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Methyl jasmonate and Echinothrips americanus regulate coumarin accumulation in leaves of Matricaria chamomilla
Biochemical Systematics and Ecology 47 (2013) 38–41
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Methyl jasmonate and Echinothrips americanus regulate coumarin accumulation in leaves of Matricaria chamomilla Miroslav Rep cák a, *, Martin Suvák b a b
Institute of Biology and Ecology, Faculty of Science, P. J. Safárik University, Mánesova 23, 041 67 Kosice, Slovak Republic Botanical Garden, P. J. Safárik University, Mánesova 23, 043 52 Kosice, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2012 Accepted 27 October 2012 Available online 8 December 2012
Matricaria chamomilla synthesize (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), the biosynthetic precursors of herniarin. During leaf development the content of (E)-GMCA decreased and no changes were found in the content of other compounds evaluating in the experiments. Methyl jasmonate elicits an increase in (E)GMCA and herniarin content in young growing leaves as well as mature and senescing leaves 48 h after treatment. In contrast, attack by the thrips, Echinothrips americanus, causes increase not only in GMCAs and herniarin content but also in the amount of coumarin, umbelliferone. The results confirmed that coumarin-like metabolites can be considered as plant defence compounds in biotic and abiotic stress conditions. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Matricaria chamomilla Echinothrips americanus Plant–pathogen interaction Methyl jasmonate (Z)- and (E)-2-ß-D-glucopyranosyloxy-4methoxy cinnamic acid Herniarin Umbelliferone
1. Introduction Plants are exposed to environmental abiotic stresses as well as being the target of various pathogens, including herbivorous arthropods. In addition to plant constitutive defences, stress inducible pathways are switched on specifically upon attack. Plant hormones such as salicylic acid, jasmonates and ethylene are the primary signalling molecules inducing defence mechanisms by activating several metabolic pathways involved in the stress response (Heil, 2009; Rasmann and Agrawal, 2009). The salicylic acid pathway is primarily linked to resistance to biotrophic pathogens, whereas the jasmonate and ethylene pathways mediate resistance to necrotrophic pathogens and wounding (López et al., 2008). The plant hormone jasmonate and its derivatives regulate various aspects of plant growth and development and play a critical role in plant responses to wounding by directly activating several mechanisms involved in healing and further defences (Browse and Howe, 2008). Exogenous application of methyl jasmonate leads to increase in the content of secondary metabolites with defensive function. Coumarins are found in some higher plants and are known to contribute to the resistance of some species to biotic and abiotic stresses (Bourgaud et al., 2006). Thrips (Thysanoptera, Thripidae) are among the most dangerous insect herbivores of cultivated plants. Echinothrips americanus Morgan is a pest species native to the eastern parts of North America, but it was recorded in Europe in 1989 and now it is present in Slovakia as well (Varga and Fedor, 2008). E. americanus is a polyphagous species forming large populations on leaves. Its life cycle is temperature dependent, and lasts 33.9 days at 20 C, but only about a third of the time (11.4 days) at 30 C (Oetting and Beshear, 1994). Infestation by thrips affects morphology of the leaf surface (Scott Brown and Simmonds,
* Corresponding author. Tel.: þ421 55 234 2310; fax: þ421 55 6337 353. E-mail address: [email protected] (M. Rep cák). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.10.009
M. Repcák, M. Suvák / Biochemical Systematics and Ecology 47 (2013) 38–41
39
2006). Application of jasmonates enhances plant resistance to thrips in Arabidopsis thaliana and Brassica rapa (Abe et al., 2008). In this paper we studied the effect of methyl jasmonate as the chemical elicitor of the accumulation of glucosidic precursors of herniarin (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA) and coumarin aglycones in developing leaves of Matricaria chamomilla. The second aim of this paper was to determine the influence of E. americanus on the quantity of the above-mentioned secondary metabolites in M. chamomilla leaves, as well as the simultaneous effect of both of these stress factors. 2. Material and methods 2.1. Cultivation of plants and experimental design Tetraploid chamomile variety (M. chamomilla L. cv. ‘Lutea’) was used in all experiments (Oravec et al., 2005). Seedlings were sown and pre-cultivated in sand. Two-week-old plants were transferred to soil in plastic pots. Plants were irrigated daily to 60% of the soil water-holding capacity until they reached desired age for experiments. The cultivation and experimental set-ups were performed under controlled light conditions: 12 h light:12 h dark (12:12 LD) regime and PAR 210 mmol m2 s1. To establish an effect of hormone methyl jasmonate (MEJA) on developing leaves twelve-week-old plants (dry weight 0.347 0.186 g) were sprayed with a water solution of 0.01% MEJA (Aldrich) in 0.1% of Tween 20. Control plants were treated with 0.1% Tween 20 only. The experimental plants were placed under a glass bell because MEJA is volatile. The plants were treated with MEJA for 48 h and then leaf rosettes were collected for analysis. Specimens of E. americanus Morgan were collected on the leaves of Acalypha hispida Burm. f. in greenhouses of the Botanical Garden, P. J. Safárik University in Kosice. Using a pooter, 2 200 images of this thrips species were picked up in two plastic cylinders. They were released under the glass bells that covered the experimental plants. Leaf rosettes of nine-weekold plants with average dry weight 0.061 0.027 g were used in the experiments with MEJA and E. americanus. The effect of both factors was tested in the course of 96 h. Four individual plants were tested for each parameter. Immediately after the experiment the leaf rosettes of plants were dried at 105 C and stored in a desiccator. 2.2. HPLC quantification of metabolites The content of (Z)- and (E)-GMCA, herniarin and umbelliferone was analysed using a gradient HPLC as described previously (Rep cák et al., 2001). Herniarin (Extrasynthése) and umbelliferone (Sigma–Aldrich) standard compounds were used. (Z)- and (E)- GMCA and were isolated from a chamomile leaf extract by semipreparative HPLC and their identity was assessed by 1H, 13C NMR spectroscopy. The quantification of (Z)- and (E)-GMCA was performed by qNMR method (Petrul’ová-Poracká et al., in preparation). Quantitative data were analysed using ANOVA and t-test. 3. Results 3.1. Effect of methyl jasmonate on developing leaves The content of (E)-GMCA decreases in the leaves of control plants during their maturation, whereas the content of other evaluated compounds did not change significantly during leaf development (Table 1). After spraying the leaves with MEJA a significant increase in (E)-GMCA and herniarin was observed in young, mature and senescing leaves after 48 h (Table 1). The
Table 1 The influence of methyl jasmonate (MEJA) spraying on (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), herniarin and umbelliferone [mg g1] accumulation in developing rosette leaves of Matricaria chamomilla after 48 h exposition. x – average; Sx – standard deviation. Growing leaves
Z-GMCA E-GMCA Herniarin Umbelliferone
Mature leaves
Senescing leaves
Control
MEJA
t-test
Control
MEJA
t-test
Control
MEJA
t-test
x
x
t-value
x
x
t-value
x
x
t-value
Sx
Sx
P
Sx
Sx
P
Sx
Sx
P
4.21 0.285 2.832 0.822 0.26 0.03 0.029 0.004
4.735 0.508 9.998 1.78 1.052 0.381 0.057 0.011
1.543 0.198 7.254 <0.001 3.587 0.023 4.088 0.015
4.286 0.069 1.389 0.222 0.294 0.045 0.031 0.003
5.01 0.859 4.905 1.254 1.114 0.077 0.055 0.01
1.455 0.219 5.674 0.002 15.923 <0.001 3.985 0.016
4.22 0.445 0.978 0.071 0.203 0.03 0.051 0.02
4.751 0.379 2.181 0.285 0.422 0.054 0.042 0.013
1.574 0.191 8.366 <0.001 6.104 0.004 0.664 0.543
Bold signifies the main results.
M. Repcák, M. Suvák / Biochemical Systematics and Ecology 47 (2013) 38–41
40
Table 2 The influence of methyl jasmonate (MEJA) spraying and Echinothrips americanus on (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), herniarin and umbelliferone [mg g1] accumulation in rosette leaves of Matricaria chamomilla after 96 h exposition. x – average; Sx – standard deviation.
Z-GMCA E-GMCA Herniarin Umbelliferone
Control
MEJA
t-test
Thrips
t-test
MEJA þ thrips
t-test
x
x
t-value
x
t-value
x
t-value
Sx
Sx
P
Sx
P
Sx
P
5.813 2.401 3.307 0.795 0.122 0.031 0.027 0.006
8.464 1.285 8.394 1.420 0.198 0.007 0.013 0.005
2.249 0.059 6.853 <0.001 4.810 0.003 3.918 0.008
9.037 2.701 10.853 2.304 0.222 0.054 0.062 0.012
2.048 0.080 6.928 <0.001 3.231 0.018 5.275 0.002
8.687 1.556 18.425 3.946 0.270 0.096 0.050 0.025
2.317 0.054 8.498 <0.001 2.954 0.026 1.756 0.130
Bold signifies the main results.
content of umbelliferone in the same experiment increased only in growing and mature leaves, changes in senescing leaves were not significant in comparison with the control (Table 1). Exposure of chamomile rosette leaves to MEJA for 96 h in next experiment caused significant increase in (Z)- and (E)-GMCA and herniarin and decrease in umbelliferone content (Table 2). 3.2. The effect of E. americanus Exposure to large number of thrips led to visible changes in experimental plants in the course of 96 h. The leaves had silver and yellow-brownish irregular diffuse spots and were distorted. Later, individual leaves started to die. The rosette leaves damaged by thrips had a significant increase in (E)-GMCA, herniarin and umbelliferone content (Table 2). The simultaneous application of MEJA and thrips caused significant (Z)-GMCA increase in comparison with rosette leaves of control plants, but not in comparison with the leaves treated by MEJA or thrips separately (Table 2). The increase in (E)-GMCA content by both factors simultaneously was approximately equal to the sum of the separate increases. The content of herniarin increased significantly in comparison with the control plants, in contrast to the minimal effect of both factors on the umbelliferone content (Table 2). 4. Discussion Several phenylpropanoid compounds including coumarins were previously identified as plant defence compounds (Dixon and Paiva, 1995). In the leaves of M. chamomilla two precursors of herniarin, (Z)- and (E)-GMCA, are synthesized, which are also components of the plant’s constitutive defences (Rep cák et al., 2001). These conjugate compounds are stored in the vacuoles. The accumulation of coumarin aglycones herniarin and umbelliferone in the leaves of M. chamomilla is regulated by various signals. Under non-stress conditions the levels of herniarin and umbelliferone are low. The quantity of GMCAs and herniarin oscillates during the day/night cycle and is regulated by the circadian clock (Repcák et al., 2009). Under stress conditions, a significant increase of the levels of coumarin aglycones was observed. After spraying the leaves with the aqueous solution of CuCl2, disruption of cellular membrane compartmentalization occurred and b-glucosidase in contact with (Z)GMCA released the instable aglycone, which is converted spontaneously into herniarin. Decrease in GMCA level was connected with an increase in herniarin content (Rep cák et al., 2001). Stress conditions which did not significantly damage the membranes, such as nitrogen deficiency, increased GMCAs and herniarin content simultaneously (Ková cik et al., 2007). Jasmonates are plant stress hormones which induce various plant defence responses, including the biosynthesis of protective secondary metabolites. MEJA induced the increase of the production of various secondary metabolites in several plant species, e.g. alkaloid pilocarpine in the leaves of Pilocarpus jaborandi (Avancini et al., 2003), N-(E)-4coumaroylputrescine and N-(E)-4-coumaroylagmatine in barley leaves (Lee et al., 1997), kalopanaxsaponin in Nigella sativa (Scholz et al., 2009) or sesquiterpene lactone artemisinin content in Artemisia annua (Wang et al., 2010). Data presented here are consistent with results of earlier studies. In response to insect attack, many plants undergo dynamic biochemical changes, leading to the induction of direct and indirect defence mechanisms. Jasmonate induced signalling pathways play a prominent and consistent role in promoting plant resistance to herbivores (Browse and Howe, 2008). In this study, methyl jasmonate as well as E. americanus evoked dramatically increased content of GMCAs. However, accumulation of umbelliferone and herniarin was affected only slightly either by E. americanus or methyl jasmonate. Acknowledgements This work was supported by the grant agency VEGA (grant no. 1/0122/09). We thank Mrs. Anna Michal cová and Mrs. Margita Buzinkaiová for their valuable technical assistance.
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References Abe, H., Ohnishi, J., Narusaka, M., Seo, S., Narusaka, Y., Tsuda, S., Kobayashi, M., 2008. Function of jasmonate in response and tolerance of Arabidopsis to thrips feeding. Plant Cell Physiol. 49 (1), 68–80. Avancini, G., Abreu, I.N., Saldaña, M.D.A., Mohamed, R.S., Mazzafera, P., 2003. Induction of pilocarpine formation in jaborandi leaves by salicylic acid and methyljasmonate. Phytochemistry 63 (2), 171–175. Bourgaud, F., Hehn, A., Larbat, R., Doerper, S., Gontier, E., Kellner, S., Matern, U., 2006. Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem. Rev. 5 (2–3), 293–308. Browse, J., Howe, G.A., 2008. New weapons and a rapid response against insect attack. Plant Physiol. 146 (3), 832–838. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7 (7), 1085–1097. Heil, M., 2009. Damaged-self recognition in plant herbivore defence. Trends Plant Sci. 14 (7), 356–363. Ková cik, J., Klejdus, B., Ba ckor, M., Rep cák, M., 2007. Phenylalanine ammonia-lyase activity and phenolic compounds accumulation in nitrogen-deficient Matricaria chamomilla leaf rosettes. Plant Sci. 172 (2), 393–399. Lee, J., Vogt, T., Schmidt, J., Parthier, B., Löbler, M., 1997. Methyljasmonate-induced accumulation of coumaroyl conjugates in barley leaf segments. Phytochemistry 44 (4), 582–592. López, M.A., Bannenberg, G., Castresana, C., 2008. Controlling hormone signaling is a plant and pathogen challenge for growth and survival. Curr. Opin. Plant Biol. 11 (4), 420–427. Oravec, V., Oravec Jr., V., Rep cák, M., Sebo, L., Jedinák, D., Varga, I., 2005. Cultivation experiences in Slovakia. In: Franke, R., Schilcher, H. (Eds.), Chamomile Industrial Profiles. Taylor & Francis, Boca Raton, pp. 121–139. Oetting, R.D., Beshear, R.J., 1994. Biology of greenhouse pest Echinothrips americanus Morgan (Thysanoptera: Thripidae). Zoology (J. Pure Appl. Zool.) 4, 307–315. Petrul’ová-Poracká, V., Rep cák, M., Vilková, M., Imrich, J., Coumarins of Matricaria chamomilla L.: aglycons and glycosides. in preparation. Rasmann, S., Agrawal, A.A., 2009. Plant defense against herbivory: progress in identifying synergism, redundancy, and antagonism between resistance traits. Curr. Opin. Plant Biol. 12 (4), 473–478. Rep cák, M., Imrich, J., Franeková, M., 2001. Umbelliferone, a stress metabolite of Chamomilla recutita (L.) Rauschert. J. Plant Physiol. 158 (8), 1085–1087. Rep cák, M., Smajda, B., Ková cik, J., Eliasová, A., 2009. Circadian rhythm of (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acids and herniarin in leaves of Matricaria chamomilla. Plant Cell Rep. 28 (7), 1137–1143. Scholz, M., Lipinski, M., Leupold, M., Luftmann, H., Harig, L., Ofir, R., Fischer, R., Prüfer, D., Müller, K.J., 2009. Methyl jasmonate induced accumulation of kalopanaxsaponin I in Nigella sativa. Phytochemistry 70 (4), 517–522. Scott Brown, A.S., Simmonds, M.S.J., 2006. Leaf morphology of hosts and nonhosts of the thrips Heliothrips haemorrhoidalis (Bouché). Bot. J. Linnean Soc. 152 (1), 109–130. Varga, L., Fedor, P.J., 2008. First interception of the greenhouse pest Echinothrips americanus Morgan, 1913 (Thysanoptera: Thripidae) in Slovakia. Plant Protect. Sci. 44, 155–158. Wang, H., Ma, C., Li, Z., Ma, L., Wang, H., Ye, H., Xu, G., Liu, B., 2010. Effects of exogenous methyl jasmonate on artemisinin biosynthesis and secondary metabolites in Artemisia annua L. Ind. Crops Prod. 31 (2), 214–218.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Methyl jasmonate and Echinothrips americanus regulate coumarin accumulation in leaves of Matricaria chamomilla Miroslav Rep cák a, *, Martin Suvák b a b
Institute of Biology and Ecology, Faculty of Science, P. J. Safárik University, Mánesova 23, 041 67 Kosice, Slovak Republic Botanical Garden, P. J. Safárik University, Mánesova 23, 043 52 Kosice, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2012 Accepted 27 October 2012 Available online 8 December 2012
Matricaria chamomilla synthesize (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), the biosynthetic precursors of herniarin. During leaf development the content of (E)-GMCA decreased and no changes were found in the content of other compounds evaluating in the experiments. Methyl jasmonate elicits an increase in (E)GMCA and herniarin content in young growing leaves as well as mature and senescing leaves 48 h after treatment. In contrast, attack by the thrips, Echinothrips americanus, causes increase not only in GMCAs and herniarin content but also in the amount of coumarin, umbelliferone. The results confirmed that coumarin-like metabolites can be considered as plant defence compounds in biotic and abiotic stress conditions. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Matricaria chamomilla Echinothrips americanus Plant–pathogen interaction Methyl jasmonate (Z)- and (E)-2-ß-D-glucopyranosyloxy-4methoxy cinnamic acid Herniarin Umbelliferone
1. Introduction Plants are exposed to environmental abiotic stresses as well as being the target of various pathogens, including herbivorous arthropods. In addition to plant constitutive defences, stress inducible pathways are switched on specifically upon attack. Plant hormones such as salicylic acid, jasmonates and ethylene are the primary signalling molecules inducing defence mechanisms by activating several metabolic pathways involved in the stress response (Heil, 2009; Rasmann and Agrawal, 2009). The salicylic acid pathway is primarily linked to resistance to biotrophic pathogens, whereas the jasmonate and ethylene pathways mediate resistance to necrotrophic pathogens and wounding (López et al., 2008). The plant hormone jasmonate and its derivatives regulate various aspects of plant growth and development and play a critical role in plant responses to wounding by directly activating several mechanisms involved in healing and further defences (Browse and Howe, 2008). Exogenous application of methyl jasmonate leads to increase in the content of secondary metabolites with defensive function. Coumarins are found in some higher plants and are known to contribute to the resistance of some species to biotic and abiotic stresses (Bourgaud et al., 2006). Thrips (Thysanoptera, Thripidae) are among the most dangerous insect herbivores of cultivated plants. Echinothrips americanus Morgan is a pest species native to the eastern parts of North America, but it was recorded in Europe in 1989 and now it is present in Slovakia as well (Varga and Fedor, 2008). E. americanus is a polyphagous species forming large populations on leaves. Its life cycle is temperature dependent, and lasts 33.9 days at 20 C, but only about a third of the time (11.4 days) at 30 C (Oetting and Beshear, 1994). Infestation by thrips affects morphology of the leaf surface (Scott Brown and Simmonds,
* Corresponding author. Tel.: þ421 55 234 2310; fax: þ421 55 6337 353. E-mail address: [email protected] (M. Rep cák). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.10.009
M. Repcák, M. Suvák / Biochemical Systematics and Ecology 47 (2013) 38–41
39
2006). Application of jasmonates enhances plant resistance to thrips in Arabidopsis thaliana and Brassica rapa (Abe et al., 2008). In this paper we studied the effect of methyl jasmonate as the chemical elicitor of the accumulation of glucosidic precursors of herniarin (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA) and coumarin aglycones in developing leaves of Matricaria chamomilla. The second aim of this paper was to determine the influence of E. americanus on the quantity of the above-mentioned secondary metabolites in M. chamomilla leaves, as well as the simultaneous effect of both of these stress factors. 2. Material and methods 2.1. Cultivation of plants and experimental design Tetraploid chamomile variety (M. chamomilla L. cv. ‘Lutea’) was used in all experiments (Oravec et al., 2005). Seedlings were sown and pre-cultivated in sand. Two-week-old plants were transferred to soil in plastic pots. Plants were irrigated daily to 60% of the soil water-holding capacity until they reached desired age for experiments. The cultivation and experimental set-ups were performed under controlled light conditions: 12 h light:12 h dark (12:12 LD) regime and PAR 210 mmol m2 s1. To establish an effect of hormone methyl jasmonate (MEJA) on developing leaves twelve-week-old plants (dry weight 0.347 0.186 g) were sprayed with a water solution of 0.01% MEJA (Aldrich) in 0.1% of Tween 20. Control plants were treated with 0.1% Tween 20 only. The experimental plants were placed under a glass bell because MEJA is volatile. The plants were treated with MEJA for 48 h and then leaf rosettes were collected for analysis. Specimens of E. americanus Morgan were collected on the leaves of Acalypha hispida Burm. f. in greenhouses of the Botanical Garden, P. J. Safárik University in Kosice. Using a pooter, 2 200 images of this thrips species were picked up in two plastic cylinders. They were released under the glass bells that covered the experimental plants. Leaf rosettes of nine-weekold plants with average dry weight 0.061 0.027 g were used in the experiments with MEJA and E. americanus. The effect of both factors was tested in the course of 96 h. Four individual plants were tested for each parameter. Immediately after the experiment the leaf rosettes of plants were dried at 105 C and stored in a desiccator. 2.2. HPLC quantification of metabolites The content of (Z)- and (E)-GMCA, herniarin and umbelliferone was analysed using a gradient HPLC as described previously (Rep cák et al., 2001). Herniarin (Extrasynthése) and umbelliferone (Sigma–Aldrich) standard compounds were used. (Z)- and (E)- GMCA and were isolated from a chamomile leaf extract by semipreparative HPLC and their identity was assessed by 1H, 13C NMR spectroscopy. The quantification of (Z)- and (E)-GMCA was performed by qNMR method (Petrul’ová-Poracká et al., in preparation). Quantitative data were analysed using ANOVA and t-test. 3. Results 3.1. Effect of methyl jasmonate on developing leaves The content of (E)-GMCA decreases in the leaves of control plants during their maturation, whereas the content of other evaluated compounds did not change significantly during leaf development (Table 1). After spraying the leaves with MEJA a significant increase in (E)-GMCA and herniarin was observed in young, mature and senescing leaves after 48 h (Table 1). The
Table 1 The influence of methyl jasmonate (MEJA) spraying on (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), herniarin and umbelliferone [mg g1] accumulation in developing rosette leaves of Matricaria chamomilla after 48 h exposition. x – average; Sx – standard deviation. Growing leaves
Z-GMCA E-GMCA Herniarin Umbelliferone
Mature leaves
Senescing leaves
Control
MEJA
t-test
Control
MEJA
t-test
Control
MEJA
t-test
x
x
t-value
x
x
t-value
x
x
t-value
Sx
Sx
P
Sx
Sx
P
Sx
Sx
P
4.21 0.285 2.832 0.822 0.26 0.03 0.029 0.004
4.735 0.508 9.998 1.78 1.052 0.381 0.057 0.011
1.543 0.198 7.254 <0.001 3.587 0.023 4.088 0.015
4.286 0.069 1.389 0.222 0.294 0.045 0.031 0.003
5.01 0.859 4.905 1.254 1.114 0.077 0.055 0.01
1.455 0.219 5.674 0.002 15.923 <0.001 3.985 0.016
4.22 0.445 0.978 0.071 0.203 0.03 0.051 0.02
4.751 0.379 2.181 0.285 0.422 0.054 0.042 0.013
1.574 0.191 8.366 <0.001 6.104 0.004 0.664 0.543
Bold signifies the main results.
M. Repcák, M. Suvák / Biochemical Systematics and Ecology 47 (2013) 38–41
40
Table 2 The influence of methyl jasmonate (MEJA) spraying and Echinothrips americanus on (Z)- and (E)-2-ß-D-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), herniarin and umbelliferone [mg g1] accumulation in rosette leaves of Matricaria chamomilla after 96 h exposition. x – average; Sx – standard deviation.
Z-GMCA E-GMCA Herniarin Umbelliferone
Control
MEJA
t-test
Thrips
t-test
MEJA þ thrips
t-test
x
x
t-value
x
t-value
x
t-value
Sx
Sx
P
Sx
P
Sx
P
5.813 2.401 3.307 0.795 0.122 0.031 0.027 0.006
8.464 1.285 8.394 1.420 0.198 0.007 0.013 0.005
2.249 0.059 6.853 <0.001 4.810 0.003 3.918 0.008
9.037 2.701 10.853 2.304 0.222 0.054 0.062 0.012
2.048 0.080 6.928 <0.001 3.231 0.018 5.275 0.002
8.687 1.556 18.425 3.946 0.270 0.096 0.050 0.025
2.317 0.054 8.498 <0.001 2.954 0.026 1.756 0.130
Bold signifies the main results.
content of umbelliferone in the same experiment increased only in growing and mature leaves, changes in senescing leaves were not significant in comparison with the control (Table 1). Exposure of chamomile rosette leaves to MEJA for 96 h in next experiment caused significant increase in (Z)- and (E)-GMCA and herniarin and decrease in umbelliferone content (Table 2). 3.2. The effect of E. americanus Exposure to large number of thrips led to visible changes in experimental plants in the course of 96 h. The leaves had silver and yellow-brownish irregular diffuse spots and were distorted. Later, individual leaves started to die. The rosette leaves damaged by thrips had a significant increase in (E)-GMCA, herniarin and umbelliferone content (Table 2). The simultaneous application of MEJA and thrips caused significant (Z)-GMCA increase in comparison with rosette leaves of control plants, but not in comparison with the leaves treated by MEJA or thrips separately (Table 2). The increase in (E)-GMCA content by both factors simultaneously was approximately equal to the sum of the separate increases. The content of herniarin increased significantly in comparison with the control plants, in contrast to the minimal effect of both factors on the umbelliferone content (Table 2). 4. Discussion Several phenylpropanoid compounds including coumarins were previously identified as plant defence compounds (Dixon and Paiva, 1995). In the leaves of M. chamomilla two precursors of herniarin, (Z)- and (E)-GMCA, are synthesized, which are also components of the plant’s constitutive defences (Rep cák et al., 2001). These conjugate compounds are stored in the vacuoles. The accumulation of coumarin aglycones herniarin and umbelliferone in the leaves of M. chamomilla is regulated by various signals. Under non-stress conditions the levels of herniarin and umbelliferone are low. The quantity of GMCAs and herniarin oscillates during the day/night cycle and is regulated by the circadian clock (Repcák et al., 2009). Under stress conditions, a significant increase of the levels of coumarin aglycones was observed. After spraying the leaves with the aqueous solution of CuCl2, disruption of cellular membrane compartmentalization occurred and b-glucosidase in contact with (Z)GMCA released the instable aglycone, which is converted spontaneously into herniarin. Decrease in GMCA level was connected with an increase in herniarin content (Rep cák et al., 2001). Stress conditions which did not significantly damage the membranes, such as nitrogen deficiency, increased GMCAs and herniarin content simultaneously (Ková cik et al., 2007). Jasmonates are plant stress hormones which induce various plant defence responses, including the biosynthesis of protective secondary metabolites. MEJA induced the increase of the production of various secondary metabolites in several plant species, e.g. alkaloid pilocarpine in the leaves of Pilocarpus jaborandi (Avancini et al., 2003), N-(E)-4coumaroylputrescine and N-(E)-4-coumaroylagmatine in barley leaves (Lee et al., 1997), kalopanaxsaponin in Nigella sativa (Scholz et al., 2009) or sesquiterpene lactone artemisinin content in Artemisia annua (Wang et al., 2010). Data presented here are consistent with results of earlier studies. In response to insect attack, many plants undergo dynamic biochemical changes, leading to the induction of direct and indirect defence mechanisms. Jasmonate induced signalling pathways play a prominent and consistent role in promoting plant resistance to herbivores (Browse and Howe, 2008). In this study, methyl jasmonate as well as E. americanus evoked dramatically increased content of GMCAs. However, accumulation of umbelliferone and herniarin was affected only slightly either by E. americanus or methyl jasmonate. Acknowledgements This work was supported by the grant agency VEGA (grant no. 1/0122/09). We thank Mrs. Anna Michal cová and Mrs. Margita Buzinkaiová for their valuable technical assistance.
M. Repcák, M. Suvák / Biochemical Systematics and Ecology 47 (2013) 38–41
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