Role of the Longest Arm of Phycobilisome Core-Membrane Linker in Assembling the Phycobilisomes From Synechocystis sp. PCC 6803

Abstract

Background:

The phycobilisomes (PBS) of cyanobacteria and red algae are unique light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. These extramembranous mega-Dalton complexes are specifically organized and anchored to photosystem II (PSII) via the multi-domain core-membrane linker (L_CM). While Arm2 is the longest segment in L_CM domain, its specific functions remain uncharacterized.

Methods:

A series of Synechocystis sp. PCC 6803 mutants with complete or partial deletions of Arm2 and its adjacent Rep domains within L_CM were constructed. The assembled PBSs were isolatedand characterized using sucrose gradient ultracentrifugation, absorption and fluorescence spectroscopy, and SDS-PAGE. Physiological functions were further assessed by analyzing growth, photosynthetic performance, state transitions, and non-photochemical quenching (NPQ).

Results:

Our results reveal that the super-secondary element of helix-turn-helix of Arm2 is critical for assembling the two longitudinal halves of PBS. The truncation of either or both helices of Arm2 results in the specific degradation of the longitudinal half harboring the terminal emitter, ApcD. Consequently, these mutants were deficient in state transitions and exhibited accelerated recovery from orange carotenoid protein (OCP)-mediated NPQ. We also identified the Arm2(37–67) motif likely involved in attaching the rods to the core, whereas the Arm2(68–129) region had no significant impact on PBS assembly.

Conclusions:

The helix-loop-helix element of Arm2 is essential for the longitudinal integrity of the PBS core and is a prerequisite for state transitions. These results suggest that state transitions may involve longitudinal rearrangements within the PBS structure, rather than lateral movements of the two halves, implicating that state transitions result from the longitudinal instead of the lateral moves of the two halves of the PBSs.

Graphical abstract

Keywords

Synechocystis / allophycocyanin / phycocyanin / linker / energy transfer / photoprotection

1. Introduction

Phycobilisomes (PBSs) are mega-Dalton light-harvesting complexes in cyanobacteria and red algae, composed of dozens of phycobiliproteins that organize hundreds of bilin chromophores [1]. These complexes absorb light across a broad spectrum (480–660 nm) and efficiently channel the energy to the photosynthetic reaction centers. This transfer occurs either directly or assisted by the chlorophylls (Chls) in the photosystems’ core complexes [2, 3]. Final energy delivery is facilitated by two terminal emitters containing phycocyanobilin (PCB): allophycocyanin (APC) B (AP-B) and the core-membrane linker (L_CM) [4, 5].

L_CM is encoded by apcE gene and comprises an N-terminal PCB-binding domain (ApcE\mathrm{\Delta }) [4] and several C-terminal repeat (Rep) domains connected by three arm-loop domains [6]. In the Synechocystis sp. PCC6803 (hereby referred to as Synechocystis), the L_CM starts from the N-terminal ApcE\mathrm{\Delta }, followed by three successive Rep domains [Rep1(230–435 amino acid (aa)), Rep2(436–664 aa), Rep3(685–885 aa)] [7]. The precise role of L_CM assembles the PBS remains to be fully elucidated. According to the structure studies of L_CM in the red alga Griffithsia pacifica [8], Rep1 and Rep2 are located at the center of the two hexamer of Cylinder 1 (a basal cylinders of the PBS cores), connecting the two APC trimers of a hexamer, respectively, while Rep3 is localized in the central cavity of the hexamer of the top Cylinder 3 [8, 9, 10]. Although the disparities observed in the L_CM topology between actual red algae and modeled cyanobacteria could mirror genuine structural disparities, the high sequence homology shared by the L_CMs of both red algae and cyanobacteria hints at a comparable topological configuration within these two types of PBS [11]. This homology enabled Liu et al. [12] to contruct a validated structural model of the PBS in Synechocystis sp. PCC6803. This model was subsequently validated through rigorous chemical cross-linking analysis [12]. Electron microscopy [7, 8] revealed that the cylinders are organized as a triangular prism with Cylinder 1 and 2 forming a base that lies on the top of the membrane and Cylinder 3 on the top of the base.

The connecting loops of Synechocystis’ L_CM include Arm1(241–249) that connects Rep1(250–400) to the PCB-binding domain (ApcE\mathrm{\Delta }), Arm3(686–714) that connects Rep2(536–685) and Rep3(715–860) [13], and Arm2 (401–534) that connects Rep1 and Rep2. The Rep domains, which are ~120 amino acids long and share high similarity with pfam_00427 (the conserved domain of the rod linker in PBS), play a vital role in fastening the APC discs [8]. Compared to the short Arm1 (9 aa) and Arm3 (29 aa), Arm2 is 134 aa in Synechocystis and effectuates like a functional domain. In Griffithsia pacifica, Arm2 consists of a super-secondary structure of helix-loop-helix followed by an unstructured extension [8], which is to the sequence in Synechocystis (Supplementary Fig. 1). Despite its prominence, the potential roles of Arm2 in PBSs are unknown.

In this study, we investigated the role of Arm2 in PBS assembly. We constructed a series of Synechocystis mutants with complete or partial truncation of Arm2 and/or deletions of its adjacent Rep2/Rep3 domains. Analysis of isolated PBSs, we found that the truncation of either one or both helices of Arm2 in L_CM split the PBSs longitudinally into a simple PBS consisting of the APC core and cyanobacterial phycocyanin (CPC) rod. Mutants with the damaged helix-loop-helix element of Arm2 were deficient in state transitions and exhibited accelerated recovery from non-photochemical quenching (NPQ) compared to wild-type (WT) and Rep3-truncated Synechocystis (\mathrm{\Delta }(rep3)).

2. Materials and Methods

2.1 Strains and Culture

Synechocystis and its mutants were grown in BG-11 medium on a shaker (130 rpm) at 30 °C under continuous white light (20 µmol photons m^-2s^-1 for LL) [14]. BG-11 medium with 1.2% (w/v) agar containing 0.3% (w/v) sodium thiosulfate was solidified for plate cultures. The photomixotrophic growth was achieved by diluting an aliquot from a photoautotrophic culture in the log phase (OD₇₃₀ ~1.0) to an OD₇₃₀ ~0.05 with BG-11 containing 10 mM glucose. The growth and cell densities were monitored at OD₇₃₀ on a Beckman DU800 spectrophotometer. The growth was evaluated based on the average of three parallel experiments. Chlorophyll a (Chl a) [15] or phycocyanin (PC) [16] concentrations were determined by absorption spectroscopy and calculated per cell [17].

2.2 Cloning and Mutant Construction

All genetic manipulations were carried out according to standard protocols [18]. The apcE gene locus (slr0335) in Synechocystis encodes the L_CM protein, which consists the following domains: the N-terminal loop(80–150), Arm1(241–249), Rep1(250–400), Arm2(401–534), Rep2(535–685), Arm3(686–714), and Rep3(715–860). To delete these domains and/or motifs, mutation plasmids were constructed as follows.

2.2.1 Plasmid for Construction of \mathrm{\Delta }(rep3)

A 0.8-kbp DNA fragment including arm2-rep2 and a 0.6-kbp DNA fragment including arm3-rep3 of apcE was amplified from Synechocystis genomic DNA by PCR using primers P1–P4 (Supplementary Table 1), as upstream and downstream targeting arms, respectively. The restriction sites XhoI plus EcoRV and EcoRV plus XbaI were introduced in the targeting arms, respectively, for cloning the fragment into pBluescript. A DNA fragment containing streptomycin resistance cassette was excised from plasmid pHB45 via the restriction site SmaI and then inserted in the plasmid containing the targeting arms via the restriction site EcoRV, thereby yielding pBlue-\mathrm{\Delta }(rep3)-str (Fig. 1C, Ref. [19]).

2.2.2 Plasmid for Construction of \mathrm{\Delta }(arm2/rep3), \mathrm{\Delta }(rep2/rep3) and \mathrm{\Delta }(arm2/rep2/rep3)

A 1.7-kbp DNA fragment including apcE\mathrm{\Delta }-arm1-rep1-\mathrm{\Delta }arm2-rep2 and a 0.6-kbp DNA fragment including arm3-rep3 was amplified from Synechocystis genomic DNA by PCR using primers P5-P10 (Supplementary Table 1) as upstream and downstream targeting arms, respectively. In the targeting arms, restriction sites of XhoI plus BamHI and BamHI plus XbaI were introduced to facilitate cloning of the targeting arms into pBluescript. A DNA fragment containing streptomycin resistance cassette was excised from plasmid pHB45(omega) via the restriction site BamHI and then inserted in the plasmid containing the targeting arms via the restriction site BamHI, yielding pBlue-\mathrm{\Delta }(arm2/rep3)-str (Fig. 1D). Similar strategies were employed for \mathrm{\Delta }(rep2/rep3) and\mathrm{\Delta }(arm2/rep2/rep3) using the primers listed in Supplementary Table 1.

2.2.3 Mutants Plasmids for Construction of Partially Arm2-Truncated Mutants Base on \mathrm{\Delta }(rep3)

A 5.3-kbp DNA fragment, including pBlue-apcE\mathrm{\Delta }(arm2(6–36)/rep3), pBlue-apcE\mathrm{\Delta }(arm2(37–67)/rep3), pBlue-apcE\mathrm{\Delta }(arm2(68–98)/rep3), pBlue-apcE\mathrm{\Delta }(arm2(99–129)/rep3), pBlue-apcE\mathrm{\Delta }(arm2(6–17)/rep3), pBlue-apcE\mathrm{\Delta }(arm2(18–28)/rep3), and pBlue-apcE\mathrm{\Delta }(arm2(29–36)/rep3), was amplified from the plasmid of pBlue-\mathrm{\Delta }(rep3) by PCR using P17–P30 primers (Supplementary Table 1). Then, these plasmids were obtained by phosphorylation reaction and standard protocols, respectively. A DNA fragment containing streptomycin resistance cassette [4] was cut from plasmid pHB45(omega) via the restriction site BamHI and then inserted in the plasmid containing the targeting arms via the restriction site BamHI, yielding pBlue-\mathrm{\Delta }(arm2(6–36)/rep3)-str, pBlue-\mathrm{\Delta }(arm2(37–67)/rep3)-str, pBlue-\mathrm{\Delta }(arm2(68–98)/rep3)-str, pBlue-\mathrm{\Delta }(arm2(99–129)/rep3)-str, pBlue-\mathrm{\Delta }(arm2(6–17)/rep3)-str, pBlue-\mathrm{\Delta }(arm2(18–28)/rep3)-str, and pBlue-\mathrm{\Delta }(arm2(29–36)/rep3)-str.

2.3 Mutation of Synechocystis and Genetic Analyses

The mutant strains were generated by transforming WT Synechocytis cells with the respective plasmids via homologous recombination [20]. Transformants were selected on BG-11 plates containing 12.5 µg/mL spectinomycin, with segregation ensured throughsuccessive rounds of selection at 25 µg/mL spectinomycin. The genotypes of all final mutant strains were confirmed by PCR using respective primer sets (Supplementary Fig. 2, Supplementary Table 1).

2.4 Absorption Spectra and Sample Concentrations

The room temperature absorption spectra of the cell samples and the isolated PBS fractions were recorded on a UV-9000S spectrophotometer with a slit width of 1 nm in a 2-mm cuvette with high scattering property. At the corresponding peak wavelengths, molar extinction coefficients of 1185 mM^-1cm^-1 and 770 mM^-1cm^-1 were used for a trimer of PC (620 nm) and APC (650 nm), respectively. According to the absorption spectra of CPC [21], the molar extinction coefficient of 266 mM^-1cm^-1 was used for a trimer of PC (650 nm), while the molar extinction coefficient of 295 mM^-1cm^-1 was used for a trimer of APC in 620 nm according to the absorption spectra of APC [22]. The concentrations of CPC and APC were calculated according to established methods [16, 23]. The equations used were:

{\mathrm{A}}_{620}={\epsilon }_{\mathrm{CPC}}^{620}{\mathrm{C}}_{\mathrm{cpc}}+{\epsilon }_{\mathrm{APC}}^{620}{\mathrm{C}}_{\mathrm{APC}}

{\mathrm{A}}_{650}={\epsilon }_{\mathrm{CPC}}^{650}{\mathrm{C}}_{\mathrm{CPC}}+{\epsilon }_{\mathrm{APC}}^{650}{\mathrm{C}}_{\mathrm{APC}}

3. Fluorescence Measurements

3.1 Fluorescence Emission Spectra

Spectra were acquired using an F-320 Fluorolog spectrofluorimeter, with slit widths set at either 5 or 10 nm. Synechocystis WT and mutant strains were cultivated under normal light until reaching an optical density of A730~0.8. For measurement, the chlorophyll a (Chl a) concentration of the cell suspensions was adjusted to either 3 or 5 µg/mL, corresponding to the excitation wavelengths of 580 nm or 430 nm, respectively. Subsequently, 800 µL aliquots of the adjusted cells were dark-adapted for 5 minutes in fresh BG11 medium prior to spectral recording. All experiments were performed with at least three independent biological replicates.

The 77-K fluorescence emission spectra were monitored using a Horiba Fluorolog spectrofluorimeter with a slit width of 8 nm. The cells at CChl = 3 µg/mL (580 nm excitation) were collected by centrifugation and suspended in an equivalent volume of fresh BG11 containing 40% (v/v) glycerol and 25 mM HEPES-NaOH (pH 7.5). In all cases, whole cells were dark-adapted for 15 min before the measurements. Then, the spectra were recorded corresponding to State II. For State I spectra, cells were illuminated with 55 µmol photons m^-2s^-1 of blue light for 5 min. Then, 800 µL suspensions were quickly frozen in quartz tubes by immersion in liquid nitrogen for 10 s. The excitation was measured at 580 nm, and emission was scanned at 600–800 nm [24, 25]. Date are from at least three biological replicates.

3.2 A Pulse Amplitude-Modulated Fluorometer (PAM) Fluorometer

The light response curves, NPQ, and state transitions were monitored on a PAM fluorometer (PAM 2500; Walz, Effelrich, Germany) [26, 27]. Mutant and WT cells were grown under similar conditions (A730 = 0.6) and estimated on dark-adapted (15 min) whole cells at a chlorophyll concentration of 3 mg/L. F0 is a minimal fluorescence level determined by illuminating dark-adapted cells with a low intensity of red-modulated light (pulses of 1 s, 1.6 kHz, 0.024 µmol photons m²s^-1). For the measurements of state transitions [28], cells were dark-adapted, irradiated with blue (55 µmol photons m²s^-1) or orange light (20 µmol photons m²s^-1), and subjected to saturating pulses (2000 µmol photons m²s^-1, 30 s) to measure the F_m^′ levels (maximum fluorescence under illumination).

All NPQ induction and recovery experiments were carried out in the presence of chloramphenicol (30 g/mL), a protein synthesis inhibitor, to inhibit protein synthesis [26]. For the measurements of the light response curve, cells (CChl = 20–30 mg/L) were irradiated with various intensities (0–1400 µmol photons m²s^-1) after dark adaptation (15 min) and then subjected to saturating pulses (2000 µmol photons m²s^-1, 30 s) to measure the F_m^′ levels. The initial slope of the rapid light response curve (alpha), the maximal rate of electron transfer (ETRmax), and the minimal saturated light intensity at semi-saturated light intensity (Ik) were calculated from the PAM data using the analysis software [29, 30]. Data are presented as mean \pm standard deviation (SD).

3.3 Protein Assay

Protein concentration was quantified using the Bradford method [31] with bovine serum albumin (BSA) as a standard. The proteins from sucrose-containing fractions were precipitated by 50% (NH4)2SO4 and collected by centrifugation to remove the sucrose. Then, the samples were diluted with distilled water and precipitated by 10% trichloroacetic acid (TCA) to remove the ammonia. The remaining TCA was removed by acetone extraction. Subsequently, the obtained samples were dried, solubilized in the sample buffer [32], and analyzed by SDS-PAGE using Laemmli buffer system [33]. Finally, the proteins were stained with Coomassie brilliant blue [34].

3.4 Isolation of PBSs

PBSs were isolated from Synechocystis cells by an ultracentrifugation-based protocol [35]. Briefly, cells were lysed with 2% (v/v) Triton X-100, and the lysate was clarified by centrifugation to remove unbroken cells, debris, and chlorophyll. The resulting supernatant (2 mL) was then layered onto sucrose step-gradients for ultracentrifugation. The gradients, prepared in 12-mL tubes with 0.8 M KPB (pH 7.2), consisted of 1 mL of 2.0 M, 2.5 mL of 1.0 M, 2 mL of 0.75 M, 2 mL of 0.5 M, and 1.5 mL of 0.25 M sucrose solutions. Centrifugation was performed at 230,000 \timesg for 13 h at 18 ℃ using a P40ST rotor (Hitachi CP80-WX ultracentrifuge). The distinct blue bands containing PBSs were collected and stored at 4 ℃ in sucrose solution for analysis within 48 hours. PBS isolations were performed from at least three independent cultures per mutant.

3.5 The Electron Microscopy (TEM)

Electron microscopy was performed on a HITACHI H-7650 transmission electron microscope operated at 100 kV. The images were recorded with a 1024 \times 1024 CCD camera at a magnification of 100,000\times. PBS preparations were negatively stained with 2% uranyl acetate by droplet method [7]. Drops of about 5 µL, containing the samples at OD at 620 nm [\mathrm{\Delta }(arm2(6-36)/rep3) was 0.02 OD, \mathrm{\Delta }(rep3) was 0.05 OD] in 0.8 M potassium phosphate (pH 7.2) were deposited on the surface of glow-discharge and carbon-coated copper grids. After 1–3 min, the droplets were stained with 2% uranyl acetate.

4. Results

4.1 The Helix-Loop-Helix Element of Arm2 in Synechocystis Mediates the Longitudinal Assembly of PBSs

Arm2 in apo-protein of L_CM (ApcE) from Synechocystis constitutes 401–534 aa. Thus, we re-numbered Arm2 as 1–134 aa (Fig. 1A). Strikingly, the amino acid sequence of Arm2 of the red alga Griffithsia pacifica was highly similar to that of Synechocystis (Supplementary Fig. 1A). Based on the 3.5 Å PBS structure from the red alga Griffithsia pacifica [8], the conservative motif of Arm2(6–36) in Griffithsia pacifica formed the super-secondary structure of \alpha-helix/loop/\alpha-helix (Supplementary Fig. 1B) and the rest of the molecule forms random coils. Arm2 in Synechocystis also forms this super-secondary structure, i.e., a loop (Arm2(18–28)) linking two \alpha-helices (Arm2(6–17) and Arm2(29–36)), followed by random coils Supplementary Fig. 1C). To investigate the functions of Arm2, we generated a series of Synechocystis mutants-\mathrm{\Delta }(rep3), \mathrm{\Delta }(arm2/rep3), \mathrm{\Delta }(rep2/rep3), and \mathrm{\Delta }(arm2/rep2/rep3)-by selectively deleting the Rep3, Arm2/Rep3, Rep2/Rep3, and Arm2/Rep2/Rep3 domains, respectively, from the ApcE protein (Fig. 1). In order to locate the functional motifs in Arm2, a set of mutants with partial truncations within Arm2 were constructed by truncating an amino acid fragment of Arm2 based on \mathrm{\Delta }(rep3) mutant (Fig. 1). Complete segregation of all mutants was confirmed by PCR analysis (Supplementary Fig. 2). The traces of the 1.5- to 2.5-kbp fragments were detected in WT but not the mutants, indicating complete segregation.

The assembly of PBSs in the absences of Rep3 remains a subject of debate. The classic model of PBSs of Synechocystis suggests that Pep3 is essential for Cylinder 3 attachment, with its lost resulting in a simple, two-cylinder core (Cylinder1 and 2) [7]. In contrast, the structural study on PBS on the red alga Griffithsia pacifica [8], Cylinder3 is lost in the absence of Rep3, and then Cylinder1 and 2 are disconnected. Given the symmetry of PBSs, Cylinder1 and 2 are same, and we would only isolate a simple PBS of Cylinder1 or 2 in the core. Next, we isolated the PBSs from \mathrm{\Delta }(rep3): the ultracentrifugation of PBSs retrieved three fractions in a molar ratio of 1.47:1:7.17 (Fig. 2A and Supplementary Table 2). The major fraction showed maximal absorption at 620 nm and maximal fluorescence at 668 nm at room temperature and at 684 nm at 77 K. Similarly, the two minor fractions absorbed maximally at 654 nm and exhibited fluorescence at 664–668 nm at room temperature and at 680–684 nm at 77 K. The data indicated the following: (1) while dismantled CPC was barely detected, a small part of the dismantled PBS cores could be isolated. In the isolated cores, the energy absorbed by APC could be efficiently transferred to the two terminal emitters, such that the cores were not damaged. (2) In the isolated PBSs, the energy absorbed by CPC could efficiently be transferred to the two terminal emitters, such that the PBSs remained intact. The SDS-PAGE (Supplementary Fig. 3A lane 3) showed that the major fraction PBSs of \mathrm{\Delta }(rep3) assembled intact similar to WT (Supplementary Fig. 3I lane 1), except for a truncated ApcE. The photosynthetic capacity parameters (Supplementary Fig. 4, Supplementary Tables 3,4) and cell fluorescence spectra at room temperature (Supplementary Fig. 5A,D and Supplementary Table 5) and 77 K (Supplementary Fig. 6A,D and Supplementary Table 5) of \mathrm{\Delta }(rep3) were similar to those of WT, further supporting the intactness and functionality of the PBSs. However, the absorption spectra of \mathrm{\Delta }(rep3) showed that this mutant had 1.3 times less phycobiliprotein per chlorophyll than WT (Supplementary Fig. 7A and Supplementary Table 5), indicating that the deletion of Rep3 of ApcE might affect the assembly of PBSs or phycobiliprotein biosynthesis [19]. Considering the smaller size observed by TEM (Supplementary Fig. 8) and the structural model from Liu et al. [12], we interpret these results as being most consistent with the isolation of a simple, one-cylinder PBS.

We next analyzed PBSs from the double mutant \mathrm{\Delta }(arm2(6–36)/rep3). Ultracentrifugation on a sucrose gradient (Fig. 2A) indicated that the PBSs of \mathrm{\Delta }(arm2(6–36)/rep3) showed a major fraction at 0.25 M and a minor fraction suspended at 0.5–0.75 M sucrose gradient; the two fractions were in 5.59:1 molar ratio (Supplementary Table 2). The major fraction had an absorbance of 620 nm and a fluorescence maxima at 658 nm at room temperature, while the 77 K fluorescence peak shifted to 666 nm (Fig. 2B–D). The minor fraction had the typical absorption and fluorescence maxima of CPC at 620 nm and 642 nm, respectively, and the 77 K fluorescence shifted to 654 nm (Fig. 2E–G). Importantly, the PBSs of \mathrm{\Delta }(arm2(6–17)/rep3) and \mathrm{\Delta }(arm2(29–36)/rep3) presented characteristics at the same sucrose gradient similar to those of \mathrm{\Delta }(arm2(6–36)/rep3) (Fig. 2). These results indicated that (1) a specific amount of the CPC rods was dismantled from the PBS cores in these mutants. These findings could question the integrity of PBSs in these mutants; (2) the major fraction contained a simple, partially dismantled PBS consisting of only CPC and APC in 1.66 \pm 0.29 molar ratio, but the energy absorbed by CPC in this simple PBS was transferred to APC. Hence, it could be speculated that in the simple PBSs, one APC hexamer binds three CPC trimers, or two APC hexamers (disks) bind three CPC hexamers (disks); (3) for the three mutants, the truncation of the helix-loop-helix of 6–36 aa, the helix of 6–17, or the helix of 29–36 from Arm2 presented a similar phenotype of PBS, i.e., the helix-loop-helix, or one of the helix exerted a similar role in PBS assembly, consistent with our previous report [11].

According to the PBS structure from the red alga Griffithsia pacifica [8], the Arm2 element of helix-loop-helix is inserted between one normal APC disk and two APC disks, one of which has a L_CM terminal emitter and the other has AP-B terminal emitter in one basal cylinder (i.e., Cylinder1 or 2). When one helix or both helices of Arm2 were truncated, a simple PBS containing only APC/CPC was generated such that the other longitudinal half PBS with the APC disks harboring the terminal emitters (L_CM and ApcD) was lost. The cell spectra showed a high fluorescence of these mutants at 660 nm (Supplementary Fig. 5A–C) that disappeared completely at 683 nm (Supplementary Fig. 5D–F). Upon excitation at 430 nm and at 580 nm, the corresponding fluorescence confirmed that the energy transfer from APC to the terminal emitters of PBSs was disrupted [19]. Moreover, due to the absence of terminal emitters, the energy transfer from the PBSs to PSs was disrupted (Supplementary Fig. 5). The photosynthetic capacities (Supplementary Table 3) were much lower than those of \mathrm{\Delta }(rep3) and WT. Consequently, the mutants \mathrm{\Delta }(arm2/rep3), \mathrm{\Delta }(arm2(6–36)/rep3), \mathrm{\Delta }(arm2(6–17)/rep3), and \mathrm{\Delta }(arm2(29–36)/rep3) of the damaged Arm2 element grew poorly under photoautotrophic conditions but are capable of growing photomixotrophically in the culture media supplemented with 10 mM glucose (Supplementary Fig. 4). Supplementary Table 4 shows that when the chlorophyll content is maintained, the level of F₀ in these mutants is 2- to 5-fold higher than that of \mathrm{\Delta }(rep3), which is consistent with the high fluorescence intensity of these mutants (Supplementary Fig. 5). Reportedly, a specific number of uncoupled PBSs may be one of the reasons for increased F₀ in cyanobacteria [19, 36, 37]. The results suggested Arm2 as a putative candidate for PBSs binding thylakoid membranes [13]. Also, the generation of small PBSs containing only APC/CPC effectuated a weak association between PBSs and thylakoid membranes.

Therefore, the Arm2 element is responsible for the longitudinal assembly of PBSs. The loss of the element results in the dissociation of half of the PBS harboring the terminal emitters, while the other half, i.e., the simple PBS with only APC and CPC is retained in the cells. The cell spectra showed the absence of 77 K fluorescence at 684–685 nm corresponding to the terminal emitters (Supplementary Fig. 6, Supplementary Table 5). Hence, the dissociated parts of the PBSs could be degraded via specific phycobiliprotein degradation routes [38, 39]. One terminal emitter, ApcD, could undergo de-chromophorylation for its degradation [22]. The other terminal emitter, L_CM, degraded slowly, and hence, a little L_CM (the small peak at ~670 nm in Fig. 2 and Supplementary Fig. 6) was detected in the isolated PBSs from some mutants of the damaged Arm2 element.

To further investigate the roles of the motif of Arm2(6–36), we isolated the PBSs of \mathrm{\Delta }(arm2/rep3), \mathrm{\Delta }(rep2/rep3), and \mathrm{\Delta }(arm2/rep2/rep3). Their ultracentrifugation (Supplementary Fig. 9) showed a similar fraction pattern compared to \mathrm{\Delta }(arm2(6–36)/rep3), and the spectra of the fractions were also similar to those of \mathrm{\Delta }(arm2(6–36)/rep3). The coincidence between \mathrm{\Delta }(arm2/rep3) and \mathrm{\Delta }(arm2(6–36)/rep3) confirmed Arm2(6–36) as the key element of Arm2. According to the PBS structure of the red alga Griffithsia pacifica [8] and a PBS core conformation similar to that suggested by Liu et al. [12], the truncation of Rep2 and 3 dismantles the APC disk of Cylinder3 and basal cylinders and retains the APC disk with L_CM of basal cylinders. Although the spectra showed 77 K fluorescence at ~680 nm (Supplementary Fig. 9D), which would be L_CM in a trimer [40], the fluorescence degree was too small compared to that at 666 nm of APC. Thus, the energy absorbed by APC could only slightly be transferred to L_CM, indicating that (1) the simple PBSs having only APC and CPC were predominant and/or (2) the APC disks with and without L_CM were not well connected.

4.2 Arm2(37–67) Facilitates CPC Rod Attachment at the Cores and Arm2(68–129) has no Remarkable Effects on the Assembly of PBSs

The ultracentrifugation of \mathrm{\Delta }(arm2(37–67)/rep3) generated a fraction pattern similar to that of \mathrm{\Delta }(arm2(6–36)/rep3). On the other hand, the minor fraction at 0.25 M sucrose gradient showed similar absorption and fluorescence at 680 nm fluorescence (Supplementary Fig. 10), the fraction suspended at the 0.5–0.75 M sucrose gradient showed maximal absorption at 620 nm, fluorescence at 650 nm at room temperature and at 656 nm at 77 K, which corresponded to CPC, as verified by SDS-PAGE (Supplementary Fig. 3J). The molar ratio of minor:major fraction was 1:1.57 (Supplementary Table 6), indicating that CPC rods mainly dissociated from the cores in \mathrm{\Delta }(arm2(37–67)/rep3) and that the 36–67-aa motif, although in random coils, might be responsible for the attachment of CPC rods at the cores (Supplementary Fig. 1).

Conversely, \mathrm{\Delta }(arm2(68–98)/rep3) and \mathrm{\Delta }(arm2(99–129)/rep3) showed an ultracentrifugation pattern different from the mutants of the damaged Arm2 but similar to that of \mathrm{\Delta }(rep3). Among the three fractions, the two minor fractions at 0.25 and 0.5 M sucrose gradient absorbed maximally at 654 nm and 620 nm with an obvious 654 nm peak that exhibited fluorescence at 661–672 nm at room temperature and at 683–684 nm at 77 K, respectively (Supplementary Fig. 10B–G). In addition, the major fraction at 0.75 M sucrose gradient had similar characteristic absorption and fluorescence at room temperature and 77 K fluorescence spectra as the PBSs from intact \mathrm{\Delta }(rep3) and WT (Supplementary Fig. 10H–J). Therefore, the energy absorbed by CPC could be transferred efficiently to the terminal emitters. The cell spectra also showed an efficient energy transfer in vivo from CPC to AP-B (Supplementary Fig. 6, Supplementary Table 5). Furthermore, due to the truncation, the fluorescence at 684–685 nm assigned to the terminal emitter, AP-B, decreased gradually according to the order of \mathrm{\Delta }(rep3), \mathrm{\Delta }(arm2(99–129)/rep3), and \mathrm{\Delta }(arm2(68–98)/rep3) (Supplementary Fig. 10J).

4.3 The Arms Helix-Loop-Helix Element is Essential for State Transitions and OCP-Associated NPQ

Cyanobacterial NPQ and state transitions affect the balance between energy transfer and dissipation within the PBSs and the distribution of excitation energy from the PBSs to the PSs [41]. Experimentally, states I and II are induced by blue LL and orange LL irradiation, respectively, such that the energy is preferentially transferred to photosystem I (PSI) and from PBS to PSII [42]. When state transitions occur at a low irradiation intensity, the high irradiation intensity quenches the non-photochemical PBSs, triggered by OCP [19, 43].

Pulse amplitude-modulated fluorometer (PAM) fluorescence technique is often used to study the energy transfer between PBSs and PSs. In PAM measurements, dark-adapted cells have a low dark maximal fluorescence (F_md), but when they are continuously irradiated by blue LL (55 µmol photons m²/s), the F_m^′ increases rapidly and arrives at a maximal F_m^′ fluorescence level (F_mb^′), indicating that the cells have undergone the transition to state I. Thus, the ratio of F_vb/F_vd (F_V = F_m – F₀) indicated the ability of mutants to transform state II\tostate I. When the Blue LL light-adapted cells are irradiated with orange light (20 µmol photons m²s^-1), the decrease in F_m^′ indicates the ability of mutants to transform from state I\tostate II [24, 44, 45]. Herein, we compared the state transition performance of WT and mutants: the ratio of F_vb/F_vd was consistent (approximately 15), and the F_m^′ decreased rapidly in WT and \mathrm{\Delta }(rep3) mutant (Supplementary Fig. 11). However, the ratio of F_vb/F_vd in \mathrm{\Delta }(arm2/rep3) and \mathrm{\Delta }(arm2(6–36)/rep3) mutants was negative (Fig. 3C,D), and that in the mutants without the Arm2(6–17) or Arm2(29–36) motif was lower than that in \mathrm{\Delta }(rep3) and WT (Fig. 3E,F), implying a strongly-weakened ability of these mutants to perform state II\tostate I. This phenomenon was supported by the results that the 77 K fluorescence emission spectra of blue-adapted WT and \mathrm{\Delta }(rep3) cells had a high PSII fluorescence band with respect to dark-adapted cells, while the PSII fluorescence peak of \mathrm{\Delta }(arm2/rep3) and \mathrm{\Delta }(arm2(6–36)/rep3) mutants or \mathrm{\Delta }(arm2(6–17)/rep3) and \mathrm{\Delta }(arm2(29–36)/rep3) mutants showed only a slight increase (Fig. 4) [19, 36, 46]. On the contrary, the ratio of F_vb/F_vd in the other mutants of the damaged Arm2 was higher than that in \mathrm{\Delta }(rep3) (Supplementary Fig. 11D–F). Interestingly, under orange LL, the F_m^′ of all mutants of the damaged Arm2 did not decrease, indicating that the transition from transition I to state II cannot occur (Fig. 3 and Supplementary Fig. 11). Based on these results, we concluded that (1) damaging the helix-loop-helix element of Arm2 produced simple APC/CPC PBSs, effectuating a weak association between PBSs and the thylakoid membrane. This prevents the state I transition from state II and the energy transfer between the two PSs; (2) damaged Arm2 disrupted the connection between APC disk and APC disk with terminal emitters, such that the energy transfer from the PBSs to PSs was perturbed and the transition from state I to state II was impaired. The terminal emitters are responsible for transferring the absorbed energy to the reaction centres and plays a crucial part in energy migration and affects state transitions [47].

Next, the NPQ performance of WT and mutants was compared (Fig. 5). The results showed strong blue light-induced PBS fluorescence quenching (about 14%) in WT, while that in \mathrm{\Delta }(arm2/rep3) or \mathrm{\Delta }(rep3) mutants was low (about 0.69% or 4.96%) (Fig. 5G). Interestingly, the mutants of the damaged helix-loop-helix element of Arm2 showed an NPQ performance similar to the WT (Fig. 5A–F), while the remaining mutants showed a phenotype similar to \mathrm{\Delta }(rep3) mutants (Supplementary Fig. 12C–E). When dark-adapted cells were exposed to white high light (HL) directly, Fluorescence quenching was reversible in all strains in the darkness (Fig. 5H and Supplementary Fig. 12F). Importantly, WT and \mathrm{\Delta }(arm2/rep3) recovered from NPQ a similar rate but 3-fold faster than \mathrm{\Delta }(rep3), while \mathrm{\Delta }(arm2(6–36)/rep3), \mathrm{\Delta }(arm2(6–17)/rep3), and \mathrm{\Delta }(arm2(29–36)/rep3) recovered faster than \mathrm{\Delta }(rep3). These results support the following findings: (1) the damaged helix-loop-helix element of Arm2 produced small APC/CPC PBSs, which had the more packed conformation increased the probability of energy transfer between different rods [47] and might allow OCP to approach the PBSs closely in some directions, such that OCP-dependent NPQ is significant; (2) the integrity of PBSs is crucial for strong binding of activated OCP. The activated OCP may be loosely bound by the small PBSs and easily released from them compared to WT PBSs.

5. Discussion

Our study establishes the critical role of the Arm2 helix-loop-helix element in the longitudinal assembly and functional integrity of the PBS in Synechocystis sp. PCC 6803. The structural and functional defects observed in our Arm2 truncation mutants provide new insights into PBS architecture, state transitions, and photoprotection.

Structural Role of Arm2 and PBS Assembly. Our data demonstrate that the helix-loop-helix element of Arm2 (residues 6–36) is essential for connecting the two longitudinal halves of the PBS core. Truncation of this element leads to the specific loss of the half-core containing the terminal emitters L_CM and ApcD, resulting in simplified APC/CPC complexes (Fig. 2, Supplementary Figs. 5,6). This phenotype aligns with the proposed location of Arm2 in the Griffithsia pacifica PBS structure [8], where it bridges APC hexamers within a basal cylinder. Our previous study, informed by homology modeling [12] and based on low-resolution molecular structure of cyanobacterial PBS [48], positions the Synechocystis Arm2 helix-loop-helix similarly, providing a structural basis for its essential role in core integrity. The CPC/APC molar ratio of ~1.66 in these simplified PBSs (Supplementary Table 2) is consistent with a structure where one APC hexamer associates with three CPC hexamers, a configuration potentially favored in the absence of the full core constraint. The subsequent degradation of the dissociated emitter-half suggests active quality control mechanisms for unassembled PBS components [38, 39]. Beyond the core, the Arm2(37–67) region, though unstructured, appears to facilitate the attachment of CPC rods to the core (Supplementary Fig. 10), highlighting the multi-functional nature of this linker domain. In contrast, the distal regions of Arm2(68–129) have minimal impact on assembly under our conditions.

Implications for State Transitions. A key finding is the abolition of state transitions in mutants lacking the Arm2 helix-loop-helix element (Figs. 3,4). The current understanding of state transitions involves redistribution of excitation energy between PSII and PSI, potentially through PBS movement [42, 49]. While some models propose lateral PBS displacement or detachment from the membrane [49, 50], our results suggest that longitudinal rearrangements within the PBS core itself, facilitated by Arm2, are crucial. The loss of state transitions in our mutants likely stems from a dual defect: (1) the physical absence of one terminal emitter-bearing half-core, which disrupts the energy delivery network to both photosystems, and (2) the weakened association of the simplified PBSs with the thylakoid membrane, potentially impeding the physical mobility or conformational changes required for state transitions. Our data do not rule out detachment/reattachment mechanisms [49, 50] but emphasize that the integrity of the core structure, maintained by Arm2, is a prerequisite for these processes.

OCP-mediated NPQ and PBS Integrity. The mutants with damaged Arm2 exhibited faster recovery from OCP-related NPQ (Fig. 5H), despite retaining significant quenching capacity. This accelerated recovery is consistent with a model where the simplified PBSs, due to their altered conformation and/or reduced size, bind the activated OCP with lower affinity, allowing for its more rapid release upon signal cessation. The core region, particularly the APC cylinders, is a proposed binding site for OCP [51, 52, 53]. Structural alterations in this region, as caused by Arm2 truncation, could directly affect OCP docking stability. While this interpretation is plausible, direct evidence from OCP binding affinity assays with purified WT and mutant PBSs would be needed to conclusively support this hypothesis, representing an important direction for future research.

In summary, the Arm2 linker in L_CM acts as a critical structural organizer for the PBS core. Its helix-loop-helix element is indispensable for longitudinal assembly, state transitions, and fine-tuning OCP-related photoprotection, underscoring the importance of linker domains in the structure and function of massive photosynthetic antenna complexes.

6. Conclusion

This study demonstrates that the helix-loop-helix element of Arm2 is critical for the longitudinal assembly of PBS. Deletion of the Rep3 domain resulted in an intact PBS core comprising only Cylinder1 or 2. Additionally, when one or both helices of Arm2 are truncated, simple PBS containing only APC/CPC could be generated, while the other longitudinal half of PBSs containing the terminal emitters (L_CM and ApcD) may be degraded and lost. Consequently, in the isolated \mathrm{\Delta }(rep3) PBSs, the energy absorbed by CPC can be transferred efficiently to the two terminal emitters, rendering the PBSs fully functional. In the mutants of the damaged helix-loop-helix element of Arm2, the energy transfer from APCs to terminal emitters to PSI and II decreases, and photosynthesis is impaired due to the generated simple PBSs APC/CPC, although the energy transfer from CPC to APC is efficient. The simple APC/CPC PBSs ascribed a weak connection between PBSs and thylakoid membrane, which impairs the energy transfer between two PSs, preventing state I transition. Conversely, when Arm2 is damaged, the APC disk and the APC disk harboring the terminal emitters are not well connected, due to which the energy transfer from the PBSs to PSs is perturbed, impairing the transition to state II. Subsequently, the activated OCP binds weakly to PBSs due to the simple APC/CPC PBSs, and post-relaxation, OCP is easily released from simple PBSs. Therefore, the mutants of the damaged helix-loop-helix of Arm2 can still perform OCP-induced-NPQ but recover quickly. In addition to the helix-loop-helix of Arm2, the motif of Arm2(37–67) mediates the association of CPC rods to the PBS core, while the other motifs, Arm2(68–98) and Arm2(99–129) have no remarkable effect on PBS assembly.

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Funding

Key Research Projects of Higher Education Institutions in Henan Province(26B230010)

Research Project of Luoyang Polytechnic(2024046)