DOI: 10.1007/s11099-015-0129-y
PHOTOSYNTHETICA 53 (3): 369-377, 2015
Structural and functional changes in the photosynthetic apparatus of Chlamydomonas reinhardtii during nitrogen deprivation and replenishment É. PREININGER*+, A. KÓSA*, ZS. LŐRINCZ*, P. NYITRAI**, J. SIMON*, B. BÖDDI*, Á. KERESZTES*, and I. GYURJÁN*† Department of Plant Anatomy, Eötvös Loránd University, Pázmány P. sétány 1/C, H-1117 Budapest, Hungary* Department of Plant Physiology and Molecular Plant Biology, Eötvös Loránd University, Pázmány P. sétány 1/C, H-1117 Budapest, Hungary**
Abstract Nitrogen is an essential factor for normal plant and algal development. As a component of nucleic acids, proteins, and chlorophyll (Chl) molecules, it has a crucial role in the organization of a functioning photosynthetic apparatus. Our aim was to study the effects of nitrogen starvation in cultures of the unicellular green alga, Chlamydomonas reinhardtii, maintained on nitrogen-free, and then on nitrogen-containing medium. During the three-week-long degreening process, considerable changes were observed in the Chl content, the ratio of Chl-protein complexes, and photosynthetic activity of the cultures as well as in the ultrastructure of single chloroplasts. The regreening process was much faster then the degradation; total greening of the cells occurred within four days. The rate of regeneration depended on the nitrogen content. At least 50% of the normal nitrogen content of Tris-Acetate-Phosphate (TAP) medium was required in the medium for the complete regreening of the cells and regeneration of chloroplasts. Additional key words: electron microscopy; nitrogen starvation; O2 evolution; 77K fluorescence.
Introduction Nitrogen deficiency is an extensively studied subject in plant physiology and phycology since it often occurs in unbalanced environmental conditions. In higher plants, nitrogen starvation is known to cause reduced leaf expansion and yellowing (Bouma 1970, Zhao et al. 2005). This is accompanied by the degradation of the chloroplast thylakoids (Kutík et al. 1993, Doncheva et al. 2001), and a decline of photosynthesis (Baszyński et al. 1975, Lawlor et al. 1989). All of these changes, together with a loss of plastid DNA, lead to leaf senescence (Scott and Possingham 1983). The green alga, Chlamydomonas reinhardtii, is a popular object in many fields of biology, e.g. for study of structure, physiology, genetics, etc. (Goodenough and Levine 1969, Gyurján et al. 1980, 1982; Davies and Grossman 1998). Fast response to many environmental changes is characteristic for this alga and it is a great advantage for experiments. A good example for the great
plasticity of C. reinhardtii is adaptation to lack of nitrogen (Fernandez and Galvan 2007, 2008), which includes a complex differentiation process. The alga acquires nitrogen by autophagy, develops mating capacity (e.g. synthesizes glycoproteins for fusion of gametes developing from the vegetative cells), and prepares itself for long-term survival (accumulates starch and lipids) (Martin and Goodenough 1975). These symptoms occur under the activation of 21 genes (Abe et al. 2005). The early responding genes are induced within 2 h, the late ones between 5 and 8 h after nitrogen removal (Abe et al. 2004). Similarly to yellowing of higher plant, a decrease of the Chl content and transformation of the photosynthetic apparatus are characteristic for nitrogen-starved cells. Nitrogen starvation unequally changes the amounts of the Chl-protein complexes that can be observed via significant changes in the fluorescence emission spectra, i.e. the dominance of the short-wavelength emission bands in the
——— Received 29 August 2014, accepted 22 January 2015. +Corresponding author; e-mail:
[email protected] Abbreviations: Fv/Fm - maximal quantum yield of PSII photochemistry; Chl - chlorophyll; CP43 and CP47 - core complex proteins of PSII; DG degreening process; P680 - primary electron donor of PSII; RG regreening process; TAP – Tris-Acetate-Phosphate. Acknowledgements: A. Kósa is grateful for the Ferenc Deák Scholarship of the Hungarian Ministry of Education and Culture (DFÖ 0021/2009). This work was supported by Richter Centennial Foundation.
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680–695 nm region (Morgan-Kiss et al. 2002). Detailed analyses (i.e. Gaussian resolution) of the emission spectra combined with gel electrophoresis showed the altered ratios of the Chl-protein complexes (Ossenbühl et al. 2004). These results showed that the PSII is markedly affected and thus the CO2 dependent O2 production is reduced (Beardall et al. 1991). Similar changes were observed in other alga species, suggesting that the unbalanced nitrogen supply generally causes the disturbance of the PSII differentiation and function (Berges et al. 1996). In addition to the changes of the photosystems and electron transport chain, the decrease of the Rubisco content occurs (Beardall et al. 1991). Moreover the general shift of the carbohydrate metabolism towards lipid accumulation has been described (Giordano et al. 2001, Work et al. 2010). Due to plasticity, most of the nitrogen-starvation
symptoms disappear and the whole C. reinhardtii culture recovers if nitrogen is resupplied. A greening process with Chl accumulation and synthesis of Chl-protein complexes takes place (Plumley and Schmidt 1989). Also the recovery of the photosynthetic activity (Fv/Fm value) was reported in Dunaliella tertiolecta culture (Young and Beardall 2003). This work aimed to study appearance of nitrogenstarvation symptoms in C. reinhardtii cultivated in nitrogen-free medium and a recovery process (regreening) after resupplying nitrogen. We compared photosynthetic properties of N-starving and recovering cultures: Chl contents, ratios of Chl-protein complexes (using 77 K fluorescence spectroscopy), the O2-evolution values, and the plastid ultrastructure. Furthermore, we searched for the minimal nitrogen content of the medium necessary for regreening.
Materials and methods Algae cultivation: Cells of the wild strain 187 (from the Strain Collection of the Chlamydomonas Center, Saint Paul, Minnesota, USA) of the unicellular green alga C. reinhardtii were grown at 25C on solid TAP medium (Sager and Granick 1953) containing NH4Cl as a nitrogen source (Sager and Granick 1953) under a 16-h light [26– 30 µmol(photon) m–2 s–1] and 8-h dark regime with monthly passages. For the degreening process (DG), the algal cultures (2–3 weeks after the last passage) were transferred to the nitrogen-free solid TAP medium. Samples were collected for pigment extraction from 0, 1, 3, 5, 8, 12, 15, and 18-d-old cultures; for fluorescence spectroscopy from 0, 1, 2, 4, 8, 12, 15, 22, and 37-d-old cultures; for the detection of oxygen evolution from 1, 3, 5, 7, 9, 15, and 21-d-old cultures, and for electron microscopy from 0, 1, 2, 5, 8, 15, 24, and 82-d-old cultures. For the regreening process (RG), the degreened, 21-dold algal cultures were transferred to the nitrogencontaining TAP medium. Samples were collected for pigment extraction from 0, 1, 3, 6, 9, 15, 24, 48, 96, and 168 h-old cultures; for fluorescence spectroscopy from 0, 1, 3, 6, 9, 24, 48, 72, 120, and 168 h-old cultures; for the detection of oxygen evolution from 0, 1, 2, 4, 6, 10, 15, 24, 48, 96, and 168 h-old cultures, and for electron microscopy from 0, 5, 14, 24, 48, 78, 120, and 168 h-old cultures. To determine the minimal nitrogen amount for the regreening RG process after the nitrogen starvation, the 21-d-old yellow algal cultures were placed onto media with a various nitrogen content. Samples were collected from 7-d-old cultures. Respiration and photosynthetic oxygen evolution: The respiration intensity and photosynthetic oxygen evolution of samples suspended in 50 mM Tricin-NaOH (pH 7.8) buffer were measured using a Hansatech OxyLab
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Medium
N content [mg l1]
TAP TAP1/2 TAP1/4 TAP1/8 TAP1/16 TAP1/32 TAP1/50 TAP1/100 TAP1/200 TAP-N
98.10 49.05 24.52 12.26 6.13 3.06 1.96 0.98 0.49 0
(Norfolk, UK) oxygen electrode. The oxygen consumption and evolution by the samples were given in mol(O2) ml–1 min–1. The samples were measured in darkness and in light, illuminated with 900 mol(photon) m–2 s–1. This photon flux density value was selected on the basis of a calibration curve. Data represent the means of three independent measurements. Chl content: Chlamydomonas reinhartdii cells (10 mg) were suspended in 0.5 ml of 0.1 M NH4OH and 4.5 ml of ice-cold acetone. After 10-min incubation, the solution was centrifuged at 12,000 × g and the supernatant was collected. Absorption was measured with a Perkin Elmer Lambda 25 (Norwalk, CT, USA) UV/VIS spectrophotometer. The Chl content of the samples was determined according to Porra et al. (1989). Data represent the means of three independent measurements. Fluorescence spectroscopy: The algal culture (1.5 mg) was suspended in 5 ml of TAP medium. Aliquots (300 l) of the diluted samples were pipetted into test tubes with a diameter of 2 mm, and frozen in liquid nitrogen. The 77 K fluorescence spectra were recorded with Fluoromax-3
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(Jobin Yvon-Horiba, France) spectrofluorometer. The excitation wavelength was 440 nm. The integration time was 0.1 s and the data frequency was 0.5 nm. The excitation and emission slits were 2 and 5 nm, respectively. For each sample, the average of three spectra was automatically calculated. The spectra were analysed with SPSERV V3.41 program (Bagyinka, Cs., Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary). The spectra were corrected for the wavelength-dependent sensitivity of the fluorometer detector. Five-point linear smoothing, baseline correction, and deconvolution into Gaussian components were performed. Electron
microscopy:
For
transmission
electron
microscopy the algal cultures were fixed in 2% (v/v) glutaraldehyde for 2 h and postfixed in 1% (w/v) OsO4 for 2 h in 70 mM K-Na phosphate buffer (pH 7.2). Samples were embedded in Durcupan resin (Fluka, Neu Ulm, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Hitachi 7100 (Hitachi Ltd., Chiyoda, Tokyo, Japan) electron microscope, operated at 75 kV. Micrographs were taken with a MegaView III camera (Soft Imaging System, Münster, Germany). Statistical analysis: The reported values are the means from at least two parallel measurements of three independent experiments (n = 6). For statistical significance, we used the Student’s t-test ( = 0,05).
Results Degreening of the algal cells on nitrogen-free medium Chl content and photosynthetic oxygen evolution: The visually detectable yellowing of the cultures on the nitrogen-free medium (Fig. 1SAD, supplement available online) resulted from gradual loss of Chl (Fig. 1). During 18 d, the Chl content decreased to 1.5% of the control and the Chl a/b ratio increased from the initial 2.1 to 4.1%. The decline of the oxygen evolution showed a curve running parallel with that of the DG (Fig. 1). In both cases, the most remarkable changes occured during the first 5 d; this was followed by a slow decline.
around 710 nm (Fig. 2, spectrum D). No fluorescence signal appeared after 37 d of cultivation (Fig. 2, spectrum E) showing the completeness of the “degreening” process. Ultrastructural characteristics: The ultrastructural alterations were slower than the biophysical and biochemical changes. Electron microscopic investigations confirmed that the well developed thylakoid system characteristic of the control cells (Fig. 3A) was gradually degraded. This
Fluorescence properties: The effect of nitrogen deficiency on the composition and relative quantity of Chl-protein complexes in C. reinhardtii cells were studied by 77K fluorescence spectroscopy. The fluorescence emission spectra of the alga culture suspensions were measured after 0, 2, 8, 15, and 37 d-long growth on the nitrogen-free medium. The spectrum of the control sample showed bands at 687–688 and 710–712 nm (Fig. 2, spectrum A), their amplitude ratio was 0.7. This ratio value gradually increased up to the 8th d (Fig. 2, spectrum C), afterwards it decreased. After 15 d of nitrogen starvation, a band of very low intensity was found around 685 nm and no characteristic band was present
Fig. 1. Chl content (solid line, ●) and photosynthetic oxygen evolution (dashed line, ▲) of algal cells maintained on nitrogenfree solid TAP medium. 100% = control green algae [Chl content: 4,180 µg g–1(FM), oxygen evolution: 2,076 µg(O2) min–1 g–1(FM)].
Fig. 2. 77 K fluorescence emission spectra of degreening cultures after 0 (A), 2 (B), 8 (C), 15 (D), and 37 (E) days on nitrogen-free solid TAP medium. Excitation wavelength was 440 nm. Numbers show the emission maxima in nm.
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Fig. 3. Electron micrographs of degreening (AD) and regreening (EH) algal cells: A 0 d, B 1 d, C 5 d, and D 15 d after starting the degreening process on nitrogen-free solid TAP medium; E 14 h, F 24 h, G 48 h, and H 120 h of regreening after placing the yellow algal cells onto N-containing medium. av autophagic vacuole, l lipid body, s starch grain, st stacked thylakoids, t thylakoids. Bars 1 µm.
could be clearly observed on the 4th d (Fig. 3C); at that time, the grana-like structures were largely disintegrated. From the 1st day of the DG, accumulation of starch grains could be detected (Fig. 3B). At the onset of nitrogen starvation, autophagic vacuoles with partly digested cell
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content were visible in the cytoplasm (Fig. 3B,C). Concomitantly, lipid globules appeared, then their number and size increased progressively (Fig. 3BD). After two weeks of nitrogen starvation, the original structure of a typical C. reinhardtii cell was hardly
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Fig. 4. Chl content (solid line, ●) and photosynthetic oxygen evolution (dashed line, ▲) of regreening algal cells transferred back onto nitrogen-containing solid TAP medium. 100% data of control green algae [Chl content: 4,180 µg g–1(FM), oxygen evolution: 2,076 µg(O2) min–1 g–1(FM)].
recognizable, as the lipid globules occupied the greater part of the cell. Grana-like membrane stacks could not be found at all, the few thylakoids occurred on the periphery of the plastid (Fig. 3D). Regreening of the algal cells on nitrogen-containing medium Chl content and photosynthetic oxygen evolution: After transfer of the 21-d-old degreened C. reinhardtii cells onto the nitrogen-containing TAP medium, their yellowish colour quickly started turning green (Fig. 1SEH). The RG of the algal cells was much faster than the DG (Fig. 3AH); the Chl content reached 20% of the control by the 15th h; after 2 d, this value was more than 60% of the control green cells, and on the 4th d, the Chl content reached 110% of the control and did not change significantly during the next 3 d (Fig. 4). In the first phase of RG (several hours), the Chl a/b ratio remained high (4.1) and then decreased gradually; on the 5th d, this value was the half (2.0) of that in the degreened algal cells. The regeneration of the oxygen evolution exceeded that of the Chl content in the first 3 d, but in the forthcoming days it saturated at 80% of the control (Fig. 4). The 100% of the oxygen evolution value could be measured only after three-week cultivation on nitrogen-containing TAP medium. After three weeks, however, the Chl content and the oxygen evolution values became similar to those of the control (stock culture). Thus DG-RG experiments could be repeated several times using the same stock culture. Fluorescence properties: A gradual increase of the fluorescence intensity was observed when the degreened cells were transferred onto the nitrogen-containing TAP medium (Fig. 5). After 6 h, a maximum at 679.5 nm appeared in their 77 K fluorescence emission spectrum (Fig. 5, spectrum B). This peak gradually shifted towards longer wavelengths during this nitrogen-induced RG. In parallel, a second fluorescence band emerged at around 710–712 nm (Fig. 5, spectrum D). The growth during 120 h resulted in a spectrum (Fig. 5, spectrum E) where the relative amplitude of the 687 nm band was lower than that
of the control spectrum (Fig. 2, spectrum A). To obtain information about the relative amounts of Chl-protein complexes, the emission spectra of the control (Fig. 2, spectrum A, Fig. 6, spectrum A) and the regreened (Fig. 5, spectrum E, Fig. 6, spectrum B) samples were resolved into the same Gaussian components (Fig. 6). Each Gaussian component corresponds to a Chl-protein complex (Ossenbühl et al. 2004). The integral ratios of 680 nm (LHCII), 686 nm (PSII-CP43), 696 nm (PSII-CP47), 704 nm (LHCI), and 716 nm (PSI-LHCI complexes) bands showed that the relative contributions of the PSII complexes were lower in the regreened cells, while the contributions of PSI complexes were higher than those of the control (Fig. 6). Ultrastructural characteristics: Transferring the 21-dold degreened C. reinhardtii cells onto the nitrogencontaining medium caused the recovery of the thylakoid membranes. The reorganization of the thylakoid system began within 5 h (not shown), and after the 14th h, distinct lamellae stacked in pairs could be observed (Fig. 3E). After 1 d, a well-organized membrane system with stacked thylakoids could be detected (Fig. 3F), the degree of stacking and the amount of thylakoids increased during the next few days (Fig. 3G,H). The regeneration of the chloroplast was almost complete on the 2nd d, and by this time the starch content decreased to the normal level
Fig. 5. 77 K fluorescence emission spectra of reegreening cultures after 0 (A), 2 (B), 8 (C), 15 (D), and 37 (E) days on nitrogen-containing solid TAP medium. Excitation wavelength was 440 nm. Numbers show the emission maxima in nm.
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Fig. 6. Comparison of 77 K fluorescence emission spectra (solid lines) of control (A) and 7 d regreened (B) Chlamydomonas cultures. The spectra were measured with 440 nm excitation and were deconvoluted into Gaussian components (dashed lines) with similar position and half-bandwidth values. The error of the fit was smaller than 1%.
(Fig. 3G). Lipid globules decreased remarkably in the cytoplasm during the first 24 h (Fig. 3E,F), then disappeared (Fig. 3G,H). Regreening of algal cells on media containing various nitrogen amount Chl content and photosynthetic oxygen evolution: To study the correlation of available nitrogen amounts, the Chl accumulation, and the oxygen evolution, the degreened cultures were transferred to media containing different amounts of nitrogen. After 7 d of cultivation, the Chl contents and the oxygen evolution values were measured (Fig. 7). These experiments showed that the TAP medium containing half of the quantity of nitrogen regenerated the Chl content over that of the control. The oxygen evolution was strongly influenced by the amount of nitrogen: at half quantity of nitrogen in the medium merely 70% of the control could be detected and the TAP
media containing 25% or less nitrogen resulted in only minimal regeneration (Fig. 7). Fluorescence properties: The intensity of the fluorescence emission spectra showed a good correlation with the nitrogen content of the culture medium after 7 d of growth (Fig. 8). Emission bands appeared at the same positions as described earlier (Figs. 2, 5), however, their intensities varied. The cultures grown on medium with 25% nitrogen of the control showed symptoms of nitrogen starvation; the shapes of their fluorescence spectra (Fig. 8, spectrum C) were very similar to those of cultures grown on nitrogenfree medium for 15 d (Fig. 2, spectrum D). However, the spectra of alga cells grown on media containing half or full quantity of nitrogen were almost identical (Fig. 8, spectra A and B).
Discussion Nitrogen is frequently limiting element in agriculture. It has direct and indirect effects on the formation and function of the photosynthetic apparatus. Nitrogen starvation of algae is a commonly used as biotechnological tool to increase storage of starch granules and lipid droplets (Wei et al. 2014). Chlamydomonas reinhardtii is a good model for studying the relationship between nitrogen supply and the photosynthetic apparatus because this alga culture reacts sensitively and quickly and, in addition, the effects of nitrogen deficiency and the recovery can be studied on the same culture. Similarly to earlier results, deprivation of nitrogen resulted in the appearance of nitrogen deficiency symptoms, e.g. loss of Chl (Ördög et al. 2012) and deterioration of the function and structure of the photosynthetic apparatus. We found here a close correlation between the Chl contents and the O2 production values during the yellowing period (Fig. 1). Similar results were published by Coleman et al. (1988), Plumley and Schmidt (1989), and Sinetova et al. (2006). This can be explained by a direct relationship between the
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amounts of Chl and the light absorbance by the LHC. However, a special aspect is the high sensitivity of PSII to nitrogen deficiency: the amounts of its constituents decrease (Berges et al. 1996). This can be considered as a self-defence mechanism: the unbalanced ratios and functions of the Chl-protein complexes can lead to photooxidation processes resulting in the fast degradation of the thylakoid membranes. The mechanism of this degradation involves NO formation from intracellular nitrite, triggering the activity of chloroplast proteases (Wei et al. 2014). The alterations in the Chl-protein complexes proceed in parallel with the decreasing amount of the thylakoids (Fig. 3AD). In the initial phase of nitrogen starvation, we could observe autophagy in vacuoles containing partly digested cell constituents. This is wellknown in nitrogen-deprived yeast (Onodera and Ohsumi 2005), as a way to maintain the necessary amino acid content in the cell. The process is controlled by the Atg7 gene; when this gene was knocked out in mice, starvationinduced autophagy became impaired (Komatsu et al. 2005).
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Fig. 7. Chl content (open columns) and photosynthetic oxygen evolution (dotted columns) of regreening cultures transferred onto solid TAP media containing different amounts of nitrogen (1/16, 1/8, 1/4, 1/2 of the normal nitrogen content and full nitrogen as a control).
Already after the first day of nitrogen starvation, the starch content considerably increased in the chloroplasts and remained high even when photosynthesis ceased (Fig. 3AD). We can suppose that at the onset of yellowing the sugar export is reduced from the chloroplast (Appenroth et al. 2003), and later the disfunctioning cell is not able to utilize starch grains (Nyitrai et al. 2007). As a result, the chloroplasts seem to be transformed into amylochloroplasts, or amyloplasts. Another prominent structural change in the cell from the onset of nitrogen starvation is the continuous increase in number of lipid globules (Fig. 3C,D). Increased lipid accumulation was observed in starchless C. reinhardtii mutants during nitrogen deprivation in the absence of starch synthesis (Work et al. 2010, James et al. 2011, Yu et al. 2013, Wase et al. 2014) and in Chlorella minutissima cultures (Ördög et al. 2012). Lipid globules are outside of the plastid, in contrast to the numerous starch grains, which remain in the plastid, distorting but not disrupting it, as found also in Chlorella fusca (Pyliotis et al. 1975). Chloroplast lipids might contribute to the rise of the cytoplasmic lipid globules, as plastoglobuli can be extruded from the chloroplast (Guiaimét et al. 1999). However, there is no change in the number of plastoglobuli during yellowing, similarly to their constancy in nitrogen-deficient Ankistrodesmus falcatus and C. fusca (Mayer and Czygan 1969). While the plastidial origin of lipid bodies seems to be improbable, autophagy and fatty acid synthesis in the cytosol, followed by fat/oil production in the endoplasmatic reticulum membranes may be the source for their formation. This is supported by isolation and analysis of C. reinhardtii lipid bodies showing the absence of galactolipids, but presence of triacylglycerols (90%) and fatty acids (10%) (Wang et al. 2009). These inclusions are characteristic also for algal cells undergoing heat stress (Semenenko et al. 1969), or senescence (McLean 1968). These results of the literature are in good agreement with the observations in this work (Fig. 3A-H) showing the alteration of the whole
Fig. 8. 77 K fluorescence emission spectra of reegreening cultures maintained on solid TAP media with different nitrogen content. A TAP full N, B TAP 1/2 N, C TAP 1/4 N, D TAP 1/50 N, E TAP 1/200 N, F nitrogen-free TAP. Excitation wavelength was 440 nm. Numbers show the emission maxima in nm.
metabolism towards accumulation of storage materials. This process ensures the surviving of the algal cells. The key factor in this shift may be the chloroplast pyruvate dehydrogenase complex catalysing acetyl-CoA formation from pyruvate (Shtaida et al. 2014). Interestingly, there was no direct correlation between the Chl content and the O2 production during the first 7 d of the regeneration of the photosynthetic apparatus after resupplying nitrogen. During the first 3 d, the relative O2 production exceeded the relative increase of the Chl content. Between the 3rd and 7th d (the observation period) the Chl content was 120%, while the relative O2 production was only 80% of that of the control (Fig.4). The culture reached the control values only three weeks later; the same culture (naturally after suitable passages on control medium) could be repeatedly used in these works. To explain the discrepancies between the Chl content and the photosynthetic activity, a detailed analysis of the photosynthetic apparatus was needed. The fast and prevailing regeneration of the PSII is a general phenomenon. In higher plants, the regeneration process of the reaction centres after yellowing is pronounced, especially in the case of PSII (Baszyński et al. 1975, Verhoeven et al. 1997, Sayed 1998). The analysis of the 77 K fluorescence emission spectra showed, however, that after the 3rd d of regeneration, Chl accumulated favourably into the PSI
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rather than into the PSII complexes (Fig. 6). The relatively low amplitude of the short-wavelength emission bands compared with the control cannot be only due to selfabsorption or reabsorption, because of the low Chl concentration in the sample and the small, 2 mm cross section of the sample holder (thus the optical pathway was maximum 1 mm). The Gaussian resolution of the emission spectra showed a relative shortage in the light-harvesting CP43, CP47, and P680 complexes in the regenerating chloroplast (Ossenbühl et al. 2004). The thylakoid system regenerated rapidly, some peripheral thylakoids stacked in pairs became visible after 14 h, granum-like assemblies appeared after 1 d. By the 2nd d, the starch content decreased considerably and the lipid globules disappeared from the cytoplasm (Fig. 3EH). Several lines of evidence support that the inner membrane of the plastid envelope gives rise to the newly forming thylakoids. The lipid composition of these membranes proved to be strikingly similar (Joyard et al. 1998). Budding of vesicles from the inner envelope membrane was inhibited in Arabidopsis thaliana mutants, preventing also the formation of thylakoids (Kroll et al. 2001, Aseeva et al. 2004). Vesicles were visualized by electron microscopy between the envelope and thylakoids in greening y1 mutant of C. reinhardtii (Hoober et al. 1991). Not only the time, but also the nitrogen availability is important in the regeneration of the photosynthetic apparatus and activity. This was observed in the
experiments, when nitrogen-starving, yellow cultures were resupplied with nitrogen in different concentrations and were compared after 7 d. The good correlation was found between the nitrogen concentrations of the culture media and the increase of the Chl contents as well as the increase of the O2 evolution values. These results showed the same tendency as in the experiments, in which the regeneration was followed in time: after 7-d recovery, the relative O2 evolution values were smaller than the relative Chl contents at each nitrogen concentration. However, the differences were more significant at low nitrogen concentrations (Fig. 7) and also the 77 K fluorescence emission spectra showed low extent of regeneration (Fig. 8). The results proved that the nitrogen availability regulates the structure of the photosynthetic apparatus in a complex way. Although there was a good correlation between the Chl content and the photosynthetic O2 evolution at certain stages of the C. reinhardtii life cycle, this alga can vary the ratio of the Chl-protein complexes preventing photodamage processes. Therefore, the first 7-d period of regeneration is only partial; despite the high Chl content, the structure and physiology of the photosynthetic apparatus differ from the control. The full regeneration needed a longer period (ca. three weeks). The fact that yellowing and regreening can occur in the same C. reinhardtii culture confirmed the high plasticity of this alga.
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