Change in Secondary Metabolites and Expression Pattern of Key Rosmarinic Acid Related Genes in Iranian Lemon Balm (Melissa officinalis L.) Ecotypes Using Methyl Jasmonate Treatments.

The medicinal herb, lemon balm (Melissa officinalis L.), which is high in rosmarinic acid (RA), has well-known therapeutic value. The goals of this study were to investigate the effects of methyl jasmonate (MeJA) on RA content, total phenolic content (TPC), and total flavonoid content (TFC), as well as changes in expression of their biosynthesis-related key genes (MoPAL, Mo4CL, and MoRAS) in Iranian lemon balm ecotypes, as first reported. Our results revealed that MeJA doses significantly increase the RA content, TPC, and TFC in both ecotypes compared with the control samples. Additionally, the higher expression levels of MoPAL, Mo4CL, and MoRAS following treatment were linked to RA accumulation in all treatments for both Iranian lemon balm ecotypes. After 24 h of exposure to 150 µM MeJA concentration, HPLC analysis showed that MeJA significantly increased RA content in Esfahan and Ilam ecotypes, which was about 4.18- and 7.43-fold higher than untreated plants. Our findings suggested that MeJA has a considerable influence on RA, TPC, and TFC accumulation in MeJA-treated Iranian M. officinalis, which might be the result of gene activation from the phenylpropanoid pathway. As a result of our findings, we now have a better understanding of the molecular processes behind RA production in lemon balm plants.

Transcriptome analysis to identify key genes involved in terpenoid and rosmarinic acid biosynthesis in lemon balm (Melissa officinalis).

Melissa officinalis (lemon balm) is a well-known pharmaceutical plant in traditional medicine around the world because of the high-value secondary metabolites. Nowadays, advances in computational biology and bioinformatics have opened new avenues to plant-based natural product drug discovery. Despite the pharmacological importance, there is low information about the genes encoding the important biosynthetic pathways related to the secondary metabolite in M. officinalis. In this study, the main genes related to the rosmarinic acid (RA) and terpenoid biosynthesis pathways were detected using transcriptome analysis. Furthermore, we isolated and characterized a novel M. officinalis Hydroxyphenylpyruvate reductase (HPPR) gene involved in RA biosynthesis pathway. An effective pipeline was used to generate 37,055 unigenes by evaluating 42,837,601 Illumina paired-end reads. Functional annotation of the unigenes revealed that 27,363 (73.84%) and 35,822 (96.67%) unigenes had significant similarity to identified proteins in the SwissProt and NR databases, respectively. Also, 10,062 (36.83%) out of 37,055 unigenes were assigned to 399 KEGG pathways. Since terpenes and RA are two prominent metabolites in this plant, the attention of this study has been on the pathways related to them. A total of 149 unigenes were found that are related to the terpenoids biosynthesis, including 75 unigenes involved in the methyl-erythritol phosphate and mevalonate pathway, terpenoid backbone biosynthesis genes, and 74 unigenes related to the terpene synthase. We also identified 144 and 30 unigenes that were associated with the biosynthesis of phenylpropanoid and the rosmarinic acid pathway. Consequently, this investigation can be a comprehensive and accurate transcriptome basis for further investigation in the metabolic engineering and detection of new genes and pathways in M. officinalis.

Eliciting effect of foliar application of chitosan lactate on the phytochemical properties of Ocimum basilicum L. and Melissa officinalis L.

An increase in the content of secondary metabolites in herbal plants is desirable due to their therapeutic and nutraceutical properties. Therefore, the effects of foliar spray of 100 mg/L or 500 mg/L of chitosan lactate (ChL) on the accumulation of selected phenolics and physiological parameters of basil and lemon balm were investigated. In basil, the concentration of rosmarinic acid (RA) increased after application of 100 mg/L of ChL. In turn, in lemon balm both ChL concentrations increased the accumulation of RA and anthocyanins, while the level of total phenolic compounds (TPC) was elevated only at the dose of 100 mg/L of ChL. Elicitation of basil with 500 mg/L of ChL increased the shoot biomass. Therefore, such an elicitor as ChL can enhance the accumulation of valuable phytochemicals in Lamiaceae species. This simple and non-laborious method can be used for elicitation of herbal plants in production of functional food.

Combined cadmium-zinc interactions alter manganese, lead, copper uptake by Melissa officinalis.

Farmland soil typical for the Polish rural environment was used in pot experiment to estimate the impact of cadmium and zinc on the manganese, lead and copper uptake by lemon balm (Melissa officinalis L). Bioavailable and total forms of investigated metals in soil and metal concentrations in plants were determined by atomic absorption spectrometry. The plant photosynthesis indicators were also examined. Intensification of photosynthesis upon the high zinc and cadmium soil supplementation was observed. This effect was not detected at low metal concentrations. ANOVA proved that cadmium and zinc treatments influenced manganese, lead and copper transfer from soil and their concentration in plants. Zinc uptake and accumulation in either roots or above-ground parts in plant was inversely proportional to cadmium concentration in soil. Manganese concentration in roots decreased upon the soil supplementation with either zinc or cadmium. It suggests that the latter ions are transported via symplastic pathways and compete with manganese for similar transporters. The opposite situation was observed for lead and copper. Soil supplementation with cadmium and zinc affects manganese, lead and copper concentrations and photosynthesis intensity in lemon balm plant. The following combined interactions in either normal or stress conditions are important indicators of the migration pathways.

Modulation of secondary metabolite profiles by biologically synthesized MgO/perlite nanocomposites in Melissa officinalis plant organ cultures.

In this work, biological synthesis of MgO/perlite nanocomposites (NCs) besides their effects on morphology and secondary metabolite profiles of Melissa officinalis plant organ cultures were evaluated. MgO NPs were immobilized on the surface of nanoperlite using M. officinalis extract as a capping agent. The as-synthesized MgO/perlite NCs were characterized by using FTIR, XRD, SEM, EDS and DLS. The average particle size of nanoperlite and MgO/perlite NCs was about 10 and 30 nm, respectively. Morphological observations showed that nanoperlite and MgO/perlite NCs had no effect on root number, as well as root and shoot length. None of the applied concentrations of perlite and MgO/perlite NCs could significantly increase the growth parameters in comparison to the control, except for 150 mg/L of nanoperlite which caused an increase in the shoot number. Although, the contents of chlorophyll and carotenoids were not affected, the maximum content of volatile compounds obtained at 100 of MgO/perlite NCs. Rosmarinic acid was detected in shoots, which treated with 25-100 mg/L of perlite and 25, 50 and 150 mg/L of MgO/perlite NCs. Our results provided the evidence that nanoperlite and MgO/perlite NCs at specific levels may act as a novel elicitor for in vitro biosynthesis of valuable secondary metabolites.

Associated Effects of Cadmium and Copper Alter the Heavy Metals Uptake by Melissa Officinalis.

Lemon balm (Melissa officinalis) is a popular herb widely used in medicine. It is often cultivated in soils with substantial heavy metal content. Here we investigate the associated effects of cadmium and copper on the plant growth parameters augmented by the manganese, zinc, and lead uptake indicators. The concentration of all elements in soil and plants was determined by the HR-CS FAAS with the ContrAA 300 Analytik Jena spectrometer. Bioavailable and total forms calculated for all examined metals were augmented by the soil analyses. The index of chlorophyll content in leaves, the activity of net photosynthesis, stomatal conductance, transpiration rate, and intercellular concentration of CO2 were also investigated. Either Cd or Cu acting alone at high concentrations in soil are toxic to plants as indicated by chlorophyll indices and gas exchange parameters. Surprisingly, this effect was not observed when both metals were administered together. The sole cadmium or copper supplementations hampered the plant's growth, lowered the leaf area, and altered the plant's stem elongation. Analysis of variance showed that cadmium and copper treatments of lemon balm significantly influenced manganese, lead, and zinc concentration in roots and above ground parts.

Can Ozone Alter the Terpenoid Composition and Membrane Integrity of in vitro Melissa officinalis Shoots?

Ozone affects volatile organic compounds that protect plants from biotic and abiotic stress. In vitro Melissa officinalis shoots were exposed to ozone (200 ppb, 3 h) in controlled environmental conditions: leaf pigments, membrane integrity and headspace composition were assayed during fumigation and after the recovery period (3 h from the beginning of the exposure, FBE). At the end of the exposure, no injury was observed in untreated and treated shoots, although an evident increase in lipid peroxidation was reported (+38.5 and +37.2% of TBARS levels in comparison with controls, respectively after 1 and 3 h FBE). The levels of total carotenoids significantly rose as a normal response mechanism to oxidative stress. SPME-GS-MS analysis showed that, as a consequence of the fumigation, the trends in non-terpenoid compounds increased after 1 and 3 h FBE. This suggests that the concentration and the duration of the treatment were enough to cause a breakdown of cells (as evidenced by increased TBARS levels) and involves an association between volatile products of the lipoxygenase pathway (LOX products) and membrane degradation.

Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L. infusions.

Melissa officinalis L. (lemon balm) is normally consumed as an infusion and presents therapeutic properties, such as sedative, carminative and antispasmodic, also being included in some pharmaceutical preparations. The phenolic profiles of different samples of lemon balm, prepared as infusions, were evaluated by HPLC-DAD-ESI/MS. The profiles were compared in order to understand the differences between cultivated, in vitro cultured and commercial (bags and granulated) samples. All the samples showed a similar phenolic profile, presenting differences only in the quantities found of each compound. Rosmarinic acid was the most abundant compound, being higher in commercial samples, especially in tea bag sample (55.68mg/g of infusion) and lower in in vitro cultured sample (15.46mg/g). Moreover, dimers, trimers and tetramers of caffeic acid were identified and quantified for the first time in lemon balm. Only one flavonoid, luteolin-3'-O-glucuronide was found in all the samples, ranging from 8.43mg/g in commercial granulate sample to 1.22mg/g in in vitro cultured sample. Overall, cultivated and in vitro cultured samples presented the lowest amounts of phenolic compounds (59.59 and 30.21mg/g, respectively); otherwise, commercial samples showed the highest contents (109.24mg/g for tea bag and 101.03mg/g for granulate sample). The present study shows that infusion of lemon balm can be a source of phenolic compounds, known for their bioactive effects.

Influência do horário de colheita sobre o rendimento e composição do óleo essencial de erva-cidreira brasileira [Lippia alba (Mill. ) N. E. Br. ] / Influence of harvest time on the yield and composition of essential oil from the Brazilian "erva-cidreira" [Lippia alba (Mill. ) N. E. Br. ]

Este trabalho teve como objetivo verificar se o horário de colheita da erva-cidreira brasileira [Lippia alba (Mill.) N. E. Br.], fenotipo carvona-limoneno, tem influência sobre a produção de massa foliar, rendimento e composição do óleo essencial. Foram avaliados cinco horários de colheita quando a cultura estava com 145 dias desde o transplante: 8:00, 10:00, 12:00, 14:00 e 16:00 h com cinco repetições, distribuídos em blocos casualizados. O experimento foi conduzido na Fazenda Experimental Lageado da FCA-UNESP/Botucatu no Setor de Horticultura do Departamento de Produção Vegetal. A colheita foi realizada a 15 cm da superfície do solo e o óleo essencial obtido através de hidrodestilação, em aparelho tipo Clevenger. Levou-se em consideração os fatores agronômicos e o rendimento obtido por hidrodestilação. O óleo essencial foi analisado em cromatógrafo gasoso acoplado a espectrômetro de massas (CG/EM). Os resultados foram submetidos à análise de variância (Teste F) e as médias comparadas pelo teste de Tukey. Não houve diferença estatística para produção de massa foliar, teor de óleo essencial e produtividade de óleo essencial em massa fresca e seca. No entanto, entre os compostos majoritários do óleo essencial das folhas, carvona e limoneno, a melhor produtividade de carvona foi obtida às 10:00 h, em matéria fresca (2,050 L ha-1) e em matéria seca (2,068 L ha-1), e para o limoneno às 16:00 h, em matéria fresca (1,068 L ha-1) e em matéria seca (1,060 L ha-1).

This study aimed to verify whether the harvest time of the "Brazilian erva-cidreira" [Lippia alba (Mill.) N. E. Br.], limonene-carvone chemotype, influences leaf mass production and essential oil yield and composition. Five different harvest times were evaluated at 145 days after transplanting: 8:00, 10:00, 12:00, 14:00 and 16:00, with five replicates, distributed in randomized blocks. The experiment was carried out at Lageado Experimental Farm of the School of Agronomical Sciences FCA-UNESP/Botucatu, Horticulture Sector, Department of Plant Production. Harvest was conducted from 15 cm above the soil surface and essential oil was obtained through steam distillation in a Clevenger-type apparatus. Both the agronomical factors and the yield obtained through steam distillation were considered. The essential oil was analyzed in a gas chromatographer coupled to a mass spectrometer (GC/MS). Results were subjected to analysis of variance (F Test) and means were compared according to Tukey's test. There was no statistical difference for leaf mass production, essential oil content and essential oil yield in fresh and dry matter. However, considering the major essential oil compounds, carvone and limonene, the best yield was obtained at 10:00 for carvone in fresh (2050 L ha-1) and dry matter (2068 L ha-1), and at 16:00 for limonene in fresh (1068 L ha-1) and dry matter (1060 L ha-1).

Global profiling of ultraviolet-induced metabolic disruption in Melissa officinalis by using gas chromatography-mass spectrometry.

Melissa officinalis contains various secondary metabolites that have health benefits. Generally, irradiating plants with ultraviolet (UV)-B induces the accumulation of secondary metabolites in plants. To understand the effect of UV-B irradiation on the metabolism of M. officinalis, metabolomics based on gas chromatography-mass spectrometry (GC-MS) was used in this study. The GC-MS analysis revealed 37 identified metabolites from various chemical classes, including alcohols, amino acids, inorganic acids, organic acids, and sugars. The metabolite profiles of the groups of M. officinalis irradiated with UV-B were separated and differentiated according to their irradiation times (i.e., 0, 1, and 2 h), using principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The PCA score plots of PC1 and PC2 showed that the three groups with different irradiation times followed a certain trajectory with increasing UV-B irradiation. HCA revealed that metabolic patterns differed among the three groups, and the 1 h-irradiated group was more similar to the control group (0 h) than the 2 h-irradiated group. In particular, UV-B irradiation of plants led to a decrease in sugars such as fructose, galactose, sucrose, and trehalose and an increase in metabolites in the tricarboxylic acid cycle, the proline-linked pentose phosphate pathway, and the phenylpropanoid pathway. This study demonstrated that metabolite profiling with GC-MS is useful for gaining a holistic understanding of UV-induced changes in plant metabolism.

Crescimento, produção de fitomassa e teor de óleo essencial de melissa (Melissa officinalis L. ) sob diferentes níveis de sombreamento / Growth, biomass production and essential oil content of lemon balm (Melissa officinalis L. ) under different shading levels

O objetivo foi avaliar a variação no teor de óleo essencial, crescimento e produção de fitomassa de Melissa officinalis L. cultivada sob diferentes níveis de sombreamento. O delineamento experimental foi inteiramente ao acaso (DIC) com quatro níveis de radiação fotossinteticamente ativa (RFA), luz plena (testemunha) e sombreamento progressivo com tela tipo sombrite (sombreamento de 75, 50 e 25%) e seis repetições. Foram avaliados: diâmetro do caule, altura da planta, massa fresca e seca da parte aérea, teor do óleo essencial, rendimento de óleo e área foliar. Os dados foram submetidos à análise de variância e análise de regressão a 1 e 5% de probabilidade de erro. As características, diâmetro do caule e altura da planta, não apresentaram diferenças significativas entre os tratamentos. A produção de massa fresca, massa seca e o teor de óleo essencial, foram similares entre todos os tratamentos diferenciando apenas do tratamento em pleno sol, onde (MF): 25%=50%, 91, 33 g e 68, 66 g; (MS): 25%=0%=50%, 11,35, 11,66, 10,66 g e 4,33 g e TO: 25%=50%, 0,48 e 0,40% respectivamente. O tratamento que resultou em menor produtividade foi o sombreado a 75%. Assim, nas condições norte-mineiras, de fevereiro a junho, o cultivo da melissa pode ser conduzido em condições de até 50% de sombreamento, favorecendo a produção de massa seca, massa fresca e teor de óleo essencial; sendo estas variáveis de interesse comercial pelos produtores de plantas medicinais e aromáticas.

The aim of this study was to evaluate the variation in essential oil content, growth and biomass production of Melissa officinalis L. cultivated under different shading levels. Experimental design was completely randomized (CRD) with four levels of photosynthetically active radiation (PAR), full light (control) and progressive shading with shade net (shading of 75, 50 and 25%) and six replicates. The following parameters were assessed: stem diameter, plant height, fresh mass and dry mass of shoot, essential oil content, essential oil yield and leaf area. Data were subjected to analysis of variance and regression analysis at 1 and 5% error probability. The characteristics stem diameter and plant height did not show significant differences among treatments. Production of fresh weight, dry mass and essential oil content was similar among all treatments, except for full light, where (FW): 25%=50%, 91.33 g and 68.66 g; (DW): 25%=0%=50%, 11.35, 11.66, 10.66 g and 4.33 g and OC: 25%=50%, 0.48 and 0.40% respectively. The treatment that resulted in the lowest productivity was 75% shading. Thus, in the north of Minas Gerais State, Brazil, from February to June, lemon balm can be cultivated under conditions of up to 50% shading, favoring the production of dry mass, fresh mass and essential oil content; these variables are of commercial interest for producers of medicinal and aromatic plants.

Potencial alelopático de extratos aquosos de Melissa officinalis L. e Mentha x villosa L. na germinação e vigor de sementes de Plantago major L. / Allelopathic effect of extracts of Melissa officinallis L. and Mentha x villosa L. on seed germination and vigor of Plantago major L.

O objetivo do trabalho foi avaliar o efeito dos extratos aquosos de hortelã e melissa na germinação e vigor de sementes de tanchagem. O experimento foi conduzido no Departamento de Fitotecnia da Universidade Federal de Viçosa, no mês de abril de 2011. O delineamento utilizado foi o inteiramente casualizado com cinco tratamentos (testemunha, 25 por cento, 50 por cento, 75 por cento e 100 por cento do extrato aquoso de hortelã e melissa) e quatro repetições. Os extratos foram preparados utilizando a parte aérea das plantas, na proporção de 100g de planta para 1 L de água destilada. Foram utilizadas 50 sementes de tanchagem em cada caixa gerbox, previamente umedecido com os extratos aquosos (correspondentes aos tratamentos), estas foram levadas para germinar em câmara de germinação, em temperatura de 20°C, fotoperíodo de 16 horas luz e 8 horas escuro, por 14 dias. Durante esse tempo foram feitas às contagens diárias das sementes germinadas para calcular o índice de velocidade de germinação e ao final dos 14 dias foi calculada a porcentagem de germinação. Sementes de tanchagem não submetidas aos extratos aquosos de melissa e hortelã apresentaram-se mais vigorosas quando comparado com as concentrações, evidenciando o potencial alelopático de melissa e hortelã sobre a sua qualidade fisiológica.

The aim of this work was to evaluate the effect of aqueous extracts of Melissa officinallis and Mentha x villosa for the germination and vigor of Plantago major seeds. The experiment was conducted at the Plant Science Department, Federal University of Viçosa, in April 2011. The design was a completely randomized design with five treatments (control, 25 percent, 50 percent, 75 percent and 100 percent of the aqueous extract of Mentha x villosa and Melissa Officinallis) and four replications. The extracts were prepared utulizando the shoots of plants at the rate of 100g of plant to 1 L of distilled water. We used 50 Plantago major seeds in each box incubator and these were subsequently placed in a germination chamber. 14 days during the counting of germinated seeds per day to calculate the index of germination speed and the end of 14 days was the germination percentage. Plantago major seeds not subjected to aqueous extracts of lemon balm and mint showed higher values in the variables studied when compared to other concentrations, showing the allelopathic potencial of Melissa officinallis and Mentha x villosa on the development of plantain, determined by germinating.

Enzymes of phenylpropanoid metabolism in the important medicinal plant Melissa officinalis L.

Lemon balm (Melissa officinalis, Lamiaceae) is a well-known medicinal plant. Amongst the biologically active ingredients are a number of phenolic compounds, the most prominent of which is rosmarinic acid. To obtain better knowledge of the biosynthesis of these phenolic compounds, two enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL) and 4-coumarate:coenzyme A-ligase (4CL), were investigated in suspension cultures of lemon balm. MoPAL1 and Mo4CL1 cDNAs were cloned and heterologously expressed in Escherichia coli and the enzymes characterised. Expression analysis of both genes showed a correlation with the enzyme activities and rosmarinic acid content during a cultivation period of the suspension culture. Southern-blot analysis suggested the presence of most probably two gene copies in the M. officinalis genome of both PAL and 4CL. The genomic DNA sequences of MoPAL1 and Mo4CL1 were amplified and sequenced. MoPAL1 contains one phase 2 intron of 836 bp at a conserved site, whilst Mo4CL1 was devoid of introns.

Are saturating pulses indeed saturating? Evidence for considerable PSII yield underestimation in leaves adapted to high levels of natural light.

The "saturating pulse" method of in vivo Chl fluorescence measurement has been widely used by physiologists and especially ecophysiologists, as it allows a simple, rapid and non-invasive assessment of PSII function and the allocation of absorbed energy into photochemical and non-photochemical processes. It is based on the accurate determination of the so-called Fm('), i.e. the fluorescence signal emitted when a "saturating" light pulse closes all PSII centers. In this methodological investigation, we examined whether the saturating pulse intensities required to obtain maximal fluorescence yields differ between leaves of various species receiving varying actinic light irradiances. It was shown that, in leaves adapted to comparatively high (yet realistic) levels of natural irradiances, the saturating pulses usually applied are not able to close all PSII reaction centers. As a result, there is a high risk of considerable Fm(') underestimation. Accordingly, the derived values of effective PSII yields and linear electron transport rates (ETR) are also underestimated, even at the highest saturation pulse levels afforded by commercial instruments. Since the extent of underestimation increases with actinic irradiance, the ETR versus light curves are considerably distorted. The possible reasons for the apparent inability of "saturating" pulses to close all PSII centers at high actinic light and the practical implications, especially in field work, are discussed.

Selective extraction of derivates of p-hydroxy-benzoic acid from plant material by using a molecularly imprinted polymer.

Selective SPE of derivates of p-hydroxybenzoic acid (pHBA) from plant extract of Melissa officinalis is presented using a molecularly imprinted polymer (MIP) made with protocatechuic acid (PA) as template molecule. MIP was prepared with acrylamide as functional monomer, ethylene glycol dimethacrylate as crosslinking monomer and ACN as porogen. MIP was evaluated towards six phenolic acids: PA, gallic acid, pHBA, vanillic acid (VA), gentisic acid (GeA) and syringic acid (SyrA), and then steps of molecularly imprinted SPE (MISPE) procedure were optimized. The best specific binding capacity of MIP was obtained for PA in ACN (34.7 microg/g of MIP). Other tested acids were also bound on MIP if they were dissolved in this solvent. ACN was chosen as solvent for sample application. M. officinalis was extracted into methanol/water (4:1, v/v), the extract was then evaporated to dryness and dissolved in ACN before application on MIP. Water and ACN were used as washing solvents and elution of benzoic acids was performed by means of a mixture methanol/acetic acid (9:1, v/v). pHBA, GA, PA and VA were extracted with recoveries of 56.3-82.1% using this MISPE method. GeA was not determined in plant extract.