Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 μM) and bergaptol (140 μM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.     

Growth Rate and Water Relations of Citrus Leaf Flushes

Growth rates of seasonal leaf flushes of ‘Valencia’orange [Citrus sinensis (L.) Osbeck] were measured and waterrelations characteristics of young (new) and over-wintered (old)citrus leaves were compared. New flush leaves had lower specificleaf weights and lower midday leaf water potentials than comparablyexposed old leaves. Spring and summer flush new leaves had higherosmotic potentials than old leaves. These differences becamenon-significant as the new leaves matured. During summer conditions,water-stressed new leaves reached zero turgor and stomatal conductancealso began to decrease in them at higher leaf water potentialsthan in old leaves. Old leaves were capable of maintaining openstomata at lower leaf water potentials. Opened flowers and newflush leaves lost more water, on a dry weight basis, than flowerbuds, fruit or mature leaves. The results illustrate differencesin leaf water potential and stomatal conductance which can beattributed to the maintenance of leaf turgor by decreases inleaf osmotic potentials as leaves mature. These changes in citrusleaf water relations are especially important since water stressresulting from high water loss rates of new tissues could reduceflowering and fruit set. Citrus sinensis (L.) Osbeck, orange, Citrus paradisi Macf., grapefruit, growth rate, leaf water relations, osmotic potential, water potential, stomatal conductance     

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.     

Ultrastructure and histochemistry of Citrus limon (L.) stigma

Citrus limon has a wet stigma which can be divided in two zones: a glandular superficial one formed by papillae, and a non-glandular one formed by parenchymatic cells. The stigmatic exudate is produced by the papillae after the latter have reached their ultimate size. The papillae of the mature pistil are of varying size and composition. Both the unicellular and multicellular ones are present. The cells at the base of the papillae are rich in cytoplasm, whereas the tip cells are vacuolated. Histochemical analysis has shown that the exudate of Citrus is composed of lipids, polysaccharides, and proteins. Our results indicate that the lipidic component is produced and secreted first, followed by production and secretion of the polysaccharidic component. The lipidic component of the exudate is produced in the basal papillae cells and accumulates as droplets in dilated parts of the smooth endoplasmic reticulum (SER). Subsequently the lipid droplets are transported to the plasma membrane, and transferred by the latter into the cell walls. Then the exudate component is accumulated in the intercellular spaces and in the middle lamellar regions of the walls. Subsequently, the polysaccharidic component of the exudate is produced and secreted by the tip cells of the papillae.Abbreviations RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Catabolism of geraniol by cell suspension cultures of Citrus limon

The addition of geraniol to cell suspension cultures of Citrus limon resulted in the rapid formation of nerol, citronellol, geranic acid and citronellic acid. Concurrently, a transient accumulation of bound forms of branched chain fatty acids, and, with a few hours delay, of regular chain C2 to C12 fatty acids was elicited. A concerted action of combined alpha/beta-oxidation enzymes on the terpenic acids, followed by an enlarged acetyl CoA pool is suggested. Terpene catabolism in plants and in vitro plant cells is discussed.     

Somatic embryogenesis from styles of lemon (Citrus limon)

Lemon [Citrus limon (L.) Burm.] styles were treated with different growth regulators for induction of somatic embryos. Styles and stigmas were dissected from flowers and cultured on a Murashige and Skoog (MS) basal medium supplemented with 4.52 M 2,4-dichlorophenoxyacetic acid and 13.3 M 6-benzyladenine. Callus was induced from the style base 2 weeks after the treatment initiation, and embryos appeared 2 months later.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

Whole-Plant Water use and Canopy Conductance of Cassava Under Iimited Available Soil Water and Varying Evaporative Demand

Cassava (Manihot esculenta Crantz), a perennial woody shrub, is known to be highly productive under favourable conditions and produce reasonably well under adverse conditions where other crops fail. Using constant heat sap flow sensors, sap flow density (F _d ) of cassava was monitored for 10 days in December 2002. Sap flow was highly correlated (R ² =0.72, P<0.05) to incoming solar radiation (R _s) than to other climatic factors. Using cross-correlation analysis, no time shift was detected between F _d and solar radiation, whereas vapour pressure deficit (VPD) lags F _d by 110 min. Solar radiation and VPD together explained 83% of diurnal variation in sap flow. Whole-plant transpiration ranged from 0.8 to 1.2 mm day⁻¹ and daily canopy conductance (g _c), computed based on the inverted Penman–Monteith model, varied between 0.7 and 2.1 mm s⁻¹ (mean = 1.4 ± 0.5 mm s⁻¹). For the measurement period, characterized by high evaporative demand coupled with low available soil water, transpiration accounted for 21% of the available energy and was only able to meet 24% of the atmospheric water demand. Average decoupling factor (Ω) of 0.05±0.02 estimated suggested that a 10% change in g _c may lead to more than 9% change in transpiration which further supports the notion that stomata play significant role in regulating cassava water use compared to other known mechanisms. Beyond light saturation (R _s >300 W m⁻²) and at higher VPD (>1.0 kPa), wind effects on the canopy transpiration under water stress condition were low, while VPD explains 94% of the observed variance in daily canopy conductance.     

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.     

Cytological changes related with salt tolerance in embryogenic callus of Citrus limon

Electron microscopy observations of salt-tolerant embrogenic calli of Citrus limon [(L.) Burm. f.] showed several changes in cell ultrastructure when compared with control calli. Both types of calli comprised clusters of meristematic cells, but salt-tolerant calli had several structural differences: thick cell walls, ring-shaped mitochondria, an increased content of lipid bodies, microbodies and parallel accumulation of rough endoplasmatic reticulum. These structural features seem to be related with salt tolerance in Citrus limon cells.     

La componente lipidica dell'essudato stigmatico di Citrus limon (L.) Burm

Abstract

The lipidic component of Citrus limon (L.) Burm. stigmatic exudate. Chromatographic methods (Gas Chromatography and Thin Layer Chromatography) were used to investigate the lipids present in the stigmatic secretion of Citrus limon.

12 fatty acids, ranging from C 12 to C 24, were identified; they form 7 classes of different lipids, 3 of them having unsaturated components.     

La composizione chimica dell'essudato stigmatico di Citrus limon (L.) Burm

Abstract

The composition of the stigmatic exudate of Citrus limon (L.) Burm. - Biochemical analyses of the stigmatic exudate of Citrus limon (L.) Burm. have revealed the presence of lipids, polysaccharides, proteins, phenols, free aminoacids and alkaloids. The role of stigma secretion is discussed in relation to pollen activation and recognition and the pollen tube growth.     

Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specificity

Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a beta-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lücker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClgammaTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of beta-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClgammaTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClgammaTS and Cl(-)betaPINS.     

柑橘木虱胁迫对柠檬叶代谢组挥发性成分的影响

柑橘木虱是柑橘黄龙病的自然传播媒介,为了解柑橘应对柑橘木虱胁迫的代谢组学变化,本文以柠檬为受体,进行柑橘木虱取食胁迫处理,分别于胁迫不同阶段(0、12、24、48、72和96 h)采集柠檬叶为试验材料,静态顶空-气相色谱-质谱联用检测其挥发性物质。主要结果如下:结合保留指数从柠檬叶中共检出37种化合物,包含15种萜类,6种醛类,6种醇类等;含量较高的挥发性成分有(+)-柠檬烯、桧烯、月桂烯等;受柑橘木虱取食胁迫后,柠檬叶中的挥发性化合物含量产生明显变化,萜类、醛类和醇类物质相对含量均呈升高趋势。桧烯、里那醇、(+)-香茅醛和橙花醇4种物质的含量在柑橘木虱取食胁迫96 h时分别升高2.1,2.9,7.8和3.2倍;α-石竹烯的含量下降1.9倍。(+)-柠檬烯、β-红没药烯和反式-α-香柑油烯等物质含量变化不明显,胁迫后的样品中新检出玫瑰呋喃氧化物、香叶醇和2-异丙基-5-甲基茴香醚等3种化合物。皮尔森相关性分析表明,桧烯、β-蒎烯、甲基庚烯酮、月桂烯、水芹烯等9种物质含量与柑橘木虱胁迫时间呈极显著正相关(P0.01),α-蒎烯、莰烯、松油烯、γ-萜品烯等10种物质含量与柑橘木虱胁迫时间呈显著正相关(P0.05),而邻甲氨基苯甲酸甲酯含量与柑橘木虱胁迫时间呈极显著负相关(P0.01)。聚类和热图进一步分析表明,胁迫不同时间的样品具有显著差异,且胁迫时柠檬叶挥发性化合物大部分呈现上升的趋势,尤其是胁迫48、72和96 h后,物质含量显著增加。研究结果为了解柑橘应对柑橘木虱等生物胁迫的代谢组学变化及柑橘木虱的生物防治提供了理论基础。     

The Influence of Glomus mosseae on Tylenchulus semipenetrans-Infected and Uninfected Citrus limon Seedlings

Greenhouse studies have shown that when rough lemon (Citrus limon) seedlings infected with TyIenchulus semipenetrans were transplanted into soil infested with Glomus mosseae, the mycorrhizal fungus infection increased seedling growth compared to nonntycorrhizal seedlings. Tylenchulus semipenetrans significantly suppressed seedling growth below that of mycorrhizal seedlings. Histological observations of nematode-free mycorrhizal roots showed that hyphae penetrated the epidermis and invaded the cortex, giving rise to arbuscules and vesicles. Nematode infection sites in T. semipenetrans-infected roots grown in soil infested with G. mosseae did not show evidence of vesicle development in the cortex but did show arbuscule development.     

Inhibition of limonoid biosynthesis in leaves of Citrus limon,by triethylamine derivatives

Both 2-(4-ethylphenoxy)triethylamine and 2-(3,4-dimethylphenoxy)triethylamine markedly inhibited the biosynthesis of limonoids in lemon leaves. However     

Effect of the desiccation tolerance and cryopreservation methods on the viability of Citrus limon L. Burm cv. Eureka seeds

Cryopreservation of the germplasm for long-term periods is of great importance to maintain the genetic resource. Argentina is one of the world's highest lemon producing country. The performance of different cooling/warming rates in the cryopreservation method of Citrus limon L. Burm cv. Eureka seeds and their influence on the interval of optimal moisture content in the desiccation stage were analyzed. Water sorption isotherm was determined and modeled using D'Arcy & Watt equation; it provided important information concerning the amounts of water associated to strong, weak and multimolecular binding sites along the sorption isotherm. Seeds tolerated a wide range of desiccation conditions (0.1<a_w<0.85) showing a high viability (>80%), however desiccation to 0.0526 g H₂O g⁻¹ d.b. (a_w = 0.0901) produced a significant loss of viability. Differential Scanning Calorimetry was used to identify the thermal transitions of lipids and water in the seed; enthalpies were used to calculate the unfrozen water fraction (0.19 g H₂O g⁻¹ d.b. corresponding to a_w = 0.64). Two cooling/warming rates were tested on desiccated seeds (0.11<a_w<0.85): i) 200 °C min⁻¹ (reached with seeds placed inside a closed cryogenic vial); ii) 1000 °C min⁻¹ (reached with aluminum-foiled seeds placed in a perforated cryogenic vial). For both methods, viability was maximum (83.3%) at a_w = 0.64. Lethal ice formation was responsible for the loss of viability at a_w>0.64 corresponding to the unfrozen water fraction. The use of higher cooling/warming rates enables a wider range of desiccation conditions (0.33<a_w<0.76) in cryopreservation procedures. This work contributes to the optimization of cryopreservation methods of economically important germplasm.     

Comparative Study of the Effect of Sample Pretreatment and Extraction on the Determination of Flavonoids from Lemon (Citrus limon)

Background

Flavonoids have shown to exert multiple beneficial effects on human health, being also appreciated by both food and pharmaceutical industries. Citrus fruits are a key source of flavonoids, thus promoting studies to obtain them. Characteristics of these studies are the discrepancies among sample pretreatments and among extraction methods, and also the scant number of comparative studies developed so far.

Objective

Evaluate the effect of both the sample pretreatment and the extraction method on the profile of flavonoids isolated from lemon.

Results

Extracts from fresh, lyophilized and air-dried samples obtained by shaking extraction (SE), ultrasound-assisted extraction (USAE), microwave-assisted extraction (MAE) and superheated liquid extraction (SHLE) were analyzed by LC–QTOF MS/MS, and 32 flavonoids were tentatively identified using MS/MS information. ANOVA applied to the data from fresh and dehydrated samples and from extraction by the different methods revealed that 26 and 32 flavonoids, respectively, were significant (p≤0.01). The pairwise comparison (Tukey HSD; p≤0.01) showed that lyophilized samples are more different from fresh samples than from air-dried samples; also, principal component analysis (PCA) showed a clear discrimination among sample pretreatment strategies and suggested that such differences are mainly created by the abundance of major flavonoids. On the other hand, pairwise comparison of extraction methods revealed that USAE and MAE provided quite similar extracts, being SHLE extracts different from the other two. In this case, PCA showed a clear discrimination among extraction methods, and their position in the scores plot suggests a lower abundance of flavonoids in the extracts from SHLE. In the two PCA the loadings plots revealed a trend to forming groups according to flavonoid aglycones.

Conclusions

The present study shows clear discrimination caused by both sample pretreatments and extraction methods. Under the studied conditions, liophilization provides extracts with higher amounts of flavonoids, and USAE is the best method for isolation of these compounds, followed by MAE and SE. On the contrary, the SHLE method was the less favorable to extract flavonoids from citrus owing to degradation.     

Isolation and characterization of microsatellites loci in the lemon (Citrus limon)

This study reports the isolation and characterization of seven polymorphic microsatellite loci in Citrus. The loci were isolated from two libraries constructed from genomic DNA nonenriched for TC and AC repeats and enriched for AC repeats. These markers yielded four to nine alleles per locus (mean 6.14) in a survey of 32 Citrus cultivars. Average observed heterozygosity ranged from 0.43 to 0.72. The levels of polymorphism found in this study suggest that these microsatellite loci can become an important tool for genetic studies in Citrus.