A malonic acid ester derivative of naringin in grapefruit

A malonic acid ester derivative of the flavanone naringin was abundant in the young leaves and fruits of grapefruit plants, but not in the mature leaves and fruits. After isolation, the structure of this compound was established as naringin 6″-malonate (naringenin 72″-O-- -rhamnosyl)-β- -glucoside 6″-malonate).     

Contrasts between Citrus species in response to salinisation: An analysis of photosynthesis and water relations for different rootstock-scion combinations

Leaf gas exchange, water relations and ion content were measured on two-year-old Valencia orange (Citrus sinensis [L.] Osbeck), Washington Navel orange (C. sinensis) and Marsh grapefruit (C. parodisi Macfad) scions budded to either Trifoliata (Poncirus infoliata [L] Raf) or Cleopatra mandarin (C. reticuLua Blanco) rootstoeks. Trees were watered with dülute nutrient solution containing either 0 or 50 _mM NaCl for 77 days. Leaf chloride concentrations (cell sap basis) were higher in all scions budded on “Trifoliata but sodium levels were lower than in equivalent foliage budded on Cleopatra mandarin rootstock. Foliar salt levels also varied according to scion. Leaves of Marsh grapefruit had higher levels of both sodium and chloride than leaves of either Valencia orange or Washington Navel orange on both rootstocks. Accumulation of sodium and chloride in salinised leaves caused a reduction in leaf osmotic potential of 0.2–1.4 MPa. and leaf water potential declined by as much as 0.5 MPa. Turgor pressure in salinised leaves was thus maintained at or above the control level. Osmotic potentials determined by psychrometry compared with pressure-volume curves were taken to imply that some accumulation of sodium or chloride in the apoplast of salinised leaves may have occurred. Despite turgor maintenance both _co2 assimilation and stomatal conductance were reduced by salinity. Following onset of leaf response to salinisation, gas exchange was impaired to a greater extent in scions budded to Cleopatra mandarin compared to those on Trifoliata. Amongst those scions. leaves of salt-treated Marsh grapefruit showed greater reductions in gas exchange than Valencia orange or Washington Navel orange budded on either rootstock. Increased sensitivity of 1Marsh grapefruit was correlated with a higher foliar sodium and chloride content in this scion. Scion differences in sensitivity of leaf gas exchange to solute concentration were independent of rootstock and appeared unrelated to leaf prolinebetaine concentrations. This implies an inherent difference between scion species with respect to salt tolerance, rather than variation in their capacity to acquire that type of compatible solute. In terms of rootstock effects, all scions proved more sensitive to salinity when budded to Cleopatra mandarin compared with Trifoliata. That response was attributed to a disproportionately higher concentration of leaf sodium in scions on Cleopatra mandarin.     

Isolation and purification of closely related citrus limonoid glucosides by flash chromatography

Several citrus limonoid glycosides have proved to be particularly difficult to purify using conventional techniques. A reversed-phase flash chromatographic technique has been developed for the separation and isolation of the closely related limonoid glucosides, nomilin 17-beta-D-glucopyranoside and nomilinic acid 17-beta-D-glucopyranoside, with confirmation of their identities by electrospray ionisation mass spectrometry. Furthermore, the semi-purification of the mixture of glucosides enriched with flavanone glucosides such as naringin, narirutin and other limonoid glucosides was obtained. The closely eluting glucosides were successfully separated to achieve a good yield and purity of 93%.     

Chiral HPLC separation and CD spectra of the C-2 diastereomers of naringin in grapefruit during maturation

Naringin is the chief flavanone glycoside of grapefruit (Citrus paradisi). It is responsible for part of the bitter taste of the fruit and can cause the inhibition of some cytochrome P450s. The direct separation of (2R)- and (2S)-naringin in the albedo of grapefruits was obtained in normal phase HPLC mode using Chiralcel OD as chiral stationary phase and n-hexane/ethanol with 0.1% of TFA as mobile phase. Chiralpak AD was almost ineffective in the separation. This procedure was used to evaluate the stereochemistry at C-2 during maturation of the grapefruit. The CD curves of (2R)- and (2S)-naringin isolated by semipreparative chiral HPLC were determined and the elution order of the chromatographic peaks was related to the absolute C-2 configuration. Partial resolution of the C-2 diastereomers of narirutin was obtained on Chiralpak AD.     

The path of photosynthate translocation into citrus fruit

Abstract The path of [¹⁴C]photosynthate translocation into citrus fruit was examined to determine which anatomical and physiological features were involved in this process. Experiments were conducted during the final pre-harvest months of 2 years grapefruit crops (Citrus paradisi Macf. cv. ‘Marsh’). A source leaf nearest the fruit was exposed to ¹⁴CO₂ for 1 h + 5 h ambient air, followed by dissection of vascular and phloem-free tissues in the fruit quarter directly aligned with the source. Radioactivity in each tissue was quantified after separation and extraction in boiling 80% ethanol. Peel (flavedo+albedo) contained an average 35% of the label in the quarter fruit, but an additional 20% was localized entirely in dorsal vascular bundles along exterior walls of juice segments. Less [¹⁴C]photosynthate was recovered from other vascular tissues and was nearly absent from adjacent mature seeds. Radioactivity in the single layer of segment epidermis, however, averaged 17% of that in the quarter fruit. Juice tissues interior to this accumulated only 17% of the total. No phloem tissue was evident in either the segment epidermis or juice tissues, but over 70% of the [¹⁴C]assimilates in the latter were localized in thread-like stalks which attach juice vesicles to dorsal vascular bundles. In addition, labelled hexose/sucrose ratios in these structures increased with distance from the vascular bundle. The majority of photosynthates, therefore, entered citrus fruit via dorsal vascular bundles and were partially hydrolysed during slow transfer through non-vascular segment epidermis and juice stalks.     

Embryo sac development and endogenous gibberellins in pollinated and unpollinated ovaries of walnut (Juglans regia)

Glycosidases were extracted from grapefruit ( Citrus paradisi Macf. cv. Ruby Red) flavedo, albedo, and juice vesicles harvested at five periods throughout the season. Flavedo β-galactosidase activity was high at the September harvest and then significantly declined by November. Thereafter, no further changes occurred in β-galactosidase activity. Flavedo α-galactosidase activity was low and unchanged throughout the study. α-Mannosidase, initially low in flavedo, steadily increased with advanced maturity. Trends in glycosidase activities of albedo were similar but attenuated. Juice vesicle β-galactosidase did not change through the study period, whereas α-galactosidase activity decreased 70% after the initial harvest period. α-Mannosidase was initially high and then decreased to 50% of the original activity. A second peak of activity was measured in March, followed by a second decline. Extractability differences of the glycosidases suggest differences in compartmentation and function. Two isozymes of α-mannosidase were separated in flavedo and one in juice vesicles, and characteristics were determined at an early and late harvest period. The results suggest that changes in the three glycosidases could be used to further define maturity and senescence in grapefruit.     

Chilling-induced oxidative stress in young sal (Shorea robusta) seedlings

Aerial parts of the chilling-sensitive young sal seedlings showed overproduction of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) in response to constant chilling exposure during November to March (9–14.1 °C) in field conditions. Almost 4–6 fold increase in ROS was observed in aerial parts of chilling exposed seedlings than the control seedlings (maintained in greenhouse). Increased formation of ROS was found to be closely associated with the rise in TBARS in leaf (5.8 fold) and shoot (4.8 fold) tissues. On the contrary the leaf and shoot of control seedling and root of both control and chilling exposed seedlings exhibited relatively very low levels of superoxide and TBARS. The chilling exposed seedlings also showed striking weakening in the free radical processing enzyme systems. The low temperatures during November to March resulted in reduced activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POX) and ascorbate peroxidase (APX) almost by 49, 26, 7 and 78 % in leaves and 65, 46, 9 and 85% in shoots respectively compared to leaves and shoots of control seedlings. Our results indicated that, substantially higher rates of liberation of superoxide and TBARS along with drastic failure of antioxidant enzyme system in chilling sensitive sal seedlings leads to oxidative bursts terminating into irreversible injury in leaves and shoot of these seedlings.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Isoenzyme pattern of superoxide dismutase in different varieties of citrus plants

Superoxide dismutases (SOD; EC 1.15.1.1) in leaves from different cultivars of citrus plants were characterized using isoelectric focusing in polyacrylamide gels. The plants studied included Citrus limonum R. (cvs Verna, Fino, and Eureka), C. paradisi Mac (cvs Red Blush and Marsh), C. aurantium L. (cv. Comun), C. sinensis L. Osbeck (cvs Navel, Valencia, and Salustiano), and C. reticulata B. (cv. Satsuma). The three molecular forms of SOD were distinguished from each other by their different sensitivity to cyanide and H₂O₂. In C. limonum leaves, four Cu,Zn-SODs, three Fe-SODs and two Mn-SODs were present. However, in leaves from different varieties of C. sinensis, C. paradisi, C. aurantium and C. reticulata the activity and number of Fe-SOD isoenzymes were lower than in lemon leaves, whereas the number of MN-SOD isozymes was increased. Cu,Zn-SODs did not show significant variations in the different species and cultivars. The identification of Fe-SODs in several species of the plant family Rutaceae extends the small number of higher plants where the presence of these Fe-containing metalloenzymes has been demonstrated. Results obtained may be useful from an evolutionary viewpoint and also in mineral nutrition studies using SOD isozymes as markers of functional metals.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

Uptake of 14C-gibberellic acid by mature grapefruit (Citrus paradisi Macf.) as affected by relative humidity and method of application

Effects of the method of application and relative humidity on the uptake of ¹⁴C-gibberellic acid (¹⁴C-GA₃) by mature grapefruit (Citrus paradisi Macf.) were examined. Uptake was higher when ¹⁴C-GA₃ was applied as a 'drying-out' solution than as a 'non-drying' solution. When ¹⁴C-GA, was applied by the 'drying-out' method, which closely imitates field conditions, rates of uptake were very high while the solution was drying out and during the first few hours after drying. Uptake from the dry residue continued in decreasing rates till the end of the experiment (72 h). Uptake from the dry residue was higher when fruits were incubated at 100% than at 50% relative humidity (RH). Transfer of fruits from 50% to 100% RH as late as 48 h after drying still increased the rate of uptake. Drying-out treatment solutions produced higher uptake rates with neutral (pH 7) as well as acid (pH 4) treatment solutions, and in the presence of triton B-1956, Triton X-100 or L-77 surfactants.     

Vacuolar citrate/H+ symporter of citrus juice cells

We have isolated a cDNA, designated Citrus sinensis citrate transporter 1 CsCit1 encoding a novel vacuolar citrate/symporter. Immunoblots using antibodies raised against CsCit1 showed that the protein is localized to the juice sac cell vacuoles. The highest expression of CsCit1 and the amount of protein in the juice sac cell vacuoles coincided with the developmental stage at which the vacuolar citrate content began declining with the concomitant increase in vacuolar pH. Vacuoles from Sacharomyces cereviseae expressing CsCit1 displayed a citrate-dependent H⁺ efflux, and our results clearly demonstrate that CsCit1 is able to mediate the electroneutral co-transport of H⁺ and citrate ions, since the citrate-dependent H⁺ fluxes are not affected by changing the electrical potential difference across the tonoplast. The roles of CsCit1 in mediating citrate efflux from the vacuole and on citric acid homoestasis in Citrus juice sac cells are discussed. T. Shimada and R. Nakano contributed equally to this work.     

Supercritical fluid extraction of naringin from the peel of Citrus paradisi

The highest yield (14.4 g/kg) of naringin, the major flavonoid from the peel of Citrus paradisi L., that could be achieved by supercritical fluid extraction was obtained using supercritical carbon dioxide modified with 15% ethanol and fresh (rather than dried) peels at 95 bar and 58.6 degrees C. This yield is higher than that attained by the conventional technique of maceration, and close to those obtained by reflux and Soxhlet methods. Furthermore, supercritical fluid extraction consumes less solvent and provides a shorter extraction time than conventional extraction methods.     

柑桔潜叶蛾对柑桔的为害及经济阈值的研究

调查福州市郊柑桔园柑桔潜叶蛾（Ｐｈｙｌｏｃｎｉｓｔｉｓｃｉｔｒｅｌａ）在雪柑、芦柑、蕉柑、福桔４个品种上的为害情况．根据其为害特性和柑桔果实产量大小年份可能给经济阈值带来的误差,采用同一树上以秋梢为计量单位,环割被测枝条（避免枝条间养分重新分配而带来误差）的方法,研究了为害秋梢的叶片光合作用强度,当年和翌年春夏梢的生长量,翌年果实品质和重量,以及叶片被害程度与柑桔潜叶蛾幼虫密度的关系．结果表明,柑桔受潜叶蛾为害后具有一定的光合补偿及组织愈伤的补偿能力;根据福州地区１９９４年柑桔生产力,以雪柑为品种确定经济阈值为１．２１头１龄幼虫／嫩叶;缩小了经济阈值受果实产量大小年份差异而带来的误差;同时考虑品种、柑桔园水热条件、产量水平、天敌作用等多种生态因子,建立了多因子经济阈值模型     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Chilling‐induced Ca2+ overload enhances production of active oxygen species in maize (Zea mays L.) cultured cells: the effect of abscisic acid treatment

Chilling (4 °C) induced a prolonged high level of intracellular Ca²⁺ (Ca²⁺ overload) and lipid peroxidation in maize (Zea mays L. cv Black Mexican Sweet) cultured cells. However, such Ca²⁺ overload and enhanced lipid peroxidation were not seen in abscisic acid (ABA)‐treated cells, which had an improved chilling tolerance. A Ca²⁺ ionophore, A23187, caused Ca²⁺ overload in both ABA‐treated maize cells and the untreated control, whereas an enhanced lipid peroxidation was detected only in the control. The high level of active oxygen species (AOS) in the control during chilling at 4 °C could be reduced by the presence of lanthanum (La³⁺), a Ca²⁺ channel blocker, in the medium. Moreover, both the A23187‐induced lipid peroxidation and AOS production in the control could be reduced by extracellular EGTA, a Ca²⁺ chelator. Laser‐scanning confocal microscopy revealed that mitochondria were one of the major AOS sources under chilling and during A23187 treatment. In vitro assays showed that superoxide production in isolated maize mitochondria was enhanced by the presence of Ca²⁺. Findings suggest that chilling‐induced Ca²⁺ influx in the control triggers a marked generation of AOS, which in turn results in the enhanced lipid peroxidation. The ability of ABA‐treated cells to avoid the chilling‐induced Ca²⁺ influx may serve as a mechanism that prevents the chilling‐induced oxidative stress and thus results in less chilling injury.     

Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags

MicroRNAs (miRNAs) are a new family of small RNA molecules found in plants and animals. We developed a comprehensive strategy for identifying new miRNA homologues by mining the repository of available citrus expressed sequence tags (ESTs). By adopting a range of filtering criteria, we identified a total of 38 potential miRNAs--nine, five, nine and 15 miRNAs in Citrus trifoliata (ctr-miRNAs), C. clementina (ccl-miRNAs), C. reticulata (crt-miRNAs) and C. sinensis (csi-miRNAs), respectively--from more than 430,000 EST sequences in citrus. Using the potential miRNA sequences, we conducted a further BLAST search of the mRNA database and found six potential target genes in these citrus species. Eight miRNAs were selected in order to verify their existence in citrus using Northern blotting, and the functions of several miRNAs in miRNA-mediated gene regulation are experimentally suggested. It appears that all these miRNAs regulate expression of their target genes by cleavage, which is the most common situation in gene regulation mediated by plant miRNAs. Our achievement in identifying new miRNAs in citrus provides a powerful incentive for further studies on the important roles of these miRNAs.     

Insulin-like growth factor I-dependent regulation of prolidase activity in cultured human skin fibroblasts

The objectives of this article are to: (i) discuss the origins and the nature of ischemic injury, (ii) identify factors influencing the evolution of injury, (iii) consider various cellular targets for ischemic injury, (iv) assess the overall importance of reperfusion injury, (v) discuss conceptual approaches to cardioprotection and (vi) to identify new ideas and approaches in the realm of myocardial protection. In the human heart, myocardial ischemia may take many forms, it may exist for periods as short as a few seconds or minutes, it may last for hours or it may persist for years. In terms of discussing interventions to combat myocardial ischemia, this article will focus on: (i) regional ischemia as occurs during evolving myocardial infarction and (ii) whole heart or global ischemia as occurs during cardiac surgery and transplantation.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.