Different carotenoid conformations have distinct functions in light-harvesting regulation in plants

Abstract

To avoid photodamage plants regulate the amount of excitation energy in the membrane at the level of the light-harvesting complexes (LHCs). It has been proposed that the energy absorbed in excess is dissipated via protein conformational changes of individual LHCs. However, the exact quenching mechanism remains unclear. Here we study the mechanism of quenching in LHCs that bind a single carotenoid species and are constitutively in a dissipative conformation. Via femtosecond spectroscopy we resolve a number of carotenoid dark states, demonstrating that the carotenoid is bound to the complex in different conformations. Some of those states act as excitation energy donors for the chlorophylls, whereas others act as quenchers. Via in silico analysis we show that structural changes of carotenoids are expected in the LHC protein domains exposed to the chloroplast lumen, where acidification triggers photoprotection in vivo. We propose that structural changes of LHCs control the conformation of the carotenoids, thus permitting access to different dark states responsible for either light harvesting or photoprotection.

Fig. 1

Spectral characterization of LHC–Asta and comparison with LHCII-WT. a Steady-state absorption spectra (normalized to the Chl content, assumed to be the same in LHC–Asta and LHCII-WT). The difference absorption spectrum (LHC–Asta minus LHCII-WT; green curve) shows features of astaxanthin in solution minus the Cars missing in LHCII-Asta (lutein, neoxanthin, and violaxanthin). The blue and the red arrows indicates the two excitation wavelengths used for the transient absorption experiments (respectively, 508 and 690 nm). b Circular dichroism (normalized to the OD) of LHC–Asta and LHCII-WT monomers. The asterisks indicate the conserved Chl excitonic bands. c A model of the protein and pigment organization of this complex based on the estimated pigment stoichiometry (about 14 Chls and three Cars per monomer, see text). In the model the protein is depicted in yellow, the Chls a and b in green and purple, respectively, and the astaxanthin molecules in orange. Native Car-binding sites in monomeric LHCs are also indicated (L1, L2, N1). d Fluorescence decay kinetics (normalized to peak value) detected at 680 nm after excitation at 470 nm, y-axis in logarithmic scale

Fig. 2

Results of the global analysis after selective excitation of Cars and Chls. a EADS estimated from the global analysis of the data collected upon 508 nm (Cars) excitation. The gray and magenta EADS associated to ultrafast (<100 fs) and long-lived (>ns) species, respectively, have been scaled for clarity as indicated in the legend. Given the short timescale (30 fs), coherent artifacts are likely present in the gray spectrum. b First three DADS normalized to their minimum for the data collected upon 690 nm (Chls) excitation. The full set of EADS and DADS for both excitation wavelengths is reported in Supplementary Figure 2a, b

Fig. 3

Target models of the excited state dynamics in LHC–Asta after Car (508 nm) and Chl (690 nm) excitations. a SADS estimated from the kinetic model for Car excitation (508 nm) shown in b. Spectrum and lifetime of S_q have been constrained to be the same for both models and for clarity the associated SADS is not shown in a. c SADS estimated from the kinetic model for 690 nm excitation shown in d. In b and d, red arrows indicate the direction of EET between the various pigments. SADS of the Car and Chl triplet states (Chl T and Car T) are assumed to be zero above 630 nm and below 630 nm, respectively. The numbers next to the arrows in b and d are rates in ns⁻¹. For clarity, the components describing unconnected astaxanthin in b, and the formation of Chl T and Car T states are not shown in b. and d. The full target models are shown in Supplementary Figure 5

Fig. 4

Dark states of astaxanthin bound to LHC–Asta. a Overlay of the SADS of the different Car dark states estimated from the target analysis reported in Fig. 3. b Simplified scheme of the potential energy surface of astaxanthin bound to LHC–Asta. In this representation, S1 is higher in energy than S* and S_q, in accordance with previously published information. S_q is positioned at the lowest level because it is found to be the only state able to quench Chls in LHC–Asta. For further explanation, see text. The red arrows indicate that S_q, similarly to S*^, , originates from twisted conformations of the Car

Fig. 5

Conformational flexibility of the Cars’ end rings in wild-type LHCII. a The dihedral distributions for the two end rings of lutein bound to site L1 and L2 of LHCII were computed over six independent MD simulations, discarding the first 400 ns of simulation per each run. The different simulations are labeled as in our previous work. The values for the dihedrals measured on three different crystals of LHCII are also reported and are indicated in the plots as Crystal-S, P or C according to the organism from which LHCII was purified (spinach, pea or cucumber). The dihedrals of the ring located at the stromal side are reported on the top panels, and those of the ring at the lumenal side are presented in the middle panels. The color scheme for all the plots follows the legend inserted in the plot associated to the L2 site, stromal side (upper right panel of a). The lower panels show the atoms used to define this dihedral angle (green for the stromal and blue for the lumenal side of the membrane). Cars are shown as orange sticks. The nearby elements of LHCII apoprotein are shown in transparent white. The left panels are for site L1 and the right for site L2. b A representation of LHCII apoprotein and of the lutein in the L2 site (depicted as in a) and, in the inset, an example of the rotations of the end-ring of lutein observed at the L2 site in one of the simulations. Different lutein conformations are depicted in different colors and aligned on top of each other