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Oct.  2021
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Fluorescence recovery after photobleaching: analyses of cyanobacterial phycobilisomes reveal intrinsic fluorescence recovery

  • Corresponding author: Hai-Nan Su, suhn@sdu.edu.cn; Yu-Zhong Zhang, zhangyz@sdu.edu.cn
  • Received Date: 2020-08-04
    Accepted Date: 2021-04-08
    Published online: 2021-06-01
  • Edited by Jiamei Li.
  • Fluorescence recovery after photobleaching (FRAP) has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997. Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome (PBS) diffusion on the surface of the thylakoid membrane. The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion. However, in red algae, another PBS-containing group, the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS, which raises questions concerning the role of FRAP in red algal PBS. Therefore, it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria. In the present study, four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics. Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains, in which the contribution of PBS diffusion to the fluorescence recovery was not possible. Moreover, the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery. These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.
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Fluorescence recovery after photobleaching: analyses of cyanobacterial phycobilisomes reveal intrinsic fluorescence recovery

    Corresponding author: Hai-Nan Su, suhn@sdu.edu.cn
    Corresponding author: Yu-Zhong Zhang, zhangyz@sdu.edu.cn
  • 1. State Key Laboratory of Microbial Technology, and Marine Biotechnology Research Center, Shandong University, Qingdao 266237, China
  • 2. College of Marine Life Sciences, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao 266003, China
  • 3. Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao 266237, China
  • 4. College of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China

Abstract: Fluorescence recovery after photobleaching (FRAP) has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997. Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome (PBS) diffusion on the surface of the thylakoid membrane. The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion. However, in red algae, another PBS-containing group, the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS, which raises questions concerning the role of FRAP in red algal PBS. Therefore, it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria. In the present study, four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics. Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains, in which the contribution of PBS diffusion to the fluorescence recovery was not possible. Moreover, the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery. These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.

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Introduction
  • Phycobilisomes (PBSs), which are anchored to the surface of photosynthetic thylakoid membranes, are the dominant light-harvesting antennae in cyanobacteria and red algae (Adir et al. 2020; MacColl 1998). The principal components of PBSs are phycobiliproteins, which covalently attach different chromophores. Different phycobiliproteins are assembled into two subcomplexes with the aid of linker polypeptides, namely the rod and the core (Li et al. 2019; Liu et al. 2005). Phycoerythrin (PE)/phycoerythrocyanin (PEC) are at the distal end of the rod, phycocyanin (PC) locates in the rod adjacent to the core, and the core is principally composed of allophycocyanin (APC) (Watanabe and Ikeuchi 2013). According to PBS composition, cyanobacteria can be largely categorized into two groups: the APC + PC type (hereafter referred as PC-type) and the APC + PC + PE type (hereafter referred as PE-type).

    The energy harvested by antennae is distributed between photosystem (PS) I and II via state transition (McConnell et al. 2002; Mullineaux and Emlyn-Jones 2005). However, the mechanism of state transition is not well understood and several hypotheses have been proposed based on different research techniques. One widely accepted hypothesis is rapid cell-wide PBS mobilization (Joshua and Mullineaux 2004; Mullineaux and Emlyn-Jones 2005), which was first proposed based on the observation of PBS fluorescence in cyanobacteria using the fluorescence recovery after photobleaching (FRAP) technique (Mullineaux et al. 1997). The FRAP technique has been widely used for studying the dynamics of biological system components (Lippincott-Schwartz et al. 2018; Rayan et al. 2010). The components of interest studied with FRAP contain fluorescent chromophores, and the fluorophores in a small area are bleached by a focused laser beam. The fluorescence recovery processes of the bleached regions are then recorded. In 1997, researchers first observed fluorescence recovery of PBS in cyanobacteria using the FRAP technique (Mullineaux et al. 1997). The fluorescence recovery was then interpreted as a rapid diffusion of PBSs in large scale on thylakoid membranes (Aspinwall et al. 2004; Mullineaux et al. 1997; Sarcina et al. 2001; Yang et al. 2007). However, in another group of PBS-containing photosynthetic organisms, the red algae, the intrinsic photoprocess was found to contribute greatly to fluorescence recovery after bleaching with the exclusion of PBS diffusion (Liu et al. 2009). Therefore, it would be useful to carry out further work to re-evaluate the nature of PBS FRAP in cyanobacteria. In the present study, four cyanobacterial strains with both PC and PE types were used in an FRAP investigation. The fluorescence recovery processes of PBS in these four cyanobacterial strains were investigated both in vivo and in vitro. The results suggested that the fluorescence in these cyanobacterial strains and isolated PBSs could be recovered after photobleaching by the intrinsic photoprocess. The findings of this study should help to interpret the FRAP nature of PBS as well as the mechanism of state transition in cyanobacteria.

Results

    Phycobiliprotein compositions in four cyanobacterial strains

  • The FRAP phenomenon was studied in four cyanobacterial strains, two filamentous, i.e., the PC-type Pseudanabaena sp. 0831 and the PE-type Pseudanabaena sp. FACHB-1277, and two unicellular, i.e., the PC-type Synechococcus sp. PCC 7002 and the PE-type Synechococcus sp. WH7805. Prior to FRAP, absorption spectra of the four strains were measured (Fig. 1). In the absorption spectra of Pseudanabaena sp. 0831 and Synechococcus sp. PCC 7002, a peak at ~640 nm from PC/APC was recorded, whereas no remarkable peaks from PE were observed. Therefore, Pseudanabaena sp. 0831 and Synechococcus sp. PCC 7002 were designated as PC-type. In the absorption spectra of Pseudanabaena sp. FACHB-1277 and Synechococcus sp. WH7805, characteristic peaks at 565 nm indicated the presence of PE. Therefore, Pseudanabaena sp. FACHB-1277 and Synechococcus sp. WH7805 were denoted as PE-type.

    Figure 1.  The absorption spectra of cells of Pseudanabaena sp. FACHB-1277 (F1277, black line), Pseudanabaena sp. 0831 (P0831, orange line), Synechococcus sp. WH7805 (WH7805, green line), and Synechococcus sp. PCC 7002 (P7002, blue line)

  • Fluorescence recovery in native cells of the four cyanobacterial strains

  • FRAP was applied to the native cells of all four strains to observe their fluorescence recovery. Figures 2 and 3 show a series of typical images acquired by post-scanning from various FRAP time-lapse experiments in all four strains. In the two filamentous strains, Pseudanabaena sp. 0831 and Pseudanabaena sp. FACHB-1277, the cells were bleached to ~30% of original fluorescence intensity, and fluorescence recovery was detected in both the wholly and partially bleached filaments (~ 4-5 cells in the middle region) (Fig. 2a-d). A quick fluorescence recovery process was observed in which the fluorescence intensity rapidly reached a plateau (about 40-45% of original fluorescence intensity) within 1 min (Fig. 2e-h). In the two unicellular strains, Synechococcus sp. PCC 7002 and Synechococcus sp. WH7805, whole-cell regions were bleached to ~30% of original fluorescence intensity, and the fluorescence partially recovered (to ~50% of original fluorescence intensity) (Fig. 3a, b). For Synechococcus sp. PCC 7002 and Synechococcus sp. WH7805, similar dynamic curves were obtained, and the fluorescence recovery process was analogous to that of Pseudanabaena sp. 0831 and Pseudanabaena sp. FACHB-1277 (Fig. 3c, d). Thus, the PBS fluorescence in all four cyanobacterial strains recovered promptly after whole-cell photobleaching.

    Figure 2.  Qualitative FRAP experiments with the filamentous cyanobacteria Pseudanabaena sp. FACHB-1277 and Pseudanabaena sp. 0831. Left panel, fluorescence images from typical sequences recorded before bleaching, immediately after bleaching, and at various time lapses. Right panel, the corresponding total fluorescence intensity of the bleached cell region as a function of time. The fluorescence recovery is presented in square spots and fitted to a single exponential function (solid line). a, b, e, and f FRAP measurements for Pseudanabaena sp. FACHB-1277; c, d, g, and h FRAP measurements for Pseudanabaena sp. 0831. a and c, partially bleached filament; b and d, wholly bleached filament. Scale bar: 10 μm

    Figure 3.  Qualitative FRAP experiments with cyanobacterial cells of Synechococcus sp. WH7805 and Synechococcus sp. PCC 7002. a and b, fluorescence images from typical sequences recorded before bleaching, immediately after bleaching, and at various time lapses. c and d, the corresponding total fluorescence intensity of the bleached cell region as a function of time. The fluorescence recovery is presented in square spots and fitted to a single exponential function (solid line). a and c FRAP measurements of Synechococcus sp. WH7805; b and d FRAP measurements of Synechococcus sp. PCC 7002. Scale bar: 5 μm

  • Fluorescence recovery of PBS-thylakoid membrane

  • PBS-thylakoid membranes were isolated from the four cyanobacterial strains for FRAP experiments. Absorption spectra and fluorescence spectra suggested that isolated thylakoid membranes kept their structural and functional integrity (Fig. 4). A square region containing PBS-thylakoid membrane vesicle aggregations in each cyanobacterial sample was bleached. The fluorescence was recovered in all tested PBS-thylakoid membranes from the four cyanobacterial strains (Fig. 5). Glutaraldehyde pretreated (1%, v/v) PBS-thylakoid membrane vesicles were also studied with the FRAP technique. It was considered that glutaraldehyde treatment could promote cross-linking between proteins, resulting in larger aggregates of thylakoid membranes that could be easily observed by microscopy. After photobleaching, the fluorescence from all glutaraldehyde pretreated PBS-thylakoid membranes recovered quickly, indicating the intrinsic photoprocess of fluorescence recovery in PBS (Fig. 5).

    Figure 4.  The spectra analysis of isolated PBSs and PBS-thylakoid membranes. Absorption spectra (first row), fluorescence emission spectra (second row), and fluorescence excitation spectra (third row) of PBS (black line) and PBS-thylakoid membrane (red line) in Synechococcus sp. WH7805 (first column), Synechococcus sp. PCC 7002 (second column), Pseudanabaena sp. FACHB-1277 (third column), and Pseudanabaena sp. 0831 (fourth column). Excitation wavelengths in fluorescence emission spectra were 545 nm (e, g) and 580 nm (f, h). Emission wavelengths in fluorescence excitation spectra were 683 nm (i-l)

    Figure 5.  FRAP analysis of isolated PBS-thylakoid membranes and glutaraldehyde-treated PBS-thylakoid membranes from four cyanobacterial strains. Detection ranged from 640 to 700 nm. Fluorescence images from sequences recorded before bleaching, immediately after PBS bleaching, and at various time lapses. a, Pseudanabaena sp. FACHB-1277 with excitation at 555 nm; b, Pseudanabaena sp. 0831 with excitation at 639 nm; c, Synechococcus sp. WH7805 with excitation at 555 nm; d, Synechococcus sp. PCC 7002 with excitation at 639 nm. Lane 1, PBS-thylakoid membranes; Lane 2, glutaraldehyde-treated PBS-thylakoid membranes

  • Fluorescence recovery of isolated PBSs

  • To further investigate the nature of PBS fluorescence recovery, PBSs from the four strains were isolated. Absorption spectra and fluorescence spectra confirmed the structural and functional integrity of the isolated PBSs (Fig. 4). Because PBSs are soluble, the isolated PBSs were pretreated with glutaraldehyde to obtain large insoluble PBS assemblages for FRAP observations. Selected PBS assemblages were wholly bleached. As shown in Fig. 6, fluorescence was recovered in all bleached PBS samples. This result further suggested that the fluorescence recovery we observed was an intrinsic photoprocess of PBS.

    Figure 6.  FRAP analysis of isolated glutaraldehyde-treated PBSs from four cyanobacterial strains. Detection ranged from 640 to 700 nm. Fluorescence images from sequences recorded before bleaching, immediately after PBS bleaching, and at various time lapses. a, Pseudanabaena sp. FACHB-1277 with excitation at 555 nm; b, Pseudanabaena sp. 0831 with excitation at 639 nm; c, Synechococcus sp. WH7805 with excitation at 555 nm; d, Synechococcus sp. PCC 7002 with excitation at 639 nm

Discussion
  • PBSs are the main light-harvesting complexes in cyanobacteria and are anchored to the surface of cyanobacterial thylakoid membranes (Adir et al. 2020; Grossman et al. 1993). Mullineaux et al. (1997) discovered the fluorescence recovery of cyanobacterial PBS after photobleaching, and interpreted the FRAP phenomenon as rapid cell-wide PBS diffusion. Yang et al. (2007) further verified PBS motility in cyanobacteria with FRAP and fluorescence loss in photobleaching (FLIP) techniques. It was suggested that PBS diffusion in cyanobacteria allowed energy redistribution between PSII and PSI (Joshua and Mullineaux 2004; Mullineaux et al. 1997).

    In red algae, another PBS-containing algal group, the intrinsic photoprocess was noticed to contribute greatly to the PBS FRAP in the unicellular form Porphyridium cruentum (Liu et al. 2009). Subsequently, the intrinsic photoprocess and PBS diffusion were both reported to exist in P. cruentum by FRAP analysis (Kaňa et al. 2014). These results showed that PBS diffusion is not the only reason for FRAP in red algae and that multiple processes may contribute simultaneously to PBS FRAP.

    The discovery of the intrinsic photoprocess of PBS in red algae prompted us to investigate the nature of cyanobacterial FRAP and in particular the role of PBS. Four cyanobacterial strains with different phenotypes and phycobiliprotein compositions were used to analyze the FRAP phenomenon. Whole-cell photobleaching could exclude the effect of PBS diffusion, and the fluorescence recovery could only be attributed to the intrinsic photoprocess. The spontaneous fluorescence recovery indicated that there is an intrinsic photoprocess of fluorescence recovery in the PBS. Moreover, investigations on isolated thylakoid membranes and PBSs further confirmed that the intrinsic photoprocess contributed greatly to the FRAP process in cyanobacteria.

    It is reasonable that the intrinsic photoprocess contributes greatly to fluorescence recovery in PBS after photobleaching, because it is supposed that all fluorescent molecules can undergo reversible photobleaching (Verkman 2002). In photosynthetic pigments, reversible photobleaching has been known for more than half a century (Goedheer 1960; Livingston and Stockman 1962; Ohki and Fujita 1979). Siebzehnrubl et al. (1989) discovered reversible photobleaching in isolated phycobiliprotein PEC and its subunit and it was suggested that this phenomenon might be related to isomerization of the chromophore (Zhao and Scheer 1995; Zhao et al. 1995). Isomerization of chromophore-induced reversible photobleaching has also been discovered in other fluorescent proteins (Henderson et al. 2007). It was suggested that fluorescence recovery is a common mechanism to all photoactivatable and reversibly photoswitchable fluorescence proteins (Henderson et al. 2007).

    The present and previous studies suggest that the intrinsic photoprocess of PBS contributes to fluorescence recovery, not only in red algae (Liu et al. 2009) but also in cyanobacteria. High-resolution images of isolated red algal thylakoid membranes show that the PBSs are densely arranged (Liu et al. 2008; Zhao et al. 2016). Although the supramolecular architecture of PBSs on cyanobacterial thylakoid membranes is lacking, observations of ultrathin sections have revealed that PBSs on cyanobacterial thylakoid membranes are densely arranged (Samsonoff and MacColl 2001). Previous FRAP analysis proposed that the diffusion coefficient for cyanobacterial PBSs on thylakoid membranes is considerably higher than that for light-harvesting complexes (Mullineaux et al. 1997). However, the dense arrangement of cyanobacterial PBSs on thylakoid membranes does not support the presence of rapid PBS diffusion. Therefore, the contribution of intrinsic fluorescence recovery of PBS must be considered when analyzing the FRAP results in cyanobacteria. Although the findings of the present study do not exclude the contribution of PBS diffusion to FRAP in cyanobacteria, there is evidence of an important role for the intrinsic photoprocess, and it is likely that both mechanisms contribute to fluorescence recovery after photobleaching in cyanobacteria. Intrinsic fluorescence recovery should therefore be taken into consideration in FRAP analysis of PBS-containing organisms in future investigations.

Materials and methods

    Cell growth

  • The Pseudanabaena sp. 0831 strain was a gift from Dr. Bo Chen at the Polar Research Institute of China, having been isolated from Arctic sea ice and grown at 10 ℃ on a 12/12-h light/dark cycle. Pseudanabaena sp. strain FACHB-1277 was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology (FACHB, China) and grown at 25 ℃ with continuous illumination. Both Pseudanabaena strains were grown in BG-11 medium (Allen 1968). The unicellular marine cyanobacterial strains Synechococcus sp. PCC 7002 and Synechococcus sp. WH7805 were obtained from the National Center for Marine Algae and Microbiota (NCMA, East Boothbay, Maine, USA) and grown in L1-Si medium on a 13/11-h light/dark cycle at 26 ℃. Synechococcus sp. PCC 7002 and Synechococcus sp. WH7805 were cultured in the presence of thiobendazole for FRAP experiments, following previous reported methods of FRAP analysis on cyanobacteria (Sarcina et al. 2001). This treatment could lengthen cyanobacterial cells without alteration in their photosynthetic function (Sarcina and Mullineaux 2000). All the cyanobacterial strains were illuminated under daylight fluorescent lamps at intensities of approximately 40-60 μE m-2 s-1.

  • Spectra measurement

  • The absorption spectra were measured with a UV-Vis V550 spectrometer (Jasco, Japan) at room temperature. Room-temperature fluorescence was recorded by a FP-6500 fluorescence spectrofluorometer (Jasco, Japan) with related excitation and emission wavelengths.

  • Preparation of intact PBSs and PBS-thylakoid membranes

  • The cells of each cyanobacterial strain were harvested for the experiments at mid-logarithmic growth phase. The intact PBSs were prepared as previously described (Glazer 1988). Isolated PBSs were dialyzed against 0.5 mol/L phosphate buffer (pH 7.0) before glutaraldehyde pretreatment. PBS-thylakoid membranes were isolated as described by Mustardy et al. (1992) with slight modifications. The harvested cells were rinsed twice in 0.5 mol/L phosphate buffer (pH 7.0) and suspended in SPC medium (0.5 mol/L sucrose, 0.5 mol/L potassium phosphate, 0.3 mol/L potassium citrate, pH 7.0). The cells were broken in a Z plus 0.75 bench top and cabinet cell disrupter (Constant System Ltd, UK) at 8, 000 psi and placed on a two-step sucrose gradient (1.0 and 1.3 mol/L) with the same SPC medium. The samples were centrifuged at 400, 000 g for 40 min, and PBS-thylakoid membranes were collected from the 1.0-1.3 mol/L sucrose interface (Arteni et al. 2008; Mustardy et al. 1992).

  • FRAP sample preparation

  • Growing cells were harvested by centrifugation, rinsed three times in sterile water, and suspended in the corresponding fresh medium (BG-11/L1-Si). The PBS-thylakoid membranes were prepared as described above and dialyzed against 0.5 mol/L phosphate buffer (pH 7.0) for the FRAP experiment. FRAP experiment samples were immobilized on a slide covered with a glass slip and observed under a Carl Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss, Germany) (Liu et al. 2009).

  • FRAP measurements

  • He-Ne lasers (555 nm, 10 mW and 639 nm, 5 mW) were used for the excitation of PE and PC at the distal end of the PBSs in PE-type and PC-type cyanobacterial cells, respectively. Their emissions were detected from 640 to 700 nm. The pinhole was adjusted to result in a 1.3-μm optical slice. After acquiring several images of pre-bleach, the filament/cell regions of interest were bleached for 1.5-2 s. The bleaching power was set to no more than 90% of the maximum. To minimize the fluorescence loss caused by successive scanning, pre- and post-scan lasers were set at 2% laser power of 639 nm He-Ne (5 mW) and 1% power of 555 nm He-Ne (10 mW), respectively. A 63-fold water-immersion objective was used for unicellular cell FRAP observations.

  • FRAP data analysis

  • The fluorescence images were aligned and the pre-bleach images were recorded as the controls. The two-dimensional bleaching profile was analyzed using an FRAP view option control block (Zeiss Corporation, Germany), which included FRAP data correction and fitted the data to an exponential equation. The fluorescence recovery curve was fitted by a single exponential function, i.e., F(t)=A-Be(-t/C), F(t)=A-Be(-t/C), where F(t) is the fluorescence intensity at time t.

Acknowledgements
  • We thank Haiyan Yu and Xiaomin Zhao from Core Facilities for Life and Environmental Sciences of Shandong University for technical help. This work was supported by the National Natural Science Foundation of China (no. 31900023), National Key R&D Program of China (no. 2018YFC1406701), Program of Shandong Taishan Scholars (no. tspd20181203), Natural Science Foundation of Shandong (no. ZR2017LD013), AoShan Talents Cultivation Program (no. 2017ASTCP-OS14), State Key Laboratory of Microbial Technology Open Projects Fund (no. M2019-07), and Young Scholars Program of Shandong University (no. 2017WLJH22).

Authors' contributions
  • YZZ and HNS conceived this work; NZ, HNS, and KL performed experiments; HNS, BBX, XLC, and BCZ analyzed data; NZ and HNS prepared the figures and wrote the manuscript. All authors approved the final manuscript.

Compliance with ethical standards

    Conflict of interest

  • The authors declare that they have no conflict of interest.

  • Animal and human rights statement

  • This article does not contain any studies with human participants or animals performed by any of the authors.

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