LHCII Populations in Different Quenching States Are Present in the

Letter pubs.acs.org/JPCL

LHCII Populations in Different Quenching States Are Present in the Thylakoid Membranes in a Ratio that Depends on the Light Conditions Lijin Tian, Emine Dinc, and Roberta Croce* Department of Physics and Astronomy, Faculty of Sciences and LaserLaB Amsterdam, VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands S Supporting Information *

ABSTRACT: LHCII is the major antenna complex of plants and algae, where it is involved in light harvesting and photoprotection. Its properties have been extensively studied in vitro, after isolation of the pigment−protein complex from the membranes, but are these properties representative for LHCII in the thylakoid membrane? In this work, we have studied LHCII in the cells of the green alga C. reinhardtii acclimated to different light conditions in the absence of the other components of the photosynthetic apparatus. We show that LHCII exists in the membranes in different fluorescence quenching states, all having a shorter excited-state lifetime than isolated LHCII in detergent. The ratio between these populations depends on the light conditions, indicating that the light is able to regulate the properties of the complexes in the membrane.

I

of the composition of the membrane in C. reinhardtii cells, making use of an inducible and repressible chloroplast gene expression system that allows inhibition of chloroplast translation by the addition of vitamins B1 and B12.19 We have used this approach to follow the degradation kinetics of the individual subunits of PSII and PSI, showing that it is possible to obtain cells in which the thylakoid membrane only contains LHCs.20 The advantage is that in these cells, the photosynthetic apparatus is assembled in normal conditions, and then, the reaction centers and the other components of the thylakoid membrane are depleted far faster than LHCII, which is stable in these membranes for several days. Here, we have taken advantage of this system to study the properties of LHCII in the membranes of C. reinhardtii. C. reinhardtii cells were acclimated to three light conditions (dark, normal light (NL), and high light (HL)) and treated with vitamins for several days. The population of functional PSII was followed by measuring the PSII yield (Fv/Fm) (Figure 1A) that, as expected, declined in all conditions during the time course of the vitamin treatment. The decline rate depends on light intensity; in HL, it drops faster than in NL and far faster than in darkness, in agreement with the fact that the D1 damage is proportional to the light intensity.21 This result is confirmed by immunoblotting, which shows that the decrease of D2 protein correlates with the decrease in Fv/Fm (Figure 1B). In agreement with previous results,20 the amount of PSI complexes also decreases during the treatment. After 8 days of darkness, 6 days in NL, or 3 days in HL, the membranes

n plants and green algae, light-harvesting complex II (LHCII) serves as antenna for the photosynthetic reaction centers (RC), substantially increasing their absorption cross section.1 Over the last years, it has become clear that several populations of LHCII, which differ in function and location, exist in the membrane. Some LHCII are tightly associated with Photosystem II (PSII), forming supercomplexes and being responsible for fast excitation energy transfer (EET),2−4 while others are only loosely associated with PSII, providing excitation energy relatively slowly5,6 and forming LHCIIenriched domains;7 a subpopulation of LHCII also acts as efficient antenna of Photosystem I (PSI).8,9 According to the common opinion, LHCII can switch to a quenched conformation in light stress conditions, dissipating a large part of the harvested energy as heat, but the molecular details of this process are not yet clear.10,11 LHCII is thus believed to be a very dynamic complex, able to fine-regulate the flow of excitation energy in the membrane. However, once isolated from the membrane and studied in detergent micelles, all LHCII, even those coming from different organisms, show identical properties (see e.g., ref 12). Thus, the question arises whether these properties are representative for LHCII in the thylakoid membrane. The excited-state lifetime of LHCII in detergent micelles is around 4 ns.13,14 Recent results suggest that this is not the case in the membranes where the lifetimes of LHCII have been shown to be shorter, although different values were reported.15−18 However, the study of LHCII in the membrane is complicated by the presence of the other photosynthetic complexes to which LHCII transfers energy, which cannot be completely eliminated even in the presence of lincomycin.16 Recently, a new approach was presented that allows for control © XXXX American Chemical Society

Received: May 7, 2015 Accepted: June 5, 2015

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Figure 1. Vitamin-treated C. reinhardtii cells grown under different light conditions. (A) Fv/Fm of cells at different stages were recorded by using a Dual-PAM 100. (B) Immunoblot analysis of photosynthetic proteins from cells in the three light conditions. Protein levels before and at the end of the vitamin treatment are compared. Rpl37 was used as a loading control in each blot; 10 μg of total protein was loaded into each well. The control and the vitamin-treated samples of the same series were detected simultaneously on the same blotting, and all blots were within the linear dynamic range of the antibody. (C) Analysis of the complexes purified from the thylakoid membranes of 3 days vitamin-treated cells under HL condition. (i) Sucrose density gradient of the thylakoid membrane, solubilized with 0.6% αDM; (ii) absorption spectra of the B2 and B3 bands from (i) compared with the absorption spectra of bands B2 and B3 purified from WT C. reihardtii cells.12

Figure 2. (A) Time-resolved fluorescence decay traces at 680 nm of NL-acclimated cells at different stages of the vitamin treatment. (B) Comparison of the lifetimes of LHCII in detergent and in cells. Time-resolved fluorescence decay kinetics of isolated LHCII trimer in 0.03% α-DDM buffer, pH 7.5 (black), dark-acclimated cells after vitamin treatment for 8 days (red), NL-acclimated cells after vitamin treatment for 6 days (green), and HLacclimated cells after vitamin treatment for 3 days (blue). Because our streak camera operates in synchroscan mode, lifetimes longer than the time window (>2 ns) can still be estimated by fitting the back-sweep signal. In each trace, the back-sweep signal is clearly present before time point 0 and is more visible for isolated LHCII because it has the longest lifetime.

contain mainly LHCII and a small amount of Lhcas and CP29, while the other proteins were below the detection level of the antibodies (see ref 20 and Figure 1B). To confirm these results, the photosynthetic complexes were isolated from the thylakoid membrane. Only three bands were observed in the sucrose gradient (Figure 1C(i)), containing free pigments, monomers, and trimers of LHC. The absorption spectrum of the B3 band was identical to that of the same band from wild-type (WT) cells (Figure 1C(ii)), confirming that it contains LHCII trimers. The spectrum of the monomeric band shows a higher Chl b

content than that of the corresponding band from WT cells, indicating that this band is strongly enriched in LHCII monomers, while it also contains minor antennas in the WT (Figure 1C). The cells were collected at the end of the vitamin treatment in the three conditions, and time-resolved fluorescence measurements were performed. For NL-acclimated cells, the fluorescence kinetics at different times following vitamin treatment is shown in Figure 2A. As 2340

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Figure 3. DAS of the cells in different conditions: (A) dark, (B) NL, and (C) HL. They are normalized to their maximum, together with the fluorescence emission spectrum of LHCII in detergent, and shown in (D−F), correspondingly.

excitation energy trapping. The other two components have lifetimes of around 90 and 700 ps, meaning that at least two additional populations of complexes in more severe quenched states are present in the membrane and can affect the trapping efficiency. The presence of quenched populations of LHCII in the membrane can also explain the relatively low Fv/Fm values observed in C. reinhardtii in comparison with plants.22 Interestingly, the relative amplitudes of the quenched components increase when going from dark via NL- to HLacclimated cells at the expense of the 2 ns component; see Table 1. This suggests that the ratio between the LHCII populations is modulated by the light conditions. It should be noted that here we are monitoring relatively long-term acclimation processes as the different populations are still

expected, the decay rate decreases during vitamin treatment, paralleling the gradual loss of RCs. The fluorescence decay kinetics of the cells at the end of the vitamin treatment are shown in Figure 2B, and the decay associated spectra (DAS) are reported in Figure 3A−C. For dark-acclimated cells, two lifetime components (460 ps and 2.2 ns) and for NL- and HL-acclimated cells three components (90 ps, 650−750 ps, and 2.0 ns) were required for a good fit of the data. The 460 ps component of the dark-acclimated cells is in between the two faster components of light-acclimated cells, suggesting that it might be a mix of them. For a direct comparison of the amplitude of the components, the three data sets were also analyzed with linked lifetimes (Supporting Information Figure S1). All components have LHCII-type fluorecence spectra, peaking at 681 nm, although they are broader and have a more intense vibronic band as compared to the LHCII fluorescence spectrum in detergent (Figure 3D−F). This is especially the case for the long component of the darkacclimated cells, which also contains the largest amount of Lhcas, suggesting that part of the red emission is due to these complexes. The longest component represents the lightharvesting state of LHCII in the membrane and has a lifetime of 2 ns, in agreement with data on plants treated with lincomycin.16 This lifetime is clearly shorter than the 4 ns observed for LHCII in detergent micelles (Figure 2B), indicating that LHCII in the membrane assumes a different and more quenched conformation. However, considering the high rates of EET from the antenna to the core,2,6,15 this shorter lifetime has only a small impact on the yield of

Table 1. Fluorescence Decay Parameters of the Cells in Different Conditionsa dark_vit_8days τ (ps)

A (%)

460 17.8 2200 82.2 τ_av = 1891 ps

NL_vit_6days τ (ps) 87 650 2000 τ_av = 1277

A (%) 13.1 35.0 51.9 ps

HL_vit_3days τ (ps)

A (%)

90 740 2000 τ_av = 857

22.5 56.6 20.9 ps

a

A is the relative population calculated based on the area under the DAS. The averaged lifetimes τ_av were calculated by weighting all components: τav = ∑ni=1 Aiτi, where n is the number of lifetime components. 2341

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disconnected LHC can also explain the low PSII efficiency (Fv/ Fm = 0.6−0.7) value measured in C. reinhardtii cells.

present after a period of dark, and thus, this effect is not related to nonphotochemical quenching. In addition, we can exclude that the presence of the short lifetimes and their relative amplitudes depends on the presence of LhcSR, the complex responsible for NPQ in C. reinhardtii23 because LhcSR is not present in NL-adapted cells (Supporting Information Figure S2). What is then the origin of the quenching? Light-induced fluorescence quenching, which was however partially reversible in the dark, has been observed in isolated LHCII monomers and trimers and lamellae aggregates.24−27 This quenching was shown to be related to the formation of LHCII macrodomains.25 Moreover fast energy relaxation has been reported in LHCII aggregates.14,28,29 The phenomenon that we observed here in cells can have a similar origin. The 77 K emission spectrum vitamin-treated cells shows a maximum of around 706 nm (Supporting Information Figure S3), which can be assigned to aggregated LHCII30 and/or to the low-energy pigments of Lhcas.31 Because the amount of Lhcas in these membrane is low (Figure 1B), EET between LHCII and Lhcas is necessary to explain the 77 K emission spectrum. This means that independently of its exact origin, the presence of a red-shifted emission at 77 K indicates that the complexes are connected in the membrane, suggesting the existence of macrodomains. However, the fact that the spectra of the dark-acclimated cells, which are the least quenched, show the highest amplitude in the red (and the highest content of Lhca) indicates that the red pigments are not involved in quenching, in agreement with previous results.32,33 Moreover, the spectra of the short components show less amplitude in the red compared to the spectra of the long components, suggesting that the quencher has a “blue” spectrum. The presence in the membrane of C. reinhardtii of several LHCII populations characterized by different lifetimes where the ratio between the populations depends on the light conditions suggests that this might be part of the strategy for long-term light-harvesting regulation. To acclimate to different light intensities, most photosynthetic organisms change the size of their antenna, especially modulating the expression of the LHCII genes. However, even in very HL, when the lightharvesting capacity of the core complexes would be sufficient for providing the necessary energy to the cells, the reduction of the antenna size is never extreme (see, e.g., refs 34−36). A minimal amount of antenna is apparently necessary for the good functioning of the photosynthetic apparatus, as also indicated by the higher sensitivity to photodamage of mutants without antenna.37 Still, for HL-acclimated conditions, the antenna size is oversized and can be a major source of photodamage, in particular in C. reinhardtii, which does not seem to be able to modulate its antenna size.38 The present results suggest that a different strategy is then used, which involves the regulation of the excited-state lifetimes; part of the antenna is permanently quenched in the membrane, and the level of quenching (and thus the ratio between the different LHCII conformations) is dictated by the light conditions. This results in an effective reduction of the functional antenna size but does not interfere with the other roles of the lightharvesting complexes in determining the structure of the membranes and assuring photoprotection, and it produces ready-to-use antenna complexes when the light conditions change. Indeed, the presence of disconnected and quenched LHCII has been observed in the WT cells of C. reinhardtii.17,39,40 Finally, the presence of a population of

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EXPERIMENTAL METHODS Strains and Cell Growth. The RR5 C. reinhardtii strain19 containing the Nac2 gene fused to the MetE promoter and the Thi4A riboswitch was grown in TAP medium under dark or continuous regular light (60 μmol photons m−2 s−1). Addition of vitamins B12 and thiamine-HCl for Nac2 repression was performed as described previously.20 Cells were allowed to grow to a density of 107 cells/mL in TAP medium without vitamins under dark and light conditions; then, they were diluted to a density of 106 cells/mL in a medium containing vitamins and subsequently every day diluted to a density of 106 cells/mL. Dark-grown cells were kept in darkness following the addition of vitamins for 8 days, and light-grown cells were kept under 60 μmol photons m−2 s−1 for 6 days or under 400 μmol photons m−2 s−1 for 3 days following vitamin repression. Their PSII activity was monitored by chlorophyll fluorescence using a Dual-PAM 100 (Walz Instruments, Germany). Thylakoid Preparation, Sucrose Gradient. Cells were harvested by centrifugation (1120 g, 5 min, 4 °C). Thylakoid membranes were prepared and separated on a discontinuous gradient (71000g, 1 h, 4 °C) in a SW41 swinging bucket rotor, as described in ref 41, with a few modifications. Thylakoids were pelleted, unstacked with 5 mM EDTA, and washed with 10 mM Hepes (pH 7.5). Membranes were then resuspended in 20 mM Hepes (pH 7.5) and 0.15 M NaCl and solubilized at a final chlorophyll concentration of 0.5 mg/mL by adding an equal volume of 0.6% α-dedocylmaltoside (α-DM). Unsolubilized material was eliminated by centrifugation (13200g 10 min at 4 °C). Supernatant was loaded on a sucrose density gradient (prepared by freezing and thawing 0.5 M sucrose, 20 mM Hepes (pH 7.5), and 0.03% α-DM buffer layered over 1 mL of 2 M sucrose). Solubilized thylakoids were separated by ultracentrifugation (287730g, 14 h, and 4 °C). The green bands visible on the sucrose gradient were harvested with a syringe. Immunoblotting. Protein extraction and immunoblot analysis were done as described in ref 20. The chemiluminescence of the immunoblots was imaged with a ImageQuant LAS-4000 (GE Healthcare). Time-Resolved Fluorescence Measurements. Time-resolved fluorescence spectra were recorded with a subpicosecond streak camera system, Hamamatsu C5680 synchroscan streak camera, combined with a Chromex 250IS spectrograph. A grating of 50 grooves/mm and blazed wavelength of 600 nm was used with the central wavelength set at 720 nm during the measurement. Each image covered a spectral width of 260 nm (for details, see ref 42). Excitation light was vertically polarized, the spot size diameter was typically ∼100 μm, and the laser repetition rate was 250 kHz. Great care was taken to minimize the path length (typical ∼100 μm) to allow measurements on high-concentration samples (OD750 nm = 3, mainly scattering) without significant self-absorption. An excitation wavelength of 400 nm was used. To avoid photodamage and significant singlet−singlet/triplet annihilations, the sample was stirred with a magnetic stirring bar, and the laser power of 400 nm of 15 μW was used. Images with a ∼2 ns time window were obtained for each sample by averaging 30−100 images in one sequence, and in the sequence, each image was taken with exposure times of 30−60 s. The averaged image was corrected 2342

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Bound to Photosystem II Is a Very Efficient Antenna for Photosystem I in State II. Plant Cell 2012, 24, 2963−2978. (10) Kruger, T. P.; Ilioaia, C.; Johnson, M. P.; Ruban, A. V.; Papagiannakis, E.; Horton, P.; van Grondelle, R. Controlled Disorder in Plant Light-Harvesting Complex II Explains Its Photoprotective Role. Biophys. J. 2012, 102, 2669−2676. (11) Ruban, A. V.; Johnson, M. P.; Duffy, C. D. The Photoprotective Molecular Switch in the Photosystem II Antenna. Biochim. Biophys. Acta 2012, 1817, 167−181. (12) Natali, A.; Croce, R. Characterization of the Major LightHarvesting Complexes (LHCBM) of the Green Alga Chlamydomonas reinhardtii. PLoS One 2015, 10, 1−18. (13) Palacios, M. A.; de Weerd, F. L.; Ihalainen, J. A.; van Grondelle, R.; Van Amerongen, H. Superradiance and Exciton (De)localization in Light-Harvesting Complex II from Green Plants? J. Phys. Chem. B 2002, 106, 5782−5787. (14) van Oort, B.; van Hoek, A.; Ruban, A. V.; van Amerongen, H. Aggregation of Light-Harvesting Complex II Leads to Formation of Efficient Excitation Energy Traps in Monomeric and Trimeric Complexes. FEBS Lett. 2007, 581, 3528−3532. (15) van Oort, B.; Alberts, M.; de Bianchi, S.; Dall’Osto, L.; Bassi, R.; Trinkunas, G.; Croce, R.; van Amerongen, H. Effect of AntennaDepletion in Photosystern II on Excitation Energy Transfer in Arabidopsis thaliana. Biophys. J. 2010, 98, 922−931. (16) Belgio, E.; Johnson, M. P.; Juric, S.; Ruban, A. V. Higher Plant Photosystem II Light-Harvesting Antenna, Not the Reaction Center, Determines the Excited-State LifetimeBoth the Maximum and the Nonphotochemically Quenched. Biophys. J. 2012, 102, 2761−2771. (17) Iwai, M.; Yokono, M.; Inada, N.; Minagawa, J. Live-Cell Imaging of Photosystem II Antenna Dissociation during State Transitions. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2337−2342. (18) Miloslavina, Y.; Wehner, A.; Lambrev, P. H.; Wientjes, E.; Reus, M.; Garab, G.; Croce, R.; Holzwarth, A. R. Far-Red Fluorescence: A Direct Spectroscopic Marker for LHCII Oligomer Formation in Nonphotochemical Quenching. FEBS Lett. 2008, 582, 3625−3631. (19) Ramundo, S.; Rahire, M.; Schaad, O.; Rochaix, J. D. Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops in Chlamydomonas. Plant Cell 2013, 25, 167−186. (20) Dinc, E.; Ramundo, S.; Croce, R.; Rochaix, J. D. Repressible Chloroplast Gene Expression in Chlamydomonas: A New Tool for the Study of the Photosynthetic Apparatus. Biochim. Biophys. Acta 2014, 1837, 1548−1552. (21) Tyystjarvi, E.; Aro, E. M. The Rate Constant of Photoinhibition, Measured in Lincomycin-Treated Leaves, Is Directly Proportional to Light Intensity. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2213−2218. (22) Bjorkman, O.; Demmig, B. Photon Yield of O2 Evolution and Chlorophyll Fluorescence Characteristics at 77 K among Vascular Plants of Diverse Origins. Planta 1987, 170, 489−504. (23) Peers, G.; Truong, T. B.; Ostendorf, E.; Busch, A.; Elrad, D.; Grossman, A. R.; Hippler, M.; Niyogi, K. K. An Ancient LightHarvesting Protein Is Critical for the Regulation of Algal Photosynthesis. Nature 2009, 462, 518−521. (24) Jennings, R. C.; Garlaschi, F. M.; Zucchelli, G. Light-Induced Fluorescence Quenching in the Light-Harvesting Chlorophyll a/B Protein Complex. Photosynth. Res. 1991, 27, 57−64. (25) Barzda, V.; Istokovics, A.; Simidjiev, I.; Garab, G. Structural Flexibility of Chiral Macroaggregates of Light-Harvesting Chlorophyll a/b Pigment−Protein Complexes. Light-Induced Reversible Structural Changes Associated with Energy Dissipation. Biochemisty 1996, 35, 8981−8985. (26) Barzda, V.; Jennings, R. C.; Zucchelli, G.; Garab, G. Kinetic Analysis of the Light-Induced Fluorescence Quenching in LightHarvesting Chlorophyll a/b Pigment−Protein Complex of Photosystem II. Photochem. Photobiol. 1999, 70, 751−759. (27) Zer, H.; Vink, M.; Keren, N.; Dilly-Hartwig, H. G.; Paulsen, H.; Herrmann, R. G.; Andersson, B.; Ohad, I. Regulation of Thylakoid Protein Phosphorylation at the Substrate Level: Reversible LightInduced Conformational Changes Expose the Phosphorylation Site of

for background and photocathode shading and then sliced up into traces of ∼4 nm width. All measurements were performed at room temperature. Data Analysis. Data obtained with the streak camera setup were first globally analyzed with the R package TIMP-based Glotaran.43 The methodology of global analysis is described in ref 44, the data were fitted as a sum of exponential decays convolved with an instrument response function (IRF). The amplitudes of each decay component as a function of wavelength are called DAS; see the details of the methodology and its applications in ref 44.

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ASSOCIATED CONTENT

* Supporting Information S

DAS with linked lifetimes, LhcSR expression, and 77 K fluorescence spectrum of the cells. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.5b00944.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +31 20 5986310. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Jean-David Rochaix for providing the Chlamydomonas reinhardtii RR5 transformant. This work was supported by the ERC consolidator Grant 281341(ASAP) to R.C.

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REFERENCES

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