Direct energy transfer from photosystem II to photosystem I confers winter sustainability in Scots Pine

Abstract

Evergreen conifers in boreal forests can survive extremely cold (freezing) temperatures during long dark winter and fully recover during summer. A phenomenon called "sustained quenching" putatively provides photoprotection and enables their survival, but its precise molecular and physiological mechanisms are not understood. To unveil them, here we have analyzed seasonal adjustment of the photosynthetic machinery of Scots pine (Pinus sylvestris) trees by monitoring multi-year changes in weather, chlorophyll fluorescence, chloroplast ultrastructure, and changes in pigment-protein composition. Analysis of Photosystem II and Photosystem I performance parameters indicate that highly dynamic structural and functional seasonal rearrangements of the photosynthetic apparatus occur. Although several mechanisms might contribute to 'sustained quenching' of winter/early spring pine needles, time-resolved fluorescence analysis shows that extreme down-regulation of photosystem II activity along with direct energy transfer from photosystem II to photosystem I play a major role. This mechanism is enabled by extensive thylakoid destacking allowing for the mixing of PSII with PSI complexes. These two linked phenomena play crucial roles in winter acclimation and protection.

Fig. 1. Seasonal dynamics of weather and photochemical performance of PSII measured by chlorophyll fluorescence in Scots pine needles.

Changes in temperature (°C) (Left Y-axis) and solar radiation (watt m⁻²) (Right Y-axis) during 2015–2016 (a), 2016–2017 (b), 2017–2018 (c) measuring seasons. Seasonal dynamics of PSII photochemistry: (d) Changes in maximal (Fm) and basic (Fo) fluorescence. (e) Maximal quantum efficiency of PSII measured as Fv/Fm (f) Effective quantum yield of PSII (Φ(II)). Energy dissipation measured as regulated and non-regulated non-photochemical quenching: (g) Changes in NPQ with increasing PAR. (h) Induction of NPQ with constant actinic light. (i) Quantum yield of non-regulated non-photochemical quenching. Recovery of pine needles under artificial conditions at 80 µmol photons m⁻² s⁻¹ of light for 48 h with 18/6 photoperiod: (j) Changes in Fo. Fm and Fv/Fm. (k) Changes in Φ(II). (l) Induction of NPQ with constant actinic light. Quantum yields were calculated at actinic light illumination of 300 μmol m⁻² s⁻¹ in the light response curve and NPQ induction was measured either at 300/800 μmol m⁻² s⁻¹ of actinic light illumination. All measurements were taken after 30 min of dark adaptation at 4 °C from winter to late spring and room temperature in summer. Error bars indicate the mean ± SD (n = 3) for (d–g), and (i); ± SD (n = 4) for (j, k). Error bars indicate the mean ± SE (n = 3) for (h, l). The statistically significant mean differences (t-test) are marked by asterisks indicating the following confidence intervals: *≤95%; **≤99%.; ***≤99.9%. Exact p values are provided in the source data file.

Fig. 2. Seasonal changes of PSI photochemistry in Scots pine needles.

Energy partitioning in PSI considering Y(I) + Y(ND) + Y(NA) = 1, where Y(I) (a), Y(NA) (b), Y(ND) (c) are the photochemical quantum yield of PSI (when P700 is reduced and A is oxidized), energy dissipation in PSI (a measure of acceptor side limitation, when P700 and A both are reduced) and energy dissipation in PSI (a measure of donor side limitation, when P700 and A both are oxidized), respectively. Recovery of PSI photochemistry under artificial conditions: (d) Y(I), (e) Y(NA), (f) Y(ND) of PSI during the recovery period. Quantum yields were calculated from 300 μmol m⁻² s⁻¹ light illumination period of a light response curve. All measurements were taken after 30 min of dark adaptation at 4 °C from winter to late spring and room temperature in summer. Error bars indicate the mean ± SD (n = 3) for (a–c); ± SD (n = 4) for (d–f). The statistically significant mean differences (t-test) are marked by asterisks indicating the following confidence intervals: * ≤95%; ** ≤99%.; *** ≤99.9%. Exact p values are provided in the source data file.

Fig. 3. Time-resolved fluorescence of intact pine needles measured using TCSPC.

(a) Fluorescence decay traces measured at −20 °C and at four characteristic wavelengths: 686 nm (mainly PSII, LHCII contributions), 698 nm (PSII, PSI contributions), 723 nm (mainly PSI contribution), and 741 nm (mainly PSI contribution). (b) Global analysis of pine needles in three states: Summer dark (S, dark-adapted summer needles, left row), ES (E.spring needles with “sustained quenching” present, middle row), Summer quenched (SQ, right row) (c) Kinetic target analysis of pine needles in the three states. See the main text for the explanations. The kinetic target analysis (SAS top, a kinetic model with rate constants in ns-1, bottom) shows the results of the detailed target modeling of the fluorescence kinetics of pine needles. The rate constants (ns⁻¹) and Species-associated emission spectra (SAS) resulted were determined from global target analysis. Species-associated emission spectra (SAS) resulted from the fit of the target kinetic model in the corresponding state. Note that fluorescence decay measurements below 680 nm to detect the decreasing short-wavelength part of the spectra were not possible due to the extremely high scattering of the pine needles. This has no effect however on the ability to distinguish the various lifetime components kinetically and spectrally.

Fig. 4. Transmission electron microphotographs depicting seasonal variations in chloroplast ultrastructure in pine needles.

a Chloroplast structure in Autumn, Winter, E. Spring, L. Spring, and Summer. b Histograms of frequency distributions of numbers of thylakoids per granum during the five distinct seasonal periods. The histograms were calculated from 80–100 electron micrographs per season, representing 2–3 chloroplasts per image [Error bars indicate mean ± SD calculated from n = 1124 (Autumn), 1235 (Winter), 521 (Early Spring), 578 (Late Spring) and 1048 (Summer) of grana stacks].

Fig. 5. Artificial induction of changes in chloroplast ultrastructure of pine needles.

a Changes in chloroplast ultrastructure in E. spring (ES), E. spring samples recovered (ER) at 18^oC for 48 h with a photoperiod of 18 h at 80 µmol m⁻² s⁻¹, ER samples treated with 800 µmol  m⁻² s⁻¹ high light for 30 min (ERQ1), for 60 min (ERQ2). Summer (S), Summer samples treated with 1200 µmol m⁻² s⁻¹ high light for 30 min (SQ1), for 60 min (SQ2). b The number of grana per chloroplasts (Error bars indicate mean ± SD (n = 75); c Histograms of frequency distributions of numbers of thylakoids per granum in different E. spring treated [n = 220 (ES), 250 (ER), 272 (ERQ1), 246 (ERQ2)]. d Summer treated [n = 576 (S), 498 (SQ1), 415 (SQ2)] samples. Error bars indicate the mean ± SD obtained from the analysis of grana stacks.

Fig. 6. Molecular model for acclimation of photosynthetic machinery under changing natural environmental conditions.

Changes in Summer unquenched (a); Winter quenched (b); Summer quenched (c) in Scots pine. The larger text indicates the process is dominating under that condition. Text colors correspond to the legend colors of the figure.