Uphill energy transfer mechanism for photosynthesis in an Antarctic alga

Abstract

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Å resolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Fig. 1. Purification of Pc-frLHC.

a Purification scheme of Pc-frLHC and other photosynthetic proteins from P. crispa’s thylakoid membranes. b Absorbance spectra (solid lines) and fluorescence spectrum (dotted line) of the thylakoids (red) and Pc-frLHC (black) measured at room temperature. c hrCN-PAGE (left) and SDS-PAGE (right) analyses of thylakoids (lane 1), PSII-LHCII (lane 2), LHCII (lane 3), PSI-LHCI (lane 4), and Pc-frLHC (lane 5). Lane numbers at the top of the gel images correspond to the italicized numbers in parentheses in (a). Bands corresponding to Pc-frLHC and the subunit of Pc-frLHC are indicated by the star and the bullet, respectively. These electrophoresis data were representative of two independent experiments. d Fitting analysis of the absorbance spectrum of purified Pc-frLHC at room temperature. The peak wavelength of each component was estimated by the second and fourth derivatives of the absorbance spectrum, and fitting analysis with Gaussian functions was performed by Magic plot 2.7.2 (Magicplot Systems, St. Petersburg, Russia). The components of LWC₇₀₈ and LWC₇₂₅ are shown by the orange and blue lines, respectively. The observed absorbance spectrum and the sum of each component of the Gaussian function are shown by black and dotted red lines, respectively. a.u.: arbitrary unit. Source data are provided as a Source Data file.

Fig. 2. Sequence alignment of Pc-frLHC from P. crispa and the closely related LHCs.

The deduced amino acid sequence of Pc-frLHC was classified into one of the LHCI groups along with Lhca2 of Chlamydomonas reinhardtii (Cr_Lhca2), Lhca-J of a marine green alga, Bryopsis corticulans (Bc_LhcaJ), and Lhca5 of a halophilic green alga, Dunaliella salina (Ds_Lhca5) with amino acid sequence identities of 32%, 32%, and 29%, respectively. Recent analyses revealed that Cr_Lhca2, Bc_LhcaJ, and Ds_Lhca5 are orthologs and loosely bound to the side of green algal PSI in a heterodimeric state together with Cr_Lhca9, Bc_LhcI, and Ds_Lhca6, respectively^–. On the basis of these results, we concluded that Pc-frLHC is phylogenetically a member of LHCI, while it transfers excitation energy to PSII. Transmembrane helices are shaded in different colors, the signal peptides are shaded, chlorophyll-binding sites (followed by the nomenclature of Liu et al.) are shown as red characters, and the sequences detected by N-terminal sequences are shown by arrows (The Mg²⁺ in Chl606 indirectly interact with Glu via a water molecule). The signal peptides and the transmembrane helices were predicted from the 3D structures registered in the PDB (Cr_Lhca2; 6JO5, 6IJO, Bc_LhcaJ; 6IGZ, Ds_Lhca5; 6SL5) and from the results of secondary structural prediction using Jpred 4 and TargetP-2.0. The Chl708 binding site in P. crispa is shown in blue.

Fig. 3. Phylogenetic tree of light-harvesting complexes (LHCs) of PSI and PSII in green algae and plants.

Evolutionary analyses of LHCs were conducted with the neighbor-joining method in MEGA7. We used amino acid sequences of only those LHCs in which 3D structural analyses had been achieved. The optimal tree with the sum of branch lengths = 17.75479796 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the Poisson correction method. The rate variation among sites was modeled with a gamma distribution (shape parameter = 3). While most LHC subunits have only three transmembrane helices (TMH), a few LHC subunits, such as Cr_Lhca2, Bc_LhcaJ (Bc_Lhca-j in the figure), and Ds_Lhca5, have been known to possess a fourth transmembrane helix. Abbreviations: Cm: Cyanidioschyzon merolae (red alga); Cr: Chlamydomonas reinhardtii; Bc: Bryopsis corticulans; Ds: Dunaliella salina; Pc: Prasiola crispa (green algae); At: Arabidopsis thaliana (plant). Blast accession numbers: Cm_Lhcr1 (XP_005538084), Cm_Lhcr2 (XP_005537362), Cm_Lhcr3 (5ZGB_3), Cr_Lhca1 (6IJJ_1), Cr_Lhca2 (XP_001691031), Cr_Lhca3 (PNW76422), Cr_Lhca4 (6IJJ_4), Cr_Lhca5 (6IJJ_5), Cr_Lhca6 (6IJJ_6), Cr_Lhca7 (AAO16495), Cr_Lhca8 (6IJJ_8), Cr_Lhca9 (XP_001692548), Cr_CP29 (XP_001697193), Cr_PSBS2 (XP_001689923.1), Cr_CP26 (XP_001695927), Cr_LHCII (XP_001700243.1), Bc_Lhca-j (6IGZ_0) (Bc_LhcaJ), Bc_Lhca-a (6IGZ_1), Bc_Lhca-c (6IGZ_2), Bc_Lhca-d (6IGZ_3), Bc_Lhca-b (6IGZ_4), Bc_Lhca-g (6IGZ_6), Bc_Lhca-h (6IGZ_7), Bc_Lhca-i (6IGZ_9), At_Lhca1 (NP_191049.1), At_Lhca2 (NP_191706.2), At_Lhca3 (NP_001185280.1), At_Lhca4 (NP_190331.3), At_Lhca5 (NP_175137.1), At_Lhca6 (NP_173349.1), At_CP26 (NP_192772.1), At_CP29 (NP_195773.1), At_CP24 (NP_173034.1), At_LHCII (NP_174286.1), At_PSBS (NP_001319163.1), Ds_Lhca1 (6RHZ_1), Ds_Lhca2 (6RHZ_2), Ds_Lhca3 (6SL5_3), Ds_Lhca4 (6QPH_4), Ds_Lhca5 (6SL5_5), Ds_Lhca6 (6SL5_6).

Fig. 4. Summary of Cryo-EM analysis.

a Angular distribution of the cryo-EM particles. b Gold standard Fourier shell correlation (FSC) curves of the refined 3D reconstruction. c The 3D reconstruction is colored according to the local resolution. Top: stromal side; bottom: lumenal side. d Structures of Pc-frLHC subunits fitted to the cryo-EM map. e Representative densities of the Chl trimer. f: Map-to-model FSC curve.

Fig. 5. Overall cryo-EM structure of Pc-frLHC.

a Top (upper panel) and side (lower panel) views from the stromal side and outside of the undecamer ring, respectively, of Pc-frLHC are shown with the cryo-EM map. Each subunit is shown in a different color. b Top and side views (the same views as in (a)) of the subunit of Pc-frLHC in rainbow colors from the N-terminus in blue to the C-terminus in red. c Chlorophyll a arrangement (the same views as in (a)) in Pc-frLHC (left panel). Each chlorophyll appears in the same color as the corresponding subunit in (a). The right panel shows the arrangement of chlorophyll a in the subunit. Chlorophylls on the stromal and lumenal sides are shown in green and cyan. Chl708 is shown in purple.

Fig. 6. Superposition of Pc-frLHC subunit (green) and Cr_Lhca2 (magenta; PDB: 7DZ7).

a Top and side views of Pc-frLHC and Cr_Lhca2 (only peptide chains) from the stromal side. Four transmembrane helices (A, B, C and F) and one amphiphilic helix (D) are labeled. b The same views as in (a) showing Chls and carotenoids. Chls common to both proteins are shown with black numbers. Chls unique to Pc-frLHC and Cr_Lhca2 are shown with green and magenta numbers, respectively. c Comparison of the two neighboring subunits of Pc-frLHC (green and cyan) with Cr_Lhca2-Cr_Lhca9 heterodimer (PDB ID: 7DZ7) (magenta and yellow). Pc-frLHC (green) is superposed with Cr_Lhca2 (magenta). d Subunit‒subunit interactions of Cr_Lhca1a-Cr_Lhca1b-Cr_Lhca8 (red, salmon, and yellow) and Pc-frLHC (green and cyan). Pc-frLHC (green) is superposed with Cr_Lhca1a (red). The bottom panel is an enlarged view of subunit‒subunit interactions of Pc-frLHC by long N-terminal loop regions from neighboring subunits. e Subunit‒subunit interactions of Cr_Lhca1a-Cr_Lhca1b-Cr_Lhca8. Transmembrane helices are labeled.

Fig. 7. The chlorophyll network in Pc-frLHC.

Connections among chlorophyll a in Pc-frLHC. Energetically connected stromal chlorophylls (including Chl708) are linked by dotted lines (Supplementary Table 3). The dotted lines are colored based on the values of exciton couplings (EC cm⁻¹ unit): EC > 60 in red, 60 ≥ EC > 30 in orange, and 30 ≥ EC > 10 in gray. Chlorophylls on the stromal and lumenal sides are shown in green and cyan, respectively. Chl708 is shown in purple. While Chls613’ and 614’ are energetically connected to Chls708, 612’, and 601’, for clarity they are shown only partially in this figure. The upper panel is viewed from the stromal side and the lower panel is viewed from outside the undecamer ring.

Fig. 8. Spectroscopic analysis of Pc-frLHC.

a Fluorescence time profiles of Pc-frLHC excited at 740 nm and monitored at 680 nm (green) observed at 273 K. The blue line shows the fitting curve to the sum of three exponential components convolved with the instrumental response function shown by red circles. b Temperature dependence of the fluorescence spectrum of Pc-frLHC excited at 460 nm. The blue lines are the fitting curves to the sum of two Gaussian functions (the filled green and orange curves). The inset shows the analysis of the Area_F713/Area_F730 ratio using the Arrhenius equation. The blue line is the fitting according to Eq. 2 (see Methods) with the energy gap fixed to 318 cm⁻¹. The error bars on the data in panel b inset were estimated from the standard errors of the fitting of the spectra to the sum of two Gaussian curves. c–e Fluorescence decay‒associated spectra of Pc-frLHC excited at 460 nm and observed at 273 K (c), 201 K (d), and 80 K (e). Source data are provided as a Source Data file.

Fig. 9. Kinetic model of the EET in Pc-frLHC.

The blue box shows the Chl b and carotenoid (Car) pool that is directly excited by the 460 nm laser. The orange, red, and dark-red boxes are the Chl pools of 680 nm‒emitting bulk Chl, the 713 nm‒emitting LWC₇₀₈ pool, and the 730 nm‒emitting LWC₇₂₅ pool, respectively. The widths of the boxes roughly express the number of pigments contained in the pools. The blue and green arrows indicate the downhill and uphill energy transfers, respectively. The rate of the uphill energy transfer is temperature dependent. The winding blue arrow shows the energy migrations between the isoenergetic LWC₇₀₈ pigments. Red arrows indicate deexcitation by the fluorescence emission, dissipation to heat, etc. The blue dotted open arrow shows a possible energy exit for PSII. Approximate time constants are given beside the arrows.