Biologia 68/5: 904—909, 2013 Section Botany DOI: 10.2478/s11756-013-0217-z
Salicylic acid regulates secondary metabolites content in leaves of Matricaria chamomilla Zuzana Dučaiová, Veronika Petruľová & Miroslav Repčák Department of Botany, Institute of Biology and Ecology, Faculty of Science, University of P. J. Šafárik, Mánesova 23, SK-04154 Košice, Slovakia; e-mail:
[email protected]
Abstract: The influence of increasing doses of salicylic acid (SA) on selected physiological parameters and the content of coumarin-related compounds of diploid and tetraploid cultivars of Matricaria chamomilla plants were studied. Forty-eight hours after treatment SA showed growth-promoting effect with decrease in tissue water content, chlorophylls and soluble proteins. High doses of SA led to an increase of reactive oxygen species (hydrogen peroxide and superoxide radical) resulted in membrane damage (measured by accumulation of malondialdehyde). Changes in secondary metabolites accumulation in leaves were also observed. The pattern of quantitative changes of studied compounds was similar in tetraploid and diploid plants. The content of herniarin and its precursor (Z )- and (E )-2-β-D-glucopyranosyloxy-4-methoxycinnamic acid increased simultaneously. A considerable increase of umbelliferone and decrease in chlorogenic acid was registered. The rise of ene-yne-dicycloether in treated plant was also detected. Key words: Matricaria chamomilla; chlorophylls; coumarin-related compounds; oxidative stress; salicylic acid Abbreviations: DW, dry weight; FW, fresh weight; GMCA, (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid; SA, salicylic acid; DIC, ene-yne-dicycloether
Introduction Plants respond to variety of physical, chemical and biological stresses through the defensive mechanisms induced by the synthesis of signal molecules. The signal molecules are involved in signal transduction systems, which trigger biosyntheses of particular enzymes catalyzing reaction forming defence compounds such as phenols, terpenoids, alkaloids or pathogenesis-related (PR) proteins (Chaman et al. 2003; Wen et al. 2005). Salicylic acid (SA) and methyl salicylate are also the most widely studied stress-signalling molecules (Raskin 1992). SA, naturally occurring phenolic compound in many plants, is considered to be a potent plant hormone because of its diverse regulatory roles in plant metabolism. SA plays a key role in the regulation of plant growth, development and flowering, in the interactions with other organisms (Hayat et al. 2010; Raskin 1992). It has been known for many years that SA is involved in the defence against pathogen attack (systemic acquired resistance). SA participates in development of stress symptoms, but it is also needed for the acclimation process and the induction of stress tolerance. Exogenous SA or methyl salicylate could induce the expression of many defence genes (Wen et al. 2005) in tobacco (Fraissinet-Tachet et al. 1998), tomato (Ding et al. 2002). The effect of exogenous SA depends on numerous factors such as developmental stage of the c 2013 Institute of Botany, Slovak Academy of Sciences
plant, the mode of application and the concentration of SA and its endogenous level in the given plant. Substantial differences in responsiveness exist among plants (Horváth et al. 2007). Chamomile (Matricaria chamomilla L.) is widely used medicinal plant and contains a large number of therapeutically active compound classes. The largest group of secondary metabolites are sesquiterpenes (chamazulene, α-bisabolol, bisabololoxides), polyacetylenes ((Z )- and (E )-ene-yne-dicycloether), flavonoids, coumarins. Phytotherapeutical effect is ascribed also to the amounts of coumarin-related compounds, which show antimicrobial and anti-inflammatory effects. The coumarins herniarin (7-methoxycoumarin) and its glucoside precursors (Z )- and (E )-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid (GMCA) are in the major amounts (Franke & Schilcher 2005). Umbelliferone (7-hydroxycoumarin) was identified as a stress metabolite (Repčák et al. 2001a). Previous results of time-dependent study showed that accumulation of coumarin-realated compounds in hydroponically cultivated chamomile was affected by exogenous SA application (Pastírová at al. 2004; Kováčik et al. 2009). Therefore the aim of this paper was to study the effect of foliar spray application of SA on the accumulation of the main coumarin-related compounds, polyacetylenes and chlorogenic acid in dose-dependent manner in the leaf rosettes of chamomile (Matricaria chamomilla L.). Consequences of high SA dosage application on main
Regulation of secondary metabolites content in chamomile leaves physiological parameters (water content, amount of chlorophylls and soluble proteins) and plant oxidative status (reactive oxygen species and membrane lipid peroxidation) were also assessed. Material and methods Cultivation and experimental design Tetraploid chamomile (Matricaria chamomilla L.), cv. ’Lutea’ and diploid cv. ’Novbona’ were used for experiments. Two-week-old plants, germinated in sand, were transferred into plastic pots filled with soil. Plants were cultivated under laboratory conditions: 12-h photoperiod with photon flux density – 210 µmol m−2 s−1 PAR at leaf level supplied by cool white fluorescent tubes (TLD 36W/33 Philips, France), with 25/20 ◦C day/night temperature and soil moisture – 60% of water holding capacity. Six-week-old plants in stage of leaf rosette were used for treatment. Leaves of cultivated plants were sprayed with 0.7 mM, 1.4 mM, 3.5 mM, 7.0 mM, 10.5 mM and 15.0 mM aq. solution of SA (Sigma-Aldrich). The parameters were measured for plants collected after 48 hours. Data obtained from fresh material were recalculated using the measured water content according to the equation [100 – (dry mass ×100/fresh mass)]. Determination of chlorophylls and soluble proteins The total chlorophyll content was estimated according to the equation proposed by Wellburn (1994). Samples were prepared by extraction of fresh leaf rosettes in methanol (w/v 1 g FW 20 mL−1 ) at laboratory temperature and analysed at wavelengths 666 nm (chlorophyll a), 653 nm (chlorophyll b) and 750 nm to correct unspecific absorption. Soluble proteins were estimated according to Bradford (1976) using 30 µL of supernatants (fresh mass was homogenized on ice bath in 50 mM potassium buffer, pH 7.0) and bovine serum albumin as the standard (595 nm). Determination of reactive oxygen species and membrane lipid peroxidation The content of hydrogen peroxide was measured by monitoring of titanium-peroxide complex (410 nm) according to Jana & Choudhuri (1981). Superoxide radical was measured as described by Elstner & Heupel (1976) by monitoring the nitrite formation from hydroxylamine (530 nm). Level of membrane lipid peroxidation was estimated as the amount
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of malondialdehyde (MDA) by the thiobarbituric acid reaction (532 nm) (Heath & Packer 1968). All measurements were done with spectrophotometer Uvi Light XTD 2 (Secomam, France). Quantification of secondary metabolite content Leaf rosettes were cut at the soil level and immediately dried at 105 ◦C to constant weight. Samples were extracted with 75% methanol (w/v; 20 mg DW mL−1 ) (Sigma Aldrich) and analysed by HPLC, under conditions: column Kromasil C18 7 µm (4.6 × 250 mm), flow rate 0.7 mL min−1 . The mobile phase was a mixture of A – acetonitrile/water/H3 PO4 (19:80:1), B – 40% acetonitrile, C – 95% acetonitrile (J.T. Barker). The gradient programme was from 100% A to 70% B (25 min.), from 70% B to 100% C (10 min.), isocratic 100% C (10 min.) and from 100% C to 100% A (5 min.). The detection was at 320 nm. Herniarin (≥ 98%; Extrasynthese), umbelliferone (≥ 99%; Fluka) and chlorogenic acid (≥ 98%; Chemika Fluka) standard compounds were used for the quantification. (Z )- and (E )-2-β-D-glucopyranosyloxy-4-methoxy cinnamic acids (GMCA) and (Z)- and (E )-ene-yne-dicycloethers were prepared and identified as described in previous papers (Repčák et al. 1999, 2001b). Statistical analysis The experiments were independently repeated twice under the same conditions. All analyses were performed in six replications. One-way analysis of variance (ANOVA) and Tukey’s test (MINITAB Release 11, Minitab Inc., State Collage, Pennsylvania) was used to evaluate the significance (P < 0.05) of differences in experiments. Errors bars of graphs represent standard deviations. Columns and lines sharing the same letters are not significantly different.
Results Effect of SA physiological attributes and plant oxidative status Various concentration of SA influenced physiological attributes in both cultivars. The rising concentration of SA caused no visually observable changes on plant habitats. Leaf rosette dry biomass increased in comparison to control plant, while the water content was simultaneously reduced (Table 1). With the increase in concentration of foliar applied SA, the significant decrease
Table 1. The influence of foliar application of different concentration of SA on growth and selected physiological parameters in rosette leaves of Matricaria chamomilla plants after 48 hours exposition. Data are means ± SDs. SA dose
M. chamomilla diploid Biomass (mg DW plant−1 ) Tissue water content (%) Chlorophyll a (mg g−1 DW) Chlorophyll b (mg g−1 DW) Soluble proteins (mg g−1 DW) M. chamomilla tetraploid Biomass (mg DW plant−1 ) Tissue water content (%) Chlorophyll a (mg g−1 DW) Chlorophyll b (mg g−1 DW) Soluble proteins (mg g−1 DW)
control
0.7 mM
1.4 mM
3.5 mM
98.0±24.3a 91.82±0.55a 17.00±0.23a 11.07±0.72a 174.6±6.95a
110.5±28.5a 91.37±0.50a 17.01±0.09a 11.28±1.27a 158.3±6.94ab
111.8±21.4a 91.48±0.49a 15.85±1.58ab 9.48±0.88ab 150.7±7.54b
104.6±16.0a 91.43±0.39a 15.06±0.95ab 9.23±0.51ab 150.6±7.33b
68.2±11.7b 92.46±0.38a 20.47±2.27a 16.15±1.31a 171.0±2.41a
139.3±17.6a 91.50±0.80ab 18.69±0.20ab 12.02±0.25b 150.7±0.96b
155.5±33.1a 91.57±0.30ab 18.29±1.01ab 11.66±1.30b 143.7±7.42bc
120.7±21.3a 91.45±0.61b 16.45±0.65bc 9.84±0.62bc 134.8±6.10bcd
7.0 mM
10.5 mM
15.0 mM
110.9±21.1a 90.35±0.59b 14.12±0.64b 8.42±0.27b 148.9±5.31b
110.0±21.5a 90.07±0.49b 14.28±0.94b 8.72±0.60b 142.2±0.75bc
104.1±32.0a 90.00±0.61b 14.02±0.71b 8.65±0.95b 128.3±4.35c
134.1±10.4a 91.72±0.32ab 16.05±0.23bc 10.24±0.34bc 131.3±10.05cd
138.2±16.6a 91.39±0.68b 15.98±0.01bc 10.00±0.35bc 121.4±7.71d
128.2±26.3a 91.84±0.59ab 14.28±0.94c 8.72±0.60c 119.7±3.86d
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Z. Dučaiová et al. monitored by MDA. In both cultivars, MDA displayed significant changes in all concentrations (Fig. 1C). The increment was more visible in tetraploid plants (more than 3 times). Effect of SA on secondary metabolites content Salicylate treatment stimulated the accumulation of coumarin-related compounds. The variability of the accumulation is outlined in Fig. 2. In diploid cultivar, the content of Z-GMCA (Fig. 2A) increased continuously around 30%, from value 10.23 to 14.35 mg g−1 DW and the content of E-GMCA (Fig. 2B) increased more than 40%, from 7.93 to 14.27 mg g−1 DW in 15.0 mM concentration. The changes in the content of Z- and E-GMCA in tetraploid cultivars were more visible, from value of 7.65 to 12.39 mg g−1 DW and from value of 6.77 to 8.61 mg g−1 DW, respectively. The increase was more than 20% in both cases. The content of herniarin (Fig. 2C) was found to increase significantly, from 0.89 to 2.68 mg g−1 DW in diploid and from 1.21 to 2.04 mg g−1 DW in tetraploid plants. The significant increase of umbelliferone (Fig. 2D) was detected, from value 0.013 to 0.072 mg g−1 DW in diploid and from value 0.015 to 0.056 mg g−1 DW in tetraploid one. The content of chlorogenic acid (Fig. 2E) simultaneously decreased from value 0.07 to 0.03 mg g−1 DW in diploid plants. On the contrary, the decreasing in tetraploid plant was less visible, from value 0.05 to 0.03 mg g−1 DW. As for the ene-yne-dicycloether (Fig. 2F), the significant increase in both cultivars was observed, from value 1.98 to 7.72 mg g−1 DW in diploid and from 2.26 to 8.61 mg g−1 DW in tetraploid plants. The results showed that exogenous application of SA can trigger the accumulation of coumarin-related compounds and depression of chlorogenic acid in Matricaria chamomilla. Discussion
Fig. 1 The influence of foliar application of different concentration of SA on hydrogen peroxide (A), superoxide radical (B) and malondialdehyde (C) content in rosette leaves of Matricaria chamomilla plants after 48 hours exposition.
in chlorophylls content was detected. The amount of chlorophyll a decreased up to 17% for diploid and up to 30% for tetraploid plants and the chlorophyll b amount decreased up to 21% for diploid and up to 46% for tetraploid one. Both, hydrogen peroxide and superoxide radical were simultaneously enhanced in treated plants. In case of hydrogen peroxide (Fig. 1A), more visible increase was observed in diploid plants (more than 4.8 times), in comparison to the tetraploid ones (only 1.4 times). The increase of superoxide radical content (Fig. 1B) was relatively similar in both cultivars. The enhanced amount of reactive oxygen species led to the membrane damage,
Salicylic acid is known to affect various biological and biochemical activities of plants and may play a key role in regulating their growth and productivity. Depending on its concentration, SA may play role as a stress factor or may provide protection against certain biotic and abiotic stresses (Horváth et al. 2007). After 48 hours of treatment, changes in biomass accumulation were observed. The similar pattern of biomass accumulation was recorded in foliar sprayed fennel (Hashmi et al. 2012) or pepper plants (Elwan & El-Hamahmy 2009). Visible increase of biomass was also detected in chamomile plants continuously cultivated in medium enriched by 2 mM SA (Pastírová et al. 2004) or with low concentration of SA (250 µM) (Kováčik et al. 2009). The similar influence of variable doses of SA on growth can be explained by different mode of application, but the time of exposure has also important impact. Recent studies suggest that environmental stresses can increase the oxygen-induced damage to cells due to
Regulation of secondary metabolites content in chamomile leaves
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Fig. 2. The influence of foliar application of different concentration of SA on selected secondary metabolite content in rosette leaves of Matricaria chamomilla plants after 48 hours exposition. A – Z-GMCA; B – E-GMCA; C – herniarin; D – umbelliferone; E – chlorogenic acid; F – ene-yne-dicycloether.
increased generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2 O2 ) and superoxide radical (. O− 2 ). The literature contains evidence of SAinduced effects on the rate of generation of ROS. At relatively low concentration (0.05–0.5 mM), SA acts a moderate oxidative damage similar to that of stressacclimating processes. Higher concentrations can lead to their accumulation and potentially interact with many cellular biomolecules like lipids, proteins and nucleic acid (Horváth et al. 2007; Hayat et al. 2010). Mungbean treated with lower concentration of SA caused no strong changes in accumulation of hydrogen peroxide (Nazar et al. 2011). On the other hand, our results showed that high doses of SA induced more visible oxidative stress. The incensement was also ob-
served in rice seedlings (Ganesan & Thomas 2001). It is known that ROS are harmful to all membrane constituents, resulting in an increased lipid peroxidation. Continuous increase of MDA, the indicator of membrane damage level, with increasing SA concentration is consistent with the work of López-Orenes et al. (2013), but low dosage of SA in Phaseolus vulgaris showed no changes (Palma et al. 2009). Photosynthetic pigments are considered to be highly sensitive to ROS, too. An increase in oxidative stress may cause decrease in total chlorophylls content. In our experiment, the higher SA concentration led to the decline in chlorophylls content. In contrast, the amount of total carotenoids increased (data not shown). The similar pattern was observed in monocot (wheat) and dicot (moong) plants. The rise of
908 carotenoid content might be explained by their protect function from oxidative stress (Moharekar et al. 2003). The production and accumulation of phenolic compounds, including coumarins, in response to stress agents has been found in various plant species. These include, e.g. caffeic acid and salvianolic acid from Salia miltiorrhiza (Dong et al. 2010), phenolic acids (cichoric acid and chlorogenic acid) from Rauvolfia serpentine (Nair et al. 2013), p-coumaroylmalic acid from Thunbergia alata (Housti et al. 2002). Due to their stress-related accumulation phenols are considered as stress metabolites. Stress metabolites were found to be elicited in plants by biotic agents (fungi, bacteria, viruses or animals) and abiotic agents (salts of heavy metals, irradiation of UV light, some plant growth regulators, such as ethylene or salicylic acid and wounding of the tissues) (Grayer & Kokubun 2001). The quantitative changes of herniarin and its precursor GMCA differ from previous work. Application of 1 mM and 2 mM SA into the Knop’s solution (Pastírová et al. 2004) as well as CuCl2 treatment (Repčák et al. 2001a) led simultaneously to an increase of herniarin and a decrease of its glucoside precursor, GMCA. However, Kováčik et al. (2009) reported their enhanced accumulation after addition of small doses of SA. The accumulation of a monosubstituted coumarin umbelliferone, considered as a stress metabolite, depended on increasing SA concentration. These results are in accord with the time course studies which show the highest accumulation of umbelliferone after 72 h in case of SA elicitation (Pastírová et al. 2004). The visible increase was detected after 48 h of methyl jasmonate elicitation, and the same trend was observed after biotic elicitation of Echinothrips americanus (Repčák & Suvák, 2013). Polyacetylenes of spiroketal enol ether type are known secondary metabolites synthesized by Asteraceae plants under stress condition. The SA-treatment enhanced the accumulation of ene-yne-dicycloethers. In previous study, the opposite trend was observed in transformed root culture of feverfew treated with 0.3mM SA (Stojakowska et al. 2002) or in CuCl2 treated chamomile plant (Eliašová et al 2004). However, in contrast, biotic elicitation led to the increase of their productivity (Repčák et al. 2001a). A group of control plants were left together with treated plants. After 48 h the increment of umbelliferone (data not shown) was noticed in both cultivars in comparison with control plants analysed before the start of SA treatment. Other studied metabolites were not significantly affected. It can be explained by air signalling of SA, e.g. volatile methyl salicylate, which is important signal in systemic acquired resistance (Ding et al. 2002). Polyploidy is one of the methods allowing to gain an increased vigour with higher biomass and adaptability of plant to the environment. This is also the case of chamomile, when tetraploid cultivar (Lutea) in this study showed higher biomass accumulation in comparison with diploid cultivar (Novbona). The comparison of content of coumarin-related compounds in non-sprayed
Z. Dučaiová et al. diploid and tetraploid plants revealed evident differences between cultivars. The content of Z-GMCA was found to be approximately two times higher in diploid plants and on the contrary, the content of umbelliferone was almost 3 times higher in tetraploid one. The increasing trend of studied metabolites was more evident for tetraploid cultivar. These data are in accordance with previous work where the content of coumarins was higher in diploid cultivars when compared to the tetraploid ones (Eliašová et al. 2004). In conclusion, SA is a plant signal molecule leading to changes at different level of plant metabolism in response to environmental stresses. In the present study, the tested SA doses showed stimulated growthpromoting responses, but simultaneously the content of chlorophylls and soluble proteins decreased. These responses may be explained by mechanism of oxidative stress induced by high doses of SA. SA also stimulated the biosynthesis of defence compounds of phenylpropanoid pathway which led to the accumulation of coumarin-related compounds. Acknowledgements This study was financially supported by Slovak Grand Agency (VEGA 1/0122/09) and the UPJS internal grant system VVGS PF 8/2011/B. We thank Mrs Anna Michalčová and Mrs Margita Buzinkaiová for their valuable technical assistance. References Bradford M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254. Chaman M.E., Copaja S.V. & Argandon V.H. 2003. Relationships between salicylic acid content, phenylalanine ammonia-lyase (PAL) activity and resistance of barley to aphid infestation. J. Agric. Food. Chem. 51: 2227–2231. Ding C.K., Wang C.Y. & Gross K.C. 2002. Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit. Planta 214: 895–901. Dong J., Wan G. & Liang Z. 2010. Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture. J. Biotechnol. 148: 99–104. Eliašová A., Repčák M. & Pastírová A. 2004. Quantitative changes of secondary metabolites of Matricaria chamomilla by abiotic stress. Z. Naturforsch. 59c: 543–548 Elstner E.F. & Heupel A. 1976. Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase. Anal. Biochem. 70: 616–620. Elwan M.W.M. & El-Hamahmy M.A.M. 2009. Improved productivity and quality associated with salicylic acid application in greenhouse pepper. Sci. Hort. 122: 521–526. Fraissinet-Tachet L., Baltz R., Chong J., Kauffmann S., Fritig B. & Saindrenan P. 1998. Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid. FEBS Lett. 437: 319–323. Franke R. & Schilcher H. 2005. Chamomila: Industrial profile. Taylor &Francis, New York, 289 pp. Ganesan V. & Thomas G. 2001. Salicylic acid responses in rice: influence of salicylic acid on H2 O2 accumulation and oxidative stress. Plant Sci. 160: 1095–1106.
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