Photosynthesis Research 30: 7-14, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves Assignment o f spectral bands N.N. Lebedev ~, A.A. Krasnovsky, Jr. 1'2 & F.F. Litvin 2

~A.N. Bakh Institute of Biochemistry Academy of Sciences of the USSR, Moscow 117071 USSR; 2Biology Department, Moscow State University, Moscow 119899, USSR Received 13 May 1991; accepted in revised form 11 July 1991

Key words: protochlorophyll, chlorophyll, phosphorescence, chlorophyll biosynthesis, chloroplast formation Abstract

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196 °C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2-20 min) illumination of etiolated leaves at the temperature range from - 3 0 to 20 °C leads to the appearance of new phosphorescence bands at about 990-1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for l h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955-960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.

Abbreviations: Pchl - protochlorophyll and protochlorophyllide; Chld - chlorophyllide; Chl - chlorophyll Introduction

Phosphorescence is a light emission caused by the direct electronic transition from the triplet to

the ground singlet state of excited molecules. Phosphorescence measurements can serve as a tool for the study of both the energy and the deactivation dynamics of the triplet state. The

8 first reliable data on phosphorescence of Pchl(ide) and Chl a in solutions at liquid nitrogen temperature were published by Krasnovsky et al. (1971, 1973, 1974, 1975) and were later confirmed by Mau and Puza (1977), Dvornikov et al. (1978) and Kleibeuker et al. (1978). Phosphorescence was also detected in etiolated and greening leaves and isolated chloroplasts at -196 ° (Krasnovsky et al. 1975, 1977, Lebedev 1976). More recently, the phosphorescence of Chl and its precursors was also observed in mature green leaves of different plants and alga cells (Krasnovsky and Kovalev 1978, Kovalev and Krasnovsky 1986, Krasnovsky and Neverov 1988). The results of the phosphorescence studies published before 1982 have been reviewed by Krasnovsky (1982) and Hoff (1986). Unlike most other conventional luminescence methods, phosphorescence measurements provide direct information on the pigment triplet state, an application of phosphorescence methods opens up additional possibilities for investigation of the spectroscopy, arrangement and mechanisms for action of the pigments in plants. To obtain such information, one needs to have a reliable assignment of the phosphorescence emission bands with respect to the pigment forms which are well known from numerous absorption and fluorescence studies. A preliminary assignment was made in our previous papers cited above. The aim of this work is to present detailed information based on joint investigation of the phosphorescence spectra, phosphorescence lifetime and phosphorescence excitation spectra in etiolated and greening leaves.

Materials and methods

Etiolated bean seedlings were grown in aqueous culture at 24-25 °C for 7-10 days under complete darkness. For their illumination, a white light (10 -4 W cm -2) from luminescent lamps was used. Phosphorescence was measured on instruments described elsewhere (Krasnovsky et al. 1974, Lebedev 1976, Krasnovsky 1979). They comprise mechanical phosphoroscopes, highpressure 1 kW xenon lamps, S-1 photomultiplier tubes, cooled to -60°C, and high-throughput

monochromators. This equipment allowed us to measure excitation spectra, emission spectra and lifetimes for the delayed luminescence with a lifetime exceeding 0.5 ms. The phosphorescence measurements were made at liquid nitrogen temperature using special metal sample-holders immersed into quartz Dewar vessels. The phosphorescence spectra obtained are corrected for the wavelength response of the detecting system which was determined using calibrated band incandescent lamps. Relative values of the phosphorescence quantum yields (~p) were measured using excitation by red light passed through a cut-off filter KS-13 (A/> 630 nm). The relative ~p values were calculated as the ratio of the phosphorescence intensity to the area under the absorption spectra of leaves or pigment solutions in the region of excitation. Fluorescence emission and excitation spectra were recorded with the same instruments using special windows in the phosphoroscopes. Absorption spectra were measured on SF-18 spectrophotometers (Leningrad, Optical Mechanics Enterprises). For low temperature measurements, a small Dewar vessel containing samples was placed inside the integrating sphere of the SF-18. This allowed us to measure the lowtemperature absorption spectra of strongly scattering leaves and solutions.

Results and discussion

Pchl phosphorescence As shown in our previous studies, the Pchl phosphorescence spectrum in etiolated leaves consists of three major bands at 870, 920 and 970 nm. The red part of the excitation spectrum for the sum of these bands has maxima at 631-636 and 648-649 nm, which are close to the absorption maxima of Pchl in etiolated leaves. However, the intensity of the 631-636 nm band is higher in the excitation spectrum. The short wavelength part of the excitation spectra, as well as fluorescence excitation spectra of PChl (Butler 1961), does not contain carotenoid bands (Krasnovsky et al. 1975, 1977, Ignatov et al. 1983). To measure the phosphorescence excitation spectra for each of the Pchl phosphorescence

9 bands we used narrow-band interference light filters with the transmission maximum at 870, 920 and 970 nm, respectively. We observed that excitation spectrum for the 870nm emission band shows a single red maximum at 627 nm and a sharp drop at longer wavelengths. Excitation spectra for the phosphorescence emission at 920 and 970 nm are rather similar. Both have main maxima at 648-649 nm and a broad band at 631-638nm with a weak shoulder at 627nm (Fig. la). If the 920nm band is registered, the maximum of the broad band is detected at 632nm. If the phosphorescence is measured at 970 nm, this maximum is shifted to 636 nm. It means that the phosphorescence maximum at 870nm is mostly associated with the 627nm absorption band of Pchl. This phosphorescence slightly contributes to the emission at 920 and 970 nm which mostly belongs to the long wavelength Pchl forms. In agreement with this, both the 627 nm excitation band and the 870 nm emission band increased after any treatments which increase the contribution of the short wavelength Pchl into the absorption spectra of etiolated leaves. This was observed after incubation of etiolated leaves at 80° for 5 rain, after treatment of etiolated seedlings with ~-aminolevulinic acid, in homogenates of etiolated leaves and in etiolated leaves genetically enriched in short wavelength Pchl

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(Krasnovsky et al. 1975, 1977, Lebedev 1976, Ignatov et al. 1983). These data correlate also with the results of the phosphorescence lifetime (Zph) measurements (Fig. lb). One can see that in the wavelength range 900-1000 nm, Zph is equal to 2.53 ms. At wavelengths shorter than 900 nm, ~'ph increases up to 10 ms, which coincides with rph for monomeric (monosolvated) Pchl in solutions (Krasnovsky et al. 1975, 1977). This means that Pchl responsible for the short wavelength phosphorescence, is probably monomeric, while the long wavelength phosphorescence is emitted by aggregated Pchl (Pchl). It also shows that there is no efficient triplet-triplet energy transfer from the phosphorescent short wavelength Pchl to longer wavelength Pchl forms which is possibly caused by a spatial disconnection. Important information comes from the measurement of the low temperature afterglow for etiolated leaves preilluminated at room temperature. Short-term (for several minutes) preillumination leads to disappearance of the 920 nm band and to development of new bands at 940 and 990nm (Fig. 2). The 870nm phosphorescence band was observed in such leaves with excitation by red light at A/> 620 nm, but it was not detected with excitation by deep red light at h ~> 650 nm. The excitation spectrum for the sum of these

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bands in preiUuminated leaves has maxima at 625, 667 and 676-678 nm and no Pchl maxima at 632-636 and 6 4 9 n m (Fig. 3). Taking into account the above dependence of the phosphorescence spectra on excitation wavelengths (Fig. 2), we can conclude that 625 nm excitation band corresponds to the 870 nm emission maximum while the phosphorescence emission at h~> 900 nm has excitation maxima at 667 and 678 nm

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that is different from the maxima of the Pchl absorption bands. Thus, it is shown that the 870 nm phosphorescence band is emitted by the short wavelength Pchl which is usually denoted as P627 according to the position of its red absorption maximum. This Pchl form is known to be inefficient in the process of photochemical Chl a formation (Kahn and Boardman 1970, Belyaeva and Litvin 1989, Ignatov et al. 1983). The 920 and 970 nm bands belong to the photochemically active Pchl forms, P637 and P650. Taking into account the absorption maxima of these forms, we can assume that the 920 nm band belongs to P637 and the 970 nm one to P650. It is known that the low temperature fluorescence of Pchl in etiolated leaves is emitted mainly by P650, while P627 and P637 have a very weak fluorescence which is hardly detectable (Belyaeva and Litvin 1989, Ignatov et al. 1983). According to our assignment the low temperature phosphorescence of P650 also dominates, but contributions of P637 and P627 to the overall phosphorescence emission is much higher• Taking into account the millisecond lifetime of the short wavelength phosphorescence, one can conclude that some fraction of Pchl molecules belonging to P627 and P637 are energetically disconnected with P650 and dissipate their excitation energy separately.

11

Pchl phosphorescence quantum yield To determine the efficiency for Pchl (P650) triplet state formation, we compared the Pchl phosphorescence intensity in etiolated leaves and in a Pchl solution in pyridine. The results of the first rough estimation was briefly reported by Krasnovsky (1982). Further analysis confirmed that under excitation with red light 2, 1>630 nm and the Pchl phosphorescence quantum yield (~p) in etiolated leaves makes 40 -+ 20% of that in solution. The phosphorescence quantum yield is given by:

~p = c~ zp/rr , where ~t is the quantum yield of the pigment triplet state formation, rp and r r are the actual and radiative lifetimes of the phosphorescence, respectively. The rp value for Pchl in pyridine is 4 ms (Krasnovsky et al. 1975, 1977) while Zp for Pchl in leaves is 2.5-3ms. Hence, one may conclude that the ~t/rr ratio in leaves is about 60% of the (I)t/T r ratio in Pchl solution. If we assume that the rr value is the same in a pigment solution and in leaves, and take into account that the ~t value for Pchl in solution is 80% (Dzagarov et al. 1977), two conclusions can be drawn: the phosphorescence emission is determined by most (or all) of the Pchl molecules involved in absorption of the exciting light, and the quantum yield for Pchld (mostly P650) triplet state formation in frozen leaves is about 50%. This result is in a good agreement with flashphotolysis measurements made by Frank and Mathis (1980).

Chld phosphorescence After the short-term illumination (for 2-20 min), absorption and fluorescence spectra of etiolated leaves in the red are known to be represented mostly by Chld (C680). At the same time we observed phosphorescence emission bands at 940-945 and 990-1000 nm (Fig. 2) (Krasnovsky et al. 1975, 1977). The 990nm maximum was observed after preiilumination during 2-5 min. It was shifted to 995-1000 nm after preillumination for 10-30 min. The lifetime was about 2 ms and 1.1ms at 940 and 1000nm, respectively. The

relative intensities of these peaks were practically independent of the preillumination time of etiolated leaves (2-20 min) and on temperature during preillumination in the range from - 3 0 up to 20 °C. As shown above, the excitation spectrum corresponding to the sum of these bands has red maximum at 676-678nm and a shoulder at 665 nm (Fig. 3). The relative intensity of the 940 nm band decreased under excitation at a/> 680 rim. This makes it possible to propose that the 990 nm phosphorescence band belongs to the Chld (C680) while the 940 nm band corresponds to shorter wavelength Chld or Chl (C665). It was shown in our previous studies that the 940nm phosphorescence band is observed in solutions of monomeric Mg-monosolvated Chl a while the 990 nm phosphorescence band corresponds to aggregated Chl in non-polar solvents (Krasnovsky et al. 1974, 1977). Thus, phosphorescence measurements demonstrate that biosynthesis of the long wavelength aggregated Chld in etiolated leaves is accompanied with formation of the monomeric pigment. This conclusion is in agreement with results of our recent fluorescence study (Lebedev et al., to be published). However, the phosphorescence method is much more efficient for detection of monomeric Chl at this stage of greening.

Chl a phosphorescence After preillumination of etiolated leaves at room temperature for 0.5h, the phosphorescence at 990nm relatively decreases and an emission band at 955-960 nm appears. After 1 h of greening, the 960 nm band becomes dominant. It has the excitation maximum at 668 nm and rp = 1.72.2 ms. This coincides with the phosphorescence spectrum and lifetime for monomeric Chl a in solutions (Krasnovsky et al. 1974, 1977). At the same time, Chl a (C670) becomes dominant in absorption and fluorescence spectra (Figs. 4 and 5). The Chl phosphorescence quantum yield in leaves at this stage of greening is about 50% of that for monomeric Chl in pyridine which indicates a very high efficiency of the triplet state formation (Egorov et al. 1985). The absorption bands of carotenoids are not detected in the phosphorescence excitation spec-

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trum (Krasnovsky et al. 1975, Lebedev 1976). This correlates with the results of the measurement of the fluorescence excitation spectra (Butler 1961, Fradkin et al. 1985). Figures 4 - 6 show how the greening influences the quantum yield, spectrum and lifetime of the

) of Chl phosphorescence.

Chl a phosphorescence• It is seen that the formation of normal photosynthetic structures and the appearance of the strong long wavelength bands in the absorption and low temperature fluores-

13 cence spectra of leaves are accompanied by the sharp decrease of the phosphorescence intensity and by minor changes in the phosphorescence lifetime and emission and excitation spectra. After 12 h of greening, it is 1% of that observed after 1 h of greening. At the same time, the 980nm band increases in the phosphorescence spectrum, but the lifetime and the red maximum of the Chl phosphorescence excitation spectrum remains practically unaltered (Figs. 5 and 6). Further greening leads to further phosphorescence quenching. In mature green leaves grown up in normal conditions, the phosphorescence quantum yield is 0.1% of that in etiolated leaves after 1 h of greening (Krasnovsky and Kovalev 1978, Krasnovsky 1982, Kovalev and Krasnovsky 1986). However, the phosphorescence lifetime and the maximum in the phosphorescence excitation spectra are the same as in etiolated leaves after 1 h of greening. In the phosphorescence spectrum of mature leaves, the 980 nm band predominates. Thus one can conclude, that at the latest stages of greening and in mature green leaves as well as in etiolated leaves after 1 h of greening, phosphorescence is likely to belong to the same group of newly synthesized Chl a molecules. Judging by the phosphorescence lifetime, the triplet state of these Chl molecules is not quenched by carotenoids, which were shown to be powerful quenchers of the Chl triplet state (Mathis and Schenck 1981). This conclusion also agrees with the fact that newly synthesized Chl a molecules are located apart from carotenoids in both etiolated leaves and completely formed photosynthetic apparatus (Fradkin et al. 1985).

Conclusion

The results obtained show that PChld phosphorescence in etiolated leaves is emitted by three forms of this pigment. The 870 nm band belongs to a short wavelength, probably monomeric, PChld (P627). The emission at 920-970 nm belongs to the long wavelength forms (P637 and P650) which are converted into Chl a. Analysis of phosphorescence data suggests that the PChld to Chl conversion occurs via intermediate formation of the long wavelength (aggregated) Chld

with the main phosphorescence maximum at 990-1000nm. This Chd is always observed together with the short wavelength Chld (Chl) with the phosphorescence maximum at 940 nm. After 60 rain of greening, both forms convert into Chl a with the phosphorescence maximum at 960 nm and the lifetime about 2ms. Further greening leads to a quick drop of this phosphorescence, but its spectra and lifetime are only weakly changed. We propose that the same Chl forms are responsible for Chl phosphorescence at the different stages of development of the photosynthetic apparatus. It is probable that they belong to a newly synthesised Chl a located in the vicinity of the centers for Chl biosynthesis. Thus, phosphorescence studies can serve as a useful tool for detection and analysis of newly synthesized Chl and its precursors in plant tissues.

References Belyaeva OB and Litvin FF (1989) Chlorophyll Photobiosynthesis. Moscow State University Publ. House, Moscow Butler WL (1961) Chloroplast development: Energy transfer and structure. Arch Biochem Biophys 92:287-295 Djagarov BM, Sagun EI, Bondarev SL, Solovyov KN and Zvirko MP (1977) Dependence of the processes of nonradiative deactivation of lowest excited states of porphyrins on their molecular structure. Biofizika 22:565-570 (in Russian) Dvornikov SS, Kniukshto VN, Sevchenko AN, Solovyov KN and Zvirko MP (1978) Polarization spectra of phosphorescence and fluorescence for chlorophylls a and b and their pheophytins. Dokl Akad Nauk SSSR 240:1457-1460 (in Russian) Egorov S Yu, Krasnovsky AA Jr and Kulakovskaia LI (1985) Investigation of the mechanism of chloroplast photodestruction: Participation of the chlorophyll triplet state. Fiziol rast 32:668-673 (in Russian) Fradkin LI, Samoilenko AG and Shlyk AA (1985) Development of energy transfer from carotenoids to chlorophyllide a in the course of Shibata shift. Dokl Akad Nauk SSSR 281:1248-1251 (in Russian) Frank F and Mathis P (1980) A short-lived intermediate in the photoenzymatic reduction of protochlorophyll(ide) into chlorophyll(ide) at a physiological temperature. Photochem Photobiol 32:799-803 Hoff AJ (1986) Triplets: Phosphorescence and magnetic resonance. In" Govindjee, Ametz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 225-266. Academic Press Inc Ignatov NV, Krasnovsky A A Jr, Litvin FF, Belyaeva OB and Walter G (1983) Low-temperature (77 K) excitation spec-

14 tra of fluorescence and phosphorescence of native forms of protochlorophyll(ide) in etiolated leaves of Phaseolus vulgaris and P. coccineous. Photosynthetica 17:352-360 Kahn A, Boardman NK and Thorne SW (1970) Energy transfer between protochlorophyllide molecules: Evidence for multiple chromophores in the photoactive protochlorophyllide complex in vivo and in vitro. J Mol Biol 48: 85-101 Kleibeuker JF, Platenkamp RI and Schaafsma TJ (1978) The triplet state of photosynthetic pigments. I. Pheophytins Chem Phys 27:51-64 Kovalev YuV and Krasnovsky AA Jr (1986) Excitation spectra of chlorophyll phosphorescence in cells of marine alga. Biofizika 31:444-448 (in Russian) Krasnovsky AA Jr (1979) Photoluminescence of singlet oxygen in pigment solutions. Photochem Photobiol 29:29-36 Krasnovsky AA Jr (1982) Delayed fluorescence and phosphorescence of plant pigments. Photochem Photobiol 36: 733-741 Krasnovsky AA Jr and Kovalev YuV (1978) Phosphorescence of chlorophyll in leaves and alga cells. Biofizika 23: 920-922 (in Russian) Krasnovsky AA Jr and Neverov KV (1988) Formation of triplet molecules of chlorophyll and its precursors in leaves treated with 5-aminolevulinic acid. Dokl Akad Nauk SSSR 302:252-255 (in Russian) Krasnovsky AA Jr, Lebedev NN and Litvin FF (1974) Spectral characteristics of phosphorescence of chlorophylls and pheophytins. Dokl Akad Nauk SSSR 216:1406-1409 (in Russian)

Krasnovsky AA Jr, Lebedev NN and Litvin FF (1975) Detection of the triplet state of chlorophyll and chlorophyll precursors from measurement of their delayed fluorescence and phosphorescence in leaves and chloroplasts. Dokl Akad Nauk SSSR 225:207-210 (in Russian) Krasnovsky AA Jr, Lebedev NN and Litvin FF (1977) Phosphorescence and delayed fluorescence of chlorophyll and its precursors in solutions, leaves and chloroplasts. Studia biophys 65:81-89 Krasnovsky AA Jr, Romaniuk VA and Litvin FF (1973) On the phosphorescence and delayed fluorescence of chlorophylls and pheophytins a and b. Dokl Akad Nauk SSSR 209:965-968 (in Russian) Krasnovsky AA Jr, Shuvalov VA, Litvin FF and Krasnovsky AA (1971) Phosphorescence and delayed fluorescence of protochlorophyll pigments. Dokl Akad Nauk SSSR 199: 1181-1184 (in Russian) Lebedev NN (1976) Phosphorescence characteristics and triplet state properties of chlorophyll in living cells and model systems. Candidate Dissertation, Moscow State University, Moscow Mathis P and Schenck CC (1981) The function of carotenoids in photosynthesis. In: Britton G and Goodwin TW (eds) Carotenoid Chemistry and Biochemistry, pp 339-351. Pergamon Press, Oxford Mau AWN and Puza M (1977) Phosphorescence of chlorophylls. Photochem Photobiol 25:601-603