Photosynthesis Resealz'h 50: 271-283, 1996. (~) 1996KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15 N i k o l a y V. I g n a t o v & F e l i x E L i t v i n Biology Department, Moscow State University, Moscow, 119899 Russia Received 15 April 1996;acceptedin revisedform22 November1996 Key words: biosynthesis of chlorophyll a, Chlorella vulgaris, green alga mutants, long-wavelength protochlorophyll Abstract

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650 > Chlide 695/684 and Pchl (ide) 640/635 > Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203-233 K). Abbreviations: Chl - chlorophyll; Chlide- chlorophyllide; Pchlide- protochlorophyllide; Pchl - protochlorophyll; PSI R C - Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band

Introduction

The activities of the two photosystems appear in the earliest stages of Chl biosynthesis both in higher plants and in green algae (Henningsen and Boardman 1973; Plesnicar and Bendall 1973; Wellburn and Hampp 1979). However, the biogenesis of the RCs pigments had practically not been studied. Methodological difficulties in the investigation of this problem stem from the fact that the amount of Chl incorporated into RCs is 2-3 orders of magnitude smaller than the total amount of Chl synthesized. For P S I to function, the formation of the pigmentprotein complex of the Photosystem I core with longwavelength absorption band (maximum at 700 nm) is required. However, both in etiolated leaves and in greening algae, the Chl (ide) formed during the photoconversion of the native forms of Pchlide 655/650 and

Pchl (ide) 640/635-637 has absorption bands of shorter wavelength, i.e. 670-684 nm (Litvin and Belyaeva 1971; Wang 1979; Shioi and Sasa 1984). Thus, it can be suggested that the previously found minor longwavelength Pchl (ide) forms with absorption bands at 670-680 nm (Litvin and Stadnichuk 1980) may be the precursors of the Chl of P S I core. But all efforts to study the photochemical reactions of those longwavelength Pchl forms and to determine into what Chl forms they are transformed have been unsuccessful because of the extremely low content of the long-wavelength Pchl forms and the pronounced overlapping of their bands with those of Chl (ide) formed from the main Pchl (ide) forms on illumination of etiolated leaves (Ignatov et al. 1983; Belyaeva et al. 1984). It seemed promising to investigate this problem on an alga mutant with a genetic block of Chl dark biosynthesis. During the growth of these mutants in dark-

272 ness a Chl precursor is accumulated which is rapidly transformed into Chl (ide) on a subsequent short-term illumination. In the fluorescence spectra of the darkgrown yellow mutant ChloreUa vulgar& B-15 a band with maxima at 682-685 nm was previously observed (Abroskina et al. 1979). It was not, however, elucidated whether that band belonged to Chl (ide) or to its precursor. In preliminary experiments with pigment extraction (see below), the dark Chl biosynthesis of this mutant was shown to be completely blocked and the band at 682 nm was shown to belong to the long-wavelength Pchl form which is accumulated in great amounts during the dark incubation of the cells. This peculiarity of the mutant allowed the study of the photoconversion of the Pchl long-wavelength form and showed it to be a direct precursor of the longwavelength form Chl 715/696, which by its spectral properties may be identified as chlorophyll of the PSI core.

Materials and methods

The UV-induced mutant strain Chlorella vulgaris B-15 was obtained from the collection of the Laboratory of Genetics of Microorganisms of Leningrad University (Kvitko et al. 1983). The alga cultures were grown in a thermostat in darkness on agar medium with glucose (Kvitko et al. 1983) at 25 °C. For spectral measurements a sample was transferred to a special sample holder without transferring it to a liquid phase. Measurements were performed on cells pre-incubated in darkness for 4 to 8 days (the precise age of the culture is indicated in the figure legends). For fluorescence measurements samples with absorbance lower than 0.1 in the 600-750 nm region were used. Fluorescence emission and excitation spectra were measured at 77 K with a KSVU-12 spectrophotometric instrument (LOMO, Russia), consisting of a xenon lamp (200 W), two high-transmission grating monochromators MDR-12 and a microcomputer for spectra correction, smoothing and calculation of derivative spectra. Spectra were measured with a resolution better than 1 nm. The emission spectra were corrected for the spectral sensitivity of the spectrofluroimoter; the excitation spectra were plotted as emission intensity per incident quantum versus an excitation wavelength. The same instrument was used to measure the low-temperature absorption spectra (using a single-beam scheme) and also difference and derivative absorbance spectra by the procedure described in

our previous paper (Ignatov et al. 1993). Absorption spectra and difference absorption spectra were also measured by the double-beam method on a standard spectrophotometer SF-18 with an integrating sphere. Samples were illuminated with an incandescent lamp having a light filter transmitting radiation in the region 400-750 nm; illumination intensity was 102 Wlm z . Pigments were extracted with dimethylformamide (saturated with MgCO3) or ethanol at room and lower temperature (203-263 K). Extraction at low temperature was applied for isolation of the labile intermediates readily undergoing transformations at room temperature. Absorption spectra of extracts were measured with a spectrophotometer Specord M 40 (Carl Zeiss, Germany). The ratio of esterified and non-esterified Pchl and Chl derivatives was determined using two modifications of the phase separation method. The first modification based on a hexane/acetone solvent partition system was developed for Chl/Chlide-containing species (Amir-Sharipa et al. 1987). The second modification based on a petroleum ether/acetone solvent partition system was developed for Pchl/Pchlideand Chl/Chlide-containing, i.e. 'greening' species (Vorobyeva et al. 1963).

Results

Protochlorophyll (ide) native forms of the dark-grown cells ofChlorella vulgaris B-15 In the absorption spectra and their second derivatives of the dark-grown cells of the mutant, absorption bands at 627, 635, 650, 672 nm were observed (Figure 1A,B). In fluorescence emission spectra under excitation at 440 nm bands at 633,640 (shoulder), 655 and 682 nm were recorded. The short-wavelength bands of fluorescence excitation spectra measured for the emission at 633, 640 and 655 nm (Figure 2), practically coincide with the fluorescence excitation spectra of Pchl (ide) native forms Pchl (ide) 633/627, Pchl (ide) 640/635, Pchlide 655/650 earlier described for etiolated leaves and for cells of dark-grown mutants Chlamydomonas reinhardtii (Ignatov et al. 1983; Lebedev et al. 1985; Lebedev et al. 1991). Under excitation at 470 nm the 633 nm band disappeared from the fluorescence emission spectra and the intensities of minor bands at 640 and 655 nm as well as of the 682 nm band markedly increased (Figure 1C). It

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Figure 1. Low-temperature (77 K) absorption spectra of dark-grown cultures of Chlorella vulgaris mutant B- 15 (A) and their second derivatives (B) measured for 4-(1), 6-(2), and 8-day-old culture (3); A4- spectrum of 8-day-old culture after freezing to 77 K and thawing to 293 K. (C) low-temperature (77 K) fluorescence emission spectra of 8-day-old dark culture of B- 15 with excitation at 440 nm(1) and 470 nm (2).

is clear from the fluorescence excitation spectra, that this effect is due to the fact that the absorption at 470 nm of the pigment with fluorescence 633 nm is negligible in comparison with that of the long-wavelength fol'nls.

Three pairs of bands in the fluorescence emission and absorption spectra (633/627, 640/635, 655/650) correlate well with the data obtained on etiolated leaves of higher plants (Litvin and Belyaeva 1971; Ignatov

et al. 1983; Lebedev et al. 1985) and algal mutants (Wang 1979; Shioi and Sada 1984; Lebedev et al. 1991), allowing these bands to be correlated with known Pchl (ide) native forms: Pchl (ide) 633/627, Pchl (ide) 640/635 and Pchl (ide) 655/650. The estimations obtained using the absorption spectra of the extracts (Figure 3A-l) showed that Chl (ide)/Pchl (ide) ratio in the dark-grown mutant cells did not exceed 1%. This allows to relate the long wave-

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Figure 2. Low-temperature (77 K) fluorescence excitation spectra measured for fluorescence emission at 633 (1), 640 (2), 655 (3) and 682 nm (4) of 8-day-old dark-grown culture of Chlorella vulgaris B-15, (5) fluorescence excitation spectrum for emission 715 nm of the same cells after illumination with white light 102 W/m 2 for 10 s at 233 K.

length pair of spectral bands 682/672 to the long wavelength protochlorophyll (|de) native form Pchl (|de) 682/672. A form with the same spectral characteristics was earlier found in small amounts in the etiolated plant leaves (Litvin and Stadnichuk 1980). The fact that the form with spectral bands 682/672 belongs to a Pchl (|de) and not Chl (|de) is confirmed by a number

of observations: in the spectra of the 8-day-old culture the area of the long wavelength band 672 nm is 2 5 30% of the total area under the absorption spectrum curve in 620-700 nm region (Figure IA-3), while Chl (ide)/Pchl (|de) ratio in the respective extracts is lower than 1% (see above). The shares of 682 and 672 nm bands in the fluorescence emission and absorp-

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Figure 3. A - absorption spectra of extracts from unilluminated (1) and illuminated (2) cells of 8-day-old culture of Chlorella vulgaris B-15. The cells were illuminated by light 102 W/m2 for 10 s at 293 K. Solvent- dimethylformamide.B - low-temperature(77 K) difference absorption spectra of 6-(1) and 8-day-old-(2) cultures of Chlorella vulgaris B-15; spectrum of the sample illuminated by light 102 W/mz for 10 s at 233 K minus spectrum of unilluminated sample. The spectra were measured by the double-beammethod using an SF-18 spectrophotometer.

tion spectra, respectively, are enhanced with the accumulation o f the extractable Pchl (ide) during the dark cultivation of cells (Figure 1A-curves 1, 2, 3; Figure 1C and Figure 4B). Finally, a disaggregating treatment (freezing to 77 K and subsequent thawing to 293 K; heating to 343 K) caused a complete transformation

of the form with spectral bands 682/672 into the short wavelength form of protochlorophyll (ide) Pchl (ide) 633/627 (Figure 1A-4). This effect is well known for protochlorophyll Ode) native forms (Butler and Briggs 1966; Dujardin 1976).

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Figure 4. A - low-temperature (77 K) difference absorption spectrum of 4-day-old culture of Chlorella vulgaris B-15: (1) spectrum of the sample illuminated for 10 s with light 102 W/m 2 at 233 K minus the spectrum of unilluminated sample: (2) reversed second derivative of spectrum 1. B - low-temperature (77 K) fluorescence spectra of the same sample before (I) and after (2) illumination. Fluorescence was excited at 470 nm.

The ratio of the esterified and non-esterified Pchl (ide) forms in the extracts is presented in Table 1. With the prolongation of dark incubation of cells from four to eight days, the share of the esterified pigment, protochlorophyll, increased from 60 to 80% of the total amount of the precursor. Judging by the absorption spectra in vivo (Figure 1A), the share of Pchl (ide) 682/672 as compared to that of the shorter-wavelength forms, also increases during dark incubation. The comparison of the share of the 672 nm absorption band area

(Figure 1A-3 and Figure 1A-l) with the data of Table 1 shows that the growth of the 672 nm band intensity during dark incubation of cells is due to the formation of the esterified precursor- protochlorophyll Pchl 682/672. The fluorescence excitation spectrum of Pchl 682/672 (Figure 2B-4), in addition to a relatively longwavelength Soret band at 444 nm, includes rather intensive bands at 470 and 482 nm with a shoulder at 493 nm. It was shown by a special experiment that

277 Tab/e 1. Change in the share of the esterifiod precursor [Pchl]/([Pchl]+ lPchlide]) and of the esterified photoeonversion product [Chl]/([Chl]+ [Chlide]) versus the duration of dark incubation of the mutantChlorellavulgarisB-15 cells Durationof dark [Pchl] % [Chll % cultivation, [Pchl]+ [Pchlide] [Chl]+ [Chlide] days Unilluminatod 10 s illumination sample I02 W/m2 at 233 K 4 6 8

60 80 90

50 70 85

Chl b was completely absent in the dark-grown cells of Chlorella vulgaris B-15: in fluorescence emission spectra of the extracts, no fluorescence of this pigment at 640-660 nm was observed, even at the selective excitation at 460-470 nm. At the same time, according to the absorption spectra of the extracts, the concentration of carotenoids in the cells of the studied mutant was h i g h - by two orders of magnitude higher than that of Pchl (ide). Therefore, the bands 470, 482 and 493 nm in the excitation spectrum measured for the fluorescence at 682 nm, should be related to the absorption of carotenoids with the subsequent migration of electron excitation energy to Pchl 682/672.

Formation of the long-wavelength chlorophyll ( ide ) native form during illumination of dark-grown cells of Chlorella vulgaris B-15 Fluorescence emission and absorption spectra of the extracts of Chlorella vulgaris B-15 dark-grown cells measured for 600-700 nm region contained only Pchl (ide) bands (Figure 3A-1). Illumination of the darkgrown cells leads to the appearance of Chl (ide) bands in the extract spectra (Figure 3A-2). However, the maximal share of the photoconversion Pchl (ide) > Chl (ide) was not higher than 10-12% even with saturating illumination doses. With those insignificant photoinduced changes in the spectra, it appeared to be convenient to observe Chl (ide) formation in vivo by the difference absorption spectra of the cells. The difference absorption spectrum 'light-minusdark' (Figure 3B) was measured during cell illumination at 233 K. The spectrum included 3 negative maxima at 635, 650, 672 nm and a single positive band belonging to the photoproduct. The position of the photoproduct absorption band depended, to some extent, upon the duration of the pre-cultivation of the

cells in darkness. The band of the 8-day-old culture was at 696 nm, that of 6-day-old culture was at 688 nm. The experiments with pigment extraction showed that the band of the photoproduct belonged to the reduced pigment, chlorophyll or chlorophyllide: the Chl (ide)/Pchl (ide) ratio was growing from 0.5 to 10% under illumination (Figure 3). Therefore, it may be concluded that illumination of the dark-grown cells of Chlorella vulgaris B-15 at 233 K leads to the formation of the long-wavelength Chl (ide) form with absorption at 688-696 nm. Several negative maxima in the difference absorption spectra show the participation of at least three forms of the Chl (ide) precursor. The dependence of the difference absorption spectrum (Figure 3B) on the age of the cells indicates that the ratio of the concentration or photoactivities of the precursor forms depends on the cell age. This made it possible to choose the optimal conditions and to observe separately the processes of photoconversion for each form of Chl (ide) precursor.

Photochemical reaction of the P chl (ide ) native forms in the dark-grown cells ofChlorella vulgaris B-15 It was most convenient to observe the photoconversion of the short-wavelength Pchl (ide) forms by the spectra of 4-day-old cells, where the Pch1682/672 content was low, whereas Pchl (ide) 633/627, Pchl (ide) 640/635, Pchl (ide) 655/650 were present in sufficient amounts (Figure 1A-l, 1B-l). Illumination of these cells at 233 K causes changes in fluorescence emission and absorption spectra (Figure 4). It can be seen in the difference absorption spectrum that Pchl (ide) 633/627 is non-photoconvertible. The positions of the maxima and the ratio of the band amplitudes of the precursor forms to those of photoproducts indicate that only two photoreactions proceed here; Pchl (ide) 640/635 Chl (ide) 680/670 and Pchl (ide) 655/650 > Chl (ide) 695/684. In the dark-grown cells cultivated 6 or 8 days, Pchl (ide) 640/635, Pchl (ide) 655/650 and Pchl 682/672 are involved into the protoconversion (Figure 3-B). We succeeded in observing the photoreactions of those three precursor forms separately, using the difference in their temperature dependences. In a special experiment a sample of dark-grown cells preliminary cooled to 77 K was heated by 10 K, then, illuminated by an intensive flash of light and cooled again in liquid nitrogen to measure the absorption and fluorescence emission spectra. Then, the same sample was heated

278 again up to the temperature 10 degrees higher than that before previous flash, illuminated again by a flash and cooled to 77 K for spectral measurements. The procedure was repeated several times to obtain a set spectral curves. The first photoinduced changes in difference absorption spectra of 6-day-old culture was observed at 203 K: the band 650 nm was decreasing and simultaneously an absorption band 684 nm was formed in the long-wavelength spectral region (Figure 5A, curve 1). At the same time in fluorescence emission spectra, the band 655 nm disappeared and a new band at 695 nm was formed (Figure 5B, curves 1 and 2). During the determination of the ratio of the esterified and nonesterified pigment forms in the extracts obtained with ethanol cooled to 203 K, we found that the product with absorption at 684 nm and fluorescence emission at 695 nm was chlorophyllide. This allows to conclude that the precursor of Chlide 695/684 in the investigated low-temperature photoreaction also is present as a non-esterified pigment form-Pchlide 655/650. Thus, in the studied mutant, as well as in other etiolated objects, Pchlide 655/650 is transformed into Chlide 695/684. Photoconversion of Pchlide 655/650 at 203 K was complete, judging by the disappearance of its bands from fluorescence emission (Figure 5B-2) and derivative absorption spectra. Therefore, further illumination of the same samples led only to photoconversion of Pchl 682/672 and Pchl (ide) 640/635. Those two processes started to manifest at 233 K: under illumination of the sample the intensities of 635 and 672 nm absorption bands decreased and the band of photoproduct at 696 nm appeared (Figure 5A-2). In the fluorescence emission spectra, a decrease in the intensity of the band 640 nm was accompanied by the formation of the bands of photoproducts at 680 and 715 nm (Figure 5B-3). The bands of Chlide 695/684 previously formed at 203 K remained unchanged. As it has been already shown, the fluorescence emission band 680 nm belongs to Chl (ide) 680/670 (Figure 4), thus, the other fluorescence band formed at 233 K at 715 nm should be related to the longer-wavelength photoproduct having the absorption maximum at 696 nm. This photoproduct, as it is shown above, presents the native form of chlorophyll (ide). The data obtained on its fluorescence emission band allow to designate it as Chl (ide) 715/696. The difference spectrum Figure 5A-2 includes two negative maxima at 635 and 672 nm, but only one band of the photoproduct at 696 nm. It was shown for 4-day-old cells that at 233 K Pchl (ide) 640/635 was

transformed into Chl (ide) 680/670 (Figure 4). This process is also observed in the 6-day-old cells: in the spectrum Figure 5A-2 the intensity of the absorption band 635 nm is decreased and the intensity of fluorescence emission at 680 nm grows (Figure 5B-3). However, judging by the negative band 672 nm in the 6-day-old cells, (Figure 5A-2), simultaneously with this process, photoconversion of Pchl 682/672 occurs as well. Therefore, the absence of positive absorption band 670 nm in he registered difference absorption spectrum Figure 5A-2 may be conditioned by the compensation of two intense bands: negative absorption band 672 nm of the transformed Pchl 682/672 and the positive absorption band 670 nm of the formed Chl (ide) 680/670. The summing up of the two successively measured difference spectra 1 and 2 of Figure 5A allowed to make the spectral curve (Figure 5C), coinciding with the above described difference absorption spectrum Figure 3B-1, registered from 6-day-old cells illumination at 233 K. A positive band of Figure 5C is found at 688 rim. The facts presented allow to assume that 688 nm absorption band does not belong to some individual photoproduct but presents a sum of the absorption bands of three photoproducts with maxima at 670, 684 and 696 nm. The dependence of the positive maximum position in the difference absorption spectra on the age of cells (Figure 3-B and 4-A) seems natural. In the 4-day-old cells, where Chl (ide) 715/696 is not formed, the positive band of the difference spectrum is localized at 670 nm with a shoulder 684 nm (Figure 4A). Contrastingly, in the difference spectrum of 8-day-old cells, where the Pchl (ide) 640/635 and Pchlide 655/650 contribution to Chl (ide) formation is low (Figure 3B-2) and, therefore, Chlide 695/684 and Chl (ide) 680/670 formation is low as well, the positive absorption band is localized at 696 nm, where the dominating photoproduct Chl (ide) 715/696 absorbs. The prolongation of dark cells incubation causes not only an increase in the share of Pchl 682/672 in the total of precursor forms and the share of Chl (ide) 715/696 in the Chl (ide) forms. The ratio Chl/(Chl + Chlide) in the extracts of illuminated cells grows in parallel (Table 1). The quantative comparison of Chl (ide) 715/696 absorption with the total Chl (ide) absorption in the cells of different age, and the data of Table 1 indicate that Chl (ide) 715/696 is an esterified pigment, i.e. chlorophyll. Thus, the studied reaction represents the photoconversion of Pchl 682/672 into Chl 715/696.

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/; Chl 715/696. Special attention was paid to elucidation of possible further transformations of the above described Chl (ide) forms formed from various precursors during cell illumination at room temperature. A short-term illumination of dark-grown cells at 293 K, as well as at 233 K, led to the appearance of new fluorescence emission bands at 680, 695 and 715 nm (Figure 6). When the illuminated sample was kept for 5 min in darkness, the band at 695 nm disappeared and the intensity of the 680 nm band increased. This effect, judging by an increase of Chl/Chlide ratio in the extracts, fits fast Shibata shift typical of green algae (Shioi and Sasa 1984); Chlide 695/684 > Chl 680/670. However, the position and intensity of the fluorescence band at 715 nm remained unchanged. Prolongation of the illumination at 293 K up to 30-40 min leads to a further transformation of the precursor into chlorophyll (ide), mainly, due to the reaction Pchl (ide) 640/635 > Chl (ide) 680/670 (Figure 6A-3). Meanwhile, the fluorescence band at 715 nm is partially masked by the increasing band 680 nm, however, judging by the derivative spectra (Figure 6-B) it remains unchanged in this time interval. Thus, the product ofphotoconversion of the long-wavelength protochlorophyll form, i.e. Chl 715/696, in contrast to the labile photoproducts of the shorter-wavelength precursor forms, presents a native pigment form, stable at 293 K i.e. most likely, the final form of this pathway of Chl biosynthesis.

Discussion

The data show that in the dark-grown cells of the heterotrophic Chlorella vulgaris mutant B-15 in addition to the known Pchl (ide) native forms, a considerable amount of a long-wavelength Pchl form appeared Pch1682/672. The Pch1682/672 differs from the previously known Pchl (ide) forms, i.e. Pchl (ide) 633/627, Pchl (ide) 640/635, Pchlide 655/650, in containing only the esterified pigment form and the stronger interaction of chromophores (bathochromic spectral shift). The fluorescence excitation spectrum of Pchl 682/672 in addition to a relatively long-wavelength Soret band at 444 nm includes rather intensive bands at 470, 482 and 493 nm. Those bands can be related to the absorption of carotenoids with the subsequent migration of electron excitation energy to Pch1682/672 (see above). Those bands are often observed in the fluores-

cence excitation spectra of the newly formed Chl in the postetiolated cells (Butler 1961; Fradkin et al. 1969). They are, however, not at all typical of the fluorescence excitation spectra of Pchl (ide) and chlorophyllide in vivo. The sensitization of Pchl 682/672 fluorescence by carotenoids detected in our experiments indicates the spatial closeness of the long-wavelength form of the precursor to carotenoid molecules. This closeness is apparently facilitated by the hydrophobic phytol in the long-wavelength form. The results presented above show that under illumination, in the dark-grown cells of Chlorella vulgaris B-15 mutant, photoreaction of three native precursor forms take place: Pchl (ide) 640/635 h~>chl(ide) 680/670, Pchlide 655/650 h~>Chlide 695/684 -----+Chl 680/670 and Pchl 682/672 hV)Chl 715/696. Two reactions of chlorophyll (ide) biosynthesis proceeding in parallel, Pchl (ide) 640/635 h~>Chl (ide) 680/670 and Pchlide 655/650 h~ Chlide 695/684 Chl 680/670, are found in the majority of greening algae (Wang 1979; Shioi and Sasa 1984). An exception is described by Lebedev et al. (1991) for mutants Chlamydomonas reinhardtii, where Pchlide 655/650 photoconversion was blocked and that of Pchl (ide) 640/635 was the only route of Chl (ide) formation. The long-wavelength photoproduct Chl 715/696 can be formed from Pchl 682/672 only. This conclusion is confirmed by the fact that Chl 715/696 was not formed when Pchl 682/672 was absent in 4-day-old cells (Figure 4). Furthermore the Chl 715/696 absorption band dominated in the difference absorption spectrum in the 8-day-old cells, where Pchl 682/672 was dominating photoactive precursor form (Figure 3B, curve 2). In Chl (ide) biosynthesis of the studied mutant not only non-esterified precursor forms, i.e. protochlorophyllide, but also the esterified pigment- protochlorophyll were photoactive. This peculiarity, in contrast to etiolated leaves, is typical of other previously studied green algae: Chlorella regularis (Sasa and Sugahara 1976), Euglena gracilis (Cohen and Schiff 1976), Scenedesmus obliquus (Kotzabasis et al. 1989). The most interesting is the question of the nature of the long-wavelength photoproduct Chl 715/696. It should be noted that the functional activity of PS-I core (the photostimulated H2 evolution) in the dark-grown cells of the Chlorella vulgaris B-15 can be observed several seconds after the onset of illumination at 293 K (Boichenko and Litvin 1990). Taking into consider-

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Figure 6. Low-temperature (77 K) fluorescence emission spectra of 8-day-old culture of Chlorella vulgaris B-15 (A). 1 - dark-grown culture after 10 s illumination by white light 102 W/m2 at 293 K; 2 - as 1 with following keeping in darkness for 5 rain at 293 K; 3 - dark sample illuminated for 40 min at 293 K. B - second derivative of the spectrum 6A-3. Fluorescence was excited at 470 nm.

ation that in our experiments Chl 715/696 was formed almost simultaneously, it seems interesting to compare the spectra of Chl 715/696 to those of the Chl of P S I core.

The absorption band of Chl 715/696 is close to the negative maximum of the difference absorption spectrum registered during photoinduced oxidation of P

700 (at 697 nm for Chlamydomonas reinhardtii (Karapetyan et al. 1980)), i.e. close to the absorption maximum of chlorophyll of PSI reaction center. The absorption band of Chl 715/696 is also close to the absorption maximum of the primary electron acceptor in PS I core - Ai,i, which is known to be at 700 nm and to represent a Chl-like pigment (Shuvalov et al. 1979).

282 The low-temperature fluorescence m a x i m u m (715 nm) coincides with the fluorescence m a x i m u m of the core antenna of P S I in Chlamydomonas reinhardtii (IshShalom and Ohad 1983). This antenna is known to be included in the CP 1 complex, which also contains P 700 and their c o m m o n apoprotein 6 6 - 6 8 kDa (IshShalom and Ohad 1983; Chua and Bennoun 1975). It should be noted, that a small amount of carotenoids is included in CP 1 complexes as well, judging by their absorption spectra (Wollmann and Bennoun 1982). This is also typical o f the complex Chl 715/696 and of its precursor Pchl 682/672, as it is clear from the above given fluorescence excitation spectra. All those data allow to suppose, as a working hypothesis only, that Chl 715/696 is very close to or identical of PS I core chlorophyll. As it has been already shown, Chl 715/696 may be formed from Pch1682/672 not only at 293 K but as well at 233 K in the course of low-temperature reaction proceeding in a rigid matrix, i.e. most likely without significant alteration of the precursor complex structure. This is testified, in particular, by preservation of energy migration from carotenoids to the porphyrin part of the complex, in the course of the low-temperature photoprocess. This implies the constance of intermolecular distances and of the mutual orientation of carotenoids and porphyrin chromophores. Here an assumption may be made that the structure of PS I core containing Pchl instead of Chl, pre-exists in the nonilluminated sample and P S I core formation is but completed in the light by means of the intracomplex photoreduction of Pchl to Chl. The dark biogenesis of the electron carriers of P S I is known (Dujardin et al. 1987). In addition this assumption is in the line with observations of darkgrown mutants Scenedesmus obliquus (Kotzabasis et al. 1991) where polypeptides of P S I core 65, 5-68 kDa - apoproteins of P 700 (Chua and Bennoun 1975) and core antenna (Ish-Shalom and Ohad 1983) are partially synthesized in darkness, though illumination can accelerate the synthesis. The proposed hypothesis requires a special study.

Acknowledgement The study was supported by the Russian Foundation for Fundamental Research (project 96-04-48443).

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