Metabolism of limonoids: nomilin to nomilinate in citrus limon

Nomilinate was found to be the major acidic limonoid present in seedlings of Citrus limon. [¹⁴C]Nomilin was converted to at least six acidic metabolites in C. limon, one of which was identified as nomilinate. The metabolism via nomilinate is the fifth metabolic pathway of nomilin shown to be present in nature.     

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.     

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.     

Phytohormone bioregulation of nomilin biosynthesis in Citrus limon seedlings

Auxins such as indoleacetic acid, indolebutyric acid, naphthaleneacetic acid and 2,4,5-trichlorophenoxyacetic acid were found to be potent inhibitors of nomilin biosynthesis in young seedlings of Citrus limon. Up to 97 % of the biosynthesis was inhibited. The auxins used were all effective and inhibited the biosynthesis of nomilin selectively. Abscisic acid was also a potent inhibitor of nomilin biosynthesis and the inhibition was reversed with a cytokinin. Gibberellic acid (GA₃) had no effect on nomilin biosynthesis.     

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.     

Metabolism of limonoids in the citrus hybrid calamondin

Radioactive tracer work showed that in young seeding of calamondin decetylnomilinate was converted to nomilin, obacunone and 6-keteb-nomilol, a new lim     

Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet

TGR5 is a member of the G protein-coupled receptor family and is activated by bile acids (BAs). TGR5 is thought to be a promising drug target for metabolic diseases because the activation of TGR5 prevents obesity and hyperglycemia in mice fed a high-fat diet (HFD). In the present study, we identified a naturally occurring limonoid, nomilin, as an activator of TGR5. Unlike BAs, nomilin did not exhibit the farnesoid X receptor ligand activity. Although the nomilin derivative obacunone was capable of activating TGR5, limonin (the most abundant limonoid in citrus seeds) was not a TGR5 activator. When male C57BL/6J mice fed a HFD for 9 weeks were further fed a HFD either alone or supplemented with 0.2% w/w nomilin for 77 days, nomilin-treated mice had lower body weight, serum glucose, serum insulin, and enhanced glucose tolerance. Our results suggest a novel biological function of nomilin as an agent having anti-obesity and anti-hyperglycemic effects that are likely to be mediated through the activation of TGR5.     

Citrus limonoid nomilin inhibits osteoclastogenesis in vitro by suppression of NFATc1 and MAPK signaling pathways

BackgroundAnimal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear.PurposeTo reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts.Study designControlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures.MethodsCell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative real-time PCR; and proteins expression was evaluated by western blot.ResultsNomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways.ConclusionThis study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.     

Biosynthesis of limonoids in Citrus seedlings

Radioactive tracer work showed that nomilinoate A-ring lactone was the predominant, if not the only, limonoid biosynthesized and accumulated in seedlings of lemon, Valencia orange, grapefruit and tangerine. Lemon seedlings were excellent tools for biosynthetic preparation of [¹⁴C]nomilin.     

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.     

Evaluation of Pomelo Seed Extracts as Natural Antioxidant,Antibacterial, Herbicidal Agents,and Their Functional Components

Pomelo seeds (PS) are important by-product of pomelo fruits (Citrus grandis Osbeck). The value-added utilization of PS remains highly challenged. This study aimed to investigate the utilization potential of PS as natural antioxidant, antibacterial, herbicidal agents, and their functional components. The ethanolic extract (EE) of PS and its four fractions as PEE (petroleum ether extract), AcOEtE (ethyl acetate extract), BTE (butanol extract), and WE (water extract), were prepared and biologically evaluated. BTE exhibited the best antioxidant activity among all these extracts, in both ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and FRAP (ferric reducing antioxidant power) assays. AcOEtE was superior to other extracts in herbicidal assay against both Festuca elata Keng (IC₅₀ of 0.48 mg mL⁻¹) and Amaranthus retroflexus L. (IC₅₀ of 0.94 mg mL⁻¹). Meanwhile, both AcOEtE and BTE demonstrated inhibitory effects against Bacillus subtilis, Escherichia coli, and Xanthomonas citri subsp. citri, with MIC ranging 2.5–5.0 mg mL⁻¹. Furthermore, the primary chemical components involving naringin, deacetylnomilin, limonin, nomilin, and obacunone, were quantified in all these extracts. PCA (principal component analysis) suggested that naringin might highly contribute to the antioxidant activity of PS, and the herbicidal activity should be ascribed to limonoids. This study successfully identified AcOEtE and BTE as naturally occurring antioxidant, antibacterial, and herbicidal agents, showing application potential in food and cosmetics industries, and organic farming agriculture.     

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.     

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.     

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.     

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     

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.     

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.     

The microRNA167 controls somatic embryogenesis in Arabidopsis through regulating its target genes ARF6 and ARF8

The aim of this study was to evaluate the influence of ethylene-releasing (ACC, Ethephon, Methionine) and -inhibiting (cobalt chloride, silver thiosulfate) compounds on ethylene production and shoot organogenesis of nodal segments, where buds were completely removed, from mature tissues of Citrus limon, Fino 49 and Verna 51 cultivars. The addition of ACC to the culture medium produced a very significant decrease of the regeneration. These results were directly related to the ethylene levels measured in the atmosphere inside the tube. Similar results were observed with ethephon and methionine; the gradual increase in ethylene levels in the tubes, with increasing ethylene-releasing compounds in the culture medium, agrees with the decrease in the regeneration rate observed, but the effect was lower than with ACC. When cobalt chloride (CoCl₂) was added to the culture medium, contrary to what was expected, the regeneration decreased in both cultivars and this decreasing was not related with the increase in ethylene production. These observations matched the occurrence of yellowish necrotic explants increasing the concentration of cobalt chloride, probably because of a toxic effect on lemon explants. The increase of silver thiosulfate (STS) in the culture medium enhanced the regeneration percentage in both Verna 51 and Fino 49 cultivars. Nevertheless, ethylene levels increased proportionally with the STS concentration and the regeneration rate. These results may be explained since the effects produced by the high ethylene levels measured in STS experiments were blocked by Ag⁺ ions, thereby increasing the regeneration percentage. Media to which STS was added produced the highest regeneration percentages. The results obtained in this study showed that ethylene plays an important role in the organogenesis of mature explants of C. limon.     

New species of pathogenic Pseudomonas isolated from citrus in Tunisia: Proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov.

A collection of Pseudomonas strains was isolated in different regions of Tunisia in the period 2016–2017 from the fruits and leaves of Citrus sinensis cv. ‘Valencia Late’ and Citrus limon cv. ‘Eureka’ plants with symptoms of blast and black pit disease. A phylogenetic analysis of the housekeeping gene rpoD was used for strain identification at the species level. The results demonstrated the affiliation of these strains with the genus Pseudomonas and revealed the presence of 11 strains representing two putative new species in two monophyletic branches. These strains were analyzed morphologically and genotypically by multilocus sequence analyses of the rpoD, gyrB and 16S rRNA (rrs) gene sequences, and their phenotypic characteristics by API 20NE and Biolog GEN III. Plant pathogenic properties were confirmed on fruits and detached leaves of C. limon cv. ‘Eureka’. Fatty acids and WC MALDI-TOF MS major protein profiles were determined. The genomes of both representatives were sequenced. The average nucleotide index and genome-to-genome distance from KC12^T and E10B^T are below the cut-off established for a described species. These results support the conclusion that the strains KC12^T, KC17, KC20, KC22, KC24A, KC25 and KC26 represent a novel species of Pseudomonas, for which the name of Pseudomonas kairouanensis is proposed. The type strain is KC12^T (=CECT9766 and CFBP 8662). The strains E10B^T, E10AB, E10CB1 and Iy3BA represent another novel species of Pseudomonas for which the name of Pseudomonas nabeulensis is proposed; the type strain is E10B^T (=CECT9765 and CFBP 8661).