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Dynamic thylakoid stacking and state transitions work synergistically to avoid acceptor-side limitation of photosystem I

Nature Plants volume 7, pages 87–98 (2021)Cite this article

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A Publisher Correction to this article was published on 29 January 2021

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Abstract

TAP38/STN7-dependent (de)phosphorylation of light-harvesting complex II (LHCII) regulates the relative excitation rates of photosystems I and II (PSI, PSII) (state transitions) and the size of the thylakoid grana stacks (dynamic thylakoid stacking). Yet, it remains unclear how changing grana size benefits photosynthesis and whether these two regulatory mechanisms function independently. Here, by comparing Arabidopsis wild-type, stn7 and tap38 plants with the psal mutant, which undergoes dynamic thylakoid stacking but lacks state transitions, we explain their distinct roles. Under low light, smaller grana increase the rate of PSI reduction and photosynthesis by reducing the diffusion distance for plastoquinol; however, this beneficial effect is only apparent when PSI/PSII excitation balance is maintained by state transitions or far-red light. Under high light, the larger grana slow plastoquinol diffusion and lower the equilibrium constant between plastocyanin and PSI, maximizing photosynthesis by avoiding PSI photoinhibition. Loss of state transitions in low light or maintenance of smaller grana in high light also both bring about a decrease in cyclic electron transfer and over-reduction of the PSI acceptor side. These results demonstrate that state transitions and dynamic thylakoid stacking work synergistically to regulate photosynthesis in variable light.

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Fig. 1: Changes in thylakoid grana diameter and excitation energy distribution between photosystems in LL and HL.
Fig. 2: Photosynthetic properties of WT, tap38, stn7 and psal Arabidopsis plants determined by infrared gas exchange, chlorophyll fluorescence and P700 absorption spectroscopy.
Fig. 3: DIRK analysis of the reduction PSI (P700+), Pc+ and oxidation of Fd−.
Fig. 4: Investigating possible causes of defective electron transfer regulation in stn7 and tap38.
Fig. 5: Comparison of PSI acceptor-side limitation and CET.

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Data availability

The datasets analysed during the current study are available from the corresponding author on reasonable request. The sequence data from this article can be found in The Arabidopsis Information Resource or GenBank/EMBL database under the following accession numbers: STN7 (At1g68830), TAP38/PPH1 (At4t27800), CURT1A (At4g01150), CURT1B (At2g46820), CURT1C (At1g52220), CURT1D (At4g38100) and PSAL (At4g12800).

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Acknowledgements

We thank D. Leister (LMU Munich) and M. Pribil (Copenhagen Plant Science Centre) for providing seeds of the psal, curt1abcd, oeCURT1A and tap38 lines and L. Eichacker (University of Stavenger) for providing seeds of stn7. C. Hill (University of Sheffield) is acknowledged for assistance with the EM. M.P.J. acknowledges funding from the Leverhulme Trust grant nos. RPG-2016-161 and RPG-2019-045 and the BBSRC White Rose DTP for a studentship to T.Z.E.M. (BB/M011151/1). The SIM imaging was performed at the University of Sheffield Wolfson Light Microscopy Facility and was partly funded by MRC grant no. MR/K015753/1.

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  1. Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, UK

    Christopher Hepworth, William H. J. Wood, Tom Z. Emrich-Mills, Matthew S. Proctor, Stuart Casson & Matthew P. Johnson

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Contributions

M.P.J. and S.C. designed the study. C.H., W.H.J.W., T.Z.E.M. and M.S.P. performed the research. C.H., W.H.J.W., T.Z.E.M. and M.P.J. analysed the data. M.P.J., C.H., W.H.J.W., T.Z.E.M., S.C. and M.S.P. wrote the paper.

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Correspondence to Matthew P. Johnson.

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Peer review information Nature Plants thanks Toshiharu Shikanai 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 PSI and PSII functional antenna size in wild-type and mutant leaves determined by absorption/chlorophyll fluorescence spectroscopy.

PSI antenna size was calculated using P700+ formation kinetics in leaves treated for 1 hour under low light (LL, 125 µmol photons m−2 s−1) or high light (HL, 1150 µmol photons m−2 s−1) followed by infiltration with 30 μM DCMU and 1 mM methyl viologen to induce a donor-limited state around PSI. PSII antenna size was calculated from chlorophyll fluorescence induction curves on LL and HL treated leaves, then infiltrated with 30 μM DCMU. The light intensity used was 12 μmol photons m−2 s−1. P700 traces were fitted with single exponential functions; the tabulated data is the average of four traces per sample. Antenna size was calculated as (WT LL t1/2÷ sample t1/2) × 100 and is expressed as a mean percentage of wild-type LL ± SD. Variable chlorophyll fluorescence was used to calculate the PSII antenna size. The letters a-d represent significant differences calculated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, a-b PSI P=<0.0001, a-c P=<0.0001, a-d P=<0.0001, b-c P=<0.0001, b-d P=<0.0001, c-d P=<0.0001.

Extended Data Fig. 2 Stomatal characterisation of Arabidopsis mutants used in this study.

a, Stomatal density in leaves; mean ± SD is shown for each sample. b, Stomatal conductance in low light (LL, 125 µmol photons m−2 s−1) treated leaves; mean ± SD is shown for each sample. c, Stomatal conductance in high light (HL, 1150 µmol photons m−2 s−1) treated leaves; n (separate plants analysed) = 5-7 for each sample, mean ± SD is shown for each sample. All differences were found to be non-significant by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Extended Data Fig. 3 Relative levels of cytb6f and PSI complexes and ratios of Pc:PSI and Fd:PSI.

Data determined by spectroscopy on thylakoids from each sample ± SD (n (separate plants analysed) = 3 for each sample); The letters a-b represent significant differences calculated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, a-b PSI P=<0.0001, a-b PSI:Pc P=<0.0001.

Extended Data Fig. 4 Fitting procedure for dark interval relaxation kinetics (DIRK) curves and initial rates of P700 and Pc reduction and Fd oxidation.

a, Half-time obtained from an example DIRK curve for P700+ reduction via a single exponential fit. The fitting residuals are shown underneath. b, The initial rate was calculated using linear fit of the DIRK data from the 3-8 ms window in a.

Extended Data Fig. 5 Investigating possible causes of defective electron transfer regulation.

a, Light-intensity dependence of proton motive force partitioning into ΔpH and ΔΨ according the method of Kramer and Sacksteder, (2000). n = n (separate plants analysed) = 3 for each sample; mean ± SD is shown for each point. b, Equilibrium plot of P700/P700+ versus Pc/Pc+ from dark interval relaxation kinetics following low light + far-red light treatment shown in Fig. 3b (low light = 125 µmol photons m−2 s−1, far-red = 740 nm, 50 μmol photons m−2 s-1). Apparent equilibrium constants (Kapp) calculated from a linear fit of the slope. c, Light-intensity dependence of proton conductivity (gH+). n (separate plants analysed) = 3 for each sample; mean ± SD is shown for each point.

Extended Data Fig. 6 Thin-section EM of membrane architectural changes.

a, EM image showing a chloroplast from WT leaf treated for 1 hour under low light (LL, 125 µmol photons m−2 s−1), b, EM image showing a chloroplast from WT leaf treated for 1 hour under high light (HL, 1150 µmol photons m−2 s−1). Scale bars 200 nm. c, Cross-section through a grana stack with repeat lumen width labelled. d, lumen width of grana layers in LL (n (grana analysed) = 21) and HL (n (grana analysed) = 21) adapted WT leaves. e, lumen width of grana layers in HL adapted WT (n (grana analysed) = 21), tap38 (n (grana analysed) = 19) and stn7 (n (grana analysed)= 23) leaves. All differences were found non-significant by one-way analysis of variance (ANOVA). Two independent sets of micrographs were obtained with similar results.

Extended Data Fig. 7 Analysis of cytb6f distribution in thylakoid membranes from WT and mutant Arabidopsis plants.

Spectroscopic analysis of chlorophyll distribution between fractions and cytochrome f/chlorophyll content (cyt f/ chl) was performed on thylakoids isolated from leaves treated for 1 hour under low light (LL, 125 µmol photons m−2 s−1) or high light (HL, 1150 µmol photons m−2 s−1). The letters a-b represent significant differences calculated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, a-b PSI P=<0.0001.

Extended Data Fig. 8 PSI function and Infiltration control experiments.

a, % functional PSI remaining after 2 hours HL (1150 µmol photons m−2 s−1) treatment. Total functional PSI calculated by amplitude of the ECS a-phase triggered by a 50 μs 635 nm pulse upon infiltration of dark-adapted leaves with 30 μM DCMU. This was compared to HL treated leaves that were subsequently infiltrated with DCMU and % remaining calculated. n (separate plants) = 5 for each, mean ± S.D. The letters a-b represent significant differences calculated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, a-b PSI P=<0.0001. b, Proton conductivity (gH+) calculated upon before (dark) and after 15 minutes treatment with FR light (740 nm, 255 µmol photons m−2 s−1) on leaves infiltrated with 20 mM Hepes pH 7.5, 150 mM sorbitol, 50 mM NaCl, 4 mM iodoacetamide (IA). n (separate plants) = 3, mean ± SD is shown for each condition. c, Rapidly-reversible NPQ (qE) following 10 minutes HL (1150 µmol photons m−2 s−1) illumination of leaves infiltrated with either 20 mM Hepes pH 7.5, 150 mM sorbitol, 50 mM NaCl (control) or the same buffer swapping NaCl for 50 mM NaNO2. n (separate plants) = 5 for each sample; mean ± SD is shown for each condition. The letters a-b represent significant differences calculated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, a-b PSI P=<0.0001.

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Hepworth, C., Wood, W.H.J., Emrich-Mills, T.Z. et al. Dynamic thylakoid stacking and state transitions work synergistically to avoid acceptor-side limitation of photosystem I. Nat. Plants 7, 87–98 (2021). https://doi.org/10.1038/s41477-020-00828-3

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