PHOSPHORESCENCE OF CHLOROPHYLL IN CHLOROPLASTS AND SUBCHLOROPLAST FRAGMENTS*

Biophysics, Vol. 25. No. 5. pp. 835-841, 1980 Translation from Russian: Biofizika 25: No. 5, 821-826,1980* CELL BIOPHYSICS PHOSPHORESCENCE OF CHLOROPHYLL IN CHLOROPLASTS AND SUBCHLOROPLAST FRAGMENTS* A. A. KRASNOVSKII, Jr., Yu. V. KOVALEV, G. P. KUKAKSKIKH and B. A. GULYAYEV Biology Faculty M.V. Lomonosov State University, Moscow (Received 21 January 1980) Spectral parameters, decay time (τ ) and quantum yield of low-temperature (-196 o C) delayed luminescence of chlorophyll were inversigted in isolated chloroplasts and subchloroplast particles obtained by differential centrifugation after treatment of chloroplasts with 0.3 % digitonin. It is shown that isolated chloroplasts, their granal fragments enriched with the photosystem 2, or granal and stromal fragments enriched with the photosystem 1 emit millisеcond delayed luminescence similar to that observed earlier in intact leaves and algal cells. In all samples phosphorescence and delayed fluorescence of chlorophyll and phosphorescence of pigments of non-chlorophyll nature were detected. It was shown that chlorophyll phosphorescence accompanies deactivation of the triplet molecules of the short wavelength chlorophyll forms, which are probably a part of antenna pigment protein complexes. The total yield of chlorophyll fluorescence in chloroplasts and subchloroplast fragments are virtually the same while the phosphorescence intensity increased with disruption of intitial chloroplast membranes and reached maximum in the lightest fragments. It is suggested that structural damage increases number of the phosphorescence-emitting chlorophyll molecules uncapable of efficient transfer of their triplet-state energy to carotenoids. It was shown that at -196 o C etiolated greening and adult leaves of normal and mutant plants as well as the cells of green, blue-green and red algae emit phosphorescence and delayed fluorescence accompanying deactivation of the triplet states of different forms of chlorophyll a and its precursors in biosynthesis [1-6]. Much less is known about this luminescence in isolated chloroplasts and their fragments. Preliminary information on isolated chloroplasts [2-4] and the photosystem 1 enriched fragments [7] pretreated by denaturing agents or dithionite is only available. In the present work we studied chlorophyll phosphorescence in chloroplasts and their fragments which were not subjected to additional destructive treatments with a goal to reveal the structures in which chlorophyll phosphorescence is expressed to the highest degree. 835

836 А. A. Krasnovskii et al. METHODS Chloroplasts and fragments were isolated as desсribed in [8,9] from 2-week old pea seedlings of the Sovershenstvo variety grown under 10,000 Ix at 25 C and 65 per cent relative humidity with a light period of 16 hr. The isolation medium contained phosphate buffer (1/15 M, ph 7.8), 0.4 M sucrose and 0.01 M KC1. To obtain subchloroplast particles (SCP), chloroplast suspensions in the isolation medium were incubated with 0.3% digitonin during 20 min at 4 C (the ratio of chlorophyll to digitonin was 1: 3). Subsequent fractionation was carried out by differential centrifugation [8, 9]. At first, large particles were sedimented at ~ 1000 g (10 min) and then, supernatant was centrifuged at 10,000 g (20 min). The pellet after the last centrifugation corresponded to the granal fraction. The supernatant contained stromal lamellae. The stromal fraction was additionally centrifuged at 100,000g (25 min). The supernatant after this centrifugation was additionally centrifuged at 144,000 g (1 hr). The sediment (SCP-144 str) contained fragments enriched with the stromal photosystem 1 [ 8-11]. The granal fraction was again treated with 0.5% digitonin for 20 min (at the chlorophyll concentration 3-4 mg/ml). The suspension was spun down for 20 min at 10,000g. The pellet was discarded. The supernatant was centrifuged for 30 min at 20,000g. The sediment (SCP-20) corresponds to the granal thylakoid fraction enriched with the photosystem 2 [8-11]. The supernatant over the SCP-20 was centrifuged for 25 min at 100,000 g, then a new supernatant was centrifuged for 1 hr at 144,000 g. The sediment (SCP-144gr,) corresponds to the particles enriched with the granal photosystem 1 [8-11]. For measurements, SCP-20, SCP-144str, and SCP-144gr were employed. The data shown in the present paper are average over the results of five independent separations. In addition, the SCP-20 and SCP-144 obtained after the fifth separation, were washed again to reduce the content of detergent and light particles not belonging to these fractions. For this purpose, they were resuspended in the medium having no detergent and sedimented at 144,000 or 20,000g respectively. The procedure was repeated twice. The washed fragments and also supernatants remaining after the last washing were used for luminescence measurements. Phosphorescence and fluorescence were detected with the previously described phosphoroscope units [1-6]. The absorption spectra were recorded using the SF-10 or SF-18 spectrophotometers placing the samples inside an integrating sphere. Usually low temperature spectra are recorded using suspensions containing 60% glycerol. Our control measurements have shown that addition of glycerol does not influence the luminescence and absorption spectra of the samples. However, analysis of afterglow is less accurate since sometimes glycerol shows its own delayed light emission in the region 700-900 nm. Therefore, in the present work, main measurements were made without glycerol on SCP suspensions frozen in the isolation medium. RESULTS The fluorescence and phosphorescence spectra of the samples at -196 e C are presented in Figs. 1-3. It is seen that in accord with the published data [9, 11-14], the SCP-144 gr and SCP-144 str show fluorescence spectra, in which the short wavelength bands (600-700 nm) are much weaker than the long wavelength band at 738 nm. In the fluorescence spectra of SCP-20, the short wavelength bands are stronger than the 738 nm band. Additional washing led to a small increase of the short wavelength bands

SCP-20 SCP-144 gr SCP-144 str Chloroplasts Pea leaves** Phosphorescence of chlorophyll in chloroplasts 837 QUANTUM YIELDS OF CHLOROPHYLL FLUORESCENCE AND CHLOROPHYLL PHOSPHORESCENCE IN CHLOROPLASTS AND THEIR FRAGMENTS AT 77K SCP-20 (wash.)*** Total yield (φ f ) Fluorescence Contribution of emission at 600-700 nm*, % Phosphorescence (φ ph ) φ sf /φ ph 0.9 50 1.6 0.5 0.9 24 4.7 3.1 0.9 10 3.5 5.9 1 15 1 1 1 15 0.3 0.3 0.9 52 1.7 0.5 SCP-144 str (wash)*** 0.9 7 1.3 3.0 Supernatant over SCP-144 gr 1 10 9 13.5 Chlorophyll a (ethanol)**** 2.3 70 200 18.5 * Contribution of the emission in the region 600-700 nm to the total fluorescence spectrum (φ sf/φ f ) ** It is supposed that the relative intensity of the short wavelength fluorescence bands in leaf is similar to that in chloroplasts; experimentally observed fluorescence spectra of leaves are strongly distorted by reabsorption. *** Data correspond to the washed particles **** Absolute quantum yields of φ f and φ ph for chlorophyll solutions are known to be 35% [3] and 10-5 [20] respectively. in the fluorescence spectra of SCP-20 and to a small decrease of the short wavelength bands in the fluorescence spectra of SCP-144 (Table). The delayed luminescence spectra of all samples (Figs. 1-3) proved to be similar. Upon excitation with red light (λ 600 nm), these spectra contained the same three components characteristic of the leaves and algae [1-6]: phosphorescence (maximum ~960 nm) and delayed fluorescence (maxima ~690 and 740 nm) of chlorophyll and also phosphorescence of the non-chlorophyll pigments (780-820 nm). Analysis of the delayed fluorescence of chlorophyll was complicated by the superposing of the non-chlorophyll luminescence. Nevertheless, it was possible to establish that the decay time (τ) of the 690 nm luminescence band is 2.5-4.5 msec, the intensity of this band is proportional to the square of the intensity of the exciting light. The ratio of the amplitudes for the bands 690 and 740 nm differ from their ratio in the fluorescence spectra (Figs. 1-3). The phosphorescence spectra of chlorophyll were the same in all samples and changed uniformly with change in the wavelength of the exciting light ( ex ). Upon excitation through the cut-off light filters with a sharp short wavelength transmission edge corresponding to 640 nm (KS-15, ex 640nm) the phosphorescence maximum is located at 956±2nm; with ex 680 nm (KS-18) the maximum was observed at 977±2 nm (Figs. 1-3). The phosphorescence lifetimes in these maxima were 1.9 and 1.45 msec, respectively. The phosphorescence lifetime strongly depended on the wavelength of recording. With ex 640 nm (KS-15): rec, nm 930 960 985 τ, msec 2.5 1.9 1.6

838 A.A. Krasnovskii et al. L/I,rel.un. 1-T Fig. 1. Delayed luminescence (1, 2) and fluorescence (3) spectra in pea chloroplasts at 77K. The absorption spectrum (4) and spectrum of phosphorescence excitation (5) in the same samples. The spectra of delayed luminescence were recorded upon excitation through the KS-17 ( ex 660 nm) (1) and KS-18 ( ex 680 nm) (2) light filters using the samples with optical density of 0.9 (1,2) and 0.5 (3) in the maximum of the red band. Excitation spectrum corresponded to the phosphorescence detected through IKS-7 light filter (transmits in the region >900 nm). Spectral width of monochromator slits was 20 nm for 1, 2; 1 nm for 3,4 and 10 nm for 5. Fig. 2. Delayed luminescence (1, 2) and fluorescence (3) spectra of SCP-20 chloroplast fragments at 77K. The absorption spectrum (4) and spectrum of phosphorescence excitation (5) of the same samples. For conditions see Fig. 1. The data indicate that in all samples, phosphorescence is emitted by similar forms of chlorophyll and the number of the phosphorescence-emitting forms is not less than two. Phosphorescence excitation spectra (Figs. 1-3). The measurement of the excitation spectra is complicated by two factors: firstly, it is the contribution of the non-chlorophyll

luminescence which has an excitation maximum at λ<600 nm. Upon excitation in the interval 600-650 nm this luminescence is comparable in intensity with the phosphorescence of chlorophyll. Secondly, because of the low intensity of the chlorophyll phosphorescence, reliable measurements were only possible if relatively dense chloroplast suspensions with optical density (OD =0.45±0.1 in the maximum of the red band) and wide monochromator slits ( 10 nm) were used. Nevertheless it was observed that in all the samples, the red maximum of the excitation spectra of chlorophyll phosphorescence is located at 669 ±1 nm with a shoulder at 650 nm, being shifted by 7-9 nm to the short wavelength side as compared with the main absorption maximum of chlorophyll. Apparently, these facts support the conclusions of our previous papers [2-6] that phosphorescence is due essentially to the short wavelength forms of chlorophyll. The presence of the shoulder at 650 nm in the phosphorescence excitation spectra indicates sensitization by chlorophyll b, hence one can suggest that the phosphorescence-emitting chlorophyll molecules are included in the normal antenna pigment-protein complex. The quantum yields of phosphorescence (φ ph ) were estimated upon excitation through a KS-15 light filter (λ 640 nm). The fluorescence yields were estimated using monochromatic excitation at 440 nm. The optical density of the samples in the red absorption maximum was 0.45±0.1. The relative quantum yields were calculated by dividing the overall phosphorescence (fluorescence) intensity of each sample by their absorption coefficient (1-T) = (1-10 -OD ) in the region of excitation (for phosphorescence we used the area under the 1-T spectrum at λ>640 nm). The absolute yields may be obtained by comparison with solutions of chlorophyll a in ethanol (Table). The Table shows that the total yields of fluorescence in the chloroplasts and their fragments are roughly the same while the values of φ ph rose with destruction of the initial chloroplast membranes reaching a maximum value in the smallest fragments. Additional washing of SCP-20 had no effect on the phosphorescence yield. In SCP-144 str after washing the phosphorescence noticeably weakened owing to passage of strongly phosphorescing fragments to the supernatant (Table). Phosphorescence of chlorophyll in chloroplasts 839 Fig. 3. Delayed luminescence (1, 2) and fluorescence (3,4) spectra of chloroplast fragments SCP-144 gr (1,3) and SCP-144 str (2,4) at 77K. The absorption spectrum (5) and spectrum of phosphorescence excitation (6) for SCP-144 gr. Phosphorescence spectra were measured upon excitation via cut-off filter KS-17 ( 640 nm). For other conditions see Fig. 1.

840 A. A. KRASNOVSKII et al. DISCUSSION Thus, isolated chloroplasts and subchloroplast particles emit millisecond low-temperature delayed luminescence similar to that observed earlier in leaves and algae. As in intact organisms, all chlorophyll forms involved in the low-temperature fluorescence are also involved in emission of delayed fluorescence, while phosphorescence is mainly emitted by the short wavelength forms of chlorophyll. Analysis of the excitation spectra shows that the phosphorescence-emitting forms of chlorophyll are probably incorporated into the chlorophyll b containing antenna pigment-protein complex. This assumption is supported by literature data on deconvolution of the absorption and fluorescence spectra of chloroplasts into the individual components [13-17]. One can note that the red maximum of the phosphorescence excitation spectrum matches the red maximum in the excitation spectrum of the short wavelength fluorescence band at 681 nm. Hence, it is not excluded that this fluorescence band might be connected with phosphorescing chlorophyll, which in this case, could be considered as a part of the functionally active pigment apparatus. The intensity of the low-temperature chlorophyll phosphorescence in the isolated subchloroplast particles was shown to be considerably higher than in the intact leaves. Only in SCP-20, the intensification of phosphorescence was observed simultaneously with appreciable rise in the yield (φ sf ) of the short wavelength fluorescence bands of chlorophyll (at 600-700 nm). In the light fragments the rise in φ ph was observed together with almost unchanged (SCP- 144) or reduced (SCP-144 and the supernatant) values of φ sf (Table). Comparison of these data suggests that in the light particles, a natural mechanism [2-6] responsible for quenching of the triplets of the phosphorescence-emitting chlorophyll is partially damaged. According to the data obtained upon investigation of the carotenoid mutants of maize [6] it is most likely that this mechanism is triplet-triplet energy transfer from chlorophyll to carotenoids [18, 19]. Consequently, it may be assumed that in the light fragments of chloroplasts and, in particular, in the smallest particles remaining in the supernatant above SCP-144, changes occur in the structure of the chlorophyll-carotenoid complex leading to the decrease of the efficiency of the triplet-triplet energy transfer causing the increase of the number of chlorophyll molecules uncapable of this process. The authors are grateful to F. F. Litvin for discussion of the results. REFERENCES I. KRASNOVSKII A. A. (Jr.) and LITVIN, F. F. Izv. Akad. Nauk SSSR, Ser. fiz. 39: 1968. 1975 2. KRASNOVSKII A. A. (Jr.) et at., Dokl. Akad. Nauk SSSR 225: 207, 1975 3. KRASNOVSKIL A. A. (Jr.), LEBEDEV, N. N. and LITVIN, F. F., Studia biophys. 65: 81, 1977 4. KRASNOVSKII A. A. (Jr.), LEBEDEV, N. N. and LITVIN, F. F. in: Proceedings of 3rd Int. Seminar on Energy Transfer in Condensed Matter (Ed. J. Fiala) p. 111, Prague. 1978 5. KRASNOVSKII A. A. (Jr.) and KOVALEV, Yu. V, Biofizika 23: 920, 1978 6. KRASNOVSKII A. A. (Jr.) et at., Dokl. Akad. Nauk SSSR 251: 1264, 1980 7. SHUVALOV, V. A. Biochim. Biophys. Acta 430: 113, 1976 8. ANDERSON, J. M. and BOARDMAN, N. Biochim. Biophys. Acta 112: 403. 1966 9. OSTROVSKAYA, L. G. et at., Dokl. Akad. Nauk SSSR 209: 1457, 1973 10. GOODCHILD, D. J. and PARK, R. В., Biochim. Biophys. Acta 226: 393 11. ARNTZEN, С J. et al., Biochim. Biophys. Acta 256: 85, 1972

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