Elevated Growth Temperature Can Enhance Photosystem I Trimer Formation and Affects Xanthophyll Biosynthesis in Cyanobacterium Synechocystis sp. PCC6803 Cells Regular Paper

¨ zge Sozer2, Kinga Kłodawska1, La´szlo´ Kova´cs2, Zsuzsanna Va´rkonyi2, Miha´ly Kis2, O Hajnalka Laczko´-Dobos2, Ottilia Ko´bori2, Ildiko´ Domonkos2, Kazimierz Strzałka1, Zolta´n Gombos2 and Przemysław Malec1,* 1

Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krako´w, Poland Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary

2

*Corresponding author: E-mail, [email protected]; Fax, +48-12-664-69-02. (Received June 24, 2014; Accepted December 9, 2014)

Keywords: Carotenoids  CD spectra  PSI trimer  PsaLdeficient mutant  Synechocystis. Abbreviations: CD, circular dichroism; CN-PAGE, clear nativePAGE; b-DM, n-dodecyl-b-D-maltoside; HRP, horseradish peroxidase; OD, optical density; PG, phosphatidylglycerol; RC, reaction center; RP-HPLC, reversed phase HPLC; WT, wild type.

Introduction PSI is one of the largest pigment–protein complexes of the photosynthetic membranes in cyanobacteria, algae and

higher plants. It catalyzes the electron transfer from reduced plastocyanin on the lumenal side of the membrane to ferredoxin or flavodoxin on the cytoplasmic/stromal membrane side (Golbeck 1992, Chitnis 2001). The PSI of algae and higher plants exists exclusively in the monomer form, consisting of a PSI reaction center (RC) which binds four peripheral light-harvesting complexes (LHCIs). LHCI complexes have not been found in cyanobacteria (Chitnis 1996). Instead, these organisms contain PSI RCs organized as monomers and trimers (Boekema et al. 1987, Rogner et al. 1990, Shubin et al. 1993). Recently, the presence of dimers and probably tetramers of PSI was observed in the cyanobacterium Anabaena 7120 and in the glaucophyte Cyanophora paradoxa (Watanabe et al. 2011). In the vast majority of cyanobacteria, PSI RCs consist of 11 protein subunits. In particular, PsaA and PsaB are the two central subunits of the heterodimeric core of the RC. Additionally, nine proteins with lower molecular weight (designated PsaC to PsaM) are involved in the formation of the PSI RC monomer (Fromme et al. 2001). PsaL, a 16 kDa hydrophobic protein subunit of the PSI RC, was identified as crucial for the formation of PSI trimers (Chitnis and Chitnis 1993, Chitnis et al. 1993, Schluchter et al. 1996). Only the monomer form of PSI RC accumulated in a PsaL-deficient Synechocystis mutant, demonstranting the importance of PsaL in the formation of PSI trimers (Chitnis and Chitnis 1993). Several carotenoid residues have been identified in the vicinity of the PsaL-containing domain within a PSI trimer structure (Fromme et al. 2001, Sozer et al. 2011). Recently, the formation of PSI trimer has been found to be repressed in a carotenoidless crtHB mutant strain (Sozer et al. 2010). It is commonly accepted that there exists a dynamic equilibrium between monomer and oligomer forms of PSI in the photosynthetic membranes of Synechocystis PCC6803 and other cyanobacteria. However, the physiological significance of PSI oligomerization remains unclear. What are known as ‘red Chls’ were identified in Spirulina platensis. They absorb light wavelengths >700 nm and were found to be preferentially associated with oligomers of PSI RCs (Karapetyan et al. 1999). It was

Plant Cell Physiol. 56(3): 558–571 (2015) doi:10.1093/pcp/pcu199, Advance Access publication on 16 December 2014, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

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In the thylakoid membranes of the mesophilic cyanobacterium Synechocystis PCC6803, PSI reaction centers (RCs) are organized as monomers and trimers. PsaL, a 16 kDa hydrophobic protein, a subunit of the PSI RC, was previously identified as crucial for the formation of PSI trimers. In this work, the physiological effects accompanied by PSI oligomerization were studied using a PsaL-deficient mutant (psaL), not able to form PSI trimers, grown at various temperatures. We demonstrate that in wild-type Synechocystis, the monomer to trimer ratio depends on the growth temperature. The inactivation of the psaL gene in Synechocystis grown phototropically at 30 C induces profound morphological changes, including the accumulation of glycogen granules localized in the cytoplasm, resulting in the separation of particular thylakoid layers. The carotenoid composition in psaL shows that PSI monomerization leads to an increased accumulation of myxoxantophyll, zeaxanthin and echinenone irrespective of the temperature conditions. These xanthophylls are formed at the expense of b-carotene. The measured H2O!CO2 oxygen evolution rates in the psaL mutant are higher than those observed in the wild type, irrespective of the growth temperature. Moreover, circular dichroism spectroscopy in the visible range reveals that a peak attributable to long-wavelength-absorbing carotenoids is apparently enhanced in the trimer-accumulating wild-type cells. These results suggest that specific carotenoids are accompanied by the accumulation of PSI oligomers and play a role in the formation of PSI oligomer structure.

Plant Cell Physiol. 56(3): 558–571 (2015) doi:10.1093/pcp/pcu199

In the present study we demonstrate that in mesophilic Synechocystis PCC6803 cells, the PSI monomer to trimer ratio depends on growth temperature. The carotenoid composition in psaL, a PSI trimer-deficient mutant, shows that PSI monomerization leads to an increased accumulation of myxoxantophyll, zeaxanthin and echinenone, irrespective of temperature conditions. Moreover, circular dichroism (CD) spectroscopy in the visible range reveals that a peak attributable to long-wavelength-absorbing carotenoids is apparently enhanced in the trimer-forming WT cells. Our results suggest that carotenoids may participate in the formation of the PSI oligomer structure.

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postulated that such oligomers may facilitate radiationless dissipation of excess energy as heat and consequently protect the photosynthetic apparatus against light stress (Karapetyan et al. 1997, Karapetyan et al. 1999, Karapetyan 2008). Other authors proposed that in Spirulina PSI these oligomerassociated red Chls can funnel light energy to P700 (van Grondelle et al. 1994, Shubin et al. 1995) and/or increase the cross-section of light absorption in the long wavelength region (Trissl 1993). However, in Synechocystis PCC6803, which contains a limited pool of red Chls, no significant differences in energy transfer were detected either between Chls within an inner PSI antenna or between PSI monomers and trimers (Turconi et al. 1996, Gobets and van Grondelle 2001). In cyanobacterial cells, the ‘state transition’ phenomenon can take the form of the movement of phycobilisomes from PSII RCs (state 1) to PSI RCs (state 2). In comparison with the wild type (WT), a Synechococcus mutant with a monomerized PSI exhibited an accelerated state 1–state 2 transition (Schluchter et al. 1996). Iron deficiency induced the monomerization of PSI, as well as affecting the energy transfer in cyanobacterial cells. Iron-stressed cells exhibited a reduced capacity for state transitions and a limited dark reduction of the plastoquinone pool (Ivanov et al. 2006). It was also suggested that the ‘energy spillover’ occurring under environmental change from light to darkness could be triggered by a dissociation of PSI trimers into monomers, with the PSI monomers re-aggregating into trimers when the cells move from darkness to light, i.e. PSI oligomerization is reversibly regulated by light or its absence (Li et al. 2006). Phosphatidylglycerol (PG) depletion, leading to a decrease in photosynthetic activity (Hagio et al. 2000, Gombos et al. 2002), resulted in a complete elimination of PSI trimers (Domonkos et al. 2004). The PG depletion-induced PSI monomerization process has been found to be accompanied by an enhanced synthesis of xanthophylls, predominantly myxoxanthophyll (Domonkos et al. 2009). Recently, the crucial role of xanthophylls as protective agents against various stresses in cyanobacteria has been confirmed (Schafer et al. 2005, Zhu et al. 2010). In particular, the double mutant of Synechocystis, crtRO, lacking echinenone, zeaxanthin and myxoxanthophyll, showed low oxygen-evolving activity accompanied by increased light sensitivity (Schafer et al. 2005). Temperature changes in the environment may induce damage in living organisms. Photoautotrophic species need to be able to acclimate to ambient temperature conditions (Berry and Bjorkman 1980). In cyanobacteria, photosynthetic activity remains constant with changing growth temperatures (Inoue et al. 2001). Two extrinsic proteins of of the PSII core complex, PsbU and PsbV, were reported to be associated with the thermostability of PSII (Nishiyama et al. 1997, Nishiyama et al. 1999). In Synechocystis PCC6803, it was found that the fluidity of thylakoid membranes plays an important role in the acclimation of PSII activity to the growth temperature (Aminaka et al. 2006). Recently, it was shown that the elimination of xanthophylls affects the fluidity of thylakoid membranes in cyanobacteria (Kłodawska et al. 2012).

Inactivation of the psaL gene results in a PSI trimer-deficient mutant with no impaired capacity for photoautotrophic growth The small subunit of PSI, PsaL, encoded by the psaL gene has previously been shown to be indispensable for trimer formation in Synechocystis PCC6803 (Chitnis and Chitnis 1993). In order to analyze the role of PSI trimer in cyanobacteria, we have created a mutant incapable of forming trimers. To this end, the psaL gene of Synechocystis was inactivated by replacing its coding region with an cassette conferring spectinomycin resistance. A fully segregated mutant was obtained (Supplementary Fig. S1). Mutant cells were grown under photoautotrophic conditions at 30 C in a medium supplemented with spectinomycin. They accumulated Chl and were not distinguishable from the WT by confocal microscopy (Fig. 1A, B). Also, the observed average cell diameter of psaL cells (2.16 ± 0.11 mm) was not significantly different from that of the WT (2.19 ± 0.12 mm). The morphology of both WT and mutant cells grown under suboptimal conditions (at either 15 or 37 C) was similar to that observed at 30 C when analyzed microscopically (data not shown). Analysis by electron microscopy revealed profound morphological changes associated with the inactivation of the psaL gene in Synechocystis grown phototropically at 30 C. First, the space occupied by thylakoid membranes within cells became relatively greater than in the WT. Further, in psaL cells, the thylakoid membranes lost the regular structure that could be observed in the WT due to the accumulation of granules expressing low electron density. These granules were localized in the cytoplasm, separating particular thylakoid layers (Fig. 1C, D). To analyze the effect of psaL deletion on PSI oligomerization, thylakoid membranes isolated from WT and psaL mutant cells taken from the late logarithmic phase of culture were solubilized with n-dodecyl-b-D-maltoside (b-DM). Under our experimental conditions, the solubilization protocol was equally effective for membranes isolated from cells grown at different temperatures, producing no visible pellet residue following the final ultracentrifugation. Equal amounts of solubilized thylakoid membranes were then loaded onto the MonoQ anion exchange column. The material was eluted with a linear 559

K. Kłodawska et al. | Photosystem-I-trimer formation in Synechocystis sp.

MgSO4 gradient to separate pigment–protein complexes. The elution was controlled by absorption at 680 nm, and fractions were collected. The resulting elution profiles of thylakoids isolated from WT and psaL cells are compared in Fig. 2A. The separation of complexes isolated from psaL cells demonstrated an abundant peak eluted at low salt concentration, similar to the PSI monomer of the WT (Fig. 2). The subsequent analysis of fractions containing the peak material by both low temperature emission fluorescence and gel filtration on a TSK column (Domonkos et al. 2004) revealed the presence of the PSI monomer (data not shown). In both the WT and the mutant, the main monomer peak was accompanied by a peak eluted at slightly higher ionic strength, containing a mixture of PSI and PSII monomers, as previously described (Domonkos et al. 2004). Moreover, as is shown in Fig. 2A, the PSI trimer, eluting in the WT as a peak at high salt concentration, was not detected in psaL thylakoids. These results confirm that the elimination of psaL leads to a trimerless phenotype. Similar elution profiles were obtained for cells grown both at 15 C and at 37 C (data not shown). The psaL mutation also resulted in a decreased accumulation of PSII monomers and dimers (compare the respective peaks in Fig. 2A). To estimate growth rates, equal amounts of cells as measured by the initial optical density recorded at 750 nm (OD750) 560

set to 0.2 were grown autotrophically at constant culture volumes. The growth rates were estimated from the slope of growth curves during 11 d of cuIture (Supplementary Fig. S2). Both at optimal (30 C) and at suboptimal (15 and 37 C) growth temperatures, both WT and psaL mutant strains showed similar growth rates and reached comparable OD750 values after 3 d of culture (Table 1).

Complementation To eliminate the possible effect of collateral mutations on the psaL phenotype, both the shuttle plasmid bearing the kanamycin resistance gene and the same vector containing the full coding psaL sequence were introduced into psaL cells. Transconjugants were selected under standard photoautotrophic conditions on medium supplemented with kanamycin, and the presence of the intact psaL gene was confirmed by PCR (data not shown). Equal amounts of solubilized thylakoid membranes isolated from WT, psaL and transconjugant lines were then resolved by clear native-PAGE (CN-PAGE). As shown in Fig. 2B, the PSI monomers were the only PSI forms observed in the psaL mutant, whereas in the WT both monomers and oligomers (trimers and supercomplexes) were clearly visible. Also, PSII dimers were not detectable in the psaL mutant by CN-PAGE. The reintroduction of a psaL copy into psaL

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Fig. 1 Representative confocal (A, B) and electron microscopic (C, D) images of the wild-type (A, C) and psaL mutant (B, D) of Synechocystis PCC6803 grown under photoautotrophic conditions at 30 C. Scale bars represent 5 mm (A, B) and 0.25 mm (C, D).

Plant Cell Physiol. 56(3): 558–571 (2015) doi:10.1093/pcp/pcu199

Table 1 Growth rates as measured by OD750 increment and by the slope of the growth curves in wild-type and psaL mutant cells at various temperatures Sample

OD750

Slope

0h

72 h

15 C

0.2

0.29 ± 0.022

0.011 ± 0.001

30 C

0.2

1.12 ± 0.273

0.25 ± 0.011

37 C

0.2

0.43 ± 0.128

0.044 ± 0.002

15 C

0.2

0.26 ± 0.056

0.006 ± 0.001

30 C

0.2

1.04 ± 0.323

0.27 ± 0.022

37 C

0.2

0.33 ± 0.099

0.048 ± 0.007

WT

psaL

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All cultures were bubbled with air containing 3% CO2. Light intensity was 60 mmol photons m–2 s–1. Error bars represent ± SE (n = 3).

Fig. 2 (A) Elution profile of b-dodecylmaltoside-solubilized thylakoid fractions from Synechocystis on an anion-exchange column (MonoQ HR 5/5). Wild-type and mutant cells were cultivated at 30 C. The peaks containing PSI oligomers and PSII are indicated by arrows. Absorbance was recorded at 680 nm. (B) CN-PAGE analysis of thylakoid Chl–protein complexes from the autotrophic cells: WT, psaL, ‘plasmid control’ (Plasmid) and the revertant (Comp.). See the text for details.

cells restored a WT-like phenotype, i.e. the ability to form PSI oligomers, whereas psaL cells transformed with an empty vector (‘plasmid control’) retained the initial (mutant) phenotype. The revertant line also showed a CD spectrum with a carotenoid band characteristic of the WT (cf. Fig. 5). These results demonstrate that the phenotype of the psaL mutant results from the inactivation of the psaL gene.

The accumulation of PSI trimer is a temperaturedependent process Temperature is one of the major factors which affects the growth and photosynthetic activity of cyanobacteria (Tandeau de Marsac and Houmard 1993). To check whether temperature has an effect on PSI oligomerization, WT cells of Synechocystis PCC6803 were cultured at various temperatures, 15, 30 and 37 C. The thylakoid membranes isolated from these cultures were solubilized and subjected to anion exchange fractionation on a MonoQ column. The presence of PSI monomers and trimers in peaks eluted with the MgSO4 gradient was confirmed by fluorescence emission at 77K and a TSK gel filtration as above (data not shown). As shown in Fig. 3, the resulting

Fig. 3 Elution profile of b-dodecylmaltoside-solubilized thylakoid fractions from wild-type Synechocystis on an anion-exchange column (MonoQ HR 5/5). Cells were cultivated at the indicated temperatures. The peaks containing PSI oligomers and PSII are indicated by arrows. Absorbance was recorded at 680 nm.

separation profiles of complexes derived from cells grown under different temperatures exhibited significant differences. The separation of complexes isolated from cells grown at 15 C resulted in a substantial peak eluting at a low salt concentration, indicating that PSI is present in these cells mainly in monomeric form (Fig. 3, bottom). In contrast, the cyanobacteria grown at higher temperatures contained significant amounts of both monomer and trimer forms of PSI, as trimer peaks eluting at a high salt concentration were found to be enhanced during anion exchange separation. Whereas the cells grown at 30 C contained comparable amounts of monomer and trimer forms of PSI (Fig. 3, middle), the trimer form dominated in cyanobacteria cultured at 37 C, as the trimer eluted at high salt comprised the most abundant peak during separation of this material (Fig. 3, top). 561

K. Kłodawska et al. | Photosystem-I-trimer formation in Synechocystis sp.

grown at 30 C (Fig. 4B). In both WT and mutant cells, the accumulation of PsaA and PsbA apparently increased with the higher growth temperature; however, significant differences between the WT and psaL mutant were not observed (Fig. 4C).

PSI monomerization results in changes in the carotenoid absorption region of the CD spectrum

The effect of PSI monomerization on the accumulation of the PSI RC To examine how the mutation affected photosynthetic light harvesting and energy transfer, we measured Chl fluorescence parameters in WT and psaL cells. Low temperature fluorescence emission spectra collected at 77K provided evidence that the energy transfer to the RCs of PSI was not distorted in the psaL mutant, as indicated by a significant emission at around 725 nm (Fig. 4A). The comparison of spectra normalized at 684 nm (the emission maximum of PSII RCs) revealed that the relative PSI/PSII ratio was not significantly affected in either WT or mutant cells grown at different temperatures, although it was slightly lower in psaL cells. The only exception was psaL grown at 15 C, where this parameter was remarkably decreased (Fig. 4A). The immunoblotting analysis indicated that psaL mutation had no remarkable effect on the accumulation of the RC core proteins PsaA and PsbA in cells 562

Oxygen-evolving activity of the "psaL mutant Non-cyclic electron transport from water to carbon dioxide was measured as oxygen-evolving activity via PSII and PSI in WT and psaL mutant cells grown at different temperatures. To assess putative differences in the overall capacity for photosynthetic electron transport in cells grown at various temperatures, all measurements were performed under standardized conditions at a constant temperature (30 C), which is close to optimum. As can be seen in Table 2, psaL mutant cells showed slightly higher oxygen-evolving activity than the WT irrespective of growth temperature. There were no statistically significant differences observed in H2O!CO2 electron transport between WT and psaL mutant cells grown at 30 and 37 C, although the values measured for the mutant were slightly higher. In WT cells grown at 15 C, oxygen-evolving activity from H2O to CO2 decreased (16%) in comparison with the values observed for cells cultivated at 30 and 37 C. In psaL mutant cells, this effect was not observed.

Pigment accumulation in the WT and "psaL mutant In comparison with the WT, the absorption spectrum of psaL cell suspensions grown at 30 C showed a relative increase in absorption level in the blue wavelength region, exhibiting bands

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Fig. 4 (A) Temperature-dependent regulation of the PSI/PSII ratio in WT and psaL mutant cells. 77K fluorescence emission spectra of Synechocystis WT (left panel) and psaL mutant (right panel) cells grown under identical growth conditions at various temperatures. The spectra were normalized at 684 nm and the fluorescence emission at 684 nm was set as 1. The excitation wavelength was 437 nm. (B and C) Immunoblotting analysis of the relative accumulation of the PsaA and PsbA proteins: in WT, psaL and revertant (Complemented) strains grown at 30 C (B); and in WT and psaL strains grown at 37 and 15 C (C). See the text for details.

The number and type of pigment molecules bound in the pigment–protein complexes as well as their structural arrangement are crucial for the functioning of the photosynthetic apparatus. To study the effect of PSI oligomerization on overall pigment organization in complexes, WT and psaL cell suspensions grown at 30 C were analyzed by CD spectroscopy in the 300–800 nm region. The CD spectra obtained were normalized to the (+)671/(–)687 excitonic signal. The results are shown in Fig. 5. In both WT and psaL cell suspensions, both the inflection point of excitonic split signals and maxima of CD bands coinciding with the corresponding absorption maxima of the main photosynthetic pigments were clearly visible. In particular, an excitonic band at +671/–687 is attributable to Chl. The maximum at 625 nm corresponds to phycobilisomes and that at 507 nm to the absorption range of carotenoids. As shown in Fig. 5, the CD band in the carotenoid absorption spectral range of psaL cells is significantly affected, indicating chirality changes associated with carotenoids. In the psaL revertant strain, this band was restored, reaching the relative amplitude and width comparable with that of the WT. It confirms that the observed changes in carotenoid-associated chirality in the psaL mutant are caused by PSI monomerization.

Plant Cell Physiol. 56(3): 558–571 (2015) doi:10.1093/pcp/pcu199

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Fig. 5 Circular dichroism spectra of Synechocystis WT (black), psaL (red) revertant (blue) and ‘plasmid control’ (green) cell suspensions normalized to the excitonic signal [(+)671/(–)687 nm]. Samples were measured at a Chl concentration of 15 mg ml–1. The peak attributable to carotenoids (maximum at around 507 nm) is indicated by an arrow.

Table 2 Oxygen-evolving activity of wild-type and psaL cells cultivated at different temperatures as measured at 30 C Sample

Oxygen evolution H2O!CO2 (mmol O2 mg Chl–1 h–1)

WT 15 C

414 ± 16

30 C

481 ± 10

37 C

483 ± 6

psaL 15 C

532 ± 1

30 C

532 ± 1

37 C

496 ± 6

The values represent the averages ± SD calculated from three independent experiments.

at 463 and 486 nm and a shoulder above 500 nm, pointing to an increase in the carotenoid content in psaL on a Chl basis (Fig. 6). Chl accumulation as measured by the Chl/protein ratio, as well as the relative content of total carotenoids, as measured by the carotenoid/Chl ratio, were determined spectrophotometrically in both WT and psaL mutant cells grown at various temperatures. The results are summarized in Fig. 7. Both WT and psaL mutant cells accumulated Chl most effectively when the cells were grown at 30 C. The Chl/protein ratios observed at this temperature were 0.99 and 0.85 mg Chl mg–1

of protein, respectively. At suboptimal growth temperatures (15 and 37 C), significant decreases in the Chl/protein ratio were noted in both the WT and psaL, up to approximately 30% of the value recorded in cells cultivated at 30 C (Fig. 7B). These results correlated with amounts of Chl measured per OD730 (Fig. 7D), indicating that psaL cells contained about the same amount of Chl as WT cells grown under the same experimental conditions. The inactivation of the psaL gene was accompanied by an enhanced accumulation of carotenoids in psaL mutant cells, in comparison with the WT. This effect was particularly noticeable in cells grown at 30 C. Whereas psaL cells grown at 15 and 37 C accumulated twice as many total carotenoids as the respective WT cells, the difference for cells cultivated at 30 C was 5-fold. Also, the total carotenoid level in both WT and psaL cells increased when cultivated at 15 C. In contrast, in cells cultivated at 37 C, the relative carotenoid accumulation decreased. This effect was particularly visible in psaL cells (Fig. 7A). Similar trends were observed for carotenoid/protein ratios (Fig. 7C).

Carotenoid composition in the "psaL mutant To evaluate putative effects of PSI trimerization on pigment composition, the accumulation of four main carotenoids: myxoxanthophyll, zeaxanthin, echinenone and b-carotene, was quantitatively analyzed by reversed phase HPLC (RP-HPLC). Carotenoid species were identified on the basis of their 563

K. Kłodawska et al. | Photosystem-I-trimer formation in Synechocystis sp.

Fig. 7 Relative pigment accumulation in wild-type and psaL cells cultivated at three different temperatures: total carotenoids (mg mg Chl–1) (A), Chl/protein ratio (mg mg–1) (B), carotenoid/protein ratio (mg mg–1) (C) and Chl/OD730 (mg) (D). The values represent the averages ± SD calculated from at least three independent experiments.

564

absorption spectra and their retention times. The relative content of the individual carotenoid species was estimated from their peak areas in the chromatograms (Table 3). The relative myxoxanthophyll and zeaxanthin contents in psaL were higher than in the corresponding WT cells, irrespective of the cultivation temperature. In contrast, the relative amounts of echinenone and b-carotene were lower in the psaL mutant than in the WT under all growth conditions. The differences were particularly noticeable in cells grown at 30 C for myxoxanthophyll and b-carotene. At this temperature, myxoxanthophyll constituted 60.4% of the main carotenoid species in psaL cells, with b-carotene at 1.1%. In WT cells, the accumulation of myxoxanthophyll and b-carotene was 19.5% and 41.5%, respectively. The amounts of carotenoid species calculated on the basis of Chl in WT and psaL cells grown at various temperatures are shown on Fig. 8. Compared with the WT, the accumulation levels of myxoxanthophyll, zeaxanthin and echinenone were significantly higher in psaL cells at all tested temperature conditions (Fig. 8A–D). In particular, the greatest differences in the concentrations of these pigments between the WT and mutant were observed at 30 C. At this temperature, the myxoxanthophyll concentration was approximately 10-fold higher in psaL cells (Fig. 8A). In contrast to this, the b-carotene concentration was approximately 10-fold lower in the mutant grown at 30 C than in the WT (Fig. 8D). At 15 C, psaL showed increased concentrations of both myxoxanthophyll and zeaxanthin (2- to 3-fold compared with the WT), whereas the concentration of echinenone slightly (but still significantly)

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Fig. 6 Absorption spectra of cell suspensions of the WT and psaL mutant, normalized at 665 nm. The maxima at 463 and 486 nm are indicated by arrows.

Plant Cell Physiol. 56(3): 558–571 (2015) doi:10.1093/pcp/pcu199

increased and the b-carotene content did not change. A similar increase in myxoxanthophyll and zeaxanthin content was observed at 37 C. However, at this temperature, the concentration of b-carotene drastically decreased in psaL, whereas the content of echinenone did not change significantly. Table 3 Distribution of the main carotenoid species in wild-type and psaL mutant cells cultivated at three different temperatures Sample

Percentage of peak areas Zeaxanthin

Echinenone

b-Carotene

15 C

42.7 ± 5.7

36.5 ± 4.1

17.5 ± 0.9

3.3 ± 0.9

30 C

19.5 ± 1.2

26.7 ± 0.5

12.2 ± 0.5

41.5 ± 1.5

37 C

20.5 ± 1.0

40.3 ± 2.3

18.8 ± 1.0

20.3 ± 1.7

15 C

53.7 ± 4.1

35.3 ± 2.8

9.5 ± 0.4

1.46 ± 0.3

30 C

60.4 ± 4.5

29.1 ± 2.6

9.3 ± 0.6

1.1 ± 0.1

37 C

34.2 ± 2.4

50.6 ± 3.3

14.4 ± 1.2

0.8 ± 0.2

WT

psaL

The values represent the averages ± SD calculated from three independent experiments.

To estimate the carotenoid composition of PSI in both WT and psaL cells, fractions from the MonoQ column containing monomer and trimer forms of PSI were lyophylized and subjected to acetone : methanol extraction and quantitantive HPLC analysis as described in the Materials and Methods. The content of total carotenoids as measured by the carotenoid/Chl ratio was also determined spectrophotometrically in these fractions. The data obtained for complexes isolated from both WT and psaL mutant cells grown at 30 C are summarized in Table 4. No significant differences were detected in total carotenoid content in either monomer or trimer forms of PSI. Also, no detectable amounts of myxoxanthophyll were found in any forms of complexes. The content of b-carotene was significantly lower in trimer forms than in monomers isolated both from the WT (16%) and the psaL mutant (33%). Also, the estimated echinenone content in PSI trimers was higher than in the corresponding WT and psaL monomers. All other observed differences were

Elevated growth temperature can enhance photosystem I trimer formation and affects xanthophyll biosynthesis in Cyanobacterium Synechocystis sp. PCC6803 cells.

In the thylakoid membranes of the mesophilic cyanobacterium Synechocystis PCC6803, PSI reaction centers (RCs) are organized as monomers and trimers. P...
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