Characterization of geraniol synthase from the peltate glands of sweet basil

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 microM were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.     

Stimulation effect of carrageenan on enzymatic defense system of sweet basil against Cuscuta campestris infection

Sweet basil is an important medicinal plant used especially for therapeutical potentials. Sweet basil is a common host for Cuscuta campestris, which has a negative effect on infected plants. Therefore, natural friendly control of C. campestris seems to be useful. It has been shown that carrageenans can act as elicitors of plant defense responses. In this work, the effect of κ-carrageenans on protection against C. campestris and suppression of its invasion in basils were studied. Basils were sprayed with a solution of κ-carrageenan (1?g?L^?1), once a week, three times in total. Infection of basils with C. campestris was performed two days after the last carrageenan treatment. C. campestris stem and the leaves of basils were collected two weeks after C. campestris inoculation for biochemical studies. Treatment with carrageenan significantly increased shoot length and leaf area of basil and decreased C. campestris infestation by about 26%. The content of malondialdehyde, other aldehydes, hydrogen peroxide and lipoxygenase (LOX) activity increased significantly in basils parasitized by C. campestris. There were significant differences in phenylalanine ammonia lyase (PAL), catalase (CAT), superoxide dismutase (SOD) and peroxidase activity of parasitized basils by C. campestris compared with healthy basils. Carrageenan treatment of basils caused a significant increase in H₂O₂ content and the activity of PAL, CAT and SOD, but not of malondialdehyde, other aldehydes content and LOX, polyphenol oxidase (PPO) and peroxidases activity. The activated enzymatic defense system (PAL, PPO, CAT, SOD and peroxidase) in carrageenan-treated basils have a vital role in alleviating oxidative stress damage in infected plants, by removing excess reactive oxygen species and inhibiting LOX activity and lipid peroxidation that was observed in this study. Our results showed that the application of κ-carrageenan-induced beneficial effects in plants, with regard to growth stimulation and the activation of enzymatic defense system. Thus, carrageenan was recommended as a natural biostimulator for protection of plants against C. campestris.     

UV-B is required for normal development of oil glands in Ocimum basilicum L. (sweet basil)

Plants of Ocimum basilicum L. grown under glass were exposed to short treatments with supplementary UV-B. The effect of UV-B on volatile essential oil content was analysed and compared with morphological effects on the peltate and capitate glandular trichomes. In the absence of UV-B, both peltate and capitate glands were incompletely developed in both mature and developing leaves, the oil sacs being wrinkled and only partially filled. UV-B was found to have two main effects on the glandular trichomes. During the first 4 d of treatment, both peltate and capitate glands filled and their morphology reflected their 'normal' mature development as reported in the literature. During the following days there was a large increase in the number of broken oil sacs among the peltate glands as the mature glands broke open, releasing volatiles. Neither the number of glands nor the qualitative or quantitative composition of the volatiles was affected by UV-B. There seems to be a requirement for UV-B for the filling of the glandular trichomes of basil.     

An investigation of the storage and biosynthesis of phenylpropenes in sweet basil

Plants that contain high concentrations of the defense compounds of the phenylpropene class (eugenol, chavicol, and their derivatives) have been recognized since antiquity as important spices for human consumption (e.g. cloves) and have high economic value. Our understanding of the biosynthetic pathway that produces these compounds in the plant, however, has remained incomplete. Several lines of basil (Ocimum basilicum) produce volatile oils that contain essentially only one or two specific phenylpropene compounds. Like other members of the Lamiaceae, basil leaves possess on their surface two types of glandular trichomes, termed peltate and capitate glands. We demonstrate here that the volatile oil constituents eugenol and methylchavicol accumulate, respectively, in the peltate glands of basil lines SW (which produces essentially only eugenol) and EMX-1 (which produces essentially only methylchavicol). Assays for putative enzymes in the biosynthetic pathway leading to these phenylpropenes localized many of the corresponding enzyme activities almost exclusively to the peltate glands in leaves actively producing volatile oil. An analysis of an expressed sequence tag database from leaf peltate glands revealed that known genes for the phenylpropanoid pathway are expressed at very high levels in these structures, accounting for 13% of the total expressed sequence tags. An additional 14% of cDNAs encoded enzymes for the biosynthesis of S-adenosyl-methionine, an important substrate in the synthesis of many phenylpropenes. Thus, the peltate glands of basil appear to be highly specialized structures for the synthesis and storage of phenylpropenes, and serve as an excellent model system to study phenylpropene biosynthesis.     

Repellent effect of sweet basil compounds on Ixodes ricinus ticks

Diseases transmitted by ticks are causing increasing concern in Europe and all around the world. Repellents are an effective measure for reducing the risk of tick bite; products based on natural compounds represent an interesting alternative to common synthetic repellents. In this study the repellency of sweet basil (Ocimum basilicum L.) was tested against the tick Ixodes ricinus L., by using a laboratory bioassay. A bioassay-assisted fractionation allowed the identification of a compound involved in the biological activity. Eugenol appeared to be as repellent as DEET at two tested doses. Linalool, which was identified in the active fraction too, failed to give any response. Repellency of eugenol was proved also in the presence of human skin odour using a convenient and practical bioassay.     

Notes on the sweet basil and its wild relatives (lamiaceae)

Artificial crossing experiments were made with three taxa of Ocimum. Plants ofO. forskolei Benth. andO. basilicum L. var. purpurascens Benth. were found to be interfertile, butO. americanum L. var.pilosum (Willd.) Paton was found to be reproductively isolated from the other two taxa. The new chromosome number 2n = 48 was counted inO. forskolei. These results suggest thatO. forskolei might be the closest relative of the Sweet Basil. The origin of the Sweet Basil is also discussed.     

Passive heat treatment of sweet basil crops suppresses Peronospora belbahrii downy mildew

Sweet basil (Ocimum basilicum) is an annual herb crop grown in polyethylene‐covered structures in Israel. It is Israel's leading herb crop, grown in warm regions of the country. Downy mildew (caused by Peronospora belbahrii) is a severe disease in Israel and in many other crop‐growing regions worldwide. Experiments were carried out to identify potential climate‐management techniques for suppression of this disease on basil in non‐heated greenhouses. Disease severity was evaluated under commercial‐like conditions in three experiments, with 8–10 walk‐in tunnels at each location. Pathogen inoculum was introduced into all walk‐in tunnels. Regression analysis was performed between the disease values and air temperature, relative humidity (RH) and soil temperature. Downy mildew severity was negatively related to high (>25°C) air temperature, RH in the range of 65–85% and high (>21°C) soil temperature. The increase in air temperature did not result in a significant increase in leaf temperature; canopy surface median temperatures only reached 30°C. Symptomless plants from relatively warmer tunnels (peak temperatures of 45–48°C) that were transferred to conditions that promote downy mildew (22 ± 2°C, RH > 95%) became severely diseased, showing sporulation of P. belbahrii, suggesting that infection occurred but at the high temperatures symptom expression/tissue colonisation was suppressed. Pot experiments in which aerial and subterranean plant organs were differentially heated revealed that treating the roots with a high temperature (26–31°C), similar to the soil temperatures in the warmer greenhouses, while maintaining the upper plant parts at ambient temperature (20°C), suppresses canopy downy mildew. The effect lasted for 1–2 weeks after the plants were removed from the heated soil treatments and maintained under optimal conditions for pathogen development. Furthermore, oospores were found in the symptomatic leaves. Oospores are minimally affected by high temperature, and therefore the high temperature presumably did not affect pathogen survival. In conclusion, the effect of high greenhouse temperature on basil downy mildew may not result from a direct negative effect of high temperature on the pathogen but from an indirect high‐temperature effect on the host, rendering it less susceptible to pathogen development.     

In vitro rosmarinic acid accumulation in sweet basil (Ocimum basilicum L.)

Leaf-derived suspension cultures of sweet basil, Ocimum basilicum L. accumulated rosmarinic acid up to 10 mg g^–1 dry wt, a value up to 11 times higher than in callus cultures or in leaves of donor plants. Immobilized cells accumulated less than 15 g rosmarinic acid g^–1.     

Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition

The potential of three arbuscular mycorrhizal fungi (AMF) to enhance the production of antioxidants (rosmarinic and caffeic acids, RA and CA) was investigated in sweet basil (Ocimum basilicum). After adjusting phosphorus (P) nutrition so that P concentrations and yield were matched in AM and non-mycorrhizal (NM) plants we demonstrated that Glomus caledonium increased RA and CA production in the shoots. Glomus mosseae also increased shoot CA concentration in basil under similar conditions. Although higher P amendments to NM plants increased RA and CA concentrations, there was higher production of RA and CA in the shoots of AM plants, which was not solely due to better P nutrition. Therefore, AMF potentially represent an alternative way of promoting growth of this important medicinal herb, as natural ways of growing such crops are currently highly sought after in the herbal industry.     

Regulation of early growth and antioxidant defense mechanism of sweet basil seedlings in response to nutrition

Basil cultivars are among most used crops worldwide, but high level of morpho-physiological variations is mainly due to the cultivation characteristics. The present study aimed to investigate the physiological changes and the accumulation of secondary metabolites as a response to prolonged nutrient deprivation in shoots and roots of sweet basil (Ocimum basilicum L. var. minimum). Sweet basil seedlings were grown in media with quarter, half and full strength of micro and macronutrients under different levels of KNO₃ alone or in combination with different levels of NH₄NO₃. Evaluated parameters included characteristics of germination, development of leaves, content of photosynthetic pigments and responses of different parameters related to oxidative stress. While the early growth is influenced mainly due to NH₄NO₃ presence, all the levels of nutrient supply were found to trigger and maintain the antioxidant defence system of sweet basil seedlings. With the variations among seedling parts, the ammonium nutrition was notably enhanced levels of activity of SOD, CAT, A-POX, G-POX and P-POX, but had no effect on total antioxidant activity and total phenolic content, including flavonoids, which is mainly accumulated in nutrient deprived roots. In addition, phenylalanine ammonia-lyase activity was analysed and potential pathways of secondary metabolites synthesis were discussed. The enzymatic and non-enzymatic system in O. basilicum var. minimum seedlings was capable to reverse the stress conditions during growth under nutrient deprivation.     

Photoprotection by foliar anthocyanins mitigates effects of boron toxicity in sweet basil (Ocimum basilicum)

Boron (B) toxicity is an important agricultural problem in arid environments. Excess edaphic B compromises photosynthetic efficiency, limits growth and reduces crop yield. However, some purple-leafed cultivars of sweet basil (Ocimum basilicum) exhibit greater tolerance to high B concentrations than do green-leafed cultivars. We hypothesised that foliar anthocyanins protect basil leaf mesophyll from photo-oxidative stress when chloroplast function is compromised by B toxicity. Purple-leafed ‘Red Rubin’ and green-leafed ‘Tigullio’ cultivars, grown with high or negligible edaphic B, were given a photoinhibitory light treatment. Possible effects of photoabatement by anthocyanins were simulated by superimposing a purple polycarbonate filter on the green leaves. An ameliorative effect of light filtering on photosynthetic quantum yield and on photo-oxidative load was observed in B-stressed plants. In addition, when green protoplasts from both cultivars were treated with B and illuminated through a screen of anthocyanic protoplasts or a polycarbonate film which approximated cyanidin-3-O-glucoside optical properties, the degree of photoinhibition, hydrogen peroxide production, and malondialdehyde content were reduced. The data provide evidence that anthocyanins exert a photoprotective role in purple-leafed basil mesophyll cells, thereby contributing to improved tolerance to high B concentrations.     

Investigation of synergistic action between coronatine and nitric oxide in alleviating arsenic-induced toxicity in sweet basil seedlings

The phytotoxin coronatine (COR) is a jasmonic acid mimic produced by several pathovars of plant pathogen. In this study, we evaluated the protective effect of COR and nitric oxide (NO) against the toxicity of sodium arsenate in sweet basil (Ocimum basilicum L.). According to the statistical analysis, arsenic had a significant adverse effect on length and biomass of plants. Seedlings that pretreated with COR and sodium nitroprusside (SNP), significantly reversed fresh and dry lose and relative water content decay induced by the metalloid. The protective effects of COR and SNP were indicated by extent of lipid peroxidation, increase glutathione (GSH), ascorbate and thiol (–SH) content, promote antioxidant enzymes and reduce H₂O₂ content in basil seedlings. The present observation suggested that reduction of excess arsenic As-induced toxicity in O. basilicum by COR and NO is through the activation of enzymes involved in ROS detoxification (CAT, SOD, POD, APX, GR) and maintenance contents of molecular antioxidant (GSH, ascorbate, non-protein thiol and protein-thiol). Moreover, the results revealed a mutually amplifying reaction between COR and NO in reducing As-induced damages.     

Microbial interference ameliorates essential oil yield and diminishes root-knot infestation in sweet basil under field conditions

Despite the vast exploration of bioinoculants for disease resistance and crop yield enhancement, knowledge about their impact on quality and quantity of essential oil as well as induction of antioxidant is scarce, especially in medicinal and aromatic crops. In the present two-year field experimentation, a subset of Bacillus spp., namely Bacillus flexus, B. subtilis, B. megaterium and B. aryabhattai, was evaluated against the most devastating root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood infestation and essential oil yield enhancement in Ocimum basilicum var. CIM-Soumya (Sweet basil). The plants treated with B. subtilis showed significant (P < 0.05) enhancement in fresh biomass (42%), essential oil content (121%), nutrient uptake (83%) and reduced root-knot infestation (54%) as compared to untreated control. The defence afforded by bioinoculants was observed based on augmentation of the phenolic (97%), flavonoid (93%), free radical scavenging activity (34%) and total antioxidant (33%) in Bacillus-treated infected plants as compared to untreated infected plants. The higher concentration of essential oil constituents, namely linalool (54%) and methyl chavicol (33%), was also recorded in same treatment. An increase in essential oil yield was reflected by induction of antioxidant machinery. The present study for the first time delineates nematicidal potentials of B. flexus and B. aryabhattai. Altogether these findings suggest that exploitation of bioinoculants could be helpful for effective management of the root-knot nematode infestation along with higher qualitative and quantitative essential oil yield with augmented antioxidants in sweet basil.     

Alginate encapsulation of axillary buds of Ocimum americanum L. (hoary basil), O. Basilicum L. (sweet basil), O. Gratissimum L. (shrubby basil), and O. Sanctum. L. (sacred basil)

Summary Propagation and conservation of four pharmaceutically important herbs, Ocimum americanum L. syn. O. canum Sims. (hoary basil); O basilicum L. (swett basil); O. gratissimum L. (shrubby basil); and O. sanctum L. (sacred basil) was attempted using synthetic seed technology. Synthetic seeds were produced by encapsulating axillary vegetative buds harvested from garden-grown plants of these four Ocimum species in calcium alginate gel. The gel contained Murashige and Skoog (MS) nutrients and 1.1-4.4 μM benzyladenine (BA). Shoots emerged from the encapsulated buds on all six planting media tested. However, the highest frequency shoot emergence and maximum number of shoots per bud were recorded on media containing BA. Of the six planting media tested, both shoot and root emergence from the encapsulated buds in a single step was recorded on growth regulator-free MS medium as well as on vermi-compost moistened with halfstrength MS medium. Rooted shoots were retrieved from the encapsulated buds of O. americanum, O. basilicum, and O. sanctum on these two media, whereas shoots of O. gratissimum failed to root. The encapsulated buds could be stored for 60 d at 4°C. Plants retrieved from the encapsulated buds were hardened off and established in soil.     

Alcohol fermentation of sweet potato. Membrane reactor in enzymatic hydrolysis

Use of Ultrafiltration membrane systems in stirred cell and in thin-Channel systems for immobilizing enzyme (sweet potato intrinsic and β-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the filtration time. The immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato β-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%.     

Epidermal coumaroyl anthocyanins protect sweet basil against excess light stress: multiple consequences of light attenuation

The putative photoprotective role of foliar anthocyanins continues to attract heated debate. Strikingly different experimental set‐ups coupled with a poor knowledge of anthocyanin identity have likely contributed to such disparate opinions. Here, the photosynthetic responses to 30 or 100% solar irradiance were compared in two cultivars of basil, the green‐leafed Tigullio (TG) and the purple‐leafed Red Rubin (RR). Coumaroyl anthocyanins in RR leaf epidermis significantly mitigated the effects of high light stress. In full sunlight, RR leaves displayed several shade‐plant traits; they transferred less energy than did TG to photosystem II (PSII), and non‐photochemical quenching was lower. The higher xanthophyll cycle activity in TG was insufficient to prevent inactivation of PSII in full sunlight. However, TG was the more efficient in the shade; RR was far less able to accommodate a large change in irradiance. Investment of carbon to phenylpropanoid biosynthesis was more in RR than in TG in the shade, and was either greatly enhanced in TG or varied little in RR because of high sunlight. The metabolic cost of photoprotection was lower whereas light‐induced increase in biomass production was higher in RR than in TG, thus making purple basil the more light tolerant. Purple basil appears indeed to display the conservative resource‐use strategy usually observed in highly stress tolerant species. We conclude that the presence of epidermal coumaroyl anthocyanins confers protective benefits under high light, but it is associated with a reduced plasticity to accommodate changing light fluxes as compared with green leaves.     

In vitro culture of sweet basil: gas exchanges,growth, and rosmarinic acid production

Five in vitro culture systems with different ventilation rates were used to investigate the influence of vessel environment on photosynthesis, dark respiration, ethylene evolution, and rosmarinic acid (RA) production in sweet basil (Ocimum basilicum L.) micropropagated shoots. The systems under comparison were two bioreactors with either temporary (RITA?) or stationary (Growtek?) immersion, and three types of vessels (Magenta?, Microbox ECO ₂ ?, and PCCV25?) that are largely used for plant micropropagation. Shoots of green-leaved cv. Genovese and purple-leaved cv. Dark Opal were cultured on a modified Murashige and Skoog medium containing 0.25 mg dm^?3 6-benzylaminopurine. The instantaneous rates of photosynthesis, dark respiration, and ethylene production were determined by gas chromatography measuring CO₂ and ethylene concentrations in vessel headspaces. The tissue RA content was determined by HPLC in HCl-methanol extracts. The explant growth and morphology were significantly affected by culture conditions and cultivars. The largest biomass production was observed under the photomixotrophic culture conditions provided by Growtek?, whereas the highest RA content in shoot tissues was found in the RITA? photomixotrophic system, where ethylene accumulated to the greatest extent.