Photoinduced delayed luminescence from certain plant tissues containing anthocyanins, betacyanins and chlorophyll

Journal of Photochemistry and Photobiology, B: Biology, 3 (1989) 259 - 267 259 PHOTOINDUCED DELAYED LUMINESCENCE FROM CERTAIN PLANT TISSUES CONTAINING ANTHOCYANINS, BETACYANINS AND CHLOROPHYLL C. STRZALKOWSKAt Institute of Physics, Pedagogical University, 30-084 Krakow (Poland) D. SLAWINSKA Department of Physics, Agricultural Academy, 60-637 Poznan (Poland) A. SLAWINSKI F. B. Biologie, Kaiserslautern Universitat, 6750 Kaiserslautern (F.R.G.) J. SLAWINSKI Institute of Physics, Pedagogical University, 30-084 Krakow (Poland) (Received April 12,1988; accepted October 1,1988) Keyurords. Photoinduced delayed luminescence, anthocyanin and betacyanin pigments, chlorophyll, fluorescence, absorption and emission spectra, energy transfer. Summary Photoinduced delayed luminescence lasting several minutes was mea- sured from several plant tissues, mainly fruits, containing various amounts of chlorophyll, anthocyanins and betacyanins. The emission spectrum, rate constants and maximum intensity of photoinduced delayed luminescence correlate with the appropriate parameters of chlorophyll and its content in the tested fruits. These correlations as well as the results of chromatographic analysis, fluorescence spectra and absorption spectra indicate that the observed photoinduced delayed luminescence originates from trace amounts of chlorophyll present, even in ripe berries and other plant tissues. It is shown that no energy transfer takes place from anthocyanins and beta- cyanins to chlorophyll. 1. Introduction Since 1951 when Strehler and Arnold [l] discovered ultraweak long- lived delayed luminescence (PDL) induced by white light in photosynthe- TAuthor to whom correspondence should be sent. loll-1344/89/$3.50 0 Elsevier Sequoia/Printed in The Netherlands 260 sizing organisms, this phenomenon has been widely studied. It has been confirmed that PDL is observed in plant cells and tissues as well as in animal cells, including human cells [2 - 51. In the case of normal and malignant animal cells the character of PDL decay is markedly different [ 4 - 61. From up-to-date studies one can conclude that the emission intensity and decay kinetics of photosynthesizing objects depend on external factors, on the development stage and, for cells, on their concentration and dif- ferentiation [2 - lo]. In many cases the PDL decay curves are hyperbolic, suggesting that the emitted radiation is coupled with the emitter (coherent rescattering) [ 7 ] . In plants, the photosynthetic light-harvesting antenna is characterized by highly efficient absorption and subsequent transport of excitation energy to the reaction centre [ 111. The antenna pigment systems, for example in cyanobacteria, have accessory pigments, phycobilins, as well as chlorophylls, which are attached to thylakoid membranes [12]. In the course of the energy transfer (ET) from the initially photoexcited phycobiliprotein to the reaction centres of the PS I and PS II, fluorescence is emitted from almost every type of pigment and can be used as a probe for investigating the mechanisms of ET inside the phycobilisome [ 131. We thought it would be interesting to investigate PDL in plant materials containing pigments of the anthocyanin and betacyanin groups widely spread in plants, and their relationship to chlorophyll. We attempted to solve this problem by studying photosynthetic delayed luminescence (PDL) from plant tissues containing anthocyanin and betacyanin pigments. Anthocyanins and betacyanins differ from one another and significantly differ from chlorophyll, which gives a very strong PDL signal Both types of pigment are present in cell vacuoles in water-soluble glucoside form and are separated from chlorophyll by at least three membranes [14,15]. 2. Materials and methods Seven different species containing anthocyanins, especially cyanidin which is the main pigment in 80% of plants [15], were tested. They are listed in Table 1. The biological samples under investigation, especially fruits, were packed into the cuvette very tightly but carefully to avoid damage. The fruits of dog rose and peach were placed in a measurement chamber on a special table. The apparatus for registration of PDL has been described by Popp et al. [6]. A white light beam generated by a 150 W halogen lamp passed through a heat filter cutting off radiation above 720 nm and below 310 nm and irradiated the 25 mm X 25 mm X 40 mm quartz cuvette . PDL was measured by two-shutter techniques; one to stop the irradia- tion and the other to open a light path to the detector. The interval (dead time) between the end of an excitation and the start of a measurement was 261 TABLE 1 Characteristics of photoinduced delayed luminescence from studied plants Sample Dye h, tir N 10 Decay W) (s) (s-l) Red rose flower (flos Rosae rubrae) CY 535 50 800 1820 Hyp., exp. Dog’s rose fruit (fructus Rosae caninae) Cy 535 50 350 442 Hyp., exp. Peaches (Z’runus persica) CY 535 50 960 4177 Hyp. Raspberry (fructi Rubi ideali) CY 535 50 210 12341 Hyp. Bilberry (fiucti Vaccini) DP 546 Mv 542 10 700 7690 Hyp. Bilberry 50 280 8970 Hyp. Bilberry 480 1700 2838 Diexp. Bilberry 600 140 10270 Diexp. CY 535 Green blackberry f. (fructi Fruticosi) Dp 546 30 500 55070 Hyp. Mv 542 Green blackberry f. 60 700 3514 Hyp., exp. Green blackberry f. 600 700 27275 Hyp., exp. Ripe blackberry f. 30 700 5372 Hyp., exp. Ripe blackberry f. 60 500 2353 Diexp. Ripe blackberry f. 600 500 3007 Diexp. Red beetroot juice (Beta Vulgaris rubra) Bet 540a 60 100 2680 Diexp. Red beetroot centrifugated pulp 60 200 1523 Diexp. Cy, cyanidin; Dp, delphinidin; Mv, malvidin; Bet, betanidin; ia, maximum absorption in methanol [16]; a, measured in acetone in the present work; tin irradiation time; N, number of experimental points (counts); Ze, initial count rate (number of initial counts per second) ; f., fruit. 300 ms. The photomultiplier was situated normal to the excitation beam. The intensity of emitted radiation was measured at time intervals (sampling times) of 10 ms, 100 ms, 200 ms or more, and registered counts were processed by an on-line computer. The PDL spectrum was determined with the same apparatus using a set of Schott interference filters. The spectrum was corrected for photocathode sensitivity and filter transmission. In the case of ripe and fresh bilberry fruits additional measurements of fluorescence and absorption spectra and chromatographic analyses were performed, to find out whether residual chlorophyll existing in the mature fruits is the source of PDL or anthocyanins, which are the main pigments in the ripe species, are the source of PDL. The fluorescence was recorded in the case of ripe bilberry fruits, juice and filtrate. The filtrate was obtained by flushing the bilberry pulp with acetone. The fluorescence was investigated from the sample of red beetroot, its juice and acetone extract. A strongly 262 monochromatized radiation beam originating from a 250 W high pressure xenon lamp, which passed through SPM2 and SPMI C. Zeiss Jena monochro- mators, was used as the source of fluorescence excitation. The fluorescence was excited at an angle of about 0” to the sample, reached the SPM2 analysing monochromator and was then measured with an EM1 photomultiplier with a sensitivity in the range 220 - 660 nm, connected to a K 201 Zeiss recorder. The fluorescence was corrected for the photo- cathode sensitivity. Chromatographic analysis of the acetone extract of bilberry pulp was performed by using a 1 m long glass pipe filled with saccharose. PDL from green and ripe blackberries containing delphinidin and malvidin, pigments similar to those to be found in bilberries, and from red beetroot containing betanidin as the main pigment, was also investigated. Absorption spectra from the juice and acetone extract of ripe bilberries and red beetroot were analysed to detect chlorophyll. These spectra were registered by means of C. Zeiss Specord UV-visible and M-40 spectropho- tometers. The chemicals used were of analytical purity. Acetone was obtained from POCH Gliwice. 3. Results Basic characteristics of PDL obtained in our experiments are gathered in Table 1. It can be seen that all the samples investigated reveal delayed luminescence, varying from a few seconds to a few minutes. This time duration of PDL coincides with the time scale of photosynthetic delayed luminescence [l, 9, lo]. The rate constants calculated from the experi- mental data are, for example, (4.5 f 27.6) X lop2 s-’ for green blackberries and (1.0 f 35) X 10e2 s-l for matured blackberries. Each decay was approximated by hyperbola using the least-squares method. A typical plot and approximate fit is shown in Fig. 1. - 2 _” _I _I _.. 7.06 - --I_ Fig. 1. Typical example of PDL decay kinetics. The bilberry fruits were irradiated for 10 s and the counting time was 100 ms. *, experimental points; ===, theoretical hyper- bolic fit. I, emission intensity; t, time. 263 The decay of PDL fits a hyperbola well when ti, < 60 s. This is clearly seen for samples 3, 5 and 9 for which the number of experimental points is relatively high (N > 500). For samples 1, 2, 10 and 12 only the initial stage of decay obeys the hyperbolic law while the next decay stage (a tail) may be approximated by an exponential. The finding that the decay of PDL is hyperbolic provides support for the hypothesis that there exists a feedback coupling between the emitter and the emitted photon field for biologically native systems [7]. For longer irradiation periods (1 min < ti, < 10 min) the emission decay is fitted better by two exponentials. The observed initial intensity IO of PDL depends on the irradiation time tir; it is high for short periods of time and decreases for longer times. The decrease in photon emission for irradiation times less than 10 min might imply that long-term strong irradiation perturbs cell biohomeostasis. Perhaps local overheating and water evaporation perturb biochemical processes which initiate addi- tional processes of photon emission. For very long irradiation times (210 min) one again observes an increase in the initial emission intensity I,,. This may be connected with the death of cells, accompanied by the so-called necrotic radiation. It is not possible to compare I, for different samples, because in every case different shapes and radiating surfaces are involved. The strong initial emission intensity IO depends on the chlorophyll contents of the samples. This is supported by the fact that emission from unmatured fruits is about ten times greater than that of matured fruits. All fruits tested contained chlorophyll, the main pigment of unmatured fruits. During ripening, the chlorophyll content decreases and the anthocyanin pigments are synthesized. The results of chromatographic and spectrophotometric analyses shed light on this problem. As is evident from Fig. 2, besides the main absorption maximum belonging to anthocyanins delphinidin and malvidin one can see an extremely weak shoulder charac- teristic of chlorophyll absorption in the region of 15 200 cm-‘. Comparison of the PDL spectral distribution with the fluorescence spectrum of ripe bilberries illustrated in Fig. 3 shows that residual chloro- phyll is responsible for PDL. The fluorescence maximum of chlorophyll a in solution is 672 nm. However, in uiuo chlorophyll shows fluorescence X,,, = 681- 685 nm and a smaller maximum h,, = 720 - 736 nm [17,18]. Using Fig. 2. Absorption spectra of acetone extract of bilberry fruits. Quartz cuvette with an optical path of 0.5 cm. A, absorbance, D, wavenumber. 264 13 15 17 I lnm) 5x103 [cm-') Fig. 3. Emission spectra of bilberry fruits. . curve 1, whole bilberries; curve 2, acetone extract from the pulp; curve 3, juice; 4, PDL spectrum from whole fruits. I, emission intensity, X, wavelength. Fig. 4. Absorption spectra of red beetroot: x , aqueous extract from the pulp; 0, acetone extract from the pulp. Conditions as in Fig. 2. a limited number of interference filters one cannot separate these bands. Because of the low sensitivity of the multiplier used in the fluorescence mea- surements it was not possible to record the second maximum of chlorophyll a in uiuo. Thus the coincidence of the PDL maximum X = 700 nm with the resulting fluorescence bands in uiuo seems to be justified. To eliminate the influence of chlorophyll, the emission from juice and centrifuged pulp of red beetroot containing betanidin was studied. Frag- ments of red beetroot from the kower part of the vegetable containing no chlorophyll or the smallest possible amounts of it were used for measure- ments. Nevertheless, in the beetroot there were also trace amounts of chloro- phyll, as indicated in Fig. 4. In the 15 200 cm-l region of the absorption spectrum of acetone extract from a beetroot pulp there is a small distortion originating from chlorophyll. Table 1 shows that the emission from an intact beetroot is nearly twice as great as that from a juiceless pulp. It is difficult to interpret the relatively strong PDL from the beetroot aqueous extract in terms of a comparison with the much weaker PDL of the pulp. The acetone extract of bilberry fruits also exhibits a higher I0 value than the intact bilberries since the extract is enriched in chlorophyll. 4. Discussion The role of anthocyanin and betacyanin pigments in plants is not yet understood. They probably take part in important oxidation-reduction processes in cells and also have a protecting function as filters elim- inating the influence of sunshine on cells, this also being an ecological factor [15]. Anthocyanin synthesis occurs only in the presence of light, and is controlled by an unstable nucleic acid analogous to the messenger RNA [16]. While the PDL phenomenon is well known in the case of chlorophyll- containing materials, analogous phenomena for plants containing anthocyanin, betacyanin and other plant pigments have not been investi- gated until now. The results of our experiments indicate that the observed PDL cannot be ascribed to the luminescence of anthocyanin and betacyanin pigments. The fluorescence spectra, the results of the chromatographic analyses and the absorption spectra of juice and acetone extract support the hypothesis of the presence of residual chlorophyll in the ripe fruits which is the source of PDL. The above facts indicate that PDL of plants containing anthocyanin and betacyanin pigments presumably results from the residual chlorophyll emission. The following considerations provide further evidence for this conclusion. (1) Energy transfer from anthocyanin pigments to chlorophyll is improbable because the two pigments are separated in plant cells. The anthocyanin pigments exist in vacuoles and are separated from chlorophyll built in the thylakoid membrane of chloroplast grana by the tonoplastid membrane of vacuoles, cytoplasm, the duplex plastid membrane and the thylakoid membrane. According to the Fijrster theory [19], energy transfer can be observed up to the critical distance of 1 pm. The cell diameter of highly organized plants is estimated to be 10 pm. Taking into account the size and morphol- ogy of the cells and the space where the pigments are located, one has to exclude the dipole-dipole-type of energy transfer from the investigated pig- ments to chlorophyll. (2) Anthocyanin pigments are water-soluble glucosides in the aqueous phase of vacuoles. It is generally known that non-radiative quenching processes dominate in this phase. (3) The process of electron (e-) transport in photosynthesis requires the coordinated interaction of PS I and PS II where absorbed light energy is utilized to enable an endergonic charge separation. This requires a lateral heterogeneity in the distribution of e- transport complexes in photo- synthetic and accessory structures. Usually chromophores are distributed in a special array to form a special channel for the sequential ET in such struc- tures [ 131. Contrary to the case of chlorophyll-lipid-protein complexes, in antho- cyanin pigments photoinduced charge separation leading to stabilized redox states of intermediates does not occur. Therefore energy storage in these redox states and the subsequent recombination radiation, i.e. PDL, as happens in thylakoid membranes, is hardly possible, if at all. 266 Although PDL has been observed in a variety of biological materials, no chromophore-specific excitation and emission spectra have been found [6,81. 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