Journal of Experimental Botany, Vol. 53, No. 375, pp. 1753-1763,
August 1, 2002
© 2002 Oxford University Press
Room temperature microspectrofluorimetry as a useful tool for studying the assembly of the PSII chlorophyll–protein complexes in single living cells of etiolated Euglena gracilis Klebs during the greening process
Received 21 September 2001; Accepted 29 April 2002
Dipartimento delle Risorse Naturali e Culturali, University of Ferrara, C.so Porta Mare, 2, I-44100 Ferrara, Italy
1 To whom correspondence should be addressed. Fax: +39 0532 208561. E-mail: fsm{at}dns.unife.it
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The assembly kinetics of the PSII chlorophyll–protein complexes was followed during the greening of Euglena gracilis by microspectrofluorimetry in vivo, at room temperature, on single living cells. The study was correlated to micro- and submicroscopic events accompanying the proplastid to chloroplast transformation and with the immunolocalization of the LHCPII. Etiolated cells of Euglena gracilis were grown in darkness in Mego’s heterotrophic liquid medium under shaking at 25±1 °C. At the stationary phase of growth, they were exposed to continuous light (330 µmol m–2 s–1) for 72 h. The analyses were carried out on samples collected at different times of illumination. Microspectrofluorimetric data were recorded in the 620–780 nm range (excitation at 436 nm) and were resolved into Gaussian components corresponding to the reaction centres (RCII) and the inner antennae (CP43–47) of the PSII and LHCPII. From the RCII/CP43–47 and LHCPII/PSII ratios, it was inferred that (1) a disconnection between RCII and CP43–47 syntheses occurs during the lag phase of chloroplast differentiation, RCII being synthesized before the inner antennae. This results in the accumulation of uncoupled PSII Chl–protein complexes; (2) after lag phase, the RCII and CP43–47 syntheses are connected one to another; (3) the freshly synthesized LHCPII complexes are immediately assembled with the PSII, suggesting that the outer antennae always maintain the form bound to PSII. Micro- and submicroscopical observations and LHCPII immunolocalization were in agreement. These data suggest that microspectrofluorimetry may constitute a useful non-destructive tool for studying the assembly kinetics of PSII, under fully physiological life conditions.
Key words: Key words: Euglena gracilis, greening, microspectro fluorimetry, PSII assembly.
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Introduction |
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Euglena gracilis is a photodependent unicellular organism for chloroplast differentiation. When grown in the dark, etiolated Euglena contains about ten small proplastids with several non-crystalline prolamellar bodies (PLBs) from which few ‘girdle-like’ thylakoids originate (Klein et al., 1972; Osafune et al., 1980). Only one protochlorophyll(ide) (PChl(ide)) form emitting at 630 nm, named PChl(ide)630, is reported to be present on the PLBs of Euglena proplastids (Cohen and Schiff, 1976; Lebedev, 1996). Indeed, the PChl(ide) forms emitting at 645 nm and 657 nm, which are associated with the crystalline PLBs and with the prothylakoids of the higher plant etioplasts (Grevby et al., 1989; Böddi et al., 1992; Sundqvist and Dahlin, 1997), are never observed in the alga (Lebedev, 1996). Upon exposure to light, proplastids are converted into photosynthetically competent chloroplasts through many co-ordinated morpho-physiological events widely described in Ben-Shaul et al. (1964), Klein et al. (1972), Osafune and Schiff (1980), Osafune et al. (1980, 1984, 1990a, b) and Schwartzbach (1990). The process presents a 12 h lag period and ends after 72 h of illumination (Schwartzbach, 1990). One of the first events of chloroplast differentiation is the dissolution of PLBs (30 min to 2 h of continuous illumination) and the beginning of the thylakoid system deposition (Ben-Shaul et al., 1964; Klein et al., 1972; Osafune et al., 1980, 1984, 1990a, b; Schwartzbach, 1990). Simultaneously, PChl(ide)630 is directly photoconverted to a chlorophyll(ide) (Chl(ide)) emitting at 675 nm (Chl(ide)675) without the occurrence of the Shibata shift (Cohen and Schiff, 1976; Yamamoto et al., 1992). The proper deposition of the inner plastid membranes is closely related to the synthesis of photosynthetic pigments and their association to the proteins to give functional photosynthetic complexes (Schwartzbach, 1990). Chls and apoproteins of the PSII antenna Chl–protein complex (CP43–47) and of the light-harvesting Chl–protein complex (LHCPII) must accumulate and arrange themselves into the thylakoid membrane in a co-ordinated fashion (Yamamoto et al., 1992; El Kaoua and Laval-Martin, 1995). During the lag period, the biosynthetic processes for chloroplast differentiation are dependent on the cytoplasmic activity (Schwartzbach and Schiff, 1983). However, the events following the lag period are the result of the co-ordinated expression of both plastid and nuclear compartments (Schwartzbach, 1990). During the first 12 h of light exposure, the rate of LHCPII synthesis increases, reaching a maximum rate at 12–24 h of illumination (Rikin and Schwartzbach, 1989). Employing the non-denaturing lithium dodecyl sulphate-polyacrylamide gel electrophoresis, Yamamoto et al. (1992) showed that, in greening E. gracilis cells, stable CP43–47 appeared after 24–48 h of illumination, while LHCPII and Chl–protein complex of PSI (CPI) appeared after 16 h and increased with illumination. Brown (1980) separated four species of Chl–protein complexes by fractionating thylakoid membranes of Euglena with a saccharose gradient and chromatography and characterizing them by 77 K fluorimetry. Up to now, the events leading to the formation of the Chl–protein complexes in Euglena have never been studied in living cells. In the laboratory, microspectrofluorimetric analyses allowed the study in vivo, at room temperature, in single cells, of the assembly kinetics of the Chl–protein complexes during the greening process in the alga. This kinetics was also correlated with the micro- and submicroscopic events accompanying the light-induced proplastid-to-chloroplast transformation and with the immunolocalization of the LHCPII, a relationship never previously reported in literature. In this work, it is reported that microspectrofluorimetric analysis provides an innovative methodology for such studies, because it is performed on single living cells and at room temperature. Since, at room temperature, most of the fluorescence originates from PSII, while only 2–5% originates from PSI (Krause and Weis, 1991; Agati et al., 1995), attention was focused on the assembly kinetics of the Chl–protein complexes of PSII.
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Materials and methods |
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Culture conditions
The organism used was Euglena gracilis Klebs strain Z, maintained in the dark in this laboratory for many years. For the greening experiments, the etiolated cells were grown in complete darkness in Mego’s heterotrophic liquid medium (Mego, 1964) under constant shaking at 25±1 °C until they reached the stationary phase of growth (2.4x106 cells ml–1). Then the suspension cultures were continuously illuminated with white fluorescent lamps (330 µmol m–2 s–1) in the same liquid medium, the other culture conditions remaining unchanged. Microspectrofluorimetric analyses and morphological observations were carried out on cell samples collected at different times of illumination from 0 to 72 h. The entire experiment was repeated four times.
Fluorescence microscopy
For ultraviolet light (UV) examinations, a photomicroscope Zeiss model Axiophot equipped with a reflected fluorescence condenser was employed. The light source was a pressure mercury vapour lamp, HBO 50 W, with BP 436/10 exciter and LP 470 filters. High speed Fujifilm Superia 400 ASA was used for photography.
Electron microscopy
Fixation, embedding and staining procedures for transmission electron microscopy (TEM) were performed according to Fasulo et al. (1983) and Pancaldi et al. (2001). In particular, the cells were harvested by centrifugation (400 g), washed with phosphate buffer 0.1 M (pH 7.2) and fixed with 3% glutaraldehyde in the same buffer at 4 °C for 2 h. After rinsing with phosphate buffer, cells were post-fixed with 1% OsO4, prepared in the same buffer, at 4 °C overnight. After further rinsing, pellets were embedded in 1% agar and then dehydrated in a graded ethanol series. The materials were then embedded in Epon–Araldite. Sections, obtained by a LKB Ultratome III ultramicrotome, were stained with uranyl acetate and lead citrate, and examined with a Hitachi H800 electron microscope (Electron Microscopy Centre, Ferrara University, Italy).
Immunocytochemistry
Cells were harvested and fixed for 2 h at 4 °C in 0.1 M phosphate buffer (pH 7.4) containing 2% paraformaldehyde and 1% glutaraldehyde. After fixation, the samples were washed for 2 h with 0.1 M phosphate buffer (pH 7.4) and left overnight in the same buffer. They were then treated with 0.5 M NH4Cl in phosphate buffer for 2 h to block free aldehydic groups. All these operations were performed at 4 °C. Fixed samples were dehydrated through a graded ethanol series (30% ethanol for 30 min at 4 °C; 50%, 70%, 95%, 100% ethanol for 1 h at –20 °C), repeatedly stirred and then infiltrated with Lowicryl K4M resin at –20 °C (ethanol-resin 1:1 for 60 min; ethanol-resin 1:2 for 60 min; Lowicryl 100% for 60 min; Lowicryl 100% overnight) (Roth et al., 1981). Polymerization at –20 °C was obtained by overnight UV irradiation (Altman et al., 1984). Thin sections, cut with a diamond knife and mounted on nickel grids, were first incubated with TBS-T buffer (20 mM Tris–HCl pH 7.4, 500 mM NaCl, 0.3% Tween 20) for 15 min and, secondly, in TBS-T buffer with the addition of 10 mg ml–1 of bovine serum albumin (TBS-TB) for 15 min. The grids were subsequently incubated with a polyclonal antibody against LHCPII 1:100 diluted in TBS-TB buffer for 15 min at room temperature, then overnight at 4 °C (Horisberger, 1981). Subsequently, they were washed with TBS-TB buffer for 15 min at room temperature, incubated with a 1:20 dilution of protein A-gold (10 nm; Sigma Chemical Co, St Louis) for 2 h and rinsed several times with TBS-TB buffer for 15 min each time. Control grids were incubated with pre-immune serum 1:100 diluted in TBS-TB buffer. The sections were finally stained with uranyl acetate and lead citrate (Roth et al., 1981; Craig et al., 1987).
Microspectrofluorimetry
Acquisition of spectra: Fluorescence emission spectra were recorded using a microspectrofluorimeter (RCS, Florence, Italy), associated with a photomicroscope Zeiss model Axiophot. All spectra were recorded in vivo, at room temperature, on single living cells that were selected at the microscope under fluorescent light (1000x magnification). For analyses, cell samples were stirred first with few drops of tepid isinglass and transferred to polylysine microslides (Menzel-Gläser, Germany), in order to limit cell movements. The excitation light at 436 nm was focused on a single cell at a time, using a 1.6 mm diaphragm. The emission light was collected by the objective lens and deviated to the detector system, which included a monochromator reticle (band pass 0.25 nm), endowed with a computer-assisted stepper-motor, and a photomultiplier tube, coupled with an analogue/digital converter for data transfer to the ‘Autolab’ software (RCS, Florence, Italy). The ‘Autolab’ software was also used to set the recording range and optimize the photomultiplier response. Fluorescence levels, measured in arbitrary units of fluorescence directly established by the setting system, were visualized as emission spectra by the same program.
Elaboration of spectra: Spectra elaboration was performed with the ‘Origin 6.0’ program (Microcal Software Inc.). For each stage of greening, experimental spectra were normalized and averaged. The resulting graph was smoothed by averaging adjacent data points, by means of the ‘Adjacent averaging’ smoothing function of ‘Origin 6.0’. The degree of smoothing was controlled by specifying the number of points (10) to be used to calculate each averaged result. The smoothed graph was resolved into Gaussian components. Peak positions were determined on the 4th derivative of the curve in order to obtain the best deconvolution. In this way, the Gaussian multipeak fitting was performed by fixing the following starting parameters: (1) the number of components and their position, resulting from the 4th derivative; (2) the halfband-width, estimated directly by the computer program. The halfband-width was eventually modified to allow the best fit, as indicated by the comparison of the experimental spectrum with the sum of Gaussian curves. Only the final result is reported.
Although the present method provides a precise definition of the components responsible for experimental curves, it should be noted that this analysis does not allow quantitative evaluations. Nevertheless, a single experimental curve yields much information from the reciprocal relations between Gaussian components, whose areas were provided by the computing system.
Since data in the literature usually refer to fluorimetric analyses performed on in vivo samples at low temperature (77 K) (Böddi et al., 1989, 1990, 1992, 1994, 1996; Schoefs and Franck, 1998), the reliability of the microspectrofluorimetric results obtained here was attested on the basis of the ascertained correspondence of the 77 K fluorimetric data reported by Schoefs and Franck (1998) in dark-grown pine primary needles with the room temperature data obtained in analogous plant samples (data not shown).
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The data reported here refer to the intervals of illumination time which proved to be of interest in the greening process of Euglena.
Dark conditions
At zero time, the dark-grown Euglena gracilis cells observed under UV light exhibited some pale red small dots corresponding to the PLBs of the proplastids to which PChl(ide) is associated (Schiff and Schwartzbach, 1982; Schwartzbach, 1990) (Fig. 1A). Microspectrofluorimetric analyses recorded a unique PChl(ide) form emitting at 628 nm, corresponding most likely to PChl(ide)630 which is associated with non-crystalline PLBs in proplastids of Euglena (Cohen and Schiff, 1976; Schiff and Schwartzbach, 1982; Lebedev, 1996) (Fig. 1A). Using TEM, the plastidial system consisted of undeveloped plastids (1–3 µm long) containing 3–5 girdle-like and/or straight thylakoids and 1–3 non-crystalline PLBs (Fig. 2A). No immunolocalization of LHCPII was detected either in the cytoplasm or inside the plastids (Fig. 2C).
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When etiolated Euglena cells were exposed to continuous light, the greening process started.
30 min light exposure
The fluorescence (Fig. 1B) and ultrastructural (Fig. 2B) aspects of the plastids were approximately the same observed at zero time. LHCPII immunolocalization was still lacking (data not shown). However, fluorescence emission spectra revealed two more emissions at 675 nm and 690 nm, in addition to the PChl(ide)628 peak (Fig. 1B). The emission at 675 nm corresponds to the first product of the PChl(ide)630 photoconversion, a Chl(ide) whose formation in Euglena occurs directly without the Shibata shift (Lebedev et al., 1995; Lebedev and Timko, 1999). The presence of one PChl(ide) only, together with its direct photoconversion product Chl(ide)675, without the appearance of intermediate steps, suggests that the photoconversion of the PChl(ide) to Chl(ide) in Euglena implies the occurrence of a pathway alternative to that reported in etiolated tissues of higher plants (Lebedev, 1996). The emission at 690 nm is not preceded by the appearance of PChl(ide)657, the aggregation product of PChl(ide)630 with NADPH-Pchlide oxidoreductase A which is characteristic of the crystalline PLBs in the etioplasts of seedlings in higher plants (Sundqvist and Dahlin, 1997). This emission in Euglena could correspond to some precursors of CPI whose appearance is precocious in the alga (Yamamoto et al., 1992). The same emissions at 675 nm and 690 nm were recorded at low temperature after a 1 ms white-light flash, in dark-grown seedlings of the det340 mutant of Arabidopsis lacking POR A but maintaining the second PChl(ide) reductase (POR B) (Lebedev et al., 1995). This analogy suggests the absence of POR A in Euglena.
2 h light exposure
Under UV light, the cells had plastids that exhibited a slightly stronger red fluorescence (Fig. 1C). Correspondingly, TEM observations revealed the presence of more elongated plastids with the visible beginning of elaboration of the inner membrane system and with PLBs still present (Fig. 2D). Accordingly, microspectrofluorimetric analyses testified to the beginning of the association process of pigments to proteins to give complexes in the making (Fig. 1C). Indeed, beside the emission peak at 628 nm, associated with the PLBs, emissions at 677 nm, 685 nm, 694 nm, and 709 nm were already noted at this stage of the greening process. The emission at 677 nm is ascribable to phaeophytin, the primary electron acceptor of the PSII reaction centre (RCII) (Omata et al., 1984), whose formation has been observed during irradiation with low light intensity as a phototransformation product of a Chl671 (Ignatov and Litvin, 1994). In this study, the greening of Euglena was achieved just by the low light intensity of 330 µmol m–2 s–1 (Váradi et al., 2000). The phototransformation may be a specific route for the formation of phaeophytin used for pigment ligation to the D1/D2 proteins in the RCII (Ignatov and Litvin, 1994). This interpretation is also supported by Franck et al. (1995) who proposed that low light intensities favour the formation of reaction centres. It was impossible to detect the Chl671 because it is a very short-lived intermediate (Sundqvist and Dahlin, 1997). The appearance of the emission peak at 677 nm after only 2 h of illumination suggests that, in Euglena, the formation of the RCII starts early. The emissions at 685 nm and 694 nm are characteristic of the PSII internal antennae (van Dorssen et al., 1987; Krause and Weis, 1991; Alfonso et al., 1994; Xiong et al., 1998; Schoefs and Franck, 1998). The precocious appearance of these 
emissions seems to be in disagreement with the electrophoretical data of Yamamoto et al. (1992), which established 24–48 h of continuous light for the formation of stable CP43–47. However, it is necessary to note that the sensitivity of microspectrofluorimetry also allows for emphasizing the emissions of the few Chl–protein complexes associated with the first thylakoid membranes deposited. The appearance of the peak at 677 nm relative to the reaction centre, together with the two peaks at 685 nm and 694 nm relative to the inner antennae, indicates that, at this time, PSII was not yet assembled and that its Chl–protein complexes emitted separately (Butler, 1978; Krause and Weis, 1991). The emission peak at 709 nm which is most likely due to LHCPII may also be an indication that assembly has not yet taken place at this point. Indeed, although the origin of the emission in the 695–715 nm spectral region in green algae and higher plants is unclear, there is some evidence pointing to the contribution of PSII peripheral antennae (Zucchelli et al., 1992; Jennings et al., 1993; Vassiliev et al., 1995). In particular, Vassiliev et al. (1995) reported that an increase of the fluorescence at 710 nm at 77 K is related to the uncoupling of LHCPII complexes and PSII reaction centres and results in a lack of the direct transfer of the excitation energy from PSII peripheral antennae to the reaction centres. After 2 h illumination, the LHCPII immunoreaction was very weak and localized on the plastidial envelope only (Fig. 2E). In fact, in Euglena, LHCPII is transported from the endoplasmic reticulum (ER) to the Golgi apparatus and from the Golgi apparatus to the chloroplast, as a membrane-bound polyprotein precursor (Rikin and Schwartzbach, 1989; Muchhal and Schwartzabch, 1992; Sulli and Schwartzbach, 1995, 1996; Enomoto et al., 1997). Within chloroplasts, mature LHCPII is proteolytically released from the polyprotein precursor (Sulli and Schwartzbach, 1995, 1996; Enomoto et al., 1997).
6 h light exposure
After 6 h illumination, no noticeable changes in the emission bands were observed (Fig. 1D). However, the relative fluorescence intensities underwent some modifications and both the RCII/CP43–47 and the LHCPII/PSII ratios varied. The RCII/CP43–47 ratio decreased from the value of 2.6 recorded after 2 h illumination, to 1.1 (Table 1). The decrease of the ratio is due to an increase of the fluorescence from the PSII internal antennae that back-transfer the excitation energy from the reaction centres. This indicates a greater stability of the PSII complexes (Butler, 1978; Krause and Weis, 1991). LHCPII/PSII ratio increased from 0.13 to 0.97 (Table 1), indicating a still inadequate energy transfer from the peripheral antennae to the PSII reaction centres (Keränen et al., 1999). At this time, a weak emission at 731 nm was also noted (Fig. 1D), probably ascribable to the PSI whose emission is very low at room temperature (Sauer and Debreczeny, 1996; Keränen et al, 1999). However, all these fluorescence emissions were still very low, because few thylakoid membranes were formed into the differentiating plastids as the ultrastructural observations testified (Fig. 2F). No emission at 630 nm was recorded, which is in agreement with the complete dissolution of the PLBs observed with TEM (Fig. 2F). At this time, the LHCPII immunolocalization was more evident but still only localized on the plastidial envelope (Fig. 2G). This suggests an incessant import of LHCPII complexes into the plastid from ER and Golgi apparatus (Enomoto et al., 1997).
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8 h light exposure
Under UV light, the Euglena cells exhibited plastids with a more intensely red fluorescence, pointing to the progression of the greening process (Fig. 1E). No fluorescence decay interfered with the observations. At TEM, plastids were more elongated with 3–5 single straight thylakoids (Fig. 2H). Emission fluorescence spectra were resolved into Gaussian components characterized by the same
observable after 6 h of light (Fig. 1E). However, the ratios between the various PSII complexes changed again. In particular, both the RCII/CP43–47 and LHCPII/PSII ratios decreased to 0.50 and 0.42, respectively (Table 1). These values are indicative of further stabilization of the assembly of the PSII components and of the beginning of a more efficient energy transfer from the peripheral antennae to PSII reaction centres (Keränen et al., 1999). Immunolocalization of LHCPII occurred mostly on the thylakoids (Fig. 2I).
12 h light exposure
After 12 h light exposure, corresponding to the end of the lag phase of the Euglena chloroplast differentiation (Schwartzbach and Schiff, 1983; Schwartzbach, 1990), the number of thylakoids in plastids increased and 4–6 lamellae, already formed in some portions by two or three apposed thylakoids, partially crossed the stroma (Fig. 2J). Under UV light, plastids emitted a more intense and better defined red fluorescence (Fig. 1F) which, however, declined after only 2–3 min during UV light observations. The emission spectra revealed an unexpected increase of the RCII/CP43–47 ratio (from 0.50 to 1.30) (Table 1) indicating a failure of the energy transfer from the inner antennae to the reaction centres, justifying the rapid decay of fluorescence under UV light. A lack of assembly of CP43–47 with RCII had already been noted during the first 6 h of illumination. Such behaviour could be due to the existence of two subsequent steps in the synthesis of the individual complexes belonging to PSII. During the first step, 2–6 h long, the developing plastids use the carbon and the energy derived from the breakdown of the storage carbohydrate paramylum to synthesize some of the Chl–protein complexes that will constitute the photosystems. In fact, the amount of chlorophyll synthesized and the length of the lag period of chlorophyll synthesis are dependent on the paramylum present prior to light exposure (Schwartzbach, 1990). The neosynthesized RCII and CP43–47 assemble immediately to give the first complete PSII complexes. In this way, the photosynthetic process may start after only a few hours of greening (Stern et al., 1964; Schwartzbach and Schiff, 1983). Subsequently (6–12 h illumination), the energy supply, deriving mostly from the photosynthetic activity, increases sufficiently to permit the accumulation of sufficient materials to allow the developing plastids to enter the period of rapid synthesis (Schwartzbach and Schiff, 1983; Schwartzbach, 1990). Fluorescence spectra revealed a massive synthesis of RCII and CP43–47 complexes (Fig. 1F) that was confirmed by the rapid elaboration of the thylakoid membrane system (Fig. 2J). The LHCPII/PSII ratio increased (Table 1). A massive synthesis of apoproteins of LHCPII probably occurred, as shown by the intense LHCPII-immunolocalization taking place at this time, both on the thylakoid and envelope membranes and on the ER adjacent the differentiating plastids (Fig. 2K). On the other hand, it is reported that the maximum rate of accumulation of LHCPII occurs after 12–24 h of illumination (Brandt and Winter, 1987; Spano et al., 1987; Weiss et al., 1988; Rikin and Schwartzbach, 1989). The shorter emitted from LHCPII (702 nm) with respect to that recorded at 8 h (about 710 nm) could be due to a still incomplete assembly of LHCPII-apoproteins with the pigments to give stable complexes.
16 h light exposure
Both the microscopical and the analytical aspects suggest an increasingly efficient photosynthetic machinery. The further increase of the red fluorescence of the chloroplasts (Fig. 1G) was justified by the numerous (8–10), sometimes apposed, straight thylakoids crossing the stroma (Fig. 3A). Fluorescence emission spectra emphasized the formation of more stable PSII complexes (Fig. 1G), and confirmed that the thylakoid formation is closely dependent upon the assembly of the proteins and the pigments (Schwartzbach, 1990). RCII/CP43–47 ratio rapidly decreased (from 1.30 detected at 12 h to 0.42 at 16 h) (Table 1), as a probable consequence of the narrow association of the inner antennae with the reaction centres, which were largely synthesized previously, to give functional PSII. The LHCPII/PSII ratio decreased accordingly from 0.52 to 0.15 (Table 1), indicating both the actual association of the LHCPII apoproteins with Chl molecules and the efficient transfer of the energy from the peripheral antennae to the PSII. The decrease of the LHCPII immunoreaction on cytoplasmic membranes and on the plastidial envelope and, conversely, its specific and more abundant presence on the thylakoid membranes (Fig. 3B) indicated a decrease of the apoprotein synthesis, but also an increased association of apoproteins with Chl molecules to constitute stable LHCPIIs.
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From this time of illumination until the completion of the greening process no substantial changes in the fluorescence emission ratio values were recorded. The ratio values (Table 1) suggest the increasing stability of the photosynthetic system. In fact, no fluorescence emission is ascribable to RCII at 72 h (Fig. 1J; Table 1). A greater emission intensity was noted in the course of time. For the sake of brevity, only the microspectrofluorimetric spectra for 24, 36 and 72 h light exposure are reported in this paper. As expected, under UV light, the organelles exhibited more and more intensely the typical red fluorescence and the aspects of the light-grown wild-type Euglena (Fig. 1H, I, J) (Pancaldi et al., 1997, 2001). With TEM, at 72 h of continuous illumination, the chloroplasts appeared as filled with several (14–16) lamellae formed by two or three apposed thylakoids regularly arranged along the longitudinal axis (Fig. 3G). In Fig. 3C and E ultrastructural aspects of chloroplasts at 24 h and 36 h of illumination, respectively, are reported. The LHCPII immunoreactions were increasingly specific and limited to the inner membranes (Fig. 3D, F, H).
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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In this work, the kinetics of the PSII complex assembly in etiolated Euglena during light-induced proplastid-to-chloroplast differentiation has been studied on the basis of the RCII/CP43–47 and LHCPII/PSII ratios obtained from microspectrofluorimetric analyses made on single living cells at room temperature.
The RCII/CP43–47 ratios indicate (a) RCII is the main chlorophyll–protein complex synthesized during the first 2 h of illumination, as shown by the very high value of the ratio; (b) the rapid decline of the ratio during the period between 2 h and 8 h of illumination testifies that the synthesis of the inner antennae and their assembly with the reaction centres has occurred; (c) however, the value of about 1.0 of the ratio after 6 h light exposure indicates that the synthesis of the inner antennae is still running and prevails over the reaction centres one; (d) the stability of the PSII complexes is reached between 6–10 h of light exposure, as a consequence of the sudden assembly of the inner antennae with the RCII complexes already accumulated; (e) the subsequent increase of the ratio which occurs at the end of the lag phase (10–12 h of light) further indicates the disconnection between RCII and CP43–47 syntheses due to a greater formation of the reaction centres with respect to that of the corresponding antennae; (f) during the following 4 h of illumination, new antenna complexes are formed and assembled into additional stable PSII, as shown by the marked decline of the ratio; (g) the constant low values of the ratios which are monitored from 16–36 h of illumination indicate that a good balance between reaction centres and inner antennae production exists, but that PSII complexes are not yet completely stable; (h) from 36 h of illumination onward PSII stability increases until it reaches the maximum level at the end of experiment (72 h), when the thylakoid system is completed.
The LHCPII/PSII ratios indicate (a) no LHCPII synthesis occurs prior to 2 h of light exposure, as shown by the extremely low value of the ratio; (b) the increase of the ratio occurring during the four subsequent h of illumination indicates that, although LHCPII is quickly synthesized, its excitation energy cannot be transferred to PSII, whose assembly has not yet been reached, as proved by the RCII/CP43–47 value; (c) between 6–8 h of light, together with the formation of some stable PSIIs, the transfer of the energy from the neosynthesized LHCPIIs to the photosynthetic complexes may occur, as suggested by the rapid decline of the LHCPII/PSII ratio; (d) a further increase of the ratio takes place during 8–12 h illumination, due to the accumulation of the peripheral antennae concomitant with the greater synthesis of RCII related to the inner antennae; (e) the LHCPII/PSII ratio declines in parallel to the decrease of RCII/CP43–47 ratio between 12 h and 16 h of light, indicating that the energy transfer may occur because of the stability of the PSII complex; (f) the low LHCPII/PSII ratio reflects a good energy transfer from peripheral antennae to stable PSII from 16 h until the completion of chloroplast differentiation (72 h). Concomitantly, no more evident imbalance between the RCII and inner antennae is observed, as proved by the RCII/CP43–47 ratio.
On the whole, it can be hypothesized that two steps of accumulation of uncoupled PSII Chl–protein complexes exist during the lag period. Moreover, the synthesis of the reaction centres precedes that of the inner antennae, which are immediately assembled, just after their formation, with the RCII to give stable PSIIs. After the lag phase, the syntheses of RCII and CP43–47 appear connected one to another, and no further accumulation of single reaction centres occurs.
With regard to the LHCPII complexes, their immediate assembly with PSII, just after their synthesis, seems to be noteworthy. In the literature regarding Euglena, no reference is made to the existence of a mobile LHCPII, as in that of higher plants. It seems that LHCPII complexes always maintain the form bound to PSII, to justify the presence of the two or three stably coupled thylakoids. This assumption could easily explain why some portions of the still incomplete straight thylakoids are already apposed at the end of the lag phase, and why, after 16 h light exposure, the complete straight thylakoids are entirely apposed on their length although the inner membranous system is far from being finished.
The present study demonstrates that microspectrofluorimetric analysis constitutes a useful non-destructive tool for studying the assembly kinetics of the Chl–protein complexes into the PSII, under fully physiological life conditions. Furthermore, the opportunity to combine a fine methodology of data acquisition and of data elaboration, together with a high system sensitivity, allows for circumscription of the field of research on a single living cell. Consequently, the fluorescence spectra reflect a more detailed picture of the situation inside the cell, especially when the organism examined belongs to an asynchronized cell population.
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Acknowledgements |
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This work was supported by grants from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) of Italy. The authors are grateful to Professor Roberto Barbato, Department of Advanced Sciences and Technologies, University of Piemonte Orientale ‘Amedeo Avogadro’, Italy, for the LHCPII antibody.
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Abbreviations: Chl(ide), chlorophyll(ide); CP43–47, antenna chlorophyll-binding proteins of photosystem II; CPI, chlorophyll-binding protein of PSI; ER, endoplasmic reticulum; LHCP, light-harvesting chlorophyll–protein complex; PChl(ide), protochlorophyll(ide); PLB, prolamellar body; POR, NADPH-protochlorophyllide oxidoreductase; PSI, photosystem I; PSII, photosystem II; RC, reaction centre; TEM, transmission electron microscope; UV, ultraviolet light..
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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