Structural determinants for red-shifted absorption in higher-plants Photosystem I

Abstract

Higher plants Photosystem I absorbs far-red light, enriched under vegetation canopies, through long-wavelength Chls to enhance photon capture. Far-red absorption originates from Chl pairs within the Lhca3 and Lhca4 subunits of the LHCI antenna, known as the 'red cluster', including Chls a603 and a609. We used reverse genetics to produce an Arabidopsis mutant devoid of red-shifted absorption, and we obtained high-resolution cryogenic electron microscopy structures of PSI-LHCI complexes from both wild-type and mutant plants. Computed excitonic coupling values suggested contributions from additional nearby pigment molecules, namely Chl a615 and violaxanthin in the L2 site, to far-red absorption. We investigated the structural determinants of far-red absorption by producing further Arabidopsis transgenic lines and analyzed the spectroscopic effects of mutations targeting these chromophores. The two structures solved were used for quantum mechanics calculations, revealing that excitonic interactions alone cannot explain far-red absorption, while charge transfer states were needed for accurate spectral simulations. Our findings demonstrate that the molecular mechanisms of light-harvesting under shaded conditions rely on very precise tuning of chromophore interactions, whose understanding is crucial for designing light-harvesting complexes with engineered absorption spectra.

Keywords: Lhca; Photosystem I; far‐red; light‐harvesting; low‐energy absorption; photosynthesis; red forms.

Fig. 1

Evolution of low‐absorbing spectral features in Viridiplantae. (a) Time tree of representative Viridiplantae species and their divergence times (in million years, Myr). The most red‐shifted fluorescence emission peak of each species' experimental spectra is highlighted in bold. Note that the PSI core complex emits at c. 720 nm. Thus, red forms associated with LHCI proteins can be readily detected only when emitting at >720 nm. The emergence of red forms (RF) can be traced back to c. 489–403 Myr co‐eval with the appearance of tall trees and canopies. (b) Low temperature (77 K) fluorescence emission spectra of representative species from green algae (Chlamydomonas reinhardtii), mosses (Physcomitrium patens), and full sun land plant angiosperms: Zea mays, Oryza sativa (Poaceae) and Arabidopsis thaliana (Brassicaceae); shade plants: Fittonia albivenis (Acanthaceae); and seagrasses (Posidonia oceanica and Cymodocea nodosa). Shade plants thrive in far‐red enriched light, while seagrasses thrive in the absence of far‐red radiation. (c) Multiple alignments of the Lhca4 (corresponding to Lhca8 for C. reinhardtii and Lhca2b for P. patens and Lhca3 protein sequences from C. reinhardtii (Cr), P. patens (Pp)), P. oceanica (Po), C. nodosa (Cn), A. thaliana (At), Z. mays (Zm), Ananas comosus (Ac), and F. albivenis (Fa). Residues binding red form Chls (Chl a603 and Chl a609) are colored in red and shown in red rectangles. Coordinating residues for Chl a615 are colored green for species with reported PDB structures. Details about the acquisition of the time tree in panel (a) and the spectra in panel (b) are reported in the Supporting Information. PS, photosystem; LHC, light harvesting complex.

Fig. 2

Spectral analysis of PSI‐LHCI and LHCI from Arabidopsis thaliana wild‐type (WT) and a603‐NH (a, c) Room temperature (RT) absorption and (b, d) 77 K fluorescence emission spectra of PSI‐LHCI (exc. λ = 440 ± 5 nm) (a, b) and LHCI (c, d) from A. thaliana WT and a603‐NH. The WT minus NH difference spectra are shown as black lines in the corresponding plots. The amplitude of the absorption difference spectra was magnified by a factor of 3, while the fluorescence difference spectra were multiplied by a factor of 0.25 in order to plot them on the same axis. Key wavelengths are indicated in nm above the respective peaks. Experiments were repeated twice independently, with similar results. PS, photosystem; LHC, light harvesting complex.

Fig. 3

Structural superposition and excitonic coupling analysis of Lhca4 and Lhca3 wild‐type (WT) and a603‐NH. (upper part) Structures of WT and a603‐NH red clusters of Lhca4 and Lhca3 from Arabidopsis thaliana PSI‐LHCI. WT structures are colored in red and orange, while a603‐NH mutant structures are colored in blue and cyan. Lines are drawn between the pigments to highlight the excitonic couplings (EC) reported in the table (lower part, values in cm⁻¹). The thickness of the line is proportional to the EC value.

Fig. 4

Spectral analysis of PSI‐LHCI and LHCI from Arabidopsis thaliana wild‐type (WT) and a603‐NH. (a) Low‐temperature (77 K) fluorescence excitation spectra of PSI‐LHCI from A. thaliana WT and a603‐NH. Far‐red fluorescence emission (734 ± 5 nm for WT and 721 ± 5 nm for the a603‐NH mutant) was followed by exciting samples from 400 to 550 nm. Spectra were normalized to the integrated area under the curve. (Solid lines represent the average of n = 3 independent biological replicates, broken lines represent the SD) The difference spectrum was magnified by a factor of 3 for better visualization. (b) circular dichroism (CD) (4°C) spectra of purified LHCI complexes from WT and a603‐NH mutants. Spectra are normalized to the same absorption in the Q_Y region. The difference spectra are shown as black lines in the corresponding plots. The experiments were independently repeated twice with similar results. PS, photosystem; LHC, light harvesting complex.

Fig. 5

Role of charge‐transfers (CTs) in enhancing far‐red absorption. (a) Schematic depiction of local excitations (left) and charge transfer excitations (right). In local excitations, electron transitions occur within orbitals of the same molecule; in charge‐transfer excitations, electrons are promoted from orbitals localized on one molecule to orbitals localized on another molecule. (b) Comparison of wild‐type (WT) and a603‐NH absorption spectra in the simulations (left) and experiments for isolated Lhca4 (right). (c) Effect of adding CT excitations to the exciton model in the a603‐NH mutant (left) and WT (right). In (b, c) the simulated spectra were rigidly shifted by −1800 cm⁻¹ to account for time‐dependent density functional theory (TD‐DFT) systematic error. (d) Energy of the lowest excited state in the a603–a609 dimer and in the a603–a609–Vio trimer. The experimental spectra of WT and a603‐NH are adapted with permission from Wientjes et al. (2012). Copyright © 2012 Elsevier B.V. All rights reserved.

Fig. 6

Spectral characteristic of different Chl‐binding mutants of Arabidopsis thaliana. (a) Fluorescence emission spectra measured on frozen intact leaves for different genotypes of A. thaliana: wild‐type (WT), koLhca3 koLhca4 lines complemented with WT sequences of Lhca3 and Lhca4 (A3WT‐A4WT), mutants lacking Chl a615 (a615‐HA and a615‐HI), koLhca3 koLhca4 (koA3A4), and a603‐NH mutant (a603‐NH), and normalized to the λ_max. (b) Barplot of the peak emission wavelength (λ_max), measured on the same genotypes. The values on the individual bars represent the mean λ_max in nm; the error bars correspond to the SD (n ≥ 5 independent biological samples). Values that are significantly different (ANOVA followed by Tukey's post hoc test at a significance level of P < 0.05) are marked with different letters. (c) PSI‐LHCI 77 K fluorescence emission spectra measured on the WT, a615‐HA, and a615‐HI genotypes (exc λ = 440 ± 5 nm). (d) Sucrose gradient fractionation of solubilized PSI‐LHCI from WT, a615‐HA, and a615‐HI plants. Five pigment‐containing bands were resolved and identified as: free‐pigments, monomeric LHCI, dimeric LHCI, PSI core complex, and PSI‐LHCI supercomplex. Experiments in panels (c) and (d) were repeated twice independently, with similar results. PS, photosystem; LHC, light harvesting complex.

Fig. 7

Comparative structural and excitonic coupling analysis of red‐shifted pigments across species. Structural superposition and excitonic coupling analysis (values in cm⁻¹) of the red cluster pigments from Fittonia albivenis (PDB 8WGH), Zea mays (PDB 5ZJI), Arabidopsis thaliana, Physcomitrium patens (PDB 7XQP), and Chlamydomonas reinhardtii (PDB 7ZQC). The analysis focused on (a) Lhca4 or the corresponding subunit, that is Lhca8 for C. reinhardtii and Lhca2b for P. patens (Yan et al., ; Gorski et al., 2022) and (b) Lhca3.