FLUORESCENCE YIELD OF CHLOROPHYLL A AND PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS

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1 Plant & Cell Physiol., 8, (1967) FLUORESCENCE YIELD OF CHLOROPHYLL A AND PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS SHIGEKI OKAYAMA 1 Department of Biology, Faculty of Science, Kyushu University, Fukuoka (Received August 22, 1966) 1. Effect of various treatments on the fluorescence yield of chlorophyll o in isolated chloroplasts was studied in relation to the activities of the HILL reaction and the ascorbate photooxidation. 2. CMU and o-phenanthroline showed no appreciable effect on the fluorescence yield at the concentration which completely inhibited the HILL reaction % SDS was sufficient to inactivate the HILL reaction completely and caused a 40% decrease in the fluorescence yield, although no shift of the absorption and fluorescence bands could be observed. At higher concentrations of SDS, the fluorescence yield increased by more than three times the original value and the absorption andfluorescencebands shifted by 5 rap toward the shorter wavelength side. 4. Ultraviolet irradiation, incubation at ph 9.2, heating at 40 and digestion with trypsin resulted in loss of the HILL activity and decrease of the fluorescence yield. 5. The DPIP-mediated photooxidation of ascorbate was enhanced by heating broken chloroplasts at 55 for 5 min, and inactivated at 70. The photooxidation of ascorbate in the absence of the dye was inhibited by 60% on heating at 55. The o-phenanthroline-sensitive photooxidation of ascorbate was inactivated at 55. The o-phenanthroline-resistant photooxidation of ascorbate was rather thermostable. The fluorescence yield was reduced by 40-50% at 55. The fluorescence yield of chlorophyll a in vivo is known to decrease in general when electrons are efficiently transported in the photosynthetic system. The fluorescence yield of isolated chloroplasts is lower in the presence of a HILL oxidant than in its absence (1-8). On the other hand, the fluorescence yield of chlorophyll is known to be highly dependent on its state. Chlorophylls dissolved in polar organic solvents are fluorescent, while chlorophylls dissolved in dry hydrocarbons or the colloidal aggregates of chlorophyll in water are not fluorescent (-4). Treatments of isolated chloroplasts with Abbreviations: CMU, 3-(p-chlorophenyl)-l, 1-dimethyl urea; DPIP, 2, 6-dichlorophenol indophenol; SDS, sodium dodecyl sulfate. 1 Present address: Biological Laboratory, General Education Department, Kyushu University, Otsubo-machi, Fukuoka. 47

2 48 S. OKAYAMA Vol. 8 (1967) lipase (5), strong light (3), organic solvents (6, 7) and detergents (7) are reported to change the steady state fluorescence yield of chloroplasts. In a previous paper (5), it was reported that the digestion of isolated chloroplasts with lipase caused a decrease in the fluorescence yield of chlorophyll a with concomitant inactivation of the photochemical oxygen evolving system, whereas the NADP photoreduction system remained unchanged. Such lipase-treated chloroplasts could photooxidize ascorbate more actively in the presence of DPIP, and less actively in the absence of the dye, than non-treated chloroplasts. In this paper, it will be described that the fluorescence yield of chlorophyll a in isolated chloroplasts is also reduced, when the HILL reaction is inactivated by various treatments other than lipase digestion. The ascorbate-photooxidizing activity of heat-treated chloroplasts was also studied in relation to the fluorescence yield. MATERIALS AND METHODS Broken choloroplasts were prepared from the leaves of Spinacia oleracea L. or Beta vulgaris L., as described in the previous paper (5). Subsequent treatments of broken chloroplasts were done with minimal exposure to visual light. Treatments with SDS (prepared from Duponol c according to the procedure described by SCHMIDT (S)) was accomplished as follows; broken chloroplasts containing 0.2 mg chlorophyll were dispersed in 2 ml of % SDS in 0.05 M phosphate buffer (ph 7.0) at 20 and the mixture was cooled immediately to 4 and left for 30 min at the same temperature. Irradiation with ultraviolet light (253.7 ran) was carried out at 4, using a " Manasru-Light Model UV-S1" ultraviolet lamp (Manasru Chemical Industry Co.) placed at a distance of 3 cm from the suspension of broken chloroplasts containing 0.5 mg chlorophyll in 5 ml of 0.1 M phosphate buffer (ph 7.0) in an open Petri dish (4cm in diameter). For the purpose of incubation at ph 9.2, broken chloroplasts containing 1 mg chlorophyll were suspended in 10 ml of 0.05 M disodium phosphate solution at 20. Digestion by trypsin was carried out at 20 in an incubation mixture containing broken chloroplasts equivalent to lmg chlorophyll and 2mg trypsin (Kanto Kagaku Co.) in 5 ml of 0.1M phosphate buffer (ph 7.0). Heat-treatment at 40 was performed on a mixture containing broken chloroplasts equivalent to 0.1 mg chlorophyll per ml of 0.1 M phosphate buffer (ph 7.0). The activities of the HILL reaction, using DPIP as the oxidant, and of the photooxidation of ascorbate were determined as described in the previous paper (5). The fluorescence was determined in a Hitachi photo-electric spectrophotometer model EPU-2 with an attachment for the fluorescence measurement. The 365 m^ mercury line was used for excitation (cf. 5). All the fluorescence yields reported in this paper are relative values of the steady state emission at m/*, and were determined on a broken chloroplast suspension containing 0.02 mg chlorophyll per 4 ml of 0.1 M phosphate buffer (ph 7.0), unless otherwise noted. Under the present experimental conditions,

3 FLUORESCENCE OF CHLOROPLASTS 49 the fluorescence intensity was directly proportional to the chlorophyll concentration of broken chloroplasts up to 0.03 mg per 4 ml. Chlorophylls were determined according to the method of ARNON (9). RESULTS CMU and o-phenanthroline, potent inhibitors of the HILL reaction, are known to attack the photochemical oxygen evolving system (10,11). Inhibition of photosynthetic electron transport by these inhibitors has been known to increase the fluorescence yield of chlorophyll a in vivo (2, 8). MURATA et al. [12,13) reported that such an increase of fluorescence by CMU in isolated chloroplasts was observed only in the presence of a HILL oxidant. In accordance with their result, CMU and o-phenanthroline at the concentration which completely inhibited the HILL reaction did not appreciably affect the fluorescence yield of broken chloroplasts without added electron acceptor, as shown in Table I. Some physicochemical properties of chloroplasts solubilized with a mixture of Duponol c and Span 80 were studied by CHIBA (U). Further investigation on the photochemical activities of the solubilized chloroplasts suggested that the treatment of chloroplasts with the surface-active agents might modify the organization of the chlorophyll molecules in the system (15). Fig. 1 shows the fluorescence spectra of broken chloroplasts, solubilized broken chloroplasts, acetone-extracted chloroplast pigments dispersed, in the buffer containing the detergents, and the extracted chloroplast pigments in 93% acetone. It may be seen that the fluorescence yields and spectra of chlorophyll a depend on the state of this pigment. The inhibition of the HILL reaction and the change in the fluorescence yield by the treatment with SDS are shown in Fig. 2. In this experiment, Duponol c was purified by recrystalization from ethanol solution and the resulting SDS (cf. 8) was used without the addition of Span 80. The chloroplast suspension gradually became visually clear at SDS concentrations higher than 0.05%. The HILL reaction was completely inhibited at a lower detergent concentration, 0.01%. These results are qualitatively TABLE I Effect of CMU and o-phenanthroline on the fluorescence yield and the Hill activity Addition None 10-'M CMU 10" 3 M o-phenanthroline HILL activity X Fluorescence yield' A*moles DPIP photoreduced per mg chlorophyll per min. ' Relative value.

4 50 S. OKAYAMA Vol. 8 (1967) 100 ft / i ' l 80 Z z z 60 oc O RELATIVE FL 20 i i i i / l. i / \, \ 1 \ / \ - i \ i > i T 3 ' - ^ 1 ',12 1 i O< WAVELENGTH [mf) Fig. 1. Fluorescence spectra of broken chloroplasts suspended in 0.1M phosphate buffer, ph 7.0 (curve 1), broken chloroplasts solubilized with a 3:1 mixture of 1% Duponol c and 1% Span 80 in the buffer (curve 2), acetone-extracted chloroplast pigments dispersed in the buffered detergent-mixture (curve 3), and the chloroplast pigments in 93% acetone (curve 4). The concentration of chlorophyll was mg per 4 ml in each case. in accordance with those of KE and CLENDENNING {16) who found that the HILL reaction was inhibited irreversibly by several surface-active agents, including SDS, at much lower concentrations than those required for chloroplast dispersion. Their data showed that 0.038% SDS was required for the complete inhibition of the HILL reaction with p-benzoquinone as the oxidant. Their value is about three times that found in the present study for the DPIP-HILL reaction. The value for the DPIP-HILL reaction coincides well with that reported by SAUER and PARK (7) for the ferricyanide-hill reaction. In the present study, however, the molar ratio of the detergent to the chlorophyll content in broken chloroplasts for 50% inhibition of the HILL i _ 800

5 FLUORESCENCE OF CHLOROPLASTS ' 0.05' CONCENTRATION OF SDS IN PER CENT Fig. 2. Effect of SDS on the fluorescence yield (open circles) and the HILL activity (closed circles) of broken chloroplasts. activity was found to be about 0.18 molecule SDS per chlorophyll molecule, although a value of 0.3 was obtained by SAUER and PARK. They reported that SDS (0.1-1%) enhanced the fluorescence yield of spinach quantasomes, but the data concerning the yield at lower concentrations of the detergent were not presented. Fig. 2 shows that the fluorescence yield decreased to 60-65% of the original level at detergent concentrations lower than 0.01%, which was required for the complete inhibition of the HILL reaction. The peak of fluorescence, situated at m^, was the same as that in the case of non-treated broken chloroplasts. No shift of the red absorption band was observed. This is also in agreement with the result of SAUER and PARK. However, the fluorescence band shifted to m// at concentrations of SDS higher than 0.05%, where the fluorescence yield began to exceed the original level. Also the red absorption band shifted from 680 m^ to 675 m/*.

6 52 S. OKAYAMA Vol. 8 (1967) Thus SDS was found to interact with chloroplasts in two different manners depending on the concentration: at lower concentrations, SDS decreased the fluorescence yield of chlorophyll, and at higher concentrations, it increased the yield. It was reported that irradiation with ultraviolet light (253.7 m/a) inactivates the HILL reaction in isolated chloroplasts without affecting the concentration of chlorophylls (17). BISHOP (18) suggested that the inactivation was due to the destruction of plastoquinone, which is known to be an essential redox component in the HILL reaction. Although this was further confirmed by SHAVIT and AVRON (19), their attempts to relieve the inhibition of the activity by readdition of plastoquinone were unsuccessful. They concluded, therefore, that the inhibition was due to the destruction of a certain TIME IN MINUTES Fig. 3. Effect of irradiation with ultraviolet light (solid lines) on the fluorescence yield (open circles) and the HILL activity (closed circles) of broken chloroplasts. The non-irradiated preparation (broken line) was left in 0.1 M phosphate buffer (ph 7.0) at 4.

7 FLUORESCENCE OP CHLOROPLASTS 53 factor (or factors) in addition to plastoquinone. HALLDAL {20) reported the ultraviolet action spectrum of photosynthetic inhibition. He interpreted the curve as indicating the inhibition attributable to destruction of a protein-like substance. Fig. 3 shows the decrease in the fluorescence yield of chlorophyll during the irradiation with ultraviolet light. No shift of the fluorescence band was observed. It seems, therefore, that the inactivation of the HILL reaction with ultraviolet light may be due to a combined effect of destruction of plastoquinone and some change in the state of chlorophyll resulting in a reduced fluorescence yield. The HILL reaction is known to be thermolabile. From a kinetic study of its thermal inactivation, BISHOP et al. {21) suggested that the inactivation was due to thermal denaturation of proteins. HlNKSON and VERNON {22) reported that the treatment of chloroplasts at ph 8.5 or 51 preferentially inactivated the HILL reaction, while leaving the photoreduction of indigocarmine and the photooxidation of ascorbate relatively unaffected. They o 8 o TIME IN MINUTES TIME IN MINUTES Fig. 4. Effect of incubation at ph 9.2 (solid lines) on the fluorescence yield (open circles) and the HILL activity (closed circles) of broken chloroplasts. The control preparations (broken lines) were incubated in 0.1 M phosphate buffer (ph 7.0) at 4. (a) and (b) are the results obtained with different preparations of broken chloroplasts.

8 54 S. OKAYAMA Vol. 8 (1967) 100 I 3.--* T-«loot o TIME IN MINUTES Fig. 5. Effect of heating at 40 (solid lines) on the fluorescence yield (open circles) and the HILL activity (closed circles) of broken chloroplasts. The control preparation (broken line) was incubated in 0.1 M phosphate buffer (ph 7.0) at TIME IN MINUTES Fig. 6. Effect of tryptic digestion (solid lines) on the fluorescence yield (open circles) and the HILL activity (closed circles) of broken chloroplasts. The control preparation (broken line) was incubated in 0.1 M phosphate buffer (ph 7.0) at 20. suggested that some components operative in the photochemical oxygen evolving system might be affected by these treatments. Figs. 4, 5 and 6 show the inactivation of the HILL reaction and the decrease in the fluorescence yield during the incubation of chloroplasts at ph 9.2, by heating at 40, and during the digestion with trypsin, respectively. As can be seen in these figures, the time courses did not coincide with each other. Thus, it is inferred that the destruction of some components, in addition to the change in the state of the fluorescent chlorophyll a, might be responsible for the inactivation of the HILL reaction. The degree of decrease in the fluorescence yield varied with different chloroplast preparations. A typical example is shown in Figs. 4-a and 4-b. Ascorbate in the presence of DPIP has been known to substitute for the photochemical oxygen evolving system, by donating electrons to the oxidizing equivalent produced by the initial photoact (15, 23). Curve 1 in Fig. 7 shows that the photooxidation of ascorbate mediated by DPIP was most markedly enhanced by heating chloroplasts at 55, where the HILL reaction was completely inactivated. These results indicate that the photooxidizing system of the DPIP-ascorbate couple is more thermostable than the photochemical oxygen evolving system and that there is a competition between water and the DPIP-ascorbate couple as the electron donor for the photochemically produced oxidizing equivalent. On the other hand, it has been known that, in the absence of DPIP, ascorbate donates its electrons to the electron transport chain at the site

9 All FLUORESCENCE OF CHLOROPLASTS 55 1 I / \ Z z TIAL i- PER 40 _ 0 1 ^ \^ / \ / \ / ' ' " Y 5 / \ \ 2 \ \ N \ > \" s \» \ \ \ \ \ \ V \ \/\ \ TEMPERATURE ( Q Fig. 7. Effect of heat-pretreatment on the photooxidation of ascorbate in the presence (curve 1) and the absence (curve 2) of DPIP, the HILL activity (curve 3), and the fluorescence yield (curve 4). The scattering of light (curve 5) was determined by the attenuation of the light of 750 mp. The chloroplast suspensions containing 0.1 mg chlorophyll per ml of 0.1 M phosphate buffer (ph 7.0) were heated for 5 min at the temperature indicated. where water supplies its electron to the chain {15, 24). Curve 2 in Fig. 7 shows that the photooxidation was inhibited by 60% at 55 in the absence of DPIP. Thus heat-treatment of chloroplasts at 55 was found to inhibit the photooxidation to almost the same extent as in the case of the inhibition with o-phenanthroline (40-55% inhibition) at the concentration which completely inhibited the HILL reaction (cf. IS). Fig. 8 shows the effect of heating on the o-phenanthroline-sensitive and -insensitive photooxidation of ascorbate in the absence of DPIP. The inhibitor-sensitive photooxidation was found to be completely inhibited at 55, whereas the resistant one was rather thermostable. The fluorescence yield of broken chloroplasts \

10 56 S. OKAYAMA Vol. 8 (1967) TEMPERATURE ( C) Fig. 8. Effect of heat-pretreatment on the photooxidation of ascorbate in the presence (curve 1) and the absence (curve 2) of o-phenanthroline (10~ 3 M), and the fluorescence yield (curve 3). The o-phenanthroline-sensitive photooxidation (curve 4) was calculated by subtracting the activity in the presence of the inhibitor from the one in its absence. Curve 5 shows the scattering of the light. The conditions of heat-pretreatment were the same as in Fig. 7, except that a different broken chloroplast preparation was used. decreased to 60-50% of the original level after heating at 55. The yield of the preparations heated at 70 and 80 might have been underestimated, since the heat coagulation of chloroplast preparations caused a large increase in light scattering, as shown in Fig. 7, curve 5 and Fig. 8, curve 5. DISCUSSION All the treatments used in the present experiments resulted in the destruction of the HILL activity and the decrease in the fluorescence yield of chlorophyll a in broken chloroplasts, except in the case of treatment with

11 FLUORESCENCE OF CHLOROPLASTS 57 SDS at higher concentrations. The pigment concentration was not changed by these treatments, and no appreciable shift of the absorption and fluorescence bands was detected when the fluorescence yield decreased. Further, the light-scattering usually decreased slightly, except in the case of heattreatments at 70 and 80 (cf. Fig. 7, curve 5 and Fig. 8, curve 5 in this paper and Fig. 5 in ref. 5). The observed decrease in the fluorescence yield, therefore, is neither due to destruction of chlorophyll a, nor due to an increase in self-absorption of fluorescence or light scattering. On the other hand, inhibitors of the HILL reaction, such as CMU or o- phenanthroline, did not appreciably affect the fluorescence yield of chlorophyll a (Table I). Thus modes of action of these inhibitors and various treatments are quite different. A possible inference from these results is that the decrease in the fluorescence yield may be related to some changes in the state of the fluorescent chlorophyll a. A part of the fluorescent chlorophyll a might be changed into less fluorescent and photochemically inactive forms by the various treatments used in the present experiments. Most of the light absorbed by such converted chlorophyll a might be degraded thermally and made inavailable for the photochemical reaction. From the results obtained by lipase-digestion of chloroplasts, it has been suggested that the lipid interphase supports the chlorophyll a molecules participating in the photochemical oxygen evolving system, to make it fluorescent and photochemically active (5). Also in the case of SDS treatment, the lipid-protein interphase in the lamellae may be attacked at lower concentrations than those required for chloroplast dispersion (cf. 16). On the other.hand, it seems that the treatments with trypsin, ultraviolet light, alkali, and heat damage proteinous components in the chloroplasts. It may, therefore, be concluded that the fluorescent chlorophyll a molecules are hold by both lipid and protein molecules in a photochemically active state. A possible mechanism of the photooxidation of ascorbate in the presence or absence of DPIP by isolated chloroplasts was discussed in the previous paper (15). o-phenanthroline-sensitive photooxidation of ascorbate was completely inhibited when the HILL reaction was inactivated by digestion with lipase (5) or by treatment at 55 for 5min (Fig. 8). Such a photooxidation might be mediated by the fluorescent chlorophyll a, which is unstable to these treatments. On the other hand, the photooxidation of ascorbate resistant to the inhibitor of the HILL reaction might be sensitized by pigments stable to these treatments. Such a pigment system might not be identical with the system responsible for the DPIP-mediated photooxidation of ascorbate, since the inhibitor-resistant photooxidation is not enhanced by heat-treatment at 55, differing from the DPIP-mediated photooxidation (compare curve 1 in Fig. 7 with curve 1 in Fig. 8). The author wishes to express his gratitude to Prof. Y. CHIBA for his kind guidance throughout the course of this study. Thanks are also due to Assoc. Prof. T. SASA for his valuable advice in preparing this manuscript, and to Dr. S. KATOH, the University of Tokyo, for his kind gift of CMU.

12 58 S. OKAYAMA Vol. 8 (1967) REFERENCES (1 ) R. LUMRY, B. MAYNE and J. D. SPIKES " Fluorescence yield against velosity relationships in the Hill reaction of 'chloroplast fragments. Disc. Faraday Soc, 27, (2) L. N. M. DUYSENS and H.'E. SWEERE." Mechanism of two photochemical reactions in algae as studied by means of fluorescence. In Studies on Microalgae and Photosynthetic. Bacteria (Special issue of Plant & Cell PhysioL). p ( 3 ) B. KOK, E. B. GASSNER and H. J. RURAINSKY Photoinhibition of chloroplast reactions. Photochem. Photobiol., 4, { 4 ) R. LIVINGSTON The photochemistry of chlorophyll. In Handbuch der Pflanzenphysiologie. 5. Edited by W. RUHLAND. p springer-verlag. Berlin. ( 5 ) S. OKAYAMA Effect of lipase-digestion on photochemical' activities of isolated chloroplasts. Plant & Cell PhysioL, ( 6 ) J. B. THOMAS and W. F. G. FLIGHT Fluorescence responces of chlorophyll in vivo to treatment with acetone. Biochim. Biophys. Acta, 79,' ( 7 ) K. SAUER and R. B. PARK Molecular orientation in quantasomes. II. Absorption spectra, Hill activity and fluorescence yields, ibid., 79, ( 8 ) G. SCHMIDT Preparation of ribonucleic acid from yeast and animal tissues. In Methods in Enzymology., 3. Edited by S.P. COLOWICK and N. O. KAPLAN, p Academic Press, New York. ( 9 ) D. I. ARNON Copper enzymes in isolated chloroplasts. Polyphenolase in Beta vulgaris. Plant PhysioL, 24, (10) H. GAFFRON o-phenanthroline and derivatives of vitamine K as stabi- ' lizers' of photoreduction in Scenedesmus. J. Gen. "PhysioL, 28, (11) N.I.'BISHOP The influence of the herbicide, DCMU,'on the oxygenevolving system of photosynthesis. Biochim. Biophys. Acta, 27, (12) N. MURATA, M. NISHIMURA and A. TAKAMIYA Fluorescence of chlorophyll in photosynthetic system. I. Analysis of " weak light effect "-in isolated chloroplasts. ibid., 112, (15) N. MURATA, M. NISHIMURA and A. TAKAMIYA Fluorescence of chlorophyll in photosynthetic system. II..Induction of fluorescence in isolated chloroplasts. ibid., 120, (H) Y. CHIBA Electrophoretic and sedimentation studies on chloroplast proteins solubilized with surface-active agents. Arch. Biochem. Biophys., 90, (2 5) Y. CHIBA and S. OKAYAMA Photooxidation reactions by chloroplasts solubilized with surface-active agents. Plant & Cell PhysioL, 3, (16) B. KE and K. A. CLENDENNING Properties of chloroplast dispersions in the presence of detergents. Biochim. Biophys. Acta, 19, (17) A. S. HOLT, I. A. BROOKS and W. A. ARNOLD Effect of 2537 A on green algae and chloroplast preparations. /. Gen. PhysioL, 34, (18) N. I. BISHOP The possible role of plastoquinone (Q-254) in the electron transport system of photosynthesis. In Ciba Foundation Symposium on Quinones in Electron Transport. Edited by G. E. W. WOLSTENHOLME and C. M. O'CONNOR, p Little, Brown and Company, Boston. (19) W. SHAVIT and M. AVRON The effect of ultraviolet light on photo-

13 FLUORESCENCE OF CHLOROPLASTS 59 phosphorylation and the Hill reaction. Biochim. Biophys. Acta, 66, (20) P. HALLDAL Ultraviolet action spectra of photosynthesis and photosynthetic inhibition in a green and a red alga. Physiol. Plant., 17, (21) N. I. BISHOP, R. LUMEY and J. D. SPIKES The mechanism of the photochemical activities of isolated chloroplasts. I. Effect of temperature. Arch. Biochem. Biophys., 58, (22) J. W. HINKSON and L. P. VERNON Comparison of three photochemical activities of chloroplasts. Plant Physiol., 34, (23) L. P. VERNON and W. S. ZAUGG Photoreductions by fresh and aged chloroplasts: Requirement for ascorbate and 2, 6-dichlorophenolindophenol with aged chloroplasts. J. Biol. Chew,., 235, (2V) A. TREBST, H. ECK and S. WAGNER Effects of quinones and oxygen in the electron transport system of chloroplasts. In Photosynthetic Mechanism of Green Plants. NAS-NRC Pub. 1145, p