Biosynthesis of obacunone from nomilin in Citrus limon

Radioactive tracer work showed that [¹⁴C]nomilin was converted to at least four metabolites in Citrus limon. One metabolite was identified as obacunone, showing that obacunone is biosynthesized from nomilin in C. limon.     

Limonin biosynthesis from obacunone via obacunoate in Citrus limon

Radioactive tracer work showed that [¹⁴C]obacunone was converted to at least four metabolites in Citrus limon. Two were identified as obacunoate and limonin. When [¹⁴C]methyl obacunoate was fed, limonin was found to be one of the metabolites. Based on these results and data accumulated thus far, biosynthetic pathways of limonoids in citrus are proposed.     

Biosynthesis of limonoids in Citrus: Sites and translocation

Sites of limonoid biosynthesis were located in Citrus limon. The stem was found to be the major site of nomilin biosynthesis from acetate. Epicotyl, hypocotyl and root tissues were also capable of biosynthesizing nomilin from acetate, but leaves, fruits and seeds did not show this capacity under the conditions used. All the tissues tested were capable of biosynthesizing other limonoids starting from nomilin. C. limon was capable of translocating nomilin from the stem to other sites.     

Biosynthesis of limonoids: Conversion of deacetylnomilinate to nomilin in Citrus limon

Radioactive tracer work showed that deacetylnomilinate was converted to nomilin in detached stems of young Citrus limon seedlings. This work and the previous findings suggest that deacetylnomilinate is the initial limonoid to be biosynthesized among the limonoids known to be present in Citrus. Possible biosynthetic pathways for the formation of limonoids in Citrus are proposed.     

Molecular characterization of a novel pathogen-responsive receptor kinase-like in <Emphasis Type="Italic">Citrus limon</Emphasis>

Infection by pathogenic microorganisms is a source of biological stress in plants. Understanding the interaction between plants and microbial infection at molecular level might contribute to our understanding for the effective control of the disease. Here, we isolated a novel putative receptor kinase-like protein (Citrus limon P5) that is the first Lec-receptor kinase-like protein isolated in lemon during a pathogen infection. C. limon P5 cDNA fragment was detected by differential display assay in C. limon during Capnodium citri (sooty mold) infection. The deduced amino acid sequence of P5 full-length cDNA revealed a 83% sequence homology with a receptor kinase-like protein from Arabidopsis thaliana characterized by a N-terminal lectin domain and C-terminal serine/threonine conserved domain. The inhibition of the pathogen-responsive P5 by the protein kinase inhibitor staurosporine and by a mitogen-activated protein kinase inhibitor (PD 98059) indicated a defense mechanism in C. limon against pathogens mediated via signal transduction pathway. To our knowledge, this is the first evidence that C. limon uses this putative defense mechanism against pathogens. The role of this protein is discussed as a starting point to understanding the molecular mechanisms in the C. limon in response to C. citri infection.     

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and ¹H/¹³C NMR spectra. The corresponding structure is a 3-(O-α-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C_16:0, C_18:0, C_18:1, and C_18:2. This α-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Δ mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.     

Accumulation of Putrescine during Chilling Injury of Fruits

Putrescine (Put) increased 68% in lemon (Citrus limon (L.) Burm. f. cv Bearss) flavedo, 39% in grapefruit (C. paradisi Macf. cv Marsh) flavedo, 49% in grapefruit juice, and 149% in pepper (Capsicum annuum L. cv Early Calwonder) pericarp when fruits were stored at chilling temperatures. In lemon flavedo, the coefficient of correlation (r²) between Put concentration with severity of chilling was 0.90 and Put levels almost doubled; the injury index going from 1 to 2 units. Pepper pericarp, which was the most chilling-sensitive tissue tested (injury index going from 1 to 3.8 units), showed the greatest difference in Put accumulation (166 to 413 nanomoles per gram fresh weight) between storage temperatures of 7.2 and 1°C. The least difference (338 to 470 nanomoles per gram fresh weight) was found in grapefruit flavedo between storage temperatures of 15.5 and 4.4°C; the injury index going from 1 to 1.3 units.     

Metabolic Pathway of alpha-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition

The uptake and metabolism of α-[5-¹⁴C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated ¹⁴C appeared in organic acids and much less in amino acids, while in the control leaves, the ¹⁴C contents of organic and amino acids were equal. In P-deficient leaves, after longer incubation times the ¹⁴C content of organic acids and amino acids increased, while that of CO₂ and residue fractions remained low. In full nutrient leaves the ¹⁴C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO₂. In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO₂ release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO₂ liberation.     

Low conductances for CO2 diffusion from stomata to the sites of carboxylation in leaves of woody species

Concurrent measurements of leaf gas exchange and on-line ¹³C discrimination were used to evaluate the CO₂ conductance to diffusion from the stomatal cavity to the sites of carboxylation within the chloroplast (internal conductance; g_i). When photon irradiance was varied it appeared that g_i and/or the discrimination accompanying carboxylation also varied. Despite this problem, g_i, was estimated for leaves of peach (Prunus persica), grapefruit (Citrus paradisi), lemon (C. limon) and macadamia (Macadamia integrifolia) at saturating photon irradiance. Estimates for leaves of C. paradisi, C. limon and M. integrifolia were considerably lower than those previously reported for well-nourished herbaceous plants and ranged from 1.1 to2.2μmol CO₂ m^?2 s^?1 Pa^?1, whilst P. persica had a mean value of 3.5 μmol CO₂ m^?2 s^?1 Pa^?1. At an ambient CO₂ partial pressure of 33Pa, estimates of chloroplastic partial pressure of CO₂ (C_c) using measurements of CO₂ assimilation rate (A) and calculated values of g_i, and of partial pressure of CO₂ in the stomatal cavity (C_st) were as low as 11.2 Pa for C. limon and as high as 17.8Pa for peach. In vivo maximum rubisco activities (V_max) were also determined from estimates of C_c. This calculation showed that for a given leaf nitrogen concentration (area basis) C. paradisi and C. limon leaves had a lower V_max than P. persica, with C. paradisi and C. limon estimated to have only 10% of leaf nitrogen present as rubisco. Therefore, low CO₂ assimilation rates despite high leaf nitrogen concentrations in leaves of the evergreen species examined were explained not only by a low C_c but also by a relatively low proportion of leaf nitrogen being used for photosynthesis. We also show that simple one-dimensional equations describing the relationship between leaf internal conductance from stomatal cavities to the sites of carboxylation and carbon isotope discrimination (Δ) can lead to errors in the estimate of g_i. Potential effects of heterogeneity in stomatal aperture on carbon isotope discrimination may be particularly important and may lead to a dependence of g_i upon CO₂ assimilation rate. It is shown that for any concurrent measurement of A and Δ, the estimate of C_c is an overestimate of the correct photosynthetic capacity-weighted value, but this error is probably less than 1.0 Pa.     

Complexity of cellulases from Trichoderma reesei with acidic isoelectric points: A two-dimensional gel electrophoretic study using immunoblotting

Antibody specific to Trichoderma reesei cellulase (65 kDa, isoelectric point, pI, 7.7) shows immuno-cross reactivity with acidic hydrolase complexes containing other cellulases, (pI_app. 3.4–4.5) when tested under conditions of 2D-electrophoresis (1^st dim. PAGIF, 2^nd dim. SDS-PAGE) together with Western blotting. Degradation pattern of ¹⁴C(U)-labeled G₁–G₅ of the 65 kDa cellulase was followed by a 2-directional oligodextrin mapping procedure.Using preparative IEF, homologous antigen portions were detected in cellulases present within acidic hydrolase complexes showing mainly identical molar weight (M_r 65 kDa and 57 kDa) but a range of charge (pI 3.4–4.5). The pattern of acidic cellulases as found after analytical 2D-electrophoresis was reconstituted by preparative IEF (pI_app. 2.7–5.1) followed by SDS-PAGE separation. Homogeneous fractions (upon IEF) gave up to 8 different polypeptides per complex upon SDS-PAGE (M_r 70−20 kDa). Charge heterogeneity of individual acidic hydrolase complexes upon IEF is discussed as one reason for ‘multiplicity’ of acidic cellulases.     

Identification of 16-hydroxyoxohexadecanoic acid monomers in plant cutins

The structure of a new hydroxyketo fatty acid which occurs as a major monomer of Citrus limon fruit cutin has been determined by IR, NMR and MS. The monomer was shown to be a mixture of positional isomers of 16-hydroxyoxohexadecanoic acid with the 10-oxo isomer predominating. Substantial amounts of the 9-oxo isomer were present together with smaller quantities of the 8- and 7-isomers. The same compounds were also found to be important constituents of the fruit cutins of Physalis peruviana and Ribes nigrum.     

Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L

Developing pea cotyledons incorporate radioactivity in vivo from [¹⁴C]glucosamine and [¹⁴C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [¹⁴C]glucosamine and [¹⁴C]mannose is also incorporated into glycopeptides. Considerable redistribution of [¹⁴C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N-acetylglucosamine to asparagine glycopeptide linkage has been isolated from [¹⁴C]glucosamine-labeled glycoproteins.     

Anti-Inflammatory Properties and Chemical Characterization of the Essential Oils of Four Citrus Species

Citrus fruits have potential health-promoting properties and their essential oils have long been used in several applications. Due to biological effects described to some citrus species in this study our objectives were to analyze and compare the phytochemical composition and evaluate the anti-inflammatory effect of essential oils (EO) obtained from four different Citrus species. Mice were treated with EO obtained from C. limon, C. latifolia, C. aurantifolia or C. limonia (10 to 100 mg/kg, p.o.) and their anti-inflammatory effects were evaluated in chemical induced inflammation (formalin-induced licking response) and carrageenan-induced inflammation in the subcutaneous air pouch model. A possible antinociceptive effect was evaluated in the hot plate model. Phytochemical analyses indicated the presence of geranial, limonene, γ-terpinene and others. EOs from C. limon, C. aurantifolia and C. limonia exhibited anti-inflammatory effects by reducing cell migration, cytokine production and protein extravasation induced by carrageenan. These effects were also obtained with similar amounts of pure limonene. It was also observed that C. aurantifolia induced myelotoxicity in mice. Anti-inflammatory effect of C. limon and C. limonia is probably due to their large quantities of limonene, while the myelotoxicity observed with C. aurantifolia is most likely due to the high concentration of citral. Our results indicate that these EOs from C. limon, C. aurantifolia and C. limonia have a significant anti-inflammatory effect; however, care should be taken with C. aurantifolia.     

The potency of lemon (Citrus limon L.) essential oil to control some fungal diseases of grapevine wood

This study aimed to evaluate the in vitro antifungal activity (AA) of the essential oil (EO) of lemon (Citrus limon L.) against three pathogenic fungi attacking grapevine wood. The composition of the EO was also studied. Ten volatile components were identified by gas chromatography–mass spectrometry. The results showed that the EO consists of volatile components where monoterpene hydrocarbons are the most abundant ones. Four major components were identified, which represent 99.9% of the total EO (limonene, neral, ß-pinene, and ?-terpinene). The AA of the EO was evaluated against three pathogenic fungi attacking grapevine wood (Eutypa sp., Botryosphaeria dothidea, and Fomitiporia mediterranea). The results showed that the EO exerts AA against all tested fungi and significantly inhibits their growth. Eutypa sp. is the most sensitive fungus. These results show, for the first time, a new use for the EO of lemon (C. limon L.) to control fungal diseases of grapevine wood.     

Changes in antioxidant enzymes and organic solutes associated with adaptation of citrus cells to salt stress

Embryogenic callus cultures of lemon (Citrus limon L. Burm f. cv Verna), were selected for resistance to salt stress (170 mM NaCl). Inorganic analysis showed that selected callus accumulated more Na⁺ and Cl^- ions than the non-selected one. Moreover, the salt-tolerant C. limon callus exhibited an increase in the activity of antioxidant enzymes involved in oxygen metabolism, with the induction of a new superoxide dismutase isozyme and an increase of the peroxidase activity while the catalase activity was unchanged. Proline and total sugar, mainly sucrose, concentrations increases significantly in salt-tolerant cells as compared to control cells. On the other hand, the selected cell line also showed an increase in choline and glycine betaine, but to lesser extent.Abbreviations BSA bovine serum albumin - P5CR pyrroline-5-carboxylated reductase - QAC quaternary ammonium compounds - SOD superoxide dismutase     

Enzymic studies of glycol and glycerol lipids containing O-alkyl bonds in liver and tumor tissues

The utilization of [1-¹⁴C]hexadecyl-[2-³H]ethyleneglycol and [1-¹⁴C]hexadecyl-[2-³H]glycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and O-alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg²⁺; although the small quantities formed precluded absolute identification, its thin-layer Chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The O-alkyl moiety of hexadecyl-ethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.     

Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100μm) was used to perturb this compartment. With control cells, incorporation of [9,10-³H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-³H]palmitic acid, 458±18pmol of [1-¹⁴C]oleic acid and 252±15pmol of [U-¹⁴C]glucose per μg of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [³H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-¹⁴C]glucose increased by 125%. Incorporation of [1-¹⁴C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.     

Citrus tissue culture: stimulation of fruit explant cultures with orange juice

In vitro growth of explant (juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice (10% v/v optimum) to a basal medium containing Murashige and Skoog salts, 50 grams per liter sucrose, 100 milligrams per liter myo-inositol, 5 milligrams per liter thiamine·HCl, 2 milligrams per liter 2,4-dichlorophenoxyacetic acid and 0.5 milligrams per liter kinetin. In analyzing this effect of orange juice on citron explant cultures, we failed to obtain increased yields by addition of appropriate concentrations of citric acid to the basal medium but obtained growth stimulation when the medium was supplemented with juice from an “acidless” orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid is involved. Addition of the inorganic ash corresponding to 10% (v/v) orange juice to the basal medium had no effect on yields. Similarly, the stimulatory effect of orange juice could not be explained based on its content of sucrose or of organic growth factors already present in the basal medium.     

Diphenylurea Derivatives Induce Somatic Embryogenesis in Citrus

The present research investigates the possibility that three diphenylurea (DPU) derivatives, N-phenyl-N′-benzothiazol-6-ylurea (PBU), N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methilendioxyphenyl)urea (3,4-MDPU), stimulate the induction of somatic embryogenesis in three Citrus species. The hypothetical embryogenic activity was assessed using stigma and styles of Citrus myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three compounds influenced the production of somatic embryos differently as regards the concentrations tested and the citrus species. PBU was able to induce somatic embryogenesis at all the concentrations tested and in all the three species with percentages that ranged from 44 (C. limon) to 85% (C. myrtifolia). 2,3-MDPU and 3,4-MDPU were completely unable to induce the production of somatic embryos in C. myrtifolia while both the compounds at the higher concentration (12 μM) acted positively in both C. madurensis and C. limon (68% of embryogenic explants). The phenylurea derivatives, used for the first time in this study to induce somatic embryogenesis in plant, showed a higher embriogenic performance when compared with 6-benzylaminopurine (BAP), a classical adenine-cytokinin, and with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), a classical DPU derivative.