. Most of this association of auxin with membranes is very sensitive to osmotic shock, high concentrations of permeable weak acids, incubation at 20° C for 20 min and to some ionophores. Long incubation times also depress the ability to accumulate radioactive IAA but this ability can be partially restored by a treatment that presumably reconstitutes the pH gradient across the membranes. Two specific inhibitors of auxin transport, N-1-naphtylphthalamic acid and 2,3,5-triiodobenzoic acid, stimulate net IAA uptake with an optimum at about 10-6 M (pH 5.0). At least two auxin carriers appear to be present in the lupin membrane vesicles. An uptake carrier seems to be saturated at 10-7 M IAA in the presence of N-1-naphtylphthalamic acid, but higher IAA concentrations are needed to saturate an efflux carrier. The uptake carrier also shows a high affinity for IAA and 2,4-dichlorophenoxyacetic acid and a low affinity for 1-naphthylacetic acid.

Selection of citrus varieties highly productive for the neohesperidin dihydrochalcone precursor

Abstract The levels of the flavanones, neohesperidin and naringin, and the neohesperidin naringin ratio in immature and mature fruit of different varieties of Citrus aurantium and the Citrus paradisi Macf. × Citrus depresssa Hayata hybrid are compared, and the flavonic content is analysed for the first time. Fruits of the hybrid, which are used to obtain neohesperidin for industrial-scale transformation into the intensely sweet neohesperidin dihycrochalcone, have two advantages: (1) although the levels of neohesperidin in the hybrid are similar to those detected in the immature fruit of Citrus aurantium , the levels of naringin detected in the immature fruit of the hybrid are lower, which means that the neohesperidin naringin ratio is greater and the need for costly neohesperidin purification processes correspondingly less; (2) unlike the mature fruit of Citrus aurantium , the mature fruits of the hybrid accumulate high levels of neohesperidin with a high neohesperidin naringin ratio, so that these too can be used to obtain neohesperidin.

Ethylene evolution during ripening of detached tomato fruit: Its relation with polyamine metabolism

Ethylene and polyamine metabolism, both sharing a common precursor, S-adenosylmethionine (SAM), were investigated during detached tomato (Lycopersicon esculentum Mill. nothovar F1 “Lorena”) fruit ripening. Putrescine (PUT) was found to be the major polyamine in the fruits, always over 100 nmols/g FW, while spermidine (SPD) was between 7% and 3% of the level of PUT. Spermine (SPM) was not detected at any stage of ripening. The level of PUT and SPD, did not change significantly during ripening in spite of the almost continuous synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, and only at the last stage of ripening was a drastic decrease in SPD content observed. The results obtained show that the onset of ACC synthesis and its accumulation within the tissue is not a consequence of a decrease in SPD synthesis.

Bioproduction of neohesperidin and naringin in callus cultures of Citrus aurantium

The accumulation of both neohesperidin and naringin as major flavonoids in callus cultures of bitter orange (Citrus aurantium) was demonstrated using high performance liquid chromatography with a diode-array detector. The identity of both compounds was confirmed by their corresponding nuclear magnetic resonance spectra. The levels of neohesperidin are higher than those of naringin in callus culture, as they are in immature fruit, and high concentrations of both are found in young tissues such as immature fruits and the outer zone of calli.

Accumulation of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus paradisi, Citrus limonia and Citrus aurantium

The production of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus species is described. The levels of these compounds were examined by gas chromatography-mass spectrometry and their yields were compared with the amounts found in mature fruits. A simultaneous increase and decrease in the levels of nootkatone and valencene, respectively, were observed with the aging of callus cultures of Citrus paradisi. These results suggest that valencene might be a possible precursor of nootkatone in this species. The high level of nootkatone detected in 9-month-old callus cultures of Citrus paradisi might be associated with the corresponding cell morphological changes observed.