Spectral Properties and Excitation Relaxation of Novel Fucoxanthin

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Spectral Properties and Excitation Relaxation of Novel Fucoxanthin Chlorophyll a/c-Binding Protein Complexes Yoshifumi Ueno, Ryo Nagao, Jian-Ren Shen, and Seiji Akimoto J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b02093 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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The Journal of Physical Chemistry Letters

Spectral Properties and Excitation Relaxation of Novel Fucoxanthin Chlorophyll a/c-Binding Protein Complexes

Yoshifumi Ueno,¶ Ryo Nagao,†,* Jian-Ren Shen,† and Seiji Akimoto¶,*

¶Graduate †Research

School of Science, Kobe University, Kobe 657-8501, Japan Institute for Interdisciplinary Science and Graduate School of Natural

Science and Technology, Okayama University, Okayama 700-8530, Japan

*Corresponding Authors: Ryo Nagao, TEL/FAX: +81-86-251-8630, E-mail: [email protected] Seiji Akimoto, TEL/FAX: +81-78-803-5705, E-mail: [email protected]

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ABSTRACT Fucoxanthin chlorophyll a/c-binding proteins (FCPs) are unique light harvesters for some photosynthetic organisms. There were several reports for the alterations of FCPs in response to light conditions. Here, we present the spectral profiles and excitation dynamics of novel FCP complexes isolated from the diatom Chaetoceros gracilis. Under a red-light condition, C. gracilis cells expressed three types of FCP complexes, two of which are very similar to FCP complexes found in the white-light grown cells, and one of which is the novel FCP complexes. The combination of steady-state absorption and fluorescence spectra, and time-resolved fluorescence spectra revealed that compared to other types of FCP complexes, the novel FCP complexes exhibited red-shifted absorption and fluorescence spectra, and fast decay of excitation. This finding will provide new insights into not only the light-harvesting strategies of diatoms but also the diversity of light adaptation machinery for photosynthetic organisms.


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To utilize the solar energy, photosynthetic organisms possess several kinds of pigments, such as chlorophylls (Chls) and carotenoids (Cars) in photosystems (PSs) and light-harvesting Chl–protein complexes (LHCs), and bilins in phycobilisomes (PBSs). The light energy captured by LHC or PBS is subsequently transferred to the reaction center in PSI or PSII.1–3 To achieve the maximum photosynthesis under the changing light environment, photosynthesis organisms have developed the regulatory mechanisms of light energy. A cyanobacterium Fremyella diplosiphon changes the composition of PBS; phycoerythrin is synthesized in PBS under green light, but not under red light.4 In a cyanobacterium Halomicronema hongdechloris, not only Chl f but also red-shifted PBS are accumulated under far-red light.5 These phenomena are known as complementary chromatic adaptation.6 In response to light conditions, cyanobacteria, red algae, and plants modify an energy transfer between PSI and PSII, called spillover, to control energy distribution between both PSs.7–9 When the organisms are exposed to high light, quenching of excitation energy is driven; the orange carotenoid protein and light-harvesting complex stress-related proteins dissipate excitation energy in cyanobacteria10 and green algae,11,12 respectively. Thus, each photosynthetic organism has a unique light-adaptation system that alters pigment contents, light-harvesting apparatus, and interactions among components of the photosynthetic apparatus, likely leading to an acquisition of ecological niches. Diatoms are a major group of microalgae living in marine and freshwater environments.13 Fucoxanthin (Fx) Chl a/c-binding proteins (FCPs) are unique light-harvesting antennas found in diatoms and brown algae.14 Recently, the crystal structure of a dimeric FCP has been solved from a marine pennate diatom Phaeodactylum tricornutum and the monomer possesses seven Chl a, two Chl c, seven 3 ACS Paragon Plus Environment


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Fx, and one diadinoxanthin (Ddx) (Chl a:Chl c:Fx:Ddx = 1.00:0.29:1.00:0.14).15 By contrast, two types of FCP complexes, FCP-A and FCP-B/C, are isolated from marine centric diatom Chaetoceros gracilis, and the pigment ratios of Chl a:Chl c1:Chl c2:Fx:Ddx

are

analyzed

to

be

1.00:0.46:0.39:1.64:0.03

for

FCP-A

and

1.00:0.29:0.31:1.88:0.10 for FCP-B/C.16 The contents of total Chl (Chl a and Chl c) and total Car (Fx and Ddx) between the two FCP complexes are comparable, although they are remarkably different from the pigment compositions in LHCs of green algae and plants (Chls:Cars = 7:2).17 It has been reported for a long time that in response to low-light or red light, P. tricornutum exhibits a fluorescence spectrum having a peak around 710 nm at room temperature.18–21 Because the fluorescence peak above 700 nm is not observed at room temperature in cyanobacteria, green algae, or land plants grown under a normal light condition, the origin of the 710 nm fluorescence at room temperature in P. tricornutum has been discussed. Recently, Herbstová et al. confirmed that the longer wavelength fluorescence components originate from a formation of the red-shifted FCP complexes.22,23 On the other hand, the C. gracilis cells grown under the low-light condition (30 μmol photons m−2 s−1) exhibit a fluorescence spectrum having two main peaks at 688 and 695 nm at liquid nitrogen temperature, which are assigned to fluorescences from CP43 and CP47 proteins, respectively,24 whereas those grown under the very low-light condition (~10 μmol photons m−2 s−1)25 or red-light conditions (Figure S1A) emit a sharp spectrum located around 690 nm. Moreover, compared with the white-light grown cells, the red-light grown cells show a red-shifted fluorescence peak at room temperature (Figure S1B). The changes of spectral shape at 77 K and peak wavelength at room temperature imply that C. gracilis could regulate its 4 ACS Paragon Plus Environment

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light-harvesting antennas in response to either light quantity or quality like P. tricornutum. In the present study, we successfully isolated novel red-shifted FCP complexes from C. gracilis cells grown under the red light, and examined its spectral properties and excitation relaxation by a combination of steady-state absorption and fluorescence spectroscopies and a time-resolved fluorescence (TRF) spectroscopy. Figure 1A shows clear native polyacrylamide gel electrophoresis (CN-PAGE) profile after solubilizing thylakoid membranes of the cells grown under the white LED (W-LED) and the red LED (R-LED). There are several bands of pigment-protein complexes from each of the cells. In the W-grown cells, PSI-FCPI, PSII dimer, FCP oligomer, and FCP trimer were isolated from the upper region of the resultant gel (bands a–d, respectively). The band pattern is consistent with our previous studies.26,27 By contrast, the R-grown cells showed unknown brown color complexes (band f) in addition to the four complexes observed in the W-grown cells. The unknown complexes are positioned between FCP oligomer (band c) and trimer (band d). Polypeptide compositions of the unknown FCP complexes are shown by sodium dodecyl sulfate-PAGE (SDS-PAGE) (Figure 1B). The FCP oligomer is mainly composed of one band corresponding to FCP-A, while the FCP trimer consists of two bands corresponding to FCP-B and FCP-C, as simply named previously.26,27 The unknown FCP showed one major subunit, which is positioned between FCP-A and FCP-B. Since the unknown complexes have the brown color without any PSII and PSI components, here we assigned the complexes as the novel FCP complexes. The FCP oligomer, unknown FCP complexes, and FCP trimer in CN-PAGE are denoted as F1, F2, and F3, respectively, and the major polypeptide band in the F2 complexes was assigned as FCP-D. 5 ACS Paragon Plus Environment


To examine the spectral properties of FCP complexes, we excised each FCP band in CN-PAGE and measured their low-temperature absorption and fluorescence spectra. Figure 2 shows steady-state absorption and fluorescence spectra of the F1, F2, and F3 complexes from the R-grown cells. Absorption profiles of the F1 and F3 complexes (Figure 2, solid line) are essentially the same as those for the W-grown cells (Figure S2, solid line); relative intensities of the Chl c Soret band (459 nm) and the Fx band (500-550 nm) are enriched in the F1 and F3 complexes, respectively.16,28 In the absorption spectrum of the F2 complexes (Figure 2, blue solid line), the Chl c Soret band the Fx band are observed with lower intensities, the carotenoid band around 490 nm (likely assigned to diadinoxanthin) is clearly found, and new bands appear at the wavelengths longer than the main peak (~669 nm) of Chl a Qy band: a peak at 686.1 nm and a shoulder around 678 nm. The F1 complexes exhibit a fluorescence spectrum having a peak at 681.2 nm with a vibronic band around 740 nm (Figure 2, red dotted line), whereas the F3 complex, a main peak at 679.6 nm with a slightly more intense vibronic band around 742 nm (Figure 2, green dotted line). This clear difference in the peak wavelength was not observed in the F1 and F3 complexes from the W-grown cells; their peak wavelengths are 680.4 and 680.6 nm, respectively (Figure S2, dotted line). The fluorescence spectrum of the F2 complexes differs much from those of other complexes (Figure 2, blue dotted line); the spectrum exhibits a red-shifted peak (689.4 nm) with a smaller vibronic band around 750 nm. In addition, the spectral width (8.6 nm FWHM) is almost half as wide as those of the F1 (16.6 nm FWHM) and F3 (15.4 nm FWHM) complexes. In the PSI complexes and the PSII core complexes, some Chls emit red-shifted fluorescence spectra with broader profiles,29 which are called the red Chls. The red-shifted but sharper spectra of the F2 complexes might be caused by a molecular 6 ACS Paragon Plus Environment

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interaction different from what brings about the red Chls in PSI and PSII. In the absorption spectra, the difference between the additional peak wavelengths of F2 and the bands also present in other complexes (F1 and F3) is ~17 nm, which is smaller than a difference between Chl a and Chl f (~40 nm),30 and fluorescence spectrum of the F2 complexes overlaps absorption spectra of F1 and F3 complexes (Figure 2). These results suggest that an uphill energy transfer from the F2 complexes to the F1 or F3 complexes is possible in protein environments. To understand the excitation relaxation of FCP complexes, we measured the TRF spectra and globally analyzed them to obtain fluorescence decay-associated (FDA) spectra. Figure 3 shows the FDA spectra of the F1, F2, and F3 complexes from the R-grown cells. The FDA spectra of the F1 and F3 complexes (Figure 3) and those from the W-grown cells (Figure S3) are essentially the same, but a few differences are recognized. First, relative magnitudes of the first to the third FDA spectra are smaller in the R-grown cells (Figure. 3), compared with those in the W-grown cells (Figure S3). Second, the peak positions of the F3 complexes from the R-grown cells are located at shorter wavelength (680 nm), which agrees well with the 77-K steady-state fluorescence spectra (Figure 2, dotted line). Relaxation processes are explained as fast- and slow-phase energy transfers from the higher- to lower-energy Chl a molecules (the first and second FDA spectra), followed by the energy dissipations (the third and fourth FDA spectra). In the second FDA spectra, magnitudes of negative amplitudes are much smaller than those of positive amplitudes, indicating that quenching occur in hundreds of picoseconds.16,28 The F2 complexes seem to show the similar relaxation processes (Figure 3, blue line), but time constants of the second to the fourth FDA spectra are considerably shorter than those of the F1 and F3 complexes (Figures 3 and S3). 7 ACS Paragon Plus Environment


To elucidate abilities of FCP complexes for light-harvesting and quenching functions, we reconstructed fluorescence decay curves at the peak wavelength of the fourth FDA spectra from positive amplitudes and time constants of the FDA spectra (Figure 4). Two remarkable results are obtained here; the F2 complexes show the fastest decay, and the F1 and F3 complexes from the R-grown cells exhibit decays slower than those of the F1 and F3 complexes from the W-grown cells, respectively (Figure S4). The fast decay of excitation energy in the F2 complexes seems a disadvantage to work as an energy donor. Instead, the F1 and F3 complexes increase the ability as an energy donor in the R-grown cells by the slowed decay of excitation energy. Therefore, the strategies of C. gracilis cells to adapt to very low-light or red-light conditions should be summarized as follows. The F1 and F3 complexes increase an efficiency of energy transfer from them, whereas the F2 complexes extend light-harvesting ability to longer wavelength region. In addition, the F2 complexes might work as a quencher when the cells adapted to very low-light condition are exposed into high light. Because the F2 complexes have the transition energy between CP43 and PSI, the complexes might mediate spillover. In summary, we isolated the novel red-shifted FCP complexes from the C. gracilis cells grown under the R-LED, and examined its spectroscopic properties. Three bands attributed to FCP complexes were detected by CN-PAGE (Figure 1A). The F1 and F3 complexes were analogous to FCP complexes found in the cells grown under the W-LED (Figure 1B). However, the excited-state lifetimes in the F1 and F3 complexes from the R-grown cells were longer than those from the W-grown cells, respectively (Figures 4 and S4). The F2 complexes are unique to the R-grown cells and exhibit a lower transition energy (Figure 2, solid line) and shorter lifetimes (Figure 4) than the F1 8 ACS Paragon Plus Environment

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and F3 complexes. The transition energy of the red-shifted FCP complexes in C. grasilis is higher than that in P. tricornutum,18–23 indicating the presence of different types of red-shifted FCP complexes. These findings will help us to understand the light-harvesting strategies of diatom species and provide insight into the diverse adaptations of photosynthetic organisms to the environmental light conditions.

EXPERIMENTAL METHODS Cells of the diatom C. gracilis were grown at 25°C with 100 rpm agitation under W-LED in air in 125-mL Erlenmeyer flasks containing 50 mL liquid medium. The cultured cells were repeatedly inoculated into fresh artificial seawater medium, at an optical density of 750 nm (OD750) = 0.04 at 7-day intervals. After the inoculated cells were grown under W-LED for 2 days, the cells were inoculated into fresh medium again and grown under different light qualities, W-LED and R-LED (660 nm), for 2 days. The intensity of LED was set to 30 μmol photons m−2 s−1 with continuous illumination. Thylakoid membranes were prepared according to the previous method31 and then suspended with a buffer containing 0.4 M sucrose, 40 mM Mes-NaOH pH6.5, and 5 mM EDTA (buffer A). CN-PAGE was performed according to the method previously.27 Thylakoid membranes were solubilized with 1% (w/v) n-dodecyl-β-D-maltoside at 0.25 mg Chl ml−1 for 10 min on ice in the dark. After centrifugation at 20,000g for 10 min, the supernatant was applied to a 4–16% polyacrylamide gel. Each sample containing 5 µg of Chl was loaded in a separate lane. Electrophoresis was performed at 4°C in electrophoresis buffers containing 25 mM imidazole (pH 7.0) as the anode buffer, and



50 mM Tricine, 7.5 mM imidazole, 0.02% (w/v) sodium deoxycholate, and 0.02% (w/v) Triton X-100 as the cathode buffer. For SDS-PAGE, the gel bands from the CN-PAGE were cut out and then denatured with 2% (w/v) lithium lauryl sulfate and 2% (v/v) 2-mercaptoethanol in buffer A for 30 min under room temperature conditions. The denatured gel samples were subjected to SDS-PAGE using a 16% polyacrylamide gel containing 7.5 M urea.32 A standard molecular weight marker (APRO Science, Japan) was used. After electrophoresis, both SDS-PAGE and CN-PAGE gels were stained with Coomassie Brilliant Blue R-250. Steady-state absorption and fluorescence spectra were measured by a spectrometer (V-650/ISVC-747; JASCO, Japan) and a spectrofluorometer (FP-8300; JASCO, Japan), respectively. The fluorescence rise and decay curves were measured by a time-correlated single-photon counting system with a wavelength interval of 1 nm/channel and with a time interval of 2.44 ps/channel.7 The excitation source was a picosecond pulse diode laser (PiL047X; Advanced Laser Diode Systems, Germany) operated with a repetition rate of 3 MHz. For both the steady-state fluorescence and TRF measurements, the excitation wavelength was set to 459 nm, which mainly brings excitations of Fx and Chl c. All the spectroscopic measurements were carried out at 77 K. The FDA spectra were constructed through the global analysis according to the previous method.33

ASSOCIATED CONTENT


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Supporting Information. Steady-state fluorescence spectra of whole cells and steady-state absorption and fluorescence spectra, FDA spectra, and fluorescence decay curves of the F1 and F3 complexes from the white-grown cells.

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS This work was supported by the Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science KAKENHI JP18J10095 (to Y.U.), JP17K07442 and JP19H04726 (to R.N.), JP17H06433 (to J.-R.S.), and JP16H06553 (to S.A.).

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FIGURE CAPTIONS

Figure 1. Oligomeric states and polypeptide compositions in the pigment-protein complexes isolated from the white- and red-grown cells. Panel A, CN-PAGE profile. Labels a-d indicate PSI-FCPI, PSII dimer, FCP oligomer (F1), and FCP trimer (F3), respectively, while label f stands for unknown FCP complexes (F2). Panel B, SDS-PAGE profile. Lanes 1-5 indicate white F1, white F3, red F1, red F2, and red F3, respectively. Characteristic FCP subunits marked as A, B, C, and D are termed as FCP-A, FCP-B, FCP-C, and FCP-D, respectively.

Figure 2. Steady-state absorption (solid line) and fluorescence (dotted line) spectra at 77 K of the F1 (red), F2 (blue), and F3 (green) complexes from the red-grown cells. The absorption and fluorescence spectra are normalized at the peak of the Chl a Qy band (~669 nm) and the peak intensity, respectively. The excitation wavelength was 459 nm for fluorescence spectra.

Figure 3. Fluorescence decay-associated spectra at 77 K of the F1 (red), F2 (blue), and F3 (green) complexes from the red-grown cells, normalized by the peak intensity of the respective 4th FDA spectrum. The excitation wavelength was 459 nm.

Figure 4. Fluorescence decay curves of the F1 (red), F2 (blue), and F3 (green) complexes from the red-grown cells, reconstructed from the positive amplitudes and time constants of the FDA spectra at 77 K (Figure 3).


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Spectral Properties and Excitation Relaxation of Novel Fucoxanthin

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