Disentangling the sites of non-photochemical quenching in vascular plants

Abstract

In nature, plants experience large fluctuations in light intensity and they need to balance the absorption and utilization of this energy appropriately. Non-photochemical quenching (NPQ) is a rapidly switchable mechanism that protects plants from photodamage caused by high light exposure by dissipating the excess absorbed energy as heat. It is triggered by the pH gradient across the thylakoid membrane and requires the protein PsbS and the xanthophyll zeaxanthin. However, the site and mechanism of the quencher(s) remain unknown. Here, we constructed a mutant of Arabidopsis thaliana that lacks light-harvesting complex II (LHCII), the main antenna complex of plants, to verify its contribution to NPQ. The mutant plant has normally stacked thylakoid membranes, displays no upregulation of other LHCs but shows a relative decrease in Photosystem I (PSI), which compensates for the decrease of the PSII antenna. The mutant plant exhibits a reduction in NPQ of about 60% and the remaining NPQ resembles that of mutant plants lacking chlorophyll (Chl) b, which lack all PSII peripheral antenna complexes. We thus report that PsbS-dependent NPQ occurs mainly in LHCII, but there is an additional quenching site in the PSII core.

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Fig. 1: Biochemical characterization of NoLHCII.
Fig. 2: Mutant phenotype.
Fig. 3: Functional antenna size of PSII.
Fig. 4: Thylakoid ultrastructure.
Fig. 5: NPQ characteristics of WT, NoLHCII and Ch1 plants.

Data availability

All data generated or analysed during this study are included in the published article and its Supplementary Information.

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Acknowledgements

We thank L. Roy for constructing the knockdown line, S. Jansson for the gift of the amiLhcb1 and amiLhcb2 seeds, A. Rubert Albiol for help in selecting the knockdown line and H. van Amerongen for helpful discussion. Electron microscopy was performed at the Vrije Universiteit Electron Microscopy Facility. R.C. received financial support from the Netherlands Organization for Scientific Research (NWO) (86510013) and the European Commission (EC) (214113). W.J.N. was supported by a European Commission Marie Curie Actions Individual Fellowship (799083). L.N. received financial support from the New Zealand Government through the Royal Society of New Zealand–Rutherford Foundation.

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R.C. conceived the project. L.N. and W.J.N. performed the experiments. Data were analysed and interpreted by L.N., W.J.N. and R.C. The manuscript was written by L.N. with contributions by W.J.N. and R.C.

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Correspondence to Roberta Croce.

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Peer Review Information Nature Plants thanks Peter Jahns and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identity of Band 4 present in NoLHCII.

a, Sucrose gradients of WT and NoLHCII thylakoids. b, Coomassie-stained SDS-PAGE of NoLHCII sucrose bands with WT thylakoids, band 3 and 4 loaded as controls. c-d, Absorption spectra of bands 3 and 4 from WT and NoLHCII, normalized to the Qy maximum. Note the increased Chl a/b ratio of the NoLHCII samples e-f, 77 K fluorescence emission spectra of bands 3 and 4 from WT and NoLHCII excited at 440 nm. Spectra are normalized to their maximum value. Note the appearance of a band peaking around 730 nm in NoLHCII samples. This indicates the presence of Lhca’s, as the two functional heterodimers, Lhca1/4 and Lhca2/3, both emit in this region, with a maximum around 730 nm at 77 K62. a-f, Experiments were repeated independently, twice with similar results.

Extended Data Fig. 2 Distribution of granal width and height in WT (a,c) and NoLHCII (b,d) thylakoids.

Transmission electron microscopy was performed on transverse sections of dark-adapted 5-week-old Arabidopsis leaves. The number of grana in each size class is presented as a percentage of the total number of grana measured. This was 203 and 235 for WT and NoLHCII, respectively. According to Student’s t-test (two-tailed), there is no significant difference in grana width; t(438) = - 1.76, p = 0.08, however there is a small but significant decrease in grana height in NoLHCII (0.09 ± 0.04) compared to WT (0.11 ± 0.05); t(438) = 4.46, p = 1.06E-5. Data represent the mean ± s.d. (n= 3 three biologically independent samples).

Extended Data Fig. 3 Carotenoid composition of WT and NoLHCII leaves normalised to 100 Chl a.

1. The data represent the mean ± s.d (n = 3 biologically independent samples) 2. A, antheraxanthin; N, neoxanthin; V, violaxanthin; L, lutein; Z, zeaxanthin; β-C, β-carotene.

Extended Data Fig. 4 Zeaxanthin dependence of NPQ. Leaves from WT and NoLHCII were vacuum infiltrated in a Hepes-sorbitol (HS) solution with and without the presence of 5 mM DTT.

a, NPQ was induced using an illumination intensity of 1287 μmol photons m−2 s−1. The data represent the mean ± s.d (n = 3 biologically independent experiments). b, Pigment analysis of DTT infiltrated leaves via HPLC shows the absence of zeaxanthin following illumination with 1287 μmol photons m−2 s−1 for 30 minutes. This experiment was repeated independently three times with similar results.

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Nicol, L., Nawrocki, W.J. & Croce, R. Disentangling the sites of non-photochemical quenching in vascular plants. Nat. Plants 5, 1177–1183 (2019). https://doi.org/10.1038/s41477-019-0526-5

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