Metabolomic approach reveals the biochemical mechanisms underlying drought stress tolerance in thyme.

Thyme as a perennial herb has been recognized globally for its antimicrobial, antiseptic and spasmolytic effects. In this investigation, we have used non-targeted metabolite and volatile profiling combined with the morpho-physiological parameters in order to understand the responses at the metabolite and physiological level in drought sensitive and tolerant thyme plant populations. The results at the metabolic level identified the significantly affected metabolites. Significant metabolites belonging to different chemical classes consisting amino acids, carbohydrates, organic acids and lipids have been compared in tolerant and sensitive plants. These compounds may take a role through mechanisms including osmotic adjustment, ROS scavenging, cellular components protection and membrane lipid changes, hormone inductions in which the key metabolites were proline, betain, mannitol, sorbitol, ascorbate, jasmonate, unsaturated fatty acids and tocopherol. Regarding with volatile profiling, sensitive plants showed an increased-then-decreased trend at major terpenes apart from alpha-cubebene and germacrene-D. In contrast, tolerant populations had unchanged terpenes during the water stress period with an elevation at last day. These results suggesting that the two populations are employing different strategies. The combination of metabolite profiling and physiological parameters assisted to understand precisely the mechanisms of plant response at volatile metabolome level.

Phytochemical investigations and antiproliferative secondary metabolites from Thymus alternans growing in Slovakia.

CONTEXT: Thymus alternans Klokov (Lamiaceae) is a neglected species of the genus Thymus (Sect. Serpyllum) endemic to Carpathian area, where it is used as a flavouring agent and for medicinal purposes. OBJECTIVE: The aim of the work was to identify antiproliferative constituents from the flowering aerial parts of this plant. MATERIALS AND METHODS: Thymus alternans extracts were analyzed by HPLC-MSn and subjected to extensive chromatographic separations. The isolated compounds (phenolics and triterpenes) were structurally elucidated by MS and 1D and 2D NMR experiments. Essential oil (EO) composition was determined by GC-FID and GC-MS. Six purified triterpenes and EO were assayed for in vitro antiproliferative activity against a panel of human cancer cells, namely, breast (MDA-MB 231), colon (HCT-15 and HCT116), lung (U1810), pancreatic (BxPC3), melanoma (A375) and cervical carcinoma (A431) cells. RESULTS: The structures of the isolated compounds were achieved on the basis of H-NMR and MS experiments. Luteolin-4'-O-ß-d-glucopyranoside (P1), chrysoeriol-7-O-ß-d-glucopyranoside (P2), chrysoeriol-5-O-ß-d-glucopyranoside (P3), apigenin-7-O-ß-d-glucopyranoside (P4), rosmarinic acid (P5), rosmarinic acid-3'-O-ß-d-glucopyranoside (P6), caffeic acid-3-O-ß-d-glucopyranoside (P7), 3α-hydroxy-urs-12,15-diene (T1), α-amyrin (T2), ß-amyrin (T3), isoursenol (T4), epitaraxerol (T5), and oleanolic acid (T6). GC-MS analysis revealed that the EO of T. alternans was devoid of phenols and belonged to the nerolidol-chemotype, that is typical of the Sect. Serpyllum. The six purified triterpenes (T1-T6) were active with IC50 ranging from 0.5 to 5 µM being comparable or better than those of reference compounds betulinic acid and cisplatin. The EO exhibited significant effects on A375, MDA-MB 231 and HCT116 cell lines with IC50 in the range of 5-8 µg/mL. CONCLUSION: The reported results suggest that T. alternans can be considered as a good source of phytoconstituents with possible importance in the pharmaceutical field.

Effect of water supply on growth and polyphenols of lemon balm (Melissa officinalis L.) and thyme (Thymus vulgaris L.).

A pot experiment was carried out with lemon balm (Melissa officinalis L.) and thyme (Thymus vulgaris L.). Different water supply was applied: 25%, 40% and 70% saturation of soil water capacity (SWC). Morphological traits, biomass and phenolic type active ingredients were investigated. Among the two species, main differences were registered in biomass and TPC. Lower SWC resulted in reduced biomass production of lemon balm, while the applied stress treatments did not effect the biomass of thyme. In lemon balm, highest TPC contents were measured in control plants both in shoots and roots but in thyme, the shoots showed a significantly increased TPC at the 25% SWC conditions. Neither the content of total flavonoids nor that of the rosmarinic acid was affected by the treatments. The antioxidant capacity proved to be in tight connection with the TPC in both species (r = 0.766-0.883). The rosmarinic acid content of lemon balm plants contributed to the antioxidant capacity, as well (r = 0.679-0.869).

Competitive effect of a native-invasive species on a threatened shrub in a Mediterranean dune system.

The canopy shade of the Retama species has been widely reported to ameliorate the environmental conditions in the understory, thus facilitating other species' establishment. The shading effect of the native-invasive leguminous shrub Retama monosperma (L.) Boiss on the endangered Thymus carnosus Boiss was analysed to determine a positive or negative net effect. Data was taken in all four seasons, representing contrasting light and water availability in a Mediterranean coastal dune ecosystem (SW Spain). The morphological and physiological status of sun-exposed T. carnosus plants growing in open areas versus shaded plants growing under R. monosperma were measured seasonally. Leaf mass area, leaf area index and pigment content showed typical sun-shade responses. In contrast, sun-exposed T. carnosus displayed higher stem water potential, transpiration rate and water use efficiency, both intrinsic and integrated, denoting low tolerance to the presence of R. monosperma. Five years after the measurements, canopy cover had decreased and mortality was higher in shaded plants, thus confirming the competitive effect of R. monosperma on T. carnosus. R. monosperma arises as a competitor for endangered T. carnosus communities, consequently reinforcing its invasive behaviour. This species-specific shrub study demonstrates that eventual beneficial effects of Retama canopy may be overridden by competition in the understory, particularly in the case of species well-adapted to high light and low water levels.

Cryopreservation of Thymus cariensis and T. vulgaris shoot tips: comparison of three vitrification-based methods.

Thymus is an important genus of the Lamiaceae family, comprising more than 400 perennial aromatic thyme species, which are used extensively for medicinal and culinary purposes. The present study focused on the development of cryopreservation procedures for Thymus vulgaris and T. cariensis, the latter being an endemic and endangered species of Turkey. For cryopreservation of T. vulgaris shoot tips, PVS2-based one-step freezing methods, i.e., PVS2 vitrification, encapsulation-vitrification and droplet-vitrification, were compared. Cold hardening and sucrose preculture were also optimized before the cryopreservation trials. For T. cariensis, a droplet-vitrification method was applied to cold-hardened shoot tips, and after sucrose preculture. In all the methods tested, PVS2 was applied for up to 120 min. The best T. vulgaris cryopreservation was achieved with a droplet-vitrification method, that involved 2-weeks cold hardening of shoot cultures, 48 h preculture of shoot tips on MS medium supplemented with 0.25 M sucrose, and a 90 min PVS2 treatment in droplets. After direct immersion in LN, thawing and plating, 80% of shoot-tips recovered. Post-thaw recovery was significantly lower when the same procedure was applied to T. cariensis shoot tips; however also here 90 min PVS2 treatment produced the highest survival (25 percent) and recovery (25 percent) levels.

Funneliformis mosseae inoculation under water deficit stress improves the yield and phytochemical characteristics of thyme in intercropping with soybean.

Intercropping of medicinal plants/legumes along with bio-fertilizer application is a relatively new sustainable practice for improving the yield and secondary metabolites production. Here, a 2-years field experiment was performed to evaluate the effects of water deficit stress and arbuscular mycorrhizal fungi (AMF) application (as bio-fertilizer) on nutrients concentration, dry matter yield, essential oil quantity and quality of thyme in intercropping with soybean. Three irrigation levels, including (i) irrigation after depletion of 20% (I20) as non-stressed, 50% (I50) as moderate water deficit and 80% (I80) available water as severe water deficit were applied as the main factor. The sub-factor was represented by different cropping patterns including thyme sole culture, replacement intercrop ratio of 50:50 and 66:34 (soybean: thyme) and the third factor was non-usage (control) and usage of AMF. According to our results, the thyme dry yield under moderate and severe water deficit stress decreased by 35 and 44% in the first year, and by 27 and 40% in the second year compared with non-stressed (I20) plants, respectively. Also, the macro- and micro-nutrients of thyme leaves increased significantly in intercropping patterns after application of AMF. The maximum essential oil percentage of thyme was achieved in 50:50 intercropping ratio treated with AMF. Under moderate and severe water deficits, the major constituents of thyme essential oil including thymol, p-cymene and γ-terpinene were increased in intercropping patterns treated with AMF. Generally, AMF application in intercropping ratio of 50:50 may be proposed to farmers as an eco-friendly approach to achieve desirable essential oil quality and quantity in thyme under water deficit stress conditions.

Cryopreservation of Thymus moroderi by droplet vitrification.

Thymus moroderi Pau ex Martinez (Labiatae) was successfully cryopreserved using the droplet vitrification method. After 20 min in loading solution at room temperature, shoot tips were dehydrated with PVS2 at 0 degree C for 30 min and immersed into LN. For thawing, shoot-tips were transferred into recovery solution for 15 min. A test of different recovery media revealed that the best results were obtained when the medium was supplement with 0.275 micromolar BA.

Comparison of vitrification and encapsulation-dehydration for cryopreservation of Thymus moroderi shoot tips.

Vitrification and encapsulation-dehydration were tested for cryopreservation of Thymus moroderi Pau ex Martínez (Labiatae), an endemic plant from south-eastern Spain. For vitrification, shoot tips were loaded in a solution containing 0.4 M sucrose + 2 M glycerol for 20 min at room temperature, dehydrated in PVS2 solution for 0-105 min at 0 degree C, then immersed in liquid nitrogen (LN) for at least 1 day and rapidly rewarmed. The highest survival (71.4 percent) was obtained after 60 min PVS2 dehydration. Encapsulation-dehydration gave slightly lower results, with up to 50 percent explants survival. In the optimal protocol, donor plants were cold-hardened at 10 degree C for 5 weeks, excised shoot tips precultured for 48 h on MS medium with 0.08 M sucrose, encapsulated, pretreated in medium with 0.75 M sucrose for 19 h, desiccated to 22 percent moisture content (fresh weight basis), and immersed in LN. Vitrification thus appears more suitable than encapsulation-dehydration for cryopreservation of T. moroderi shoot tips.

Thymol, carvacrol, and antioxidant accumulation in Thymus species in response to different light spectra emitted by light-emitting diodes.

The essential oils and antioxidant activity of four Thymus species were evaluated under five light spectra (namely, red, blue, red-blue, white, and greenhouse condition). The highest essential oil yield (4.17%) was observed under red light in T. migricus, while the lowest (1.05%) was observed in T. carmanicus under greenhouse conditions. Light quality also led to difference in essential oil constituents. The highest thymol (66%) was found in T. migricus exposed to blue light, while the least (1.69%) was observed in T. kotschyanus grown under red-blue light. The LED treatments did not induce any significant effect on carvacrol of Thymus species in comparison to the greenhouse condition. Finally, the analysis of variance indicates that the effect of light varied with the studied species. T. migricus performed the highest antioxidant capacity (IC50 = 176.8 µg/mL) under blue light. Overall, essential oil components as well as antioxidants showed significant responses to light emitting diodes wavelengths.

Native bacteria promote plant growth under drought stress condition without impacting the rhizomicrobiome.

Inoculation of plants with beneficial plant growth-promoting bacteria (PGPB) emerges a valuable strategy for ecosystem recovery. However, drought conditions might compromise plant-microbe interactions especially in semiarid regions. This study highlights the effect of native PGPB after 1 year inoculation on autochthonous shrubs growth and rhizosphere microbial community composition and activity under drought stress conditions. We inoculated three plant species of semiarid Mediterranean zones, Thymus vulgaris, Santolina chamaecyparissus and Lavandula dentata with a Bacillus thuringiensis strain IAM 12077 and evaluated the impact on plant biomass, plant nutrient contents, arbuscular mycorrhiza fungi (AMF) colonization, soil rhizosphere microbial activity and both the bacterial and fungal communities. Inoculation with strain IAM 12077 improved the ability of all three plants species to uptake nutrients from the soil, promoted L. dentata shoot growth (>65.8%), and doubled the AMF root colonization of S. chamaecyparissus. Inoculation did not change the rhizosphere microbial community. Moreover, changes in rhizosphere microbial activity were mainly plant species-specific and strongly associated with plant nutrients. In conclusion, the strain IAM 12077 induced positive effects on plant growth and nutrient acquisition with no impact on the rhizosphere microbiome, indicating a rhizosphere microbial community resilient to native bacteria inoculation.

Is the plant-associated microbiota of Thymus spp. adapted to plant essential oil?

We examined whether the microbiota of two related aromatic thyme species, Thymus vulgaris and Thymus citriodorus, differs in relation to the composition of the respective essential oil (EO). A total of 576 bacterial isolates were obtained from three districts (leaves, roots and rhizospheric soil). They were taxonomically characterized and inspected for tolerance to the EO from the two thyme species. A district-related taxonomic pattern was found. In particular, high taxonomic diversity among the isolates from leaves was detected. Moreover, data obtained revealed a differential pattern of resistance of the isolates to EOs extracted from T. vulgaris and T. citriodorus, which was interpreted in terms of differing chemical composition of the EO of their respective host plants. In conclusion, we suggest that bacterial colonization of leaves in Thymus spp. is influenced by the EO present in leaf glandular tissue as one of the selective forces shaping endophytic community composition.

Effects of meteorological conditions and plant growth stage on the accumulation of carvacrol and its precursors in Thymus pulegioides.

The effects of meteorological conditions (temperature, rainfall, photosynthetically active solar radiation (PAR) and sunshine duration) and plant growth stages on the quantitative composition of a secondary metabolite - essential oil and its main compounds, in the carvacrol chemotype of Thymus pulegioides L. (Lamiaceae) cultivated in open ground were studied under the same micro-edaphoclimatic environmental conditions for six years. The essential oil was isolated by hydrodistillation, the analysis of monoterpenic phenol carvacrol and the biogenetic precursors (monoterpene hydrocarbons p-cymene and γ-terpinene) were carried out annually using GC-FID and GC-MS. In the carvacrol chemotype investigated in this study, the yield of essential oil varied from 0.72% to 0.98% (CV = 12%) at full flowering stage. Regression analysis showed a significant negative relationship between the amount of essential oil and both temperature and rainfall during T. pulegioides flowering (July) and the period from April (beginning of vegetation) to July, but a strong positive relationship with photosynthetically active solar radiation during April-July (beta = 0.658, p < 0.05). The percentage of carvacrol, p-cymene and γ-terpinene ranged between 16.88 and 29.29% (CV = 18%), 5.54-11.33% (CV = 23%) and 20.60-24.43% (CV = 6%) respectively. Regression analysis showed the significant positive relationship between the percentage of carvacrol and sunshine duration at the flowering stage (in July) (beta = 0.699, p < 0.05); while the negative relationship was established between the percentages of precursors of carvacrol and photosynthetically active solar radiation and sunshine duration. The accumulation of p-cymene, the percentage of which varied most strongly from all investigated chemical compounds, showed significant positive relationships with temperature and rainfall during the period April-July and temperature in July (beta = 0.617, beta = 0.439 and beta = 0.429 respectively, p < 0.05). The analysis of plant growth stages showed that the highest amount of essential oil in carvacrol chemotype of T. pulegioides accumulated at ripening/seed maturity stage. The percentage of carvacrol increased gradually from the stem elongation to the post-flowering/seed maturity stage, overtaking the full flowering stage by 22%. The highest carvacrol content in the T. pulegioides carvacrol chemotype coincided with the highest oil yield during the fruiting stage. The amount of essential oil was also measured at the second full flowering stage, because after cutting the aerial part of the plant at the full flowering stage T. pulegioides often bloom for a second time: the carvacrol chemotype accumulated by 21% lower yield of essential oil at this stage compared to the first flowering, however, only traces of carvacrol and its precursors were found at the second full flowering stage.

Plants belonging to the genus Thymus as antibacterial agents: from farm to pharmacy.

In traditional medicine, plants have been used since ancient times for the prevention and/or protection against infectious diseases. In recent years, the use of herbal medicines and food supplements containing botanical ingredients, as alternative therapy for infectious diseases, has been intensified due to their high content of antimicrobial agents such as polyphenols, i.e. flavonoids, tannins, and alkaloids. Plants from the genus Thymus are important medicinal herbs, which are known to contain antimicrobial agents, and are rich in different active substances such as thymol, carvacrol, p-cymene and terpinene. In this review, we summarise the available literature data about the in vitro antibacterial effects of the main plants belonging to the genus Thymus. We also provide information about cultivation, chemical composition of the essential oils obtained from these plants, and their use for medicinal purposes.

Influence of nitrogen fertilizers on the yield and composition of thyme (Thymus vulgaris).

The influence of nitrogen fertilizers on the yield of crop, as well as on the production and composition of the essential oil and some other chemical characteristics of thyme, was investigated. Different levels of fertilizers (N = 0, 45, 90, and 135 kg x ha(-)(1)) were applied. It was found that fertilizers increase thyme crop, but differences in the yield of essential oil were not remarkable. However, the use of certain amounts of nitrogen fertilizers resulted in higher yields of essential oil obtainable from the cultivation area unit (dm(3) ha(-)(1)). Totally, 61 constituents were identified in thyme essential oil by capillary GC and GC-MS. Thymol was the dominating compound in the all analyzed oils (44.4-58.1%), followed by p-cymene (9.1-18.5%), gamma-terpinene (6.9-18.9%), and carvacrol (2.4-4.2%). Differences in the percentage of these and other compounds in thyme herb cultivated under different fertilization doses were not significant; very slight changes in the percentage composition were detected after drying. Some variations in the amount of individual constituents expressed in arbitrary units per kilogram of herb (which is almost equivalent to mg x kg(-)(1)) were observed. The highest amounts of sugars and sucrose, in particular, were determined in the second year of thyme cultivation. Differences in the content of dry soluble substances were not meaningful, and there was no effect of nitrogen fertilizers on this chemical characteristic. Some effect of fertilization on the content of vitamin C and carotenes was observed in the first year of thyme cultivation. It was determined that nitrogen fertilizers influence the amount of nitrates, which was highest in the second-year-first-harvest.

Watering level effect on Thymus hyemalis Lange essential oil yield and composition.

Thymus hyemalis Lange (chemotype thymol) was cultivated as an experimental crop under different watering level conditions in order to achieve 80, 60, 40, and 20% of the local potential evapotranspiration (Eto). Two harvesting periods were considered, winter and spring. As a consequence of the great variability among plants, essential oil yield percentages did not show statistically significant differences among seasons and different levels of water supply. Capillary GC-MS analysis of the essential oils permitted the detection of 84 volatile components. Among them, 54 are described for the first time as volatile constituents of the chromatographic profile of this Thymus species. Winter harvesting showed high concentrations in thymol percentage (25.92 +/- 4.39), the 40% Eto watering level being the best with respect to obtaining the optimum quality of this essential oil. However, in spring this thyme species needs a greater water supply (80% Eto) to achieve the same amount of thymol in the essential oil (29.20 +/- 2.83). From this, it was concluded that winter harvesting could be used for the extraction of the essential oil, with a low level of water supply, whereas spring harvesting could be employed for collection of leaves as a food condiment.

Chemical composition and antibacterial activity of essential oils from Thymus spinulosus Ten. (Lamiaceae).

The chemical composition of essential oils from aerial parts of Thymus spinulosus Ten. (Lamiaceae) is reported. Four oils from plants growing in different environmental conditions were characterized by GC and GC-MS methods; the oils seem to indicate a new chemotype in the genus Thymus. Influences of soil and altitude characteristics on the essential oil composition are discussed. The oils showed antibacterial activity against Gram-positive (Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, and Bacillus cereus) and Gram-negative (Proteus mirabilis, Escherichia coli, Salmonella typhimuium Ty2, and Pseudomonas aeruginosa) bacteria.

GC/MS evaluation of thyme (Thymus vulgaris L.) oil composition and variations during the vegetative cycle.

Capillary GC/MS analysis based on polar and non-polar columns has been applied to evaluation of the volatile oils hydrodistilled from thyme (Thymus vulgaris L.) plants. The adopted methodology has been used to monitor seasonal variations in the composition of the oil obtained from thyme herbs harvested at different periods during the plant vegetative and life cycles. Oils from thyme plants of young (2 years) and old (5 years) cultivations have been evaluated from four and two collections, respectively, effected throughout May/December growth period. Generally, the oil was found to be rich in the active monoterpene phenols (thymol and carvacrol) and their corresponding monoterpene hydrocarbon (HC) precursors (p-cymene and gamma-terpinene), which collectively showed synchronized patterns of variation during the different collection periods and in different seasons. The oil from old plant collected in May/June period (0.15% v/w) was characterized by significantly lower levels of monoterpene HCs (mainly gamma-terpinene) and the highest levels of the oxygenated monoterpenes (linalool and borneol), monoterpene phenols (mainly thymol) and their derivatives (mainly carvacrol methyl ether), sesquiterpenes (mainly beta-caryophyllene) and their oxygenated derivatives (e.g. caryophyllene oxide) in comparison with all other samples. A characteristic presence of camphor and thymodihydroquinone was also observed in the old plant oils. On the other hand, the young plant, collected in June/July just before the end of the vegetative cycle, provided the best oil yield (1.2%) with also the highest % content of the monoterpene phenols (thymol: 51.2% and carvacrol: 4%). This latter growth period can represent the best harvest time of young thyme plants in order to obtain an essential oil with better quality and quantity.

Comparison of antibacterial activity of natural and hydroformylated essential oil of Thymus capitatus growing wild in north Sardinia with commercial Thymus essential oils.

Thymus capitatus growing wild in Sardinia showed different essential oil composition if grown surrounding Cagliari than in north Sardinia. Here we verify the composition and antimicrobial activity of the oil to make it suitable for the cosmetic and confectionery industries. With the aim of improving the scent and the antimicrobical activity of T. capitatus essential oil, a hydroformylation reaction was carried out to transform the unsaturated components of the oil into the corresponding aldehydes. The essential oil of T. capitatus exhibited a significant antibacterial activity (MIC 0.125-0.5 mg/mL), and was also found effective on C. albicans (MIC 0.125 mg/mL). After hydroformylation, several new irregular terpenoid aldehydes were detected. The perfume of the new terpenic-like aldehydes is very agreeable and, therefore, the acceptability of the aroma is remarkably improved, but the antimicrobial activity was not increased.

Influence of growth regulators in biomass production and volatile profile of in vitro plantlets of Thymus vulgaris L.

In vitro shoots of thyme (Thymus vulgaris L.) were established, and the effects of the auxin indole-3-acetic (IAA) acid and the cytokinins benzyladenine (BA), zeatin (ZEA), and kinetin (KIN) at 1.0, 5.0, and 10.0 microM on rooting, biomass production, and volatile compounds production by these plants were investigated. The volatiles were extracted by solid phase microextraction (SPME) and analyzed by gas chromatography. The highest biomass shoot growth was obtained with BA at 5.0 microM, while IAA at all concentrations tested achieved 100% rooting frequency. The three major compounds were gamma-terpinene (22.8-38.8%), p-cymene (13.8-27.9%), and thymol (6.5-29.0%). Quantitative changes of these compounds were observed in response to the effect of varying growth regulators concentrations in the culture medium. Growing Thymus vulgaris L. plants in media supplemented with IAA at 1.0 microM increased volatile compounds such as thymol by 315%. Nevertheless, the same major compounds were produced in all treatments and no qualitative changes were observed in the volatile profile of thyme plants.

Portuguese Thymbra and Thymus species volatiles: chemical composition and biological activities.

Thymbra capitata and Thymus species are commonly known in Portugal as thyme and they are currently used as culinary herbs, as well as for ornamental, aromatizing and traditional medicinal purposes. The present work reports on the state of the art on the information available on the taxonomy, ethnobotany, cell and molecular biology of the Portuguese representatives of these genera and on the chemotaxonomy and antibacterial, antifungal and antioxidant activities of their essential oils and other volatile-containing extracts.