Plant Cell Rep (2009) 28:1137–1143 DOI 10.1007/s00299-009-0708-0
BIOTIC AND ABIOTIC STRESS
Circadian rhythm of (Z)- and (E)-2-b-D-glucopyranosyloxy-4methoxy cinnamic acids and herniarin in leaves of Matricaria chamomilla Miroslav Repcˇa´k Æ Benˇadik Sˇmajda Æ Jozef Kova´cˇik Æ Adriana Eliasˇova´
Received: 5 March 2009 / Revised: 20 April 2009 / Accepted: 20 April 2009 / Published online: 9 May 2009 Ó Springer-Verlag 2009
Abstract Chamomile (Matricaria chamomilla) in the above-ground organs synthesizes and accumulates (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acids (GMCA), the precursors of phytoanticipin herniarin (7-methoxycoumarin). The diurnal rhythmicity of the sum of GMCA (maximum before daybreak) and herniarin (acrophase at 10 h 21 min of circadian time) was observed under artificial lighting conditions LD 12:12. The acrophase is the time point of the maximum of the sinusoidal curve fitted to the experimental data. In continuous light, the circadian rhythms of both compounds were first described with similar acrophases of endogenous rhythms; a significantly different result from that in synchronized conditions. The rhythms’ mesor (the mean value of the sinusoidal curve fitted to the experimental data) under freerunning conditions was not influenced. Abiotic stress under synchronized conditions decreased the average content of GMCA to half of the original level and eliminated the rhythmicity. In contrast, the rhythm of herniarin continued, though its content significantly increased. Nitrogen deficiency resulted in a significant increase in GMCA content, which did not manifest any rhythmicity while the rhythm of herniarin continued. Circadian control of herniarin could be considered as a component of the plant’s specialized defence mechanisms.
Communicated by M. Petersen. M. Repcˇa´k (&) B. Sˇmajda J. Kova´cˇik A. Eliasˇova´ Institute of Biology and Ecology, Faculty of Science, P. J. Sˇafa´rik University, Ma´nesova 23, 041 67 Kosice, Slovak Republic e-mail:
[email protected]
Keywords Circadian rhythm Herniarin Matricaria chamomilla (Z)- and (E)-2-b-Dglucopyranosyloxy-4-methoxy cinnamic acids Abbreviations GMCA (Z)- and (E)-2-b-D-glucopyranosyloxy-4methoxy cinnamic acids LD Light–dark LL Continuous light MDA Malondialdehyde
Introduction A wide range of physiological activities in plants is controlled by internal biological clocks, which could be governed by regular changes in environmental factors, e.g. light or temperature. The genes involved in the regulation of growth and metabolism are also subject to control by these clocks (Dowson-Day and Millar 1999; Nozue et al. 2007). Clock control of the transcriptome is widespread (Covington et al. 2008). The circadian clock has a regulatory role in nearly all aspects of plants’ life (Yakir et al. 2007). Diurnal oscillations of metabolite content are less dramatic (McClung 2008). The production of some secondary metabolites with a defensive role is rhythmical as well. Inducible defensive volatiles have been found to follow a diurnal rhythm with maximum levels during the light period (Martin et al. 2003). A diurnal rhythm of emission of phenylpropanoidderived volatiles from flowers has been observed in Nicotiana silvestris Speg. and Comes and N. suaveolens Lehm. (Loughrin et al. 1991). In Arabidopsis, 23 genes have been found encoding enzymes of phenylpropanoid biosynthesis
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which are co-ordinately regulated, oscillating with mRNApeaks about 4 h before subjective dawn (Harmer et al. 2000). The transcription of pap1 (production of anthocyanin pigment) oscillates in harmony with these genes and probably acts as a master regulator of their clock-controlled transcripts. The clock probably regulates pap1 expression, which in turn regulates the entire phenylpropanoid biosynthetic pathway (Borevitz et al. 2000). The circadian system has a role in regulating responses to both abiotic and biotic stresses (Yakir et al. 2007). Endogenous circadian rhythmicity of antioxidative enzymes and low-molecular antioxidants has been described in different organisms, including plants (Hardeland et al. 2003; Barros et al. 2005; Kolarˇ and Macha´cˇkova´ 2005). Many stress-induced phenylpropanoids (including coumarins) have been observed in plants under pathogen attack and wounding (Dixon and Paiva 1995). The medicinal plant Matricaria chamomilla, in its aboveground organs, synthesizes and accumulates phenylpropanoids (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acids (GMCA), the precursors of phytoanticipin herniarin (7-methoxycoumarin, Repcˇa´k et al. 2001). Phytoanticipins are low-molecular weight, antimicrobial compounds that are present in plants prior to challenges by microorganisms or are produced after infection solely from preexisting constituents (vanEtten et al. 1994). Under stress conditions, namely foliar Cu2? application, the level of these precursors decreases and the content of herniarin and of other stress metabolite umbelliferone increases (Repcˇa´k et al. 2001; Eliasˇova´ et al. 2004). Z-GMCA, after hydrolysis by a specific b-glucosidase, releases aglycone which spontaneously lactonises to herniarin. The variable content of herniarin in commercial chamomile drugs is due to its formation as an artefact after disruption of cellular compartmentalization during post-harvest processing (Tosi et al. 1995). Both GMCA and herniarin increase in response to nitrogen deficiency (Kova´cˇik et al. 2007), while hydroponical Cu2? application (Kova´cˇik et al. 2008) causes less pronounced effect when compared to foliar application as mentioned above. Coumarin derivatives have been described as antimicrobial and anti-inflammatory active substances (Mares 2005). Herniarin after activation with ultraviolet light (320–400 nm) is a natural antimicrobial and antimycotic compound. No activity of this coumarin has been found in the dark. Some coumarin derivatives possess antioxidant activity, which correlates with the number of hydroxyl groups (Lin et al. 2008). A similar relation has been suggested also for other phenolic metabolites, phenolic acids, which are strongly induced by Cu excess in chamomile roots (Kova´cˇik and Klejdus 2008). In dermatophyte Microsporum cookie Ajello, herniarin may affect
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morphogenesis through destabilization of the cytoskeleton and consequent inhibition of vesicle transport and a strong slowing down of apical growth (Ceska et al. 1992; Mares 2005). The presence of herniarin has been identified in all above-ground parts of chamomile. Herniarin is a nonvolatile compound and has never been found as a component of chamomile scent (Brunke et al. 1993; Surburg et al. 1993). The physiological functions of coumarin derivatives in plants are still unknown (Bourgaud et al. 2006). The aim of the present investigation was to study the rhythmicity of GMCAs and their product herniarin in leaves of M. chamomilla. The first aim was to confirm the endogenous character of the rhythms of both compounds. The second aim was to study the influence of two abiotic stress factors, foliar Cu2? application and nitrogen deficiency, on the diurnal rhythmicity (both these factors significantly modulate the level of the above-mentioned metabolites).
Materials and methods Cultivation of plants and experimental design Tetraploid chamomile (Matricaria chamomilla L. cv. ‘Lutea’) was used for all experiments. Seedlings were pre-cultivated in sand. Two-week-old plants were transferred to soil and daily moistened to 60% of the soil water-holding capacity. Nine-week-old plants in leaf rosette phase with average dry weight of 0.0859 ± 0.0433 g were used. The cultivation and experiments were performed under controlled lighting conditions: PAR 210 lmol m-2 s-1 with a lighting regime of 12-h light:12-h dark (LD 12:12). In all experiments, leaf rosettes of plants were collected at 3-h intervals over a 3-day period and immediately dried at 95°C and stored in a desiccator. Different sets of plants were used for the different time points. In the experiment with continuous light (LL), plants were illuminated for 24 h before the start. Abiotic stress was induced by spraying the leaves with 1% aqueous solution of CuCl2 in 5 mg/L Tween 80. Spraying the leaves with water containing Tween was also tested. Plants for the nitrogen-deficient experiment were cultivated in Hoagland hydroponic solution for 3 weeks. Seven days before the start of the experiment, the Hoagland solution was replaced with a modified solution without nitrogen [Ca(NO3)2 9 4H2O was replaced with CaCl2 9 2H2O, NH4H2PO4 with KH2PO4 and KNO3 with K2SO4; Kova´cˇik et al. 2007]. Nitrogen content in the leaves was estimated using the Kjeldahl method in four individual plants (Allen 1989).
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Membrane lipid peroxidation The level of lipid peroxidation caused by the tested stress factors was estimated as the amount of malondialdehyde using the thiobarbituric acid reaction from four individual plants as described in detail previously (Kova´cˇik et al. 2006). HPLC quantification of metabolites For the estimation of the sum of (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acids, herniarin and umbelliferone dry ligulate flowers were homogenised and extracted with methanol. Gradient HPLC was used for quantification of metabolites as described in detail in our previous paper (Repcˇa´k et al. 2001). A typical chromatogram is shown in Fig. 5.
are then used to arrive at an estimation of population rhythm parameters, together with their statistical verification, by constructing a two-dimensional confidence area (the error ellipse). The ellipse is centred at the top of a vector constructed in a polar graph, with a length corresponding to the estimate of the population amplitude and its orientation to the estimation of the population acrophase. If the error ellipse does not overlap the origin of the graph, the zero-hypothesis about the non-existence of a rhythm of the given period length in data can be rejected at a given probability level. For more details see, for example Bingham et al. (1982). For performing computations with this method, a self-made computer programme created in C-language in Java-environment was used. Student’s t test (P \ 0.001) was used for evaluation of selected data, which are shown as mean ± standard deviation in the text.
Statistical analyses
Results
The leaf rosettes from a total of 1,750 plants were collected and analysed. The numbers of plants sampled at each time point in individual experiments are given in Table 1. The presence of rhythmicity in each time series of data was statistically evaluated using the cosinor method (Cornelissen et al. 1980; Halberg et al. 1967) and F test of sinusoidality (Bingham et al. 1982). The cosinor method is based on fitting the individual sets of data (measurements on individual subjects during one or more cycles) by means of least square fit with a harmonic (sinusoidal) curve of a pre-set period length (e.g. 24 h). The received parameters of individual curves (mesors, amplitudes and acrophases)
Circadian rhythms of coumarins Rhythmic fluctuations in GMCA and in herniarin were observed under light/dark (12/12 LD) conditions. The F test of sinusoidality showed that the shape of all experimental curves did not significantly differ from the sinusoidal curve, so the use of the cosinor test is appropriate for this case (Figs. 1, 2; Table 1). The results show a significant diurnal rhythm of the GMCA sum, with increasing content during the night (maximum before daybreak) (Table 1; Fig. 1a). A significant diurnal rhythm of herniarin was also confirmed, with content increasing during the
Table 1 Diurnal and circadian rhythms of (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acids (GMCA) and herniarin in rosette leaves of Matricaria chamomilla Condition and compound
Amplitude
Acrophase
CI
CI
Mesor ± SEM
Rhythmicity (%)
Cosinor significance P
LD 12:12 (n = 22) GMCA
0.88
0.54-1.22
23 h 50 m
22 h 12 m-1 h 37 m
8.759 ± 0.156
44.77
\0.05
Herniarin LL (n = 10)
0.03
0.02-0,04
10 h 21 m
9 h 12 m-11 h 37 m
0.146 ± 0.003
64.56
\0.05
GMCA
0.68
0.44-0.92
18 h 28 m
17 h 15 m-19 h 34 m
9.643 ± 0.569
36.47
\0.05
Herniarin
0.02
0.01-0.03
19 h 15 m
16 h 59 m-21 h 54 m
0.162 ± 0.003
39.92
\0.05
20 h 56 m
15 h 46 m-20 h 49 m
1.533 ± 0.058
46.55
\0.05
6 h 39 m
5 h 05 m-8 h 26 m
0.926 ± 0.038
46.03
\0.05
LD 12:12, abiotic stress (n = 28) herniarin
0.29
0.17-0.41
LD 12:12, N-deficiency (n = 10) Herniarin
0.32
0.21-0.43
Results of cosinor testing for 24-h period length. Abiotic stress induced by spraying plants with 1% CuCl2 Acrophase, the time point of the maximum of the sinusoidal curve fitted to the data (h-min of circadian time); mesor, the mean value of the fitted curve (mg g-1 dm), rhythmicity, the part of overall variability in experimental data explained by the sinusoidal model of the rhythm, in %; amplitude in mg g-1 dm; LD 12:12–12 h light:12 h dark; LL continuous light; CI confidence interval
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Fig. 1 Chronograms of (Z)- and (E)-2-b-D-glucopyranosyloxy-4methoxy cinnamic acids (a) and herniarin (b) in rosette leaves of Matricaria chamomilla under light/dark regimen 12 h/12 h and in continuous light (given as arithmetic mean ± SEM); black boxes correspond to the dark period; CT circadian time, leaf rosettes of 22 (LD) and 10 (LL), respectively; plants were analysed per time point
morning hours and peaking in the afternoon (acrophase at 10 h 21 min of circadian time) (Table 1; Fig. 1b). Umbelliferone was present in very low concentrations (average 0.04 mg g-1) and no rhythm was observed (data not shown). Under continuous light (LL) regular variations were observed for both compounds, testifying to a circadian regulation of their level. Similar acrophases of endogenous rhythms were observed for GMCA and herniarin. Their values were significantly different from those found in synchronized conditions (Table 1; Figs. 1a, b, 2a–d). The rhythms’ mesor under free-running conditions were not modified. Stress influence on diurnal rhythms Abiotic stress (spraying with CuCl2) caused a specific reaction in chamomile leaves as described in a previous paper (Eliasˇova´ et al. 2004). Accumulation of analysed compounds did not differ significantly in plants sprayed with water containing Tween when compared to control (non-sprayed) plants (GMCA: control 6.30 ± 0.42; water with Tween 7.39 ± 0.38 mg g-1 of leaves dry mass;
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Fig. 2 Circadian rhythmicity of (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acids (a, b) and herniarin (c, d) in rosette leaves of Matricaria chamomilla. Cosinor diagrams: the circle denotes the period length of 24 h. Black boxes correspond to the dark period. The length of the line segment from the origin of coordinates is proportional to the amplitude and its orientation gives the acrophase of the rhythm. The ellipse represents the joined confidence interval (for P \ 0.05) of rhythm acrophase and amplitude
herniarin: control 0.188 ± 0.01; water with Tween: 0.190 ± 0.02 mg g-1 of leaves dry mass). Malondialdehyde (MDA) is a widely-used indicator of membrane damage level and increased amounts of it were observed in our previous experiments preferentially in the roots of Cuexposed or N-deficient plants (Kova´cˇik and Bacˇkor 2007; Kova´cˇik et al. 2008). Content of MDA in the present experiment was measured 48 h after spraying. Its accumulation in Cu-sprayed leaves was significantly higher (332 ± 14 lmol g-1 dry mass) in comparison with control (217 ± 33 lmol g-1 dry mass). It was the aim of the subsequent experiment to find out if the stress interfered with the newly identified circadian regulation. Abiotic stress under synchronized conditions decreased the average content of GMCA to 4.9 mg g-1 of leaves dry mass. No rhythm was observed (Table 1; Fig. 2a). The diurnal rhythm in herniarin continued with the acrophase shifted to the middle of the dark period. The effect of stress increased the values of the mesor and also of the amplitude coincidently about 10 times (Table 1; Fig. 2b). However, under stress condition the differences between peaks and troughs appear to be reduced when compared to non-stress conditions (Figs. 1b, 3b). The content of umbelliferone increased ten times after 24 h, reaching values of 0.3–0.4 mg g-1, but showing no rhythmicity (data not shown).
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Fig. 3 Chronograms of (Z)- and (E)-2-b-D-glucopyranosyloxy-4methoxy cinnamic acids (a) and herniarin (b) in rosette leaves of Matricaria chamomilla under conditions of abiotic stress induced by spraying plants with 1% CuCl2 (arrow); leaf rosettes of 28 plants were analysed per time point. Other details as in Fig. 1
Malondialdehyde accumulation in the leaves of N-deficient plants after 7 days of starvation (733 ± 38 lmol g-1 dry mass) was not altered significantly when compared to control (827 ± 98 lmol g-1 dry mass). This indicates no detectable effect of nitrogen deficiency on lipid peroxidation status, corresponding to our previous observations (Kova´cˇik et al. 2007). Control plants cultivated in hydroponics contained higher amounts of MDA in comparison with control plants cultivated in soil. The difference between control values in these two experiments may reflect either different age (9 and 4 weeks in Cu-spray and N-deficient experiments, respectively) or the influence of cultivation conditions, and further studies are warranted. Eliminating nitrogen from the culture medium caused a gradual decrease of its content in leaves from 59.8 to 38.5 mg g-1 dry mass during the first 7 days. The average content of GMCA under conditions of nitrogen starvation significantly increased (23.1 mg g-1 dry mass), but no
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Fig. 4 Chronograms of (Z)- and (E)-2-b-D-glucopyranosyloxy-4methoxy cinnamic acids (a) and herniarin (b) in rosette leaves of Matricaria chamomilla under conditions of nitrogen deficiency; leaf rosettes of 10 plants were analysed per time point. Other details as in Fig. 1
rhythmic fluctuations in its level were found. The content of herniarin in leaves was increased in comparison with control plants, but diurnal fluctuations with significant sinusoidality were observed (Table 1; Fig. 4a, b).
Discussion Circadian rhythms in the phenylpropanoid pathway have been confirmed in Arabidopsis thaliana L. at transcript abundance level (Harmer et al. 2000). Circadian changes have also been found in the transcript abundance of lignin biosynthetic genes, a specialized branch of phenylpropanoid metabolism (Rogers et al. 2005). No data about circadian rhythmicity of coumarin-like compounds and their precursors were found in the literature. Phytoanticipin compounds are sequestrated by storage inside the vacuole in glycosylated forms representing biosynthetic intermediates. Upon decompartmentalization caused by cellular
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Fig. 5 HPLC chromatogram of the methanol extract of Matricaria chamomilla leaf rosettes. 1 (Z)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acid, 2 (E)-2-b-D-glucopyranosyloxy-4-methoxy cinnamic acid, 3 umbelliferone, 4 herniarin
disintegration, they are released from the conjugates and further cleaved by b-glucosidases into their active form (Morant et al. 2008). The rhythms of b-glucosidase activity have rarely been studied, although in tobacco leaves its activity was found to increase during the light period (Nova´kova´ et al. 2005). In Artemisia dracunculus L., (E)-2b-D-glucopyranosyloxy-4-methoxy cinnamic acids release (E)-2-hydroxy-4-methoxy cinnamic acid which is a UVunstable compound spontaneously cyclizing into herniarin (Hofer et al. 1986). The present study showed that the synthesis and/or degradation of GMCA and herniarin in leaves of chamomile (Matricaria chamomilla L.) have circadian rhythms. The endogenous rhythm of both compounds in continuous light manifested the same acrophase. Under light–dark regime, the acrophases of both compounds were significantly different in comparison with synchronized conditions. The vacuolar conjugated precursor peaked during the dark period. Photosensitive aglycone with a defense role was released with a peak in the light period. Both stress factors disrupted the control of GMCA synthesis by the circadian oscillator. In contrast, the diurnal rhythm of herniarin continued in this condition. Higher tissue levels of this compound may participate in defence mechanisms to biotic and abiotic stresses during the light period. It will be the objective of further research to achieve elucidation of the biological role of increased GMCA and herniarin accumulation in chamomile tissue by combined abiotic– biotic stress treatment. Acknowledgment This work was supported by the grant agency VEGA (project no. 1/0444/03). We thank Mrs. Anna Michalcˇova´ and Mrs. Margita Buzinkaiova´ for their valuable technical assistance.
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