Flavonoid Compounds Are Enriched in Lemon Balm (Melissa officinalis) Leaves by a High Level of Sucrose and Confer Increased Antioxidant Activity

in HortScience
View More View Less
  • 1 College of Life Sciences and Biotechnology, Korea University, Anam-Dong, Sungbuk-ku, Seoul 136-713, Republic of South Korea

Medicinal plants are widely used in traditional medicine because plant secondary metabolites have been shown to benefit a broad spectrum of health conditions. Lemon balm, Melissa officinalis L., a member of the mint family, is native to Europe and is well known for its ability to reduce stress and anxiety, promote sleep, and ease pain and discomfort associated with digestion. In various plant species, strong anthocyanin induction is triggered by sucrose, but not by other sugars or osmotic stress; however, the mechanisms that induce anthocyanin accumulation in lemon balm leaves in response to sucrose and phytohormones remain unclear. In this study, we investigated the mechanisms that lead to increased levels of flavonoids in lemon balm plants. We observed that sucrose significantly increases the level of flavonoids in lemon balm plants and that sucrose induction appears to be mediated by the phytohormones abscisic acid and ethylene. We also identified delphinidin as the anthocyanidin that is primarily enriched in leaves grown in high-sucrose medium. Finally, we observed that reactive oxygen species levels are positively correlated with sucrose-mediated anthocyanin accumulation. Taken together, our results demonstrate that the level of flavonoids in lemon balm can be increased significantly and that plants such as lemon balm could potentially be used to prevent diseases that have been purported to be caused by free radical damage. Chemical abbreviations used: ABA, (+)-cis, transabscissic acid; ACC, 1-aminocyclopropane-carboxylic acid; CHI, chalcone isomerase; CHS, chalcone synthase; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; GA, gibberellic acid; IAA, indole-3-acetic acid.

Abstract

Medicinal plants are widely used in traditional medicine because plant secondary metabolites have been shown to benefit a broad spectrum of health conditions. Lemon balm, Melissa officinalis L., a member of the mint family, is native to Europe and is well known for its ability to reduce stress and anxiety, promote sleep, and ease pain and discomfort associated with digestion. In various plant species, strong anthocyanin induction is triggered by sucrose, but not by other sugars or osmotic stress; however, the mechanisms that induce anthocyanin accumulation in lemon balm leaves in response to sucrose and phytohormones remain unclear. In this study, we investigated the mechanisms that lead to increased levels of flavonoids in lemon balm plants. We observed that sucrose significantly increases the level of flavonoids in lemon balm plants and that sucrose induction appears to be mediated by the phytohormones abscisic acid and ethylene. We also identified delphinidin as the anthocyanidin that is primarily enriched in leaves grown in high-sucrose medium. Finally, we observed that reactive oxygen species levels are positively correlated with sucrose-mediated anthocyanin accumulation. Taken together, our results demonstrate that the level of flavonoids in lemon balm can be increased significantly and that plants such as lemon balm could potentially be used to prevent diseases that have been purported to be caused by free radical damage. Chemical abbreviations used: ABA, (+)-cis, transabscissic acid; ACC, 1-aminocyclopropane-carboxylic acid; CHI, chalcone isomerase; CHS, chalcone synthase; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; GA, gibberellic acid; IAA, indole-3-acetic acid.

More than 30,000 different secondary metabolites are produced exclusively by plants. One such class of compounds, the flavonoids (polyphenolic compounds), are classified according to their chemical structures into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins, and chalcones. These metabolites have a broad spectrum of action in plants; for example, they protect against pathogen attack, act as attractants for pollinators, and have been used as colorants, as scents, and for allelopathy. Flavonoids also contain chemical structural elements that are responsible for antioxidation, and their antioxidant activities have been well established biochemically.

Lemon balm, Melissa officinalis, is a member of the mint family that is native to Europe. Its use as a medicinal herb dates from the Middle Ages, and it is very well known for its ability to reduce stress and anxiety, promote sleep, improve appetite, and ease pain and discomfort associated with digestion. Moreover, several studies suggest that lemon balm is beneficial for a wide variety of human disorders such as cancer, HIV-1, Alzheimer's disease, attention deficit hyperactivity disorder, indigestion, gas, insomnia, and hyperthyroidism (de Sousa et al., 2004; Galasinski et al., 1996; Geuenich et al., 2008; Kennedy et al., 2004, 2006; Muller and Klement, 2006; Yamasaki et al., 1998).

Because plants are autotrophic organisms, they need to synthesize sugars for growth and storage. Sugars appear to act as hormone-like signaling molecules in plant cells because they can regulate plant metabolism, growth, and development (Rolland et al., 2002; Rook and Bevan, 2003; Smeekens, 2000). For example, in grape skins, sugars induce most of the genes involved in anthocyanin synthesis (Gollop et al., 2002). Recently, sucrose-specific induction of anthocyanin biosynthesis was also examined in Arabidopsis (Solfanelli et al., 2006; Teng et al., 2005), Torenia (Nagira et al., 2006), and Camptotheca (Pasqua et al., 2005). Exogenous growth regulators, especially abscisic acid (ABA), were also reported to enhance anthocyanin accumulation in Arabidopsis (Loreti et al., 2008) and Torenia (Nagira et al., 2006) when plants were supplied with sucrose. Moreover, several studies have shown that phytohormones are involved in the sucrose-regulated expression of genes encoding anthocyanin biosynthetic enzymes in Arabidopsis seedlings (Chen et al., 2007; Devoto et al., 2005; Loreti et al., 2008; Tonelli et al., 2007). However, the mechanisms of anthocyanin accumulation remain unclear in the leaves of lemon balm. Therefore, in this study, we intended to understand the role of sucrose and phytohormones leading to the increased level of anthocyanins in this plant.

Materials and Methods

Plant growth conditions and chemicals.

Explants from containerized lemon balm (Melissa officinalis L.; Lamiaceae) plants grown in sterile Murashige and Skoog (MS) medium (4 g·L−1 MS, 50 mm sucrose solidified with 7 g·L−1 agar at pH 5.7) were used for this study. Explants ≈2 cm in length, including the tip and one pair of leaves, were transferred to the culture/test media and incubated in a controlled environment with a 15-h light/9-h dark regime. To examine the effects of sucrose concentration on plant growth, anthocyanin accumulation, flavonoid biosynthesis, chlorophyll content, and antioxidant activity, explants were grown in MS agar medium for 20 d. To examine the effects of plant growth regulators on anthocyanin production, the explants were grown in MS agar medium containing 50 mm sucrose for 12 d and then were transferred to liquid MS medium containing 50 or 300 mm sucrose. This medium was supplemented with various concentrations of growth regulators. After transfer, the explants were incubated for a further 3 d before analysis.

All of the chemicals used in this study were purchased from Sigma Aldrich (St. Louis, MO) except for the ABA [(+)-cis, transabscissic acid], ACC, indole-3-acetic acid (IAA), fluridon, and silver nitrate, which were purchased from Duchefa Biochemie (Postbus, Haarlem, The Netherlands), and the methanol and ethanol, which were purchased from Merck (Darmstadt, Germany).

Quantification of anthocyanins and chlorophyll content.

Anthocyanins were extracted as described by Neff and Chory (1998) with minor modifications. Briefly, 100 mg of fresh leaf samples were ground in liquid nitrogen, added to 250 μL of 1% HCl (v/v) in methanol, and extracted in the dark at 4 °C overnight. Distilled water (250 μL) was added to each tube. Extracts were recovered, and the chlorophyll was removed by adding 500 μL chloroform, mixing the samples, and centrifuging at 3000 rpm for 2 min. The aqueous phase (200 μL) from each sample was then placed in a well of a 96-well plate and the absorbance was determined using a microplate spectrophotometer at 530 nm. The chlorophyll content was measured as described by Porra et al. (1989) with slight modifications. Briefly, two leaf discs (0.5 cm2 each) were weighed and placed into 1 mL of dimethylformamide (DMF) before extraction in the dark (at 4 °C) for 24 h. Absorbances were determined against a blank (1 mL DMF) at 647 nm and 664 nm using a spectrophotometer.

Liquid chromatography–mass spectrometry analysis.

Leaves of lemon balm were ground and homogenized in liquid nitrogen using a mortar and pestle. Sample preparation and extraction were carried out as described by Tolstikov et al. (2007). Methanol (1 mL) was added to 100 mg of the ground sample in a microcentrifuge tube and mixed by vortexing. Reserpine dissolved in methanol at 0.2 mg·ml−1 (50 μL) was added as an internal reference to the sample tube with 50 μΛ of water, and the tube was vortexed for 5 min. The sample was then centrifuged for 5 min at 14,000 g, and the supernatant was transferred to a glass sample vial (2 mL; Agilent, Wilmington, DE) with a screw cap lined with Teflon.

Sample analysis was conducted using a high-performance liquid chromatography (HPLC) Surveyor MS Pump Plus (ThermoFisher Scientific, San Jose, CA) coupled to an LTQ Orbitrap mass spectrometer (MS) with an electrospray ionization (ESI) source (ThermoFisher Scientific). The compounds were separated using a BetaBasic-18 HPLC column (150 × 2.1 mm i.d., 5 μm; ThermoFisher Scientific) with aqueous 0.2% (v/v) formic acid as solvent A and 1% (v/v) formic acid in methanol as solvent B. The solvent gradient was programmed in the following sequence: 0 min, 80% solvent A; 40 min, 20% solvent A; 45 min, 80% solvent A; and 60 min, 80% solvent A. The flow rate was 0.2 mL·min−1, and the sample injection volume was 20 μL. ESI conditions were as follows: spray voltage of 4.2 kV, capillary temperature of 200 °C, capillary voltage of 240, sheath gas flow rate of 20 arb with nitrogen, aux gas flow rate of 12 arb with nitrogen, and sweep gas flow rate of 10 arb with nitrogen. MS data were acquired over an m/z range of 230 to 1200. The resolution of the mass spectra was set to 60,000 to measure the masses of the compounds.

Data sets organized in matrix form were subsequently exported to SIMCA-P software (Version 11.5; Umetrics, Umea, Sweden) for principal component analysis (PCA). PCA is an unsupervised clustering method that is able to retain the maximum number of variations present in a data set consisting of a large number of interrelated variables while reducing the dimensionality of the data set. Therefore, it is possible to observe any groupings of the data set in a score plot. Coefficients multiplied with the original variables to obtain the principal components (PCs) are called “loadings,” and the numerical value of a loading for a given variable on a PC indicates the relationship of the variable with that component (Ma et al., 2007).

Determination of flavonoid content and free radical scavenging activity.

Samples for determining the flavonoid content and antioxidant activity of lemon balm leaves were prepared as described by Faudale et al. (2008) with slight modifications. Briefly, 100 mg of fresh leaf samples from each treatment were ground in liquid nitrogen and extracted with 10 mL of water/ethanol (80:20 v/v) three times in 20 min with sonication. After centrifuging at 7600 g for 10 min, the supernatant was adjusted to a final volume of 10 mL and was filtered with a 0.45-nm filter. Extracted leaf samples were quantified immediately after extraction to avoid possible degradation.

The flavonoid contents of lemon balm leaves grown in various concentrations of sucrose were determined using a colorimetric method as described by Faudale et al. (2008) with minor modifications. Briefly, 0.1 mL of phytochemical extract was diluted with 0.5 mL of distilled water. Thirty microliters of a 5% NaNO2 solution were added and the solution was thoroughly mixed by inverting. Six minutes later, 60 μL of a 10% AlCl3·6H2O solution was added and the mixture was left to stand for 5 min. Two hundred microliters of 1 M NaOH were added, and the total volume was brought up to 1 mL with distilled water. After mixing the solution thoroughly, the absorbance was measured immediately against a prepared blank at 510 nm using a microplate spectrophotometer. The flavonoid content was calculated using the standard equation Y = 0.006X + 0.003 (R2 = 0.999), which was derived from a standard curve generated with 2, 4, 10, and 20 μg·mL−1 quercetin.

The radical scavenging activities of lemon balm leaves grown in various concentrations of sucrose were determined using the stable radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (Brand-Williams et al., 1995) as described by Faudale et al. (2008). Briefly, each leaf extract was diluted to various concentrations ranging from 10 to 200 μL·mL−1 in 0.5 ml (methanol for control) and each sample was added to 1.0 mL of 20 mg·L−1 DPPH dissolved in methanol. After 20 min, the absorbance was measured at 517 nm with a microplate spectrophotometer. The percentage of DPPH in the sample was calculated according to the following equation: % decolorization = [1 – (Abs sample/Abs control)] × 100. Decoloration was plotted against the sample extract concentrations, and a logarithmic regression curve was established (using GraphPad Prism 5 software; GraphPad Software, Inc., La Jolla, CA) to calculate the IC50, which is the amount of sample necessary to decrease the absorbance of DPPH by 50%.

Reactive oxygen species (H2O2) staining.

Young leaves of lemon balm from each treatment were washed with 50 μM PBT (potassium phosphate) buffer in a 24-well plate on a rotary shaker for 10 min. The PBT was replaced with 3, 3′-diaminobenzidine (DAB) solution in PBT (0.3 mg·mL−1), and samples were incubated for 10 min at room temperature. For the color reaction, 1 μL of 30% H2O2 (1:1 in PBT) was added to each leaf, and reactions were shaken vigorously for 10 min. Then, the DAB was replaced with PBT to stop the reaction. The leaves were washed twice in PBT for 5 min and dehydrated in 100% methanol overnight for imaging (Fester and Hause, 2005). Stained leaves were stretched on water in six-well plates (Corning Inc., New York, NY), and images were captured with a digital still camera (Sony Corp., Tokyo, Japan).

Statistical analysis.

Each experiment was repeated at least three times to confirm reproducibility. All data were statistically analyzed using the SPSS program (Version 13.00; SPSS Inc., Chicago, IL). Analysis of variance and Duncan's multiple range test were performed to assess the possible significant differences among the treatments at the P ≤ 0.05 level.

Results

Sucrose dramatically increases anthocyanin levels in lemon balm leaves.

There is little information in the literature about optimal growth conditions for cultured lemon balm plants. Therefore, we used trial and error to determine that lemon balm grows optimally in an MS medium supplemented with at least 50 mm sucrose (data not shown). To examine whether sucrose increases anthocyanin levels in lemon balm, we grew lemon balm plants in MS medium supplemented with 50, 100, 150, 200, 250, or 300 mm sucrose for 20 d and measured the levels of anthocyanins in the leaves. As demonstrated in Figure 1, lemon balm leaves accumulated a high level of anthocyanins in response to sucrose in a dose-dependent manner: on treatment with 300 mm sucrose, the level of anthocyanins was increased up to 30-fold compared with samples treated with 50 mm sucrose.

Fig. 1.
Fig. 1.

Sucrose-induced accumulation of anthocyanins in lemon balm leaves. Plants were grown in full-strength Murashige and Skoog medium with 50, 100, 150, 200, 250, or 300 mm sucrose and 0.7% agar for 20 d and anthocyanin levels were determined. Data represent the mean ± se from nine independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test). (Inset: Leaf extracts obtained for anthocyanin determination.)

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

As shown in Figure 2A, lemon balm grew well in medium containing 150 or 200 mm sucrose. However, plants grown in 300 mm sucrose showed lower total dry weight as well as lower total fresh weight (Fig. 2B). Similar patterns were observed when we measured the leaf fresh weight and leaf dry weight of lemon balm plants in medium supplemented with 50, 100, 150, 200, 250, or 300 mm sucrose. Plant growth performance was severely damaged when the sucrose concentration was increased to 350 or 400 mm (Fig. S1; view supplemental figures online at http://hortsci.ashspublications.org). In our study, we observed that the relative chlorophyll content increased with sucrose treatment up to 200 mm, after which it dropped sharply (Fig. 2C). However, we noticed that lemon balm leaves grown in high levels of sucrose became thicker compared with leaves grown in 50 mm sucrose. We therefore decided to determine the specific leaf weight (leaf weight per unit area) of the plants grown in different concentrations of sucrose and found a positive correlation between the level of sucrose treatment and the specific leaf weight (Fig. 2D).

Fig. 2.
Fig. 2.

Growth performance of lemon balm grown in Murashige and Skoog (MS) medium with various concentrations of sucrose. (A) Plants were grown in MS medium with 50, 100, 150, 200, 250, or 300 mm sucrose for 20 d. (B) Total fresh weight (TFW), total dry weight (TDW), leaf fresh weight (LFW), and leaf dry weight (LDW) per plant. (C) Chlorophyll content of the leaves. (D) Specific leaf weights measured as leaf weight per unit leaf area. Values are mean ± se from nine independent experimental replicates.

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

A high level of sucrose increases the overall flavonoid content in lemon balm.

Because there are other types of flavonoids in plants besides anthocyanins, we wondered if sucrose would also increase the total amount of flavonoids in lemon balm. Like anthocyanin, the highest flavonoid content [16.9 μg quercetin equivalents (QE)·mg−1] was recorded in leaves treated with 300 mm sucrose followed by 15.9 μg QE·mg−1 in the leaves treated with 250 mm sucrose. The lowest flavonoid content (3.8 μg QE·mg−1) was observed in plants grown in 50 mm sucrose (Fig. 3). The flavonoid content in plants treated with 300 mm sucrose was approximately fourfold higher than that in control plants. This result indicates that the overall flavonoid content can also be increased by sucrose treatment.

Fig. 3.
Fig. 3.

Total flavonoid levels in lemon balm leaves increase following sucrose treatment in a dose-dependent manner. Plants were grown in Murashige and Skoog medium supplemented with 50, 100, 150, 200, 250, or 300 mm sucrose for 20 d and the total flavonoid concentration was determined according to established methods (Faudale et al., 2008). Values are means ± se from three independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test).

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

To confirm this result, we used liquid chromatography (LC)/MS to analyze the specific metabolites produced by plants treated with different sucrose concentrations. Among the various metabolites detected by LC/MS, those that showed significant differences in quantity among the plant groups were selected for PCA. A major separation between the lemon balm sample groups was easily achieved by combining the principal components PC1 and PC2, where the ellipse marked the 95% confidence level on the Hotelling T2 control chart. Because PC1 and PC2 accounted for 66.4% and 12.0% of the total variance, respectively, the majority of the variables (78.4% of the variance) were well described by the first two PCs. The PC values acquired from samples grown in different sucrose concentrations showed a clear counterclockwise trajectory on the score plot for PC1 and PC2, which followed the increments in sucrose concentration (Fig. 4A). On the loading plot corresponding to PC1 and PC2, the different metabolites corresponding to the scores were clearly distinguishable. In other words, the location of each sample in the score plot was affected by an identical placement of metabolites in the loading plot. As shown in Figure 4B, metabolites with molecular weights (MWs) of 523.129 and 617.153 were more abundant than other metabolites in lemon balm grown in 300 mm sucrose. A metabolite with MW 615.714 was found at a higher concentration in lemon balm treated with 50 mm sucrose, whereas metabolites with MW 540.063 and 435.13 (presumptively delphinidin-3-arabinoside) were found to be more abundant in plants grown in 250 mm sucrose.

Fig. 4.
Fig. 4.

Score plot of principal component analysis (PCA) of lemon balm extracts by combining PC1 and PC2. (A) Ellipse represents the Hotelling T2 with 95% confidence. The labels 50, 100, 150, 200, 250, and 300 indicate lemon balm leaves grown in Murashige and Skoog media with 50, 100, 150, 200, 250, and 300 mm sucrose, respectively, for 20 d. (B) The loading plot of the PCA for PC1 and PC2. PC1 has an explained variation of 0.67 and a predicted variation of 0.417.

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

Based on the observation that the levels of anthocyanins in lemon balm leaves increased with increasing sucrose concentrations, we next assessed the reactive oxygen species (ROS) scavenging activities in the lemon balm leaves. In this assay, we observed that the DPPH scavenging activities in the leaf extracts varied from 27.3% to 73.9% depending on the concentration of sucrose tested (Table 1). The maximum scavenging activity (73.9%) was obtained from leaves grown in 300 mm sucrose, whereas the lowest activity (27.3%) was in leaves grown in 50 mm sucrose. As shown in Table 1, the IC50 values decreased significantly when plants were grown in 200, 250, or 300 mm sucrose. Lower IC50 values correlate with higher antioxidant activities in plant extracts (Patro et al., 2005). The lowest IC50 value (52.9 μL·mL−1) was recorded from plants grown in 300 mm sucrose. The antioxidant activity of plants grown in 300 mm sucrose was 10 times higher than that in plants grown in 50 mm sucrose.

Table 1.

Antioxidant activities of lemon balm leaf extracts from plants grown in various concentrations of sucrose.z

Table 1.

Effects of growth regulators on sucrose-mediated anthocyanin accumulation.

To examine the effects of phytohormones on sucrose-mediated anthocyanin accumulation in lemon balm, we altered the treatment conditions. A shorter period of incubation was used to avoid the degradation of hormone activity under long exposures to light. Instead of using plants grown directly in high sucrose medium for 20 d, explants were allowed to grow in normal medium (50 mm sucrose) for 12 d and then were transferred to a liquid medium containing 50 or 300 mm sucrose. This medium was supplemented with one of five different growth regulators at concentrations of 0, 5, or 10 μM. The compounds tested were the ethylene precursor ACC and the hormones kinetin, ABA, IAA, and gibberellic acid (GA). After transfer to the liquid medium, the explants were incubated for a further 3 d before analysis. Figure 5 shows that treatments with ACC and ABA were able to enhance the anthocyanin accumulation significantly when combined with sucrose. However, GA negatively affected anthocyanin accumulation even at a high sucrose concentration (300 mm) (Figs. 5 and S2).

Fig. 5.
Fig. 5.

The levels of anthocyanins in lemon balm plants grown in 50 or 300 mm sucrose in the presence of phytohormones. Plants grown in Murashige and Skoog medium with 50 mm sucrose for 12 d were transferred to liquid medium with 50 mm (open bar) or 300 mm (closed bar) sucrose in combination with 0, 5, or 10 μM of the ethylene precursor 1-aminocyclopropane-carboxylic acid (ACC) or one of the phytohormones, kinetin, (+)-cis, transabscissic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA). The plants were incubated for a further 3 d before anthocyanin measurements. Values are means ± se from nine independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test).

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

Next, we examined whether ABA or ethylene is involved in sucrose-mediated anthocyanin accumulation. Lemon balm plants were exposed for 3 d to 50 or 300 mm sucrose combined with the ethylene action inhibitor silver nitrate (AgNO3), or with the ABA biosynthesis inhibitor fluridon, and anthocyanin levels were determined. The inhibitors were used at concentrations of 0, 5, 10, or 20 μM. The levels of anthocyanin decreased gradually in lemon balm leaves as the concentrations of fluridon or AgNO3 increased (Fig. 6). The results also indicate that AgNO3 is a more efficient inhibitor than fluridon in sucrose-mediated anthocyanin accumulation (Fig. 6). To examine whether these inhibitors also prohibit antioxidant activity in lemon balm grown at a high sucrose level, we measured the DPPH scavenging activities of leaves that had been exposed for 3 d to 0, 5, 10, or 20 μM AgNO3 or fluridon in combination with 300 mm sucrose and observed increasing IC50 values with increased inhibitor concentrations (Table 2).

Table 2.

DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging activities of leaf extracts from plants treated with 50 or 300 mm sucrose and 0, 5, 10, or 20 μM fluridon or AgNO3 for 3 d.z

Table 2.
Fig. 6.
Fig. 6.

The effect of fluridon and AgNO3 on anthocyanin accumulation in lemon balm leaves in the presence of 300 mm sucrose. Lemon balm plants grown in 50 mm sucrose for 12 d were incubated with 50 mm (open bar) or 300 mm (closed bar) sucrose together with fluridon (Fl) or AgNO3 (Sn) for 3 d. Homogenized samples were analyzed using a microplate spectrophotometer to obtain anthocyanin contents. Values are means ± se from nine independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test).

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

Because ROS are involved in osmotic stress, ROS production levels were assessed in lemon balm plants grown in 50, 100, 150, 200, 250, or 300 mm sucrose. As shown in Figure 7A, the production of ROS became increasingly evident in the leaves as the concentration of sucrose increased. We did not observe any significant ROS staining in leaves grown with up to 150 mm sucrose, and the leaves grown in 200 mm sucrose were only slightly stained. Because it was easy to detect ROS production with the 300 mm sucrose treatment, we confined our analysis to this condition for the remaining experiments. To determine the effects of various phytohormones on ROS production, plants were treated for 3 d with 50 or 300 mm sucrose combined with the ethylene precursor ACC or one of the four phytohormones, kinetin, ABA, IAA, and GA (each at 0, 5, or 10 μM). As shown in Figure 7B, dark ROS staining was evident in leaves grown in 50 mm sucrose and 10 μM ABA. ROS staining intensified when plants were grown with 300 mm sucrose combined with 5 or 10 μM ACC or ABA. On the other hand, we observed that the ROS staining gradually decreased with increasing concentrations of GA, although sucrose was present at a concentration of 300 mm (Fig. 7B).

Fig. 7.
Fig. 7.

Determination of reactive oxygen species (ROS) levels in lemon balm leaves grown in the presence of sucrose and phytohormones. Brown-colored staining obtained by the incubation of lemon balm leaves with 3, 3′-diaminobenzidine (DAB) was used as a marker for endogenous ROS (H2O2) production. (A) ROS staining of lemon balm leaves grown in various concentrations of sucrose for 20 d. (B) Lemon balm leaves were treated with 0, 5, or 10 μM 1-aminocyclopropane-carboxylic acid (ACC), kinetin, (+)-cis, transabscissic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA) in the presence of 50 or 300 mm sucrose in liquid medium for 3 d. Shown are the representative results from three independent experimental replicates.

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

Discussion

Sugar-induced anthocyanin accumulation has been reported in various plant species (Nagira et al., 2006; Nagira and Ozeki, 2004; Solfanelli et al., 2006). One recent report suggests that strong anthocyanin induction can be triggered by sucrose but not by other sugars or by osmotic stress in Arabidopsis (Teng et al., 2005). However, the mechanisms controlling the induction of anthocyanin accumulation in lemon balm leaves in response to sucrose or to phytohormones remain unclear. In the experiments presented here, lemon balm leaves were exposed to high levels of sucrose, and in response, they accumulated anthocyanins as well as other flavonoids, which correlated with DPPH scavenging activity (Table 1). Moreover, delphinidin-3-arabinoside, which was found to be the primary anthocyanin by PCA analysis, was enriched in lemon balm grown in a high concentration of sucrose (Fig. 4B). Recently, Azuma and colleagues assessed the ROS scavenging activities of five purified anthocyanins using radical substrates such as the DPPH radical and the linoleic acid radical. The authors showed that delphinidin (delphinidin 3RGcaf5G) had a higher ROS scavenging activity than petunidin (petunidin 3RGc5G) (Azuma et al., 2008). Moreover, another recent report suggests that flavonoids can replace vitamin E as chain-breaking antioxidants in liver microsomal membranes (van Acker et al., 2000). This indicates that the delphinidin enriched in lemon balm leaves grown in 300 mm sucrose likely contributed to the enhanced ROS scavenging activity of the samples.

We observed a positive synergistic effect on anthocyanin accumulation in lemon balm leaves when ABA or ACC were combined with sucrose in the growth medium. However, the phytohormones themselves slightly increased anthocyanin levels in plants grown in 50 mm sucrose (Fig. 5). When combined with 300 mm sucrose, ABA and ACC upregulated the accumulation of anthocyanins to similar extents. Consistent with this, the ABA biosynthesis inhibitor and the ethylene action inhibitor reduced the levels of sucrose-induced anthocyanin accumulation (Fig. 6). These results suggest positive roles of ABA and ethylene in the accumulation of anthocyanins in response to sucrose treatment. IAA also exhibited a positive effect on the accumulation of anthocyanins with sucrose treatment, but it did not reach the level triggered by ABA or ACC. It seems that plants harbor different systems to trigger the accumulation of anthocyanins in response to hormones or sucrose. For example, in the Camptotheca acuminata cell culture system, anthocyanin production was significantly increased by the addition of kinetin (Pasqua et al., 2005), whereas we observed no significant effect with kinetin.

In our study, GA greatly reduced the accumulation of anthocyanins in a dose-dependent manner (Fig. 5). Reductions in anthocyanin biosynthesis by GA have been reported in seedlings of tomato (Khan, 1980), radish (Jain and Guruprasad, 1989), and maize (White and Rivin, 2000). In another study, gibberellins were also shown to counteract the induction of anthocyanin biosynthesis by sucrose in Arabidopsis (Loreti et al., 2008). These authors demonstrated that GA3 repressed the induction of the PAP1 and PAP2 genes, which are responsive to sucrose in anthocyanin biosynthesis (Solfanelli et al., 2006; Teng et al., 2005). These reports suggest the existence of similar crosstalk mechanisms between sucrose and GA in the vegetative tissues of lemon balm and other species. In contrast, GA has been implicated as having a positive effect on the induction of flavonoid-specific genes, including CHS and CHI, in flowers (Moalem-Beno et al., 1997; Neta-Sharir et al., 2000; Weiss et al., 1990). Therefore, different plant organs may harbor different signaling systems for mediating anthocyanin biosynthesis in response to phytohormones. It will be of interest to identify what components account for the variation in responses to phytohormones in different species and different plant parts.

ROS are regarded as toxic byproducts of aerobic metabolism that accumulate to a greater extent in cells under conditions of stress (Gratão et al., 2005, 2008). On the other hand, ROS have also been implicated as important second messenger molecules in the signaling and regulation of genes that are governed by various stimuli (Mittler, 2002). These contradictory roles are indicative of a complex modulation between divergent stress signals and ROS production for the integration of various cell signaling activities. In our study, the accumulation of anthocyanins was positively correlated with the level of ROS production (Fig. 7A). This clearly indicates that ROS may function as signaling molecules for the accumulation of anthocyanins in lemon balm plants. Both ABA and ACC increased ROS accumulation in lemon balm leaves grown with high levels of sucrose (Fig. 7B). ABA is also known to increase the activity of antioxidant enzymes, which can cause a decrease in oxidative stress. Thus, our results support the role of ABA in oxidative stress responses. We also observed that GA reduced the appearance of ROS in a dose-dependent manner, which was accompanied by a decrease in anthocyanin accumulation.

In conclusion, we have demonstrated for the first time that lemon balm plants respond to sucrose with increased levels of anthocyanins. Our studies revealed that sucrose significantly enhances the levels of flavonoids in lemon balm plants and that sucrose induction appears to be mediated by phytohormones, primarily ABA and ethylene. We identified delphinidin-3-arabinoside as the primary anthocyanin compound that accumulates in leaves grown in a high-sucrose medium. Our results also showed that ROS levels are positively correlated with sucrose-mediated anthocyanin accumulation. These findings could be the foundation for future work in lemon balm at the molecular level.

Literature Cited

  • Azuma, K., Ohyama, A., Ippoushi, K., Ichiyanagi, T., Takeuchi, A., Saito, T. & Fukuoka, H. 2008 Structures and antioxidant activity of anthocyanins in many accessions of eggplant and its related species J. Agr. Food Chem. 56 10154 10159

    • Search Google Scholar
    • Export Citation
  • Brand-Williams, W., Cuvelier, M.E. & Berset, C. 1995 Use of a free radical method to evaluate antioxidant activity Lebensm.-Wiss. Technol. 28 25 30

  • Chen, Q.F., Liang-Ying, D., Shi, X., Yun-Sheng, W., Xiong-Lun, L. & Guo-Liang, W. 2007 The COI1 and DFR genes are essential for regulation of jasmonate-induced anthocyanin accumulation in Arabidopsis J. Integr. Plant Biol. 49 1370 1377

    • Search Google Scholar
    • Export Citation
  • de Sousa, A.C., Alviano, D.S., Blank, A.F., Alves, P.B., Alviano, C.S. & Gattass, C.R. 2004 Melissa officinalis L. essential oil: Antitumoral and antioxidant activities J. Pharm. Pharmacol. 56 677 681

    • Search Google Scholar
    • Export Citation
  • Devoto, A., Ellis, C., Magusin, A., Chang, H.S., Chilcott, C., Zhu, T. & Turner, J.G. 2005 Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defense, and hormone interactions Plant Mol. Biol. 58 497 513

    • Search Google Scholar
    • Export Citation
  • Faudale, M., Viladomat, F., Bastida, J., Poli, F. & Codina, C. 2008 Antioxidant activity and phenolic composition of wild, edible, and medicinal fennel from different Mediterranean countries J. Agr. Food Chem. 56 1912 1920

    • Search Google Scholar
    • Export Citation
  • Fester, T. & Hause, G. 2005 Accumulation of reactive oxygen species in arbuscular mycorrhizal roots Mycorrhiza 15 373 379

  • Galasinski, W., Chlabicz, J., Paszkiewicz-Gadek, A., Marcinkiewicz, C. & Gindzienski, A. 1996 The substances of plant origin that inhibit protein biosynthesis Acta Pol. Pharm. 53 311 318

    • Search Google Scholar
    • Export Citation
  • Geuenich, S., Goffinet, C., Venzke, S., Nolkemper, S., Baumann, I., Plinkert, P., Reichling, J. & Keppler, O.T. 2008 Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density Retrovirology 5 27

    • Search Google Scholar
    • Export Citation
  • Gollop, R., Even, S., Colova-Tsolova, V. & Peri, A. 2002 Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region J. Expt. Bot. 53 1397 1409

    • Search Google Scholar
    • Export Citation
  • Gratão, P.L., Monteiro, C.C., Peres, L.E.P. & Azevedo, R.A. 2008 The isolation of antioxidant enzymes from mature tomato (cv. Micro-Tom) plants HortScience 43 1608 1610

    • Search Google Scholar
    • Export Citation
  • Gratão, P.L., Polle, A., Lea, P.J. & Azevedo, R.A. 2005 Making the life of heavy metal-stress plants a little easier Funct. Plant Biol. 32 481 494

  • Jain, V.K. & Guruprasad, K.N. 1989 Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings Physiol. Plant. 75 233 236

    • Search Google Scholar
    • Export Citation
  • Kennedy, D.O., Little, W., Haskell, C.F. & Scholey, A.B. 2006 Anxiolytic effects of a combination of Melissa officinalis and Valeriana officinalis during laboratory induced stress Phytother. Res. 20 96 102

    • Search Google Scholar
    • Export Citation
  • Kennedy, D.O., Scholey, A.B., Tildesley, N.T., Perry, E.K. & Wesnes, K.A. 2004 Attenuation of laboratory-induced stress in humans after acute administration of Melissa officinalis (lemon balm) Psychosom. Med. 66 607 613

    • Search Google Scholar
    • Export Citation
  • Khan, M.I. 1980 Gibberellic acid bioassay based on the inhibition of anthocyanins production in tomato seedlings Biol. Plant. 22 401 403

  • Loreti, E., Povero, G., Novi, G., Solfanelli, C., Alpi, A. & Perata, P. 2008 Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis New Phytol. 179 1004 1016

    • Search Google Scholar
    • Export Citation
  • Ma, H.L., Qin, M.J., Qi, L.W., Wu, G. & Shu, P. 2007 Improved quality evaluation of Radix Salvia miltiorrhiza through simultaneous quantification of seven major active components by high-performance liquid chromatography and principal component analysis Biomed. Chromatogr. 21 931 939

    • Search Google Scholar
    • Export Citation
  • Mittler, R. 2002 Oxidative stress, antioxidants and stress tolerance Trends Plant Sci. 7 405 410

  • Moalem-Beno, D., Tamari, G., Leitner-Dagan, Y., Borochov, A. & Weiss, D. 1997 Sugar-dependent gibberellin-induced chalcone synthase gene expression in petunia corollas Plant Physiol. 113 419 424

    • Search Google Scholar
    • Export Citation
  • Muller, S.F. & Klement, S. 2006 A combination of valerian and lemon balm is effective in the treatment of restlessness and dyssomnia in children Phytomedicine 13 383 387

    • Search Google Scholar
    • Export Citation
  • Nagira, Y., Ikegami, K., Koshiba, T. & Ozeki, Y. 2006 Effect of ABA upon anthocyanin synthesis in regenerated torenia shoots J. Plant Res. 119 137 144

  • Nagira, Y. & Ozeki, Y. 2004 A system in which anthocyanin synthesis is induced in regenerated torenia shoots J. Plant Res. 117 377 383

  • Neff, M.M. & Chory, J. 1998 Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development Plant Physiol. 118 27 35

    • Search Google Scholar
    • Export Citation
  • Neta-Sharir, I., Shoseyov, O. & Weiss, D. 2000 Sugars enhance the expression of gibberellin-induced genes in developing petunia flowers Physiol. Plant. 109 196 202

    • Search Google Scholar
    • Export Citation
  • Pasqua, G., Monacellia, B., Mulinacci, N., Rinaldi, S., Giaccherini, C., Innocenti, M. & Vinceri, F. 2005 The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures Plant Physiol. Biochem. 43 293 298

    • Search Google Scholar
    • Export Citation
  • Patro, B.S., Bauri, A.K., Mishra, S. & Chattopadhyay, S. 2005 Antioxidant activity of Myristica malabarica extracts and their constituents J. Agr. Food Chem. 53 6912 6918

    • Search Google Scholar
    • Export Citation
  • Porra, R.J., Thompson, W.A. & Kriedmann, P.A. 1989 Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy Biochim. Biophys. Acta 975 384 394

    • Search Google Scholar
    • Export Citation
  • Rolland, F., Moore, B. & Sheen, J. 2002 Sugar sensing and signaling in plants Plant Cell 14 S185 S205

  • Rook, F. & Bevan, M.W. 2003 Genetic approaches to understanding sugar response pathways J. Expt. Bot. 54 495 501

  • Smeekens, S. 2000 Sugar-induced signal transduction in plants Annu. Rev. Plant Physiol. Plant Mol. Biol. 51 49 81

  • Solfanelli, C., Poggi, A., Loreti, E., Alpi, A. & Perata, P. 2006 Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis Plant Physiol. 140 637 646

    • Search Google Scholar
    • Export Citation
  • Teng, S., Keurentjes, J., Bentsink, L., Koornneef, M. & Smeekens, S. 2005 Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene Plant Physiol. 139 1840 1852

    • Search Google Scholar
    • Export Citation
  • Tolstikov, V.V., Fiehn, O. & Tanaka, N. 2007 Application of liquid chromatography-mass spectrometry analysis in metabolomics 141 155 Weckwerth W. Metabolomics methods and protocols Humana Press Totowa, NJ

    • Search Google Scholar
    • Export Citation
  • Tonelli, C., Cominelli, E., Allegra, D. & Galbiati, M. 2007 Plant tolerance to drought and salinity: Modulation of transcription factors Proc. 18th International Conference on Arabidopsis Research 176

    • Search Google Scholar
    • Export Citation
  • van Acker, F.A., Schouten, O., Haenen, G.R., van der Vijgh, W.J. & Bast, A. 2000 Flavonoids can replace alpha-tocopherol as an antioxidant FEBS Lett. 473 145 148

    • Search Google Scholar
    • Export Citation
  • Weiss, D., Tunen, A.J.V., Halevy, A.H., Mol, J.N.M. & Gerats, A.G.M. 1990 Stamens and gibberellic acid in the regulation of flavonoid gene expression in the corolla of Petunia hybrida Plant Physiol. 94 511 515

    • Search Google Scholar
    • Export Citation
  • White, C.N. & Rivin, C.J. 2000 Gibberellins and seed development in maize. II. Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured embryos Plant Physiol. 122 1089 1097

    • Search Google Scholar
    • Export Citation
  • Yamasaki, K., Nakano, M., Kawahata, T., Mori, H., Otake, T., Ueba, N., Oishi, I., Inami, R., Yamane, M., Nakamura, M., Murata, H. & Nakanishi, T. 1998 Anti-HIV-1 activity of herbs in Labiatae Biol. Pharm. Bull. 21 829 833

    • Search Google Scholar
    • Export Citation
Fig. S1.
Fig. S1.

Inhibition of plant growth by high levels of sucrose. Plants were grown in Murashige and Skoog medium supplemented with 350 or 400 mm sucrose for 20 d before the photograph was taken. Shown are the representative results from three independent experimental replicates.

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

Fig. S2.
Fig. S2.

Pigments of anthocyanins extracted from lemon balm leaves treated for 3 d with 0, 5, or 10 μM 1-aminocyclopropane-carboxylic acid (ACC), kinetin, (+)-cis, transabscissic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA) along with 50 mm or 300 mm sucrose in a liquid medium. Shown are the representative results from three independent experimental replicates.

Citation: HortScience horts 44, 7; 10.21273/HORTSCI.44.7.1907

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

This work was supported by a grant from ARPC (to H.L., Grant # 107100-03-1-SB010) and in part by a grant from ARPC (to H.L., Grant # 108066-03-1-HD120).

To whom reprint requests should be addressed; e-mail lhojoung@korea.ac.kr.

  • View in gallery

    Sucrose-induced accumulation of anthocyanins in lemon balm leaves. Plants were grown in full-strength Murashige and Skoog medium with 50, 100, 150, 200, 250, or 300 mm sucrose and 0.7% agar for 20 d and anthocyanin levels were determined. Data represent the mean ± se from nine independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test). (Inset: Leaf extracts obtained for anthocyanin determination.)

  • View in gallery

    Growth performance of lemon balm grown in Murashige and Skoog (MS) medium with various concentrations of sucrose. (A) Plants were grown in MS medium with 50, 100, 150, 200, 250, or 300 mm sucrose for 20 d. (B) Total fresh weight (TFW), total dry weight (TDW), leaf fresh weight (LFW), and leaf dry weight (LDW) per plant. (C) Chlorophyll content of the leaves. (D) Specific leaf weights measured as leaf weight per unit leaf area. Values are mean ± se from nine independent experimental replicates.

  • View in gallery

    Total flavonoid levels in lemon balm leaves increase following sucrose treatment in a dose-dependent manner. Plants were grown in Murashige and Skoog medium supplemented with 50, 100, 150, 200, 250, or 300 mm sucrose for 20 d and the total flavonoid concentration was determined according to established methods (Faudale et al., 2008). Values are means ± se from three independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test).

  • View in gallery

    Score plot of principal component analysis (PCA) of lemon balm extracts by combining PC1 and PC2. (A) Ellipse represents the Hotelling T2 with 95% confidence. The labels 50, 100, 150, 200, 250, and 300 indicate lemon balm leaves grown in Murashige and Skoog media with 50, 100, 150, 200, 250, and 300 mm sucrose, respectively, for 20 d. (B) The loading plot of the PCA for PC1 and PC2. PC1 has an explained variation of 0.67 and a predicted variation of 0.417.

  • View in gallery

    The levels of anthocyanins in lemon balm plants grown in 50 or 300 mm sucrose in the presence of phytohormones. Plants grown in Murashige and Skoog medium with 50 mm sucrose for 12 d were transferred to liquid medium with 50 mm (open bar) or 300 mm (closed bar) sucrose in combination with 0, 5, or 10 μM of the ethylene precursor 1-aminocyclopropane-carboxylic acid (ACC) or one of the phytohormones, kinetin, (+)-cis, transabscissic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA). The plants were incubated for a further 3 d before anthocyanin measurements. Values are means ± se from nine independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test).

  • View in gallery

    The effect of fluridon and AgNO3 on anthocyanin accumulation in lemon balm leaves in the presence of 300 mm sucrose. Lemon balm plants grown in 50 mm sucrose for 12 d were incubated with 50 mm (open bar) or 300 mm (closed bar) sucrose together with fluridon (Fl) or AgNO3 (Sn) for 3 d. Homogenized samples were analyzed using a microplate spectrophotometer to obtain anthocyanin contents. Values are means ± se from nine independent experimental replicates. The same letters indicate no significant difference at P ≤ 0.05 (analysis of variance and Duncan's multiple range test).

  • View in gallery

    Determination of reactive oxygen species (ROS) levels in lemon balm leaves grown in the presence of sucrose and phytohormones. Brown-colored staining obtained by the incubation of lemon balm leaves with 3, 3′-diaminobenzidine (DAB) was used as a marker for endogenous ROS (H2O2) production. (A) ROS staining of lemon balm leaves grown in various concentrations of sucrose for 20 d. (B) Lemon balm leaves were treated with 0, 5, or 10 μM 1-aminocyclopropane-carboxylic acid (ACC), kinetin, (+)-cis, transabscissic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA) in the presence of 50 or 300 mm sucrose in liquid medium for 3 d. Shown are the representative results from three independent experimental replicates.

  • View in gallery

    Inhibition of plant growth by high levels of sucrose. Plants were grown in Murashige and Skoog medium supplemented with 350 or 400 mm sucrose for 20 d before the photograph was taken. Shown are the representative results from three independent experimental replicates.

  • View in gallery

    Pigments of anthocyanins extracted from lemon balm leaves treated for 3 d with 0, 5, or 10 μM 1-aminocyclopropane-carboxylic acid (ACC), kinetin, (+)-cis, transabscissic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA) along with 50 mm or 300 mm sucrose in a liquid medium. Shown are the representative results from three independent experimental replicates.

  • Azuma, K., Ohyama, A., Ippoushi, K., Ichiyanagi, T., Takeuchi, A., Saito, T. & Fukuoka, H. 2008 Structures and antioxidant activity of anthocyanins in many accessions of eggplant and its related species J. Agr. Food Chem. 56 10154 10159

    • Search Google Scholar
    • Export Citation
  • Brand-Williams, W., Cuvelier, M.E. & Berset, C. 1995 Use of a free radical method to evaluate antioxidant activity Lebensm.-Wiss. Technol. 28 25 30

  • Chen, Q.F., Liang-Ying, D., Shi, X., Yun-Sheng, W., Xiong-Lun, L. & Guo-Liang, W. 2007 The COI1 and DFR genes are essential for regulation of jasmonate-induced anthocyanin accumulation in Arabidopsis J. Integr. Plant Biol. 49 1370 1377

    • Search Google Scholar
    • Export Citation
  • de Sousa, A.C., Alviano, D.S., Blank, A.F., Alves, P.B., Alviano, C.S. & Gattass, C.R. 2004 Melissa officinalis L. essential oil: Antitumoral and antioxidant activities J. Pharm. Pharmacol. 56 677 681

    • Search Google Scholar
    • Export Citation
  • Devoto, A., Ellis, C., Magusin, A., Chang, H.S., Chilcott, C., Zhu, T. & Turner, J.G. 2005 Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defense, and hormone interactions Plant Mol. Biol. 58 497 513

    • Search Google Scholar
    • Export Citation
  • Faudale, M., Viladomat, F., Bastida, J., Poli, F. & Codina, C. 2008 Antioxidant activity and phenolic composition of wild, edible, and medicinal fennel from different Mediterranean countries J. Agr. Food Chem. 56 1912 1920

    • Search Google Scholar
    • Export Citation
  • Fester, T. & Hause, G. 2005 Accumulation of reactive oxygen species in arbuscular mycorrhizal roots Mycorrhiza 15 373 379

  • Galasinski, W., Chlabicz, J., Paszkiewicz-Gadek, A., Marcinkiewicz, C. & Gindzienski, A. 1996 The substances of plant origin that inhibit protein biosynthesis Acta Pol. Pharm. 53 311 318

    • Search Google Scholar
    • Export Citation
  • Geuenich, S., Goffinet, C., Venzke, S., Nolkemper, S., Baumann, I., Plinkert, P., Reichling, J. & Keppler, O.T. 2008 Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density Retrovirology 5 27

    • Search Google Scholar
    • Export Citation
  • Gollop, R., Even, S., Colova-Tsolova, V. & Peri, A. 2002 Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region J. Expt. Bot. 53 1397 1409

    • Search Google Scholar
    • Export Citation
  • Gratão, P.L., Monteiro, C.C., Peres, L.E.P. & Azevedo, R.A. 2008 The isolation of antioxidant enzymes from mature tomato (cv. Micro-Tom) plants HortScience 43 1608 1610

    • Search Google Scholar
    • Export Citation
  • Gratão, P.L., Polle, A., Lea, P.J. & Azevedo, R.A. 2005 Making the life of heavy metal-stress plants a little easier Funct. Plant Biol. 32 481 494

  • Jain, V.K. & Guruprasad, K.N. 1989 Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings Physiol. Plant. 75 233 236

    • Search Google Scholar
    • Export Citation
  • Kennedy, D.O., Little, W., Haskell, C.F. & Scholey, A.B. 2006 Anxiolytic effects of a combination of Melissa officinalis and Valeriana officinalis during laboratory induced stress Phytother. Res. 20 96 102

    • Search Google Scholar
    • Export Citation
  • Kennedy, D.O., Scholey, A.B., Tildesley, N.T., Perry, E.K. & Wesnes, K.A. 2004 Attenuation of laboratory-induced stress in humans after acute administration of Melissa officinalis (lemon balm) Psychosom. Med. 66 607 613

    • Search Google Scholar
    • Export Citation
  • Khan, M.I. 1980 Gibberellic acid bioassay based on the inhibition of anthocyanins production in tomato seedlings Biol. Plant. 22 401 403

  • Loreti, E., Povero, G., Novi, G., Solfanelli, C., Alpi, A. & Perata, P. 2008 Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis New Phytol. 179 1004 1016

    • Search Google Scholar
    • Export Citation
  • Ma, H.L., Qin, M.J., Qi, L.W., Wu, G. & Shu, P. 2007 Improved quality evaluation of Radix Salvia miltiorrhiza through simultaneous quantification of seven major active components by high-performance liquid chromatography and principal component analysis Biomed. Chromatogr. 21 931 939

    • Search Google Scholar
    • Export Citation
  • Mittler, R. 2002 Oxidative stress, antioxidants and stress tolerance Trends Plant Sci. 7 405 410

  • Moalem-Beno, D., Tamari, G., Leitner-Dagan, Y., Borochov, A. & Weiss, D. 1997 Sugar-dependent gibberellin-induced chalcone synthase gene expression in petunia corollas Plant Physiol. 113 419 424

    • Search Google Scholar
    • Export Citation
  • Muller, S.F. & Klement, S. 2006 A combination of valerian and lemon balm is effective in the treatment of restlessness and dyssomnia in children Phytomedicine 13 383 387

    • Search Google Scholar
    • Export Citation
  • Nagira, Y., Ikegami, K., Koshiba, T. & Ozeki, Y. 2006 Effect of ABA upon anthocyanin synthesis in regenerated torenia shoots J. Plant Res. 119 137 144

  • Nagira, Y. & Ozeki, Y. 2004 A system in which anthocyanin synthesis is induced in regenerated torenia shoots J. Plant Res. 117 377 383

  • Neff, M.M. & Chory, J. 1998 Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development Plant Physiol. 118 27 35

    • Search Google Scholar
    • Export Citation
  • Neta-Sharir, I., Shoseyov, O. & Weiss, D. 2000 Sugars enhance the expression of gibberellin-induced genes in developing petunia flowers Physiol. Plant. 109 196 202

    • Search Google Scholar
    • Export Citation
  • Pasqua, G., Monacellia, B., Mulinacci, N., Rinaldi, S., Giaccherini, C., Innocenti, M. & Vinceri, F. 2005 The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures Plant Physiol. Biochem. 43 293 298

    • Search Google Scholar
    • Export Citation
  • Patro, B.S., Bauri, A.K., Mishra, S. & Chattopadhyay, S. 2005 Antioxidant activity of Myristica malabarica extracts and their constituents J. Agr. Food Chem. 53 6912 6918

    • Search Google Scholar
    • Export Citation
  • Porra, R.J., Thompson, W.A. & Kriedmann, P.A. 1989 Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy Biochim. Biophys. Acta 975 384 394

    • Search Google Scholar
    • Export Citation
  • Rolland, F., Moore, B. & Sheen, J. 2002 Sugar sensing and signaling in plants Plant Cell 14 S185 S205

  • Rook, F. & Bevan, M.W. 2003 Genetic approaches to understanding sugar response pathways J. Expt. Bot. 54 495 501

  • Smeekens, S. 2000 Sugar-induced signal transduction in plants Annu. Rev. Plant Physiol. Plant Mol. Biol. 51 49 81

  • Solfanelli, C., Poggi, A., Loreti, E., Alpi, A. & Perata, P. 2006 Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis Plant Physiol. 140 637 646

    • Search Google Scholar
    • Export Citation
  • Teng, S., Keurentjes, J., Bentsink, L., Koornneef, M. & Smeekens, S. 2005 Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene Plant Physiol. 139 1840 1852

    • Search Google Scholar
    • Export Citation
  • Tolstikov, V.V., Fiehn, O. & Tanaka, N. 2007 Application of liquid chromatography-mass spectrometry analysis in metabolomics 141 155 Weckwerth W. Metabolomics methods and protocols Humana Press Totowa, NJ

    • Search Google Scholar
    • Export Citation
  • Tonelli, C., Cominelli, E., Allegra, D. & Galbiati, M. 2007 Plant tolerance to drought and salinity: Modulation of transcription factors Proc. 18th International Conference on Arabidopsis Research 176

    • Search Google Scholar
    • Export Citation
  • van Acker, F.A., Schouten, O., Haenen, G.R., van der Vijgh, W.J. & Bast, A. 2000 Flavonoids can replace alpha-tocopherol as an antioxidant FEBS Lett. 473 145 148

    • Search Google Scholar
    • Export Citation
  • Weiss, D., Tunen, A.J.V., Halevy, A.H., Mol, J.N.M. & Gerats, A.G.M. 1990 Stamens and gibberellic acid in the regulation of flavonoid gene expression in the corolla of Petunia hybrida Plant Physiol. 94 511 515

    • Search Google Scholar
    • Export Citation
  • White, C.N. & Rivin, C.J. 2000 Gibberellins and seed development in maize. II. Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured embryos Plant Physiol. 122 1089 1097

    • Search Google Scholar
    • Export Citation
  • Yamasaki, K., Nakano, M., Kawahata, T., Mori, H., Otake, T., Ueba, N., Oishi, I., Inami, R., Yamane, M., Nakamura, M., Murata, H. & Nakanishi, T. 1998 Anti-HIV-1 activity of herbs in Labiatae Biol. Pharm. Bull. 21 829 833

    • Search Google Scholar
    • Export Citation
All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 564 161 25
PDF Downloads 123 59 9