Diffusion and Electric Mobility of KCI within Isolated Cuticles of Citrus aurantium

Fick's second law has been used to predict the time course of electrical conductance change in isolated cuticles following the rapid change in bathing solution (KCI) from concentration C to 0.1 C. The theoretical time course is dependent on the coefficient of diffusion of KCI in the cuticle and the cuticle thickness. Experimental results, obtained from cuticles isolated from sour orange (Citrus aurantium), fit with a diffusion model of an isolated cuticle in which about 90% of the conductance change following a solution change is due to salts diffusing from polar pores in the wax, and 10% of the change is due to salt diffusion from the wax. Short and long time constants for the washout of KCI were found to be 0.11 and 3.8 hours, respectively. These time constants correspond to KCI diffusion coefficients of 1 × 10⁻¹⁵ and 3 × 10⁻¹⁷ square meters per second, respectively. The larger coefficient is close to the diffusion coefficient for water in polar pores of Citrus reported elsewhere (M Becker, G Kerstiens, J Schönherr [1986] Trees 1: 54-60). This supports our interpretation of the washout kinetics of KCI following a change in concentration of bathing solution.     

Movement of Cations through Cuticles of Citrus aurantium and Acer saccharum: Diffusion Potentials in Mixed Salt Solutions

We examined some biophysical mechanisms of ion migration across leaf cuticles enzymatically isolated from Acer saccharum L. and Citrus aurantium L. leaves. Diffusion potential measurements were used to calculate the permeabilities of Cl^-, Li⁺, Na⁺, and Cs⁺ ions all as a ratio with respect to the permeability of K⁺ in cuticles. In 2 millimolar ionic strength solutions the permeability sequence from high to low was K = Cs > Na > Li » Cl. When the outer and inner surfaces of cuticles were bathed in artificial precipitation and artificial apoplast, respectively, diffusion potentials ranging from −52 to −91 millivolts were measured (inside negative). The Goldman equation predicted that the measured potentials were enough to increase the driving force on the accumulation of heavy metals by a factor of 4 to 7. Other ions migrate with forces 3 to 10 times less than predicted by the Goldman equation for concentration differences alone. Our analysis showed that Ca²⁺, and perhaps Mg²⁺, might even be accumulated against concentration gradients under some circumstances. Their uptake was apparently driven by the diffusion potentials created by the outward migration of monovalent salts. We feel that future models predicting leaching of nutrients from trees during acid rain events must be modified to account for the probable influence of diffusion potentials on ion migration.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

中药“枳壳”的组织培养与植株再生（简报）

以酸橙的茎段为外植体,进行离体再生体系研究.结果表明,培养基MS + 6-BA 1.0 mg/L + IAA 0.5 mg/L + CH 400 mg/L适宜不定芽诱导;培养基MS + 6-BA 0.5 mg/L+ IAA 0.25 mg/L + CH 400 mg/L适宜不定芽增殖;培养基1/2MS + NAA 1.0 mg/L + IBA 2.0 mg/L生根效果较好,生根率达80%以上.     

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.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FID flame-ionisation detector - FW fresh weight - GLC gas liquid chromatography - K Kinetin - NAA naphthalene acetic acid     

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of ³H-labeled water (³H₂O) and the ¹⁴C-labeled organic acids benzoic acid ([¹⁴C]BA), salicylic acid ([¹⁴C]SA), and 2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (³H₂O/[¹⁴C]BA, ³H₂O/[¹⁴C]SA, and ³H₂O/[¹⁴C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination ³H₂O/[¹⁴C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of ³H₂O of all 12 investigated species were highly correlated with the permeances of [¹⁴C]BA (r² = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.     

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.Abbreviations DMSO dimethylsulphoxide - DW dry weight - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - Rt retention time - V/UV visible/ultraviolet     

枳实中辛弗林和橙皮苷的联合提取工艺研究

以辛弗林和橙皮苷的收率及纯度为指标,研究了含辛弗林的枳实提取物及橙皮苷的综合制备工艺.先采用酸液渗漉法提取枳实中的辛弗林,再采用碱提酸沉法提取原料中的橙皮苷,考察盐酸浓度及用量、流速等因素的影响.辛弗林的最佳提取工艺为:药材加5倍水浸泡过夜,渗漉盐酸液的浓度为0.02 mol/mL,渗漉料液比为1:4,以5 mL/min的流速渗漉;碱提酸沉法提取橙皮苷的优化工艺为:于提取辛弗林之后的药渣中加入65%乙醇,于60℃搅拌条件下加碱调节pH 13,过滤后的滤液以稀盐酸调节pH 5,过滤后再用碱回调pH7,放置过夜.橙皮苷的收率为11.3%,HPLC测定橙皮苷的纯度为86.7%.     

Simultaneous extraction of bioactive limonoid aglycones and glucoside from Citrus aurantium L. using hydrotropy

Citrus limonoids were demonstrated to possess potential biological activities in reducing the risk of certain diseases. Limonoids are present in citrus fruits in the form of aglycones and glucosides. At present, limonoid aglycones and limonoid glucosides are extracted in multiple steps using different solvents. In order to understand their potential bioactivity, it may be beneficial to isolate and purify these compounds using environment friendly methods. A new method of extraction and purification of limonoids was established using a hydrotrope polystyrene adsorbent resin. Extraction of aglycones and glucosides was achieved in a single step, using an aqueous solution of sodium cumene sulphonate (Na-CuS). Sour orange (Citrus aurantium L.) seed powder was extracted with 2 M Na-CuS solution at 45 degrees C for 6 h. The filtered extract was diluted with water and loaded on an SP 700 adsorbent column. The column was washed with distilled water to remove the hydrotrope and then eluted using water and methanol in different compositions to obtain three compounds. The structures of the isolated compounds were confirmed by NMR spectroscopy as deacetyl nomilinic acid glucoside (DNAG), deacetyl nomilin (DAN) and limonin (LIM).     

Studies on Octylphenoxy Surfactants : III. Sorption of Triton X-100 by Isolated Tomato Fruit Cuticles

Sorption characteristics of a polyethoxy (EO) derivative of octylphenol (OP) were determined for enzymically isolated mature tomato (Lycopersicon esculentum Mill. cv Sprinter) fruit cuticles at 25°C. Sorption was followed using ¹⁴C-labeled OP + 9.5EO (Triton X-100). Solution pH (2.2-6.2) did not affect surfactant sorption by tomato fruit cuticular membranes (CM). Surfactant concentration (0.001-1.0%, w/v) had a marked impact on sorption. Sorption equilibrium was reached in 24 hours for OP + 9.5EO concentrations below the critical micelle concentration (CMC), whereas 72 to 120 hours were required to reach equilibrium with concentrations greater than the CMC. Regardless of when equilibrium was attained, initial sorption of OP + 9.5EO occurred rapidly. Partition coefficients (K) of approximately 300 were obtained at pre-CMC concentrations, whereas at the highest concentration (1.0%), K values were approximately 15- to 20-fold lower. Sorption was higher for dewaxed CM (DCM) than for CM. At OP + 9.5EO concentrations below the CMC, the amount (millimoles per kilogram) sorbed by CM and DCM increased sharply as the CMC was reached. After an apparent plateau in the amount sorbed at concentrations immediately below and above the CMC, sorption by CM and DCM increased dramatically with OP + 9.5EO concentrations greater than the CMC (0.5 and 1.0%). In contrast, sorption of OP + 5EO (Triton X-45) by CM and DCM differed from one another at relatively high (0.5 and 1.0%) concentrations, where sorption by DCM increased with increasing concentration, but plateaued for the CM. Sorption of OP + 9.5EO was also related to CM concentration, with an inverse relationship existing between sorption and CM at concentrations less than 3.33 milligrams per milliliter.     

Steady-State Kinetics of NADH:coenzyme Q Oxidoreductase Isolated from Bovine Heart Mitochondria

Steady-state kinetics of the bovine heart NADH:coenzyme Q oxidoreductase reaction were analyzed in the presence of various concentrations of NADH and coenzyme Q with one isoprenoid unit (Q₁). Product inhibitions by NAD⁺ and reduced coenzyme Q₁ were also determined. These results show an ordered sequential mechanism in which the order of substrate binding and product release is Q₁–NADH–NAD⁺–Q₁H₂. It has been widely accepted that the NADH binding site is likely to be on the top of a large extramembrane portion protruding to the matrix space while the Q₁ binding site is near the transmembrane moiety. The rigorous controls for substrate binding and product release are indicative of a strong, long range interaction between NADH and Q₁ binding sites.     

Insecticidal activity of Citrus aurantium peel extract against Bactrocera oleae and Ceratitis capitata adults (Diptera: Tephritidae)

Citrus aurantium (L.) peel extracts in petroleum ether were evaluated for toxicity against olive fruit fly, Bactrocera oleae (Gmelin) and medfly, Ceratitis capitata (Wiedemann) adults. Bactrocera oleae flies were more susceptible to the extract than C. capitata in contact and residual bioassays. Fumigation bioassay had no effect. Both sexes of B. oleae were equally susceptible in both types of bioassays. However, males of C. capitata were more susceptible than the conspecific females. LD₅₀ values (concentration causing 50% mortality) after 96 h for the males and females of B. oleae were 44.8 and 40.1 μg/insect in contact bioassay through topical application. Whereas, LD₅₀ values for the males and females of C. capitata were 38.8 and 67.8 μg/insect respectively. LC₅₀ values after 96 h for the males and females of B. oleae were 18.8 and 17.8 μg/cm² in Petri dish residual bioassay. Whereas, LC₅₀ values for the males and females of C. capitata were 70.6 and 147.1 μg/cm² respectively.
Fractionation of the extract on a silica gel column with three different polarity solvents resulted in three fractions with only the intermediate polarity solvent fraction having substantial insecticidal activity. Toxicity of the mixtures of active and inactive fractions was equal to the original extract. Our results indicate that C. aurantium has potential for controlling insect pests.     

High efficiency in vitro organogenesis from mature tissue explants of Citrus macrophylla and C. aurantium

A simple and efficient protocol for obtaining organogenesis from mature nodal explants of Citrus macrophylla (alemow) and Citrus aurantium (sour orange) has been developed by optimizing the concentrations of the plant growth regulators, the incubation conditions, the basal medium and by the choice of explant. In order to optimize the plant growth regulator balance, explants were cultured in the regeneration medium supplemented with several N ⁶-benzyladenine (BA) concentrations or with 2 mg?l^?1 BA in combination with kinetin (KIN) or 1-naphthaleneacetic acid (NAA). The presence of BA was found to be essential for the development of adventitious buds; the best results were obtained using BA at 3 and 2 mg?l^?1 for alemow and sour orange, respectively. The combination of BA with KIN or NAA in the culture medium decreased the regeneration frequency, with respect to the use of BA alone. The effect of three different basal media was rootstock-dependent. For C. macrophylla the best results were obtained with Woody Plant Medium or Driver and Kuniyuki Walnut Medium (DKW). However, for C. aurantium, although high percentages of regenerating explants were obtained independently of the basal medium used, the highest number of buds per regenerating explants was obtained with DKW medium. Attempts were made to identify the type of explants which had a higher regeneration ability using particular regions along the mature shoots of C. macrophylla. When nodal segments, where the buds were completely removed, and internode segments were compared, the highest percentage of responsive explants was obtained with nodal segments. The existence of a morphogenetic gradient along the shoot was observed and the organogenic efficiency was highest when explants from the apical zone were used. Incubation in darkness for 3 or 4 wk was essential for regeneration process in both rootstocks.     

Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus aurantium

The distribution of the flavanones naringin and neohesperidin has been analyzed during the development of the leaves, flower buds, and fruits of Citrus aurantium. These flavonoids are at maximum concentration in the organs studied during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the naringin and neohesperidin concentration in leaves, flower buds, and fruits is due to a dilution of the flavonoids caused by cell growth, because total content per organ continues to increase. The levels of neohesperidin are always greater than those of naringin, although the ratio between the relative concentrations is different in the three organs studied. Leaves have the highest ratios, varying between 8.83 and 5.18, followed by flowers (3.15-1.85), and fruits (2.23-1.02). These observations suggest different relationships between the respective enzymic activities in their biosynthetic pathway.     

Physiological and ultrastructural responses of sour orange (Citrus aurantium L.) clones to water stress

Water stress is a major abiotic constraint leading to serious crop losses. Recently, in the Mediterranean region, water stress has become markedly sensed, especially in Citrus orchards. This study investigated the physiological responses of local sour orange (Citrus aurantium L.) clones to severe water stress. Water stress was applied by withholding irrigation during weeks, followed by a rewatering phase during three months. Under water stress, sour orange clones decreased their stomatal conductance, net photosynthetic rate, and transpiration rate. On the contrary, biomass was stable, especially in the Kliaa clone. In addition, reduced leaf water potentials (-3 MPa) and water contents were measured in most of the clones, except Kliaa which kept the highest water potential (-2.5 MPa). After rewatering, all clones recovered except of the Ghars Mrad (GM) clone. Ultrastructural observations of leaf sections by transmission electron microscopy did not reveal marked alterations in the mesophyll cells and chloroplast structure of Kliaa in comparison to the sensitive clone GM, in which palisade parenchyma cells and chloroplasts were disorganized. This contrasting behavior was mainly attributed to genetic differences as attested by molecular analysis. This study highlighted GM as the drought-sensitive clone and Kliaa as the tolerant clone able to develop an avoidance strategy based on an efficient stomatal regulation. Although a high percentage of polyembryony characterizes C. aurantium and justifies its multiplication by seeds, heterogeneous water-stress responses could be observed within sour orange plants in young orchards.     

Plastids in the Quiescent Embryo and Young Seedlings of the Chloroembryophyte Citrus nobiisxCitrus aurantium amara pumila

Plastid ultrastructure has been investigated in green clementineembryos. Research has been extended to both dark- and light-grownseedlings in order to study the fate of the chioroplasts showinglarge membrane stacks which had been found in the more differentiatedembryo tissues. During germination in the dark the chloroplastslose their membrane stacks and prolamellar bodies are visible.In the light their thylakoid system grows more abundant andis well arranged. Therefore in both cases the embryo chloroplastsdevelop internal organization similar to that of the plastidsdifferentiated from the proplastids of the embryo meristems.The possible significance and function of the embryo chloroplastsis discussed. Citrus nobilis x C, auranhium amara pumila, clementine, embryo, plastid, ultrastructure     

The Chemical Constituents of the Essential Oil from the Flowers,Leaves and Peels of Citrus aurantium

Citrus aurantium L. var. amara Engl., is a better species of sour oranges. There are essential oils in the flowers, the peels, the leaves and the branches of C. aurantium. The flower oil can be used in the preparation of perfumes of high quality. The peel oil is used mainly for the flavor-endowing of soft drinks, alcoholic drinks, bread, confectionaries and cakes. In order to control the quality of the essential oils and to improve them, we have systema- tically studied the chemical constituents of the flowers, the leaves and the peals of C. aurantium with our preparation. 12 main components were separated by silica gel column chromatography. The following 33 chemical components were identified by IR, GC-MS and GC retention index: α-thujene, α-pinene, camphene, β-pinene, myrcene, limonene, β-ocimene, trans-linalooloxide (furanoid), cis-linalooloxide (furanoid), linalool, 1,4-p-methadien-7-ol, trans-pinocarveol, camphor, terpinen-4-ol α-terpineol, nerol, citral-b, geraniol, linalylacetate, citrala, trans-linalooloxide (pyranoid), methyl anthranilate, terpinyl acetate, cis-linalooloxide (pyranoid), neryl acetate. geranyl acetate, nonanal, β-caryophyllene, α-humulene, γ-muurolene, β-nerolidol, farnesol, α- nerolidol. GC retention index of 33 compounds were measured. A fast method for routine determination is presented.     

Citrus aurantium, an ingredient of dietary supplements marketed for weight loss: current status of clinical and basic research

Seville orange (Citrus aurantium) extracts are being marketed as a safe alternative to ephedra in herbal weight-loss products, but C. aurantium may also have the potential to cause adverse health effects. C. aurantium contains synephrine (oxedrine), which is structurally similar to epinephrine. Although no adverse events have been associated with ingestion of C. aurantium products thus far, synephrine increases blood pressure in humans and other species, and has the potential to increase cardiovascular events. Additionally, C. aurantium contains 6',7'-dihydroxybergamottin and bergapten, both of which inhibit cytochrome P450-3A, and would be expected to increase serum levels of many drugs. There is little evidence that products containing C. aurantium are an effective aid to weight loss. Synephrine has lipolytic effects in human fat cells only at high doses, and octopamine does not have lipolytic effects in human adipocytes.     

Effects of soil phosphorus and Glomus intraradices on growth,nonstructural carbohydrates,and photosynthetic activity of Citrus aurantium

Sour orange (Citrus aurantium L.) grown in low-P (9–12 ppm) and high-P (420 ppm) soil inoculated with or without Glomus intraradices (G.i.), were evaluated for biomass, carbohydrates, ribulose bisphosphate carboxylase (RuBPCase), phosphoenolpyruvate carboxylase (PEPCase) activity, leaf ¹⁴CO₂ incorporation, and other physiological parameters. Growth of plants in the low-P, noninoculated soil was lowest, with total dry biomass reduced up to half of the low-P, inoculum treatment. Total nonstructural carbohydrates were 40% lower in leaves of plants in the low-P, noninoculated soil, compared with the other treatments. Inoculation of the low-P soil enhanced leaf ¹⁴CO₂ incorporation by 67%, total chlorophyll content by 28%, and RuBPCase activity by 42%, compared with low-P, noninoculated treatment. Improved P-use efficiency by G.i. in low-P soil was comparable to high-P nutrition in improving leaf ¹⁴CO₂ incorporation and concentration of major leaf photosynthetic products that include starch and sucrose. Leaf PEPCase activity in the low-P, noninoculated treatment, however, was at least threefold higher than the other treatments, suggesting a possible alteration in organic acid metabolism in sour orange leaves as a result of P deficiency.